The Phylageography of Hypentelium etowanum In The Southeastern US
Josh Blair
Dr. Banford
Department of Biology
Abstract
The historical basis of our research is a phylogeographic study by
Bermingham and Avise (1986). They used restriction fragment length polymorphism
(RFLP) analysis of mitochondrial DNA (mtDNA) of several species of southeastern
U.S. freshwater fishes. Their study indicated genetic breaks between eastern and
western populations of fishes, suggesting geographic population structuring limiting
dispersal and gene. Importantly, there were strong patterns of congruence across
species in the geographic placements of the genetic breaks with most occurring in
western Georgia between the rivers we include in our study. A genetic break is a fixed
genetic difference that can be as small as a single base pair substitution between
groups.
The fish we have chosen for study is Hypentelium etowanum, commonly
known as the Alabama hog sucker. They belong to the family Catostomidae, which
are fishes known as suckers that are largely restricted to North American waters. Hog
suckers are freshwater fishes and can grow up to 25 cm in length (Mettee et al.). Hog
suckers are bottom dwellers that attach themselves to rocks and other underbrush by
their suction-cup like mouths so they are not swept away in rivers. They are not
widely fished for recreationally because they provide little sport for anglers and are
not highly prized in the US for their flesh (Mettee et al.).
Our objective is to investigate the population genetics of the Alabama hog
sucker in the Tallapoosa and Chattahoochee River systems, a species that was not
included in Bermingham and Avises’ 1986 study. The Little Tallapoosa River is a
part of the greater Alabama/Mobile River system, whereas the Chattahoochee River
lies to the east and flows directly into Apalachicola Bay on the Gulf coast of the U.S.
We will determine the entire DNA sequence of a mtDNA gene cytochrome b (cyt b)
to determine if there is genetic structure at the population level in these fishes. The
techniques that we are employing (i.e. DNA sequencing) are more sensitive than the
techniques utilized by Bermingham and Avise (1986) over two decades ago. While in
the field we have learned how to electro-fish and sample tissue from fishes. This
tactic uses a Smith-Root backpack apparatus with two electro-shocking wands to stun
fishes long enough to net and capture them. We sample target species of fishes for
tissue, then tagged and preserved both the entire fish as a voucher specimen in 10%
formalin, and tissue (usually gill) separately in cryotubes in 70% ethanol from which
we extract DNA. Each fish tagged and cryotube have the same sample number which
links voucher specimen to DNA sequence data that will be generated in the future.
Tallapoosa collection locations included Little Tallapoosa R. and Buck Cr., and
Chattahoochee R. specimens were collected from Yellowdirt Cr. Individuals were
collected in a variety of habitats.
Once we collected tissue from several individuals from each drainage, we used
a Quiagen DNeasy Blood and Tissue Kit® to extract genomic DNA. It uses column
chromatography to selectively bind DNA while removing other molecules. The DNA
is eluted with ethanol in the final step. After extraction we electrophoretically ran 4µl
of each sample product with 1µl of loading dye out on a 1.5% agarose gel to estimate
quality and quantity of DNA extracted. DNA was stained with ethidium bromide to
visualize DNA under fluorescent light.
In total we collected 29 individuals of H. etowanum and two specimens of a
closely related confamilial the blacktail redhorse Moxostoma poeciliurum. Blacktail
redhorse sequence will represent interspecific genetic variation in contrast to the
intraspecific levels found in Alabama hog suckers. We have successfully extracted
DNA from 9 individuals of Alabama hogsucker from the Little Tallapoosa R. and 6
individuals from the Chattahoochee R, including 1 blacktail redhorse from the Little
Tallapoosa R.
With the continuation of this project we will be sequencing the mtDNA gene
cytochrome b (cyt b). We will PCR amplify the gene using primers specifically
designed for catostomids by Schmidt and Gold (1993). Primer L14724CYP (5” –
GTGACTTGAAAACCACCGTTG- 3’) lies at position 14724 (I in Fig. 2) of the
mtDNA genome within the glutamine t-RNA gene flanking cyt b, and primer
H15915CYP (5’ – CAACGATCTCCGGTTTACAAGAC- 3”) lies at poisiton 15915
within the threonine t-RNA gene (Schmidt and Gold, 1993; Palumbi et al., 1991).
Amplification products will be sent to a commercial facility for sequencing.
Literature Cited
Bermingham, E. and J. Avise. 1986. Molecular zoogeography of freshwater fishes in the
southestern United States. Genetics 113: 939-965.
Boschung, H.T. and R.L. Mayden. 2004. Fishes of Alabama. Smithsonian Insitution,
Washington.
Mettee, M.F., P.E. O’Neil, and J.M. Pierson. 1996. Fishes of Alabama and the Mobile
Basin. Oxmorehouse, Birmingham.
Palumbi, S., A. Martin, S. Romano, W.O. McMillan, L. Stice, and G. Grabowski.1991.
The Simple Fool Guide to PCR. V 2.0.=20
Schmidt, T.R. and Gold, J.R. 1993. Complete sequence of the mitochondrial cytrochrome
b gene in the Cherry Fin Shiner, Lythrusus roseipinnis (Teleostei Cyprindae). Copeia,
880-883.