1. Phylogeography of the Alabama hog sucker,
Hypentelium etowanum, the continuing saga II.
Tom Bohrer, Adrianna Hall, and Dr. Heidi Banford
Department of Biology, University of West Georgia, Carrollton, Georgia 30118
Materials and Methods
This project began in the summer of 2007 and is a continuing project in our lab that has involved a number of
undergraduate students from past research. Collections of fish were provided by Dr. Heidi Banford and students using
electrofishing techniques. Tissue samples were extracted from the gill region of fish and preserved in 95% ethanol. In each
instance the entire fish were preserved in 10% formalin to server as voucher specimens. DNA was extracted from each
individual using Qiagen DNA extraction kit and following manufacturers protocol for “tissue extraction” (QIAGEN, July 2006).
Genomic DNA was visualized on a 1.5% agarose gel.
The genomic DNA extracted served as a template for amplification by polymerase chain reaction (PCR). For each
individual we amplified the mtDNA cyt b gene using 20µl HotStarTaq mastermix (QIAGEN, 2005), and specific primers
(Schmidt and Gold, 1993; Kocher et al.,1989; and Meyer et al., 1990) (Table 2) to provide sufficient DNA for sequencing of
the cyt b mtDNA gene. PCR amplification was preformed as given by manufacturer’s protocol for 70µl reactions (QIAGEN,
October 2005). A touchdown PCR increment annealing program was performed using an Eppendorf Mastercycler Gradient.
PCR products were visualized on a 1.5% agarose gel (Fig. 3) using size standard ladder (QIAGEN, 2006) to determine the
size (base pairs, bp) of the PCR products .
PCR products were purified using QIAquick gel extraction kit (QIAquick Spin Handbook, 2006). Purified PCR products were
electrophoresed on a 1.5% agarose gel and visually compared to a known standard of DNA (50ng/ml) to quantify the amount
of DNA in the purified product. DNA sequencing was performed by a commercial facility, Functional Bioscience INC.
Sequences were aligned and screened for errors using Geneious DNA analysis software (ver. 3.6.2, Biomatters Ltd.).
Information
Hypentelium etowanum, also known as
the Alabama hog sucker, was originally
described in 1877 by D.S. Jordan. The
Alabama hog sucker is a freshwater fish
that ranges in the Southeastern United
States from the Chattahoochee River in
Georgia, through the Mobile River
drainage in Alabama, Mississippi, and
Tennessee (Page and Burr, 1991) (Figure
1). The fish are found mainly above the fall
line and rarely below as in the Fall Line
Hills District of the East Gulf Coast Plains
(Mettee et al, 1996). Hypentelium
etowanum are benthic fish that prefer
sand, cobble, or gravel substrate in sub
fast paced currents typical of sub high
gradient streams (Page and Burr, 1991).
Bermingham and Avise (1986)
conducted a study using restriction
fragment length polymorphisms (RFLP) of
mitochondrial DNA (mtDNA) to reconstruct
the expansion of the relationships of
conspecific populations in four species of
freshwater fish collected from river
drainages across the southeastern United
States. They observed that phylogenetic
differences in mtDNA between same-
species populations were structured
geographically. These findings suggested
that historical geographic processes were
the primary factor determining population
structure by limiting the effects of dispersal
and gene flow within the species
(Bermingham and Avise, 1986). The
geographic location of genetic breaks were
largely consistent across the four species
surveyed, occurring between drainages
ranging from central Georgia to central
Alabama.
The objective of our study was to
determine the extent of genetic differences
between populations of H. etowanum. Our
aim was to hypothesize how earth history
and geography have had a hand in
shaping genetic structure between
populations of this species by assessing
genetic variation in mtDNA cytochrome b
gene region. Direct sequencing of DNA
provides a more refined analysis of these
fishes population structure than the
techniques (RFLP) used over twenty years
ago by Bermingham and Avise (1986).
Results and Discussion
We collected about 1150 base pairs from the amplified PCR
of the mtDNA fragment of the taxa. After gene cleaning the
DNA from the taxa of the different regions, we concluded that
there are gradual genetic variations that occur in the
Hypentelium Etowanum. Possible mutations may have
occurred over time in order to have created this outcome in
differences. These preliminary results indicate that
geographic variation within H. etowanum may be consistent
with that observed by Bermingham and Avise (1986). We
furthered the research of this experiment by adding on to the
phylogenetic neighbor joining tree with the taxa from the
Little Tallapoosa Drainage. Our research indicates that the
taxa from the Tallapoosa Drainage was closely related to
that of the Chatahoochee Drainage. The Nigricans Cyt
B(Figure 4) indicates the taxa located in the northern region
of the United States. This is just a reference to provide the
information from a different geographical region of the same
taxa. This is currently an ongoing study, where the number of
fish from different locations and individuals sequenced will be
increased and added to the phylogenetic tree.
Acknowledgements
This project was funded by a National Science Foundation STEP
grant #DUE-0336571.We wish to thank J. Akins for assistance in the
laboratory and Dr. Leos Kral for technical advice.
Table 2. Primers used for PCR reaction and sequencing (Schmidt and
Gold, 1993; Kocher et al., 1989; and Meyer et al., 1990) .
Gene Primer Sequence
Cyt b L14724CYP
5’-TGACTTGAARAACCAYCGTTG-3’
H15915CYP
5’-GGCAAATAGGAARTATCATTC-3’
Literature Cited
Avise, C. John, and Bermingham, Eldredge. (1986). Molecular Zoogeography of Freshwater
Fishes in the Southeastern United States. Department of Genetics, University of Georgia, Genetics
113,939-965.
Burr, Brooks M., and Page, Lawrence M. (1991). Perterson’s Field Guides: Freshwater fishes of North
America and North of Mexico. Houghton Mifflin, New York.
Froese, Rainier et. al. (1991). Hypentelium etowanum Alabama Hog Sucker. March, 17, 2008.
http://www.fishbase.org
Figure 1. Map of southeastern U.S. indicating
distribution of H. etowanum (brown)
Figure 3. PCR products of cyt b
visualized on a 1.5% agarose gel.
Size standard is to the right
indicating fragment length of
approximately 1,200 bp.
Figure 2. Chromatographs of a portion of mtDNA cyt b sequence. Nucleotides
highlighted in color and indicated by black arrows illustrate genetic variation observed
in different haplotypes from Yellowdirt Cr. (Chattahoochee River) and Little Tallapoosa
River.
Yellowdirt
Yellowdirt
Yellowdirt
Little Tallapoosa
Species Location samples (n) cyt b
H. etowanum
Buck Cr. (T) 10 6
Indian Cr. (T) 6 2
Little Turkey Cr. (T) 3 3
UWG Campus (T) 2 2
Yellowdirt Cr. (C) 20 10
Snake Cr. (C) 6 4
Walker Cr. (LT) 7 7
Table1. Represents the locations and number of individuals from which the tissue has been collected
from the taxa. (T), ( C ), and (LT) represent Tallapoosa R., Chattahoochee R., and Little Tallapoosa
Drainage systems respectively. These samples indicate the number of individuals which tissue has been
collected as well the number of cyt b individuals from which the DNA has been sequenced.
QuickTime™ and a
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are needed to see this picture.
.0845
.0028
.0001
.0004
.0004
.0007
.0009
.0007
.0011
.0008
.00001
.00007
.0001
.0024
.00005
.0009
.0008 .0021
.0004
.0005
Nigricans Cyt B
UWG Campus
Walker Creek
Walker Creek
Walker Creek
Walker Creek
Walker Creek
Walker Creek
Yellow Dirt
Yellow Dirt
Yellow Dirt
Yellow Dirt
Yellow Dirt
Yellow Dirt
Yellow Dirt
Yellow Dirt
Yellow Dirt
Buck Creek
Buck Creek
Buck Creek
Buck Creek
Tallapoosa Drainage
Little Tallapoosa Drainage
Chattahoochee Drainage
Figure 4. Neighbor Joining Phylogenetic tree showing evolutionary distance from
each of the taxa.