Site by Bennett Web & Design Co.
2006 SOUTHEASTERN NATURALIST 5(1):85–92
Population Structure of the
Tallapoosa Shiner (Cyprinella gibbsi) and the
Tallapoosa Darter (Etheostoma tallapoosae)
HEATHER M. CONNELLY1,2, CHRISTOPHER R. TABIT1, AND LEOS G. KRAL1,*
Abstract - Cyprinella gibbsi (Tallapoosa shiner) is sympatric with Etheostoma
tallapoosae (Tallapoosa darter) and both species are endemic to the Tallapoosa River
system of Georgia and Alabama. The darter population has been shown to be divided
into genetically divergent populations. In this study, mitochondrial ND4L sequences
were analyzed for 10 populations of the Tallapoosa shiner from throughout its
distribution. Phylogenetic analysis and analysis of molecular variance show that the
shiner population is also divided into genetically divergent populations. These can be
designated as management units for future monitoring of the species. The distributions
of the genetically divergent populations of the shiner and the darter are similar,
and indicate that the two species share a common biogeographic history.
Cyprinella gibbsi (Howell and Williams) (Tallapoosa shiner) is endemic
to the Tallapoosa River system in northwestern Georgia and eastern Alabama,
and this system is tributary to the Alabama River. This shiner, a midwater
dweller, is confined to that part of the Tallapoosa River system above
the fall line (Howell and Williams 1971).
Etheostoma (Ulocentra) tallapoosae Suttkus and Etnier (Tallapoosa
darter), a benthic species, is also endemic to the piedmont portion of the
Tallapoosa River system (Suttkus and Etnier 1991). Brogdon et al. (2003) have
shown that the Tallapoosa darter population is highly structured genetically,
and that genetically divergent populations are confined to specific regions of
the Tallapoosa system.
This structuring of the darter population is not surprising, since species
of the subgenus Ulocentra are mostly allopatrically distributed among and
within drainages (Bauer et al. 1995, Porter et al. 2002), presumably due to
their limited dispersal abilities. Since the Tallapoosa shiner and its sister
species, Cyprinella trichroistia (Jordan and Gilbert) (Jordan and Brayton
1878; tricolor shiner) (Broughton and Gold 2000, Mayden 1989), are allopatric
in neighboring tributary systems of the Alabama River, it is likely that
the Tallapoosa shiner is subject to the same barriers to dispersal as is the
sympatric Tallapoosa darter, and thus both may have a similar genetically
divergent population structure.
1Department of Biology, University of West Georgia, Carrollton, GA 30118. 2Current
address - School of Genome Science and Technology, University of Tennessee/
Oak Ridge National Laboratory, Oak Ridge, TN 37831. *Corresponding author -
86 Southeastern Naturalist Vol. 5, No. 1
The purpose of this study is to ascertain the degree of genetic structuring
present in populations of the Tallapoosa shiner. Significant structure will
require that future monitoring of the species be focused on particular population
groups to ensure preservation of maximum genetic heterogeneity.
Materials and Methods
A total of 98 Tallapoosa shiners was collected by seine and electrofishing
from 10 locations throughout the distribution of this species (Table 1, Fig.1).
The sample size reflects approximate species abundance at each site. Our
goal was to obtain at least 10 specimens from each site, but this was not
possible despite an increased collection effort at some sites. Individuals
were immediately preserved in 95% ethanol, and voucher specimens deposited
in the University of West Georgia collection.
Genomic DNA was isolated and quantified as previously described
(Brogdon et al. 2003). Single stranded conformation polymorphism (SSCP)
Table 1. Site numbers (in reference to Figs. 1 and 3), capture locality, and size of sample of
Cyprinella gibbsi sampled in this study.
Site Collection locality Sample size
1 Tallapoosa River, Haralson County, GA 11
2 Silas Creek, Cleburne County, AL 12
3 Lockhelooge Creek, Cleburne County, AL 11
4 Buck Creek, Carroll County, GA 9
5 Wedowee Creek, Randolph County, AL 6
6 Cornhouse Creek, Randolph County, AL 10
7 Chikasanoxee Creek, Chambers County, AL 16
8 Jay Bird Creek, Tallapoosa County, AL 10
9 Enitachopco Creek, Clay County, AL 10
10 Oakachoy Creek, Coosa County, AL 3
Figure 1. Cyprinella gibbsi sample
sites (circles) in the Tallapoosa River
system above the fall line (FL). Numbers
outside of circles refer to sample
sites and numbers inside circles refer
to sample size at each site, both as
listed in Table 1. Inset shows the extent
of the Tallapoosa River system
and the range of the Tallapoosa shiner
above the fall line (shaded oval).
Other abbreviations: TR = Tallapoosa
River, LT = Little Tallapoosa River,
HR = Harris Reservoir, and ML =
2006 H.M. Connelly, C.R. Tabit, and L.G. Kral 87
detection of ND4L haplotypes was performed in 20-μl reactions containing
10 ng of genomic DNA, 10-μl Qiagen HotstarTaq Master Mix, 0.5-μM
concentrations of primer tsND4LF, 5' AAA ATT GTG GTT TAA GTC CAC
GG 3', and primer tsND4LR, 5' AAG ATT AAG GTT TTG TAA GCG GTC
3' under the following temperature profile: 15-minute hot start at 95 oC
followed by 35 cycles of 30 seconds at 94 oC, 1 minute at 55 oC, and 2
minutes at 72 oC. The final cycle was followed by a 10-minute incubation at
72 oC. These primers amplify a 312-bp fragment, and enable the detection of
variation within 259 nucleotides of the 297 basepair-long ND4L gene. The
amplified products were denatured, fractionated by electrophoresis
(Sunnucks et al. 2000) and visualized with SYBRgold staining.
Representative samples of SSCP-detected ND4L haplotypes were characterized
by DNA sequencing. The entire mitochondrial ND4L gene and a
portion of the ND2 gene were PCR-amplified in 50-μl reactions containing 50
ng of genomic DNA, 25-μl Qiagen HotstarTaq Master Mix, 0.5-μM concentrations
of primer Arg B-L, 5' CAA GAC CCT TGA TTT CGG CTC A 3' and
primer NAP2, 5' TGG AGC TTC TAC GTG RGC TTT 3' (Broughton and
Gold 2000) under the same conditions as above. Prior to DNA sequencing,
PCR products were gel purified utilizing the Qiagen Qiaquick Gel Extraction
Kit. Automated sequencing of the ND4L gene was performed at Davis
Sequencing, Davis, CA (www.davissequencing.com), utilizing the Arg B-L
amplification primer as the sequencing primer. Sequence alignments were
carried out with GeneJockey II DNA analysis software (Biosoft). Only the
portions of the DNA sequence data that represented the SSCP discernable
portion of the ND4L gene were used to define haplotypes.
Haplotype cladogram analysis (Templeton et al. 1992) was conducted
using TCS version 1.13 (Clement et al. 2000). Statistical analysis of genetic
structuring was conducted by analysis of molecular variance (AMOVA) of
SSCP-identified haplotypes utilizing ARLEQUIN version 2.000 (Schneider
et al. 2000).
In all populations of the Tallapoosa shiner sampled, a total of 12 haplotypes
of the mitochondrial ND4L gene was identified (Table 2). All but two nucleotide
variants are synonymous substitutions that do not alter the amino acid
sequence. Position 11 variant (haplotypes C and L) results in a substitution that
replaces a valine with an alanine. Position 186 variant (haplotypes H and I)
results in a conserved amino acid substitution that replaces a leucine with a
phenylalanine. The maximum sequence divergence observed among the
haplotypes is 2.02%. This is within the range of intraspecies sequence variation.
The ND4L gene sequence divergence between the Tallapoosa shiner and
the most closely related species (Broughton and Gold 2000), Cyprinella
trichroistia (GenBank accession number: AF111249) is about 4%.
Analysis of molecular variance of all populations yielded a FST of 0.54 (P less than
0.001). This indicates a significant degree of genetic structuring of the
Tallapoosa shiner populations. The distribution of haplotypes among sample
88 Southeastern Naturalist Vol. 5, No. 1
sites is shown in Table 3. The phylogenetic relationship among the haplotypes
is represented as a gene genealogy network in Figure 2. When populations are
grouped by haplotype composition and geographic distribution and these
various groupings are tested by AMOVA, four groups of populations are
identified that maximally partition the molecular variance among the populations
(Table 4). Group 1 is comprised of populations located in the upper
Tallapoosa River north of Harris Reservoir (sample sites 1, 2, and 3), the Little
Tallapoosa River (sample sites 4 and 5), and the Tallapoosa River south of
Harris Reservoir (sample sites 6 and 7). Groups 2, 3, and 4 are comprised of
individual creek populations near Martin Lake (sample sites 8, 9, and 10,
respectively). Those haplotypes found either exclusively or at highest frequency
in any one of the four groups are shade-coded in Figure 2, and the
geographic distribution of these haplotype clusters is shown in Figure 3. In this
grouping of populations, 71.25% of genetic variation is among the four groups,
1.00% of the variation is among populations within groups, and 27.74% of the
variation is within the populations (FCT = 0.7125, P < 0.01).
While not supported by the overall AMOVA analysis, it is possible that
population group 1, as described above, may be comprised of two populations.
Haplotypes B and D comprise 19% of all haplotypes observed in
Table 2. Mitochondrial ND4L haplotypes for Cyprinella gibbsi from the Tallapoosa River
system. Only variable sites are shown at their respective base numbers. GenBank accession
Haplotype 11 51 55 93 102 111 132 156 186 189 195 246 249
A T A C A G G A G A A T T C
B T A C A G A A G A G T T T
C C A C A G G A G A A T T C
D T A C G G G A G A A T T C
E T A C A G A A A A G T T T
F T A C A G G A G A A C T C
G T A C A A G A G A A T T C
H T A T A G G A G T A T T C
I T A C A G G A G T A T T C
J T A C A G G G G A A T T C
K T G C A G A A G A A T T C
L C A C A G G A G A A T G C
Table 3. Distribution of Cyprinella gibbsi mitochondrial ND4L haplotypes at Tallapoosa River
system collection sites as listed in Table 1.
Site Haplotype (frequency)
2 A(11) F(1) G(1)
4 A(6) B(2) D(1)
6 A(6) B(1) D(1) F(1) H(1)
7 A(10) B(3) I(1) J(1) K(1)
8 B(7) E(3)
9 C(8) D(1) L(1)
2006 H.M. Connelly, C.R. Tabit, and L.G. Kral 89
samples from the Tallapoosa River tributaries south of the Harris Reservoir
and the Little Tallapoosa River (sample sites 4, 5, 6, and 7: population 1A).
These haplotypes are absent in samples from the Tallapoosa River and its
tributaries north of the Harris Reservoir (sample sites 1, 2, and 3: population
1B). When these two population groupings alone are compared by AMOVA
analysis, weak statistical support is obtained for this subdivision (FCT =
0.085, P = 0.0469 ± 0.0056).
At least four genetically divergent populations of the Tallapoosa shiner
have been identified in this study. These populations can be designated as
separate Management Units (MUs) by the criteria that these populations
differ significantly in mitochondrial allele frequencies, which indicates very
low levels of gene flow between populations (Moritz 1994a, b).
Table 4. Analysis of molecular variance (AMOVA) among populations of the Tallapoosa shiner.
Sum of Variance % of
df squares components variation
Among groups 3 34.361 0.86136 Va 71.25
within groups 6 2.772 0.01213 Vb 1.00
Within populations 87 29.176 0.33536 Vc 27.74
Total 96 66.309 1.20885
FSC 0.03490 P = 0.0000 ± 0.0000
FST 0.03490 P = 0.2053 ± 0.0118
FCT 0.03490 P = 0.0088 ± 0.0000
Figure 2. Gene genealogy network
of Tallapoosa shiner ND4L
haplotypes shown in Table 2. Shading
represents geographic prevalence
of haplotypes as listed in
Table 3. The diameter of the circles
is proportional to the relative frequency
of each haplotype among all
of the haplotypes present in the entire
Tallapoosa River system.
90 Southeastern Naturalist Vol. 5, No. 1
The Tallapoosa River system north and south of the Harris Reservoir
delineates the geographical boundary of a historically large interbreeding
population that can be considered as one MU (but see discussion below).
Potential loss of a small number of populations in western Georgia within
this MU as a result of habitat degradation (mostly siltation and impoundments;
Etnier 1997) due to urbanization is of lesser conservation concern
since these populations would not likely be genetically unique.
The portion of the Tallapoosa River system in the area of Martin Lake
contains at least three genetically divergent populations, each of which can
be considered a separate MU. It should be noted that one of these populations
(Oakachoy Creek—sample site 10) is defined only by a sample size of
3, and thus the designation of this population as a MU is tentative.
Future census data of the Tallapoosa shiner should be interpreted in terms
of stability of the MUs identified in this study. Particular attention should be
paid to the MUs present in individual streams draining into the Martin Lake
portion of the Tallapoosa River. These genetically distinct populations could
potentially harbor sequestered components of genetic diversity not present in
the larger interbreeding population north of Martin Lake.
The genetic population structure of the Tallapoosa shiner is similar to
that of the Tallapoosa darter (Brogdon et al. 2003). In particular, in the
Martin Lake area, individual creek populations of both the shiner and the
darter are nearly or completely homogeneous for individual alleles of the
Figure 3. Sample sites
(circles) and associated
haplotypes of Cyprinella
gibbsi in the Tallapoosa
River system above the
fall line (FL). Numbers
refer to sample sites as
listed in Table 1. Shades
of gray within circles represent
haplotypes as shadecoded
in Figure 2. Inset
shows the extent of the
Tallapoosa River system
and the range of the
Tallapoosa shiner above
the fall line (shaded oval).
Other abbreviations: TR =
Tallapoosa River, LT =
Little Tallapoosa River,
HR = Harris Reservoir,
and ML = Martin Lake.
2006 H.M. Connelly, C.R. Tabit, and L.G. Kral 91
mitochondrial loci studied. This suggests that the main channel of the
southern portion of the Tallapoosa River (historically) and Martin Lake
(over the last 76 years) have served as a barrier to migration in both of these
species. It is likely, therefore, that other individual creek populations of the
shiner within this portion of the Tallapoosa River drainage are also genetically
unique, and should be monitored as separate MUs.
Further comparison of the genetic structures of the Tallapoosa shiner and
the Tallapoosa darter populations indicates an additional similarity. For each
species, a historically interbreeding population has been identified by mitochondrial
allele distribution to span both the Little Tallapoosa River system
north of the Harris Reservoir and the Tallapoosa River system south of the
Harris Reservoir. This suggests that the main river channel upstream from
Martin Lake in conjunction with the Little Tallapoosa River tributary do not
have water flow rates high enough to act as a migration barrier over time for
both species. While Harris Reservoir probably is now a migration barrier, its
existence for only 20 years would not have affected the current distribution
of mitochondrial haplotypes.
Different population structures of these two species are observed in the
upper Tallapoosa River north of the Harris Reservoir (Brogdon et al. 2003).
The darter population in the upper Tallapoosa River is genetically unique
compared to the population in the Little Tallapoosa River and the Tallapoosa
River south of the Harris Reservoir. This indicates that the confluence of the
Upper Tallapoosa River and the Little Tallapoosa River served as a migration
barrier for the Tallapoosa darter prior to the construction of Harris Reservoir.
In contrast, the shiner populations compared in these same waters are not
unique. The A haplotype is the predominant haplotype in all of the Tallapoosa
shiner populations in this entire portion of the Tallapoosa River system.
However, examination of the distribution of shiner haplotypes reveals that
some of the minor haplotypes present in the population range of the Little
Tallapoosa River and the Tallapoosa River south of Harris Reservoir do not
occur in the populations of the upper Tallapoosa River. While statistical
support for genetic differentiation between these populations is not strong, it
suggests that the confluence of the Upper Tallapoosa River and the Little
Tallapoosa River have served as a barrier to migration for the shiner as well.
In conclusion, at least four genetically divergent populations of the
Tallapoosa shiner have been identified that can be designated as MUs for
future monitoring. From analysis of the distribution of the genetically distinct
populations identified in this study, it is likely that populations in other
creeks in the Martin Lake area are also genetically distinct. The haplotype
distribution of these populations should be determined.
The genetic population structures of the Tallapoosa shiner and the
Tallapoosa darter suggest that the two species have a common biogeographic
history. This is interesting since these species occupy different niches, with
the darter being a benthic species and the shiner a mid-water species. Due to
their strongly different life histories, it is unexpected that both the shiner and
the darter would be impeded by the same barriers to dispersal.
92 Southeastern Naturalist Vol. 5, No. 1
The authors thank Stephen Brogdon, William Bouthillier, Lahna McGee, Richard
Childers, and Heidi Banford for help with stream collections. We thank Heidi
Banford, Rudolf Arndt, and four anonymous reviewers for providing valuable comments
on the manuscript. This study was supported by Faculty Research Grant funds
obtained from the University of West Georgia.
Bauer, B.H., D.A. Etnier, and N.M. Burkhead. 1995. Etheostoma (Ulocentra) scotti
(Osteichthyes: Percidae), a new darter from the Etowah River system in Georgia.
Bulletin of the Alabama Museum of Natural History 17:1–16.
Brogdon, S.M., C.R. Tabit, and L.G. Kral. 2003. Population structure of the
Tallapoosa darter (Etheostoma tallapoosae). Southeastern Naturalist 2:487–498.
Broughton, R.E., and J.R. Gold. 2000. Phylogenetic relationships in the North
American cyprinid genus Cyprinella (Actinopterygii: Cyprinidae) based on mitochondrial
ND2 and ND4L gene sequences. Copeia 2000:1–10.
Clement, M., D. Posada, and K. Crandall. 2000. TCS: A computer program to
estimate gene genealogies. Molecular Ecology 9:1657–1660.
Etnier, D.A. 1997. Jeopardized southeastern freshwater fishes: A search for causes.
Pp. 87–104, In G.W. Benz and D.E. Collins (Eds.). Aquatic Fauna in Peril.
Special Publications 1, Southeast Aquatic Research Institute, Lenz Design and
Communication, Decatur, GA. 554 pp.
Howell, W.M., and J.D. Williams. 1971. Notropis gibbsi, a new cyprinid fish from
the Tallapoosa River system in Alabama and Georgia. Copeia 1971:55–64.
Jordan, D.S., and A.W. Brayton. 1878. Contributions to North American ichthyology.
No. 3. A. On the distribution of the fishes of the allegany region of South
Carolina, Georgia, and Tennessee, with descriptions of new or little known
species. Buletin of the US National Museum 12:1–95.
Mayden, R.L. 1989. Phylogenetic studies of North American minnows, with emphasis
on the genus Cyprinella (Teleostei: Cypriniformes). University of Kansas,
Museum of Natural History, Miscellaneous Publication 80:1–189.
Moritz, C. 1994a. Defining “evolutionarily significant units” for conservation.
Trends in Ecology and Evolution 9:373–375.
Moritz, C. 1994b. Applications of mitochondrial DNA analysis in conservation: A
critical review. Molecular Ecology 3:401–411.
Porter, B.A., T.M. Cavender, and P.A. Fuerst. 2002. Molecular phylogeny of the
snubnose darters, subgenus Ulocentra (genus Etheostoma, family Percidae).
Molecular Phylogenetics and Evolution 22:364–374.
Schneider, S., D. Roessli, and L. Excoffier. 2000. Arlequin: A software for population
genetics data analysis. Ver 2.000. Genetics and Biometry Lab, Department
of Anthropology, University of Geneva, Geneva, Switzerland.
Sunnucks, P., A.C.C. Wilson, L.B. Beheregaray, K. Zenger, J. French, and A.C.
Taylor. 2000. SSCP is not difficult: The application and utility of single-stranded
conformation polymorphism in evolutionary biology and molecular ecology.
Molecular Ecology 9:1699–1710.
Suttkus, R.D., and D.A. Etnier. 1991. Etheostoma tallapoosae and E. brevirostrum,
two new darters, subgenus Ulocentra, from the Alabama river drainage. Tulane
Studies in Zoology and Botany 28:1–24.
Templeton, A.R., K.A. Crandall, and C.F. Sing. 1992. A cladistic analysis of phenotypic
associations with haplotypes inferred from restriction endonuclease mapping
and DNA sequence data. III. Cladogram estimation. Genetics 132:619–633.