Redeye Bass (Micropterus coosae) and Alabama Spotted Bass (M. punctulatus henshalli) Hybridization in Keowee Reservoir
D. Hugh Barwick, Kenneth J. Oswald, Joseph M. Quattro, and Robert D. Barwick
Southeastern Naturalist, Volume 5, Number 4 (2006): 661–668
Full-text pdf (Accessible only to subscribers.To subscribe click here.)
Site by Bennett Web & Design Co.
2006 SOUTHEASTERN NATURALIST 5(4):661–668
Redeye Bass (Micropterus coosae) and Alabama Spotted
Bass (M. punctulatus henshalli) Hybridization
in Keowee Reservoir
D. Hugh Barwick1,*, Kenneth J. Oswald2, Joseph M. Quattro2,
and Robert D. Barwick3
Abstract - Keowee Reservoir has supported an abundant population of native
Micropterus coosae (redeye bass) for over 30 years. Recently, redeye bass
abundance in this reservoir declined concomitantly with the establishment of anglerintroduced
Micropterus punctulatus henshalli (Alabama spotted bass). We suspected
declines in redeye bass abundance may be related to their hybridizing with the
Alabama spotted bass resulting in offspring that are difficult to differentiate from the
Alabama spotted bass. Thus, we collected tissue for genetic analyses from what was
thought to be pure redeye bass (Jocassee Reservoir, SC), the original source of
Alabama spotted bass (Lake Lanier, GA) stocked in Lake Keowee, and suspected
redeye bass x Alabama spotted bass hybrids (Keowee Reservoir, SC) to determine if
hybridization might be occurring. These analyses confirmed that hybridization
among species of Micropterus had occurred in Keowee Reservoir.
Keowee Reservoir has supported a unique and significant population of
Micropterus coosae Hubbs and Bailey (redeye bass) for more than 30 years
(Barwick and Moore 1983; Barwick et al. 1995; Duke Power, Huntersville,
NC, unpubl. data). Redeye bass are unique in Keowee Reservoir because they
are reported to survive in southeastern US reservoirs only temporarily following
impoundment (Webb and Reeves 1975, Wood et al. 1956). Barwick and
Moore (1983) speculated that the continued long-term survival of redeye bass
in Keowee Reservoir might be related to the absence of Micropterus
punctulatus (Rafinesque) (spotted bass). Both Wood et al. (1956) and Webb
and Reeves (1975) reported declines in redeye bass abundance in reservoirs
where sympatric populations of redeye bass and spotted bass occurred.
In the mid-1980s, anglers introduced (reportedly from Lake Lanier, GA)
a subspecies of the spotted bass (M. p. henshalli [Alabama spotted bass])
into Keowee Reservoir, and by the mid-1990s, this fish was the most frequently
caught sport fish in the impoundment (Duke Power, unpubl. data).
Prior to this introduction, spotted bass were not present in Keowee Reservoir
or other areas of the Savannah River Drainage.
1Duke Power, 13339 Hagers Ferry Road, Huntersville, NC 28078. 2Department of
Biological Sciences, Marine Science Program, School of the Environment, University
of South Carolina, Columbia, SC 29208. 3North Carolina Wildlife Resources
Commission, 1721 Mail Service Center, Raleigh, NC 27699. *Corresponding author -
662 Southeastern Naturalist Vol. 5, No. 4
While introduction of the Alabama spotted bass created a significant
fishery in Keowee Reservoir, their presence and establishment corresponded
with declines in redeye bass abundance. Results of electrofishing surveys
conducted from 1996 through 2002 suggested an 83% decline in redeye bass
catch rates, while Alabama spotted bass catch rates increased four-fold
(Duke Power, unpubl. data). However, growth rates of redeye bass in 1999
remained unchanged in Keowee Reservoir (Duke Power, unpubl. data) from
that reported earlier by Barwick and Moore (1983). This prompted us to
investigate hybridization rather than competition for food as a factor affecting
changes in the Micropterus community in Keowee Reservoir.
Because hybridization among species of Micropterus is common (e.g.,
Morizot et al. 1991, Pierce and Van Den Avyle 1997, Whitmore 1983), we
suspected that the black bass community in Keowee Reservoir could be
altered by hybridization if hybrid offspring typically possessed characteristics
of one of the parents as suggested by Pierce and Van Den Avyle
(1997). In as much as redeye bass x Alabama spotted bass hybridization
has been suspected in areas where their ranges overlap (Kassler et al.
2002), it has not been confirmed and was not possible in Keowee Reservoir
prior to introduction of the Alabama spotted bass. Our objective in this
study was to determine if hybridization between redeye bass and Alabama
spotted bass had occurred in Keowee Reservoir.
Keowee Reservoir is a 7435-ha impoundment built by Duke Power in the
upper Savannah River Drainage of northwestern South Carolina. This reservoir
was built primarily to serve as a source of condenser cooling water for
the 2580-MW Oconee Nuclear Station and a source of water for the 610-
MW Jocassee Pumped Storage Hydroelectric Station and the 140-MW
Keowee Hydroelectric Station. This reservoir reached full pool (243.8 m
above mean sea level) in 1971.
For comparison of genetic information in our investigation regarding
potential hybridization between redeye bass and Alabama spotted bass in
Keowee Reservoir, we sampled individuals of Alabama spotted bass from Lake
Lanier, Georgia (n = 10, 185–388 mm TL), “pure” redeye bass from Jocassee
Reservoir, South Carolina (n = 10, 112–226 mm TL), and suspected redeye
bass x Alabama spotted bass hybrids from Keowee Reservoir (n = 9, 139–250
mm TL). All specimens were collected in October 2002. Alabama spotted bass
were captured using gill nets, while redeye bass and putative redeye bass x
Alabama spotted bass hybrids were collected using a boat-mounted
electrofisher. Alabama spotted bass and redeye bass were differentiated using
taxonomic characters described by Etnier and Starnes (1993) along with the
absence of pigment in the anal and caudal fins of Alabama spotted bass and
presence of pigment in the anal and caudal fins of redeye bass (D.H. Barwick,
pers. observ.); suspected putative redeye bass x Alabama spotted bass hybrids
were identified using the presence of anatomical characters normally
2006 D.H. Barwick, K.J. Oswald, J.M. Quattro, and R.D. Barwick 663
diagnostic for each species of which white caudal lobe plus an unpigmented
anal fin or the absence of a white caudal lobe associated with a pigmented anal
fin were most useful. For all fish used in the genetic analyses, the lower lobe of
the caudal fin was removed from each individual and preserved in ethanol for
later DNA extraction.
Total nucleic acids were isolated from fin tissue with Qiagen tissue
extraction columns following the manufacturer’s protocol. A 750-base-pair
(bp) portion of the mitochondrial cytochrome-b gene was amplified from the
extracted DNA using the universal primers GLUDGL and CB3H (Palumbi
1996). Because the mitochondrial genome is inherited maternally, and thus
indicative only of the maternal parent in matings between species, hybridization
was further investigated using bi-parentally inherited nuclear loci.
Two nuclear introns were sequenced from each individual: the sixth intron
of the lactate dehydrogenase A gene (LDHA6) and the seventh intron of the
creatine kinase M locus (CKM7). Introns LDHA6 and CKM7 were amplified
with primers and conditions described in Quattro and Jones (1999);
however, no variation within or between species was observed at these two
loci. Subsequently, a third intron from the b-actin locus was obtained from
all individuals using primers and methods described in Bostrom et al.
(2002). Amplification products were precipitated, and a 20–50-ng aliquot
was used as template in ABI Dye Terminator cycle sequencing reactions.
Reactions were run on an ABI 377 automated sequencer.
Sequences were sufficiently homologous to be aligned by eye and no
gaps were necessary. Phylogenetic relationships among cytochrome-b
haplotypes were estimated in Molecular Evolutionary Genetics Analysis
(MEGA version 2.1; Kumar et al. 2001) using the neighbor-joining (NJ)
algorithm (Saitou and Nei 1987) and uncorrected pairwise differences as a
distance metric. Bootstrapping (Felsenstein 1985) was used to estimate the
reliability of phylogenetic reconstructions (1000 replicates). Sequence
divergence among observed b-actin alleles was minimal, thus no allelic
phylogeny was constructed.
About 250 basepairs (bp) of the mitochondrial cytochrome-b gene were
sequenced from a sample of 29 individuals: 10 of Alabama spotted bass from
Lake Lanier, 10 “pure” redeye bass from Jocassee Reservoir, and 9 suspected
putative redeye bass x Alabama spotted bass hybrids from Keowee Reservoir
(Table 1). A neighbor-joining phylogeny relating observed cytochrome-b
haplotypes is shown in Figure 1. Bootstrapping strongly supported the existence
of two distinct clades that included cytochrome-b sequences sampled
from Alabama spotted bass and redeye bass; spotted bass and redeye bass
sequences formed monophyletic groups. Cytochrome-b sequences sampled
from the suspected putative hybrids were not monophyletic; three of nine
individuals clustered with “pure” redeye bass, while six sequences clustered
with those collected from Alabama spotted bass (Table 1, Fig.1).
664 Southeastern Naturalist Vol. 5, No. 4
Hybridization events themselves cannot be supported unequivocally by
uni-parentally inherited genomes such as mtDNA; e.g., the mitochondrial
sequence results can be explained by morphological misdiagnosis of “pure”
redeye bass and Alabama spotted bass individuals or ancestral polymorphism.
Although morphological examination suggests that these individuals are of
hybrid origin, genetic assays of an unlinked bi-parentally inherited nuclear
locus could potentially support hybridization between species, not misidentification,
as the most likely explanation of the mtDNA patterns uncovered. To
this end, approximately 400 bp of b-actin sequence was assayed in all individuals.
Only two polymorphic sites were uncovered that differentiate redeye bass
and Alabama spotted bass individuals assayed. One site showed a putative
fixed difference between the two taxa (site 203), while the other (77) was fixed
in redeye bass but polymorphic in Alabama spotted bass (Table 1). None of the
putative hybrid individuals were heterozygous for the fixed difference at
position 203 that would indicate an F1 hybrid individual. However, three
“hybrid” individuals carried redeye bass mtDNA sequences and b-actin alleles
sampled only from the Lake Lanier Alabama spotted bass, and can be assigned
as of hybrid origin. The remaining six individuals carried Alabama spotted bass
mtDNA and Alabama spotted bass b-actin alleles, and thus cannot be differentiated
from Lake Lanier Alabama spotted bass individuals.
Table 1. Mitochondrial DNA cytochrome-b and nuclear b-actin sequence variation observed in
samples of Alabama spotted bass (SPB), redeye bass (REB) and putative hybrids (HYB). Only
variable sites are shown. Cytochrome-b sites are numbered relative to a sequence from largemouth
bass (LGMB, GenBank accession L14074). b-actin positions were taken from a manual
alignment (redundancy codes: Y = C/T, R = A/G). Alignments are available from JMQ.
Composite Cytochrome-b b-actin # observed
11 1111111122 2222222 2
5566788900 1233689900 1222359 70
6709214928 1325567847 9568251 73
REB1 TCTCACACTG AGGTCCTCTC CTGCTTT CA 3
REB4 C . . . . . . .C . . . . . . . . . . . . . . . . . . . . 2
REB6 .T . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
REB7 CA . . . .T .C . . . . . . . .G . . . . . . . . . . . 1
REB8 C . . . . .T . . . . . . . . . . . . . . . . . . . . . . 2
REB10 CA . . . . . .C . . . . . . . . . . . . . . . . . . . . 1
SPB1 . .C .CT .TCC . . . . . . .G .T TC .T .CC TG 4
SPB4 . .C .CT .TCC . . . . . . .G .T TC .T .CC YG 3
SPB5 . .C .CT .TCC . . . . . . .G .T TCAT .CC TG 1
SPB6 . .C .CT .TCC . . . . . . .G .T TC . . .CC YG 1
SPB9 . .C .CT .TCC . .A . . . .G .T TC . . .CC TG 1
HYB1 . . . . . . . . . . . . . . . . . . . . . . . . . . . TG 3
HYB2 . .C .CT .TCC . . . . . . .G .T TC .T .CC YG 2
HYB5 . .C .CT .TCC . . . . . . .G .T TC .T .CC TG 2
HYB9 . .C .C . .TCC . .C . . . .G .T TC .T .CC TG 1
HYB10 . .C .CT .TCC . . . . . .AG .T TC . . .CC TG 1
LGMB . .CT . .G .C . GAACTA.TCT AC .TC . . — 1
2006 D.H. Barwick, K.J. Oswald, J.M. Quattro, and R.D. Barwick 665
Our genetic analyses, although limited in scope, suggest that hybridization
has occurred between redeye bass and Alabama spotted bass in Keowee
Reservoir. Three putative “hybrid” individuals of nine sampled carried
redeye bass mitochondrial genomes and Alabama spotted bass nuclear alleles
at the b-actin locus. Interestingly, these individuals were homozygous for
Alabama spotted bass b-actin alleles, not heterozygous as expected of F1
hybrids (e.g., Dowling et al. 1996). Nonetheless, backcrossed individuals
would be expected to have mitochondrial genomes indicative of one taxon
Figure 1. Neighbor-joining phylogram relating cytochrome-b sequences observed in
largemouth bass (LGMB), Alabama spotted bass (SPB), redeye bass (REB) and
putative hybrids (HYB). The phylogeny was rooted with a comparable sequence
from M. dolomieu (GenBank accession AY225685), although for clarity, this branch
was omitted. Numbers represent bootstrap support for individual nodes; only those
nodes supported at greater than 50% are shown. Taxa names are as in Table 1; letters
following taxa names are b-actin genotypes (see Table 1 for detail).
666 Southeastern Naturalist Vol. 5, No. 4
but carry two alleles indicative of the other, precisely the pattern we observe
in Keowee Reservoir hybrids. Subsequent crosses between these F1 hybrids
and spotted bass would produce individuals with redeye bass mitochondrial
genomes that are homozygous for Alabama spotted bass nuclear alleles. It
follows then that F1 hybrids of the two parental species must be somewhat
fertile, suggesting hybridization is ongoing.
Our nuclear gene analyses are somewhat limited since only one of three
loci examined yielded diagnostic differences between the two species. It
would be desirable to survey a suite of diagnostic nuclear gene loci to
assay the extent of backcrossing in this system. Similarly, it is not clear if
the putative hybrid individuals with Alabama spotted bass mitochondrial
genomes and Alabama spotted bass nuclear alleles are backcrossed hybrids,
misidentified “parentals,” or represent ancestral polymorphism. We
presume the former, given our morphological criteria, but further genetic
analyses would be necessary to differentiate between these hypotheses. It
is clear, however, that our hypothesis of hybridization between introduced
Alabama spotted bass and native redeye bass in Keowee Reservoir is
supported by the current genetic data.
Hybridization and genetic introgression between redeye bass and Alabama
spotted bass in Keowee Reservoir are major concerns for the native
redeye bass population. It is likely that morphologically and genetically
“pure” populations of redeye bass will become rare in Keowee Reservoir,
particularly if backcrossing is common. Hybridization and introgression
between redeye bass and spotted bass appear to best explain the decline in
redeye bass abundance in Keowee Reservoir and may explain similar declines
of redeye bass populations in other reservoir systems (e.g., Webb and
Reeves 1975, Wood et al. 1956). Although redeye bass in Keowee reservoir
may be increasingly found only in atypical combinations with Alabama
spotted bass alleles, they are not the “pure” redeye bass genotypic combinations
that predate the Alabama spotted bass introduction. After the demise of
native redeye bass genotypes, detection of redeye bass phenotypes will
likely become difficult during field sampling (our field experiences lead us
to believe that this is presently the case in Keowee Reservoir). Of course, the
creation of atypical genotypic combinations in Keowee Reservoir is not
restricted to just the redeye bass population, since redeye bass genes have
presumably introgressed into the Alabama spotted bass population as well.
We can only speculate at this point whether a loss of fitness or outbreeding
depression might jeopardize the survival, reproduction, and growth of
Keowee Reservoir basses (Philipp et al. 2002).
The harm caused to this unique population of redeye bass in Keowee
Reservoir by introduction of Alabama spotted bass is permanent and may
impact other areas of the Savannah River Drainage that currently support
native redeye bass. Alabama spotted bass from Keowee Reservoir are expanding
their range to nearby reservoirs, including Jocassee and Hartwell
reservoirs (Dan Rankin, SC Department of Natural Resources, Pendleton, SC,
2006 D.H. Barwick, K.J. Oswald, J.M. Quattro, and R.D. Barwick 667
pers. comm.). We suspect that hybridization with redeye bass is a likely
outcome of this expansion, and that, ultimately, redeye bass populations in the
Savannah River Drainage might soon be restricted to isolated tributary
streams separated from the reservoirs by falls that prevent the upstream
movement of spotted bass. Successful management of isolated, rare populations
will be critical to ensure the long-term persistence of what was once a
high-quality redeye bass fishery in the Savannah River Drainage.
We thank C. Baker, A. Rabern, and R. Weaver with the Georgia Department of
Natural Resources for providing Alabama spotted bass from Lake Lanier, and K. Baker,
D. Coughlan, and M. Rash with Duke Power for their help in collecting redeye bass
from Jocassee Reservoir and suspected redeye bass x Alabama spotted bass hybrids
from Keowee Reservoir. Funding for the genetic portions of this project was provided,
in part, by grants from the Cooperative Institute for Fisheries Molecular Biology
(FISHTEC; NOAA/NMFS (RT/F-1)) and SC Sea Grant (R/MT-5) to J.M. Quattro.
Barwick, D.H., and P.R. Moore. 1983. Abundance and growth of redeye bass in two
South Carolina reservoirs. Transactions of the American Fisheries Society
Barwick, D.H., L.E. Miller, W.R. Geddings, and D.M. Rankin. 1995. Fish biomass
and angler harvest from a South Carolina cooling reservoir. Proceedings of the
Annual Conference Southeastern Association of Fish and Wildlife Agencies
Bostrom, M.A., B.B. Collette, B.E. Luckhurst, K.S. Reece, and J.E. Graves. 2002.
Hybridization between two serranids, the coney (Cephalopholis fulva) and the
creole-sh (Paranthias furcifer), at Bermuda. US National Marine Fisheries
Service Fishery Bulletin 100:651–661.
Dowling, T.E., C. Moritz, J.D. Palmer, and L.H. Rieseberg. 1996. Nucelic acids III:
Analysis of fragments and restriction sites. Pp. 249–320, In D.M. Hillis, C.
Moritz, and B.K. Mable (Eds.). Molecular Systematics. Sinauer Associates, Inc.,
Etnier, D.A., and W.C. Starnes. 1993. The fishes of Tennessee. The University of
Tennessee Press, Knoxville, TN. 681 pp.
Felsenstein, J. 1985. Confidence limits on phylogenies: An approach using the
bootstrap. Evolution 39:783–791.
Kassler, T.W., J.B. Koppelman, T.D. Near, C.B. Dillman, J.M. Levengood, D.L.
Swofford, J.L. VanOrman, J.E. Claussen, and D.P. Phillipp. 2002. Molecular and
morphological analyses of the black basses: Implications for taxonomy and
conservation. Pp. 291–322, In D.P. Philipp and M.S. Ridgway (Eds.). Black
Bass: Ecology, Conservation, and Management. American Fisheries Society,
Symposium 31, Bethesda, MD.
Kumar, S., K. Tamura, and M. Nei. 2001. MEGA: Molecular evolutionary genetics
analysis, version 2.1. The Pennsylvania State University, University Park, PA.
Morizot, D.C., S.W. Calhoun, L.L. Clepper, J.H. Williamson, and G.J. Carmichael.
1991. Multispecies hybridization among native and introduced centrarchid
basses in central Texas. Transactions of the American Fisheries Society
668 Southeastern Naturalist Vol. 5, No. 4
Palumbi, S.R. 1996. Nucleic acids II: The polymerase chain reaction. Pp. 205–247,
In D.M. Hillis, C. Moritz, and B.K. Mable (Eds.). Molecular Systematics.
Sinauer Associates, Inc., Sunderland, MA.
Philipp, D.P., J.E. Claussen, T.W. Kassler, and J.M. Epifanio. 2002. Mixing stocks
of largemouth bass reduces fitness through outbreeding depression. Pp. 349–363,
In D.P. Philipp and M.S. Ridgway (Eds.). Black Bass: Ecology, Conservation,
and Management. American Fisheries Society, Symposium 31, Bethesda, MD.
Pierce, P.C., and M.J. Van Den Avyle. 1997. Hybridization between introduced
spotted bass and smallmouth bass in reservoirs. Transactions of the American
Fisheries Society 126:939–947.
Quattro, J.M., and W.J. Jones. 1999. Amplification primers that target locus-specific
introns in actinopterygian fishes. Copeia 1999:171–176.
Saitou, N., and M. Nei. 1987. The neighbor-joining method: A new method for
reconstructing phylogenetic trees. Molecular Biology and Evolution 4:406–425.
Webb, J.F., and W.C. Reeves. 1975. Age and growth of Alabama spotted bass and
northern largemouth bass. Pp. 204–215, In H. Clepper (Ed.). Black Bass Biology
and Management. Sport Fishing Institute, Washington, DC.
Whitmore, D.H. 1983. Introgressive hybridization of smallmouth bass (Micropterus
dolomieui) and Guadalupe bass (M. treculi). Copeia 1983:672–679.
Wood, R., R.H. Macomber, and R.K. Franz. 1956. Trends in fishing pressure and
catch, Allatoona Reservoir, Georgia, 1950–1953. Journal of the Tennessee Academy
of Science 31:215–223.