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. Introduction 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 - dhbarwic@duke-energy.com. 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. Methods 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. Results 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. Site 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 Discussion 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. Acknowledgments 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. Literature Cited 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 112:216–219. 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 49:129–139. 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., Sunderland, MA. 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 120:283–289. 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.