S.S. Taylor, S. Woltmann, A. Rodriguez, and W.E. Kelso 2013 Southeastern Naturalist Vol. 12, No. 3 514 2013 SOUTHEASTERN NATURALIST 12(3):514–522 Hybridization of White, Yellow, and Striped Bass in the Toledo Bend Reservoir Sabrina S. Taylor1,*, Stefan Woltmann1, Andrew Rodriguez1, and William E. Kelso1 Abstract - Long-term stocking of non-native Morone saxatilis (Striped Bass) in Toledo Bend Reservoir may have had adverse effects on the integrity of the native Morone chrysops (White Bass) and M. mississipiensis (Yellow Bass) genome through introgression. We examined microsatellite genotypes for evidence of hybridization in a sample of Striped, White, and Yellow Bass but found only four potential hybrids. Despite the introduction of millions of Striped Bass over four decades, there is no evidence for either a hybrid swarm or substantial introgression. Low numbers of hybrids may be the result of poor hybrid survival, little reproduction between species, or a combination of both. Introduction The Toledo Bend Reservoir on the border of Louisiana and Texas supports native populations of Morone chrysops Rafinesque (White Bass) and M. mississippiensis Jordan and Eigenmann (Yellow Bass), and was stocked from 1967 to 2009 with ≈9.37 million M. saxatilis Walbaum (Striped Bass) to enhance recreational fishing opportunities. Stocking ceased when surveys indicated a lack of interest in the fishery. Introduced Striped Bass can hybridize and produce fertile offspring with White Bass in the wild (Avise and Van Den Avyle 1984). Similarly, introduced Striped Bass can hybridize with Yellow Bass, producing 100% females in hatchery crosses (Wolters and DeMay 1996). We were interested in whether the integrity of the White and Yellow Bass genome had been compromised by introgression over the past four decades. The introgression of genes from one species into another is a serious concern because it has caused or contributed to the extinction of many species, including several fishes (Allendorf and Luikart 2007, Rhymer and Simberloff 1996). Moreover, White and Yellow Bass may waste reproductive effort by mating with Striped Bass, which can depress population growth rates and decrease population size (Leary et al. 1993, Rhymer and Simberloff 1996). From a hatchery perspective, hybridization may be a problem if hybrids cannot be distinguished from Striped Bass and are subsequently used by hatcheries as brood stock during annual spawning efforts (Fries and Harvey1989, Woods et al. 1995). Hatchery operations may also unintentionally release hybrid bass via drains, which may contribute to the overall numbers of White Bass x Striped Bass hybrids (B. Reed, Louisiana Department of Wildlife and Fisheries [LDWF], Baton Rouge, LA, pers. comm.). In Toledo Bend Reservoir, qualitative observations by the LDWF indicate that putative Striped Bass egg color, egg morphology, and broodstock size have changed through time (R. Yeldell, LDWF, Anacoco, LA, 1 School of Renewable Natural Resources, Louisiana State University AgCenter, RNR Building, Baton Rouge, LA 70803. *Corresponding author - staylor@agcenter.lsu.edu. 515 S.S. Taylor, S. Woltmann, A. Rodriguez, and W.E. Kelso 2013 Southeastern Naturalist Vol. 12, No. 3 pers. comm.). Furthermore, preliminary allozyme studies indicate that at least F1 hybrids are present in the reservoir (W.E. Kelso, unpubl. data). Collectively, these observations suggest that hybrids are present in the reservoir, which may have ultimately affected the genetic integrity of both native and introduced Morone species. However, data collected to date cannot quantify the degree of backcrossing between hybrids and the parental species. To assess whether a hybrid swarm is present, analyses of multiple, co-dominant loci such as microsatellites are necessary (Boecklen and Howard 1997). In this paper, we assess whether long-term stocking of Striped Bass has created hybrid bass swarms and altered the genetic make-up of native Morone species in Toledo Bend Reservoir. We also examine whether identification of Striped Bass and hybrid Striped Bass based on morphological characteristics is reliable; an important concern related to broodstock collection (Avise and Van Den Avyle 1984, Kerby 1979; Scribner et al. 2000). To address these issues, we present data on the frequency of genetically identified hybrids observed in a sample from the reservoir, and whether hybrids were correctly identified in the field. Methods White Bass (n = 25), Yellow Bass (n = 20), Striped Bass (n = 27), and morphologically identified hybrid White Bass x Striped Bass (n = 3) were captured in 2011 by electrofishing on the Sabine River above the Toledo Bend Reservoir, or by rod and reel in the Reservoir near Highway 1215. Blood was drawn from the caudal vein for DNA analysis. Additional whole Yellow Bass (n = 40) were collected from Poverty Point (Richland Parish, LA), where Striped Bass have never been present, to obtain Yellow Bass allele frequencies without the potential influence of Striped Bass hybridization. White Bass and Striped Bass allele frequencies given in the literature (Couch et al. 2006) were used as reference data because no pure White or Striped Bass populations exist in Louisiana as far as we are aware. Blood was stored in Queen’s lysis buffer (Seutin et al. 1991) and whole Poverty Point bass were frozen. DNA from blood or caudal fin tissue was extracted with the Qiagen DNEasy kit (Valencia, CA). Samples were genotyped with 14 previously developed microsatellite loci as follows: MSM 1075, 1078, 1097, 1102, 1106, 1107, 1137, 1138, 1144, 1149, 1154, 1157, 1177, 1246 (Couch et al. 2006). These loci had non-overlapping, species-specific allele size ranges for White and Striped Bass (Couch et al. 2006). DNA was amplified via polymerase chain reaction (PCR) with 5–10X buffer (New England Biolabs), 0.16 mM dNTPs (Qiagen), 2.0 mM MgCl2, 0.5 U Taq polymerase (New England Biolabs), 0.16 μM primers tagged with M13 forward or reverse tails (Operon), 0.008 μM M13 Forward or Reverse IRDye 700 or 800 (Li-COR Biosciences), and nanopure water for a total reaction volume of 10 μL. Some reactions also included 0.5 M betaine and 3% by volume DMSO. Thermocycling conditions consisted of 2 min at 94 °C followed by 35 cycles of 94 °C for 30 s, 55–61 °C for 30 s, and 72 °C for 30 s with a final extension step of 72 °C for 7 min. After PCR, 3 μL of stop dye was added to the reactions, followed by S.S. Taylor, S. Woltmann, A. Rodriguez, and W.E. Kelso 2013 Southeastern Naturalist Vol. 12, No. 3 516 a 4 min denaturation step at 94 °C, and then 0.8 μL of the mixture was electrophoresed on a Li-COR 4200 automated DNA analyzer with size-standard IRDyes of 50–350 bp (Li-COR Biosciences). Potential hybrid genotypes were verified with replicate PCR reactions. Alleles were scored with Saga® software (v. 3.2; Li-Cor Biosciences), and verified by eye. Statistical analyses Allele frequency distributions were calculated with Genetix v. 4.05 (Belkhir et al. 1999). A bayesian approach was used to examine genetic clustering among the three species and hybrids based on the program Structure v. 2.3.2 (Pritchard et al. 2000). For this analysis, a burn-in period of 100,000 was used with 100,000 repetitions for each of five simulations with K = 3 populations. Prior information on the species identity or source population was excluded in order to obtain results based solely on the genetic data. Individuals with less than 95% membership in a species were considered to be hybrids. Results Alleles for individual species were largely distinct in Toledo Bend Reservoir and identification of hybrids was straightforward (Tables 1, 2). Allele frequency distributions indicated that four hybrids were present among the individuals sampled (Table 1). One individual was a potential Yellow x Striped Bass hybrid from Toledo Bend. Four loci did not amplify in this individual, but at the remaining loci, all alleles were consistent with Yellow Bass allele frequencies except for one locus that was homozygous for a Striped Bass allele (Table 1). The remaining three hybrids were White Bass x Striped Bass hybrids that were morphologically Table 1. Genotypes of the four potential hybrids. Bold formatting indicates genotypes consistent with F1 hybrids, and underlining indicates genotypes consistent with backcrossing or mating among hybrids. Bass species White x Striped White x Striped White x Striped Yellow x Striped MSM1075 247219 247219 233219 205205 MSM1078 163147 163145 159147 169169 MSM1102 161145 161145 161145 165165 MSM1137 168140 168140 166140 152152 MSM1138 203183 203181 203181 187187 MSM1154 174000 216174 200174 165165 MSM1149 210198 216198 210198 0 MSM1144 198163 196151 196141 171171 MSM1157 203161 161161 161161 165157 MSM1177 209209 209209 209209 209209 MSM1107 170154 154154 154154 0 MSM1097 160150 166150 166150 0 MSM1106 167165 165165 165165 159159 MSM1246 256214 214000 214214 0 517 S.S. Taylor, S. Woltmann, A. Rodriguez, and W.E. Kelso 2013 Southeastern Naturalist Vol. 12, No. 3 Table 2. Bass allele frequencies for 14 microsatellite loci. Potential hybrids (n = 4) are not included in the allele frequencies given (see Table 2 for hybrid genotypes). Freq. = frequency. Yellow Bass: Yellow Bass: White Bass Striped Bass Toledo Bend Poverty Point Allele Allele Allele Allele Locus size (bp) Freq. size (bp) Freq. size (bp) Freq. size (bp) Freq. MSM1075 219 1.0000 225 0.0385 205 1.0000 205 1.0000 227 0.1538 229 0.0577 231 0.0577 233 0.2308 235 0.1346 237 0.0385 239 0.1154 247 0.1538 321 0.0192 MSM1078 159 0.0600 145 0.0926 169 1.0000 163 0.1316 163 0.9400 147 0.9074 169 0.8684 MSM1097 150 1.0000 160 0.3462 182 1.0000 182 1.0000 162 0.1920 164 0.0385 166 0.0385 168 0.5577 MSM1102 145 1.0000 161 1.0000 165 1.0000 165 1.0000 MSM1106 202 0.8571 165 0.3889 159 1.0000 159 1.0000 204 0.1429 167 0.5556 171 0.0556 MSM1107 154 1.0000 170 0.9524 200 1.0000 200 1.0000 172 0.0476 MSM1137 140 1.0000 142 0.4167 150 0.3947 150 0.2375 160 0.0278 152 0.6053 152 0.7625 168 0.1389 170 0.0556 172 0.0833 174 0.2500 181 0.0278 MSM1138 177 0.1000 183 0.1296 187 1.0000 187 1.0000 181 0.8400 203 0.1667 183 0.0600 205 0.1667 207 0.3184 209 0.1667 211 0.0556 S.S. Taylor, S. Woltmann, A. Rodriguez, and W.E. Kelso 2013 Southeastern Naturalist Vol. 12, No. 3 518 identified in the field as hybrids. These fish had genotypes consistent with F1 offspring at 9–12 loci (one allele from each parental species) but showed evidence for backcrossing or mating among hybrids at 2–5 loci (both alleles from a single parental species; Table 1). Structure v. 2.3.3 analyses clustered White Bass and Striped Bass as separate groups with their hybrids as intermediates between the two clusters (Fig. 1). Yellow Bass from Toledo Bend Reservoir and Poverty Point clustered Table 2, continued. Yellow Bass: Yellow Bass: White Bass Striped Bass Toledo Bend Poverty Point Allele Allele Allele Allele Locus size (bp) Freq. size (bp) Freq. size (bp) Freq. size (bp) Freq. MSM1144 141 0.0208 141 0.3184 171 1.0000 171 1.0000 194 0.0208 147 0.0370 196 0.7292 151 0.1481 198 0.0833 153 0.0185 199 0.0208 157 0.1481 200 0.1250 159 0.1111 161 0.0741 163 0.1111 171 0.0370 MSM1149 198 1.0000 210 0.8704 200 1.0000 200 1.0000 214 0.0185 216 0.1111 MSM1154 174 1.0000 200 0.0370 165 1.0000 165 1.0000 216 0.8148 218 0.1481 MSM1157 161 1.0000 161 0.0400 157 0.6053 157 0.0256 185 0.0200 165 0.3684 165 0.5769 193 0.1000 169 0.0263 169 0.3974 203 0.2000 205 0.1600 211 0.0200 217 0.4200 223 0.0400 MSM1177 237 1.0000 207 0.0962 215 1.0000 215 1.0000 209 0.8846 211 0.0192 MSM1246 214 1.0000 224 0.0185 214 0.0357 226 0.0132 236 0.0926 262 0.9643 262 0.8816 244 0.1111 266 0.1053 256 0.6296 262 0.1481 519 S.S. Taylor, S. Woltmann, A. Rodriguez, and W.E. Kelso 2013 Southeastern Naturalist Vol. 12, No. 3 closely together and were proximal to the Yellow x Striped Bass hybrid (Fig. 1). Structure v. 2.3.3 results giving the proportion of membership in each of three species are provided in Table 3. Table 3. Proportion of membership in each of three species based on Structure results. n = number of individuals. Inferred cluster Species or hybrid White Bass Yellow Bass Striped Bass n White Bass 0.997 0.001 0.002 25 White x Striped Bass 0.484 0.001 0.515 3 Striped Bass 0.001 0.001 0.998 27 Yellow x Striped Bass 0.001 0.903 0.095 1 Yellow Bass Toledo Bend 0.003 0.996 0.001 19 Yellow Bass Poverty Point 0.001 0.998 0.001 40 Figure 1. Analysis of bass genotypes using Structure v. 2.3.2. Black circle = Striped Bass, grey circle = White Bass, vertically striped circle = White x Striped Bass hybrids, white circle = Poverty Point Yellow Bass, horizontally striped circle = Toledo Bend Yellow Bass, spotted circle = Yellow x Striped Bass hybrid. S.S. Taylor, S. Woltmann, A. Rodriguez, and W.E. Kelso 2013 Southeastern Naturalist Vol. 12, No. 3 520 Discussion Despite many years of stocking with Striped Bass, Toledo Bend Reservoir shows no evidence of hybrid bass swarms despite considerable potential for this to occur both through hybridization in the wild following the release of millions of Striped Bass, and inadvertent stocking of hybrids via hatchery escapees. Although more extensive collections and larger sample sizes may produce different numbers, our results suggest that, given the small number of hybrids present, introgression-related impacts on native species’ genomes appear to be minimal. Of 75 fish sampled, only 4 hybrid individuals were identified, and three of these were recognized through morphological differences. Our genetic analyses identified only one Yellow x Striped Bass hybrid, suggesting that hybridization rates between these species may be low and, therefore, introduced Striped Bass are not causing introgression in Yellow Bass. Three White Bass x Striped Bass hybrids were present and had genotypes consistent with F1 hybrids at 9–12 loci, and genotypes at 2–5 loci that indicated backcrossing with Striped Bass (one individual at two loci) or crossing among hybrids (two individuals at four and five loci, respectively). Reproduction between White and Striped Bass and among Striped Bass hybrids has been documented previously in several areas including Toledo Bend Reservoir (Avise and Van Den Avyle 1984, Crawford et al. 1984, Forshage et al. 1986). However, White Bass x Striped Bass hybrids have a shorter life span and poorer reproductive success than White Bass (Bartley et al. 2000, Champeau 1984, Ross 2001), and temperatures in the Toledo Bend Reservoir have caused summer die-offs of large Striped Bass, which do not tolerate temperatures above 22 °C (Matthews 1985). Together, these factors may limit the number of White Bass x Striped Bass hybrids in Toledo Bend, especially following LDWF’s decision to cease stocking efforts in 2009, which eliminated new inputs of Striped Bass. The low frequency of bass hybrids in Toledo Bend Reservoir may be attributable to differences in reproductive strategies and poor hybrid survivorship as outlined above in published studies. However, other evidence suggests that the frequency of hybridization may depend on population size. When one species is abundant and the other is rare, females of the rare species may mate with males of the abundant species because they do not encounter males of their own species. For example, Avise and Saunders (1984) reported that genetically identified sunfish (Lepomis spp.) hybrids were crosses between males of the most common species (L. macrochirus Rafinesque [Bluegill Sunfish] and L. auritus L. [Redbreast Sunfish]) and females of the rarest species (L. cyanellus Rafinesque [Green Sunfish], L. gulosus Cuvier in Cuvier and Valenciennes [Warmouth], and L. microlophus Günther [Redear Sunfish]). Given that native White and Yellow Bass populations in the Toledo Bend Reservoir are probably very large, there may be little risk of females mating with Striped Bass and therefore little risk of introgression in the native Morone genomes. Finally, morphological identification of White Bass x Striped Bass hybrids corresponded to hybrids identified through genetic techniques. These individuals 521 S.S. Taylor, S. Woltmann, A. Rodriguez, and W.E. Kelso 2013 Southeastern Naturalist Vol. 12, No. 3 were genetically very similar to F1 hybrids, and may in fact have been F1s if some loci had null alleles in hybrids (i.e., one species’ allele preferentially amplifies). 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