Copeia 103, No. 2, 2015, 272–280 Lack of Hybridization between Naturally Sympatric Populations of Red and Blacktail Shiner (Cyprinella lutrensis and C. venusta) in Texas, but Evidence of Introgression among Three Lineages of the C. lutrensis Species Group Christopher L. Higgins1, Allison Love-Snyder1, Wesley Wiegreffe1, and Russell S. Pfau1 Hybridization is more common in actinopterygian fishes than any other group of vertebrates. This is especially true for members of the family Cyprinidae; for example, Hubbs found 68 different combinations of inter-generic and intrageneric hybridization among cyprinids collected east of the continental divide. Hybridization between two cyprinid species, the red shiner and the blacktail shiner (Cyprinella lutrensis and C. venusta), has been described in detail in Georgia where C. lutrensis is an introduced species. However, hybridization has not been thoroughly assessed where the two species are naturally sympatric. Our specific objectives were to determine the extent of ongoing hybridization between the two species using nuclear markers and morphometrics and to determine the extent of historical introgression using mitochondrial DNA (mtDNA). We collected 100 individuals from four different locations along the Bosque River and an additional 100 individuals from four locations along the Paluxy River. We used amplified fragment length polymorphism (AFLP) to verify species identification and determine hybrid status of each individual. A total of 56 AFLP fragments were scored, with 82.14% (47 fragments) being polymorphic (95% criterion). Based on these nuclear markers, we only identified two hybrids out of the 200 specimens analyzed; one from the Bosque River, Texas and one from the Paluxy River, Texas. There were no instances of introgression of mitochondrial DNA (mtDNA) from one species into the nuclear background of the other species. We did, however, discover the sympatric occurrence of three mtDNA lineages of C. lutrensis within a single nuclear gene pool; there was only one mtDNA lineage of C. venusta. Overall body shape was assessed using a truss network and was found to be statistically different with red shiner having the shorter and deeper body. The minimal amount of hybridization inferred from AFLP data, in combination with the absence of mtDNA introgression and limited morphological overlap, indicates that pre- or postzygotic isolating mechanisms effectively minimize genetic exchange between naturally sympatric populations of C. lutrensis and C. venusta within these two river systems. F RESHWATER ecosystems are some of the most diverse in the world. They contain over 10,000 fish species, almost 40% of global fish diversity and 25% of total vertebrate diversity, despite covering less than 1% of the Earth’s surface (Dudgeon et al., 2006). However, the distributions of many actinopterygians (i.e., ray-finned fishes) in North America are shrinking and their abundances dwindling. In fact, recent estimates suggest that 39% of all freshwater and diadromous species are imperiled (i.e., vulnerable, threatened, or endangered) and in need of conservation (Jelks et al., 2008). The declines in distribution and abundance of most North American species are a result of habitat degradation, pollution, flow regulation and water extraction, fisheries overexploitation, and the introduction of non-native species (Strayer and Dudgeon, 2010). Although each of these anthropogenic activities contribute to the decline in fish diversity, the introduction of non-native species is considered one of the greatest threats to native biodiversity (Vitousek et al., 1997; Rahel, 2000). Introduced species threaten native biodiversity by altering the habitat, increasing predation pressure, increasing interspecific competition, and/or hybridizing with native species (Fridley et al., 2007). Hybridization is more common in actinopterygians than any other group of vertebrates, largely because of competition for limited spawning habitat, external fertilization, weak behavioral isolating mechanisms, and unequal abundances of parental species (Hubbs, 1955; Scribner et al., 2001). These underlying factors can result in four main outcomes, including the formation of hybrid swarms, 1 hybrid speciation, introgression, and reinforcement of reproductive isolating mechanisms (Osterberg and Rodriguez, 2006). The specific outcome may depend on many factors including whether the parental species are naturally allopatric, parapatric, or sympatric (Woodruff, 1973). When populations are naturally sympatric, reproductive incompatibility is often strengthened through reinforcement of pre- and postzygotic barriers (Noor, 1999). When species are naturally allopatric, but come into secondary contact through the introduction of non-native species, these reproductive barriers may not be sufficient to prevent hybridization (Rhymer and Simberloff, 1996). For example, Weigel et al. (2003) reported finding hybridization between introduced rainbow trout (Oncorhynchus mykiss) and native cutthroat trout (Oncorhynchus clarkii) in 64% of their 80 sampling localities distributed throughout the Clearwater River Basin, Idaho. In Montana, hybridization between introduced O. mykiss and native O. clarkii resulted in a 50% decrease in reproductive success of native cutthroat trout in only a four-year time span (Muhlfeld et al., 2009). The loss of biodiversity in Cyprinidae, the most speciose family in North America (Nelson et al., 2004), is of particular concern because 46% of cyprinids are considered imperiled; 49 are listed as vulnerable, 20 are listed as threatened, 47 are listed as endangered, and 11 are considered extinct (Jelks et al., 2008). Hybridization among cyprinids is widespread and can be intra-generic (i.e., between different species within a genus) or inter-generic (i.e., between different genera). For example, Hubbs (1955) found 68 different combinations of intra-generic and inter-generic hybridization among cyprinids Department of Biological Sciences, Box T-0100, Tarleton State University, Stephenville, Texas 76402; E-mail: (CLH) [email protected]. Send reprint requests to CLH. Submitted: 17 March 2014. Accepted: 1 December 2014. Associate Editor: T. J. Near. DOI: 10.1643/CG-14-046 Published online: April 30, 2015 F 2015 by the American Society of Ichthyologists and Herpetologists Higgins et al.—Hybridization and introgression in cyprinids collected east of the continental divide. Hybridization within the genus Cyprinella, the second most diverse genera within Cyprinidae comprised of 30 species (Nelson et al., 2004), is particularly common. Interspecific hybridization within the genus Cyprinella is believed to be a threat to diversity in the southeastern United States (Walters et al., 2008). The primary hybridizing species is the introduced red shiner (C. lutrensis). Cyprinella lutrensis is an extremely tolerant species that can aggressively colonize degraded habitats and thrives under harsh environmental conditions (Matthews and Hill, 1977; Marsh-Matthews et al., 2011). The native distribution of C. lutrensis extends throughout much of the Great Plains, including populations in the Mississippi River basin and Gulf Coast drainages to the west (Page and Burr, 2011). However, they have been introduced into at least five drainages in the southeastern United States, successfully establishing populations throughout the region (Fuller et al., 1999) and hybridizing with native congeners including the blacktail shiner (C. venusta; Walters et al., 2008). Cyprinella venusta has a native distribution that extends throughout the southeastern United States, including drainages east and west of the Mississippi River Basin (Page and Burr, 2011). Much of what we know about hybridization in Cyprinella is based on introduced populations of C. lutrensis in Georgia (Walters et al., 2008; Blum et al., 2010; Ward et al., 2012), but hybrids have been reported elsewhere. Broughton et al. (2011) identified C. lutrensis 3 C. venusta hybrids in two out of 20 rivers in Texas and Oklahoma: the North Fork of the Guadalupe River in Kerr Co., TX and Sycamore Creek in Edwards Co., TX. Additionally, Jurgens (1951) and Hubbs et al. (1953) reported hybrid swarms in the San Marcos and Guadalupe rivers, respectively. The latter two studies documented hybrids based on phenotype only. The documented hybrids in Georgia and Texas suggest that C. lutrensis is capable of hybridizing across the extent of the geographic distribution of C. venusta. However, the Texas studies did not use multilocus genetic techniques necessary to address the degree of hybridization, which may vary depending on whether the parental populations are naturally allopatric, parapatric, or sympatric (Woodruff, 1973). The overall goal of our study was to examine patterns of hybridization and introgression in C. lutrensis and C. venusta from two adjacent tributaries of the Brazos River in Texas, where the two species are naturally sympatric. Observed levels of hybridization were compared with those from Georgia where C. venusta is native and C. lutrensis is introduced. Our specific objectives were to determine the extent of ongoing hybridization between C. lutrensis and C. venusta using nuclear markers and morphometrics and to determine the extent of historical introgression using mtDNA as an indicator. MATERIALS AND METHODS Study sites.—We surveyed four sites along the Paluxy River (P1–P4) and four sites along the Bosque River (B1–B4; Fig. 1). The Paluxy River is located in north central Texas and flows 47 km through Erath, Hood, and Somervell counties before merging with the Brazos River just east of Glen Rose, Texas. The Bosque River is a 185 km river that also originates in Erath Co. and flows through Hamilton, Bosque, and McLennen counties before emptying into Lake Waco. Because the Paluxy River covers a fraction of the distance the Bosque River does, we only sampled sites on the North Bosque River. Sampling sites along the Paluxy River and the 273 Fig. 1. Map depicting locations of the eight collecting sites on the Bosque and Paluxy rivers. Bosque River were located at road crossings, were approximately equidistant from one another, and represented a gradient of coexistence between C. lutrensis and C. venusta with proportionately more C. lutrensis in downstream reaches (Linam and Kleinsasser, 1989; Jones, 2000; Stone, 2012). Sampling method.—All specimens were collected on 15 August 2011 under permit #SPR-0403-284. At each of the eight sampling localities, we used a seine (3 mm 3 5 mm mesh) to harvest the first 50 Cyprinella without regard to species identification. Because we wanted to determine whether hybrids were indeed intermediate in phenotype, we did not attempt to distinguish among C. lutrensis, C. venusta, or their putative hybrids a priori. Specimens were immediately stored in 95% ethanol. Upon returning to the lab, individuals were arranged from smallest to largest in regard to standard length and numbered 1 through 50. A random number generator was used to subsample 25 individuals per site for genetic and morphological analyses. Each individual selected was given a unique number code for identification purposes (e.g., B2-01 is the first specimen from the second sampling site along the Bosque River). Amplified fragment length polymorphisms (AFLP).—We extracted DNA from pectoral fin tissue using phenol:chloroform: isoamyl alcohol (25:24:1) and ethanol precipitation. Amplified fragment length polymorphism (AFLP) was used to verify species identification and determine hybrid status of each individual. The AFLP protocol was a modified version (Phillips et al., 2007) of the original protocol of Vos et al. (1995). DNA was digested with the restriction enzymes EcoRI and MseI followed by ligations using EcoRI and MseI adapters. A subset of ligated fragments were then amplified by polymerase chain reaction (PCR) using the following preselective primer pairs: EcoRI-C (59–ACTGCGTACCAATTCC– 39) and MseI-A (59–GATGAGTCCTGAGTAAA–39). A subset of the resulting preselective PCR products was amplified in a second PCR reaction using the following three selective primer pairs: EcoRI-CAC (59–ACTGCGTACCAATTCCAC–39) paired with MseI-ACC (59–GATGAGTCCTGAGATAACC–39), MseI-ATT (59–GATGAGTCCTGAGTAAATT–39) and MseIAGC (59–GATGAGTCCTGAGTAAAGC–39). The EcoRI primer was fluorescently labeled to allow detection by a Beckman- 274 Coulter CEQ8000 Genetic Analysis System (Beckman-Coulter Inc., Fullerton, CA). The size of fragments was based on an internal size standard, and fragments were automatically placed into bins (one base pair in size) using BeckmanCoulter software. Results of automated scoring was verified visually and corrected when necessary. Only fragments that were unambiguously present or absent across all specimens were retained. We conducted admixture analyses of AFLP data using two different Bayesian clustering approaches known to provide complementary results (Burgarella et al., 2009), NEWHYBRIDS 1.1 (Anderson and Thompson, 2002) and STRUCTURE 2.3.4 (Pritchard et al., 2000), along with Principal Coordinate Analysis. NEWHYBRIDS uses Markov chain Monte Carlo sampling to determine deviation from HardyWeinberg equilibrium among multilocus genotypes. Computations were performed without prior information on population or allele frequencies and with a burn-in of 100,000 steps and 50,000 iterations with Jeffreys-like priors for the mixing proportions and allele frequencies. We used the posterior probabilities obtained from NEWHYBRIDS to quantify the likelihood that each specimen was categorized as pure C. lutrensis, pure C. venusta, F1 hybrid, F2 hybrid, C. lutrensis backcross, or C. venusta backcross. Different criteria and thresholds can be used to actually classify parental and hybrid individuals based on these probabilities (Burgarella et al., 2009). One criterion requires that the posterior probability for each separate category must be greater than or equal to some a priori threshold. Under this conservative criterion, it is possible that some of the individuals are not classified at all if the probabilities are fairly low for all categories. A second criterion involves setting a threshold for the purebred categories only (i.e., in order for an individual to be classified as a pure species the posterior probability must be greater than some set value) and categorizing all other individuals as hybrids. Although this criterion is liberal at identifying hybrid individuals, it does classify each individual as either a parental species or a hybrid; this could be important if maintaining sample size is an important component of the experimental design. A third criterion involves summing the probabilities from the four hybrid categories and comparing that total to some threshold. This particular criterion has been shown to perform well with simulated data in regard to power and accuracy (Burgarella et al., 2009). For our study, we used this last criterion and classified an individual as being a hybrid if the sum of the posterior probabilities across the four hybrid categories was $ 0.5, making the probability that it is a pure species less than 50%. STRUCTURE uses model-based algorithms for clustering genetic data assuming Hardy-Weinberg and linkage equilibrium (Pritchard et al., 2000). We used STRUCTURE to estimate admixture proportions and assign 90% credibility intervals (Bayesian analogs of confidence intervals) to identify parental and hybrid individuals Simulations were carried out with K 5 2 (two distinct genetic entities) under the admixture model assuming independent allele frequencies. A burn-in of 50,000 steps followed by 50,000 iterations was used. We considered individuals to be hybrids if the 90% credibility interval was completely contained within the range of 0.10 and 0.90, which includes admixture proportions expected for F1 and F2 hybrids (0.50) as well as backcrosses (0.25, 0.75). Copeia 103, No. 2, 2015 Principal Coordinate Analysis (PCoA) was used to visualize patterns of divergence among all 200 specimens using the software GenAlEx 6.4 (Peakall and Smouse, 2006). PCoA uses a pairwise, similarity matrix, based on Hamming distances (Choi et al., 2010), to arrange individuals in principal coordinate space so that specimens with similar genetic profiles will be grouped together. Assuming parental species are distinct and hybrids intermediate in genotype, one would expect to find hybrid individuals between the clusters of individuals representing parental species. Mitochondrial DNA (mtDNA).—Partial cytochrome b (cyt b) DNA sequences were obtained using polymerase chain reaction (PCR). Amplifications were carried out in 25 ml reactions containing 1X Buffer, 1.5 mM MgCl2, 0.8 mM deoxynucleotide triphosphates, 2.5 mM of both primers, 1.25 units Taq DNA polymerase, and 0.5 ml of DNA. Primer pairs used were LA (59–TGACTTGAAAAACCACCGTTG–39) and HA (59–CAACGATCTCCGGTTTACAAGAC–39; Schmidt et al., 1998). Amplification conditions included an initial step of 95uC for 5 minutes, followed by 20 cycles of 95uC for 30 s, 56uC (20.5uC per cycle) for 60 s, 72uC for 90 s, and 20 cycles of 95uC for 30 s, 46uC for 60 s, 72uC for 90 s, then held at 72uC for 10 minutes. PCR products were prepared for sequencing using ExoSAP-IT and sequenced using a Beckman-Coulter CEQ8000 Genetic Analysis System. Cyt b sequences were aligned using ClustalX as implemented in BioEdit along with comparison sequences selected from Scho ¨ nhuth and Mayden (2010) and obtained from GenBank. They include two from the C. venusta species group (GQ275207, CV374; GQ275205, CVFC1) and nine from the C. lutrensis species group (GQ275187, CL112434; GQ275186, CL767; GQ275201, CL0637; GQ275190, 2433; GQ275189, CLBC2; DQ324102, CG7891; EU082522, 1499; GQ275176, DSP0633; GQ275177, CL8811). Cyprinella proserpina and C. labrosa were used as outgroups (DQ324101, CPR8814; GQ275181, CL38541). Numbers indicate GenBank accessions followed by cyt b designations of Scho ¨ nhuth and Mayden (2010). The best-fit model of nucleotide substitution (TN93+I) was identified using MEGA 5.2 (Tamura et al., 2011) and used to create a Maximum Likelihood tree in MEGA 5.2. A neighbor-joining tree was also constructed using the TN93 model. Morphometrics.—We used a digital camera to photograph the lateral view of all 200 specimens, paying close attention to consistent positioning to obtain unbiased measurements (Strauss and Bond, 1990). We then digitized 11 anatomic landmarks (Fig. 2A) on each image using TPSDIG (http:// life.bio.sunysb.edu/morph). We used the landmarks to form a truss network, which is a systematically arranged set of distances among a preselected set of anatomical landmarks (Strauss and Bookstein, 1982), comprised of 20 interlandmark distances (Fig. 2B); the resulting distances served as a multivariate data set characterizing overall body morphology. We used a truss network because trusses generally ensure complete coverage of the body and allow measurement error to be partitioned statistically from the variables (Strauss and Bond, 1990). Using the species/hybrid identities determined by NEWHYBRIDS as a fixed factor, we conducted a multivariate analysis of variance to determine whether species/hybrids were morphologically different from one another. We performed discriminate function analysis (DFA) on log- Higgins et al.—Hybridization and introgression in cyprinids Fig. 2. Location of anatomical landmarks (closed circles) and corresponding distances (dashed lines) used to quantify overall body morphology (A). Map of morphometric distances used to create truss network obtained from digitized anatomical landmarks (B). transformed measurements to ascertain which morphological characters were the most distinguishing features among species/hybrids and to estimate classification functions to predict group membership based on phenotypic variation rather than genotypic differences. DFA provided discriminate scores, which allowed us to visually examine the maximum separation between species and produced loadings (i.e., vector correlations between morphological variables and discriminate functions) to indicate how well each variable separated the species. Because all loadings on the first discriminate axis were positive and of similar magnitude, we performed a ‘‘size-free’’ DFA to determine the degree to which species could be optimally distinguished independent of size variation. This size-free DFA was conducted by finding the pooled within-group principal components, regressing the first principal component from each character independently, and using the regression residuals in a canonical discriminate analysis (Strauss, 1995). RESULTS A total of 56 AFLP fragments were included in the dataset, with 83.9% (47 fragments) being polymorphic (95% criterion) among the 200 individuals. Seven fragments were fixed in C. lutrensis and absent in C. venusta, and two were fixed in C. venusta and absent in C. lutrensis. Proportions of polymorphic fragments in pure C. lutrensis and pure C. venusta were 69.64% and 44.6%, respectively. Overall, C. lutrensis was more widely distributed and abundant within the Bosque River than the Paluxy River (Fig. 3). Cyprinella venusta was found at all four localities in both rivers, but in decreasing numbers in the downstream localities, whereas C. lutrensis was not observed in upstream localities. Of the 200 specimens examined, only two individuals were identified as being possible hybrids based on results from both NEWHYBRIDS and STRUCTURE (Fig. 4); one from the Bosque River and one from the Paluxy River. B3–15 had a 0.99 probability of being a hybrid with the probability of being classified as an F2 equal to 0.69; the 90% credibility interval was completely within the 0.10–0.90 admixture range (Fig. 3A). P4–14 had a 0.99 probability of being a hybrid with the probability of being a C. lutrensis backcross 275 Fig. 3. Admixture proportions (qi) from STRUCTURE indicating membership of individual specimens of C. lutrensis and C. venusta from the (A) Bosque and (B) Paluxy rivers. Individuals with low q-values are C. venusta. Bars indicate 90% credible regions. Individuals with C. lutrensis cyt b haplotypes are indicated with filled circles whereas those with C. venusta haplotypes are indicated with open circles. equal to 0.88; the 90% credibility interval was completely within the 0.10–0.90 admixture range (Fig. 3B). PCoA revealed two distinct clusters of C. venusta and C. lutrensis with the two hybrids being the most centrally located of all 200 individuals (Fig. 4). The positions of hybrids along the primary axis, which accounted for 80.3% of the genetic variation among individuals, were more indicative of backcrosses than F1 hybrids. There was no evidence of mitochondrial introgression as all individuals possessed the mtDNA sequence that was expected based on the proportion of nuclear DNA that they exhibited. Both phylogenetic trees placed our specimens and reference sequences within the same clades, so only the maximum likelihood tree is shown (Fig. 5). Our specimens were closely allied with the reference specimens of Scho ¨ nhuth and Mayden (2010), and species identifications based on mtDNA matched those based on AFLP in all cases. Additionally, we documented three mtDNA lineages of C. lutrensis in the Paluxy River and two in the Bosque River (clades A, B, and C in Fig. 5). The most abundant lineage (clade A; found in both rivers) is allied with a specimen from the Rio Grande River of New Mexico in the phylogeny of Scho ¨ nhuth and Mayden (2010:fig. 6). The second lineage (clade B; also found in both rivers, but represented by only seven specimens) is allied with the specimens from the Mississippi and Rio Grande drainages in the phylogeny of Scho ¨ nhuth and Mayden (2010:fig. 6). The third lineage (clade C) is represented in our study by only one specimen from the Paluxy River and is most closely related to a specimen of C. lutrensis from the Pecos River of Texas (CL0637) and C. garmani from Durango, Mexico (CG7891, 1449) in the phylogeny of Scho¨ nhuth and Mayden (2010:fig. 6). Cyprinella venusta ranged in size from 2.09 cm to 8.23 cm (mean6std; 4.2561.36), whereas C. lutrensis ranged in size from 1.77 cm to 4.47 cm (mean6std; 3.0560.61). The two species differed in overall body morphology (Wilks’ lambda 276 Fig. 4. Principal coordinate plot based on AFLPs of C. lutrensis (open symbols) and C. venusta (closed symbols) from the Bosque (squares) and Paluxy (circles) rivers showing distinct genetic differences between species. Individuals identified as hybrids are identified with stars. 5 0.101, F3,192 5 12.75, P , 0.001), with the first discriminant axis accounting for 87.5% of morphological variation among individuals. The key morphological characters that separated the two species were standard length (character 20; Fig. 2B) with C. venusta being the longer species and measures of body depth (characters 11 and 12; Fig. 2B) in which C. lutrensis has the deeper body. Morphologically, individuals classified as hybrids based on results from NEWHYBRIDS and STRUCTURE were not intermediate in reference to parental species. Based on the discriminant functions of morphological characters, individuals genetically identified as C. lutrensis from the Paluxy River were correctly classified 100% of the time whereas those from the Bosque River were correctly classified 92% of the time. Individuals genetically identified as C. venusta from the Paluxy River were correctly identified based on morphology with 97.4% accuracy; C. venusta from the Bosque River were classified with 93.6% accuracy. DISCUSSION Cyprinella lutrensis and C. venusta represent two species groups consisting of multiple lineages, only some of which have been formally recognized taxonomically (Scho ¨ nhuth and Mayden, 2010). Because of current taxonomic uncertainties, we use the names C. venusta and C. lutrensis in a broad sense to include all lineages not currently described as unique species by Scho ¨ nhuth and Mayden (2010). Within this context, the number of individuals classified as hybrids (2 out of 200 specimens) in our study was unexpectedly low and appeared to be later generation backcrosses. Furthermore, there were no instances of mtDNA introgression of one species into the nuclear background of the other species. The minimal amount of hybridization inferred from Copeia 103, No. 2, 2015 AFLP data, in combination with the lack of mtDNA introgression, indicates that pre- or postzygotic isolating mechanisms effectively prevent genetic exchange between C. lutrensis and C. venusta within the Bosque and Paluxy rivers. The finding of minimal hybridization at our study sites is in contrast to those of Walters et al. (2008) and Ward et al. (2012) who reported extensive introgressive hybridization between C. lutrensis and C. venusta in the upper Coosa River, Georgia where C. lutrensis is thought to have been introduced in 1974, and Broughton et al. (2011) who reported hybridization in two drainages in Texas where the two species are likely to be naturally sympatric. Walters et al. (2008), using morphological, microsatellite, and mtDNA data, found that 34% of their total catch was represented by hybrids, with only 1.2% represented by C. lutrensis. Most individuals having hybrid genotypes were phenotypically indistinguishable from C. venusta. Ward et al. (2012), using the same morphological and genetic methodology, found hybrids at all but the uppermost reach of their transect, with some sites consisting of .20% hybrids. The majority of hybrids were later-generation, with C. venusta backcrosses being predominant. Broughton et al. (2011) sampled small numbers of individuals from multiple drainages across Oklahoma and Texas and used coloration along with two mtDNA loci and one nuclear locus to identify hybrids. They reported a mismatch between nuclear and mtDNA sequences in two out of 19 specimens from the two rivers in which hybridization was observed: one from Sycamore Creek, a tributary of the Rio Grande, on the county line between Val Verde and Kinney counties, TX (erroneously indicated as being from Cooke Co., in their table 1 [Broughton, pers. comm.]) and one from the North Fork of the Guadalupe River in Kerr Co., TX, representing a hybrid frequency of 11% and 10%, respectively. Most individuals from these two rivers were reported as having intermediate or mixed phenotypes, and several individuals had nuclear or mtDNA sequences that did not match their morphology (though not all specimens were represented by both mtDNA and nuclear sequences). No specimens from other rivers were documented as having mixed or intermediate morphologies or mismatched DNA sequences; however, rivers in their study were represented by only one to six specimens. Earlier reports of hybrids in the San Marcos and Guadalupe rivers of Texas were documented by Jurgens (1951) and Hubbs et al. (1953) but were based only on phenotype. Furthermore, the authors did not state which phenotypic characteristics were relied upon to identify hybrids, thus the validity of their findings cannot be assessed. Field guides, such as Freshwater Fishes of Texas: A Field Guide (Thomas et al., 2007) and Fishes of Oklahoma (Miller and Robinson, 2004), typically indicate that breeding males of C. venusta have blue dorsal and lateral regions with yellow-white or yellow fins, respectively. However, the Peterson’s field guide (Page and Burr, 2011) describes C. venusta in Texas as having red-orange fins. Within the Bosque and Paluxy rivers, we routinely observed specimens with the distinct caudal spot typical of C. venusta in combination with reddish-orange fins. We examined 16 of these brightly colored specimens, and they were clearly C. venusta based on AFLP data and mtDNA cyt b sequences (data not shown). These results indicate that the bright nuptial coloration typical of male C. lutrensis (Dugas and Franssen, 2011) can also occur in C. Higgins et al.—Hybridization and introgression in cyprinids 277 Fig. 5. Maximum likelihood tree of cyt b haplotypes of C. lutrensis and C. venusta from the Bosque and Paluxy rivers along with reference specimens of other species within these species groups. Only individuals representative of unique haplotypes are shown. Bootstrap values for major clades are indicated. Reference specimens are indicated by the first three letters of the specific epithet (ven 5 C. venusta, gar 5 C. garmani, lut 5 C. lutrensis, lep 5 C. lepida, sua 5 C. suavis, pro 5 C. proserpina, lab 5 C. labrosa) followed by the cyt b designations (in brackets) given in Scho¨nhuth and Mayden (2010). Cyprinella proserpina and C. labrosa are outgroups. 278 venusta. Given that Jurgens (1951) and Hubbs et al. (1953) may have relied on this combination of characteristics to identify hybrids, their reports of hybrid swarms should be viewed with caution. Although hybridization unquestionably has been documented at other locations (Walters et al., 2008; Broughton et al., 2011; Ward et al., 2012), we clearly documented that hybridization is not an inevitable consequence of the two species occurring sympatrically. Lower hybridization rates in the Bosque and Paluxy rivers compared to the Coosa River in Georgia (Walters et al., 2008; Ward et al., 2012) and Sycamore Creek and the Guadalupe River in Texas (Broughton et al., 2011) can be explained by several factors, including differing environmental conditions, population structure, and prezygotic isolating mechanisms. Postzygotic barriers seem to be a less likely explanation given the evidence of intrinsic hybrid viability reported by Blum et al., 2010, unless hybrids have a selective disadvantage in certain environments. Increased hybridization may occur under certain environmental conditions not currently present in the Bosque and Paluxy rivers. River systems can have substantial environmental differences, such as stream discharge and turbidity, which affect reproductive behavior, altering the ability to discriminate between species. Environmental changes are thought to be responsible for increased hybridization beyond what historically occurred in naturally sympatric populations of trout (Young et al., 2001; Heath et al., 2010). Alternatively, differences in population structure where one species differs greatly in abundance may increase hybridization rates (Avise and Saunders, 1984; Dowling et al., 1989). Such differences certainly occur between our study sites where both species are abundant and spatially segregated and the Coosa River, Georgia, where the native species, C. venusta, greatly outnumbers the introduced species, C. lutrensis (Walters et al., 2008; Ward et al., 2012). Additionally, regional differences in hybridization rate could result from differences in prezygotic isolation between sympatric and allopatric populations evolved through selective mechanisms (Albert and Schluter, 2004; Crispo et al., 2011), including reinforcement (Dobzhansky, 1940), adaptation to different niches (Kilias et al., 1980), direct selection on mating preferences (Servedio, 2001), or through neutral processes (Edmands, 2002). Differences in premating isolation between allopatric and sympatric populations have been observed in other fishes including sticklebacks (Rundle and Schluter, 1998) and trout (Rubidge and Taylor, 2004) and have been attributed to reinforcement or spatiotemporal reproductive segregation. Although assortative mating between C. venusta and C. lutrensis was documented by Blum et al. (2010), native populations of C. venusta from the Coosa River, which have only encountered introduced populations of red shiner since the 1970s (Fuller et al., 1999), may not possess the same degree of prezygotic incompatibility as populations that have been in sympatry with C. lutrensis for longer periods of time. Furthermore, differences in reproductive compatibility among populations may have evolved by neutral as opposed to selective processes (Demuth and Wade, 2005). As lineages diverge, changes in mating behaviors or genetic differences resulting in epistasis among derived alleles eventually lead to reproductive incompatibility even in the absence of reinforcement or other selective mechanisms. Cyprinella venusta and C. lutrensis in the Coosa River of Georgia may Copeia 103, No. 2, 2015 not be of the same genetic lineages as those in Texas and, if so, they may have accumulated neutral genetic differences leading to reproductive isolation. Scho ¨ nhuth and Mayden (2010) showed polyphyletic C. venusta and C. lutrensis mtDNA clades and reported a highly divergent lineage of C. venusta (basal to other C. venusta, C. spiloptera, and C. whipplei) from the Apalachicola and Ochlocknee rivers of Georgia and Florida (near the Coosa River). Kristmundsdo´ttir and Gold (1996) previously identified this divergent clade, along with another that included specimens from the Coosa River, and suggested that C. venusta could be separated into four different species. Within the Bosque and Paluxy rivers, we identified three mtDNA lineages of C. lutrensis and one of C. venusta; however, Walters et al. (2008) did not provide DNA sequences that could be used to determine the genetic lineage of their specimens relative to ours. Given the likelihood that genetic lineages of one or both species in Texas are different from those in the Coosa River, Georgia, and that these lineages have been evolving separately for a considerable length of time, geographic differences in reproductive compatibility caused by neutral processes cannot be ruled out as an explanation for differences in hybridization rates. Another unexpected finding of our study was the sympatric occurrence of divergent mtDNA lineages of C. lutrensis in both the Bosque and Paluxy rivers. These three lineages occurred within a single, homogenous nuclear background, demonstrating that they do not represent three sympatric species. The existence of multiple, divergent mtDNA lineages of C. lutrensis and paraphyly of the C. lutrensis species group has been recognized by Broughton ¨ nhuth and Mayden (2010), and and Gold (2000), Scho Broughton et al. (2011); however, these lineages have never been reported as being sympatric. Broughton et al. (2011) examined specimens from throughout Texas and Oklahoma and identified four mtDNA clades of C. lutrensis, which appeared to be geographically restricted to the Red River drainaige of Oklahoma and Texas, the Arkansas River drainage of Oklahoma, Croton Creek of the Brazos River drainage (represented by one specimen), and rivers of southern Texas and Mexico. The latter clade most likely represents C. suavis of Scho ¨ nhuth and Mayden (2010), which did not occur among our samples suggesting that C. suavis may not occur as far north as hypothesized by Scho ¨ nhuth and Mayden (2010:fig. 7). Although the four clades documented by Broughton et al. (2011) appeared to occur allopatrically, this may simply be an artifact of limited sampling. The sympatric occurrence of multiple, divergent mtDNA lineages in the Bosque and Paluxy rivers may be the product of recent bait bucket introductions or historical (natural) introgression among members of the C. lutrensis species group. Our finding of three, highly divergent mtDNA lineages of C. lutrensis within a single nuclear gene pool emphasizes a major weakness in using mtDNA to delimit and identify species (sometimes referred to as DNA taxonomy and DNA barcoding, respectively)—namely, the problem of mtDNA introgression. Although mtDNA studies play a valuable role in identifying patterns of genetic diversity, multilocus techniques such as AFLP (and microsatellites) are better able to delimit gene pools and species (Creer et al., 2004; Dasmahapatra et al., 2010). Single-nucleotide polymorphisms (SNPs) will be increasingly valuable in this regard as the use of massively parallel sequencers (using techniques Higgins et al.—Hybridization and introgression in cyprinids such as RAD-seq) has become cost effective (Peterson et al., 2012; Rocha et al., 2013). Because of widespread transport of Cyprinella for the baitfish trade (e.g., bait distributors in Texas can obtain C. lutrensis from hatcheries in Missouri) and bait bucket introductions, it is likely that the historical geographic range of mtDNA lineages within the C. lutrensis species group will remain largely unknown. Furthermore, if introgression among lineages of the C. lutrensis species group (as reported herein) occurs widely, it may be difficult to fully resolve longstanding taxonomic issues within this group. ACKNOWLEDGMENTS We thank J. Munz for assistance in the field, and acknowledge the Office of Student Research & Creative Activities at Tarleton State University for monetary support. 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