2008 SOUTHEASTERN NATURALIST 7(4):637–650 Population Genetics of the Blue Shiner, Cyprinella caerulea Anna L. George1,2,*, John B. Caldieraro1,3, Kathryn M. Chartrand1,4, and Richard L. Mayden1 Abstract - Cyprinella caerulea (Blue Shiner) is a federally threatened minnow endemic to the Mobile Basin that is currently restricted to four disjunct populations. We examined the population structure in the Blue Shiner by sequencing the mitochondrial ND2 gene in 37 individuals. We recovered eleven haplotypes, with only one shared between populations, for an overall haplotype diversity of 0.768. Genetic differentiation between populations was significant, accounting for 26% of the variability found within the species. One individual morphologically identified as a Blue Shiner had a haplotype resolved with the sympatric Cyprinella trichroistia (Tricolor Shiner) in our phylogenetic analysis. Long-term management of the Blue Shiner should focus on restoring connectivity between populations in order to restore natural patterns of gene fl ow. Introduction The diverse aquatic communities of the southeastern United States are threatened by a broad array of human activities, including urbanization, sedimentation, pollution, and alteration of flow regimes (Etnier 1997). Since the 1970s, the percentage of southeastern fishes that are considered jeopardized has more than doubled (Warren et al. 2000). Cyprinella caerulea (Jordan) (Blue Shiner) is a small minnow endemic to the Mobile Basin in Alabama, Georgia, and Tennessee (Fig. 1). Although the Blue Shiner was formerly widespread in the Cahaba and Coosa rivers upstream of the Fall Line, the species has not been collected from the Cahaba River since 1971 (Ramsey 1976). Due to concerns about its declining range, the Blue Shiner was listed as federally threatened in 1992 (US Fish and Wildlife Service 1992). While the exact cause of the decline is unknown, it is thought to be linked to habitat degradation and degraded water quality from urbanization, pollution, and sedimentation (Stephens and Mayden 1999, US Fish and Wildlife Service 1995). In the Coosa River drainage, Blue Shiners historically occupied Weogufka Creek, Choccolocco Creek, Big Wills Creek, and Little River in Alabama; the Coosawattee River and one tributary, and unspecified tributaries of the Oostanaula River in Georgia; and the Conasauga River and three tributar- 1Department of Biology, Saint Louis University, 3507 Laclede Avenue, St. Louis, MO 63103. 2Tennessee Aquarium Research Institute, 5385 Red Clay Road, Cohutta, GA 30710. 3103 Calstrada, Staunton, IL 62088. 4Department of Biology and Marine Biology, Center for Marine Science, University of North Carolina Wilmington, 5600 Marvin Moss Lane, Wilmington, NC 28409. *Corresponding author - alg@tnaqua. org. 638 Southeastern Naturalist Vol. 7, No. 4 ies in Georgia and Tennessee (US Fish and Wildlife Service 1995). Extant populations are now restricted to Weogufka and Choccolocco creeks, Little River and an adjacent tributary to Weiss Reservoir, and the Conasauga River. The watersheds of Choccolocco Creek and the Conasauga River are partially contained in National Forests, and Little River has been designated a National Preserve, managed by the National Park Service. The population of Blue Shiners in Weogufka Creek is found on forested land privately owned by a timber company (US Fish and Wildlife Service 1995). Populations in the lower Coosa River drainage are highly localized, and further isolated from Figure 1. Distribution of the Blue Shiner in the Mobile Basin. The black circles represent extant populations, and site numbers correspond with those listed in Table 1 that were sampled for genetic analysis. The grey circles represent localities from which the species is extirpated. The open circle represents the type locality for the Blue Shiner, from which it is now extirpated. 2008 A.L. George, J.B. Caldieraro, K.M. Chartrand, and R.L. Mayden 639 each other by a series of large impoundments on the mainstem Coosa River. Currently, the largest population occupies just over 32 km of the Conasauga River (US Fish and Wildlife Service 1995). Studies of Blue Shiner movement suggest that, while the majority of individuals occupy the same habitat patch from late spring to early fall, they are capable of both upstream and downstream dispersal between adjacent habitat patches of up to 332 m over a 3-month time frame, with an average distance of 130 m (Johnston 2000). These distances suggest that there is no longer any chance for gene fl ow between extant populations, as there are large intervening stream reaches without suitable habitat patches. Three other native congeners occur sympatrically with Blue Shiners: Cyprinella callistia (Jordan) (Alabama Shiner), Cyprinella trichroistia (Jordan and Gilbert) (Tricolor Shiner), and Cyprinella venusta Girard (Blacktail Shiner). All four native Cyprinella species spawn in crevices (Stephens and Mayden 1999), and Blue Shiners and Tricolor Shiners have even been observed to share crevices (J.R. Shute, Conservation Fisheries, Inc., Knoxville, TN, pers. comm.). The spawning habitat and behavior is similar for Blue Shiners and Tricolor Shiners, but differs from Alabama Shiners in the number of males and type of crevices used (Johnston and Shute 1997). Blue Shiner spawning season extends from late April until the end of August, peaking in late May and early June (Krotzer 1990). Because successful spawning requires behavioral interactions including visual displays by both males and females, and requires woody debris with suitable crevices or grooves in the bark, clear water and forested land are essential habitat requirements (Johnston and Shute 1997, Stephens and Mayden 1998). While habitat destruction is the proximate cause of decline in many southeastern fishes, the introduction of exotic species is also a troubling long-term problem. Once established, nonindigenous aquatic taxa are nearly impossible to eradicate, and may affect native taxa through numerous interactions, many of which may not be predictable prior to introduction (e.g., Findlay et al. 2000, Townsend and Crowl 1991, Whittier and Kincaid 1999). Cyprinella lutrensis (Baird and Girard) (Red Shiner), is native to much of the Great Plains and parts of the Mississippi River basin, but has been widely introduced both to the east and west of its native range (Boschung and Mayden 2004, Jenkins and Burkhead 1994, Timmons et al. 1977). For two decades after the discovery of the Red Shiner from the Mobile Basin in the early 1980s (Timmons 1982), the distribution of Red Shiners in the Coosa River did not appear to pose a threat to Blue Shiners, and were not even mentioned in their recovery plan (US Fish and Wildlife Service 1995). However, surveys by the US Geological Survey (USGS) during 2000 revealed that Red Shiners had become abundant above Weiss Reservoir, which had been the main barrier preventing their dispersal into the Conasauga River, home to the most robust population of Blue Shiners (N.M. Burkhead, USGS, Gainesville, FL, pers. comm.). The Red Shiner has been observed to hybridize readily with other 640 Southeastern Naturalist Vol. 7, No. 4 species of Cyprinella, both in its native range (Hubbs and Strawn 1956, Page and Smith 1970, Sorensen 1981) and where introduced (Wallace and Ramsey 1982). In addition, invasive Red Shiners have been found to prey on juvenile native fish, significantly impacting recruitment (Gido et al. 1999). The recent expansion of Red Shiners within the range of Blue Shiners represents an immediate threat not addressed by current recovery actions (US Fish and Wildlife Service 1995). Two of the recommended actions under the recovery plan are periodic monitoring of the status of Blue Shiner populations, and reintroduction into former habitats (US Fish and Wildlife Service 1995). While reintroduction has successfully been used as a tool in the recovery of imperiled southeastern fishes (J.R. Shute et al. 2005, P.W. Shute et al. 1998), it is imperative to use genetic data to identify appropriate source broodstock and assess the health of source and reintroduced populations. Our goal for this study was to provide an assessment of mitochondrial genetic variability in the Blue Shiner in partial fulfillment of these recovery actions. We view this work as a necessary component of periodic monitoring and reintroduction actions, and particularly critical prior to the impending widespread dispersal of Red Shiners throughout the range of the Blue Shiner. Field-site Description Six collection sites were located in the Coosa River Basin in Alabama and Tennessee (Table 1, Fig. 1). Sampling was conducted during the winter and summer of 2001, and fishes were captured with either 3.7 x 1.8 m seines or a backpack electrofisher. Two sites were sampled in the Choccolocco Creek system on 23 February 2001. Site 1, at Murray Spring Run, is 11 rkm downstream from Site 2, Choccolocco Creek in the Talladega National Forest. Water was clear, and the substrate was silted at both sites, with more gravel and cobble occurring upstream. Two sites were sampled in the Weogufka Creek system on 20 July 2001, with Site 3 located 9 rkm downstream of Site 4. Water was clear to slightly turbid, and the substrate consisted primarily of bedrock and boulders, with smaller gravel patches. Justicia americana (L.) (American Water Willow), was present along the Table 1. Collection data for specimens used in genetic analyses. Tissues and specimens or voucher photos are accessioned at the University of Alabama Ichthyological Collection (UAIC). Number Site Latitude Longitude Accession No. analyzed Locality number (°N) (°W) UAIC 13012.03 6 Murrays Spring Run, Calhoun County, AL 1 33.72 85.69 UAIC 13022.03 8 Choccolocco Creek, Calhoun County, AL 2 33.80 85.65 UAIC 13328.01 2 Weogufka Creek, Coosa County, AL 3 32.94 86.36 UAIC 13329.01 1 Weogufka Creek, Coosa County, AL 4 32.93 86.39 UAIC 13330.01 10 Little River, Cherokee County, AL 5 34.28 85.67 UAIC 13464.01 10 Conasauga River, Polk County, TN 6 35.01 84.72 2008 A.L. George, J.B. Caldieraro, K.M. Chartrand, and R.L. Mayden 641 margins of the gravel bars. Blue Shiners were extremely scarce at these two sites. Site 5, the Little River, was also sampled on 20 July 2001, near the mouth of the Little River at the southern end of the Little River Wildlife Management Area. Water was clear, and the substrate included gravel and cobble, with no vegetation. Blue Shiners were exceptionally abundant at this site. The Conasauga River was sampled on 25 July 2001, approximately 4.5 rkm downstream of the western boundary of the Cherokee National Forest. Water was turbid and high, substrate included cobble, gravel, and boulder, with American Water Willow along gravel bars. Blue Shiners were abundant at this site. Methods A total of thirty-seven Blue Shiners was collected from four extant populations (Table 1). Whole specimens or tissue samples were either frozen or preserved in 95% ethanol. DNA was extracted from tissue samples using DNEasy kits (QIAGEN, Valencia, CA) and used as templates to amplify the complete mitochondrial ND2 gene via polymerase chain reaction (PCR) with the primers of Broughton and Gold (2000). Amplifications consisted of 35 cycles, with a 40 s denaturation at 94 °C, 60 s annealing at 55 °C, and 90 s extension at 72 °C. PCR products were purified with QIAGEN Gel Extraction kits (QIAGEN, Valencia, CA). Sequencing reactions used a dye-labeled terminator cycle sequencing kit (Beckman-Coulter DTCS Quick Start Kit) and were visualized on a Beckman-Coulter CEQ 2000 XL sequencer. Light and heavy strands were sequenced for all samples. Sequences were verified by consensus between the two strands, edited, and aligned by eye using BioEdit vers. 5.0.9 (Hall 1999). Veracity of all mutations was assessed via comparative alignment and examination of the electropherograms using Bio- Edit. Sequences from this study are available on GenBank under accession numbers EU153045–EU153060. Relationships among haplotypes were inferred under the parsimony criteria using the heuristic search option in PAUP* (Swofford 2000) with ACCTRAN and tree-bisection-reconnection during 100 replicates of random sequence addition. Maximum parsimony (MP) analyses were conducted with molecular characters unweighted and unordered. All minimal-length trees were kept, and zero-length branches collapsed. Support for individual nodes was assessed by performing 1000 jackknife replicates with 37% data deletion in each replicate and JAC emulation selected. Outgroup taxa included Hybopsis winchelli Girard (Clear Chub), Notropis atherinoides Rafinesque (Emerald Shiner), and Lythrurus roseipinnis (Hay) (Cherryfin Shiner), GenBank numbers AF111231–AF111233. All other members of the genus Cyprinella (Broughton and Gold 2000) were included in the phylogenetic analysis to screen for potential hybrids. Sequences used included GenBank numbers DQ306610, NC008103, and AF111205–AF111230. 642 Southeastern Naturalist Vol. 7, No. 4 Haplotype networks were constructed using TCS 1.13 (Clement et al. 2000). Haplotype diversity (Nei and Tajima 1981) and nucleotide diversity (Nei 1987) were calculated using DNAsp (Rozas and Rozas 1999). DNAsp was also used to test for historical population changes and neutral mutation using Tajima’s (1989) and Fu and Li’s (1993) tests. Arlequin (Schneider et al. 2000) was used to examine nucleotide variation, substitution patterns, and pair-wise φST values, and to perform an analysis of molecular variance (AMOVA) under a distance model of sequence evolution using pair-wise differences (Excoffier et al. 1992). The AMOVA measured φST, the genetic variation within populations relative to the species as a whole (Excoffier et al. 1992). Figure 2. Sample phylogram from 40 trees recovered by parsimony analysis. Jackknife values above 80 are shown above nodes; Blue Shiner haplotypes are labeled as in Table 2. Length = 2782, CI = 0.335, RCI = 0.233. 2008 A.L. George, J.B. Caldieraro, K.M. Chartrand, and R.L. Mayden 643 Results Of the 1047 positions in the ND2 gene, 575 were variable, and 482 were parsimony-informative. Maximum parsimony analysis recovered 40 trees (length = 2782 steps, CI = 0.335, RCI = 0.233; Fig. 2), in which all Blue Shiner haplotypes formed a monophyletic group with 100% jackknife support. Two weakly supported clades of haplotypes were recovered wherein branches were very shallow. One individual from Choccolocco Creek (UAIC 13022.03) identified as a Blue Shiner based on morphology was recovered sister to the sympatric Tricolor Shiner (GenBank number EU153049). This individual was discarded for all subsequent intraspecific analyses. Eleven haplotypes were recovered from the 36 individuals of Blue Shiner sequenced (Table 2). The haplotype network consists of two major groups, separated by a minimum of 5 base pair differences (Fig. 3). Three populations contained haplotypes found in both groups, while the two haplotypes recovered from Weogufka Creek were members of only one of the groups. All but one haplotype was found in a single population (Table 2); haplotype A was sampled from a total of 16 individuals across three of the four populations including Little River (3), Conasauga River (5), and Choccolocco Creek (8). Overall haplotype diversity (h) for the species was 0.768, and nucleotide diversity (π) was 0.00426. Haplotype diversity at the population level ranged from 0.467 (Little River) to 0.778 (Conasauga River) and nucleotide diversity ranged from 0.00255 (Weogufka Creek) to 0.00381 (Choccolocco Creek). Neither Tajima’s D (0.050), nor Fu and Li’s D* (-0.012) or F* (-0.18) values were significant. The AMOVA was significant, with 26% of the variation found among populations (φST = 0.26, P < 0.01). Four of the six pair-wise φST values were significantly different (Table 3). Table 2. Distribution of haplotypes among sampled populations of Blue Shiners. Site numbers correspond with those in Table 1. Haplotype abbreviations correspond with those in Figure 3. GenBank Choccolocco Weogufka Little Conasauga accession Creek Creek River River number (sites 1 and 2) (sites 3 and 4) (site 5) (site 6) caeA EU153050 8 0 3 5 caeB EU153051 0 0 0 1 caeC EU153052 0 0 0 1 caeD EU153053 0 0 0 1 caeE EU153054 0 0 0 1 caeF EU153055 0 1 0 0 caeG EU153056 0 0 7 0 caeH EU153057 0 0 0 1 caeI EU153058 0 2 0 0 caeJ EU153059 2 0 0 0 caeK EU153060 3 0 0 0 ccaxtri EU153049 1 0 0 0 Total 14 3 10 10 644 Southeastern Naturalist Vol. 7, No. 4 Figure 3. Haplotype network for Blue Shiners representing 11 haplotypes with 36 individuals. Circle or rectangle size refl ects the frequency of haplotypes; any haplotype recovered from multiple individuals is labeled with the total number recovered. The rectangular haplotype represents the most likely ancestral haplotype as estimated by TCS. Solid lines connecting haplotypes represent one mutational event, and small black circles represent missing or theoretical haplotypes. Haplotypes are as labeled in Table 2. Table 3. Pairwise φST values between all four populations of Blue Shiners. Values followed by an asterisk are significant at P < 0.05. Choccolocco Weogufka Little Conasauga Creek Creek River River Choccolocco Creek - Weogufka Creek 0.40* - Little River 0.21* 0.26 - Conasauga River 0.06 0.54* 0.35* - Discussion Our analyses suggest that while Blue Shiners are currently locally stable, as supported by Tajima’s and Fu and Li’s tests, continued loss of connectivity between these remnant populations will likely limit recovery of the species due to attrition of genetic diversity. The population of Blue Shiner in the Conasauga River, the largest stream reach left in the species’ range, had the highest haplotype diversity (0.778) and second highest nucleotide diversity (0.00321). This is not surprising, as genetic diversity is positively correlated with abundance (Boessenkool et al. 2007, Franklin and Frankham 1998), which has also been correlated with range size (Blackburn et al. 1997, Pyron 1999). The population of Blue Shiners in Choccolocco Creek had the highest nucleotide diversity (0.00381). These are the only populations in national forests, where habitat protection of the surrounding watersheds has not only facilitated the persistence of Blue Shiners, but also maintained sufficient habitat to ensure high levels of genetic diversity. 2008 A.L. George, J.B. Caldieraro, K.M. Chartrand, and R.L. Mayden 645 One factor that affects our estimation of genetic diversity within Blue Shiners is a limited sample size (only 3 individuals) of the population in Weogufka Creek. A rough estimate of our catch per unit effort at Weogufka Creek was the lowest, indicating that Blue Shiners are relatively scarce at this site, consistent with previous studies (Stephens and Mayden 1999). This population was the only one that contained members of only one haplogroup; the nucleotide diversity was subsequently the lowest among surveyed populations. While it is possible that the absence of the common haplotype A from the population in Weogufka Creek is due to our limited sampling, it may have been lost due to the combined effects of restricted gene flow and local bottleneck events, reflected in the current scarcity of Blue Shiners at this site. The results of our AMOVA, short branch lengths in our phylogenetic analysis, and the presence of a common shared haplotype in three of the four populations suggest the recent loss of connectivity between the populations. Because the Tajima’s and Fu and Li’s tests for neutrality do not detect any demographic changes within the species, we hypothesize that the underlying genetic signature within the Blue Shiner is not an overall population bottleneck, but the steady loss of connectivity. It is intriguing, however, that two distinct haplogroups were recovered from the haplotype network (Fig. 3). This situation may be due to historical periods of isolation, leading to differentiation, followed by connectivity and gene fl ow, leading to shared haplotypes among populations. Because both haplogroups are recovered in the three most northerly populations, conservation programs must be careful to preserve all genetic diversity recovered in the species, ideally through increasing range size and restoring connectivity to allow for natural recruitment. Although restoring historical patterns of gene fl ow among populations of Blue Shiners should be the ultimate goal of conservation activities, the destruction of main-channel habitat and the relatively poor dispersal ability of the species makes it unlikely that this will occur naturally throughout the species’ current range. Due to these circumstances, effective recovery will require stream and riparian restoration to improve habitat. The Conasauga River is home to not only the largest and most genetically diverse population of Blue Shiner, but also a generally threatened endemic fauna including two other federally protected fishes and 9 federally protected mussels (Burkhead et al. 1997). Habitat improvements should begin with the goal of expanding stream reaches that can support larger populations of Blue Shiner within this drainage. The lack of reservoirs on the Coosa River above Weiss Lake means that habitat improvement throughout the upper basin could allow for the natural movement of Blue Shiner into restored streams, including the formerly occupied type locality. For tributaries at and downstream of Weiss Lake, habitat restoration alone will not be sufficient to augment gene flow and natural recovery. Captive propagation for reintroduction or translocation may become an 646 Southeastern Naturalist Vol. 7, No. 4 important recovery tool. In this case, care must be taken to ensure that individuals used for propagation reflect the levels of variation present in the species. Our discovery of a Tricolor Shiner haplotype in a morphologically diagnosed Blue Shiner suggests that additional markers will need to be developed for screening all broodstock prior to captive propagation efforts. The presence of a viable population of Blue Shiner in the Cahaba River is considered necessary for delisting (US Fish and Wildlife Service 1995). Blue Shiners were last collected in the Cahaba River in 1971 and likely declined from extensive development in Birmingham, simultaneously with other disturbance-sensitive cyprinids (Onorato et al. 2000). This population is widely considered to have been extirpated over 30 years ago (Krotzer 1984, Ramsey 1976), and was not observed during extensive surveys in the 1980s (Pierson et al. 1989). Regardless, we cannot recommend reintroduction efforts until after an exhaustive survey of stream reaches identified through ecological modeling has been conducted. Until such a survey has been undertaken, we recommend that recovery actions should focus on expanding the range of Blue Shiner where it persists in the Coosa Basin, and perhaps reintroducing the species into other high-quality streams within the formerly occupied portion of the basin. Successful habitat improvements and recovery of the Blue Shiner must address the specific habitat needs for spawning. Reducing the amount of suspended sediment in streams would likely have a greatly beneficial effect on the reproductive success, and thus the viability, of populations of Blue Shiner (Burkhead and Jelks 2001). In addition, woody debris is necessary to provide suitable crevices for spawning (Johnston 1999, Johnston and Shute 1997) and has become limited throughout the range of the Blue Shiner (Stephens and Mayden 1998). The deterioration of water quality through sedimentation and loss of riparian zones and associated woody debris is likely already impacting Blue Shiner reproduction as evidenced by the Blue Shiner x Tricolor Shiner hybrid recovered in this study from Choccolocco Creek. With fewer sites available for spawning, Blue Shiners and Tricolor Shiners may be forced to share more crevices (Johnston and Shute 1997), leading to an increase in hybridization of native Cyprinella. Alternatively, there may be naturally low levels of hybridization between these two native species. Regardless, the potential for hybridization with the Red Shiner makes periodic monitoring even more crucial. Since our samples were collected, Red Shiners have been detected just south of the range of Blue Shiners in the Conasauga River (N.M. Burkhead, pers. comm.) Ex situ behavioral research indicates that male Red Shiners court female Blue Shiners (N.M. Burkhead, pers. comm.). If hybridization were also unidirectional between the species in the wild, it would be silent in the matrilineally inherited mitochondrial genome, and microsatellite or other nuclear markers will be necessary to detect hybridization. Further movement 2008 A.L. George, J.B. Caldieraro, K.M. Chartrand, and R.L. Mayden 647 of Red Shiners upstream will only increase the likelihood of introgressive swamping across all native Cyprinella, making survey work and genetic monitoring necessary throughout the range of Blue Shiners. The data presented here represent a baseline understanding of the population genetics of the Blue Shiner and indicate widespread fragmentation, but with retention of both local and global genetic diversity. While introduced species represent a real threat to the continued existence of the Blue Shiner, widespread habitat alteration and loss of gene fl ow between populations increases the likelihood of local extirpation and could quickly erode the genetic diversity of this imperiled species. Acknowledgments We thank B.R. Kuhajda, N.J. Lang, D.A. Neely, and J.M. Pierson for assistance in the field. We also thank D.A. Neely, L.S Friedlander, and two anonymous reviewers for providing helpful suggestions on the manuscript. This study was supported by US Forest Service Grant CS1180100006. Specimens were collected under Tennessee permit number 713 and Alabama permit number 2548. Literature Cited Blackburn, T., K. Gaston, R. Quinn, H. Arnold, and R. Gregory. 1997. Of mice and wrens: The relation between abundance and geographic range size in British mammals and birds. Philosophical Transactions of the Royal Society of London B, Biological Sciences 352:419–427. Boessenkool, S., S.S. Taylor, C.K. 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