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Testing for Genetic Divergence Within and Among Isolated Populations of a Threatened Species in Georgia and Alabama, Percina aurolineata (Percidae; Goldline Darter)
Steven L. Powers, Sarah E. Ahlbrand, Bernard R. Kuhajda, and Kelsey E. Wests

Southeastern Naturalist, Volume 14, Issue 4 (2015): 675–684

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Southeastern Naturalist 675 S.L. Powers, S.E. Ahlbrand, B.R. Kuhajda, and K.E. West 22001155 SOUTHEASTERN NATURALIST 1V4o(4l.) :1647,5 N–6o8. 44 Testing for Genetic Divergence Within and Among Isolated Populations of a Threatened Species in Georgia and Alabama, Percina aurolineata (Percidae; Goldline Darter) Steven L. Powers1,*, Sarah E. Ahlbrand1, Bernard R. Kuhajda2, and Kelsey E. West1 Abstract - Percina aurolineata (Percidae: subgenus Hadropterus) (Goldline Darter) is a federally threatened species that currently exists in disjunct populations in the Coosawattee River, GA, and Cahaba River, AL. These 2 Mobile Basin drainages are home to considerable endemism, and these disjunctions may actually represent cryptic diversity within Goldline Darter. We examined sequence data from the mitochondrial cytochrome b gene (cyt b) and nuclear recombination-activation gene exon 1 (RAG1) from specimens (n = 34) collected from 4 streams in the Coosawattee River drainage and 4 streams in the Cahaba River drainage for the purpose of assessing phylogenetic structure and genetic divergence to test the hypothesis that the disjunct populations of Goldline Darter represent a single species. Specimens from each of the rivers sampled were not resolved as a clade in any analysis. For cyt b, divergence within the Coosawattee was 0.8%, divergence within the Cahaba was 0.3%, and net divergence between populations was 0.4%. For RAG1, divergence within the Coosawattee was 0.0%, divergence within the Cahaba was 0.1%, and net divergence between populations was 0.0%. We detected a unique allele for RAG1 with a frequency of 0.559 in the Cahaba specimens. No clades were resolved that contained specimens representative of only one locality and the difference between mean divergence among and within rivers was low; thus, these results support the hypothesis that the disjunct populations of Goldline Darter represent a single species and an evolutionarily significant unit. The divergence of allele frequencies among Cahaba and Coosawattee for RAG1 qualifies them as separate management units, and future conservation efforts should manage them as such. Introduction Percina aurolineata Suttkus and Ramsey (Percidae: subgenus Hadropterus) (Goldline Darter) is a threatened species protected under the Endangered Species Act (Federal Register 1992). It is found in riffles 10–100 cm deep on streams 15–60 m wide in moderate to swift current among bedrock, boulder, and cobble substrate often covered with Podestemum ceratophyllum Michx. (Riverweed) and adjacent to Justicia sp. (a water willow) beds (Suttkus and Ramsey 1967). Spawning occurs from April to June (Powers and Mayden 2002). Eggs are deposited in fast-moving water over sand to gravel substrate downstream of boulders in ~0.6-m deep water (Stiles and Ramsey 1986). Larval Chironomidae and Simulidae are primary food sources, with Ephemeroptera, Trichoptera, and Plecoptera more rarely consumed (Powers and Mayden 2002). 1Biology Department, Roanoke College, 221 College Lane, Salem, VA 24153. 2Tennessee Aquarium Conservation Institute, 201 Chestnut Avenue, Chattanooga, TN 37402. *Corresponding author - powers@roanoke.edu. Manuscript Editor: Andrew Rypel Southeastern Naturalist S.L. Powers, S.E. Ahlbrand, B.R. Kuhajda, and K.E. West 2015 Vol. 14, No. 4 676 Suttkus and Ramsey (1967) hypothesized that the Goldline Darter was once distributed throughout the upper Alabama River drainage, but was extirpated from all but the Cahaba and Coosawattee drainages where populations persist. They suggested that Goldline Darters may still persist in as yet undiscovered populations in unsampled tributaries to the Alabama River. They identified extensive habitat alteration by impoundments and point and nonpoint pollution in eastern Alabama and northwest Georgia were identified as likely causes of this extirpation from other Alabama River tributaries. Goldline Darters appear to be sensitive to degraded water quality; for example, their abundance in the Cahaba River increased following improvements in water quality associated with upgrades to wastewatertreatment facilities that occurred as the result of a lawsuit against Jefferson County, AL in 1993–1994 for violating the Clean Water Act with unpermitted discharges into the Cahaba and Black Warrior rivers (PARCA 2001). Impoundments such as Carters Lake, and point and nonpoint-source pollution from urbanization and agriculture have limited the range and abundance of the Goldline Darter within the Coosawattee River drainage (Albanese et al. 2013) supporting the hypothesized once-continuous distribution of the species throughout the Alabama River drainage. Despite extensive sampling of the Alabama River drainage system over the last 4 decades, no other Goldline Darter populations have been discovered (Albanese et al. 2013, Boschung and Mayden 2004, Mettee et al. 1996). Alternatively, the extant populations could have been initially isolated by the lack of ideal habitat in the Alabama River on the coastal plain thousands of years before humans reached North America. These 2 disjunct populations not only occur in different tributaries to the Alabama River but are also in distinct physiographic provinces—the Cahaba River in the Valley and Ridge province and the Coosawattee River of the upper Coosa on the junction of the Piedmont and Blue Ridge provinces (Mettee et al. 1996, USGS 1992). These disparate drainage and geologic settings suggest that vicariance may have led to speciation, with at least 2 and 8 fish species endemic to the Cahaba and upper Coosa River drainages, respectively (Baker et al. 2013, Boschung and Mayden 2004, Jelks et al. 2008, Mettee et al. 1996). Thus differentiation may have occurred within Percina aurolineata, leaving behind cryptic biodiversity currently considered disjunct populations of a single species. Captive propagation techniques for Goldline Darter have been investigated using specimens from the Cahaba River, AL (Rakes and Shute 2003). Understanding the genetic structure of the 2 disjunct populations is key to effective maintenance of this imperiled species. While there are no current plans to use propagated or translocated specimens for conservation of this species (P.A. Rakes, Knoxville, TN, pers. comm.), that strategy is commonly considered for critically imperiled species (George et al. 2009). Captive propagation and translocation efforts that ignore genetic structure within and among populations have well-documented negative impacts (Ferguson 1990, Leary et. al. 1993, Meffe and Vrijenhoek 1988, Philipp 1991, Storfer, 1999). Therefore, identifying phylogeographic and genetic patterns within and among populations of an imperiled species is a necessary precursor to making informed conservation decisions for that species. Southeastern Naturalist 677 S.L. Powers, S.E. Ahlbrand, B.R. Kuhajda, and K.E. West 2015 Vol. 14, No. 4 The primary objective of this study was to test the hypothesis that, as currently recognized, the Goldline Darter represents a single evolutionary species. Secondarily, we examined patterns of genetic variation within and among populations for evidence of recent and historical barriers to gene flow in order to better inform conservation actions. Field-site Description We obtained Goldline Darter specimens for this project from the following localities (with the number of specimens examined from each locality in parentheses; Fig. 1): Mountaintown Creek along Stillwell Road, Gilmer County, GA (n = 4); Mountaintown Creek at Highway 52, Gilmer County, GA (n = 1); Mountaintown Creek at Fisher Trail, Gilmer County, GA (n = 1); Mountaintown Creek at Craigtown Road, Gilmer County, GA (n = 1); Coosawattee River at Ellijay, Gilmer County, GA (n = 2); Coosawattee River at Seawall Haste near Ellijay, Gilmer County, GA (n = 1); Cartecay River at Blackberry Falls, Gilmer County, GA (n = 2); Cartecay River at Lower Cartecay Road, Gilmer County, GA (n = 2); Cartecay River along Highway 52, Gilmer County, GA (n = 2); Ellijay River at Pinson Road, Gilmer County, GA (n = 2); Ellijay River at Highway 52, Gilmer County, GA (n = 1); Shades Creek, Bibb Figure 1. Distribution map of Percina aurolineata (Goldline Darter). Dots represent localities from which we collected specimens for this study. Southeastern Naturalist S.L. Powers, S.E. Ahlbrand, B.R. Kuhajda, and K.E. West 2015 Vol. 14, No. 4 678 County, AL (n = 5); Schultz Creek at Highway 219, Bibb County, AL (n = 2); Little Cahaba River at County Road 65, Bibb County, AL (n = 1); Cahaba River at Centreville, Bibb County, AL (n = 4); Cahaba River at County Road 24 near Piper, Bibb County, AL (n = 1); Cahaba River at County Road 52, Shelby County, AL (n = 4); Cahaba River at County Road 26 at Pratt’s Ferry, Bibb County, AL (n = 1); and Cahaba River at Marvel Slab, Bibb County, AL (n = 1). Methods and Materials We obtained sequence data for the mitochondrially encoded cytochrome b gene (cyt b) and the nuclear recombination-activation gene exon 1 (RAG1) from Goldline Darters (n = 34) from across the range of the species in northern Georgia and central Alabama (Fig. 1). Our samples included individuals from Mountaintown Creek, Coosawattee River, Cartecay River, and Ellijay River, GA; and Schultz Creek, Shades Creek, Little Cahaba River, and Cahaba River, AL. We collected specimens between July 2007 and June 2013 using a Smith-Root Model 24 backpack electrofisher (Smith-Root, Vancouver, WA) and a 1.5 m x 3.3 m seine with 5-mm mesh and preserved them in 95% ethanol as whole specimens or as fin-clips from captured and released specimens. We did not attempt to utilize formalin-fixed museum specimens due the lack of effective protocols for DNA extraction. We extracted whole genomic DNA from ethanol-preserved specimens using standard phenol-chloroform methods (Hills et al. 1996). We amplified cyt b and RAG1 genes separately with 30 cycles of PCR using primers designed by Song et al. (1998) and Lopez et al. (2004), respectively. Denaturation, annealing, and extension temperatures and times were: 95 oC, 40 sec; 55 oC, 60 sec; and 72 oC, 90 sec, respectively. We purified the amplified PCR products by centrifugal filtration using the GenElute® PCR Clean-Up Kit (Sigma-Aldrich Inc., St. Louis, MO) following manufacturers’ directions. Technicians at Virginia Bioinformatics Institute, Blacksburg, VA, conducted the sequencing. We aligned sequences by eye, checked them for accuracy against chromatograms, and examined RAG1 for multiple alleles using BioEdit (Hall 1999); no gaps were needed for alignment. We deleted ambiguous data at the beginning and end of each sequence, leaving 958 bases of cyt b and 1446 bases of RAG1 for analyses. Sequence data are available at GenBank (accession numbers KP698231-KP698298). We used sequence data from Sander canadense (Griffin and Smith) (Sauger) and Etheostoma cinereum Storer (Ashy Darter) as outgroups allowing for polarization of characters and rooting of the trees. We examined genetic variation within and among river drainages by calculating pairwise distances using MEGA4 (Tamura et al. 2007). We discovered 2 alleles for RAG1 in the Cahaba population which prompted us to examine chromatograms to determine whether individuals were homozygous for either allele or heterozygous. We employed a chi-square test to determine if the 2 alleles in the Cahaba population are in Hardy-Weinberg equilibrium. Phylogenetic hypotheses were generated with maximum parsimony in NONA (vers. 2, Goloboff, P., NONA, Tucumán, Argentina). We conducted heuristic searches with all characters equally weighted and 50 replications of the randomSoutheastern Naturalist 679 S.L. Powers, S.E. Ahlbrand, B.R. Kuhajda, and K.E. West 2015 Vol. 14, No. 4 addition sequence option. Branches with lengths of zero were collapsed. We evaluated support for hypotheses by performing 1000 bootstrap replicates (Felsenstein 1985) in NONA. We performed phylogenetic analysis of RAG1 sequences employing data for heterozygotes as reported by the sequencer. Results For cyt b, divergence within the Coosawattee was 0.8%, divergence within the Cahaba was 0.3%, and net divergence between populations was 0.4%. For RAG1, divergence within the Coosawattee was 0.0%, divergence within the Cahaba was 0.1%, and net divergence between populations was 0.0%. Two different alleles for RAG1 were present in the Cahaba River Goldline Darter population, while a single allele was present in the Coosawattee River population. This unique allele appears to be due to a 2nd-position transition (C to T) at base 1268 that caused amino acid 423 to change from serine to phenylalanine. The unique allele in the Cahaba population had a frequency of 0.559, and the 2 alleles were not out of Hardy-Weinberg equilibrium (P = 0.965) in the individuals examined from the Cahaba drainage. Maximum-parsimony analysis of cyt b data produced 11 equally parsimonious trees of 336 steps in length with a consistency index (CI) of 0.91 and retention index (RI) of 0.86. A strict consensus of those alleles contains a clade of Coosawattee specimens with high bootstrap support in a polytomy containing other specimens from the Coosawattee and Cahaba rivers (Fig. 2). Maximum-parsimony analysis of RAG1 data identified a single tree with a length of 67 steps having a CI and RI of 1 (Fig. 3). All Goldline Darter specimens were recovered as a clade with bootstrap support of 100, and 12 specimens from the Cahaba River drainage were recovered as a clade with bootstrap support of 60. All other specimens from the Cahaba were left unresolved in a polytomy with all specimens from the Coosawattee River drainage. Neither cyt b nor RAG1 revealed strict concordance between clades and geographic distribution. Discussion The greater pairwise divergence of cyt b data from within the Coosawattee specimens than among specimens from the Coosawattee and Cahaba drainages does not suggest long-term isolation of the populations. Similarly, the greater pairwise divergence of RAG1 data from within the Cahaba specimens than among specimens from the Coosawattee and Cahaba drainages does not suggest long-term isolation of these populations. The overall similarity of within- versus among-population pairwise divergence is consistent with a recent extirpation of a once more broadly distributed Goldline Darter having gene flow throughout the Alabama River drainage as suggested by Suttkus and Ramsey (1967). The lack of phylogenetic structure in either the cyt b or RAG1 analyses congruent with geographic distribution (i.e., drainages and clades matching up) also suggests that the isolation of populations is a relatively recent phenomenon. Without some other data suggesting otherwise, our analysis fails to reject the hypothesis of Suttkus and Ramsey (1967) that the Southeastern Naturalist S.L. Powers, S.E. Ahlbrand, B.R. Kuhajda, and K.E. West 2015 Vol. 14, No. 4 680 disjunct populations in the Cahaba and Coosawattee river drainages represent a single species under species concepts commonly used by taxonomists such as the biological species concept (Mayr 1996), diagnosable version of the phylogenetic species concept (Nixon and Wheeler 1990), or the monophyly version of the phylogenetic species concept (Rosen 1978). Despite the absence of a pattern indicative of speciation, the unique allele for RAG1 within the Cahaba River leaves us with detectable genetic differences between populations. This unique allele also suggests that some restrictions to gene flow between the Cahaba and Coosawattee populations may have been present Figure 2. Strict consensus of 11 equally parsimonious trees, 336 steps in length with a consistency index of 0.91 and retention index of 0.86 based on 958 bases of cyt b data from Goldline Darter specimens (n = 34) labeled by the stream from which they were collected. Bootstrap support is listed above the branch. Southeastern Naturalist 681 S.L. Powers, S.E. Ahlbrand, B.R. Kuhajda, and K.E. West 2015 Vol. 14, No. 4 prior to habitat alteration in the Alabama River drainage. The >300-rkm distance between these localities would likely have allowed for some genetic differentiation to occur at opposite extremes of the range as predicted by an isolation-by-distance model (see Hedrick 2005) even if they were once distributed throughout the Alabama River drainage as hypothesized by Suttkus and Ramsey (1967). The apparent Hardy-Weinberg equilibrium of specimens from the Cahaba (P = 0.965) suggests that few if any barriers to gene flow occur between collection localities in the Cahaba River drainage. The sampled localities are scattered throughout the range Figure 3. Most parsimonious tree of 67 steps in length with a consistency index and retention index of 1 based on 1446 bases of RAG1 data from Goldline Darter specimens (n = 34) labeled by the stream from which they were collected. Bootstrap support is listed above the branch. Southeastern Naturalist S.L. Powers, S.E. Ahlbrand, B.R. Kuhajda, and K.E. West 2015 Vol. 14, No. 4 682 of the Golden Darter in the Cahaba; thus it appears that a single, large, panmictic population occurs within this drainage. Numerous discussions of protecting genetic diversity within an imperiled species have led to identification of and several definitions and critiques of the evolutionarily significant unit (ESU; Crandall et al. 2000; Mayden and Wood 1995; Moritz 1994; Pennock and Dimmick 1997; Ryder 1986; Waples 1991, 1995). Despite the lack of agreement on what constitutes an ESU among authors, less controversy surrounds the management unit (MU) as defined and discussed by Moritz (1994, 2002). The MU is described as having “… divergence of allele frequencies at nuclear or mitochondrial loci, regardless of phylogenetic distinctiveness of the alleles.” These genetically divergent Goldline Darter populations contain unique alleles that may represent adaptations to local environments essential for survival of local populations, and provide the necessary components for evolutionary processes to continue in future generations. Although the cyt b and RAG1 data we examined in this study lack phylogenetic structure congruent with geographic distribution expected for recognition as unique species or ESUs, the high frequency of the unique RAG1 allele (0.559) in the Cahaba drainage and apparent absence of the allele in the Coosawattee drainage qualifies the Coosawattee and Cahaba populations as separate MUs. As separate MUs, the maintenance of this genetic diversity may be key to preserving the Goldline Darter as a species; therefore, the Coosawattee and Cahaba populations should be managed independently. If more-active management strategies such as propagation, translocation, reintroduction, and augmentation are implemented for the Goldline Darter, these efforts should follow the guidelines of George et al. (2009). Captured individuals should not be moved out of the drainage from which they were collected. Brood stock for captive propagation should also come only from the drainage in which the offspring are to be released, and resource managers should make an effort to conserve the genetic diversity within each MU. Acknowledgments Fishes were collected under collecting permits issued by the Georgia Department of Natural Resources, the Alabama Department of Conservation and Natural Resources, and the US Fish and Wildlife Service. We thank the Georgia Department of Natural Resources, Wildlife Resources Division for providing funding, and Brett Albanese for help obtaining specimens for this study. Sarah Hazzard of the Tennessee Aquarium Conservation Institute assisted with delineation of physiographic provinces. Literature Cited Albanese, B., T. Litts, M. Camp, and D.A. Weiler. 2013. Using occupancy and speciesdistribution models to assess the conservation status and habitat use of the Goldline Darter (Percina aurolineata) in Georgia, USA. Ecology of Freshwater Fish 23:347–359. Baker, W.H., R.E. Blanton, and C.E. Johnston. 2013. Diversity within the Redeye Bass Micropterus coosae (Perciformes: Centrarchidae) species group, with descriptions of four new species. Zootaxa 3635:379–401. Boschung, H.T, Jr., and R.L. Mayden. 2004. Fishes of Alabama. Smithsonian Institution, Washington, DC. 736 pp. Southeastern Naturalist 683 S.L. Powers, S.E. Ahlbrand, B.R. Kuhajda, and K.E. 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