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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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
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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.
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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.
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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
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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
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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.
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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.
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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.
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