EPA/600/R-07/124 

Red Shiner Invasion of the Upper Coosa River System: 

Dynamics and Ecological Consequences

blacktail 

hybrid 

red 

RESEARCH AND DEVELOPMENT 
EPA/600/R-07/124 

Red Shiner Invasion of the Upper Coosa River System: 

Dynamics and Ecological Consequences

David M. Walters and Michael J. Blum 

U.S. EPA Office of Research and Development 

United States Environmental Protection Agency 

Office of Research and Development 

National Exposure Research Laboratory 

26 West Martin Luther King Jr. Blvd. 

Cincinnati, OH 45268 
NOTICE 

The research described in this document has been funded by the United States Environmental Protection Agency. It has 

been subjected to Agency peer and administrative review and approved for publication as an EPA document. 

Mention of trade names or commercial products does not constitute endorsement or 

recommendation for use. 

The correct citation for this document is: 

Walters, D.M. and M.J. Blum. 2007. Red shiner invasion of the upper Coosa River System: Dynamics and Ecological 

Consequences. EPA/600/R-07/124. U.S. Environmental Protection Agency, Cincinnati, Ohio. 

ii 
Executive Summary: 

Background: The red shiner (Cyprinella lutrensis) has been widely introduced across 11 states 

outside its native range, presumably through bait-bucket and aquarium releases. Its native range 

includes Great Plain and Central Lowland tributaries of the Mississippi River and western Coastal 

Plain drainages of the Rio Grande River. This species thrives under harsh conditions (e.g., low 

flow, high turbidity, poor water quality) and aggressively colonizes severely degraded habitats. 

Introduced populations spread rapidly, often displacing native Cyprinids. Red shiners readily 

hybridize with congeners, sometimes causing widespread displacement of native species. 

Hybridization is a significant threat to Southeastern Cyprinella, as red shiner hybrids have been 

reported for nine native taxa. NERL scientists, in collaboration with researchers at U.S. Geological 

Survey (USGS), The University of Georgia, and Duquesne University, conducted three related 

studies on the dynamics of red shiner invasion and hybridization with native blacktail shiners, C. 

venusta stigmatura, in the upper Coosa River System (UCRS). The overall goals of these studies 

were to identify environmental drivers of red shiner invasion and to determine genetic and 

environmental factors promoting hybridization. 

Key Findings: The first study investigated the role of anthropogenic disturbance in red shiner 

invasion and hybridization with blacktail shiners in tributary streams draining to mainstem rivers of 

the UCRS. Human disturbance increases the invasibility of lotic ecosystems and the likelihood of 

hybridization between invasive and native species. Historical collection records indicated that red 

shiners and hybrids have rapidly (up to 31 km y

-1) dispersed in the UCRS via large, mainstem rivers 

since the mid to late 1990s. We measured the occurrence and abundance of parental species and 

hybrids near tributary-mainstem confluences and characterized populations at these incipient 

contact zones by examining variation across morphological traits and molecular markers. Red 

shiners represented only 1.2% of total catch in tributaries yet introgression was widespread with 

hybrids accounting for 34% of total catch. Occurrence of red shiners and hybrids was highly 

correlated with occurrence of blacktail shiners, indicating that streams with native populations are 

preferentially colonized early in the invasion and that hybridization plays a key role in the 

establishment and expansion of invasive red shiners and their genome into new habitats. Tributary 

invasion was driven primarily through advanced backcross (post F1) individuals exhibiting 

asymmetry (in genetic and morphological traits) favoring blacktail shiner. Occurrence of red 

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shiners and hybrids and the relative abundance of hybrids all significantly increased with measures 

of human disturbance including turbidity, catchment agricultural land use, and low dissolved 

oxygen concentration. Red shiners pose a serious threat to southeastern Cyprinella species 

diversity, given that 41% of these species hybridize with red shiner, that five major Southeastern 

River drainages have been invaded, and that southeastern river systems are increasingly disturbed 

by urbanization. 

A second study focused on population dynamics and genetics of hybrid swarms in the mainstem 

UCRS in Georgia and Alabama (Conasauga River downstream to Weiss Lake) and the Terrapin 

Creek/Dead River system in Alabama. A detailed report is in preparation, but preliminary analyses 

revealed two key results. First, the hybrid swarm is comprised primarily of later generation (postF1) hybrids. F1 hybrids were rarely collected in sample populations suggesting that interactions 

between parental red shiner and blacktail shiner are infrequent. In contrast, advanced generation 

hybrids (F2 and backcross generations) were common, indicating that hybrids are viable and fertile. 

Second, the large-scale distribution of parent species and hybrids fit the classic tension zone model 

(a spatial model of species’ distributions describing a clinal transition from species A through a 

hybrid zone to species B). Populations in the southern UCRS (near Weiss Lake) had high relative 

abundance of red shiners, intermediate river reaches of the UCRS (Oostanuala River and lower 

Conasauga River) were dominated by hybrids, and upstream populations (upper Conasauga River) 

were dominated by blacktail shiners. At a finer scale, patterns of hybridization depart from the 

traditional tension zone model due to the dendritic structure of river-stream networks. Data from the 

Dead River/Terrapin Creek system revealed that (1) hotspots of hybridization occur at mainstemtributary confluences, and that (2) hybridization markedly attenuates in tributaries above 

confluences. 

The third study was a laboratory experiment to characterize prezygotic and postzygotic reproductive 

isolating mechanisms and to measure fitness among parent taxa (red and blacktail shiners) and their 

hybrid progeny. This information may enable timely management that aids or reinforces pre- and 

postzygotic isolation to prevent hybridization and curtail the loss of native congeners. We 

conducted conservative no-choice laboratory trials to measure mating preferences, and raised 

broods generated from intra- and interspecific crosses to assay hybrid viability through early 

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juvenile development. Females of both species were significantly more responsive to conspecific 

versus heterospecific mates, although blacktail shiner females responded more often to 

heterospecific mates than did red shiner females. Heterospecific crosses resulted in lower 

fertilization and egg hatching rates, but we found no other evidence of inviability. Rather, we found 

comparatively low larval mortality of hybrids, which is suggestive of heterosis. Considering prior 

studies that have linked high water turbidity to the formation of C. lutrensis x C. venusta hybrid 

swarms, a prescription of water quality improvement with a focus on reducing turbidity could 

reinstate sexual isolation and prevent further hybridization between introduced red shiner and 

endemic blacktail shiner in the Coosa River basin. 

Results of these studies provide an integrated picture of the processes driving red shiner invasion in 

the UCRS that may be applicable to other southeastern rivers. At the basin scale, large mainstem 

rivers serve as key corridors of dispersal for red shiners and their hybrid progeny. Tributary streams 

(particularly those with native populations) are subsequently invaded, further dispersing the red 

shiner genome into the drainage network. Anthropogenic stressors (e.g., detrimental land use 

practices, elevated turbidity) exacerbate the problem, increasing the vulnerability of streams to 

invasion and the vulnerability of native populations to hybridization. Effects on native populations 

are widespread and rapid. Assuming our reconstructed timeline of invasion is reasonable, 

introgressive hybridization has compromised native populations in hundreds of kilometers of lotic 

habitats over the course of a single decade. Evidence from tributary and mainstem populations 

indicates that post-F1 hybrids are driving the invasion. Most of these hybrids are physically 

indistinguishable from parental blacktail shiners, complicating efforts to monitor the impact of red 

shiner on native populations or to assess the efficacy of mitigation efforts. The prevalence of postF1 hybrids is indicative of backcrossing, suggesting that hybrids are both viable and fertile. This 

hypothesis was strongly supported by laboratory spawning experiments showing that heterspecific 

crosses were commonly successful and that F1 hybrids demonstrated vigor relative to parental 

species. 

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5 

CHAPTER 1. 

Red shiner invasion and hybridization with blacktail shiner in the upper Coosa River, 

USA 

D.M. Walters

1*, M.J. Blum1,2

, B. Rashleigh3, B.J. Freeman

4

, B.A. Porter 5, and N.M. 

Burkhead6 

1 U.S Environmental Protection Agency, National Exposure Research Laboratory, 26 

West MLK Blvd. Cincinnati, OH. 45268 

2

 Department of Ecology and Evolutionary Biology, Tulane University New Orleans, LA 

70118 

3

 U.S. Environmental Protection Agency, National Exposure Research Laboratory, 960 

College Station Road, Athens, GA 30605 

4

 Georgia Museum of Natural History and Odum School of Ecology, University of 

Georgia, Athens, GA 30602 

5

 Duquesne University, Department of Biological Sciences, 600 Forbes Avenue, 

Pittsburgh, PA 15282 

6 U.S. Geological Survey, Florida Integrated Science Center, 7920 NW 71st Street 

Gainesville, Florida 32653 

*corresponding author: walters.davidm@epa.gov; 513-569-7302 

running head: Red shiner invasion and hybridization 

key words: land use, turbidity, Cyprinella, hybrid swarm, introgression, Southeastern 

fishes, urbanization. 

To be submitted to: Conservation Biology 

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6 

Abstract: Human disturbance increases the invasibility of lotic ecosystems and the 

likelihood of hybridization between invasive and native species. We investigated the role 

of disturbance in the invasion of red shiner (Cyprinella lutrensis) and their hybridization 

with native blacktail shiner (C. venusta stigmatura) in the upper Coosa River System 

(UCRS). Historical collection records indicated that red shiners and hybrids have rapidly 

(up to 31 km y-1) dispersed in the UCRS via large, mainstem rivers since the mid to late 

1990s. We measured the occurrence and abundance of parental species and hybrids near 

tributary-mainstem confluences and characterized populations at these incipient contact 

zones by examining variation across morphological traits and molecular markers. Red 

shiners represented only 1.2% of total catch in tributaries yet introgression was 

widespread with hybrids accounting for 34% of total catch. Occurrence of red shiners 

and hybrids was highly correlated with occurrence of blacktail shiners, indicating that 

streams with native populations are preferentially colonized early in the invasion and that 

hybridization plays a key role in the establishment and expansion of invasive red shiners 

and their genome into new habitats. Tributary invasion was driven primarily through 

advanced backcross (post F1) individuals exhibiting asymmetry (in genetic and 

morphological traits) favoring blacktail shiner. Occurrence of red shiners and hybrids 

and the relative abundance of hybrids all significantly increased with measures of human 

disturbance including turbidity, catchment agricultural land use, and low dissolved 

oxygen concentration. Red shiners pose a serious threat to southeastern Cyprinella 

species diversity, given that 41% of these species hybridize with red shiner, that five 

major Southeastern River drainages have been invaded, and that southeastern river 

systems are increasingly disturbed by urbanization. 

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INTRODUCTION 

Invasive species are a primary threat to aquatic biodiversity (Allan & Flecker 

1993; Richter et al. 1997). Habitat disturbance facilitates fish invasion in lotic 

ecosystems (Gido & Brown 1999; Marchetti et al. 2004; Moyle & Light 1996) and 

increases the likelihood of hybridization between fishes (see studies reviewed in Rhymer 

& Simberloff 1996). Hybridization is a serious and under-appreciated aspect of species 

invasion, and can lead to genetic extinction of native species or the loss of locally adapted 

gene complexes (Hitt et al. 2003; Rhymer & Simberloff 1996). Hybridization can have 

the perverse effect of enhancing invasion success (Allendorf et al. 2001; Ellstrand & 

Schierenbeck 2000; Hitt et al. 2003; Rhymer & Simberloff 1996) by mitigating the 

constraint of propagule pressure, a key factor in successful colonization by invasive 

fishes and other species (Kolar & Lodge 2001; Ruesink 2005). 

Red shiners, Cyprinella lutrensis, have been introduced into at least five 

Southeastern drainages since the 1970s, presumable through bait-bucket or aquarium 

releases (Fuller et al. 1999). Red shiners thrive under harsh conditions (e.g., low flow, 

high turbidity, poor water quality) and aggressively colonize severely degraded habitats 

(Cross & Cavin 1971; Matthews 1985; Matthews & Hill 1977, 1979). Introduced 

populations spread rapidly, often displacing native congeners and other Cyprinids 

(Greger & Deacon 1988; Minckley & Deacon 1968; Moyle 2002). They readily 

hybridize with congeners, sometimes causing widespread displacement of native species 

(Larimore & Bayley 1996; Page & Smith 1970). Hybridization is a significant threat to 

Southeastern Cyprinella diversity, as red shiner hybrids have been reported for nine 

native species and subspecies (DeVivo 1996; Hubbs & Strawn 1956; Johnson 1999, W.C. 

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Starnes personal communication, Burkhead unpublished data; Page & Smith 1970; 

Wallace & Ramsey 1982). 

Prior studies have hypothesized that habitat disturbance increases both the 

likelihood of red shiner colonization and the likelihood of hybridization with congeners 

(Hubbs et al. 1953; Larimore & Bayley 1996; Page & Smith 1970). Examples of 

degraded lotic habitats colonized by red shiners include drainage ditches and severely 

altered agricultural and urban streams (DeVivo 1996; Moyle 2002; Page & Smith 1970). 

Likewise, red shiner hybrid swarms in Texas and Illinois were attributed to poor water 

quality and high turbidity (Hubbs et al. 1953; Hubbs & Strawn 1956; Larimore & Bayley 

1996; Page & Smith 1970). However, these prior accounts of habitat disturbance effects 

on red shiner invasions were observational, and empirical tests of these disturbance 

hypotheses are lacking. 

We investigated red shiner colonization and hybridization with native blacktail 

shiner, C. venusta stigmatura, in the upper Coosa River System (UCRS, Georgia, 

Alabama and Tennessee). We have observed rapid dispersal of red shiners and 

hybridization with blacktail shiners in mainstem rivers of the UCRS since the 1990s. 

Along with establishing a timeline of UCRS invasion, we measured the occurrence and 

abundance of parental species and hybrids near tributary-mainstem confluences. We then 

characterized the genetic composition of populations at these incipient contact zones by 

examining variation across morphological traits and molecular markers. This approach 

enabled us to determine whether the occurrence of native congeners inhibits (e.g., via 

competition) or enhances (e.g., via hybridization) dispersal of red shiners into new 

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habitats. Finally, we tested the hypotheses that disturbance (assessed at the basin and 

reach scales) promotes red shiner dispersal and hybridization with congeners. 

METHODS 

Invasion History and Site Selection 

We used an extensive database of fish distributions maintained by the Georgia 

Museum of Natural History (GMNH) to develop a chronology of red shiner and C. 

lutrensis x C. venusta stigmatura hybrids (hereafter hybrids) dispersal in the Upper Coosa 

River system (UCRS). All specimens, including putative hybrids, were identified on the 

basis of morphological characters (Boschung & Mayden 2003). 

Collection records indicated that red shiners or hybrids were distributed in 

mainstem rivers of the UCRS including Coosa River upstream of Weiss Lake, 

Oostanaula River, Conasauga River upstream to Dalton, GA, and downstream reaches of 

the Etowah and Coosawattee rivers (Figure 1). The sample population of tributaries 

entering these mainstem rivers included 43 second, third, and fourth order streams. 

Sample reaches within streams were located at the first or second most downstream road 

crossing, since reaches closer to mainstem populations are most likely to be colonized 

first. In the instances where neither location was suitable for sampling (e.g., too deep for 

wading), the stream was excluded. Due to these constraints, only 33 of the 43 available 

sites were sampled. 

Collection and characterization of fishes 

Reach-length for fish sampling was scaled to 25X stream width, and all reaches 

included both riffle and pool habitats. Representative habitats were sampled using a total 

of 30 kick-sets and/or seine hauls along with a backpack electrofisher. Samples were 

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collected between June and August 2005, and all Cyprinella were anesthetized and 

preserved in 95% ethanol for genetic analyses. 

Morphological Characterization 

We characterized all individuals ≥ 30mm standard length (SL) using three traits 

that differentiate red from blacktail shiners (Boschung & Mayden 2003): 1) caudal spot 

intensity, 2) the number of lateral line scales, and 3) the ratio of body standard length to 

depth (length:depth). The caudal spot is large and intense in blacktail shiners but absent 

in red shiners. Blacktail shiners have more lateral line scales (36-48 versus 33-36 for red 

shiners), and a higher length:depth ratio. Caudal spot intensity was assessed on a 

phenetic scale scored from zero (absent) to two (intense), with a faint or muted spot 

scored as a one. Specimens classified as hybrids exhibited obvious morphological 

intermediacy or incongruency (i.e., morphological traits of both parental species). For 

example, an incongruent specimen had a high lateral line scale count and length:depth 

ratio (blacktail shiner traits), but lacked a caudal spot (red shiner trait). We did not assess 

morphology of individuals < 30mm SL (n = 10, 2.4% total catch) due to the difficulty of 

counting scales on juveniles. 

Genetic Characterization 

DNA extraction and mtDNA RFLP assay: Genomic DNA was extracted from ~0.05 g of 

preserved fin tissue from each specimen using DNeasy kits (Qiagen, Inc., Valencia, CA). 

Approximately 10-50 ηg of DNA was then used as template for 15μl polymerase chain 

reaction (PCR) mixtures that also included 2.5 mM MgCl2, 2.5 mM each dNTP, 0.5 units 

Taq DNA polymerase (Invitrogen, Carlsbad, CA), 0.5 μM each of a pair of 

oligonucleotide primers and PCR buffer (Invitrogen, Carlsbad, CA) to a final 1X 

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concentration. The complete cytochrome b gene (1140bp) was amplified with primers HA 

and LA as described in Schmidt et al. (1998) under a thermal regime of 35 cycles of 94°C 

for 30 seconds, 49°C for 30 seconds, and 72°C for 90 seconds, followed by a final 

extension stage at 72oC for 5 minutes with a MJ Dyad thermocycler (MJ Research, Inc., 

Waltham, MA). 

Prior to PCR amplification of the cytochrome b gene from specimens in this 

study, we examined cytochrome b sequence variation in each species from an alignment 

of sequences previously obtained from 34 blacktail shiners from the upper Conasauga 

River (TN), Raccoon Creek (Etowah River, GA) and Sipsey Creek (Black Warrior River, 

AL) as well as sequences from 26 red shines from Peachtree Creek (Chattahoochee 

River, GA), and from the Canadian River (OK) (Blum et al., unpublished data). These 

data revealed that HinfI restriction sites of cytochrome b amplified from blacktail shiners 

generate ~ 130bp, 480bp and 530bp fragments versus ~ 95bp, 130bp, 350bp and 570bp 

fragments from red shiners. These differences formed the basis for a PCR-RFLP 

approach to establish species-level mtDNA ancestry of specimens in this study. The 

PCR-RFLP approach involved Hinf I restriction digestion of each cytochrome b PCR 

amplicon as recommended by the enzyme manufacturer and scoring fragment size 

profiles by agarose gel electrophoresis of the restricted amplicons. 

Microsatellite PCR amplification and analysis: Individuals were genotyped at seven 

polymorphic microsatellite markers developed for other target species. We used the 

following loci (modified annealing temperature given in parentheses): Can6EPA (53°C ) 

developed for Campostoma anomalum (Dimsoski et al. 2000); Nme 25C8.208 (55°C), 

Nme 18C2.178 (52°C), Nme 24B6.191 (57°C), and Nme 24B6.211 (57°C) developed for 

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Notropis mekistocholas (Burridge & Gold 2003); and Rhca20 (54°C ) and Rhca24 (52°C 

) developed for Rhinichthys cataractae (Girard & Angers 2006). PCR mixtures for 

amplifying microsatellite loci were identical to those designed for amplifying the 

cytochrome b gene. The PCR regime for all loci was 25 cycles of 60 seconds at 95°C, 60 

seconds at the locus-specific annealing temperature, and 90 seconds at 72°C, followed by 

a final extension stage of 7 minutes at 72°C. All reactions were run on an MJ Research 

Dyad with fluorescently labeled forward primers. Labeled PCR amplicons were 

characterized using a MJ Research Basestation Genetic Analyzer and Cartographer© 

software. 

Microsatellite allelic variation was analyzed using Structure v2.2 (Falush et al. 

2007) to construct a multi-locus admixture profile for all specimens. Few putative C. 

lutrensis specimens were collected, so admixture profiles were based on an expanded 

dataset that included additional “learning samples” (Montana & Pritchard 2004) of 50 C. 

lutrensis from Proctor Creek (Chattahoochee River) and genotyped for a related study 

(Blum et al. unpublished data). After several intermediate-length trial runs, we chose a 

burn-in period of 30,000 iterations and collected data from an additional 106 iterations for 

five replicate runs where K (the number of populations) was set at two, which is 

representative of the two parental species potentially contributing to the ancestry of each 

specimen. Each run was parameterized following a model of admixture and correlated 

allele frequencies, and average assignment values to each cluster were subsequently 

calculated for all specimens. Admixture categories reflected average assignment values 

to the first cluster as follows: (i) red shiner for values 0.90-1.0; (ii) backcross to red 

shiner (Hr) for values 0.76-0.89; (iii) F2 hybrids for values of 0.26-0.39 and 0.61-0.75; 

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(iv) F1 hybrids for values 0.40-0.6; (v) backcrosses to blacktail shiner (Hbt) for values 

0.11-0.25; and (vi) blacktail shiner for values 0-0.10. 

Integrative characterization of specimens 

Summary determinations of mixed ancestry for individuals ≥ 30mm SL reflected 

incongruency or intermediacy of an individual’s morphology, mtDNA haplotype and 

microsatellite genotype. Characterization of individuals <30mm SL was restricted to 

comparison of mtDNA haplotype and nuclear genotype. Comparison of phenotypic 

variation to mtDNA haplotype and nuclear genotype enabled us to differentiate 14 hybrid 

categories from red to blacktail shiner (e.g., Costedoat et al. 2007). For example, 

individuals were classified as hybrid if they exhibited blacktail shiner morphology and 

multi-locus microsatellite genotype, but expressed a red shiner mtDNA haplotype. 

Similarly, hybrid classification extended to individuals with a hybrid phenotype, but with 

blacktail mtDNA profile and microsatellite genotype. Hybrids were categorized as either 

putative F1 hybrids, or later generation hybrids with asymmetry favoring red shiner (i.e., 

red shiner backcross) or blacktail shiner traits. 

Environmental Characterization 

Water Quality and Geomorphology: Environmental variables and summary values are 

provided in Table 1. Baseflow turbidity was measured on three occasions using a handheld turbidity meter (Hach 2100P). Baseflow conditions were met if no rain was 

observed in the region for the preceding 72 hours and if USGS gauges on nearby streams 

indicated stable, low flow. Turbidity measurements were made at least two weeks apart 

from late May through early October, 2005, which overlaps with the spawning season for 

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red and blacktail shiner. Conductivity, pH and dissolved oxygen concentration were 

measured once per site using a Hydrolab Datasonde 4a. 

Depth, velocity, particle size, and large woody debris (LWD, >10 cm diameter) 

were measured along the centerline of the stream at intervals equal to 0.25 times the 

channel width (Walters et al. 2003b). The centerline transect length was 25X channel 

width, with n=100 observations per site. Mean width was calculated from five randomly 

selected locations within the first 100 m of the reach. Velocity was measured at 60% 

depth using a top-setting wading rod and velocity meter (Marsh-McBirney Flo-Mate). 

Maximum velocity was also measured in the area of highest flow observed within the 

reach. Substrate particle size was determined by visually estimating the dominant 

particle size class in a 50 cm diameter patch at each sample point (Walters et al. 2003b). 

Size classes were based on the phi scale (Gordon et al. 1992) and values were recorded 

as whole phi intervals (-log2 of intermediate axis in mm). These size classes were used to 

calculate the percentage of major substrate types (e.g., sand) for the reach. Each 

sampling point was visually estimated as either erosional (as in riffles) or depositional (as 

in pools) habitat. The presence of LWD in the cross section of the stream perpendicular 

to the centerline at the sampling point was also noted. Percentage open canopy was 

measured using a spherical densiometer at every 20th sampling location. Stream 

gradient (average gradient projected through the tops of riffles in the reach) was 

measured using an electronic total station. 

Spatial Analysis 

Land use for the basin upstream of sites was calculated from a 1998, 18-class 

land-use layer (NARSAL 2005a). Classifications were further grouped into 6 classes: 

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open water, urban (including high and low intensity urban and transportation areas), 

forest (including evergreen, deciduous and mixed forests), cleared land (including clear 

cuts, bare rock and quarries), agriculture (including pasture and row crops), and wetlands 

(including forested and non-forested wetlands). Only the three dominant land use types, 

urban, forest, and agriculture, were considered for analyses. For basins that extended 

beyond the Georgia border, data were patched in from the 1992 National Land Cover 

Dataset (Multi-Resolution Land Characteristics Consortium 1992). Percent of 

impervious surface area (ISA) in a 1 km radius surrounding the sampling location was 

also measured to provide an indicator of local land use conditions. ISA was calculated 

from 2001 color infrared photos with 1m resolution (NARSAL 2005b). Basin area and 

distance from the sample reach to mainstem tributaries were calculated using ArcMap 9.0 

(ESRI, Redlands, CA). 

Statistical Analysis of Distributional Patterns 

Exploratory analysis was conducted to prepare data for regression analysis. We used 

SAS (SAS Institute, Cary, NC) to examine variables for normality and skewness prior to 

regression analysis, and non-normal variables were transformed accordingly (Table 1). 

Boxplots were used to identify extreme outliers (>3 interquartile ranges away from either 

the sample 25th or 75th percentiles) (Jongman et al. 1995). Three variables (pH, CV-V, 

IA) had extreme outliers and were excluded from analyses. We calculated Spearman 

correlations among variables and retained only those correlated at |ρ|<0.80 in order to 

reduce multicollinearity (Glantz & Slinker 1990). Drainage area (DA) and width (W) 

were highly correlated (ρ=0.89), and W was retained. Particle size (PS) was highly 

correlated with percent sand (SAND, ρ=-0.85), and SAND was retained. 

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We used the remaining 20 environmental variables to develop multiple logistic 

regression models predicting the probability of occurrence for red shiner, blacktail shiner, 

and hybrids. We considered all models containing ≤ 3 variables to avoid overfitting 

models relative to sample size (Burnham & Anderson 2002). Models were constructed 

using variables within each of three variable classes (landscape, reach, water quality) 

both alone and in combination, for a total of seven categories of models. We used 

Akaike’s Information Criterion corrected for small sample size (AICc) to assess model 

goodness-of-fit, where smaller AICc indicates a more parsimonious model (Burnham & 

Anderson 2002). For each model i, difference (Δi) was calculated between the model’s 

AICc and the minimum AICc value within the set. Values of Δi < 2 are considered the 

most parsimonious (Burnham & Anderson 2002). A weight (wi) was calculated for each 

model according to Burnham and Anderson (2002). The weight can be interpreted as the 

probability that model i is the best model within the set of models considered. We 

calculated the percent of correctly predicted presence, absence, and overall occurrences. 

In order to examine the intensity of hybridization relative to environmental variables, we 

used multiple linear regression to relate hybrid shiner abundance to environmental 

variables for the subset of sites that contained hybrids, blacktail shiners, or both (n=18). 

We used the AIC approach for model selection and reported the r2

 of the final model. 

RESULTS 

Time-line of red shiner invasion of the Upper Coosa River System (UCRS) 

Red shiners were first collected in Weiss Lake in 1974 (Figure 1A) with 

additional populations collected in Terrapin Creek, a tributary of the “Dead River” arm of 

the Coosa River, in 1982. The earliest records upstream of Lake Weiss were two hybrids 

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collected in 1992 in Coahulla Creek and a single red shiner collected in 1993 from the 

lower Etowah River. Collections in the Oostanaula River in 1998 revealed an extensive 

hybrid swarm extending upstream from Lake Weiss to the confluence with the 

Conasauga River. The hybrid swarm rapidly dispersed into the Conasauga River 

between 2000-2003. We conducted annual surveys of the Conasauga River from 2000 

and 2003 to map the upstream extent of the hybrid swarm and documented that it 

extended 31 river km upstream between August 2000 and August 2001 alone. Four fish 

collections were made in the Oostanaula and lower Conasauga rivers between 1993 and 

1997 (Figure 1B). Blacktail shiners were collected at three of these sites, but neither red 

shiners nor hybrids were present, suggesting that most of the upstream dispersal of red 

shiners in the UCRS has occurred since the mid-to-late 1990s. A 2005 survey confirmed 

that a hybrid swarm currently extends from Lake Weiss north to Dalton (M. Blum and B. 

Porter, unpublished data). 

The absence of red shiners and hybrids from localities between Coahulla Creek 

and Lake Weiss from 1974 to 1992 suggests the potential for separate introductions in the 

southern and northern parts of the UCRS. Samples collected from 1993 to 1998 from 

Coahulla Creek, Conasauga River, and their tributaries failed to uncover additional 

specimens in the northern part of the UCRS prior to the upstream expansion of the swarm 

from the Oostanaula River in 2000 (Figure 1B). We draw four conclusions from 

historical records: 1) the spread of red shiners and hybrids in the upper Coosa River 

system likely began from the southern part of the UCRS in the vicinity of Weiss Lake; 2) 

red shiners and hybrids are dispersing upstream in the system via large, mainstem rivers; 

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3) much of the dispersal has occurred since the mid-to-late 1990s; and 4) the rate of 

dispersal is high (up to 31 km y

-1). 

It is important to note that red shiners and hybrid were previously collected from 

Cane Creek, a tributary of the lower Coosa River system, in 1970 and 1971 (R.D. 

Suttkus, Tulane University, retired, personal communication). Cane Creek is a tributary 

of Lake Logan Martin, a Coosa River impoundment. Lake Logan Martin and Weiss Lake 

are separated by another impoundment, Lake H. Neely Henry. The occurrence of red 

shiners in 1970 suggests that they could have been introduced to the UCRS earlier than 

museum records indicate. It is unlikely that red shiners from Cane Creek traversed two 

dams and two reservoirs spanning 97 river km (between Cane Creek and Weiss Lake) in 

four years. Rather, the association of red shiners with different reservoirs suggests 

multiple introductions within the Coosa River system, probably through bait-bucket 

releases. 

Genetic and morphological assessment of populations 

Red shiners, blacktail shiners, or hybrids were collected at 18 of 33 sites (Table 2, 

Figure 2). Red shiners were rarely collected, occurring at 5 sites and accounting for only 

1.2% of the total catch. They only occurred at sites that contained both hybrids and 

blacktail shiners. Hybrids and blacktail shiners each occurred at sixteen sites. Hybrid 

occurrence was significantly correlated with blacktail shiner occurrence (Χ2 = 18.9, p < 

0.0001) with hybrids occurring at all but two of the sites occupied by blacktail shiners 

and vice-versa. Overall, hybrids accounted for approximately 36% of the total catch, 

with the vast majority exhibiting asymmetry favoring blacktail shiners (Hbt, Table 2). 

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19 

Hybrids were well distributed among sites, with no clear trend in relative abundance with 

respect to upstream distance from Weiss Lake (Figure 2). 

Morphological traits distinguished between red shiners, blacktail shiners and 

several categories of hybrids (Figure 3). Red shiners and hybrids with asymmetry 

favoring red shiners (Hr) had fewer lateral line scales, lower length:depth, and a less 

intense or absent caudal spot. Values for all three measurements were higher for Hr 

individuals than red shiner, which is likely a reflection of blacktail shiner genomic 

contributions. F1 hybrids were intermediate for all morphological measurements, but 

showed more phenotypic overlap with blacktail shiners than red shiners. Blacktail 

shiners and hybrids showing asymmetry favoring blacktail shiners (Hbt) overlapped in all 

morphological measurements and were indistinguishable from one another based on the 

selected traits. 

Environmental regulation of occurrence and hybridization 

Overall percent correctly predicted occurrences was >75% for the best-fit models 

(Table 3), which is considered satisfactory model performance (Hurley 1986). Models 

constructed using variables from multiple environmental categories (i.e., landscape, reach 

geomorphology, and water quality) consistently outperformed single category models. 

Land use variables were generally absent from best-fit models, except for the negative 

relationship between hybrid occurrence and relative abundance with basin agriculture. 

Turbidity (NTU) was consistently selected in occurrence models that included water 

quality variables. However, NTU was not selected for models of hybrid relative 

abundance. Stream size and water velocity were consistent predictors of parental species 

and hybrids. All shiners were more likely to occur in larger, higher velocity streams. 

19 
20 

The most parsimonious model for blacktail shiners showed that they were more 

likely to occur in wider, more turbid streams with higher velocity (Table 4). None of the 

confidence intervals for the model parameters contained zero. Despite the similarity of 

the distribution patterns of blacktail shiners and hybrids, their best-fit models differed. 

Hybrids were more likely to occur at sites with higher turbidity and agricultural land 

cover that were closer to mainstem rivers. Only the confidence intervals for the intercept 

parameter contained zero (Table 3). Hybrid relative abundance was positively related to 

agriculture and negatively related to dissolved oxygen concentration. Relative abundance 

was also positively associated with gradient in this model. However, the confidence 

interval for this parameter contained zero, so there is uncertainty about this relationship. 

The AIC approach identified parsimonious models for red shiner occurrence, but even the 

most parsimonious model had poor explanatory power and confidence intervals on all 

model parameters contained zero. Low power for this model was related to infrequent 

occurrence of red shiners (n = 5 sites). 

DISCUSSION 

Dispersal of invasive red shiner 

Biological invasions generally follow a pattern of introduction, permanent 

establishment, and range expansion (Kolar & Lodge 2001). Prior reports of red shiner 

introductions compared pre- and post-introduction data collected decades apart (Moyle 

2002; Page & Smith 1970), and thus provide limited information on early stages of 

invasion and factors contributing to establishment and spread. We identified four 

patterns of red shiner dispersal in the upper Coosa River system that may be generalized 

to other red shiner invasions in other systems with native congeners. Red shiners and 

20 
21 

hybrids rapidly disperse (up to 31 river km y

-1) via large, mainstem rivers with 

subsequent invasion of tributaries. Tributaries with populations of native congeners were 

the first to be colonized and the incipient invasion of tributary streams was driven 

primarily by hybrids. The strong affinity of red shiners and hybrids for streams with 

blacktail shiner populations suggests that hybridization plays a key role in the 

establishment and expansion of red shiners and their genome into new habitats, 

supporting prior findings that introgressive hybridization facilitates the establishment and 

spread of invasive species (Allendorf et al. 2001; Ellstrand & Schierenbeck 2000; Hitt et 

al. 2003; Rhymer & Simberloff 1996). This mechanism of dispersal is particularly 

relevant for invasive fishes, which are more prone to hybridization than other vertebrates 

(Allendorf et al. 2001; Rhymer & Simberloff 1996). 

Hybridization often complicates taxonomic identification, hindering the 

assessment of native and introduced populations (Allendorf et al. 2001; Rhymer & 

Simberloff 1996). Populations in the UCRS expressed a mosaic of parental phenotypes 

and genotypes and are best characterized as hybrid swarms (Allendorf et al. 2001). The 

majority of hybrids exhibited asymmetry favoring blacktail shiners (Hbt). These were 

morphologically indistinguishable from parental species, suggesting that the degree of 

hybridization could be underestimated without complementary data from multi-locus 

genetic analyses. Thus, our reconstructed timeline conservatively estimates the 

geographic extent and expansion of the hybrids, since it depended on morphological 

identification of specimens. 

The nature of pre- and postzygotic reproductive isolation between red and 

blacktail shiners may influence patterns in phenotypic and genetic variation observed in 

21 
22 

the UCRS. Artificial crosses (where eggs and sperm were mixed to produce hybrids) and 

backcrosses of laboratory reared hybrids to red and blacktail shiners have produced 

viable and fertile hybrids (Hubbs and Strawn 1956). The UCRS hybrid swarm is 

dominated by later generation hybrids. F1 hybrids are relatively uncommon, suggesting 

that interactions between red and blacktail shiners are infrequent. If so, then prezygotic 

reproductive isolation is stronger than postzygotic isolation between red and blacktail 

shiners (Mendelson 2003). The predominance of Hbt individuals among hybrids is also 

suggestive of asymmetric pre- or postzygotic barriers that promote introgression. The 

comparatively low abundance of red shiners likely augments asymmetric backcrossing 

between hybrids and blacktail shiners (Taylor and Hastings 2005). Thus, undiminished 

hybrid fitness and persistent backcrossing under demographic conditions that favor 

introgression could be driving progressive genetic assimilation of blacktail shiners with 

few (if any) external indications of hybridization. 

The role of disturbance in red shiner invasion and hybridization 

Regression models supported prior hypotheses that disturbance increases 

colonization by red shiners and hybridization with congeners (e.g., Hubbs et al. 1953; 

Larimore & Bayley 1996; Page & Smith 1970) and other studies linking disturbance with 

fish invasion of lotic ecosystems (Gido & Brown 1999; Marchetti et al. 2004; Moyle & 

Light 1996). Best-fit models indicated that hybrid and red shiner occurrence increased 

with turbidity and agricultural land use. These findings have two limitations. First, red 

shiner models had low power due to small sample size, limiting their interpretation. 

Second, we cannot separate the role of disturbance from the presence of blacktail shiners 

in determining the distribution of red shiners and hybrids. That is, red shiners and 

22 
23 

hybrids both had strong affinity for blacktail shiner streams, but blacktail shiners were 

also positively correlated with turbidity, an indicator of stream disturbance. Hybrid 

relative abundance was also positively related to disturbance, particularly lower dissolved 

oxygen concentration and increased agricultural land use. This suggests that rates of 

hybridization or hybrid survival increase with disturbance, thereby facilitating the spread 

of red shiner genome into native populations. Occurrence and hybrid relative abundance 

models using variables from multiple environmental categories (i.e., landscape, reach 

geomorphology, and water quality) consistently outperformed single-category models. 

Superior performance of these models indicate that red shiner colonization and 

hybridization are influenced by factors operating at multiple spatial scales and that 

physical habitat and water quality are both influential at the reach-scale. 

We expected positive correlations between turbidity and hybrid relative 

abundance since turbidity has been associated with other red shiner hybrid swarms 

(Hubbs et al. 1953; Hubbs & Strawn 1956; Larimore & Bayley 1996). Turbidity is 

thought to weaken prezygotic reproductive barriers by impairing visual recognition and 

assortative mating among Cyprinella species (Page and Smith 1970, Hubbs et al. 1956). 

This hypothesis is consistent with other studies demonstrating that turbidity can weaken 

sexual selection and promote hybridization (Candolin et al. 2007; Seehausen et al. 1997). 

Contrary to our expectations, turbidity was not a leading predictor of hybrid relative 

abundance. The influence of turbidity on hybridization rates may be obscured in the 

UCRS because blacktail shiners appear to be moderately tolerant to elevated turbidity 

(i.e., blacktail shiner occurrence was positively correlated with turbidity in this study). 

Alternatively, other aspects of mating behavior unaffected by turbidity could 

23 
24 

counterbalance the negative effects of turbidity on assortative mating. Controlled 

spawning experiments under different turbidity regimes are needed to test the hypothesis 

that light limitation increases hybridization between red shiners and congeners. 

Conservation implications for Southeastern Rivers and Cyprinella diversity 

Hybridization with red shiners is a serious threat to Southeastern Cyprinella 

diversity. Twenty-two Cyprinella occur in the Southeast (Warren et al. 2000), and nine 

(41%) hybridize with red shiners in the wild (C. analostana, C. camura, C. callitaenia, C. 

spiloptera, C. venusta cercostigma, C. v. stigmatura, C. v. venusta, and C. whipplei; 

(Hubbs & Strawn 1956; Johnson 1999, and W.C. Starnes personal communication; Page 

& Smith 1970; Wallace & Ramsey 1982)) or under laboratory conditions (C. caerulea, 

Burkhead, unpublished data). Southeastern rivers colonized by red shiners include the 

Pee Dee, Roanoke, Mobile, Apalachicola, and Altamaha river drainages (Fuller et al. 

1999), and hybrids have been documented in three of these systems (DeVivo 1996, W.C. 

Starnes personal communication, this study; Wallace & Ramsey 1982). Southeastern 

fishes are characterized by high rates of endemism and imperilment, and nine Cyprinella 

are endemic to a single southeastern river drainage (Warren et al. 2000). These species 

are particularly vulnerable to red shiner invasion considering their limited distribution 

and relatively small populations (Allendorf et al. 2001; Rhymer & Simberloff 1996). 

While the blacktail shiner is not a protected species, their displacement via 

hybridization in the Coosa system underscores the conservation challenges facing other 

southeastern Cyprinella species. Hybridization is difficult to stop if, as our data suggest, 

hybrids are fertile and capable of backcrossing with parental taxa (Allendorf et al. 2001; 

Rhymer & Simberloff 1996). Native populations decline rapidly under these conditions 

24 
25 

as hybrid swarms form within a few generations and the proportion of parental 

individuals progressively declines in successive generations (Allendorf et al. 2001). 

Through this process, native species are lost or relegated to isolated parts of their former 

range. Our data support this model of native species decline, with widespread 

introgression of populations in lower reaches of mainstem tributaries. 

Urbanization will likely play an increasing role in red shiner invasions of 

Southeastern rivers. The Southeast is among the most rapidly developing regions of the 

U.S. (U.S. Department of Agriculture 2000), and the UCRS epitomizes development 

pressures facing these river systems. Human population in the UCRS increased 37% 

(270000 to 330000) from 1990 to 2006 with 7400 residential building permits issued 

from 2000-2006 in one county (Bartow) centrally located in the UCRS (U.S. Census 

Bureau 2007). Red shiners thrive in urban streams (DeVivo 1996) whereas endemic and 

other native fishes decline (Walters et al. 2003a). It is reasonable to assume that 

urbanization will increase vulnerability of Southeastern streams and native Cyprinella to 

red shiner invasion, barring management actions to mitigate the detrimental effects of 

urbanization on stream ecosystems. 

ACKNOWLEDGEMENTS 

We thank D. Homans for assisting in study design and supervising sample collection, B. 

Dakin, T. Crum, and C. Tepolt for generating the genetic data, C. Straight and M. Reif 

for compiling and mapping collection data, R.D. Suttkus and N. Rios for providing 

additional collection records, and K. Oswald for reviewing the manuscript. Although this 

work was reviewed by US EPA and approved for publication, it may not necessarily 

25 
26 

reflect official Agency policy. Mention of trade names or commercial products does not 

constitute endorsement or recommendation for use. 

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29 

Table 1. Environmental variables collected from mainstem tributaries in the upper Coosa River system. 

acronym variable description units transform range mean (1SD) 

landscape 

DTMS distance to mainstem (m) m log 0.2-7.8 2.3 (1.6) 

DA drainage area (km2

) km2 log 4.6-456.3 46.7 (80.6) 

URB basin urban area % arcsine square-root 3.1-56.1 15.3 (13.2) 

FOR basin forested % arcsine square-root 18.7-86.7 55.8 (16.3) 

AG basin agriculture % arcsine square-root 2.9-67 21.5 (12.9) 

IA impervious area, 1-km radius % arcsine square-root 0.2-26.1 4.1 (6.5) 

reach habitat 

GR gradient % arcsine square-root 0.01-1.4 0.3 (0.3) 

W average width m log 2.8-12.5 6.4 (2.5) 

V maximum velocity m sec

-1 log 0.03-1.12 0.57 (0.26) 

V-m mean velocity m sec-1 log 0-0.3 0.11 (0.1) 

D average depth m log 0.2-1 0.4 (0.2) 

PS average particle size phi log2 1.6-8 4.1 (1.3) 

SAND proportion sand % arcsine square-root 0-59 12.8 (15.1) 

GRAV proportion gravel % arcsine square-root 4.8-96.3 62.5 (22.8) 

COBB proportion cobble % arcsine square-root 0-74 9.5 (18.2) 

BR proportion bedrock % arcsine square-root 0-67.9 15.2 (18.5) 

LWD proportion large woody debris % arcsine square-root 0-66 13.3 (13.7) 

EH proportion of erosional habitat % arcsine square-root 6-56.2 28.1 (14.7) 

CV-D CVa

 depth % none 31-99.5 61.2 (18.2) 

CV-V CV velocity % none 0-612.3 147.2 (117.1) 

OC % open canopy % arcsine square-root 1.8-72.7 27.1 (19.5) 

water quality 

CON conductivity μS cm-2 log 88-573 232.8 (84.8) 

NTU mean turbidity NTUb log 1.4-25.1 6.6 (5.3) 

DO dissolved oxygen mg l-1 log 2.4-9.7 7.1 (1.8) 

pH pH NA none 3.8-8.7 7.8 (0.8) 

a

 coefficient of variation 

b

 nephelometric turbidity units 

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30 

Table 2. Percent population contribution of red shiner (RS), hybrids exhibiting 

asymmetry favoring red shiner (Hr), F1 hybrids, hybrids exhibiting asymmetry favoring 

blacktail shiner (Hbt), and blacktail shiner (BTS) collected in the study area. Sample 

locales are shown in Figure 2. 

Site code Site name n RS Hr F1 Hbt BTS 

1 Coahulla Cr. 73 0 9.6 0 12.3 78.1 

2 Little Cr. 2 0 0 0 50 50 

3 Drowning Bear Cr. 1 0 0 0 0 100 

4 Jobs Cr. 70 0 2.9 4.3 28.6 64.3 

5 Swamp Cr. 61 0 11.5 4.9 23 60.7 

6 Polecat Br. 5 20 20 0 20 40 

7 Town Cr. 47 4.3 2.1 6.4 34 53.2 

8 Dry Cr. 35 2.9 5.7 5.7 25.7 60 

9 Oothkalooga Cr. 11 0 9.1 9.1 27.3 54.5 

10 Snake Cr. 26 0 3.8 3.8 19.2 73.1 

11 Bow Cr. 6 16.7 16.7 0 0 66.7 

12 Rocky Cr. 6 0 16.7 33.3 0 50 

13 Johns Cr. 29 0 0 3.4 20.7 75.9 

14 Lovejoy Cr. 6 0 0 33.3 66.7 0 

15 Woodward Cr. 16 0 0 6.3 25 68.8 

16 Silver Cr. 12 0 0 0 8.3 91.7 

17 Webb Cr. 2 0 0 0 0 100 

18 Kings Cr. 1 0 100 0 0 0 

Totals 409 1.2 6.1 4.6 22.7 65.3 

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Table 3. Predictive habitat models for presence of blacktail shiner (BTS), hybrids, and 

red shiner (RS) as well as relative abundance of hybrids presented in rank order. K is the 

number of estimable parameters in the model; AICc is Akaike’s Information Criterion 

corrected for small sample size (smaller is better); Δi is the difference in AICc from the 

best-fit model; and w is the model weight (models with higher weights are more 

parsimonious). Explanations of predictor variables are given in Table 1. Variable 

categories: reach = reach habitat; wq = water quality; land = landscape. 

Model Predictor variables K AICc ΔAICc w % Correctly predicted 

Presence Absence Overall 

BTS presence= 

(reach+wq) 

(reach) 

(reach+land) 

(reach+land+wq)

(land+wq) 

(wq) 

(land) 

Hybrid presence= 

(land+wq) 

(reach+wq) 

(reach) 

(reach+land+wq)

(wq) 

(reach+land) 

(land) 

RS presence= 

V, W, NTU 

V, V-m, COBB 

W, V, DTMS 

 W, NTU, DTMS 

NTU, URB, CON 

NTU, CON, DO 

AG, FOR, DTMS 

AG, NTU, DTMS 

D, NTU, CON 

V, EH, GRAV 

 W, NTU, DTMS 

NTU, CON 

D, W, DTMS 

URB, DTMS 

4 

4 

4 

4 

4 

4 

4 

4 

4 

4 

4 

3 

4 

3 

29.04 

29.37 

32.95 

33.00

37.55 

38.62 

50.47 

25.00 

26.64 

27.55 

29.11

32.54 

32.69 

41.06 

0 0.19 81.3 82.4 81.8 

0.33 0.19 75.0 76.5 75.8 

3.91 0.16 81.3 82.4 81.8 

 3.96 0.16 68.8 76.5 72.7 

8.51 0.12 75.0 82.4 78.8 

9.58 0.12 81.3 70.6 75.8 

21.43 0.07 43.8 47.1 45.5 

0 0.18 87.5 88.2 87.9 

1.64 0.17 75.0 70.6 72.7 

2.55 0.16 81.3 82.4 81.8 

 4.11 0.15 81.3 88.2 84.8 

7.54 0.13 81.3 88.2 84.8 

7.69 0.13 75.0 70.6 72.7 

16.06 0.08 75.0 76.5 75.8 

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32 

(reach+wq) V-m, NTU, CV-D 4 15.49 0 0.21 60.0 89.3 84.8 

(reach+land+wq) V-m, FOR, NTU 4 18.94 3.45 0.17 40.0 92.9 84.8 

(reach+land) V-m, FOR, W 4 22.67 7.18 0.14 60.0 92.9 87.9 

(reach) D, V-m 3 23.44 7.95 0.14 40.0 96.4 87.9 

(land+wq) DTMS, NTU 3 25.32 9.83 0.13 20.0 89.3 78.8 

(wq) NTU, DO 3 26.49 11.0 0.12 40.0 96.4 81.8 

(land) FOR, DTMS 3 32.27 16.78 0.09 0.0 92.9 78.8 

Hybrid abundance 

(reach+land+wq) DO, AG, GR 4 -5.9 0 0.17 

(reach+wq) GR, DO, EH, 4 -4.4 1.5 0.15 

(land+wq) FOR, URB, DO 4 -3.9 2.0 0.15 

(reach+land) GR, FOR, V-m 4 -3.4 2.5 0.15 

(land) AG, FOR 3 -2.4 3.5 0.14 

(reach) LWD, EH, GR 4 -1.1 4.8 0.13 

(wq) DO, CON 3 1.4 7.3 0.12 

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33 

Table 4. Parameter estimates for best-fit models from Table 3. Logistic and linear 

regression models are based on occurrence and relative abundance, respectively. 

Logistic 

model 

BTS 

N 

33 

Wald chisquare 

8.1 

P 

0.0448 

Parameter Parameter 

estimate 

Standard 

error 

95% Confidence 

Interval 

Lower Upper 

Intercept -17.3 6.3 -29.6 -5.0 

NTU 6.6 3.2 0.3 12.8 

V 7.7 3.3 1.2 14.2 

W 10.7 5.1 0.7 20.6 

Hybrid 33 6.9 0.0760 

Intercept 15.0 11.0 -6.6 36.5 

DTMS -13.5 5.7 -24.7 -2.2 

AG 20.9 8.9 3.4 38.4 

NTU 13.7 5.3 3.3 24.1 

RS 33 1.8 0.6176 

Intercept -22.3 19.5 -60.7 16.0 

V-mean 140.1 120.7 -96.4 376.6 

NTU 27.8 27.7 -24.4 82.0 

CV-D -0.3 0.3 -0.7 0.3 

Linear model r

2

 F, Pr>F 

Hybrid 18 0.50 4.6 (0.02) 

Intercept 0.9 0.5 -0.3 2.0 

DO -1.5 0.5 -2.6 -0.4 

AG 0.9 0.4 0.1 1.6 

GR 0.4 2.9 -5.9 6.7 

33 
34 

List of Figures 

Figure 1. A) Historical spread of red shiners and C. lutrensis x C. venusta hybrids in the 

upper Coosa River system. Key locales and dates illustrate the earliest known collections 

in the system and the rapid upstream migration of phenotypic red shiner and hybrids. For 

illustration purposes, 2001-2003 records for the Conasauga River only include the 

upstream-most site where red shiner or hybrids were collected. B) Extent of fish 

collections 1993-98 (when the hybrid swarm was first discovered) and 1998-2001 

(corresponding with the period of rapid upstream dispersal). Blacktail shiners are widely 

distributed in 3rd order and larger streams, but red shiners and hybrids were collected at 

only three locales upstream of Lake Weiss prior to 1998 (see Figure 1A). No red shiners 

or hybrids were collected in the Coahulla Creek or Conasauga River systems from 1993 – 

1998, suggesting that the red shiners and hybrids did not radiate southward from a 

northern introduction. Samples collected from 1998-2001 confirm that red shiners and 

hybrids upstream of Weiss Lake were limited to the mainstem Oostanaula and Conasauga 

rivers. 

Figure 2. Collection locales in mainstem tributaries. Relative abundance (percent) of red 

shiner, blacktail shiner, and hybrids are provided in pie charts. Open circles indicate 

locales where both species and hybrids were absent. Site codes are in Table 2. 

Figure 3. Box and whisker plots of caudal spot intensity, lateral line scales, and lengthto-depth ratio of for red shiners (RS), hybrids exhibiting asymmetry favoring red shiner 

(Hr), F1 hybrids, hybrids exhibiting asymmetry favoring blacktail shiner (Hbt), and 

34 
35 

blacktail shiner (BTS) collected in the study area. Top and bottom boundaries of the box 

indicate 75th and 25th percentiles, respectively, and the line within the box indicates the 

median. Whiskers above and below the box indicate the 90th and 10th percentiles. Points 

outside the boxes are outliers (observations beyond the 10th and 90th percentiles). 

35 
36 

Figure 1. 

A 

B 

36 
37 

Figure 2. 

37 
Figure 3. 

0 

1 

2 

3 

caudal spot intensity 

3 

3 

4 

4 

5 

lateral line scales 3. 

3. 

4. 

4. 

5. 

length:depth 

RS Hr F1 Hbt BTS 

38
CHAPTER 2. 

Preventing loss of native species following hybridization with an invasive species; 

Lessons from prezygotic and postzygotic reproductive isolation between introduced red 

shiner and native blacktail shiner (Cyprinidae: Cyprinella) 

Michael J. Blum1,2*, David M. Walters1, Noel M. Burkhead3, 

Byron J. Freeman

4

, Brady A. Porter5

1 National Exposure Research Laboratory, US Environmental Protection Agency, 

 Cincinnati, OH 45268 

2 Department of Ecology and Evolutionary Biology, Tulane University, New Orleans, LA 

70118 

3

 Florida Integrated Science Center, US Geological Survey, Gainesville, FL 32653 

4

 Georgia Museum of Natural History and Odum School of Ecology, University of 

Georgia, Athens, GA 30602 

5

 Department of Biological Sciences, Duquesne University, Pittsburgh, PA 15282 

*corresponding author 

Contact information: Department of Ecology and Evolutionary Biology, Tulane 

University, New Orleans, LA 70118 

Phone: 504-862-8295 

Fax: 504-862-8076 

Email: mjblum@tulane.edu 

Word count: ca. 5,400 (including references) 

Article type: Original article 

Key words: hybrid fitness, mate preference, biological invasion, hybrid swarm,

freshwater fish, aquatic biodiversity 

Running title: Patterns of reproductive isolation between invasive and native cyprinids 

To be submitted to: Conservation Genetics 

39
ABSTRACT

Biological invasions involving hybridization can lead to rapid displacement and loss of 

native species. Timely management that aids or reinforces pre- and postzygotic isolation 

could be effective for preventing hybridization and curtailing the loss of native species. 

Such strategies should account for how isolating mechanisms vary across species 

interactions and environments. Here we present a study of prezygotic and postzygotic 

reproductive isolation between non-native red shiner (Cyprinella lutrensis) and blacktail 

shiner (C. venusta stigmatura) from the Coosa River basin where a hybrid swarm is 

rapidly expanding. We conducted conservative no-choice spawning trials to measure 

mating preferences and raised broods from intra- and interspecific crosses to assay hybrid 

viability through early juvenile development. Females of both species were significantly 

more responsive to conspecific versus heterospecific mates, although blacktail shiner 

females responded more often to heterospecific mates than did red shiner females. 

Heterospecific crosses resulted in lower fertilization and egg hatching rates, but we found 

no other evidence of inviability. Rather, cumulative post-fertilization and larval mortality 

of hybrids were significantly lower than parent species, which is suggestive of heterosis. 

Considering prior studies that have linked high water turbidity to the formation of C. 

lutrensis x C. venusta hybrid swarms, a water quality improvement plan focused on 

turbidity reduction could potentially reinstate sexual isolation and prevent further 

hybridization between introduced red shiner and endemic blacktail shiner in the Coosa 

River basin. 

40
INTRODUCTION 

Finding evidence of hybridization between an invasive species and a native 

congener has become tantamount to tolling a death knell for the native species. 

Hybridization can provide a foothold for the establishment of non-native species, and can 

initiate widespread invasions (Ellstrand and Schierenbeck, 2000; Sakai et al., 2001; Lee, 

2002). Biological invasions resulting from hybridization can lead to rapid displacement 

and loss of native species via genetic dilution or assimilation (Rhymer & Simberloff, 

1996). Wolf et al. (2001) estimated that the genetic extinction of an endemic species can 

occur as quickly as five generations following an initial hybridization event. Under such 

potentially dire circumstances, can anything be done to prevent the loss of native species? 

Is it possible to avoid genetic extinction by preventing or even eliminating hybridization 

between non-native species and native congeners? 

Timely management that aids or reinforces pre- and postzygotic isolation could be 

effective for preventing hybridization and curtailing the loss of native species. Such 

strategies must account for how isolating mechanisms vary across species interactions 

and environments. In some sexually dimorphic freshwater fishes, for example, prezygotic 

isolation can be sufficient to prevent hybridization between congeners (Mendelson, 

2003). However, elevated turbidity due to eutrophication or excessive sedimentation can 

weaken sexual selection and result in hybridization (Hubbs & Strawn, 1956; Seehausen et 

al., 1997; Järvenpää & Lindström, 2004). Similarly, the spread of hybrids is often 

constrained by environmental conditions that favor parental species, but disturbance can 

create marginal habitats where selection against hybrids is comparably weak (Anderson, 

1949). 

41
Developing management approaches intended to prevent hybridization requires 

identifying causal factors of pre- and postzygotic isolation, particularly when it is unclear 

how environment influences species interactions or the success of hybrids. Hubbs et al. 

(1953) observed that episodes of hybridization between sexually dimorphic red shiner 

(Cyprinella lutrensis) and blacktail shiner (C. venusta) in the Guadalupe River coincided 

with increased turbidity resulting from excessive sedimentation. Jurgens (1951) 

discovered a second C. lutrensis x C. venusta hybrid swarm in a turbid reach of the San 

Marcos River adjacent to active oil fields. Hybridization in the San Marcos River abated 

as water quality improved, leading Hubbs & Strawn (1956) to conclude that turbidity 

disrupts species recognition during spawning, and by extension, that hybridization abates 

because prezygotic isolation is reinstated as turbidity declines. Although this is consistent 

with other studies that show increased turbidity weakens or alters sexual selection 

(Järvenpää & Lindström, 2004; Candolin et al., 2006; Engström-Öst & Candolin, 2007) 

some evidence suggests that C. lutrensis x C. venusta F1 and F2 hybrids may be inviable 

or have comparably low fitness (Hubbs & Strawn, 1956). Identifying causal factors of 

pre- and postzygotic isolation between red shiner and congeners would help clarify 

whether mitigating environmental disturbance improves species recognition or eliminates 

conditions favoring the persistence of hybrids, thereby laying the groundwork for 

management strategies to curtail hybridization. 

The spread of red shiner and hybridization with endemic blacktail shiner (C. 

venusta stigmatura) in the Coosa River basin presents an opportunity to implement novel 

management practices to control a nuisance species if more were known about the nature 

of reproductive isolation among Cyprinella species. Recent surveys of the upper Coosa 

42
River basin have documented extensive hybridization between introduced red shiner and 

endemic blacktail shiner. The hybrid swarm formed around 1998, following at least 6 

years of intermittent hybridization (Walters et al., unpublished data). Subsequent surveys 

have tracked the rapid upstream expansion of the swarm (Walters et al., unpublished 

data). The hybrid swarm now overlaps with the largest remaining population of federally 

Threatened blue shiner (C. caerulea) in the Conasauga River. Continued expansion of red 

shiners is a viable threat to this population, since they electively hybridize with blue 

shiner under laboratory conditions (Burkhead et al., 2006). Considering that prior studies 

(Jurgens, 1951; Hubbs & Strawn, 1956; Page & Smith, 1970) of red shiner and blacktail 

shiner have linked hybridization with deteriorated water quality, it might be possible to 

modify existing water quality management practices to prevent hybridization. Greater 

understanding of isolation mechanisms among red shiner and blacktail shiner would help 

guide development and application of such modifications. 

Here we present a study of prezygotic and postzygotic reproductive isolation 

between non-native red shiner and blacktail shiner from the Coosa River basin. Prior field 

and laboratory studies (Hubbs & Strawn, 1956) found that red shiners electively 

hybridize with blacktail shiners, but no studies have documented the strength or 

symmetry of prezygotic isolation between the two species. Similarly, it has been shown 

that C. lutrensis x C. venusta hybrids can be fertile (Hubbs & Strawn, 1956), but hybrid 

fertility and inviability have not been quantified. We conducted conservative no-choice 

laboratory trials with wild-caught fish to measure mating preferences, and raised broods 

generated from intra- and interspecific crosses in common garden conditions to assay 

hybrid viability from post-fertilization to early juvenile development. Comparison of pre43
and postzygotic isolation enabled us to infer what range of management practices could 

potentially prevent further hybridization between introduced red shiner and native 

congeners. 

METHODS 

Field collection, transport and care 

Individuals used in trials were collected between July 2004 and October 2005. 

Blacktail shiners were collected from the upper Conasauga River in Whitfield County 

(GA), and Bradley County (TN) more than 28 river km above the upstream front of the 

C. lutrensis x C. venusta hybrid swarm in the Coosa River system (Walters et al., 

unpublished data). Blacktail shiners did not exhibit any intermediate or incongruent 

morphological or molecular genetic attributes indicative of hybridization (Blum et al., 

unpublished data), but it remains possible that all blacktail shiners in the system are 

nominally affected by introgression due to the extent of hybridization within the upper 

Coosa River basin. The C. lutrensis x C. venusta hybrid swarm encompasses all sites 

where red shiners are known to occur in the upper Coosa River basin. To avoid 

complications arising from introgression, we collected non-native red shiners from 

Proctor Creek (Dekalb Co., GA) in the adjacent Chattahoochee River basin. Red shiners 

are abundant and widespread in Atlanta metropolitan area streams, like Proctor Creek 

(Couch et al., 1995; DeVivo, 1996). No evidence of persistent hybridization between red 

shiner and blacktail shiner (C. venusta cercostigma) has been documented in this region, 

and blacktail shiners are rare in these highly urbanized streams (Couch et al., 1995; 

DeVivo, 1996; Herrington, 2004). 

44
Fishes were collected by seine and transferred to live wells containing local, 

temperature-equilibrated water prepared with 2mg/L metomidate, 0.13ml/L Ammo Lock 

(Aquarium Pharmaceuticals), and 0.26ml/L Stress Coat (Aquarium Pharmaceuticals) to 

reduce stress during transport. In the laboratory, fishes were transferred to 38L tanks on a 

flow-through, well-water system. Density per tank was limited to 2.5 cm of fish per 3.8L 

of water. Individuals were segregated to prevent all contact between species and 

prophylactically treated using oxytetracyclin medicated flake food. Following treatment, 

individuals were transferred to 120L circular, flow-through tanks and maintained on a 

diet of 43% protein pellet food. 

Prezygotic reproductive isolation: Mating experiments 

Upon onset of male nuptial coloration (e.g. red and yellow fin coloration in male 

red and blacktail shiners, respectively) in May 2006, individuals of both species were 

sexed and segregated. Photoperiod was adjusted to a 12:12 day-night cycle to promote 

spawning behavior. 

When females of both species appeared gravid, we transferred nuptial pairs to 

individual spawning tubs. Nuptial pairs consisted of one male and one female in four 

cross configurations: red shiner male x red shiner female, red shiner male x blacktail 

shiner female, blacktail shiner male x red shiner female, blacktail shiner male x blacktail 

shiner female. Twenty unique pairs of each configuration were established. No individual 

was used twice. This no-choice trial design (i.e., where a single male and female are 

allowed to interact) provides a conservative estimate of mating preferences (Coyne, 1993; 

Hatfield & Schluter, 1996). Each nuptial pair was kept for an initial period of seven days 

in a 66.5L black plastic, circular tub equipped with an Aquaclear powerhead (Model 402) 

45
to create circulation, a 4cm air diffuser (0.5cm slip, medium pore) to maintain aeration, 

and a single spawning tower. Towers consisted of stacked large (6cm x 6cm) alternating 

with small (5cm x 5cm) black squares of 3mm thick plexi-glass arrayed onto a 0.6cm 

stainless steel rod attached to a clear plexi-glass base. Spawning tubs were inspected 

daily for eggs in and around the spawning tower. After seven days, nuptial pairs were 

transferred to separate 38L flow-through tanks containing a spawning tower and 

monitored for another 120 days. 

Males of both species maintain spawning territories. Females inspect territories, 

and choose whether to deposit eggs within crevices in a male’s territory. Thus, we 

measured female preference as whether or not a female deposited eggs in crevices. We 

did not measure male preference because it was impossible to distinguish between 

milting runs and visually similar behaviors such as inspection of spawning crevices. 

Post-mating prezygotic reproductive isolation and postzygotic reproductive isolation: 

Fertilization, hatching success and larval mortality 

Spawning towers containing eggs were transferred to tubs containing temperatureequilibrated well water. Eggs were dislodged by gently disassembling the spawning 

towers. Eggs adhering to plates were removed using an extra-fine paint brush. Eggs were 

counted and checked for fertilization as a measure of postmating prezygotic isolation 

(Mendelson et al., 2006). Eggs from each cross were placed in separate rearing cones, 

and towers were returned to their original spawning tank. Eggs were held in rearing cones 

(#4 Brew Rite permanent gold-tone coffee filters set in Styrofoam platforms) until 

hatching. The platforms floated in 38L tanks with air stones placed under each cone to 

oxygenate the water around the eggs and to increase circulation enough to gently roll the 

46
eggs. Eggs were inspected daily to determine development times to the eyed stage and to 

first hatch, as well as time between the first and last hatch. Eggs were checked daily for 

fungal infections and mortalities. Dead and fungal-infected eggs were counted and 

discarded to protect the health of adjacent eggs. 

We recorded hatch mortalities (when a larva is unable to completely exit its egg) 

as hatching commenced. Surviving hatchlings were transferred into cross-specific 20L 

white tubs filled to a depth of 10cm with well water slightly aerated with an air stone. 

After yolksacs were absorbed, larvae were fed on a mixed diet of concentrated brine 

shrimp nauplii and rotifers. Flake food supplements were added when larvae began 

surface feeding. Each tub was checked daily and all mortalities were recorded as 

occurring at one of the following stages: early yolksac, late yolksac, post-yolksac, or 

young. Larvae were transferred to separate 38L tanks upon attaining lengths of 

approximately 1cm and were fed flake food for a period of 180 days, after which the 

study was terminated. All mortalities during this time were removed and recorded as 

having occurred at the young stage. 

Statistical analyses 

Mating preference was assessed as the number of crosses where females deposited eggs 

and as the number of eggs laid by females in conspecific and heterospecific pairings since 

maternal investment can vary according to mate quality (Mousseau & Fox, 1998). We 

used chi-squared tests to determine if, overall, females deposited eggs more often with 

conspecific versus heterospecific mates. Chi-squared tests were also used to determine if 

red shiner and blacktail shiner females differed in response to conspecific males and 

heterospecific males (Prohl et al., 2006). Two-tailed t-tests were used to test whether 

47
females deposited more eggs with conspecific versus heterospecific mates. We used 

analysis of variance (ANOVA) with a post-hoc Tukey test to determine if the number of 

eggs deposited varied among all cross configuration (Bolnick & Near, 2005; Mendelson 

et al., 2006). 

The proportion of fertilized eggs in conspecific versus heterospecific crosses 

served as our measure of postmating prezygotic isolation (Mendelson et al., 2006). 

Postzygotic isolation was assessed by comparing development rates, hatching success 

and larval mortality at each growth stage in broods resulting from conspecific versus 

heterospecific crosses. We used two-tailed t-tests to determine whether fertilization rates, 

development rates and measures of larval mortality differed between conspecific and 

heterospecific crosses. We used ANOVA routines with post-hoc Tukey tests to assess 

differences across the four cross configurations. All statistical tests were performed using 

Systat v.10 (SPSS, Inc.). 

RESULTS 

Prezygotic reproductive isolation and fecundity 

Nineteen conspecific red shiner and 14 conspecific blacktail shiner crosses 

resulted in females depositing eggs. Females deposited eggs in five red shiner male x 

blacktail shiner female crosses and only one of the blacktail shiner male x red shiner 

female crosses. Females were significantly more likely to deposit eggs when paired with 

a conspecific male (x

2

= 36.5, p < 0.0001). Within conspecific crosses, red shiner females 

were more likely to deposit eggs than were blacktail shiner females (x

2

= 4.32, p < 0.038). 

Blacktail shiner females deposited eggs with heterospecific males more often than red 

shiner females, but the difference was not significant (x

2

= 3.13, p = 0.077). Females 

48
deposited more eggs when paired with conspecific males than with heterospecific males 

(Table 1), but the difference was not statistically significant (t=1.52, p= 0.13). Within 

conspecific crosses, red shiner females deposited more eggs than did blacktail shiner 

females (pairwise mean difference = 262.3, p = 0.018; Figure 1A). Red shiner conspecific 

crosses had significantly higher spawning frequency than other configurations and 

pairwise differences among these other configurations were not significant (Figure 1B) 

Post-mating prezygotic and postzygotic reproductive isolation 

Comparisons of outcomes from conspecific versus heterospecific crosses 

demonstrated that hybrid progeny were equally or more viable than controls with two 

exceptions (Table 1). Eggs from heterospecific crosses were less likely to be fertilized (t= 

-3.38, p= 0.002; Figure 2A) and fertilized eggs were less likely to hatch (t= -2.57, p= 

0.014; Figure 2B). However, the proportion of eggs that hatched from blacktail shiner 

conspecific crosses was comparable to the outcome of heterospecific crosses, whereas red 

shiner conspecific crosses exceeded other categories (Figure 2C). Days to eyed stage (t= 

1.28, p= 0.21) and first hatch (t= 0.82, p= 0.41) did not differ between conspecific and 

heterospecific crosses (Table 1), although eggs from red shiner conspecific crosses 

reached the eyed stage faster than eggs from other cross configuration (Figure 3A). Eggs 

from red shiner conspecific crosses hatched faster compared with blacktail shiner 

conspecific crosses (pairwise mean difference=2.75, p= 0.015; Figure 3B) but not 

compared to heterospecific crosses. Larval mortality across developmental stages was 

similar between all cross configurations except during the post-yolksac stage (Figure 4AE). Post-yolksac larvae from red shiner conspecific crosses suffered greater mortality 

than larvae from blacktail shiner conspecific crosses and red shiner female x blacktail 

49
shiner male crosses (Figure 4D). Cumulative larval mortality of hybrid progeny was 

significantly lower than larval mortality of progeny from control crosses (t= -2.60, p= 

0.014). Total post-fertilization mortality, which includes hatching mortality, was also 

significantly lower for hybrid progeny (t= -4.08, p< 0.0001; Figure 4F). 

DISCUSSION 

Patterns of reproductive isolation 

Variation in sexual dimorphism, phylogenetic relationships and genetic distance between 

congeners provide some basis for inferring general patterns of reproductive isolation 

among Cyprinella species (Coyne & Orr, 1989; Sasa et al., 1998; Mendelson, 2003; 

Mendelson et al., 2004; Moyle et al., 2004; Bolnick & Near, 2005). Hybrid viability 

generally decreases with genetic distance and time since divergence (Coyne & Orr, 1989; 

Bolnick & Near, 2005). For example, Mendelson (2003) showed that sexual isolation 

evolves to completion faster than hybrid inviability between species pairs of Etheostoma 

darters. Cyprinella are similar to Etheostoma in that they commonly exhibit striking 

sexual dimorphism, suggesting that sexual isolation could be a more potent isolating 

mechanism than hybrid inviability among members of the genus (Mendelson, 2003). 

Evidence from Centrarchid fishes (Bolnick & Near, 2005) also suggests that hybrid 

inviability is a weak isolating mechanism among Cyprinella. Bolnick & Near (2005) 

found an initial lag time before the onset of hybrid inviability among Centrarchids, after 

which a hatching success declined 3.13% per million years of divergence. Thus, complete 

hybrid inviability has yet to evolve between the most divergent Centrarchid species 

(Bolnick & Near, 2005). Hybrid inviability among Cyprinella may evolve over a 

comparably slow rate. Fossil-calibrated divergence times have not been accurately 

50
estimated for Cyprinella, but the viability of hybrids from crosses between red shiner and 

blue shiner (Burkhead et al., 2006) suggests that complete hybrid inviability has not 

evolved among distantly related congeners (Broughton & Gold, 2000). 

Our study supports the hypothesis of strong sexual isolation among species of 

Cyprinella. We found that egg fertilization rates were lower among heterospecific crosses 

than conspecific crosses, and the no-choice trials we conducted indicate females are 

significantly more responsive to conspecific versus heterospecific mates. More research 

into why fertilization rates differ is warranted, especially to determine whether sperm 

precedence is a contributing factor (Mendelson, 2003). More extensive mate preference 

trials also will be necessary to assay what cues females use when choosing mates since 

females can refer to single or multiple cues (Hankison & Morris, 2002; Rosenfield & 

Kodric-Brown, 2003). However, it is likely that mate choice by Cyprinella females 

depends on several sexually selected courtship cues, including male aggression 

(Rosenfield & Kodric-Brown, 2003; Burkhead, unpublished data). If aggression is a 

courtship cue in both species, our finding that blacktail shiner females are more prone to 

choose heterospecific mates than are red shiner females could be a result of red shiner 

males being more aggressive than blacktail shiner males (Burkhead, unpublished data). 

By extension, this would suggest that factors impeding species recognition but not 

courtship cues could promote hybridization (Hankison & Morris, 2002). 

Postzygotic reproductive isolation appears to be weak between red shiner and 

blacktail shiner. Lower egg hatching rates from heterospecific crosses is indicative of 

postzygotic reproductive isolation, but we found no other evidence of inviability. Rather, 

the comparatively low larval mortality of C. lutrensis x C. venusta hybrids is suggestive 

51
of heterosis, where hybrids exhibit greater viability than controls. This finding is 

consistent with other studies of recently diverged taxa that have found evidence of hybrid 

vigor (Rosenfield et al., 2004; Bolnick & Near, 2005). However, we cannot exclude the 

possibility that some aspects of hybrid inviability are dependent upon ecological context 

and therefore are not measurable under laboratory conditions (Mendelson, 2003). We 

also cannot exclude the possibility that C. lutrensis x C. venusta F1 hybrids are partially 

or completely infertile because we concluded our study prior to progeny from the crosses 

reaching sexual maturity. Additional work will be necessary to address this and other 

possibilities, such as the likelihood that F2 offspring are inviable. 

Evidence of sexual isolation suggests that heterospecific mating between 

introduced red shiner and native blacktail shiner in the Coosa River basin should be 

infrequent, but the frequency of interactions probably varies because the strength of 

sexual isolation can change according to environmental conditions (Seehausen et al., 

1997; Järvenpää & Lindström, 2004; Candolin et al., 2006; Engström-Öst & Candolin, 

2007). Prior studies suggest that elevated turbidity increases the frequency of interactions 

and the propensity of Cyprinella to hybridize (Jurgens, 1951; Hubbs & Strawn, 1956; 

Page & Smith, 1971). High turbidity obscures and potentially eliminates color differences 

among freshwater fishes (Seehausen et al., 1997; Seehausen & van Alphen, 1998). Since 

species of Cyprinella likely discriminate by color, elevated turbidity might reduce the 

efficacy of species recognition cues (Hubbs & Strawn, 1956), whereby hybridization 

proceeds because females favor sexually selected traits expressed in both conspecific and 

heterospecific mates. Similarly, hybridization could be a consequence of increased 

turbidity favoring heterospecific mates that expend greater energy (Engström-Öst & 

52
Candolin, 2007) or compensatory plasticity in display tactics during courtship (Luyten & 

Liley, 1991). Carrying out additional experiments will be necessary to determine if one or 

more of these mechanisms promotes hybridization between red shiner and blacktail 

shiner in the Coosa River basin. 

Preventing hybridization and loss of native species 

Introductions of red shiner have put many congeners at risk of genetic extinction 

(Mayden, 1989; Fuller et al., 1999). No where is this more of a concern than in speciose 

southeastern US river basins that harbor the greatest number of Cyprinella species in 

North America. Red shiner have hybridized with at least nine of the 22 Cyprinella taxa 

found in southeastern drainages (Hubbs & Strawn, 1956; Page & Smith, 1970; Wallace & 

Ramsey, 1982; Johnson, 1999; Warren et al., 2000; W.C. Starnes, personal 

communication), and hybridize with federally Threatened blue shiner under laboratory 

conditions (Burkhead et al., 2006). Can measures be taken to limit the breadth or 

intensity of impact introduced red shiner are having on native congeners? Observational 

studies suggest that hybridization between red shiner and other congeners is mediated by 

environment (Jurgens, 1951; Hubbs & Strawn, 1956; Page & Smith, 1970). If hybrid 

swarms form and dissipate as environmental conditions decline and improve (Hubbs & 

Strawn, 1956), it could be possible to implement management practices to eliminate 

conditions favoring hybridization. 

Hybridization does not necessarily cease following eradication or decline of a 

non-native species. The introduction of non-native smooth cordgrass (Spartina 

alterniflora) to San Francisco Bay (California, USA) led to the formation of a hybrid 

swarm with the endemic California cordgrass, S. foliosa. Smooth cordgrass has since 

53
become exceedingly rare in San Francisco Bay while S. alterniflora x S. foliosa hybrids 

are spreading rapidly and overtaking marshes once occupied by S. foliosa (Ayres et al., 

2004). Similarly, a single introduction of a very small number of non-native sheepshead 

minnow (Cyprinodon variegatus) into the Pecos River drainage led to hybridization with 

the endemic Pecos pupfish (C. pecosensis). Hybrids replaced Pecos pupfish throughout 

more than half of the species’ range in less than five years (Echelle & Conner, 1989), and 

a C. pecosensis x C. variegatus hybrid swarm now exceeds the historic range of Pecos 

pupfish in the Pecos River (Wilde & Echelle, 1992; Childs et al., 1996). 

Management involving steps beyond eradication has prevented the loss of some 

native species following hybridization. In two cases where hybridization has occurred 

between a second endemic pupfish (C. bovinus) and sheepshead minnow in the Pecos 

River drainage, a combination of trapping, application of piscicides, and reintroductions 

prevented the loss of the endemic pupfish (Echelle & Echelle, 1997; Rosenfield et al., 

2004). The success of these strategies likely hinged on the comparatively small 

geographic extent of the C. bovinus x C. variegatus hybrid swarms (Rosenfield et al., 

2004), which reinforces the idea that early detection and rapid response can help prevent 

loss of native congeners (Hobbs & Humphries, 1995). However, the success of the 

control strategies also depended on there being some degree of reproductive isolation 

between C. bovinus and C. variegatus. Rosenfield et al. (2004) argue that similar 

management strategies would be unlikely to prevent the loss of Pecos pupfish because 

female Pecos pupfish preferably breed with male sheepshead minnows (Rosenfield & 

Kodric-Brown, 2003) and because C. pecosensis x C. variegatus hybrids can exhibit 

greater vigor than either parental species (Rosenfield et al., 2004). 

54
Management that strengthens prezygotic isolation between red shiner and 

blacktail shiner could help prevent the loss of blacktail shiner in the Coosa River basin. If 

additional work on mate choice confirms observations linking hybridization to elevated 

turbidity, then a prescription of eradication and water quality improvement with a focus 

on reducing turbidity would be in order. In the Coosa River basin, turbid conditions 

generally correspond to high levels of suspended sediments (Burkhead & Jelks, 2001; 

Walters et al., 2003; Roy et al., 2005). Improving capture of urban runoff or reducing soil 

loss in agricultural landscapes are just two among many potential strategies that could 

reduce sediment loads. Although reducing excessive sedimentation is a well recognized 

management priority for sustaining salmonids fisheries, it is under-appreciated for 

conserving non-game fishes (Burkhead & Jelks, 2001; Walters et al., 2003). Besides 

potentially leading to hybridization with invasive species, excessive sedimentation 

contributes to biotic homogenization of fish assemblages by creating habitat conditions 

favored by cosmopolitan species like red shiner (Matthews & Hill, 1979; Walters et al., 

2003). Burkhead & Jelks (2001) also demonstrated that elevated suspended sediments 

can reduce the fecundity of native Cyprinella in southeastern US streams. Competition, 

hybridization and reduced fecundity could all be contributing to the decline of native 

Cyprinella in turbid southeastern US streams. Thus, improving water quality by reducing 

sedimentation could potentially eliminate one or more major invasion pathways and 

broadly benefit at-risk aquatic biodiversity. 

ACKNOWLEDGEMENTS 

We thank J. Fontaine and G. Glotzbecker for assisting with the care and maintenance of 

fishes, J. Fontaine, E. Derryberry, R. Hamilton, C. Storey, C. Tepolt and others for 

assisting with field collections of brood stock. Although this work was reviewed by US 

EPA and approved for publication, it may not necessarily reflect official Agency policy. 

55
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Table 1: Measures of prezygotic, post-mating prezygotic, and postzygotic reproductive 

isolation between non-native red shiner and native blacktail shiner. Comparisons are 

provided for conspecific versus heterospecific crosses, and among each of the four 

separate cross configurations. Values in the first column for each measure = range, values 

in the second column = mean (standard deviation). 

Prezygotic Post-mating Prezygotic 

Cross N #spawns Eggs Laid % Fertilization 

Conspecific 33 1-6 2.4 (1.6) 7-1059 252 (280) 0.09-1 0.67 (0.26) 

Heterospecific 6 1 1 (0) 19-330 151 (113) 0.03-0.75 0.27 (0.31) 

R x R 19 1-6 3.2 (1.6) 53-1059 364 (314) 0.28-0.95 0.74 (0.17) 

B x B 14 1-3 1.3 (0.6) 7-327 101 (119) 0.09-1 0.58 (0.33) 

R♀ x B♂ 1 1 19 0.58 

R♂ x B♀ 5 1 1 (0) 55-330 176 (104) 0.03-0.75 0.21 (0.31) 

Postzygotic (prehatch development) 

Cross Days to eyed Days to first hatch >Days to final hatch 

Conspecific 1-8 3.7 (1.7) 2.8-14 7.1 (2.7) 2-10 4.8 (1.9) 

Heterospecific 2-7 4.8 (1.9) 7-9 8 (0.6) 0-6 3.4 (2.3) 

R x R 1-5 3 (1) 2.8-12.4 6 (2.1) 2-10 4.8 (2.2) 

B x B 3-8 5.4 (1.8) 5-14 8.8 (2.8) 3-7 4.9 (1.3) 

R♀ x B♂ 2 8 6 

R♂ x B♀ 4-7 5.5 (1.3) 7-9 8 (0.71) 0-5 2.8 (2.1) 

Postzygotic (posthatch development) 

Cross % Egg hatch % Hatch mort % early yolksac mort % late yolksac mort 

Conspecific 0-1 0.61 (0.30) 0-1 0.16 (0.28) 0-1 0.04 (0.18) 0-0.17 0.007 (0.03) 

Heterospecific 0.03-0.75 0.27 (0.31) 0-0.12 0.02 (0.06) 0 0 0 0 

R x R 0.28-0.95 0.75 (0.17) 0-0.74 0.13 (0.19) 0-0.11 0.02 (0.04) 0-0.17 0.01 (0.04) 

B x B 0-1 0.43 (0.34) 0-1 0.21 (0.33) 0-1 0.09 (0.3) 0 0 

R♀ x B♂ 0.58 0 0 0 

R♂ x B♀ 0.03-0.75 0.21 (0.31) 0-0.12 0.02 (0.06) 0 0 0 0 

Postzygotic (posthatch development) 

Cross % post yolksac mort % young mort % cumul larval mort % total mort 

Conspecific 0-0.76 0.24 (0.24) 0-0.83 0.22 (0.24) 0-1 0.51 (0.27) 0-1 0.67 (0.25) 

Heterospecific 0-0.37 0.09 (0.15) 0.02-0.2 0.13 (0.07) 0.07-0.44 0.21 (0.14) 0.13-0.44 0.23 (0.12) 

R x R 0.01-0.76 0.35 (0.24) 0-0.52 0.17 (0.17) 0.25-0.99 0.55 (0.2) 0.26-1 0.67 (0.21) 

B x B 0-0.18 0.05 (0.08) 0-0.83 0.29 (0.33) 0-1 0.43 (0.36) 0-1 0.64 (0.33) 

R♀ x B♂ 0 0.18 0.18 0.18 

R♂ x B♀ 0-0.37 0.1 (0.16) 0.02-0.2 0.11 (0.07) 0.07-0.44 0.21 (0.15) 0.13-0.44 0.24 (0.13) 

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FIGURE LEGENDS

Figure 1: The number of eggs laid per spawn (A) and the number of spawns (B) for each 

cross configuration. A bold asterisk indicates a significant pairwise difference (Tukey, p 

< 0.05) to red shiner conspecific crosses. Axis legend: B = blacktail shiner; R = red 

shiner. 

Figure 2: Fertilization rates (A) egg hatching rates per spawn for conspecific and 

heterospecific crosses (B) and for each cross configuration (C). A bold asterisk indicates 

a significant pairwise difference (Tukey, p < 0.05) to red shiner conspecific crosses. Axis 

legend: B = blacktail shiner; R = red shiner. 

Figure 3: The number of days for eggs to reach eyed stage (A) and to first hatch (B) for 

each cross configuration. A bold asterisk indicates a significant pairwise difference 

(Tukey, p < 0.05) to red shiner conspecific crosses. Axis legend: B = blacktail shiner; R = 

red shiner. 

Figure 4: Mortality rates at five progressive stages of development among all cross 

configurations (A-E) and total larval mortality of conspecific versus heterospecific 

crosses (F). A bold asterisk indicates significant pairwise difference (p < 0.05) to red 

shiner conspecific crosses. Axis legend: B = blacktail shiner; R = red shiner. 

61
Figure 1. 

62
Figure 2. 

63
Figure 3. 

64
Figure 4. 

65