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.
-------
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 FI) 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.
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 (post-
FI) hybrids. FI 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^ through a
hybrid zone to species E). 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 mainstem-
tributary 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
in
-------
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-Fl 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 post-
FI 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 FI hybrids demonstrated vigor relative to parental
species.
IV
-------
CHAPTER 1.
Red shiner invasion and hybridization with blacktail shiner in the upper Coosa River,
USA
D.M. Walters1*, MJ. Blum1'2, B. Rashleigh3, BJ. Freeman4, B.A. Porter5, andN.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
6U.S. Geological Survey, Florida Integrated Science Center, 7920 NW 71st Street
Gainesville, Florida 32653
* corresponding author: waiters. 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
-------
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 stigmaturd) 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 FI) 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.
-------
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.
-------
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
-------
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 offish 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 & May den 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
-------
10
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 lengtkdepth 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 andmtDNA 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 r|g of DNA was then used as template for 15|il polymerase chain
reaction (PCR) mixtures that also included 2.5 mM MgCb, 2.5 mM each dNTP, 0.5 units
Tag DNA polymerase (Invitrogen, Carlsbad, CA), 0.5 jiM each of a pair of
oligonucleotide primers and PCR buffer (Invitrogen, Carlsbad, CA) to a final IX
10
-------
11
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 72°C 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 Hinfl 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 Hinfl 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
11
-------
12
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;
12
-------
13
(iv) Fl 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 FI 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 hand-
held turbidity meter (Hach 21 OOP). 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
13
-------
14
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 (-Iog2 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:
14
-------
15
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, Gary, 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 |p|<0.80 in order to
reduce multicollinearity (Glantz & Slinker 1990). Drainage area (DA) and width (W)
were highly correlated (p=0.89), and W was retained. Particle size (PS) was highly
correlated with percent sand (SAND, p=-0.85), and SAND was retained.
15
-------
16
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 /', difference (A,) was calculated between the model's
AICc and the minimum AICc value within the set. Values of A. < 2 are considered the
most parsimonious (Burnham & Anderson 2002). A weight (Wj) was calculated for each
model according to Burnham and Anderson (2002). The weight can be interpreted as the
probability that model / 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 1 A) 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
16
-------
17
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 IB). 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 IB). 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;
17
-------
18
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 (X2 = 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).
18
-------
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 lengtkdepth, 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. FI 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. FI 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. vemista, 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.
REFERENCES
Allan, J. D., and A. S. Flecker. 1993. Biodiversity conservation in running waters. BioScience 42:32-43.
Allendorf, F. W., R. F. Leary, P. Spruell, and J. K. Wenburg. 2001. The problems with hybrids: setting
conservation guidelines. Trends in Ecology & Evolution 16:613-622.
Boschung, H. T., Jr., andR. L. Mayden2003. Fishes of Alabama. Smithsonian Books, Washington D.C.
Burnham, K. P., and D. R. Anderson 2002. Model Selection and Multimodel Inference: A Practical
Information-Theoretic Approach. Springer, New York, NY.
Burridge, C. P., and J. R. Gold. 2003. Conservation genetic studies of the endangered Cape Fear Shiner,
Notropis mekistocholas (Teleostei: Cyprinidae). Conservation Genetics 4:219-225.
Candolin, U., T. Salesto, and M. Evers. 2007. Changed environmental conditions weaken sexual selection
in sticklebacks. Journal of Evolutionary Biology 20:233-239.
Costedoat, C., N. Pech, R. Chappaz, and A. Gilles. 2007. Novelties in hybrid zones: Crossroads between
population genomic and ecological approaches. PLoS ONE 4:1-17.
Cross, F. B., andL. M. Cavin. 1971. Effects of pollution, especially from feedlots, on fishes in the upper
Neosho River basin. Contribution 79. Kansas Water Resources Institute, Manhattan, KS.
DeVivo, J. C. 1996. Fish assemblages as indicators of water quality within the Apalachiacola-
Chattahoochee-Flint (ACF) River basin. Institute of Ecology. The University of Georgia, Athens,
GA.
Dimsoski, P., G. P. Toth, and M. J. Bagley. 2000. Microsatellite characterization in central stoneroller
Campostoma anomalum (Pisces: Cyprinidae). Molecular Ecology 9:2187-2189.
Ellstrand, N. C., and K. A. Schierenbeck. 2000. Hybridization as a stimulus for the evolution of
invasiveness in plants? Proceedings of the National Academy of Sciences of the United States of
America 97:7043-7050.
Falush, D., M. Stephens, and J. K. Pritchard. 2007. Inferences of population structure using multilocus
genotype data: dominant markers and null alleles. Molecular Ecology Notes 7:574-578.
Fuller, P. L., L. G. Nico, and J. D. Wiliams 1999. Nonindigenous fishes introduced into inland waters of
the United States. American Fisheries Society, Special Publication 27, Bethesda, MD.
Gido, K. B., and J. H. Brown. 1999. Invasion of North American drainages by alien fish species.
Freshwater Biology 42:387-399.
Girard, P., and B. Angers. 2006. Characterization of microsatellite loci in longnose dace (Rhinichthys
cataractae) and interspecific amplification in five other Leusciscinae species. Molecular Ecology
Notes 6:69-71.
Glantz, S. A., and B. K. Slinker 1990. Primer of Applied Regression and Analysis of Variance. McGraw-
Hill, Inc., New York, NY.
Gordon, N. D., T. A. McMahon, andB. L. Finlayson 1992. Stream Hydro logy: An Introduction for
Ecologists. John Wiley and Sons, Chichester.
Greger, P. D., and J. E. Deacon. 1988. Food partitioning among fishes of the Virgin River. Copeia
1988:314-323.
Hitt, N. P., C. A. Frissell, C. C. Muhlfeld, andF. W. Allendorf. 2003. Spread of hybridization between
native westslope cutthroat trout, Oncorhynchus clarki lewisi, and nonnative rainbow trout,
Oncorhynchus mykiss. Canadian Journal of Fisheries and Aquatic Sciences 60:1440-1451.
Hubbs, C., R. A. Kuehne, and J. C. Ball. 1953. The fishes of the upper Guadalupe river, Texas. Texas
Journal of Science 5:216-244.
Hubbs, C., and K. Strawn. 1956. Interfertility between two sympatric fishes, Notropis lutrensis and
Notropis venustus. Evolution 10:341-344.
Hurley, J. F. 1986. Summary: development, testing, and application of wildlife-habitat models - the
manager's viewpoint. Pages 151-153 in A. Verner, M. L. Morrison, and C. J. Ralph, editors.
Wildlife 2000: Modeling Habitat relationships of Terrestrial Vertebrates. University of Wisconsin
Press, Madison, WI.
26
-------
27
Johnson, C. E. 1999. The relationship of spawning mode to conservation of North American minnows
(Cyprinidae). Environmental Biology of Fishes 55:21-30.
Jongman, R. H. G., C. J. F. t. Braak, and O. F. R. v. Tongeren, editors. 1995. Data Analysis in Community
and Landscape Ecology. Cambridge University Press, Cambridge, U.K.
Kolar, C. S., andD. M. Lodge. 2001. Progress in invasion biology: predicting invaders. Trends in Ecology
& Evolution 16:199-204.
Larimore, R. W., and P. B. Bayley. 1996. The fishes of Champaign County, Illinois, during a century of
alterations of a prairie ecosystem. Illinois Natural History Survey Bulletin 35:53-183.
Marchetti, M. P., T. Light, P. B. Moyle, and J. H. Viers. 2004. Fish invasions in California watersheds:
Testing hypotheses using landscape patterns. Ecological Applications 14:1507-1525.
Matthews, W. J. 1985. Distribution of midwestern fishes on multivariate environmental gradients with
emphasis on Notropis lutrensis. American Midland Naturalist 113:225-237.
Matthews, W. J., and L. G. Hill. 1977. Tolerance of the red shiner, Notropis lutrensis (Cyprinidae) to
environmental parameters. Southwestern Naturalist 22:89-98.
Matthews, W. J., and L. G. Hill. 1979. Influence of physico-chemical factors on habitat selection by red
shiners, Notropis lutrensis (Pisces: Cyprinidae). Copeia 1979:70-81.
Mendelson, T. C. 2003. Sexual isolation evolves faster than hybrid inviability in a diverse and sexually
dimorphic genus offish (Percidae : Etheostoma). Evolution 57:317-327.
Minckley, W. L., and J. E. Deacon. 1968. Southwestern fishes and the enigma of "endangered species".
Science 159:1424-1432.
Montana, G., and J. K. Pritchard. 2004. Statistical tests for admixture mapping with case-control and cases-
only data. American Journal of Human Genetics 75:771-789.
Moyle, P. B. 2002. Inland fishes of California University of California Press, Berkeley, CA.
Moyle, P. B., and T. Light. 1996. Biological invasions of fresh water: Empirical rules and assembly theory.
Biological Conservation 78:149-161.
Multi-Resolution Land Characteristics Consortium. 1992. National Land Cover Dataset (NLCD 1992).
http://landcover.usgs.gov/natllandcover.php. Access date April, 2005.
Natural Resource Spatial Analysis Laboratory. 2005a. Georgia GAP land cover database.
http://narsal.ecologv.uga.edu/index.html. Access date April, 2005.
Natural Resource Spatial Analysis Laboratory. 2005b. Metro Atlanta land cover methodology: Mapping
impervious surface area, http://narsal.ecologv.uga.edu/atl landcover/methodologv.html. Access
date April, 2005.
Page, L. M., and R. L. Smith. 1970. Recent range adjustments and hybridization of Notropis lutrensis and
Notropis spilopterus in Illinois. Transactions Illinois Academy of Science 63:264-272.
Rhymer, J. M., and D. Simberloff. 1996. Extinction by hybridization and introgression. Annual Review of
Ecology and Systematics 27:83-109.
Richter, B. D., D. P. Braun, M. A. Mendelson, and L. L. Master. 1997. Threats to imperiled freshwater
fauna. Conservation Biology 11:1081-1093.
Ruesink, J. L. 2005. Global analysis of factors affecting the outcome of freshwater fish introductions.
Conservation Biology 19:1883-1893.
Schmidt, T. R., J. P. Bielawski, and J. R. Gold. 1998. Morphological phylogenetics and evolution of the
cytochrome b gene in the cyprinid genus Lythrurus (Actinopterygii: Cypriniformes). Copeia
1998:14-22.
Seehausen, O., J. J. M. vanAlphen, and F. Witte. 1997. Cichlid fish diversity threatened by eutrophication
that curbs sexual selection. Science 277:1808-1811.
U.S. Census Bureau. 2007. State and County QuickFacts - Georgia.
http://quickfacts.census.gov/qfd/states/13000.html. Access date, May 2007.
U.S. Department of Agriculture. 2000. 1997 National Resource Inventory. Percent Change in Developed
Land Area 1982-1997. http://www.nrcs.usda.gov/technical/land/meta/m5008.html. Access date,
August 2007.
Wallace, R. K., and J. S. Ramsey. 1982. A new cyprinid hybrid, Notropis lutrensis and N. callitaenia, from
the Apalachicola draingage in Alabama. Copeia 1982:214-217.
Walters, D. M., D. S. Leigh, and A. B. Bearden. 2003a. Urbanization, sedimentation, and the
homogenization offish assemblages in the Etowah River Basin, USA. Hydrobiologia 494:5-10.
Walters, D. M., D. S. Leigh, M. C. Freeman, B. J. Freeman, and C. M. Pringle. 2003b. Geomorphology and
fish assemblages in a Piedmont river basin, U.S.A. Freshwater Biology 48:1950-1970.
27
-------
28
Warren, M. L., Jr., B. M. Burr, S. J. Walsh, J. H.L. Bart, R. C. Cashner, D. A. Etnier, B. J. Freeman, B. R.
Kuhajda, R. L. Mayden, H. W. Robison, S. T. Ross, and W. C. Starnes. 2000. Diversity,
distribution, and conservation status of the native freshwater fishes of the southern United States.
Fisheries 25:7-31.
28
-------
29
Table 1. Environmental variables collected from mainstem tributaries in the upper Coosa River system.
acronym
DIMS
DA
URB
FOR
AG
IA
GR
W
V
V-m
D
PS
SAND
GRAY
COBB
BR
LWD
EH
CV-D
CV-V
oc
CON
NTU
DO
pH
variable description
distance to mainstem (m)
drainage area (km2)
basin urban area
basin forested
basin agriculture
impervious area, 1-km radius
gradient
average width
maximum velocity
mean velocity
average depth
average particle size
proportion sand
proportion gravel
proportion cobble
proportion bedrock
proportion large woody debris
proportion of erosional habitat
CVa depth
CV velocity
% open canopy
conductivity
mean turbidity
dissolved oxygen
pH
units transform
landscape
m log
km2 log
% arcsine square-root
% arcsine square-root
% arcsine square-root
% arcsine square-root
reach habitat
% arcsine square-root
m log
m sec"1 log
m sec"1 log
m log
phi Iog2
% arcsine square-root
% arcsine square-root
% arcsine square-root
% arcsine square-root
% arcsine square-root
% arcsine square-root
% none
% none
% arcsine square-root
water quality
uS cm"2 log
NTUb log
mg I"1 log
NA none
range
0.2-7.8
4.6-456.3
3.1-56.1
18.7-86.7
2.9-67
0.2-26.1
0.01-1.4
2.8-12.5
0.03-1.12
0-0.3
0.2-1
1.6-8
0-59
4.8-96.3
0-74
0-67.9
0-66
6-56.2
31-99.5
0-612.3
1.8-72.7
88-573
1.4-25.1
2.4-9.7
3.8-8.7
mean (1SD)
2.3 (1.6)
46.7 (80.6)
15.3 (13.2)
55.8(16.3)
21.5 (12.9)
4.1 (6.5)
0.3 (0.3)
6.4 (2.5)
0.57 (0.26)
0.11(0.1)
0.4 (0.2)
4.1(1.3)
12.8(15.1)
62.5 (22.8)
9.5 (18.2)
15.2(18.5)
13.3(13.7)
28.1 (14.7)
61.2(18.2)
147.2(117.1)
27.1 (19.5)
232.8 (84.8)
6.6 (5.3)
7.1(1.8)
7.8 (0.8)
a coefficient of variation
b nephelometric turbidity units
29
-------
30
Table 2. Percent population contribution of red shiner (RS), hybrids exhibiting
asymmetry favoring red shiner (Hr), FI 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
1
2
o
J
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Totals
Site name
Coahulla Cr.
Little Cr.
Drowning Bear Cr.
Jobs Cr.
Swamp Cr.
Polecat Br.
Town Cr.
DryCr.
Oothkalooga Cr.
Snake Cr.
Bow Cr.
Rocky Cr.
Johns Cr.
Lovejoy Cr.
Woodward Cr.
Silver Cr.
Webb Cr.
Kings Cr.
n
73
2
1
70
61
5
47
35
11
26
6
6
29
6
16
12
2
1
409
RS
0
0
0
0
0
20
4.3
2.9
0
0
16.7
0
0
0
0
0
0
0
1.2
Hr
9.6
0
0
2.9
11.5
20
2.1
5.7
9.1
3.8
16.7
16.7
0
0
0
0
0
100
6.1
Fl
0
0
0
4.3
4.9
0
6.4
5.7
9.1
3.8
0
33.3
3.4
33.3
6.3
0
0
0
4.6
Hbt
12.3
50
0
28.6
23
20
34
25.7
27.3
19.2
0
0
20.7
66.7
25
8.3
0
0
22.7
BTS
78.1
50
100
64.3
60.7
40
53.2
60
54.5
73.1
66.7
50
75.9
0
68.8
91.7
100
0
65.3
30
-------
31
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); A, 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
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=
Predictor variables
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, GRAY
W, NTU, DTMS
NTU, CON
D, W, DTMS
URB, DTMS
K
4
4
4
4
4
4
4
4
4
4
4
3
4
3
AICc
29
29.
32
33
37
.04
.37
.95
.00
.55
38.62
50.47
25.00
26
27
29
32.
32
41.
.64
.55
.11
.54
.69
.06
AAICc
0
0.33
3.91
3.96
8.51
9.58
21.43
0
1.64
2.55
4.11
7.54
7.69
16.06
w
0.
0.
0
0
0.
0.
0.
0.
0
0.
0
0.
0
0.
.19
.19
.16
.16
.12
.12
.07
.18
.17
.16
.15
.13
.13
.08
% Correctly
Presence
81.3
75.0
81.3
68.8
75.0
81.3
43.8
87.5
75.0
81.3
81.3
81.3
75.0
75.0
predicted
Absence
82
76
82
76
82
70
47.
88
70
82
88
88
70
76
.4
.5
.4
.5
.4
.6
.1
.2
.6
.4
.2
.2
.6
.5
Overall
81.8
75.8
81.8
72.7
78.8
75.8
45.5
87.9
72.7
81.8
84.8
84.8
72.7
75.8
31
-------
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
32
-------
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 N Wald chi- P Parameter Parameter Standard 95% Confidence
model square estimate error Interval
Lower Upper
BTS 33 8.1
Hybrid 33 6.9
RS 33 1.8
Linear model r2
Hybrid 18 0.50
0.0448
Intercept
NTU
V
w
0.0760
Intercept
DIMS
AG
NTU
0.6176
Intercept
V-mean
NTU
CV-D
F, Pr>F
4.6 (0.02)
Intercept
DO
AG
GR
-17.3
6.6
7.7
10.7
15.0
-13.5
20.9
13.7
-22.3
140.1
27.8
-0.3
0.9
-1.5
0.9
0.4
6.3
3.2
3.3
5.1
11.0
5.7
8.9
5.3
19.5
120.7
27.7
0.3
0.5
0.5
0.4
2.9
-29.6
0.3
1.2
0.7
-6.6
-24.7
3.4
3.3
-60.7
-96.4
-24.4
-0.7
-0.3
-2.6
0.1
-5.9
-5.0
12.8
14.2
20.6
36.5
-2.2
38.4
24.1
16.0
376.6
82.0
0.3
2.0
-0.4
1.6
6.7
33
-------
34
List of Figures
Figure 1. A) Historical spread of red shiners and C. lutremis 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 offish
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 1 A). 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 length-
to-depth ratio of for red shiners (RS), hybrids exhibiting asymmetry favoring red shiner
(Hr), FI 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
,th
median. Whiskers above and below the box indicate the 90 and 10 percentiles. Points
outside the boxes are outliers (observations beyond the 10th and 90th percentiles).
-------
36
Figure 1.
Features
• Red Shiner
i Hybrid
» Red Shiner + Hybrid
10 5 a 10
B
36
-------
37
Figure 2.
Relative Abundance
Red Shiner
Hybrid
Blacktail Shiner
^M
O None Present
10 5 0
Kilometers
37
-------
Figure 3.
3
.
"o
a
1
I 4
1
"03
T
T
•
•
•
5.
4.
oo
s
3.
3.
:
t I
T i
i
i i
-i.
i T
1 v *
•
•
:
i
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. Freeman4, 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 stigmaturd) 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; Jarvenpaa & Lindstrom, 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. venustd) 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
(Jarvenpaa & Lindstrom, 2004; Candolin et al., 2006; Engstrom-Ost & Candolin, 2007)
some evidence suggests that C. lutrensis x C. venusta FI 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 pre-
43
-------
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 cercostigmd) 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 offish 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-matingprezygotic reproductive isolation andpostzygotic reproductive isolation:
Fertilization, hatching success and larval mortality
Spawning towers containing eggs were transferred to tubs containing temperature-
equilibrated 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
Systatv.lO(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 = 36.5, p < 0.0001). Within conspecific crosses, red shiner females
were more likely to deposit eggs than were blacktail shiner females (x2= 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 (x2= 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=l.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 IB)
Post-mating prezygotic andpostzygotic 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,/?= 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,/?= 0.21) and first hatch (t= 0.82,/?= 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 3 A). Eggs
from red shiner conspecific crosses hatched faster compared with blacktail shiner
conspecific crosses (pairwise mean difference=2.15.,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 4A-
E). 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,/K 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 ofEtheostoma
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 FI 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; Jarvenpaa & Lindstrom, 2004; Candolin et al., 2006; Engstrom-Ost & Candolin,
2007). Prior studies suggest that elevated turbidity increases the frequency of interactions
and the propensity ofCyprinella 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 ofCyprinella 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 (Engstrom-Ost &
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
alterniflord) 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 offish 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
-------
REFERENCES
Anderson, E. 1949. Introgressive Hybridization. John Wiley & Sons, Inc., New York,
NY, USA.
Ayres, D.R., D.L. Smith, K. Zaremba, S. Klohr, & D.R. Strong. 2004. Spread of exotic
cordgrasses and hybrids (Spartina sp.) in the tidal marshes of San Francisco Bay,
California, USA. Biological Invasions 6: 221-231.
Bolnick, D.L, & TJ. Near. 2005. Tempo of hybrid inviability in Centrarchid fishes
(Teleostei: Centrarchidae). Evolution 59: 1754-1767.
Broughton, R.E., & J.R. Gold. 2000. Phylogenetic relationships in the North American
cyprinid genus Cyprinella (Actinopterygii: Cyprinidae) based on sequences of the
mitochondrial ND2 andND4L genes. Copeia 2000:1-10.
Burkhead, N.M., & H.L. Jelks. 2001. The effects of suspended sediment on the
reproductive success of the tricolor shiner, a crevice-spawning minnow. Transactions of
the American Fisheries Society 130: 959-968.
Burkhead, N.M., MJ. Blum, D.M. Walters, B.A. Porter, BJ. Freeman, & J.A. Fontaine.
2006. Hybrid swarms, aggressive hybridization, and the red shiner: Miscues or mystery?
Proceedings of the American Fisheries Society Annual Conference, Lake Placid, N.Y.,
U.S.A.
Candolin, U., T. Salesto, & M. Evers. 2007. Changed environmental conditions weaken
sexual selection in sticklebacks. Journal of Evolutionary Biology 20:233-239.
Childs, M.R., A. A. Echelle, & T.E. Bowling. 1996. Development of the hybrid swarm
between Pecos pupfish (Cyprinodontidae: Cyprinodonpecosensis) and sheepshead
minnow (Cyprinodon variegatus): a perspective from allozymes and mtDNA. Evolution
50:2014-2022.
Couch, C.A., J.C. DeVivo, & B.J. Freeman. 1995. What fish live in the streams of
metropolitan Atlanta? U.S. Geological Survey, National Water Quality Assessment
Program, Fact sheet FS-091-95, Atlanta, GA, USA.
Coyne, J.A. 1993. The genetics of an isolating mechanism between two sibling species of
Drosophila. Evolution 47:778-788.
Coyne, J.A., & H.A. Orr. 1989. Patterns of speciation in Drosophila. Evolution 43:362-
381.
56
-------
DeVivo, J. C. 1996. Fish assemblages as indicators of water quality within the
Apalachiacola-Chattahoochee-Flint (ACF) River basin. Masters Thesis. The University
of Georgia, Athens, GA, USA.
Echelle, A.A., & PJ. Connor. 1989. Rapid geographically extensive genetic introgression
after secondary contact between two pupfish species (Cyprinodon, Cyprinodontidae).
Evolution 43: 717-727.
Echelle, A. A., & A.F. Echelle. 1997. Genetic introgression of endemic taxa by non-
natives: a case study with Leon Springs pupfish and sheepshead minnow. Conservation
Biology 11: 153-161.
Ellstrand, N. C., & K. A. Schierenbeck. 2000. Hybridization as a stimulus for the
evolution of invasiveness in plants? Proceedings of the National Academy of Sciences of
the United States of America 97:7043-7050.
Engstrom-Ost, J., & U. Candolin. 2007. Human-induced water turbidity alters selection
on sexual displays in sticklebacks. Behavioral Ecology 18:393-398.
Hankison, S.J., & M.R. Morris. 2002. Sexual Isolation and species recognition in the
pygmy swordtail, Xiphophoruspygmaeus: conflicting preferences. Behavioral Ecology &
Sociobiology 51: 140-145.
Hatfield, T., & D. Schluter. 1996. A test for sexual selection on hybrids of two sympatric
sticklebacks. Evolution 50: 2429-2434.
Herrington, SJ. 2004. Ecology and impacts of nonindigenous red shiner in the
Chattahoochee River drainage, Georgia. Doctoral Thesis, Auburn University, Auburn,
AL, USA.
Hobbs, RJ. & S.E. Humphries. 1995. An integrated approach to the ecology and
management of plant invasions. Conservation Biology 9: 761-770.
Hubbs, C., R. A. Kuehne, & J. C. Ball. 1953. The fishes of the upper Guadalupe river,
Texas. Texas Journal of Science 5:216-244.
Hubbs, C., & K. Strawn. 1956. Interfertility between two sympatric fishes, Notropis
lutremis and Notropis venustus. Evolution 10:341-344.
Jarvenpaa, M., & K. Lindstrom. 2004. Water turbidity by algal blooms causes mating
system breakdown in a shallow-water fish, the sand goby Pomatoschistus minutus.
Proceedings of the Royal Society B: Biological Sciences 271: 2361-2365.
Johnson, C.E. 1999. The relationship of spawning mode to conservation of North
American minnows (Cyprinidae). Environmental Biology of Fishes 55:21-30.
57
-------
Jurgens, K.C. 1951. The distribution and ecology of the fishes of the San Marcos River.
Masters Thesis, The University of Texas, Austin, Texas, USA.
Lee, C.E. 2002. Evolutionary genetics of invasive species. Trends in Ecology &
Evolution 17: 386-391.
Luyten, P.H., & N.R. Liley. 1991. Sexual selection and competitive mating
success of males guppies (Poecilia reticulata) from four Trinidad
populations. Behavioral Ecology & Sociobiology 28: 329-336.
Matthews, W.J., & L.G. Hill. 1979. Influence of physico-chemical factors on habitat
selection by red shiners, Notropis lutrensis (Pisces: Cyprinidae). Copeia 1979:70-81.
Mendelson, T.C. 2003. Sexual isolation evolves faster than hybrid inviability in a diverse
and sexually dimorphic genus offish (Percidae: Etheostoma). Evolution 57: 317-327.
Mendelson, T.C., B.D. Inouye, & M.D. Rausher. 2004. Quantifying patterns in the
evolution of reproductive isolation. Evolution 58: 1424-1433.
Mendelson, T.C., V.E. Imhoff, & M. Kathryn lovine. 2006. Analysis of early
embryogenesis in Rainbow and Banded Darters (Percidae: Etheostoma) reveals
asymmetric postmating barrier. Environmental Biology of Fishes 76: 351-360.
Mousseau, T.A., & C.W. Fox, 1998. The adaptive significance of maternal effects.
Trends in Ecology and Evolution 13: 403-407.
Moyle, L. C., M.S. Olson, & P. Tiffin. 2004. Patterns of reproductive isolation in three
angiosperm genera. Evolution 58: 1195-1208.
Page, L.M., & R.L. Smith. 1970. Recent range adjustments and hybridization of Notropis
lutrensis and Notropis spiloptems in Illinois. Transactions Illinois Academy of Science
63:264-272.
Prohl, H., R.A. Koshy, U. Mueller, A.S. Rand, & MJ. Ryan. Geographic variation of
genetic and behavioral traits in northern and southern Tungara frogs. Evolution 60: 1669-
1679.
Rhymer, J. M., & D. Simberloff. 1996. Extinction by hybridization and introgression.
Annual Review of Ecology and Systematics 27:83-109.
Rosenfield, J.A., & A. Kodric-Brown. 2003. Sexual selection promotes hybridization
between Pecos pupfish, Cyprinodonpecosensis and sheepshead minnow, C. variegatus.
Journal of Evolutionary Biology 16: 595-606.
58
-------
Rosenfield, J.A., S. Nolasco, S. Lindauer, C. Sandoval, & A. Kodric-Brown. 2004. The
role of hybrid vigor in the replacement of Pecos pupfish by its hybrids with sheepshead
minnow. Conservation Biology 18: 1589-1598.
Roy, A. H., M. C. Freeman, B. J. Freeman, S. J. Wenger, W. E. Ensign, and J. L. Meyer.
2005. Investigating hydrologic alteration as a mechanism offish assemblage shifts in
urbanizing streams. Journal of the North American Benthological Society 24:656-678.
Sakai, A.K., F.W. Allendorf, J.S. Holt, D.M. Lodge, J. Molofsky, K.A. With, S.
Baughman, RJ. Cabin, I.E. Cohen, N.C. Ellstrand, D.C. McCauley, P. O'Neil, I.M.
Parker, J.N. Thompson, & S.G. Weller. 2001. The population biology of invasive species.
Annual Review of Ecology and Systematics 32: 305-332.
Sasa, M.M., P.T. Chippindale, & N.A. Johnson. 1998. Patterns of postzygotic isolation in
frogs. Evolution 52:1811-1820.
Seehausen, O., J. J. M. vanAlphen, and F. Witte. 1997. Cichlid fish diversity threatened
by eutrophication that curbs sexual selection. Science 277:1808-1811.
Seehausen O, & J.M. van Alphen. 1998. The effect of male coloration on female mate
choice in closely related Lake Victoria cichlids (Haplochromis nyererei complex).
Behavioral Ecology & Sociobiology 42:1-8.
Vives, S.P. 1993. Choice of spawning substrate in red shiner with comments on crevice
spawning in Cyprinella. Copeia 1993: 229-232.
Wallace, R.K., & J.S. Ramsey. 1982. A new cyprinid hybrid, Notropis lutremis and N.
callitaenia, from the Apalachicola draingage in Alabama. Copeia 1982: 214-217.
Walters, D.M., D.S. Leigh, & A.B. Bearden. 2003. Urbanization, sedimentation, and the
homogenization offish assemblages in the Etowah River Basin, USA. Hydrobiologia
494: 5-10.
Warren, M.L., Jr., B.M. Burr, S.J. Walsh, H.L. Bart, R.C. Cashner, D.A. Etnier, B.J.
Freeman, B.R. Kuhajda, R.L. Mayden, H.W. Robison, S.T. Ross, & W.C. Starnes. 2000.
Diversity, distribution, and conservation status of the native freshwater fishes of the
southern United States. Fisheries 25:7-31.
Wilde, G.R., & A. A. Echelle. 1992. Genetic status of Pecos pupfish populations after
establishment of a hybrid swarm involving an introduced congener. Transactions of the
American Fisheries Society 121: 277-286.
Wolf, D.E., N. Takebayashi, & R.H. Rieseberg. 2001. Predicting the risk of extinction
through hybridization. Conservation Biology 15: 1039-1053.
59
-------
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).
Cross
Conspecific
Heterospecific
RxR
BxB
R? xBc?
Re! xB?
Prezygotic
N #spawns
33 1-6 2.4(1.6)
6 1 1 (0)
19 1-6
14 1-3
1 1
5 1
3.2(1.6)
1 .3 (0.6)
1 (0)
Eggs Laid
7-1059 252(280)
19-330 151 (113)
53-1059
7-327
19
55-330
364(314)
101 (119)
176(104)
Post-mating
Prezygotic
% Fertilization
0.09-1 0.67 (0.26)
0.03-0.75 0.27 (0.31)
0.28-0.95
0.09-1
0.58
0.03-0.75
0.74(0.17)
0.58 (0.33)
0.21 (0.31)
Postzygotic (prehatch development)
Cross
Conspecific
Heterospecific
RxR
BxB
R? x Be?
Re? x B?
Days to eyed
1-8 3.7(1.7)
2-7 4.8 (1 .9)
1-5 3(1)
3-8 5.4 (1 .8)
2
4-7 5.5 (1 .3)
Days to first hatch
2.8-14 7.1 (2.7)
7-9 8 (0.6)
2.8-12.4
5-14
8
7-9
6(2.1)
8.8 (2.8)
8(0.71)
>Days to final hatch
2-10 4.8(1.9)
0-6 3.4 (2.3)
2-10
3-7
6
0-5
4.8 (2.2)
4.9(1.3)
2.8(2.1)
Postzygotic (posthatch development)
Cross
Conspecific
Heterospecific
RxR
BxB
R? xBe!
Re! xB?
% Egg hatch
0-1 0.61 (0.30)
0.03-0.75 0.27(0.31)
0.28-0.95 0.75(0.17)
0-1 0.43 (0.34)
0.58
0.03-0.75 0.21 (0.31)
% Hatch
0-1
0-0.12
0-0.74
0-1
0
0-0.12
mort
0.16(0.28)
0.02 (0.06)
0.13(0.19)
0.21 (0.33)
0.02 (0.06)
% early yolksac mort
0-1 0.04(0.18)
0 0
0-0.11
0-1
0
0
0.02 (0.04)
0.09 (0.3)
0
% late yolksac mort
0-0.17 0.007(0.03)
0 0
0-0.17
0
0
0
0.01 (0.04)
0
0
Postzygotic (posthatch development)
Cross
Conspecific
Heterospecific
RxR
BxB
R? xBe!
Re! xB?
% post yolksac mort
0-0.76 0.24 (0.24)
0-0.37 0.09(0.15)
0.01-0.76 0.35(0.24)
0-0.18 0.05(0.08)
0
0-0.37 0.1 (0.16)
% young
0-0.83
0.02-0.2
0-0.52
0-0.83
0.18
0.02-0.2
mort
0.22 (0.24)
0.13 (0.07)
0.17(0.17)
0.29 (0.33)
0.11 (0.07)
% cumul larval mort
0-1 0.51 (0.27)
0.07-0.44 0.21 (0.14)
0.25-0.99
0-1
0.18
0.07-0.44
0.55 (0.2)
0.43 (0.36)
0.21 (0.15)
% total
0-1
0.13-0.44
0.26-1
0-1
0.18
0.13-0.44
mort
0.67 (0.25)
0.23(0.12)
0.67 (0.21)
0.64 (0.33)
0.24(0.13)
60
-------
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,/?
< 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,/? < 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.
IZUU
to 1000
to
2
" 800
CD
Q.
1 600
to
en
iff 4°°
=tt
200
-
-
~
0
i
*
I
i i
-
-
-
__
1
o -
i 1
r^-^r^n r-»r^ r^^r-lO
B
to
to
2
o
(D
Q.
to
Q.
w
/
6
5
4
1
0
i i i
* *
-
-
-
•
-
-
i
i i i
BxB B^xR? RxR
62
-------
Figure 2.
1.0
0-8
I 0.4
0.2
I -
1.0
0.8
0.6
0.4
02
O h*t*r
-------
Figure 3.
8
,
re
Q
"•H* O
n
1 I
*
T
1
-
i
1
i
i i
[
i
*
I
1
1
-
-
-
IO
_.
j? 10
(/)
o
+->
B
n
* ' ' '
o
o
o
T
I ' 0
. I
I
I I I I
BxB B^xR? RxR R^xB?
BxB B3xRl RxR
64
-------
Figure 4.
I.U
& 0.8
ro
~t!
o
E 0.6
£""
O
-t-J
TO
•s- o 4
O)
LU
# 0.2
A o
,-, 1.0
o
TO
to
js£
£0.8
>,
~
TO
,£. 0.6
5
o O-4
E
"ro
£ 0.2
TO
B * o
1f\
^ .0
'tj
TO
to
f, °'8
TO
~ 0.6
"TO
o 0.4
E
"TO
I 0.2
C * n
T 1 f 1
-
O
o
-
o
-
T
T
i
i
o
1 I 1 — i— 1 |
? I 1 1
- -
— -
—
8
o
1 1 1 1
-
~ "~
-
o
1 1 £ 1
^ I.U
0
TO
.*:
£0.8
. .
to
o
^
"TO
o 0.4
E
TO
TO °'2
° * 0
1.0
f s
0)
§ 0.8
o
^
•^06
0
E 0.4
ro
£
ro
-1 0.2
^
E 0
1f\
.0
0.8
^
^ 0.6
0
*"•«
0.2
F n
i i
-
-
* *
-
o
T
I • 1
1 1 1 1
—
—
T -
1 1
CP
1 T
BxB B^xR? RxR R^xB?
O heterospecific r \
x conspeclfic _ « \ rn
------- |