Download Sexual selection promotes hybridization between Pecos pupfish

Sexual selection promotes hybridization between Pecos pupfish,
Cyprinodon pecosensis and sheepshead minnow, C. variegatus
J. A. ROSENFIELD & A. KODRIC-BROWN
Biology Department, Castetter Hall University of New Mexico, Albuquerque, NM, USA
Keywords:
Abstract
female choice;
fish;
genetic introgression;
hybrid zone;
male competition;
mating system;
Pecos River;
reproductive isolation;
sexual selection;
speciation.
Rapid and extensive genetic introgression has occurred between Pecos pupfish
(Cyprinodon pecosensis) and sheepshead minnow (Cyprinodon variegatus) in the
wild. We studied both female mate choice and male–male competition for
mates among C. pecosensis, C. variegatus, and their F1 hybrids to determine
what role these behaviours played in the formation of the hybrid swarm.
Female C. pecosensis preferred male C. variegatus to conspecific males,
C. variegatus females displayed no significant preference when given a choice
between purebred males, and neither C. pecosensis nor C. variegatus females
discriminated against F1 hybrid males. We found no evidence for female
olfactory recognition of mates. Male F1 hybrids and C. variegatus were more
aggressive than C. pecosensis males, achieving greater reproductive success under
two different experimentally-induced mating systems. Hybrids were superior to
C. variegatus when only two males competed (dominance interactions), but the
two types were competitively equivalent in a territorial mating system. Our
results indicate that active inter- and intra-sexual selection contributed to the
accelerated hybridization between these two species. By including the possibility that some aspects of a hybridization and introgression event may be under
positive selection, researchers may better understand the dynamics that lead to
hybrid zone stability or the spread of introgressed genetic material.
Introduction
When allopatric populations are brought into secondary
contact, the mechanisms that retard gene flow between
them are of interest because they provide insights into
the processes of speciation (Hewitt, 1988; Arnold, 1997;
Boake, 2000). Divergent sexual selection (e.g. WestEberhard, 1983; Boake, 2000; Uy & Borgia, 2000),
natural selection (e.g. Schluter, 1998), and random
genetic divergence (Dobzhansky, 1940; Muller, 1942;
Orr, 1995) are each expected to reduce reproductive
compatibility between geographically isolated populations (but see Rice & Hostert, 1993). The reproductive
isolating mechanisms produced by these processes can be
Correspondence: Jonathan A. Rosenfield, Wildlife, Fish, and Conservation
Biology Department, One Shields Avenue, University of California –
Davis, Davis, CA 95616-8751, USA.
Tel.: +1 530 752 8409; fax: +1 530 752 4154;
1 e-mail: [email protected]
assigned to two broad categories (1) premating isolating
mechanisms, which limit the number of matings between heterospecifics, and (2) post-mating isolating
mechanisms, which limit the survival and reproduction
of hybrid offspring.
In contrast to the two well-studied outcomes of
hybridization (random genetic mixing and slower-thanrandom gene flow between hybridizing populations), the
mechanisms that drive faster-than-random fusion of
genomes have received little study. Sexual selection
could promote gene flow between populations if hybrids
benefit from female mate choice preferences or asymmetries in male–male competition for mates. Both processes,
acting alone or in combination, would promote heterospecific matings and could result in faster-than-random
fusion of genomes. Pre- and post-mating processes that
promote greater inter-population cohesion than intrapopulation cohesion (sensu Templeton, 1989) have been
documented. Pre-existing sensory bias (e.g. Kaneshiro,
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J. A. ROSENFIELD AND A. KODRIC-BROWN
1976; Ryan & Wagner, 1987; Ryan & Rand, 1993, 1995;
Pfennig, 2000), resulting in female preferences for
heterospecific males, is one phenomenon that may
produce higher than random rates of heterospecific
matings. Heterosis (hybrid vigor) exemplifies a postmating mechanism that could promote gene flow
between divergent populations (e.g. Scribner, 1993).
Either pre-existing sensory bias or heterosis could lead to
rapid genetic introgression (fusion of gene pools; Anderson, 1949) when formerly allopatric populations are
brought into secondary contact. Because the role of
sexual selection in promoting inter-population cohesion
is poorly understood, its operation during the formation
of hybrid zones may lead to incorrect interpretation of
the forces producing genetic structure in hybrid swarms.
We conducted a series of experiments to determine the
role of female mating preferences and male competitive
interactions in promoting the extremely rapid and
extensive genetic introgression that occurred in the wild
between Pecos pupfish (Cyprinodon pecosensis Echelle and
Echelle) and sheepshead minnow (Cyprinodon variegatus
Lacepede). We studied female visual and olfactory mate
preferences because of their importance in mate recognition in other fishes (e.g. swordtails: McLennan & Ryan,
1999; pupfish: Strecker & Kodric Brown, 1999, 2000). In
species with territorial mating systems, male competitive
interactions are indicative of male vigor and reproductive
success (examples in Andersson, 1994).
Study System
Cyprinodon pecosensis is endemic to the southern Pecos
River drainage of New Mexico and Texas (Echelle &
Echelle, 1978). Cyprinodon variegatus has a much broader
geographic range, stretching from the coast of Massachu2 setts to the Yucatán Peninsula (Lee et al. 1980); the
species is widely introduced into inland water bodies
where it has become established. Sometime during the
early 1980’s, C. variegatus were introduced to the Pecos
River of Texas (Echelle & Connor, 1989). In <5 years,
hybrids between C. pecosensis and C. variegatus had
completely replaced the endemic (C. pecosensis) throughout more than 500 Km of the Pecos River. From the
initial, presumably localized site of introduction, the
geographic range of the hybrids has expanded beyond
the historical southern limit of C. pecosensis (Echelle &
Connor, 1989). Genetic analyses of the hybrid swarm
(Echelle & Connor, 1989; Childs et al., 1996) suggest that
the introduction of C. variegatus occurred only once, from
a naturalized source population (probably in Lake
Balmorhea, Reeves County, TX), and that only a small
number of individuals were originally introduced. The
proportion of introduced genetic material exceeds 50%
throughout most of the hybrid population and both sexes
of both species have contributed to formation of the
hybrid swarm (Echelle & Connor, 1989; Childs et al.,
1996). Pure populations of Pecos pupfish remain in two
main areas: Bitter Lake National Wildlife Refuge
(BLNWR) and Bottomless Lakes State Park (BLSP), both
in Chavez County, New Mexico (Echelle et al., 1997).
These populations are physically isolated from the Pecos
River and the hybrid swarm.
Materials and methods
Study organisms
Cyprinodon pecosensis and C. variegatus are closely related,
but C. pecosensis has probably been isolated from other
Cyprinodon species for approximately 10 000 years
(Echelle & Echelle, 1978, 1992). Cyprinodon pecosensis
differs from C. variegatus in scalation, morphology,
emphasis of male mating colours, and shape of ventrolateral markings; the two species also display fixed
differences in allozyme markers and mtDNA sequences
(Echelle & Echelle, 1978; Echelle & Connor, 1989; Childs
et al., 1996). The two species are not sister taxa (Echelle &
Echelle, 1992). Both species have a promiscuous breeding system in which males compete to establish breeding
territories where they court females (Itzkowitz, 1977,
1981; Kodric-Brown, 1977, 1983). Females evaluate
males and their territories and indicate a willingness to
mate by swimming close to the substrate where spawning takes place. Females lay eggs on the substrate and
may mate with several males each day (Kodric-Brown,
1977, 1983; Itzkowitz, 1978). There is no parental care of
eggs other than the incidental product of males guarding
territories. Male courtship displays are similar between
the two species.
We collected C. pecosensis from Figure 8 Lake in BLSP
3 (1997–1999) and from BLNWR (1999). Cyprinodon
variegatus were purchased from a commercial breeder
(1997–1999; Aquatic Biosystems, Ft. Collins, CO, USA);
collected from Sea Rim State Park, Jefferson County, TX
(1997) and from Lake Balmorhea, TX (1999); and
donated by Dr P. Klerks, Louisiana State UniversityLaFayette, LA (2000). All of these populations had
genetic ancestry from populations in the northern coast
of the Gulf of Mexico. These fish and their offspring,
including C. variegatus · C. pecosensis (Cv · Cp) and
C. pecosensis · C. variegatus (Cp · Cv) F1 hybrids, were
used in experiments. All F1 hybrids were bred and reared
on the UNM campus.
All fish were allowed to acclimate to laboratory conditions and our animal care protocol for at least 3 weeks
before use in a trial. Fish were housed in 37.85 and
75.70 L aquaria at similar densities in single-sex groups
and fed ad libitum on flake food mixed with freeze-dried
brine shrimp. Aquaria were filled with dechlorinated tap
water mixed with artificial sea salt to 10 ppt; healthy wild
populations of adult C. pecosensis are found in salinities
ranging from 3.5 to 50 ppt (New Mexico Department
of Game and Fish, 1999) and C. variegatus also have
extremely broad salinity tolerances (Haney, 1999).
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Sexual selection drives genetic assimilation
Female choice
Visual preference experiment
Three 37.85 L aquaria (50 · 25.5 · 29 cm) were placed
parallel to each other, 1 cm apart. The two lateral aquaria
were divided into five equal sections with 0.57 mm
transparent plexiglas dividers. The centre aquarium was
undivided. Aquaria were illuminated from above using
three full-spectrum, fluorescent bulbs (model: GE,
F20T12-Pl/AQ, General Electric, Fairfield, CT, USA,
http://catalog.gelighting.com). Fish in the aquaria were
observed from above through holes in a horizontal
screen. Cardboard dividers were placed between the
three aquaria during the 15-min acclimation period, and
removed before each trial.
A single mature female was placed in the centre
aquarium. Each female was used in only one trial.
A single male was placed in each of the five compartments of the two divided aquaria. We measured the total
length of the female and each male. Males of one type
(C. pecosensis, C. variegatus, Cp · Cv hybrid, or Cv · Cp
hybrid) were placed in one of the divided aquaria and
males of another type were placed in the other divided
aquaria. To avoid side-bias, males of a particular type
were placed on different sides of the female aquarium in
different trials. The transparent dividers in lateral aquaria
allowed males to see and display to conspecific males but
prevented physical contact between males. Males were
selected at random with respect to coloration, but we
tried to match opposing males (those in the opposite
sections of the two lateral aquaria) so that they were of
roughly equal mass. The relationship between mass and
length differs among the parental species and hybrids, so
mass was estimated using species-specific length-mass
relationships (J.A. Rosenfield, unpublished data). Males
were stored separately from females and thus had no
intercourse for at least 1 week prior to the trial.
Male coloration intensity and behaviour are affected
by interactions with neighbouring males and breeding
status (Kodric-Brown, 1983, 1996; Kodric-Brown &
Mazzolini, 1992). We rearranged and replaced males
between trials to expose each female to a different
combination of male phenotypes. Between trials, one
pair of competing males was removed from the lateral
aquaria and replaced with a different pair of competing
males. At least two males in each aquarium were moved
to different sections of their aquarium between trials.
This protocol could not completely eliminate the potential effect of pseudoreplication as there was some overlap
in the males that certain females were exposed to.
Completely replacing all 10 males for each trial would
have required far more males than we were able to
support. Compared with an experimental design where
all females are exposed to the same males, our methodology substantially reduced any impact of pseudoreplication. In addition, by exposing each female to five
conspecific (10 total) males simultaneously, we reduced
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the possibility that species-level preferences were
obscured by the particular attributes of individual males.
For each male, coloration intensity was scored for the
dorsal fin, caudal fin, and dorsum on a scale from 0 to 6
(0 ¼ coloration absent, 6 ¼ peak coloration) before and
after each trial. The summed pre- and post-trial scores
were averaged for each male.
Female C. pecosensis were exposed to male C. pecosensis
competing against either male C. variegatus (n ¼ 20) or
male F1 hybrids (n ¼ 18). Female C. variegatus were
exposed to male C. variegatus competing against either
C. pecosensis (n ¼ 20) or F1 hybrids (n ¼ 20). A female
was judged to be inspecting a particular male when
she oriented towards that male while swimming with
her snout <2 cm from the glass (a ‘contact’). In the wild,
such active investigation of males by females would
usually result in a mating (Kodric-Brown, 1977, 1983).
Studies of other fish species have found that female
association behaviour of this type is a good predictor of
female mating preferences (e.g. Ryan & Wagner, 1987;
Kodric-Brown, 1993). Trials continued for 15 min after
the first contact. The species identity and aquarium
section of contacted males and duration of each contact
were recorded. Trials where females did not contact any
male within 25 min or those where females contacted
males for a total of <90 s during the 15-min trial period,
were discarded. All C. variegatus females met these
minimum standards of activity, but 10 female C. pecosensis
did not. We controlled female side-bias by switching
competing male types between sides of the aquaria,
between trials and by observing females for 5 min prior
to the trial (i.e. with screens in place). Wilcoxon Matched
Pairs tests were used to test an overall effect of female
side-bias. Individual females that displayed a side-bias
during the pretrial observation period were not used in
subsequent trials.
Analysis
For each combination of male and female types, a
Wilcoxon Matched Pairs test was used to determine
whether females preferred conspecifics or heterospecifics.
Because the two species are known to hybridize in the
wild, we employed a two-tailed test.
The effect of interspecific differences in female behaviour on total contact time was evaluated using a Mann–
Whitney U-test. Only those trials in which females had
a choice between purebred males (C. pecosensis vs.
C. variegatus) were used in this analysis. The effect of
male species on female activity level was evaluated
within female species. We compared female activity
levels in trials where they chose between purebred males
only to those where females were given a choice between
conspecifics and hybrids. This allowed us to compare the
activity levels of females exposed to hybrid heterospecifics with those exposed to purebred heterospecifics.
Finally, we used a Mann–Whitney U-test to compare
differences in female response to the two reciprocal
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crosses of hybrid males (individual females were exposed
to one hybrid cross or the other, not a mixture of the
two).
Expression of female preferences for male coloration
and length were evaluated in two ways. Females might
prefer males that displayed an extreme of either of these
two characteristics. To detect such a preference, we
studied the individual males that females contacted the
most (‘preferred’ males) within each trial and ranked
them for length and colour score as either high (among
the five largest or most colourful males) or low (among
the five smallest or least colourful males). A chi-square
goodness of fit test was used to determine if preferred
males were more colourful or larger than males they
competed against. Our second analysis of female preference for male colour and length was designed to
determine if females displayed a graded response to
increasing coloration intensity or length. Because we
observed species-specific differences in length (caused by
controlling for mass) and coloration, this analysis could
confound length or colour intensity preferences with
preferences for other species-specific attributes. Thus, in
this analysis, we ranked males within species for length,
colour, and female preference (contact time). We generated a probability distribution for the relationship
between female preference ranks and colour or length
rank by producing 1000 randomized replicates of actual
female preference ranks and colour or length ranks. We
then compared the Spearman Rank Correlation between
female preference and male colour or length in the actual
data set to the randomized distribution to determine the
probability that our observations could occur by chance
alone. In this analysis, a significant correlation between
contact time and colour or length would reveal a female
preference for colour or length independent of male
species.
Olfactory preference experiment
Female olfactory preference was evaluated using methods modified from McLennan & Ryan (1999) and
Strecker & Kodric Brown (1999). A single female was
placed in a 303 L aquarium (91 · 41 · 38 cm) half-filled
with dechlorinated, salt water (10 ppt). Two lines drawn
on the outside of the aquarium divided its length into
three equal sections. The aquarium was surrounded by
opaque, white plexiglass to eliminate external visual
signals. Females were given 15 min to acclimate to this
environment. Approximately 1 L of water (treated
water) was taken from one of two 76 L aquaria containing 15 to 25 mature conspecific males. To reduce the
potentially attractive scent of food, treated water was
taken from aquaria where males had not fed for at least
40 h. Treated water was placed into a clean intravenous
(IV) bag and dripped into one end of the female’s
aquarium. The drip rate was set at two drops per second
using an IV drip-regulator. Into the other side of the
female’s aquarium, dechlorinated salt water (untreated
water) was supplied from an IV at the same rate as
treated water. At this drip rate, coloured waters dripped
into opposing ends of the female tank were observed to
mix after about 15 min. Female behaviour was videotaped for 15 min and data were recorded from the tape.
Female preference for a given type of water was scored as
the amount of time she spent in the third of the tank
closest to each of the two water sources. Females were
observed for 15 min prior to the trial in order to detect
side-bias; no side-bias was detected. The side of the
aquarium to which treated water was supplied was
randomly assigned for each trial. The female’s aquarium
was cleaned between trials as per McLennan & Ryan
(1999).
Analysis
A Wilcoxon Matched Pairs test was used to determine
whether females of a given species preferentially associated with the treated or untreated water source.
Male competition
We evaluated male competitive ability by tallying male–
male aggressive interactions and mating behaviours and,
where appropriate, estimating the size of male territories.
All male competition trials were conducted outdoors on
the UNM campus, Albuquerque, NM between 17 May
and 2 August of 1998–2001. Aggressive behaviours
included (1) males chasing after, (2) lunging at, and (3)
fighting with their opponents. Mating behaviours included male pursuit of females and spawning (a jerking
motion of the male and female when their bodies are in
contact). Individual males were used in only one trial.
In order to provide a comprehensive picture of male–
male competitive dynamics, we employed two different
experimental protocols: (1) dominance trials, where two
males competed for access to females but males did not
establish or defend territories, and (2) territoriality trials,
where four males competed for spawning territories and
access to females. These two different scenarios allowed
us to record different types of valuable information. In
the Dominance Experiment, we were able to observe the
behaviours of individual males. In the Territoriality
Experiment (where four males competed), we were not
able to track the behaviour of individuals between
observations; however, competition between multiple
neighbouring males is normal in the wild, so the
Territoriality Experiments represented a more typical
context than the Dominance Experiment. The protocols
reflected an effort to produce experimental conditions
that allow for complete enumeration of all relevant
behaviours (simplicity) and those that represent somewhat natural conditions (complexity). The Cyprinodon
mating system is affected by local population density and
habitat size (Soltz, 1974; Kodric-Brown, 1981, 1988a,b).
By staging male–male competition under different population density conditions, we were also able to observe
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Sexual selection drives genetic assimilation
whether the outcome of male–male competition for
mates was influenced by changes in the local mating
system. The protocols are described below in detail.
Dominance experiment
A single male from each of two types (C. pecosensis,
C. variegatus, or F1 hybrids) competed for mating access
to three or six females in a 1.8-m diameter stock tank
that was divided with opaque plexiglass into two semicircular arenas. Independent trials were conducted
simultaneously in each arena (area ¼ 1.27 m2 each,
population density ¼ 3.94 or 6.30 m)2). Females in each
arena represented a range of sizes and were a mixture of
the three genotypes (i.e. C. variegatus, C. pecosensis, and F1
hybrid). A cinder block, with travertine rock glued over
its holes, was placed in the centre of each arena to
provide a focal territory. Competing males were placed in
the arena on the morning of a trial (females remained in
the arenas between trials) and, after a 15-min acclimation period, were observed from behind a blind in two to
four 15-min observation periods conducted between
1000 and 1700 hours. We recorded the frequencies of
male aggressive behaviour (chases, lunges, and fights)
and mating behaviour (pursuits of females and spawnings). Observation periods were discarded if no aggressive
or courtship activity was observed; trials were discarded if
aggressive or courtship behaviours were not observed in
at least two observation periods. We conducted trials
with the following combinations of male types: C.
pecosensis vs. C. variegatus (n ¼ 21), C. pecosensis vs. F1
hybrids (n ¼ 21), C. variegatus vs. F1 hybrids (n ¼ 24).
Trials were completed within 24 h because dominance
was quickly established by one of the competing males.
Analysis
For each male, an aggressiveness score was calculated as
the number of lunges at and chases of the opposing male;
fights (aggressive events in which there was no clear
winner) were analysed separately. The number of male
pursuits of females and spawnings were analysed separately. Data were standardized by dividing each variable
by the total number of minutes in a trial. These
standardized counts were square-root transformed to
produce distributions that met the assumptions of parametric statistics. We used the transformed data and twotailed, paired t-tests to test whether male types differed in
the frequency of agonistic behaviours or in courtship and
mating success. In trials where C. variegatus males
competed with F1 hybrid males, the data for male
pursuits of females were non-normal; for this comparison
only, a Wilcoxon Matched Pairs test was used. We looked
for significant correlations between the estimated difference in mass of competing males and differences in
(1) aggressive, (2) courtship, and (3) mating behaviour of
competing males using Kendall’s Tau. For these correlations, a Bonferroni adjustment of alpha (adjusted
a ¼ 0.017) was performed because we searched for
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correlations between the mass differences of competing
males and each of three independent variables. To
determine if certain combinations of male types engaged
in fights more often than others, A N O V A was performed
with the combination of competing males as the independent variable and the number of fights (square-root
transformed) as the dependent variable.
Territoriality experiment
A pair of males from each of two types (C. pecosensis,
C. variegatus, or F1 hybrids) competed for territories and
mating access to six females of one type (either C.
pecosensis or C. variegatus) in 1.2-m diameter stock tanks
(area ¼ 1.13 m2, population density ¼ 8.85 m)2). Four
trays of gravel and travertine chips were placed equidistant from each other to serve as focal territories for males
and spawning substrates for females. We conducted 28
trials with C. variegatus males and C. pecosensis males
competing (n ¼ 17 with C. pecosensis females, n ¼ 11 with
C. variegatus females). Twelve trials were conducted with
C. pecosensis males and F1 hybrid males competing; female
C. pecosensis were used in each of these trials. Twelve trials
were conducted with C. variegatus and F1 hybrids competing; C. variegatus females were used in each of these
trials. As it was difficult to distinguish C. variegatus males
from F1 hybrid males when they competed with each
other, males in this treatment were first marked according to type with white or yellow latex paint injected
subcutaneously into the caudal peduncle and the area
behind the dorsal fin (males were anaesthetized with
aqueous MS-222 prior to injections). Males were marked
2 days prior to use in trials allowing sufficient time for
them to fully recover from the injection. These identifying marks were clearly visible for over a week and had no
adverse effect on the males’ behaviour (see Results).
Treatments involving C. pecosensis males did not require
marking males because they are easily distinguished from
C. variegatus and F1 hybrid males.
After a 24-h period, during which they established
territories, competing males were observed from behind
a blind in 30-min observation periods conducted
between 1000 and 1700 hours. Trials were continued
for approximately 1 week in order to evaluate the
stability of male territories. We recorded spawning events
for each male; observation periods were discarded if no
males spawned and trials were discarded if spawning was
not observed in at least two observation periods. We
tallied agonistic encounters (fights and chases) between
conspecifics separately from those between heterospecific
males. We also recorded territory size using gravel trays
and other marks in the stock tanks to define territory
boundaries.
Analysis
Species-specific differences in spawning success were
detected by using paired t-tests to compare the number of spawning attempts (square-root transformed)
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attributable to each type of male in a trial. For this
analysis, results for conspecific males were summed
across the two observation periods within each trial
because the identity of conspecific individuals in the
same trial could not be tracked between observation
periods. Using A N O V A , the sum of fight frequency and
chase frequency (square-root transformed) was compared among treatments to determine whether aggression was greater in one combination of male types than
others.
Results
Female preference – visual experiment
Female C. pecosensis spent more time in contact with male
C. variegatus than conspecific males (Wilcoxon Matched
Pairs: z ¼ 2.016, n ¼ 20, P < 0.05; Fig. 1). C. variegatus
females also spent more time in contact with C. variegatus
males, but the preference was not statistically significant
(z ¼ 1.792, n ¼ 20, P ¼ 0.072). Neither C. pecosensis
females nor C. variegatus females discriminated between
conspecific males and F1 hybrid males (z ¼ 0.9799,
n ¼ 18, ns and z ¼ 0.7467, n ¼ 20, ns, respectively).
Side-bias was not detected among C. pecosensis
females (Wilcoxon matched pairs test: z ¼ 0.7396,
n ¼ 38, ns) or C. variegatus females (z ¼ 1.665, n ¼ 40,
P ¼ 0.096).
Female activity did not vary significantly between the
two species or with regard to the species of competing
males. When females were given a choice between
purebred males, the activity levels of C. variegatus females
and C. pecosensis females were not significantly different
(mean total contact time: 500.1 and 412.5 s, respectively;
Mann–Whitney U-test: U20,20 ¼ 155, ns). Activity levels
of C. pecosensis females exposed to purebred males
were not significantly different from those where
females chose between C. pecosensis and F1 hybrid males
(311.3 s; Mann–Whitney U-test: U20,18 ¼ 139, ns). Likewise, C. variegatus females exposed to competing purebred males displayed total activity levels similar to those
of females exposed to male conspecifics competing
against F1 hybrids (407.4 s; U20,20 ¼ 164, ns). Neither
C. variegatus nor C. pecosensis females displayed a significant preference for one cross of hybrid males as
compared with hybrid males of the reciprocal cross
(variegatus females: U13,7 ¼ 29, ns; pecosensis females:
U11,7 ¼ 20, P ¼ 0.09).
Female preference was not related to male coloration
intensity or male length. For both species of females,
preferred males were not significantly more colourful
than the other competing males (Chi-square for pecosensis
females: v21 ¼ 0.947, ns; variegatus females: v21 ¼ 1.256,
ns; Fig. 2). Similarly, there was no significant correlation
between male colour rank (ranked within male species,
for this analysis) and female preference rank when our
data were compared with a distribution of 1000 randomized replicates (Spearman’s correlation: C. pecosensis
females, r ¼ )0.031, ns; C. variegatus females,
r ¼ )0.024, ns). The coloration of male C. variegatus
was generally more intense than that of male C. pecosensis
in the visual choice trials (mean scores ¼ 3.6 and 2.5,
respectively). Also, hybrid males had higher colour
intensity scores than C. pecosensis males (mean
scores ¼ 2.9 and 2.2, respectively) but lower scores than
C. variegatus males (mean scores ¼ 3.3 and 3.9, respectively). The distribution of length ranks among preferred
males did not deviate significantly from random for
female C. pecosensis (Chi-square: v21 ¼ 0.105, ns; Fig. 2) or
C. variegatus (v21 ¼ 0.9, ns). No significant correlation
between male length rank and female preference rank
was detected when our data were compared to a
distribution of 1000 randomized replicates (Spearman’s
correlation: C. pecosensis females, r ¼ 0.018, ns; C. variegatus females, r ¼ 0.07, ns).
Female preference – olfactory experiment
Neither female C. pecosensis nor female C. variegatus
distinguished between water taken from an aquarium
with conspecific males and plain, untreated water. In 14
trials, seven C. pecosensis females ‘preferred’ male-treated
water (Wilcoxon Matched Pairs test: z ¼ 0.878, ns). In 10
trials, five C. variegatus females ‘preferred’ male-treated
water (z ¼ 0.801, ns).
Fig. 1 Mean duration (+SE) of females’ contact with competing
male types in visual choice experiments. Significance values are
indicated by asterisks (*, P < 0.05). Cyprinodon pecosensis are represented by clear bars, Cyprinodon variegatus are represented by black
bars, and F1 hybrids are represented by half-tone bars. Twenty trials
were conducted with each male-female species combination, except
for C. pecosensis males vs. F1 hybrid males (n ¼ 18).
Male competition – dominance experiment
No significant difference in competitive balance between
types was detected for dominance trials involving six
females and those where males competed for access to
three females (t-test for independent samples:
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Sexual selection drives genetic assimilation
601
Fig. 2 Distribution of the colour and length
ranks of preferred males (the single male that
an individual female contacted for the
longest time). No significant preference for
either male length or male coloration intensity was detected among female Cyprinodon
pecosensis or Cyprinodon variegatus.
t22 ¼ 0.093, ns) and the results of the two types of trials
were combined for further analyses. F1 hybrid males
were significantly more aggressive than either male
C. pecosensis (two-tailed t-test for paired samples:
t20 ¼ 3.385, P < 0.005) or male C. variegatus
(t23 ¼ 2.321, P < 0.05, Fig. 3). Hybrid males also pursued
females more frequently than C. pecosensis males or
C. variegatus males (t20 ¼ 4.771, P < 0.001, and Wilcoxon
Matched Pairs test: z ¼ 2.411, n ¼ 24, P < 0.05, respectively) and spawned more often as a result (t20 ¼ 3.022,
P < 0.01 and t23 ¼ 2.421, P < 0.05, respectively; Fig. 3).
Cyprinodon variegatus males were more aggressive
(t20 ¼ 3.702, P < 0.005) and pursued females more often
(t20 ¼ 2.254, P < 0.05) than competing male C. pecosensis;
however, the two species had similar spawning success in
this experiment (t20 ¼ 2.254, P ¼ 0.086; Fig. 3). Fewer
fights occurred in dominance trials involving C. pecosensis
than in those where C. variegatus competed against F1
hybrids (A N O V A planned contrast: F1,64 ¼ 12.328,
P < 0.001, Fig. 4). No significant correlations were detected between the mass difference of competing males and
the outcome of their competitive interaction.
Male competition – territoriality experiment
Cyprinodon pecosensis males were inferior to both
C. variegatus and F1 hybrid males at obtaining territories
and they spawned less frequently as well. In trials where
C. variegatus males competed against C. pecosensis males,
C. variegatus established significantly larger territories
than C. pecosensis (t-test for dependant samples:
t27 ¼ 2.509, P < 0.05; Fig. 5). In this experiment,
C. variegatus obtained significantly greater spawning
access to females than their C. pecosensis competitors
(t27 ¼ 2.6734, P < 0.05). The proportion of spawnings
(arc-sin transformed) obtained by C. pecosensis males did
not vary significantly between trials involving female
C. pecosensis and those where C. variegatus females were
used (t-test for independent samples: t25 ¼ 1.397, ns).
C. variegatus and F1 hybrids were equally successful in
establishing territories (t11 ¼ 0.544, ns) and obtaining
spawnings (t11 ¼ 0.434, ns; Fig. 5). F1 hybrid males were
more successful at establishing and defending territories
than C. pecosensis males (t11 ¼ 2.521, P < 0.05) and
obtained more spawnings as well (t11 ¼ 2.951, P < 0.05;
Fig. 5). Different treatments (combinations of male types)
did not produce significantly different frequencies of fights
in this experiment (A N O V A : F1,48 ¼ 0.926, ns).
Discussion
Female choice
The results provide insight into the role of inter-sexual
selection in the initial hybridization between C. vaiegatus
and C. pecosensis following the introduction of C. variegatus
to the Pecos River. Female C. pecosensis displayed a visual
preference for C. variegatus. Cyprinodon variegatus females
did not discriminate between purebred types although
there is some suggestion of a preference for conspecifics
(Fig. 1). In a mixed population, these visual preferences probably created a reproductive advantage for
C. variegatus males. The attraction of C. pecosensis females
for C. variegatus males represents more than a simple
failure of allopatric populations to develop premating
isolating mechanisms as such a failure would, by definition, not produce a preference for heterospecifics. Female
mating preferences probably played an important role in
the extremely rapid genetic fusion between Pecos pupfish
and sheepshead minnow in the Pecos River.
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J. A. ROSENFIELD AND A. KODRIC-BROWN
Fig. 4 Frequency of fights between different combinations of male
types in the Dominance Experiment. Values are means (+SE) for a
15-min observation period. Trials involving Cyprinodon pecosensis
males (a) had significantly fewer fights (P < 0.001) than those where
Cyprinodon variegatus males competed against F1 hybrid males (b).
Fig. 3 Behavioural rates of competing males in the Dominance
Experiment. All values are means (+SE) for a 15-min observation
period. Male aggression included males chasing or lunging at the
opposing male. In all categories, male F1 hybrids were significantly
more active than their competitors. Significance values are indicated
by asterisks (*P < 0.05; **P < 0.01; ***P < 0.005). Values for each
species are represented by the bar patterns described in Fig. 1.
We were not able to identify particular characters that
allow female discrimination between male C. variegatus
and C. pecosensis. No evidence of preference for males on
the basis of length or colouration intensity was detected.
Kodric-Brown (1983) found a preference among
C. pecosensis females for colour intensity in male conspecifics. In our visual choice trials, males did not attain the
intensity of coloration seen among breeding males in the
wild or those seen in the male competition experiments
(A. Kodric-Brown & J.A. Rosenfield, unpublished data).
The most intense male colourations appear after males
spawn for the first time on a given day (Kodric-Brown,
1996, 1998). Males in our trials had not reproduced for
several days prior to exposure to females. With respect to
male colouration then, our visual choice experiment
forced females to evaluate male attractiveness before
Fig. 5 Average spawning frequency and territory size (cm2; +SE) of
competing male types in the Territoriality Experiment. In each trial,
four males (a pair from each species) were observed in two, 30min
observation periods. Values reported here reflect average performance of each species [i.e., (total spawnings for the species)/(two
males)/(two observation periods)]. Statistically significant differences are indicated by an asterisk (*P < 0.05). Values for each species
are represented by the bar patterns described in Fig. 1.
males mated. This experiment could not eliminate the
possibility that females respond to visual signals that
were not measured (for example, evaluation of male
courtship behaviour or territory defense).
J. EVOL. BIOL. 16 (2003) 595–606 ª 2003 BLACKWELL PUBLISHING LTD
Sexual selection drives genetic assimilation
Females of neither parental species discriminated
visually between conspecifics and F1 hybrids. Failure of
both parental species to discriminate against hybrids
would facilitate introgression in the wild. The lack of a
clear female preference for hybrid or conspecific males
suggests that the rate and extent of genetic introgression
likely depended on the outcome of male–male competition for mates.
Females of neither species discriminated between maletreated water and untreated water. This negative result
may reflect some deficiency in our experimental design;
however, other Cyprinodon species displayed olfactory
recognition of conspecifics and discrimination between
conspecifics and heterospecifics in identical experiments
(Strecker & Kodric Brown, 1999, 2000). Also, allopatric
fish species in other genera discriminate between conspecific and heterospecific mates when exposed to a
similar protocol (e.g. McLennan & Ryan, 1999). Therefore, it is unlikely that either C. variegatus or C. pecosensis
females use olfactory cues for mate recognition.
Male competition
Our behavioural studies reveal a significant role for intrasexual selection in promoting hybridization and genetic
introgression between the two parental species in the
Pecos River. Male C. variegatus and F1 hybrids were
superior to male C. pecosensis in competition for mates
under each of the protocols we used. This competitive
asymmetry almost certainly promoted the rapid replacement of C. pecosensis by hybrids in the Pecos River because
territorial male pupfish achieve much higher mating
success than nonterritorial males (Itzkowitz, 1977;
Kodric-Brown, 1977, 1988a) and females are attracted
to vigorous male courtship (a factor not measured in our
female preference experiments; Itzkowitz, 1978; KodricBrown, 1983, 1996).
Unlike competitive trials involving C. pecosensis, the
outcome of male competition between C. variegatus and
F1 hybrids was affected by the different experimental
protocols we used. Our results indicate that hybrids have
an advantage over both parental types in dominance
situations where individual males control large areas of
spawning territory and receive the vast majority of
spawning attempts (a ‘dominance hierarchy’; Fig. 3).
These conditions occur in the wild when habitat size is
not limiting and population density is low (Soltz, 1974;
Kodric-Brown, 1981, 1988a). Under more typical conditions, where breeding habitat is limited and the density of
competing males is high, hybrids are competitively
equivalent to C. variegatus, although they still outcompete
C. pecosensis for territories and mates. This interaction
between ecology and behaviour may help to explain why
pure C. variegatus were eliminated from the Pecos River so
soon after their introduction. Future studies that focus on
behavioural mechanisms operating in hybrid zones
should help us to understand how ecological conditions
603
(Hubbs, 1955, 1961; Mayr, 1963) interact with mate
choice systems and hybrid genotypes to promote hybridization and introgression.
Male–male competitiveness is only one measure of the
viability of hybrid offspring. The rapid introgression
observed in the wild would only have occurred if hybrid
offspring were also ecologically viable. Under laboratory
conditions, growth rates among F1 hybrid juveniles
are intermediate to those of C. variegatus and C. pecosensis
(J.A. Rosenfield, unpublished data). Also, mature F1
hybrids and backcross hybrids display greater swimming
endurance than mature C. pecosensis (J.A. Rosenfield,
unpublished data). The assumption that hybrids are
competitively inferior to purebred parentals does not
hold in this case; F1 hybrid males are competitively
superior to C. pecosensis males in several experimental
contexts and backcross hybrids have replaced C. pecosensis
in all habitats to which they had access.
Reproductive compatibility and the ‘Species
Problem’
Our results would lead some biologists to conclude that
C. pecosensis and C. variegatus are not different species
(sensu Mayr, 1963). Female C. pecosensis’ choice of mating
partners and the success of F1 hybrid males actively
promoted hybridization and introgression between the
two parental species; thus, our results reveal cohesive
forces operating beyond ‘species’ boundaries, something
that cannot (or should not) occur under most species
concepts (see Arnold, 1997). However, C. pecosensis and
C. variegatus, have diverged in morphometric and meristic
measures, coloration patterns, and display fixed differences in allozymes and mtDNA (Echelle & Echelle, 1978;
Echelle & Connor, 1989; Childs et al., 1996). Additionally,
the behavioural and ecological differences between parental species (current study; J.A. Rosenfield, unpublished
data) demonstrate that these two taxa are not ecologically
or demographically interchangeable (Van Valen, 1988;
Templeton, 1989; Smith et al., 1995). Finally, the behaviour and ecology of hybrids (Echelle & Connor, 1989;
current study; J.A. Rosenfield, unpublished data) reveals a
synergy between the parental genomes; this positive
interaction is evidence of genetic divergence for the same
reasons that partial or complete reproductive isolation
(negative interactions between genomes) would be interpreted as evidence of divergence. The lack of pre- or postmating reproductive isolation between such divergent
forms reveals that the mechanisms that produce lineage
cohesion may remain intact despite substantial evolution
and differentiation in other areas (Smith et al., 1995).
Mating behaviour and the study of genetic
introgression
Both premating mechanisms (female mate choice
and male competition for mates) and a post-mating
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604
J. A. ROSENFIELD AND A. KODRIC-BROWN
mechanism (hybrid vigour) promoted rapid genetic
fusion between these two allopatric pupfish species upon
secondary contact. These findings contradict the assumption that individuals will always mate preferentially with
conspecifics when a choice is available (e.g. Tinbergen,
1953; Paterson, 1985). Examples of females in geographically isolated lineages expressing an attraction for
heterospecific males are becoming more common
(Kaneshiro, 1976; Ryan & Wagner, 1987; Pierotti &
Annett, 1993; Ryan & Rand, 1993, 1995; Grant & Grant,
1997; Pfennig, 2000; current study). Our results also
stand in contrast to the common finding that hybrid
offspring are inferior to parental species in obtaining
mating opportunities. This inferiority may occur because
hybrids are less viable ecologically (Dobzhansky, 1940;
Barton & Hewitt, 1985; Hatfield & Schluter, 1999) or
because they are discriminated against by potential mates
(e.g. Vamosi & Schluter, 1999). However, hybrid
inferiority across habitats is far from universal (e.g.
Scribner, 1993; Arnold & Hodges, 1995; Arnold, 1997)
and some studies show that hybrid organisms may
actually have an advantage in intra-sexual competition
for mates (e.g. Pierotti & Annett, 1993; Good et al., 2000;
current study). If hybrid offspring are fertile and outcompete one or both parental species for access to mates,
rapid and extensive introgression is likely.
The mechanisms that contribute to lineage cohesion
between formerly allopatric populations require additional study. For example, the visual preference of
C. pecosensis females for heterospecific males suggests
the possibility that these females retain a preference for
ancestral male sexual signals (e.g. Kaneshiro, 1976;
McLennan & Ryan, 1999) displayed by C. variegatus. All
but one species in the Gulf Coast assemblage of
Cyprinodon species, including C. pecosensis, are believed
to derive from a C. variegatus-like ancestor (Echelle &
Echelle, 1992). Female preference for ancestral male
characteristics raises the question: if sexual selection
favours ancestral characters, why were those characters
lost or diminished during the evolution of C. pecosensis?
The ecological success of hybrids argues against correlated evolution of sexual characters with other characters
under strong natural selection (Paterson, 1985). The
secondary loss of characteristics under positive sexual
selection may be a common phenomenon among
isolated species (e.g. Kaneshiro, 1976; Ryan & Rand,
5 1995) and Cyprinodon species in particular (Loiselle,
1982).
Similarly, the role of aggressive behaviour, and
hybrid behaviour in general, in the population
dynamics of hybrid swarms should be investigated.
Our results indicate that, because of their extreme
aggressiveness, hybrid males are competitively superior
to males of both parental species under certain
conditions. As a result, this example of hybrid vigour
is a cohesive force in both the premating (hybrids will
get more matings than purebreds) and post-mating
(hybrid offspring are more vigorous than some parentals) realms. Female selection for male aggressiveness
may drive hybridization and introgression in other
hybridizing species pairs (Brodsky et al., 1988; Pierotti
& Annett, 1993; Good et al., 2000; McDonald et al.,
2001) and elevated aggressiveness among hybrid offspring has been documented in other hybrid zones,
particularly those involving avian species pairs (Rohwer
& Wood, 1998; Good et al., 2000; McDonald et al.,
2001). Additional research is necessary to determine
whether elevated aggressiveness among hybrids is a
general phenomenon.
Studies of the dynamics of natural hybridization and
genetic introgression have largely neglected mating
behaviours and have instead focused on the ecological
forces that limit expansion of the hybrid swarm
(Moore, 1977; Barton & Hewitt, 1985; Harrison,
1986; Howard, 1986). Many recent studies of hybrid
zones have attempted to infer the behavioural processes that produce patterns in the population genetic
structure of the hybrid swarm (e.g. Rosenfield et al.,
2000 and references cited therein). However, inferences drawn purely from descriptions of gene frequencies may provide an incomplete view of the
evolutionary dynamics within hybrid zones if mating
system structure and sexual selection behaviours are
not reflected in population genetic models (e.g. Childs
et al., 1996). In the context of speciation and hybridization studies, mating behaviours have historically
been viewed only in terms of their potential for
decreasing reproductive compatibility. The alternative
possibility, that behaviours increase the likelihood of
interspecific mating and thereby promote hybridization
and introgression, has rarely been considered (but see
Good et al., 2000; McDonald et al., 2001). Ecological
conditions contribute to hybridization in many animal
systems (Hubbs, 1955), but, their effects are likely
mediated through the mating system and the behavioural responses of individuals (Grant & Grant, 1997).
Researchers investigating other cases of hybridization
and introgression should explore the role of mating
behaviours and the response of these behaviours to
changes in ecological conditions.
Acknowledgments
The authors wish to thank K. Howard, J.H. Brown,
D. Schluter, O. Seehausen and an anonymous referee for
providing valuable comments on this manuscript. S. Cain
conducted the olfactory preference experiments.
C.M. Sandoval, S.Cain, S. Lindauer, and T. Angón
assisted in conducting the male competition trials.
A. Allen helped to conduct the resampling analyses
of male colour and length. P. Nicoletto and P. Klerks
provided invaluable assistance in procuring C. variegatus
for this study. Funding was provided by the National
Science Foundation (#IBN-9806483), New Mexico
J. EVOL. BIOL. 16 (2003) 595–606 ª 2003 BLACKWELL PUBLISHING LTD
Sexual selection drives genetic assimilation
Department of Game and Fish, and Graduate and Biology
Research Allocation Committees of the University of New
Mexico. J.A.R. was supported by a STAR fellowship from
the United States Environmental Protection Agency. This
research was conducted under animal care protocols
approved by the University of New Mexico Animal Care
Committee (Permit No. 97001).
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Received 24 November 2002; revised 25 February 2003; accepted 4
March 2003
J. EVOL. BIOL. 16 (2003) 595–606 ª 2003 BLACKWELL PUBLISHING LTD