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Color, Body Size, and Genitalia Size Are Correlated Traits in Eastern Mosquitofish (Gambusia holbrooki) Lisa Horth1, Christopher Binckley1, Rebecca Wilk1, Pranav Reddy1, and Abhinav Reddy1 The effect of natural selection acting upon correlated traits can alter mating success and relative fitness dramatically. Eastern Mosquitofish (Gambusia holbrooki) harbor a genetic body-pigmentation polymorphism where pigmentation color is associated with particular correlated traits. About ninety-nine percent of all males are silver and about one percent are melanic (or black spotted). Here we demonstrate that these pigmentation morphs also differ in correlated life-history traits that affect fitness. Melanic males are larger than silver males in nature, and larger than silver siblings in a controlled environment. Melanic males have relatively larger gonopodia (mating organ) than silver males and greater survival during conspecific competition. This is indicative of a genetic correlation between body color, body size, growth rate, and relative gonopodium size (after controlling for body size). Our past work demonstrates that melanic males also have much higher survival with predators. Thus, while a relatively larger gonopodium is said to result in a cost with respect to predation in two Gambusia species, in G. holbrooki the fitness effects of correlated traits appear to far outweigh this cost for melanic males.
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OST animal taxa harbor pigmentation polymorphisms where the expression of eumelanin (black pigment) varies among morphs (Majerus et al., 1998). The particular type of melanic patterning (spotted versus spotless in Ladybird Beetles; Dobzhansky, 1933; Majerus et al., 1998) and the color tone (dark versus light in Pocket Mice, Chaetodipus intermedius; Nachman et al., 2003; Hoekstra et al., 2005; and humans; Shriver et al., 2003) are often adaptive. Molecular differences associated with melanism have recently been uncovered in a few genes, including Mc1r (for birds; Mundy et al., 2004; Mundy, 2005; for mammals; Nachman et al., 2003; for fish; Parichy et al., 1999; Rawls et al., 2003; reviewed in Horth, 2005). Positive selection has also now been identified for a melanic mutant allele at the K locus in Gray Wolves (Canis lupus; Anderson et al., 2009). Like pigmentation polymorphisms, body-size polymorphisms are widespread in nature, and there is ample evidence that large size is often adaptive. Large size is advantageous for individual survival (Ryan, 1985; Gilburn et al., 1992; Altwegg and Reyer, 2003) as a direct benefit for females’ choosing larger mates (Downhower and Brown, 1980), and for progeny, as a result of greater defense of broods by larger males (Bisazza and Marconato, 1988). Nevertheless, the association between body-size and melanic pigmentation has been quantified in just a few organisms (turtles, Chrysemys picta bellii; Gronke et al., 2006; and Digger Wasps, Bembecinus quinquespinosus; O’Neill and Evans, 1983). Despite the fact that melanic polymorphisms are relatively common in poeciliid species (Poecilia latipinna; Angus, 1983; P. latipunctata, Xiphophorus helleri, X. montezumae; Axelrod and Wishnath, 1991; Gambusia holbrooki; Horth, 2003), the body-size: pigmentation association has been more thoroughly studied in the species with more colorful polymorphisms. For example, larger Dwarf(X. pygmaeus) and High-backed Pygmy Swordtail (X. multilineatus) males have more blue coloration than smaller ones (Kallman, 1989; Kingston et al., 2003), and larger Green Swordtail (X. helleri) males have a red body-stripe, whereas smaller males have a black stripe (Franck et al., 2003). 1
Additionally, body size and mating strategy are correlated in several poeciliid species (X. ingress; Ryan and Causey, 1989; P. latipinna; Travis and Woodward, 1989; Ptacek and Travis, 1996; X. multilineatus; Rosenthal and Evans, 1998), and there is a general trade-off, where larger, more ornamented males tend to court and be preferred by females, and smaller, less decorated males tend to sneak copulations (Rosen and Tucker, 1961). This occurs in Sailfin Mollies (P. latipinna) where the larger males have bright, multi-colored nuptial fin- and body-coloration, which they use for courtship displays (Snelson, 1985). These males are preferred by females over the relatively drab silver to olivaceous-gray males that perform forced copulations (Snelson, 1985). Not all poeciliid species have particular size-classes that are associated with distinct mating behaviors. Even in species without distinct size-classes, behavioral differences often emerge that reinforce the fact that large size is adaptive. For example, in Belonesox belizanus and Girardinus falcatus larger males overwhelmingly dominate smaller ones for access to females in competition trials (Bisazza et al., 1996). In poeciliids such as the Eastern Mosquitofish (G. holbrooki, Fig. 1A), the body-size, body-color association has not yet been evaluated, although the body-color, mating-behavior association has (Horth, 2003). The primary mating tactic generally considered to be used by mosquitofish is forced copulation (Bisazza and Marin, 1995), and while this remains true, recent work has shown distinct differences in the silver and melanic male morphs’ mating behavior, as well as in females’ responses to mating attempts (Horth, 2003). Melanic males are more aggressive and attempt more matings than size-matched, silver siblings (Horth, 2003). In addition, they spend almost twice as long in close pursuit of, or swimming alongside, females, during which time they also chase away other potential suitors (Horth, 2003). Females approach and follow silver males, in a non-aggressive fashion, nearly twice as often as they do melanic males. During mating attempts by males, females tilt their bodies, and hop out of the water away from melanic males, nearly twice as often as they do from silver
Department of Biological Sciences, Old Dominion University, Norfolk, Virginia 23529; E-mail: (LH)
[email protected]; (CB) cbinckle@ odu.edu; (RW)
[email protected]; (PR)
[email protected]; and (AR)
[email protected]. Send reprint requests to LH. Submitted: 15 February 2009. Accepted: 23 November 2009. Associate Editor: J. M. Quattro. DOI: 10.1643/CG-09-044 F 2010 by the American Society of Ichthyologists and Herpetologists
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Fig. 1. Eastern Mosquitofish (Gambusia holbrooki). (A) Melanic male morph (left) and silver male morph (right). (B) Male gonopodium (left) and apical portion of gonopodium (under 10X magnification, light microscope) to show hook near gonopodium tip.
males (Horth, 2003). In Tamesı´ Mollies (P. latipunctata), melanic males display to females more than silver males (Ptacek et al., 2005). In Poeciliidae, the male anal fin has evolved into a gonopodium that serves as an internal insemination organ (Fig. 1B). A correlation among body-size: gonopodium size also exists among poeciliids, although the direction of this correlation varies among species, and by circumstance. In Gila Topminnows (Poeciliopsis occidentalis), large territorial males have relatively short gonopodia, whereas small sneaker males have longer gonopodia (Constantz, 1975). Similarly, larger Sailfin Molly males (defined as .45 mm; Snelson, 1985) have showy display ornaments (longer, higher dorsal fins; Travis, 1994; brighter nuptial coloration; Snelson, 1985) but relatively smaller gonopodia (Ptacek and Travis, 1997). Small males display no distinctive coloration or enhanced morphological development (Snelson, 1985). Trinidadian Guppies (Poecilia reticulata), which perform courtship displays as well as conduct sneaky matings, lack discrete body-size classes, but all males have colorful, sexually selected spots, and there is a positive association between male body size and gonopodium length (Kelly et al., 2000). In guppies (Kelly et al., 2000) and Brachyrhaphis episcope (Jennions and Kelly, 2002), relative gonopodium length is also greater in higher predation areas. Greater gonopodium length is presumably adaptive for sneaky matings in an environment where courtship is conspicuous to predators (Endler, 1987). For two Gambusia species (G. hubbsi, G. affinis), relative gonopodium length also differs by predator regime, but in stark contrast to guppies, males in these two Gambusia species with relatively larger gonopodia are found in predator-free environments (Langerhans et al., 2005). In
mosquitofish, larger gonopodia are indicated to be preferred by females and to result in decreased burst swim speed (Langerhans et al., 2005). As is generally true for poeciliids in Western Mosquitofish (G. affinis), gonopodium length is set at maturity although body size may increase slightly after maturation (Turner, 1941; Angus et al., 2005). In Eastern Mosquitofish (G. holbrooki), a stable male color polymorphism persists where 99% of males are silver and 1% are melanic (or black patterned, Fig. 1A; Horth and Travis, 2002). Melanism appears to be controlled by a Y-linked gene and an autosomal modifier, although temperature can also affect expression (Horth, 2006). Prior work has revealed higher survival for melanic males in the presence of predators in mark–recapture (Horth, 2003, 2004) and mesocosm studies, where melanic males were established at both relatively low (3.3%) and high frequency (8.3%; Horth and Travis, 2002). The low and high frequencies established spanned the average frequency of melanism (4%) identified in the north Florida population from where these fish were collected. In this study, females demonstrated much higher survival when melanic males were present at lower frequency (Horth and Travis, 2002). Here we investigate the relationship between melanism, body size, and genitalia size in G. holbrooki in nature and in a laboratory setting. Since mating behavior and aggression are associated with color, we also evaluate whether the relative fitness of the two male color morphs differs in mating competition. MATERIALS AND METHODS We haphazardly collected a total of 763 male Eastern Mosquitofish by dipnet from native Florida (USA) habitat
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during 17 sampling events. Sixteen of these events occurred seasonally between 1996 and 1999, with an additional sampling event of one population in 2007. From 1996 to 1999, we sampled northern Florida at four locations: Newport Springs (Wakulla Co., USA, sulphur spring), Lake Bradford (Leon Co., freshwater lake), Picnic Pond (Wakulla Co., brackish, open-water), and Wakulla Springs (Wakulla Co., freshwater spring). In 2007, we also sampled the major southern Florida ecosystem (Sawgrass Park, Broward Co., FL, brackish open-water). A histogram of the frequency of melanism from a much larger, multi-year study of 45 populations can be found in Horth and Travis (2002). The mean frequency of melanism across these 45 populations was 0.01 6 0.02 SEM (Horth and Travis, 2002). We transported live fish from northern Florida to Florida State University (Tallahassee, FL), and from southern Florida to Old Dominion University (ODU, Norfolk, VA, USA). Representative voucher specimens (#3214–3216) for this work are held at the Old Dominion University Museum, Norfolk, VA, USA. Body size difference among unrelated color morphs in nature.— In the laboratory, we measured standard body length (snout to caudal peduncle) of each fish twice (to the nearest 0.5 mm). To demonstrate that length is a proxy for size, we measured the body length and the body height (from the ventral point where the gonopodium attaches to the body, up to the dorsal crest) of 40 fish (20 silver, 20 melanic, from Sawgrass Park). For subsequent analyses, data were transformed, as needed, to meet the ANOVA assumption of normality. To test whether melanic and silver males collected in nature differ in body size, we used an ANOVA. Source population was the blocking factor to remove variation in body size among populations before testing whether the two color morphs differed statistically in body length. The analysis was performed using SAS 9.1 with Type III sums of squares. Next, we repeated the above analysis, except that here we generated a single value per sampling event (population, season, year) by using the average body size for each color morph. Thus, 33 values were generated for the average melanic and silver body size from 17 total sampling events (there are 33 not 34 events because at Wakulla Springs in Spring 1998, no melanic fish were captured, so this collection has no value generated). Body size difference among sibling color morphs in the laboratory.—Since melanic males sire melanic and silver sons, to identify whether size differs in genetically related black and silver males, we measured 73 (42 melanic and 31 silver) siblings sired by melanic males from Sawgrass Park. Siblings were reared in one laboratory under constant conditions to minimize effects other than genotype. To test whether sibling color morphs differ in body size, we used an ANOVA with sibling group as our blocking factor. This block removes variation in body size among the different sib groups before testing whether color morphs differ in size. The analysis was performed using SAS 9.1 with Type III sums of squares. Body size/gonopodium length association among unrelated fish in nature.—Sixty-nine fish (35 silver, 34 melanic) were measured from Sawgrass Park. Each fish was placed in a 400-ml beaker of water, tranquilized with tricaine (MS-222), then placed on a microscope slide and observed through a
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light microscope. A micrometer was placed parallel to the fish to measure standard body length and gonopodium length (to nearest 0.10 mm). Two authors (AR, PR) conducted both measurements on each fish. The average of their measurements was used. We log transformed our data, then performed two analyses. We tested whether a relationship between body length (mm) and gonopodium length (mm) existed for unrelated melanic and silver color morphs collected from nature using linear regression. We evaluated allometry as it relates to these two traits by testing whether regression slopes differed from one. We also tested whether the two color morphs differ in gonopodium length after controlling for body length using an ANCOVA. We arcsin transformed the gonopodium length: body length ratio data and then ran an ANOVA to evaluate whether this ratio differed for melanic and silver males, since this ratio is sometimes referred to in the poeciliid literature as a measure reflective of sexual selection. Body size/gonopodium length association among unrelated fish in the laboratory.—Thirty-five fish (15 silver and 20 melanic) were measured in the laboratory from three populations (Wakulla Springs, Rock Spring, and Sawgrass Park). Parent fish were housed in 18.93-liter (5-gal) tanks at 23uC in a 14:10 L/D light cycle until young were born. After a week, young were transferred to individual 9.47-liter (half-gallon) jars and checked weekly for maturation (completely formed gonopodium), at which point they were measured. As with field fish, we log transformed these data then tested whether a relationship between body length (mm) and gonopodium length (mm) existed for both melanic and silver morphs using linear regression. We evaluated allometry between traits by testing whether the regression slopes differed from one. Next, we tested whether the two color morphs differ in gonopodium length, after controlling for body length, using an ANCOVA. We used an arcsin transformation on the gonopodium length: body length ratio and then used an ANOVA to evaluate whether the ratio differed for silver and melanic males. Male competition trials.—We established mating trials in the laboratory using field fish from Picnic Pond. A trial was comprised of a pair of size-matched males (body-length difference #0.5 mm) and one virgin female, all three of which were housed together in a 75.7-liter (20-gal) tank within a temperature controlled room (31uC) with a 14:10 L/ D light cycle. Each control tank housed two silver males and one virgin female. Each experimental tank housed one silver male, one melanic male, and one virgin female. Sixteen control and 16 experimental replicates were successfully established. If a male was killed in the first few days of an experiment, a second size-matched male of the same color was placed in the tank. In some cases, this male was also killed and the color ratio of the young in these tanks was not evaluated. To evaluate whether each color morph sires 0.50 of all F1 male offspring, a Pearson chi-square analysis was conducted using the observed ratio for all F1 males (31 M: 39 S). The fact that melanic males produce silver sons must be considered to calculate the expected values for each color morph. We proceed with two calculations. First, results from genetic analysis from a large study (Horth, 2006) indicate
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silver F1 males are expected from the melanic sire, and 35 silver males from the silver sire. The expected ratio for all trials is 25.2 M: 44.8 S. RESULTS Body size differences among unrelated color morphs in nature.— Comparing the mean of all field-collected males (628 silver and 135 melanic) demonstrated that melanic males (mean 5 21.97 6 0.19 mm) were larger than silver males (mean 5 19.77 6 0.11 mm; F1,757 5 115.54; P , 0.001). When average morph body size per sampling event was evaluated, the difference remained significant (F1,27 5 20.29; P , 0.001). The correlation between body length and body height for 40 males was high and positive (r 5 0.94; P , 0.001), indicating that length is an appropriate proxy for body-size in these fish (Fig. 2). Body size differences among sibling color morphs in the laboratory.—Both sibling group and color morph were significant. Sibling groups differed in body size, and melanic males (mean 5 20.67 6 0.49 mm) were larger than their silver male siblings (mean 5 18.78 6 0.51 mm; F1,59 5 7.79; P 5 0.0071).
Fig. 2. The relationship between (log) body- and (log) gonopodiumlength for (A) 69 field males (35 silver, 34 melanic) and (B) 35 lab fish (15 silver, 20 melanic).
that in multiple populations, 0.19 of F1 males sired by melanic males are silver (Horth, 2006). Data from the Picnic population differ, and indicate a higher frequency (0.28) of silver F1 males (Horth, 2006). Unlike other populations evaluated by us, melanism is temperature sensitive in the Picnic population and requires exposure to cold temperatures (18uC) for $12 weeks for expression. This temperature change (from 31uC to 18uC) and the additional monitoring period (and an equipment failure during the experiment) resulted in a higher number of deaths for males in this population than for other populations. If a silver F1 male were to die at 12 weeks, he was scored as silver. However, any variance in timing of expression of melanism might mean this male could have been a melanic genotype that had not yet expressed black pigmentation. Thus, the frequency of melanism is likely underrepresented in the Picnic population. Given this information, we perform two chi-square analyses for F1 males. For the first analysis, we use the null expectation that each sire color morph produces 0.50 of the 70 male offspring, recognizing that approximately 0.19 of melanic males’ sons are expected to be silver, as seen for populations other than Picnic that do not require extended time in a cold room (Horth, 2006). In this case, melanic sires are expected to produce 28.35 melanic and 6.65 silver sons, and silver sires are expected to produce 35 silver sons. Here, the expected melanic: silver color ratio for all trials is 28.35 M: 41.65 S. For the second analysis, we use the Picnic population expression rate where 0.28 silver F1 males arise from melanic sires. Here 25.2 melanic and 9.8
Body size/gonopodium length association among unrelated fish in nature.—For both morphs, there was a positive and statistically significant relationship: bigger fish had larger gonopodia (melanic r2 5 0.66, F1,32 5 61.47, P , 0.001; silver r2 5 0.48, F1,33 5 30.87, P , 0.001, Fig. 2). The slope of the relationship describing how gonopodium length increased with body length differed between the two color morphs (P , 0.001); however, neither slope was significantly different from one (melanic P 5 0.99, silver P 5 0.20), indicative of isometry. Melanic fish (6.48 6 0.09 mm) had larger gonopodia than silver fish (5.85 6 0.09 mm) even after removing variation produced by differences in body size (F1,66 5 21.91, P , 0.001). Melanic male gonopodia were 32.1% (60.004) of these males’ body length, which is greater (F1,67 5 21.64, P , 0.001) than this measure for silver males (29.2% 6 0.004). Body size/gonopodium length association among unrelated fish in the laboratory.—For both morphs, there was a positive and statistically significant relationship as bigger fish had larger gonopodia (melanic r2 5 0.43, F1,18 5 13.37, P , 0.002; silver r2 5 0.39, F1,13 5 8.45, P , 0.01). Again, the slope of the relationship describing how gonopodium length increased with body length differed between the two color morphs (P 5 0.0002). This slope was ,1.0 for melanic fish (P , 0.001) but not for silver fish (P 5 0.11), indicative of negative allometry and isometry, respectively. Melanic fish (7.59 6 0.11 mm) had longer gonopodia than silver fish (6.91 6 0.12 mm), even after removing the variation produced by differences in body size (F1,32 5 17.29, P , 0.001). The melanic male gonopodia were 34.9% (6SEM 0.007) of the body length of these fish, and the silver male gonopodia were 32.4% (6SEM 0.008); these values differed significantly (F1,33 5 4.34, P , 0.04). Male competition trials.—Experimental trials demonstrated an extreme amount of aggression, especially by melanic males, and this resulted in mortality of silver males. In 11 of 16 (68.75%) experimental trials, silver males were killed by melanic males within a few days. In three of 16 (18.75%)
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trials, the melanic male was killed, and in two of 16 (12.5%) trials, neither male was killed. In most cases the replacement silver male was also killed by the melanic male. Where the replacement fish was killed (and in tanks where more than two replacements were necessary—which were tanks in addition to the 16 ‘successful trials’ described above), tanks were not assessed for progeny color-ratio comparison. Six experimental tanks did result in progeny. A total of 31 melanic (M) and 39 silver (S) male progeny survived to maturity (tank 1: 2 M + 8 S, tank 2: 3 M + 7 S, tank 3: 4 M + 6 S, tank 4: 6 M + 4 S, tank 5: 7 M + 8 S, tank 6: 9 M + 6 S). Control tanks never produced any melanic progeny and had less aggression (death) overall. Using the multi-population expression rate (0.19 silver) comparing the expected and observed melanic: silver ratio (expected, 28.35 M: 41.65 S; observed, 31 M: 39 S) values for all 70 F1 male offspring with a Pearson chi-square analysis resulted in x2(0.05,1) 5 0.416, which was not significant (P 5 0.5188). Using the Picnic population expression rate (0.28 silver), comparing expected (25.2 M: 44.8 S) and observed (31 M: 39 S) values resulted in x2(0.05,1) 5 2.086, which was also not significant (P 5 0.1487). DISCUSSION In stark contrast to the trade-off associated with long gonopodium length (preferred by females, but considered costly in burst swim speed) identified in G. affinis and G. hubbsi (Langerhans et al., 2005), we find that in G. holbrooki, melanic males are the larger morph with the faster growth rate and relatively larger genitalia, and they demonstrate higher survival with predators (Horth, 2004). Thus, in G. holbrooki the benefits of these correlated traits outweigh their cost during predation. This is particularly noteworthy, given that G. holbrooki hybridizes with G. affinis, and the silver males of these species appear nearly indistinguishable phenotypically, except for fin-ray counts. Our results indicate that the relationship between body color, body size, and genitalia size occur in the field and the laboratory, suggestive of a heritable component for these correlated traits. We find that melanic males demonstrate more aggression toward silver males than vice versa in these mating trials. This trait association is consistent with our previous work where color and behavior were correlated in sibling male G. holbrooki (Martin, 1977; Horth, 2003). For teleosts, a size advantage generally allows for acquisition of greater resources, faster predator evasion, and/or greater longevity (Barlow, 1961). Large males are often dominant, and grow at a faster rate, than subordinate ones (Nakano, 1995). Larger G. holbrooki males, independent of color, routinely dominate smaller males in mesocosm populations (Horth, pers. obs.). Further, in several poeciliids, females prefer large males (Ryan and Causey, 1989), and there is some evidence for this in Eastern Mosquitofish (Bisazza et al., 2001), suggestive of advantages associated with size, but not melanic pigmentation. Poeciliid males grow until sexual maturity, when growth slows or ceases (Yan, 1987; Morris and Ryan, 1990). Thus, our finding that melanic genotypes are larger than silver siblings in a controlled environment suggests a genetic basis for size and growth rate. In X. nigrensis, small males actually mature approximately 46 days faster than larger ones (Morris and Ryan, 1990). Larger melanic Eastern Mosquitofish may result from greater time to maturity, resource acquisition, predator evasion, and/or longevity. Resource
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acquisition may be affected by size and aggression. Predator evasion and longevity may be affected by aggression, size, color, and gonopodium length. Future studies will compare otolith measurements to determine age at maturation, to identify whether melanic males are generally older than silver males in nature, and whether size-matched males of each color are the same age. Evidence for sexual selection is weak and variable in Eastern Mosquitofish (Pilastro et al., 1997). There is evidence that coercive mating by males is the primary mating tactic (Bisazza et al., 2001), and there is also evidence that females prefer larger, silver males over smaller ones (McPeek, 1992) as well as silver males over melanic ones (Nelson and Planes, 1993; Taylor et al., 1996). While Langerhans et al. (2005) indicate female preference for longer gonopodia in two species of Gambusia, our evaluation of allometry for G. holbrooki indicates no evidence of sexual selection (defined as slope .1 for the regression of log gonopodium length on log body length). Further, we see no obvious mating advantage in terms of number of F1 males sired (for the Picnic population the F1 sex ratio is 1:1; Horth, 2006). Additionally, when evaluating the fraction of total body length that the gonopodium comprises (where a larger value is associated with a decreased likelihood of sexual selection), G. holbrooki do not show strong evidence of sexual selection. For comparison, three non-courting (thrusting only) species in this fish family have gonopodia that are between approximately 30% and 45% of their body length (Girardinus falcatus, 45%; Heterandria formosa, 35–40%; and Phalloceros caudimaculatus, 30–35%; Bisazza and Pilastro, 1997). We show that gonopodia measurements for G. holbrooki demonstrate a range from 29% to 35% (with this value being greater for melanic males than silver males, and with a relatively small sample size for laboratory fish). This range is most similar to the non-courting P. caudimaculatus. A combined evaluation of large and small (thus, courting and thrusting, respectively) males of Poecilia latipinna demonstrates the gonopodia of this species to be 28.3% of total body length (calculated from Ptacek and Travis, 1998). For Sailfin Mollies, however, it would be most useful for a study of males with high versus low courtship display rates to be analyzed separately to supplement the analysis we provide. Sexually selected guppies (P. reticulata) have gonopodia that are #25% of their body length (Bisazza and Pilastro, 1997). Additionally, while larger body-size and larger gonopodium size are associated in melanic G. holbrooki, melanic males are rejected by females twice as often as silver sibling males (Horth, 2003), suggesting that color (and/or associated aggressive behavior) overrides female preference (if present) that has been indicated for other species of Gambusia (Langerhans et al., 2005). The larger gonopodium size of melanic males could result for several reasons, including the relatively relaxed predation pressure on this color morph, combined with the potential fitness benefit from forced copulations, or simply from selection to increase reproductive fitness via coercive mating, consistent with the concept that sneaker males in other species have longer gonopodia than do courting males (Rosen and Tucker, 1961). However, since the gonopodium also has a hook (Fig. 1B) that tears the female’s urogenital opening, can leave her bloody, and that is sometimes lethal (pers. obs.), testing whether this hook is more costly to females when males have larger gonopodia and are more aggressive
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would be valuable. In experimental mesocosm populations, female numbers (and melanic male survival) decrease when melanic males are present in relatively high frequency. Yet when melanic males are established in relatively low frequency, females as well as melanic males have higher survival. Additionally, melanic males contribute relatively more melanic sons to the F1 generation than they do when established at high frequency (Horth and Travis, 2002). Thus, density and frequency are essential parameters necessary to evaluate the overall fitness of these male morphs. Here, in approximately 70% of the mating trials, silver males were killed by melanic competitors. Since aggression can be inflated in the laboratory, these survival differences are not likely precisely reflective of nature. A more conservative evaluation is that melanic males are more aggressive than silver ones during male–male competition, which corroborates past findings (Horth and Travis, 2002; Horth, 2003) and indicates potential fitness benefits during male–male interactions. Given what is known regarding pigmentation inheritance patterns in these fish (Horth, 2006), melanic F1 males were not produced in numbers greater than expected by chance, so no fitness advantage is gained through production of F1 melanic males (although further work on this is warranted because of the small sample sizes here). In conclusion, the association between body size, color, and genitalia size may afford melanic males substantial fitness benefits with respect to predator evasion and male–male interactions. ACKNOWLEDGMENTS We are grateful to J. Travis for laboratory space use and for financial support from NSF (DEB 99-02312). We are also grateful to the Jeffress Memorial Trust (760776) for financial support. This work was approved by animal use committee (IACUC #06-004). LITERATURE CITED Altwegg, R., and H. U. Reyer. 2003. Patterns of natural selection on size at metamorphosis in water frogs. Evolution 57:872–882. Anderson, T. M., B. M. vonHoldt, S. I. Candille, M. Musiani, C. Greco, D. R. Stahler, D. W. Smith, B. Padhukasahasram, E. Randi, J. A. Leonard, C. D. Bustamante, E. A. Ostrander, H. Tang, R. K. Wayne, and G. S. Barsh. 2009. Molecular and evolutionary history of melanism in North American gray wolves. Science 323:1339–1343. Angus, R. A. 1983. Genetic analysis of melanistic spotting in sailfin mollies. Journal of Heredity 74:1–84. Angus, R. A., J. Stanko, R. L. Jenkins, and R. D. Watson. 2005. Effects of 17a-ethynylestradiol on sexual development of males western mosquitofish (Gambusia affinis). Comparative Biochemistry and Physiology, Part C 140:330–339. Axelrod, H. R., and L. Wischnath. 1991. Swordtails and Platies. TFH Publications, Neptune City, New Jersey. Barlow, G. W. 1961. Social behavior of the desert pupfish, Cyprinodon macularius, in the fields and in the aquarium. American Midland Naturalist 65:339–359. Bisassa, A., and M. Marconato. 1988. Female choice, male– male competition and parental care in the river bullhead
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