The maintenance of genetic variation in the face of natural selection is a long-standing question in evolutionary biology. In the blueﬁn killiﬁsh Lucania goodei, male coloration is polymorphic. Males can produce either red or yellow coloration in their anal ﬁns, and both color morphs are present in all springs. These 2 morphs are heritable and how they are maintained in nature is unknown. Here, we tested 2 mechanisms for the maintenance of the red/yellow color morphs. Negative frequency- dependent mating success predicts that rare males have a mating advantage over common males. Spatial variation in ﬁtness predicts that different color morphs have an advantage in different microhabitat types. Using a breeding experiment, we tested these hypotheses by creating popula- tions with different ratios of red to yellow males (5 red:1 yellow; 1 red:5 yellow) and determining male mating success on shallow and deep spawning substrates. We found no evidence of negative frequency-dependent mating success. Common morphs tended to have higher mating success, and this was particularly so on shallow spawning substrates. However, on deep substrates, red males enjoyed higher mating success than yellow males, particularly so when red males were rare. However, yellow males did not have an advantage at either depth nor when rare. We suggest that preference for red males is expressed in deeper water, possibly due to alterations in the lighting en- vironment. Finally, male pigment levels were correlated with one another and predicted male mat- ing success. Hence, pigmentation plays an important role in male mating success. Key words: carotenoid, color polymorphism, environmental heterogeneity, melanin, negative frequency dependence, pterin. The ubiquity of pronounced variation among individuals within theory tells us that natural and sexual selection should reduce gen- populations represents a paradox that how can such variation exist etic variation in coloration, resulting in a single color morph within when selection and drift are constantly acting to remove variation a population (Lewontin 1974; Bradbury et al. 1987). The mainten- within populations (Mitchell-Olds et al. 2007)? Variation in animal ance of variation in coloration is even more problematic especially coloration is particularly perplexing because coloration can affect when the color pattern is controlled by a few alleles (Rosenblum many aspects of an organism including its ability to thermoregulate, et al. 2004; Hoekstra et al. 2006; van’t Hof et al. 2011). to avoid predators, and to attract a mate and/or defend a mate from There are multiple forms of balancing selection that can, in competitors (Andersson 1994; Ruxton et al. 2004). Hence, animal theory, maintain color polymorphisms. Polymorphisms can be coloration should be under intense natural and sexual selection. Yet, preserved by negative frequency-dependent selection (NFDS), V C The Author(s) (2018). Published by Oxford University Press. 733 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact email@example.com Downloaded from https://academic.oup.com/cz/article-abstract/64/6/733/4921187 by Ed 'DeepDyve' Gillespie user on 11 January 2019 734 Current Zoology, 2018, Vol. 64, No. 6 overdominance, habitat-dependent selection, or trade-offs between with small environmental effects (Antoniazza et al. 2010; Roulin different fitness components. Here, we examine the extent to which and Ducrest 2013; Saino et al. 2013). Therefore, the extent to which 2 mechanisms, negative frequency dependence and microhabitat melanic traits are honest signals is unclear. Melanic traits might variation in mating success, contribute to the maintenance of 2 dis- serve as honest badges of status and indicate male aggressiveness, or crete color morphs in a freshwater killifish. NFDS has received con- they might be driven by frequency-dependent selection or local siderable empirical support in maintaining color polymorphisms adaption (see Roulin 2016 for a review). (Horth and Travis 2002; Fitzpatrick et al. 2007; Gray and Carotenoid-derived ornaments are assumed to honestly reflect McKinnon 2007; Roulin and Bize 2007; Dijkstra and Border 2018). the animals’ diet since they cannot synthesize carotenoids de novo. Negative frequency dependence occurs when rare genotypes have a Hence, they could truly reflect male foraging ability to potential fitness advantage over common genotypes. An advantage to rare mates especially when carotenoids are limited in habitats (Olson genotypes can involve a number of fitness components including and Owens 1998; Grether 2000). Moreover, carotenoids are anti- mating success, fecundity, survival, and/or a reduction in predation. oxidants and benefit the immune system, so carotenoid-derived or- In guppies, rare color morphs attract more attention from females naments are also signals of health (Johnson and Fuller 2015; Megı ´a- and suffer less predation (Olendorf et al. 2006; Bond 2007). In cich- Palma et al. 2017). lids, sticklebacks, and darters, male aggression is more intensive to- Pterins have received less attention than melanins or carotenoids. wards competitors with similar coloration, such that rare male color Pterins can be synthesized de novo (as can melanin) and have poten- morphs experience reduced intrasexual competition (Seehausen and tial immune and antioxidant function (McGraw 2005). However, Schluter 2004; Pauers et al. 2008; Dijkstra et al. 2009; Sluijs et al. there are few studies that examine the influence of pterins on male 2013; Lehtonen 2014; Martin and Mendelson 2016; Moran and mating success (Johnson and Fuller 2015), and even fewer that con- Fuller 2018; Tinghitella et al. 2018). In a scale-eating cichlid sider the effects of all 3 pigment types. Perissodus microlepis, where animals are curved to either the left or This study focuses on a pronounced color polymorphism present right to pick scales off of other fish, rare morphs have higher forag- among males in bluefin killifish Lucania goodei. In clear spring popu- ing rates than common morphs (Hori 1993). lations, nearly all males have either solid red or solid yellow anal fins Genetic variation in coloration can also be maintained within (Fuller 2002). There is no evidence that these color morphs represent populations if there is microhabitat variation such that each color different alternative mating strategies. They do not differ in size or in morph can outcompete the others in a particular set of conditions time to sexual maturation, and neither morph acts as a “sneaker” (Hedrick 2006; Dreiss et al. 2012; Burri et al. 2016). The perception (Fuller 2001, 2002; Johnson and Fuller 2015). Breeding studies have of coloration is dependent on lighting environment. Lighting envir- shown that this variation is largely controlled by a single locus where onments are particularly variable in aquatic habitats as the inherent yellow alleles are dominant to red alleles (Fuller and Travis 2004). optical properties of water (e.g., materials dissolved or suspended in Yellow and red color morphs are present in all investigated clear water) alter the distribution and intensity of the ambient light spec- water populations (Fuller 2002), which raises the question of how trum (Lythgoe and Partridge 1989). In addition, within any given these alleles are maintained within populations in nature. The anal fin population, lighting environments differ as a function of time of day can also be blue or a combination of blue and either red or yellow. and depth. Here, we focus on variation caused by depth. Depth Males with blue coloration are found primarily in swamps. All males alters the lighting environment due to the absorption of different possess the ability to express either red or yellow anal fins, but this wavelengths of light (Lythgoe 1988). Studies of cichlids have shown coloration is essentially displaced by blue provided that animals have that speciation has occurred along a depth gradient where red color the right genetics and rearing environment (Fuller and Travis 2004). morphs are favored in deeper water and blue color morphs are In this study, we focus solely on the red and yellow color morphs and favored in shallower water (Seehausen et al. 2008). Speciation along how they are maintained within populations. depth clines has also observed in rockfish (Sebastes)(Ingram 2011). A previous study in bluefin killifish showed no evidence of In addition to rarity and microhabitat types, other aspects of NFDS between yellow and red males. While red males sired more male coloration may influence mating success. A variety of pigment offspring when rare, yellow males did not (Fuller and Johnson types contribute male coloration and their expression levels are 2009). Hence, red males had higher mating success than expected, influenced by both internal and external factors. Hence, males with but yellow males did not. Fuller and Johnson (2009) suggested that identical coloration may still differ in phenotype (Ligon and a female mating preference was present in the study population. McCartney 2016). Males of most species possess multiple traits that Rare red males benefited from this preference, but common males can signal different aspects of male quality. In terms of coloration, did not because the preference was, in essence, diluted by the pres- the 3 main pigment types are melanin, carotenoid, and pterin. Red, ence of many other red males. Subsequent work has suggested that orange, and yellow ornaments in coloration are primarily composed spring females have a weak preference for red males (Fuller and Noa of carotenoids and pterins (McGraw et al. 2004). In some species, 2010), but other studies have failed to find such an effect (McGhee males possess all the 3 types of pigments. et al. 2007; Mitchem et al. in review). The biology of the melanin pathway is well known. The melano- The original test for negative frequency-dependent mating suc- cortin system involves many biological functions such as the im- cess by Fuller and Johnson (2009) was not perfect. The experiment mune system, energy homeostasis, and sexual behavior. Hence, did not maintain good water quality as algal blooms occurred in selection on melanin might also involve selection on other traits some, but not all, of the experimental tanks. This may have altered (Ducrest et al. 2008). Despite our depth of knowledge concerning fish perception of male anal fin morph. In addition, the paternity the melanocortin system, the meaning of melanin-based coloration analysis only allowed assignment of offspring to the rare or common is debated. Some studies indicate that the degree of melanism is quite morphs in the tank as a class, rather than to specific fathers and plastic and is determined by male condition and/or the outcome of mothers. The latter would have allowed for a more detailed analysis past contests (Keka ¨la ¨ inen et al. 2010; Piault et al. 2012; Henschen of factors influencing levels of parentage. Fuller and Johnson (2009) et al. 2016), while others show that melanism is highly heritable only considered 2 additional traits, body length and condition, as Downloaded from https://academic.oup.com/cz/article-abstract/64/6/733/4921187 by Ed 'DeepDyve' Gillespie user on 11 January 2019 Johnson et al. The maintenance of color polymorphism in killifish 735 covariates that could potentially explain male mating success. Experimental setup Finally, Fuller and Johnson (2009) did not examine mating success Our first goal was to determine whether negative frequency-depend- as a function of depth, which may also contribute to the mainten- ence or spatial variation in mating success could potentially main- ance of the color polymorphism. tain both color morphs within populations. To do this, we In bluefin killifish, the anal fin (red vs. yellow) is controlled by manipulated the ratio of red males to yellow males in each tank. We the expression of 2 pterin pigments (likely xanthopterin and drosop- also stocked tanks with spawning substrates (yarn mops) at 2 depths terin) (Johnson and Fuller 2015). Red males express both pterin (surface and bottom) to determine whether red and yellow males dif- types, whereas yellow males only express xanthopterin. Throughout fered in spawning location. We created 2 experimental treatments - this article, we refer to these pigments as yellow (likely xanthop- one where red males were rare (1 red male, 5 yellow males, and 6 fe- terin) and red pterin (likely drosopterin). The anal fin also has a mel- males) and another where yellow males were rare (1 yellow male, 5 anic black border that predicts the outcome of male/female red males, and 6 females). We performed 7 replicates of each treat- competition. Males with larger black borders are clearly more dom- ment resulting in 14 experimental breeding populations in stock inant (Johnson and Fuller 2015), which is in keeping with predic- tanks (Supplementary Table 1). In each stock tank, animals could tions based on the biology of the melanocortin pathway (Ducrest spawn on substrates that were floating on the surface or were on the et al. 2008). The caudal fin also has reddish/orange coloration, bottom of the tank (approximately 50 cm deep). For the remainder which is due to carotenoid pigments. Previous work in this system of this article, we refer to these as “floating mops” and “bottom indicates that both carotenoid and pterin expression are predictive mops.” Hence, the experiment also allowed us to examine whether of health and overall mating success (Johnson and Fuller 2015). there is spatial variation in relative fitness. Hence, pigment expression (in addition to color morph identity) The yarn mops in each tank were searched at least 3 times a may be critical in male reproduction. week for eggs. Eggs were removed and maintained in a dilute solu- The goal of this study was to determine whether negative fre- tion of methylene blue (about 12 ppm) to preventing fungal infec- quency-dependent mating success and/or spatial variation in mating tion until the fry hatched. Fry were fed baby Artemia for an success as a function of depth could account for the maintenance of additional 3 weeks after hatching. They were then stored in ethanol red and yellow color morphs. Negative frequency-dependent mating and frozen until DNA could be extracted using a standard protocol. success predicts that rare color morphs have a mating advantage At the conclusion of the experiment, all the adult fish were euthan- over common color morphs. Spatial variation in mating success pre- ized with 0.025% MS-222. For each individual, standard length was dicts that each color morph must have a microhabitat where it out- recorded using a laminated piece of engineering grid paper (nearest performs the other. In addition to testing these 2 hypotheses, we to 1 mm), and wet mass was recorded using an electronic balance also asked whether the size, condition, and pigmentation of the male (nearest to 0.0001 g). (melanin, carotenoid, and pterin) could account for male mating Our second goal was to determine whether the degree of pigment success. expression in the anal and caudal fins influenced male reproductive success (whether a male had offspring or not) in L. goodei.To do this, we extracted and measured pterins from the anal fin and carot- Materials and Methods enoids from the caudal fin. We also used photography to measure the amount of black coloration (i.e., melanin) on the anal fin. We Study system and fish collection examined correlations between the continuous variation in pigmen- The bluefin killifish is a freshwater fundulid that is native to the tation and male mating success. At the end of each trial, males were southeastern United States of America. Its distribution range is placed against a white background with a color standard, and a digi- mainly in Florida. During the breeding season (mainly March to tal picture was taken of the left side of each male using a Nikon mid-summer) (Lee et al. 1980), males protect territories of aquatic D3300 camera. A Camera PictoColor 4.5 Photoshop plug-in was vegetation, where spawning and egg attachment occurs. Females can subsequently used to standardize the light and color levels of each deposit eggs on vegetation throughout the water column ranging picture. The caudal and anal fin were removed and spread out on a from floating vegetation to bottom substrate vegetation (< 1.5 m glass slide. Rough measurements to the nearest 1 mm of each fin depth) (Fuller 2001). Females spawn their eggs in small batches were taken by treating the fin as a parallelogram and noting the across multiple males’ territories (Fuller and Travis 2001). Both fe- length of its proximal and distal ends and the distance between the male choice and male-male competition contributes to male mating 2. The fins were stored at 80 C until pigment could be quantified. success (McGhee et al. 2007). The caudal peduncles of all adults were removed and stored in etha- The fish used in this experiment was captured with a seine in nol at 80 C until DNA was extracted. May of 2011 from the Upper Bridge population of the Wakulla River, near Tallahassee, Florida. This population is polymorphic in male coloration. Males with blue anal fins are very rare. Both yellow Parentage analysis and red males are abundant in this population (Fuller 2002). The Parents and offspring were typed at the following 3 highly poly- fish was transported back to the University of Illinois and housed morphic microsatellite loci: CA (Fuller and Johnson 2009), AC17 briefly in a communal oval stock tank (1.85 m in length 0.86 m in (Burg et al. 2002), and Lg1 (Creer and Trexler 2006). Forward pri- width 0.65 m in height) before being moved to 12 experimental mers were labeled with VIC (CA), 6FAM (AC17), or Pet (Lg1). The oval stock tanks (1.85 m in length 0.86 m in width 0.65 m in loci were amplified in 1 multiplex reaction according to the standard height), which were housed in a glass greenhouse where the tem- protocol in the QIAGEN Multiplex Polymerase chain reaction (PCR) perature was 20 C 30 C and exposed to natural lighting condi- Kit. The PCR products were run on an ABI Prism 3730xl Analyzer at tions. UV sterilizers were attached to the tanks to prevent algal the University of Illinois’ W.M. Keck Center for Comparative and blooms and maintain water clarity. The following experiments were Functional Genomics. Fragment sizes were scored using GeneMapper approved by the Institutional Animal Care and Use Committee at software (Applied Biosystems) and verified manually. We then used the University of Illinois (protocol numbers #11143 and #08183). CERVUS V 3.0.3 (fieldgenetics.com) to assign parentage to the fry Downloaded from https://academic.oup.com/cz/article-abstract/64/6/733/4921187 by Ed 'DeepDyve' Gillespie user on 11 January 2019 736 Current Zoology, 2018, Vol. 64, No. 6 (Kalinowski et al. 2007). Each stock tank was analyzed separately, pictures were corrected for by scaling each image to a size standard. and offsprings were assigned parentage based on 80% likelihood. The anal fin was isolated using the freehand selection tool, and the Only a small number of offspring (38 of 1051) failed to have parent- image was converted to black and white using the adjust threshold age assigned to them, due to either unresolvable parentage or poor function and selecting black and white threshold color. The image DNA quality. was then converted to a binary image, and the area of the black Many replicates experienced adult mortality. These individuals band was calculated with the measurement tool. were included in the CERVUS parentage simulations as un-sampled potential parents. With the exception of 1 deceased female, dead fe- Statistical analysis males did not contribute any offspring, and we treated the replicate We first report basic statistics on paternity, the skew in reproduction in as having been formed without them. However, 3 deceased males males and females, and general associations between size, condition, did leave a notable number of offspring. We were able to reconstruct and mating success. We measured reproductive skew (S) separately for his or her genotype, which helped further identify parentage, and de- males and females in each replicate using the formula presented in duce the color morph of the missing males by examining the body Keller (1993) that results in a value from 0 (no skew) to 1: and/or deducing it from the other morphs in the tank. However, we v N þ N were unable to measure pigmentation, and our sample sizes reflect S ¼ N þ N this. In other cases, individuals with pale fin coloration were initially misidentified as the wrong sex or morph. This altered our gender where, N is the number of adults that bred at least 1 offspring, N b n and morph ratios (Supplementary Table 1), but it did not affect our is the number of individuals assigned 0 offspring, and v is the stand- ability to detect the effect of pigmentation on paternity, and in fact ard deviation among breeders that have at least 1 offspring in the more accurately represents the pigment variation found in nature. proportion of total offspring assigned parentage (Supplementary Table 1). Reproductive skew was measured for males and females in Coloration analysis each tank (2 genders 14 tanks¼ 28 values total). We then tested The methods here follow Johnson and Fuller (2015), where the pig- for differences in reproductive skew between genders using analysis ments were extracted and identified. Briefly, to quantify pterins and of variance (ANOVA) and also for differences in male reproductive carotenoids, we used 2 solvents, 1% NH OH and 1: 1 mixture of skew between our treatments (red rare/yellow common vs. yellow hexane: tert-butyl methyl ether, to extract these 2 pigments from rare/red common, Supplementary Table 1). fins and partition carotenoids from pterins. This method of identify- We first asked whether negative frequency dependence in mating ing pterins and carotenoids has been widely applied to coloration success could potentially maintain both color morphs within popu- studies in different animals (Kikuchi et al. 2014; Steffen et al. 2015; lations. Negative frequency-dependent mating success predicts that Cuervoa et al. 2016). Individual anal and caudal fins were thor- rare males have a mating advantage over common males. For each oughly ground with a mortar and pestle in 1% NH OH, and then a male, we calculated the total mating success (% of total offspring 1: 1 mixture of hexane: tert-butyl methyl ether was added when sired by a male), the mating success on floating mops (% of off- eluting carotenoids. The absorption spectra of these 2 solvent layers spring from floating mops sired by a male), and the mating success were examined to determine pigment class. While eumelanic and on bottom mops (% of offspring from bottom mops sired by a structural coloration did not go into solution, pterins could be iden- male). We then calculated the average mating success for yellow and tified by a strong UV absorption in the NH OH layer (Hill and red males for each tank. We used linear models to determine McGraw 2006). Carotenoids were identified by a characteristic pat- whether the average male mating success of red and yellow males tern of absorbance in the hexane: tert-butyl methyl ether solvent varied depending on male color morph (red vs. yellow), rarity status (McGraw 2005). (rare vs. common), and the interaction between the male color The caudal fins were homogenized in 1 ml 1% NH OH. The morph and rarity status for each of the 3 measures of male mating ground material and solvent were transferred to a fresh tube and an success (% total offspring, % offspring from floating mops, and % equivalent volume of a 1: 1 hexane: tert-butyl methyl ether solvent offspring from bottom mops). To do this, we used the “lmer” func- was added. The solution was vortexed, centrifuged, and the 2 solv- tion in R (lme4 package). The experimental tank was treated as a ents were separated. Carotenoids were present in the top layer, the random effect in all 3 models. We used a Type 3 analysis in the hexane: tert-butyl methyl ether layer. The absorption of the hexane: “car” package to determine the effects of each term. tert-butyl methyl ether layer was measured on a spectrophotometer, We next asked whether males varied in where they spawned and the height of the absorption peak at 445 nm was used to quan- their offspring. Here, we measured the number of offspring that tify carotenoid levels (Johnson and Fuller 2015). males sired on bottom mops relative to the number that they sired For measurements of pterins in anal fins, the anal fins were on floating mops. This analysis used individual males as the level of homogenized in 400 mL1%NH OH, centrifuged, and the resulting observation and, by default, excluded males that did not sire any off- supernatant was collected. The height of the absorption peak at spring. We asked whether male color morph (red vs. yellow), rarity 398 nm was used to quantify yellow pterin pigment (xanthopterin) (rare vs. common), and the interaction between rarity and color and that at 498 nm was used to quantify red pterin pigment (drosop- morphs affected where males spawned their offspring. Experimental terin) (Johnson and Fuller 2015). Total anal fin pterin was measured tank was a random effect. To do this, we used binomial model in R as red and yellow pterin (absorption) summed. Yellow males express using the “glmer” function from the “lme4” package. We used a Type only the yellow pterin. Red males express both the yellow and red 3 analysis in the “car” package to determine the effects of each term. pterin. Our initial model suffered from over-dispersion, so we included Anal fin melanin could not be analyzed using absorption spec- individual ID as an additional random effect (Harrison 2014). troscopy, so digital picture analysis in ImageJ (U.S. National Finally, we examined the effect of male size, condition, and pig- Institutes of Health, Bethesda, Maryland, USA, imagej.nih.gov/ij/) ments levels on male mating success. We first examined Pearson cor- was used instead. Small differences in magnification between relations between standard length, condition, anal fin size, caudal Downloaded from https://academic.oup.com/cz/article-abstract/64/6/733/4921187 by Ed 'DeepDyve' Gillespie user on 11 January 2019 Johnson et al. The maintenance of color polymorphism in killifish 737 fin size, pigmentation (melanin, red pterin, yellow pterin, and total Table 1. Mating success (proportion of offspring sired) as a func- tion of male color morph, rarity, and their interaction pterin carotenoid levels), and the 3 measures of male mating success (% total offspring, % offspring from floating mops, and % offspring A: Total mating success (proportion of offspring sired) from bottom mops). The condition of each fish was calculated as the Term FDF (num, denom) P residuals of the log of weight regressed on the log of standard 10 10 (Intercept) 66.8 1, 12 <0.0001 length (Bolger and Connoly 1989). Color 0.83 1, 12 0.3805 We then asked whether inclusion of these traits altered our inter- Rarity 5.1 1, 12 0.0433 pretation of our experimental treatments. Because many of the char- Color Rarity 0.64 1, 12 0.4408 B: Mating success on ﬂoating mops acters were significantly correlated (see results), we used principal Term FDF (num, denom) P components analysis to obtain composite scores of 6 male characters (Intercept) 38.1 1, 12 <0.0001 (standard length, condition, caudal fin size, yellow pterin, caroten- Color 0.1 1, 12 0.7239 oid, and melanin). We excluded red pterin, total pterin, and anal fin Rarity 6.8 1, 12 0.0225 size from the analysis because these traits varied between yellow and Color Rarity 0.1 1, 12 0.7378 red males. We examined the results of the principal components C: Mating success on bottom mops analysis and retained the first 3 principal components. We then per- Term FDF (num, denom) P formed 4 analyses. The first analysis simply asked whether male (Intercept) 78.7 1, 12 <0.0001 color morph, rarity, the interaction between rarity and color morph, Color 7.7 1, 12 0.0169 and the first 3 principal components explained whether or not males Rarity 4.4 1, 12 0.0584 Color Rarity 4.0 1, 12 0.0674 mated, which we refer to as mating status. For this analysis, we cate- gorized males as either having mated or not. We used a binomial The analysis considers the tank means of mating success for red and yellow model in R using the “glmer” function from the “lme4” package. males (and their associated rarity status) across the 14 tanks. Tank is treated We then performed another 3 analyses where we examined the ef- as a random effect. “num” refers to numerator, and “denom” refers to de- fects of male color morph, rarity, their interaction, the first 3 princi- nominator. Terms with P< 0.05 in bold. P< 0.10 but P> 0.05 in italics. pal components on total male mating success (% of total offspring sired), male mating success on floating mops (% of offspring from floating mops sired), and male mating success on bottom mops between male coloration and rarity. We found nearly identical re- (% of offspring from bottom mops sired). Here, we used a linear sults for mating success on floating mops (Figure 1B, Table 1B). model using the “lmer” function from the “lme4” package. For all 4 This was not surprising as 84% of the offspring came from floating models, experimental tank was treated as a random effect. Type 3 mops. A significant effect of rarity was present (P ¼ 0.0225), where models were used throughout. common males had higher mating success than rare males, and the The raw data for this experiment have been deposited in Dryad pattern became much stronger after the removal of a large outlier (number to be entered upon acceptance). (rarity: F ¼ 111.1, P< 0.0001). The effect of rarity had a similar 1, 11 effect on males of both color morphs (average mating success on floating mops: common-yellow¼ 0.18, rare-yellow¼ 0.06, com- Results mon-red ¼ 0.18, rare-red¼ 0.09). Removal of this data point also re- Testing for negative frequency-dependent mating sulted in a marginally significant (F ¼ 4.5, P ¼ 0.0575) of color 1, 11 success where yellow color morphs had slightly higher mating success on We identified parentage in a large number of fry (Supplementary floating mops. Table 1). In total, 1,560 eggs were collected across the experiment. A different pattern emerged from bottom mops. Rare males had From those eggs, 1,060 fry hatched and survived long enough to slightly higher mating success than common males (Table 1C, have DNA extracted. A subset of those (1,051) were typed, and of Figure 1C, P ¼ 0.058). This was particularly so for red males. There those, 1,011 (96%) were successfully assigned parentage by was a statistically significant affect male coloration (P ¼ 0.0169), CERVUS at 80% confidence level or above. Reproductive skew did where red males had higher mating success than yellow males. A not differ between males and females (paired t-test on male-female marginally significant interaction was also present, where red males skew across the 14 tanks: t ¼ 0.951, P ¼ 0.359), nor did male re- were more likely to have high mating success on bottom mops when productive skew vary between tanks in which red or yellow males they were rare (P¼ 0.0674, average mating success: common-yel- were rare (F ¼ 3.01, P ¼ 0.1083) (Supplementary Table1). There 1, 12 low¼ 0.13, rare-yellow¼ 0.14, common-red ¼ 0.17, and rare- was no difference between treatments in the number of eggs laid in red¼ 0.35). Red males had 2X greater mating success on bottom the tanks after correcting for experimental duration and the number mops when rare than when they were common, and>2X greater of females in the tanks (F ¼ 2.48, P ¼ 0.1434). 1, 12 mating success on bottom mops than either common-yellow or rare- There was no evidence for negative frequency-dependent mating yellow males. success when considering all of the data. Rarity status had a margin- ally significant effect on total male mating success (Table 1A, Testing for differences in spawning location due to P ¼ 0.043) but common males had slightly higher mating success than rare males (Figure 1A). This effect was present for males of depth both color morphs (average mating success: common-yellow¼ 0.14, Here, we asked whether color morph and rarity affected where rare-yellow¼ 0.07, common-red¼ 0.18, rare-red¼ 0.13). Removal males spawned. Eleven of 87 males in the experiment did not suc- of a large outlier rendered the pattern even more significant (rarity: cessfully reproduce, so they were excluded from the analysis. The F ¼ 63.8, P< 0.0001) with common males having higher mating results largely matched the patterns found for male mating success 1, 11 success than rare males. There was little evidence that mating suc- on bottom mops. Rarity influenced where males spawned cess differed due to male coloration or due to the interaction (Table 2, Figure 2, P ¼ 0.0011). Rare males spawned more of their Downloaded from https://academic.oup.com/cz/article-abstract/64/6/733/4921187 by Ed 'DeepDyve' Gillespie user on 11 January 2019 738 Current Zoology, 2018, Vol. 64, No. 6 A All of offspring B Offspring from floating mops (84%) 0.6 0.6 0.4 0.4 0.2 0.2 0.0 0.0 Common morph Rare morph Common morph Rare morph Rarity status Rarity status C Offspring from bottom mops (16%) 0.6 0.4 0.2 0.0 Common morph Rare morph Rarity status Figure 1. The average mating success of red and yellow males as a function of rarity. Lines denote averages of red and yellow males from the same experimental tank. Red ﬁll denotes red males. Yellow ﬁll denotes yellow males. (A) Average mating success (percentage of the total offspring sired for a tank). (B) Average mat- ing success on ﬂoating mops (percentage of the offspring sired from ﬂoating mops). (C) Average mating success on bottom mops (percentage of the offspring sired from bottom mops). Note that 84% of all offspring were spawned on ﬂoating mops and 16% were spawned on bottom mops. 1.00 Male phenotypical characters and reproductive success Here, we asked whether red and yellow males differed in other traits Male color that might affect male mating success. Three males with incomplete 0.75 morph data were excluded from this analysis, leaving us with 84 males. In Red addition, a large outlier was also present among the carotenoid 0.50 data, which was excluded for analyses of carotenoid levels. Yellow Males in each tank were assigned a color morph (red/yellow by AJ). Visual assignment matched the absorption spectroscopy data 0.25 from the anal fins. Red and yellow males did not differ in standard length (F ¼ 1.59, P¼ 0.211), condition (F ¼ 0.16, P¼ 0.691), 1, 82 1, 82 0.00 or caudal fin size (F ¼ 0.01, P¼ 0.936), but red males did have 1, 82 Common morph Rare morph larger anal fins than yellow males (F ¼ 12.12, P¼ 0.001). We cal- 1, 82 culated the residuals of a regression of anal fin size on standard length Rarity status to determine whether this was a genuine pattern or simply an effect due to subtle differences in size. The analysis revealed that the re- Figure 2. Male spatial distribution of offspring as a function of rarity status (common vs. rare) and color morph (red vs. yellow). The y-axis shows the siduals were larger for red males than yellow males (F ¼ 16.67, 1, 82 proportion of offspring on the bottom mop versus the total of offspring indi- P¼ 0.0001), indicating that red males have larger anal fins regardless vidual males. N¼ 76. Eleven males were excluded because they did not sire of body size. Red and yellow males did not differ in the amount of ca- any offspring. rotenoid (F ¼ 1.30, P¼ 0.258), melanin (F ¼ 0.97, P¼ 0.327), 1, 81 1, 82 or yellow pterin (F ¼ 0.15, P¼ 0.696). However, not surprisingly, 1, 82 offspring on the bottom mops than on the floating mops relative red males had significantly more red pterin (F ¼ 64.10, 1, 82 to common males. There was also a significant effect of male col- P< 0.0001) and more total pterin (F ¼ 5.39, P¼ 0.023) than yel- 1, 82 oration, where red males spawned more of their offspring on bot- low males. tom mops than on floating mops relative to yellow males We next asked whether there were correlations between con- (P ¼ 0.035). Finally, there was a marginally significant interaction tinuously varying traits: standard length, condition, anal fin size, due to the fact that the rare-red males placed more offspring on caudal fin size, carotenoid, yellow pterin, red pterin, total pterin, the bottom mops than common-red, common-yellow, and rare- melanin, the proportion of total offspring sired, the proportion of yellow males (Figure 2, Table 2). offspring sired from floating mops, and the proportion of % of offspring on bottom mops Average mating success Average mating success bottom mops Average mating success floating mops Downloaded from https://academic.oup.com/cz/article-abstract/64/6/733/4921187 by Ed 'DeepDyve' Gillespie user on 11 January 2019 Johnson et al. The maintenance of color polymorphism in killifish 739 Table 2. General linearized model examining the proportion of eggs laid on bottom mops versus ﬂoating mops by individual males Term v df P Intercept 9.02 1 0.0027 Color 4.45 1 0.0348 Rarity 10.66 1 0.0011 Color Rarity 3.01 1 0.0827 The model assumes a binomial distribution with a logit link function. Tank and individual identity are treated as random effects. Terms with P< 0.05 in bold. P< 0.10 but P> 0.05 in italics. N ¼ 76. Eleven males (out of 87 total) did not sire any offspring and were excluded from the analysis. offspring sired from bottom mops. Our analysis revealed that there were several statistically significant correlations among these variables. The 3 pigment classes (carotenoid, pterin, and melanin) were loosely correlated with one another. Melanin was correlated with yellow pterin, red pterin, and total pterin (Supplementary Figure 1A–C). Carotenoid was correlated with yellow pterin and total pterin (Supplementary Figure 2A–B). Red andyellowpterinwerecorrelatedwithone another (Supplementary Figure 2C) and with total pterin. All 3 pigment classes were loosely correlated with male mating success (mel- anin: Supplementary Figure 3A–C; carotenoid: Supplementary Figure 4A–C; yellow pterin: Supplementary Figure 5 A–C;red pterin: Supplementary Figure 6A–C). Carotenoid was loosely cor- related with the proportion of total offspring sired and the pro- portion of offspring sired on floating mops. Both yellow pterin and melanin were correlated with the proportion of total off- spring sired, the proportion of offspring sired from floating mops, and the proportion of offspring sired from bottom mops. Red pterin was loosely correlated with the proportion of total off- spring sired and strongly correlated with the proportion of off- spring sired on bottom mops. This result is in keeping with the result that red males have higher mating success on bottom mops than do yellow males. We next asked whether incorporation of pigment levels, stand- ard length, condition, and fin sizes altered the results of our treat- ments. We used a principal components analysis to summarize the broad patterns of covariation among these traits and then asked whether or not inclusion of the principal component scores dramat- Figure 3. (A) The relationship between PC1 and whether or not a male spawned any offspring. (B–D) The relationship between PC1 and (B) the pro- ically altered our treatment effects. We included standard length, portion of total offspring sired, (C) the proportion of offspring sired on ﬂoat- condition, caudal fin size, carotenoid, yellow pterin, and melanin ing mops, and (D) the proportion of offspring sired on bottom mops. N¼ 84 values in the principal components analysis. Red pterin and anal fin for all three graphs. Graphs B and C indicate whether males were common size were excluded because they differed between yellow and red (open circles) or rare (dark circles). Graph D indicates whether males were males. Supplementary Table 3 shows the result of the principal com- red or yellow color morphs. ponents analysis. The first 3 principal components accounted for over 50% of the variation in the traits. PC1 loaded strongly onto all traits except standard length. PC2 loaded strongly onto standard for male mating success on floating mops. Higher levels of PC1 were length, caudal fin size, and carotenoid but negatively onto yellow loosely associated with increased mating success (Table 4C, pterin and melanin. PC3 loaded strongly onto condition and caudal Figure 3C, P¼ 0.0528), and common males had an advantage over fin size, but negatively onto standard length, yellow pterin, and rare males (P¼ 0.0235). Finally, the analysis of male mating success melanin. on bottom mops again indicated that red males had an advantage Table 4 shows the results of our analyses. PC1 had a strong effect over yellow males (Table 4C, Figure 3D, P¼ 0.0258). The interaction on whether or not males mated (Table 4A, Figure 3A). Males that between color and rarity was marginally significant (P¼ 0.0678) due failed to mate had low PC1 values. Not surprisingly, PC1 also affected to the fact that red males had higher mating success when rare (mating total male mating success (Table 4B, Figure 3B). This analysis was success on bottom mops: rare-red¼ 0.347, common-red¼ 0.169, similar to the previous analysis on tank means (Table 1A). Here, there rare-yellow¼ 0.124, common-yellow¼ 0.139). There were also mar- was a marginal effect of rarity (P¼ 0.071) where common males had ginal effects of PC1 where higher levels of PC1 were loosely associated higher mating success than rare males. The same patterns were seen with increases in mating success on bottom mops. Downloaded from https://academic.oup.com/cz/article-abstract/64/6/733/4921187 by Ed 'DeepDyve' Gillespie user on 11 January 2019 740 Current Zoology, 2018, Vol. 64, No. 6 Table 3. Pearson’s correlation coefﬁcients (above the diagnoal) and P-values (below the diagonal). SL Condition Anal Fin Caudal Carot Yellow Red Total Mel % offspring % offspring % offspring Area fin area pterin pterin pterin (total) (bottom) (top) SL 0.008 0.269 0.090 0.291 0.110 0.059 0.070 0.018 0.073 0.067 0.109 Condition 0.943 0.144 0.349 0.141 0.379 0.171 0.361 0.336 0.090 0.121 0.081 Anal ﬁn area 0.013 0.193 0.157 0.035 0.210 0.339 0.279 0.122 0.078 0.065 0.070 Caudal ﬁn area 0.416 0.001 0.153 0.313 0.216 0.106 0.208 0.140 0.064 0.086 0.051 Carotenoid 0.008 0.204 0.754 0.004 0.324 0.003 0.261 0.174 0.229 0.119 0.224 Yellow pterin 0.319 0.000 0.055 0.049 0.003 0.471 0.959 0.590 0.267 0.222 0.235 Red pterin 0.596 0.119 0.002 0.339 0.975 0.000 0.702 0.275 0.221 0.333 0.160 Total pterin 0.528 0.001 0.010 0.057 0.017 0.000 0.000 0.564 0.287 0.286 0.242 Mel 0.874 0.002 0.269 0.205 0.115 0.000 0.011 0.000 0.219 0.215 0.182 Percentage offspring 0.507 0.415 0.481 0.564 0.038 0.014 0.043 0.008 0.045 0.520 0.978 (total) Percentage offspring 0.543 0.271 0.559 0.438 0.285 0.043 0.002 0.008 0.050 0.000 0.346 (bottom) Percentage offspring 0.322 0.461 0.529 0.642 0.042 0.031 0.146 0.027 0.097 0.000 0.001 (top) N¼ 84, except for correlations involving carotenoid where a single, large outlier was removed. Carot ¼ carotenoid, mel ¼ melanin. Values in bold denote P< 0.05. Discussion possibly provided an area where females can exert preference with- out being disrupted by competing males. Common males had higher Mechanisms maintaining variation mating success than rare males on floating mops (Figure 1B), but By manipulating the ratios of red and yellow morphs in bluefin killi- rare males had higher mating success than common males on bot- fish, we were able to test whether rare morph males have a mating tom mops (Figure 1C). In addition, more eggs were obtained from advantage that results in increased paternity. We show here, in cor- floating mops than from bottom mops. Why this occurs is unclear? roboration with previous results (Fuller and Johnson 2009), that One possibility is that fish prefer to place their eggs on floating rare males have no overall mating advantage. In fact, rare-morph mops and that common males compete intensely over these sub- males actually sired significantly fewer offspring than common- strates. However, Sandkam and Fuller (2011) asserted that bluefin morph ones in our experimental setup, particularly on the floating killifish have no clear preference for spawning on either floating mops. Theoretically, our results could have been affected by mortal- mops or bottom ones. However, in their experiment, there was only ity in eggs or fry that prevented us from assigning parentage to 1 male and 1 female in each trial. Therefore, male-male competition 100% of the offspring. However, this scenario would require that for spawning substrates was precluded. However, there were mul- the offspring of rare color morphs suffer higher mortality than the tiple males in an experimental tank in our study. Hence, the spawn- offspring of common color morphs, and this is extremely unlikely. ing substrates could have become limited when males competed to We can, therefore, be reasonably certain that negative frequency- establishing their own territories. The spatial difference in mating dependent sexual selection is not operating to maintain the red/yel- success between common and rare males might imply that bluefin low anal fin polymorphism in bluefin killifish. killifish prefer floating mops to bottom ones and display stronger These findings are in keeping with previous work. Fuller and male aggression towards oppisite-morph. Such a scenario has been Johnson (2009) performed a similar experiment testing for negative shown in a cichlid, Astatotilapia burtoni, and in the white-throated frequency-dependent mating success between yellow and red males. sparrow (Korzan and Fernald 2006; Horton et al. 2012). They found that red males (but not yellow males) had a mating ad- An alternative hypothesis is that spatial variation in mating success vantage when rare. The current experiment produced a similar pat- allows for the maintenance of the 2 color morphs. Here, we did find tern in that red males had an advantage when rare, but only on the that on bottom mops, red males had higher mating success on bottom bottom mops. The 2 studies are also similar in that yellow males mops and that this was particularly so when they were rare never had a mating advantage when rare. The explanation proposed (Figure 1C, Table 3). This finding is in keeping with Fuller and by Fuller and Johnson (2009) was that a female mating preference Johnson (2009) who found that red males had increased mating suc- was present in this spring population favoring red males. cess when rare, but that there was no reciprocal effect for yellow Subsequent work by Fuller and Noa (2010) showed that, indeed, males (Figure 1C). Likewise, here, there was no evidence that yellow there is a slight mating preference for red males over yellow and males had increased mating success on floating mops (Figure 1B)nor blue males for spring females. The finding that red males have an ad- that they had heightened mating success when rare (Figure 1A). In vantage when rare suggests that red males receive a disproportionate order for negative frequency dependence to maintain the variation in share of matings with females when rare, but that this advantage is coloration, both morphs must have increased fitness when rare. This diluted when females have many red males to choose among. Fuller was clearly not the case. Likewise, in order for spatial variation in and Johnson (2009) also did not maintain crystal clear water, which mating success to maintain the variation, each color morph must have might have made it easier to females to exert mating preferences a microhabitat where it outperforms the other. While red had higher without being disrupted by competing males. The bottoms of our mating success than yellow males on bottom mops, the reverse was stock tanks had more nooks and crannies where animals are less vis- not true for yellow males. Yellow males did not outperform red males ible to competing males. Hence, bottom substrates may have on floating mops. Downloaded from https://academic.oup.com/cz/article-abstract/64/6/733/4921187 by Ed 'DeepDyve' Gillespie user on 11 January 2019 Johnson et al. The maintenance of color polymorphism in killifish 741 Table 4. Type 3 analyses on the effects of male color, rarity, male Another potential explanation for why rare-red males sired more color rarity, PC1, PC2, and PC3 on (A), (B), (C) and (D) offspring on the bottom mops is that rare-red males simply spent more time at the bottom. This might occur if common-yellow males A: Mating status (yes or no) expelled the rare-red males from floating mops or if rare-red males actively chose to stay at bottom. These 2 reasons are not mutually Term X DF P exclusive. The different offspring spatial composition between (Intercept) 10.8812 1 0.0010 common-red and rare-red males suggests a potentially fruitful av- Color 0.7557 1 0.3847 enue of future research on the color morph spatial distribution along Rarity 0.0015 1 0.9691 the water depth in the wild populations, and more importantly Color Rarity 2.3678 1 0.1239 points out that environmental heterogeneity may assist in maintain- PC1 9.4215 1 0.0021 ing the color polymorphism of bluefin killifish. PC2 0.2651 1 0.6067 The pattern in bluefin killifish stands in contrast to the guppy PC3 0.2983 1 0.5849 B: Total mating success (proportion of offspring sired) Poecilia reticulata, where NFDS occurs through at least 2 known Term F DF (num, denom) P mechanisms (mating and predation) (Hughes et al. 1999; Olendorf (Intercept) 40.3449 1, 23.8 <0.0001 et al. 2006). The source of this disparity might stem from the differ- Color 0.3998 1, 70.2 0.5292 ent effective population sizes of the 2 species. Guppy populations Rarity 3.366 1, 69.1 0.0709 are small and can become quite isolated, especially during the dry Color Rarity 0.2456 1, 24.8 0.6246 season (Griffiths and Magurran 1997). In addition, numerous stud- PC1 4.8099 1, 58.5 0.0323 ies have demonstrated that guppies can suffer from inbreeding de- PC2 0.0191 1, 63.8 0.8907 pression (Mariette et al. 2006; Pitcher et al. 2008; Johnson et al. PC3 2.0448 1, 52.6 0.1587 2010). These factors may favor behaviors that facilitate inbreeding C: Mating success on ﬂoating mops avoidance, such as a preference for rare males. In contrast, bluefin Term F DF (num, denom) P (Intercept) 27.5609 1, 23.8 <0.0001 killifish have extremely large population sizes (Turner et al. 1999), Color 0.0482 1, 70.2 0.8269 and females actively allocate their eggs across multiple males (Fuller Rarity 5.3638 1, 69.1 0.0235 2001), which lessens the potential consequences of inbreeding. Color Rarity 0.0103 1, 24.8 0.9201 Without the potential for inbreeding depression, female bluefin killi- PC1 3.9061 1, 58.5 0.0528 fish might not benefit from avoiding mating with common-morphs. PC2 0.0354 1, 63.8 0.8515 On the contrary, if rare males are rare because they have low fitness, PC3 2.1978 1, 52.6 0.1442 c then preference for rare males might be maladaptive. D: Mating success on bottom mops Term F DF (num, denom) P (Intercept) 55.125 1, 23.8 0.0000 Effects of male phenotypical characters Color 5.1907 1, 70.2 0.0258 Males with high levels of pigmentation (yellow pterin, red pterin, Rarity 2.3244 1, 69.1 0.1319 total pterin, melanin, and carotenoid) were more likely to sire off- Color Rarity 3.6466 1, 24.8 0.0678 spring. All 3 types of pigmentation (melanin, pterin, carotenoid) PC1 3.3457 1, 58.5 0.0725 were strongly associated with whether or not a male had offspring PC2 1.2204 1, 63.8 0.2734 (Table 3, Figure 3) and were correlated with mating success. Our PC3 0.0603 1, 52.6 0.8070 previous work looked at the effect of pigmentation on dominance N¼ 84 for all 4 tables. Table 4A shows a generalized linear model that as- and found a strong effect of anal fin melanin on dominance, and sumes a binomial distribution with a logit link function. Analyses 4B–4D are thus access to females (Johnson and Fuller 2015). The results of the linear models of proportional data. All analyses include tank as a random ef- current study confirm those results and suggest that dominance in fect. DF ¼ degrees of freedom. num ¼ numerator, denom ¼ denominator. this species can directly translate into increased likelihood of mating, Terms with P< 0.05 in bold. P< 0.10 but P> 0.05 in italics. N¼ 76., even when females can presumably avoid aggressive males by hiding a b Percentage of total offspring spawned., Percentage of offspring spawned on or preferentially mating with other males. Males with high levels of ﬂoating mops., Percentage of offspring spawned on bottom mops. carotenoids and pterins were also more likely to sire offspring. Exactly what these 2 pigments are signaling is unclear. We argued previously that higher levels of carotenoids and pterins may be at- While unable to explain the maintenance of genetic color tractive to females because these pigments signal condition in the morphs, our study does shed light on some of the determinants of case of carotenoid, and parasite load in the case of both pterin and male mating success. Our results suggest that microhabitat variation carotenoid abundance (Johnson and Fuller 2015). In this study, con- in light quality affects the mating success of the red color morph. dition was significantly correlated with pterin and melanin, but the Our previous research suggests that bluefin killifish have preferences correlation between carotenoid and condition was not statistically for colors that contrast with available light (Fuller et al. 2010; Fuller significant (Table 3). Possible reasons for these discrepancies include and Noa 2010; Johnson et al. 2013). In clear spring water, (like that the fact that the fish used in this experiment were smaller, younger, of our source population and which our stock tanks mimicked), lon- and more heavily parasitized than the fish used in Johnson and ger wavelengths are attenuated more quickly than shorter wave- Fuller (2015). Hence, the value of pigmentation as a signal of health lengths. Thus, there are relatively fewer red wavelengths in and condition may vary due to natural conditions. spawning substrates at the bottom of the water column than at the In conclusion, this study found little evidence that negative fre- top. Of course, this would indicate that red males should be found quency-dependent mating success can account for the maintenance more often at depth, which is not readily apparent in the wild of the yellow-red genetic color polymorphism in bluefin killifish. (Fuller 2001). Nonetheless, this suggests a potentially fruitful avenue Red males did have a mating advantage on bottom spawning sub- of future research. strates when they were rare, but yellow did not have a reciprocal Downloaded from https://academic.oup.com/cz/article-abstract/64/6/733/4921187 by Ed 'DeepDyve' Gillespie user on 11 January 2019 742 Current Zoology, 2018, Vol. 64, No. 6 Burri R, Antoniazza S, Gaigher A, Ducrest A-L, Simon C et al., 2016. The advantage. Hence, negative frequency-dependent mating success genetic basis of color-related local adaptation in a ring-like colonization cannot account for the maintenance of the 2 color morphs. Red around the mediterranean. Evolution 70:140–153. males may benefit from a female mating preference when they are Creer DA, Trexler JC, 2006. New polymorphic microsatellite loci in two ﬁsh rare, particularly when spawning on bottom substrates. This obser- species: Blueﬁn killiﬁsh Lucania goodei and yellow bullhead Ameiurus nata- vation does suggest that variation in the lighting environment might lis. Mol Ecol Resour 6:167–169. alter either the attractiveness of the males or the ability of females to Cuervoa JJ, Belliure J, Negro JJ, 2016. Coloration reﬂects skin pterin concen- exert mating preferences. tration in a red-tailed lizard. Comp Biochem Physiol B-Biochem Mol Biol The question remains as to what maintains such striking levels of 193:17–24. polymorphism across multiple populations. Negative frequency- Dijkstra PD, Border SE, 2018. How does male-male competition generate neg- dependent fitness could emerge from other selective forces such as ative frequency-dependent selection and disruptive selection during specia- predation. Another possibility is that balancing selection is present in tion? Curr Zool 64:89–99. the form of overdominance (heterozygote advantage). Transcriptomes Dijkstra PD, Hemelrijk C, Seehausen O, Groothuis TGG, 2009. Color poly- morphism and intrasexual competition in assemblages of cichlid ﬁsh. Behav and a linkage map for bluefin killifish have been published (Kozak Ecol 20:138–144. et al. 2014; Berdan et al. 2018). Fuller is currently assembling the blue- Dreiss AN, Antoniazza S, Burri R, Fumagalli L, Sonnay C et al., 2012. Local fin killifish genome. Hence, it may be feasible to identify the red/yellow adaptation and matching habitat choice in female barn owls with respect to locus and test for overdominance. This study did find that the mating melanic coloration. J Evol Biol 25:103–114. success of red males is heightened on deeper spawning substrates, Ducrest A-L, Keller L, Roulin A, 2008. Pleiotropy in the melanocortin particularly when they were rare, and also found positive correlations system, coloration and behavioural syndromes. Trends Ecol Evol 23: between overall pigmentation and reproductive success, reaffirming 502–510. the importance of coloration on mating dynamics in this species. Fitzpatrick MJ, Feder E, Rowe L, Sokolowski MB, 2007. Maintaining a behaviour polymorphism by frequency-dependent selection on a single gene. Nature 447:210–212. Funding Fuller RC, 2001. Patterns in male breeding behaviors in the blueﬁn killiﬁsh Lucania goodei: a ﬁeld study (Cyprinodontiformes: Fundulidae). Copeia The corresponding author was financed by a grant from the Ministry of 2001:823–828. Science and Technology, Taiwan (MOST 105-2917-I-564-075). This work Fuller RC, 2002. Lighting environment predicts the relative abundance of was funded by the National Science Foundation (NSF) (DEB #0953716 and male colour morphs in blueﬁn killiﬁsh Lucania goodei populations. Proc R DEB #1011369). Soc B 269:1457–1465. Fuller RC, Johnson AM, 2009. A test for negative frequency-dependent mat- ing success as a function of male colour pattern in the blueﬁn killiﬁsh. Biol J Author contributions Linn Soc 98:489–500. The experiment was designed by A.M.J. and R.C.F., and carried out Fuller RC, Noa LA, 2010. Female mating preferences, lighting environment, and a test of the sensory bias hypothesis in the blueﬁn killiﬁsh. Anim Behav by A.M.J. The data were collected by A.M.J. and analyzed by C-H.C. 80:23–35. The manuscript was prepared by C-H.C. and supervised by R.C.F. Fuller RC, Noa LA, Strellner RS, 2010. Teasing apart the many effects of light- ing environment on opsin expression and foraging preference in blueﬁn killi- ﬁsh. Am Nat 176:1–13. Conflict of interests Fuller RC, Travis J, 2001. A test for male parental care in a fundulid, the blue- The authors report no conflict of interests. ﬁn killiﬁsh Lucania goodei. Environ Biol Fish 61:419–426. Fuller RC, Travis J, 2004. Genetics, lighting environment, and heritable responses to lighting environment affect male color morph expression in blueﬁn killiﬁsh Lucania goodei. Evolution 58:1086–1098. Supplementary Material Gray SM, McKinnon JS, 2007. Linking color polymorphism maintenance and Supplementary material can be found at https://academic.oup.com/cz. speciation. Trends Ecol Evol 22:71–79. Grether GF, 2000. Carotenoid limitation and mate preference evolution: a test of the indicator hypothesis in guppies Poecilia reticulata. Evolution 54: References 1712–1724. Grifﬁths SW, Magurran AE, 1997. Schooling preferences for familiar ﬁsh vary Andersson M, 1994. Sexual Selection. Princeton (NJ): Princeton University with group size in a wild guppy population. Proc R Soc B 264:547–551. Press. Antoniazza S, Burri R, Fumagalli L, Goudet J, Roulin A, 2010. Local adapta- Harrison XA, 2014. Using observation-level randomeffects to model overdis- tion maintains clinal variation in melanin-based coloration of european persion in count data in ecology and evolution. PeerJ 2:e616. barn owls Tyto alba. Evolution 64:1944–1954. Hedrick PW, 2006. Genetic polymorphism in heterogeneous environments: Berdan EL, Kozak GM, Ming R, Rayburn AL, Kiehart R et al., 2018. Insight the age of genomics. Annu Rev Ecol Evol Syst 37:67–93. into genomic changes accompanying divergence: Genetic linkage maps and Henschen AE, Whittingham LA, Dunn PO, 2016. Oxidative stress is related to synteny of Lucania goodei and Lucania parva reveal a robertsonian fusion. both melanin- and carotenoid-based ornaments in the common yellow- G3-Genes Genomes Genet 8. doi: 10.1534/g3.114.012096. throat. Funct Ecol 30:749–758. Bolger T, Connolly PL, 1989. The selection of suitable indexes for the meas- Hill GE, McGraw KJ, 2006. Bird coloration: mechanisms and measurements. urement and analysis of ﬁsh condition. J Fish Biol 34:171–182. Cambridge (MA): Harvard University Press. (Mechanics of uncommon col- Bond AB, 2007. The evolution of color polymorphism: crypticity, searching ors: Pterins, porphyrins, and psittacofulvins.) images, and apostatic selection. Annu Rev Ecol Evol Syst 38:489–514. Hoekstra HE, Hirschmann RJ, Bundey RA, Insel PA, Crossland JP, 2006. A Bradbury JW, Andersson MB, Heisler L, 1987. Sexual Selection: Testing the single amino acid mutation contributes to adaptive beach mouse color pat- Alternatives. Berlin: Wiley. tern. Science 313:101–104. Burg TM, Wilcox JL, Martin A, 2002. Isolation and characterization of poly- van’t Hof AE, Edmonds N, Dalı´kova ´ M, Marec F, Saccheri IJ, 2011. Industrial morphic microsatellite loci in pupﬁsh (genus Cyprinodon). Conserv Genet melanism in British peppered moths has a singular and recent mutational 3:197–204. origin. Science 332:958–960. Downloaded from https://academic.oup.com/cz/article-abstract/64/6/733/4921187 by Ed 'DeepDyve' Gillespie user on 11 January 2019 Johnson et al. The maintenance of color polymorphism in killifish 743 Hori M, 1993. Frequency-dependent natural selection in the handedness of and brown feathers in swallows, bluebirds, penguins, and domestic chick- scale-eating cichlid ﬁsh. Science 260:216–219. ens. Condor 6:390–395. Horth L, Travis J, 2002. Frequency-dependent numerical dynamics in mosqui- Megı´a-Palma R, Martı´nez J, Merino S, 2017. Manipulation of parasite load toﬁsh. Proc R Soc B 269:2239–2247. induces signiﬁcant changes in the structural-based throat color of male Horton BM, Hauber ME, Maney DL, 2012. Morph matters: aggression bias Iberian green lizards. Curr Zool. doi: 10.1093/cz/zox036. in a polymorphic sparrow. PLoS ONE 7:e48705. Mitchell-Olds T, Willis JH, Goldstein DB, 2007. Which evolutionary proc- Hughes KA, Du L, Rodd FH, Reznick DN, 1999. Familiarity leads to female esses inﬂuence natural genetic variation for phenotypic traits? Nat Rev mate preference for novel males in the guppy Poecilia reticulata. Anim Genet 8:845–856. Behav 58:907–916. Moran RL, Fuller RC, 2018. Male-driven reproductive and agonistic character Hughes KA, Houde AE, Price AC, Rodd FH, 2013. Mating advantage for rare displacement in darters and its implications for speciation in allopatry. Curr males in wild guppy populations. Nature 503:108–110. Zool 64:101–113. Ingram T, 2011. Speciation along a depth gradient in a marine adaptive radi- Olendorf R, Rodd FH, Punzalan D, Houde AE, Hurt C et al. 2006. ation. Proc R Soc B 278:613–618. Frequency-dependent survival in natural guppy populations. Nature 441: Johnson AM, Chappell G, Price AC, Rodd FH, Olendorf R et al. 2010. 633–636. Inbreeding depression and inbreeding avoidance in a natural population of Olson VA, Owens IPF, 1998. Costly sexual signals: are carotenoids rare, risky guppies Poecilia reticulate. Ethology 116:448–457. or required? Trends Ecol Evol 13:510–514. Johnson AM, Fuller RC, 2015. The meaning of melanin, carotenoid, and Pauers MJ, Kapfer JM, Fendos CE, Berg CS, 2008. Aggressive biases towards pterin pigments in the blueﬁn killiﬁsh Lucania goodei. Behav Ecol 26: similarly coloured males in lake malawi cichlid ﬁshes. Biol Lett 4:156–159. 158–167. Piault R, van den Brink B, Roulin A, 2012. Condition-dependent expression of Johnson AM, Stanis S, Fuller RC, 2013. Diurnal lighting patterns and habitat melanin-based coloration in the eurasian kestrel. Naturwissenschaften 99: alter opsin expression and colour preferences in a killiﬁsh. Proc R Soc B 391–396. 280:20130796. Pitcher TE, Rodd FH, Rowe L, 2008. Female choice and the relatedness of Kalinowski ST, Taper ML, Marshall TC, 2007. Revising how the computer mates in the guppy Poecilia reticulata. Genetica 134:137–146. program cervus accommodates genotyping error increases success in pater- Rosenblum EB, Hoekstra HE, Nachman MW, 2004. Adaptive reptile nity assignment. Mol Ecol 16:1099–1106. color variation and the evolution of the mc1r gene. Evolution 58: Keller L, 1993. Queen Number and Sociality in Insects. Oxford: Oxford 1794–1808. University Press. Roulin A, 2016. Condition-dependence, pleiotropy and the handicap prin- Keka¨la ¨ inen J, Valkama H, Huuskonen H, Taskinen J, 2010. Research paper: ciple of sexual selection in melanin-based colouration. Biol Rev 91: multiple sexual ornamentation signals male quality and predicts female 328–348. preference in minnows. Ethology 116:895–903. Roulin A, Bize P, 2007. Sexual selection in genetic colour-polymorphic spe- Kikuchi DW, Seymoure BM, Pfennig DW, 2014. Mimicry’s palette: wide- cies: a review of experimental studies and perspectives. J Ethol 25:99–105. spread use of conserved pigments in the aposematic signals of snakes. Evol Roulin A, Ducrest A-L, 2013. Genetics of colouration in birds. Semin Cell Dev Dev 16:61–67. Biol 24:594–608. Kozak GM, Brennan RS, Berdan EL, Fuller RC, Whitehead A, 2014. Ruxton GD, Sherratt TN, Speed MP, 2004. Avoiding Attack: The Functional and population genomic divergence within and between two spe- Evolutionary Ecology of Crypsis, Warning Signals and Mimicry. Oxford: cies of killiﬁsh adapted to different osmotic niches. Evolution 68:63–80. Oxford University Press. Korzan WJ, Fernald RD, 2006. Territorial male color predicts agonistic behav- Sandkam BA, Fuller RC, 2011. The effects of water depth and light on oviposi- ior of conspeciﬁcs in a color polymorphic species. Behav Ecol 18:318–323. tion and egg cannibalism in the blueﬁn killiﬁsh Lucania goodei. J Fish Biol Lee DS, Platania SP, Burgess GH, 1980. Atlas of north american freshwater 78:967–972. ﬁshes. Raleigh (NC): North Carolina State Museum of Natural History. Saino N, Romano M, Rubolini D, Teplitsky C, Ambrosini R, Caprioli M, Lehtonen TK, 2014. Colour biases in territorial aggression in a neotropical Canova L, Wakamatsu K, 2013. Sexual dimorphism in melanin pigmenta- cichlid ﬁsh. Oecologia 175:85–93. tion, feather coloration and its heritability in the barn swallow Hirundo rus- Lewontin RC, 1974. The Genetic Basis of Evolutionary Change. New York tica. PLoS ONE 8:e58024. and London: Columbia University Press. Seehausen O, Schluter D, 2004. Male-male competition and nuptial-colour Ligon RA, McCartney KL, 2016. Biochemical regulation of pigment motility displacement as a diversifying force in Lake Victoria cichlid ﬁshes. Proc R in vertebrate chromatophores: a review of physiological color change mech- Soc B 271:1345–1353. anisms. Curr Zool 62:237–252. Seehausen O, Terai Y, Magalhaes IS, Carleton KL, Mrosso HDJ et al., 2008. Lythgoe JN, 1988. Light and vision in the aquatic environment. In: Atema J, Speciation through sensory drive in cichlid ﬁsh. Nature 455:620–626. Fay RR, Popper AN, Tavolga WN, editors. Sensory Biology of Aquatic Sluijs I, Dijkstra PD, Lindeyer CM, Visser B, Smith AM et al. 2013. A test of Animals. Berlin/Heidelberg/New York: Springer, 57–82. genetic association among male nuptial coloration, female mating prefer- Lythgoe JN, Partridge JC, 1989. Visual pigments and the acquisition of visual ence, and male aggression bias within a polymorphic population of cichlid information. J Exp Biol 146:1–20. ﬁsh. Curr Zool 59:221–229. Mariette M, Kelley JL, Brooks R, Evans JP, 2006. The effects of inbreeding on Steffen JE, Learn KM, Drumheller JS, Boback SM, McGraw KJ, 2015. male courtship behaviour and coloration in guppies. Ethology 112:807–814. Carotenoid composition of colorful body stripes and patches in the painted Martin MD, Mendelson TC, 2016. Male behaviour predicts trait divergence turtle Chrysemys picta and red-eared slider Trachemys scripta. Chelonian and the evolution of reproductive isolation in darters (Percidae: Conserv Biol 14:56–63. Etheostoma). Anim Behav 112:179–186. Takahashi Y, Yoshimura J, Morita S, Watanabe M, 2010. Negative McGhee KE, Fuller RC, Travis J, 2007. Male competition and female choice frequency-dependent selection in female color polymorphism of a damselﬂy. interact to determine mating success in the blueﬁn killiﬁsh. Behav Ecol 18: Evolution 64:3620–3628. 822–830. Tinghitella RM, Lehto WR, Lierheimer VF, 2018. Color and behavior differ- McGraw KJ, 2005. The antioxidant function of many animal pigments: are ently predict competitive outcomes for divergent stickleback color morphs. there consistent health beneﬁts of sexually selected colourants? Anim Behav Curr Zool 64:115–123. 69:757–764. Turner AM, Trexler JC, Jordan CF, Slack SJ, Geddes P et al. 1999. Targeting McGraw KJ, Wakamatsu K, Ito S, Nolan PM, Jouventin P et al. 2004. You ecosystem features for conservation: standing crops in the ﬂorida everglades. can’t judge a pigment by its color: carotenoid and melanin content of yellow Conserv Biol 13:898–911.
Current Zoology – Oxford University Press
Published: Dec 1, 2018
It’s your single place to instantly
discover and read the research
that matters to you.
Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.
All for just $49/month
Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly
Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.
Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.
Read from thousands of the leading scholarly journals from SpringerNature, Wiley-Blackwell, Oxford University Press and more.
All the latest content is available, no embargo periods.
“Hi guys, I cannot tell you how much I love this resource. Incredible. I really believe you've hit the nail on the head with this site in regards to solving the research-purchase issue.”Daniel C.
“Whoa! It’s like Spotify but for academic articles.”@Phil_Robichaud
“I must say, @deepdyve is a fabulous solution to the independent researcher's problem of #access to #information.”@deepthiw
“My last article couldn't be possible without the platform @deepdyve that makes journal papers cheaper.”@JoseServera