Color and behavior differently predict competitive outcomes for divergent stickleback color morphs

Color and behavior differently predict competitive outcomes for divergent stickleback color morphs Our knowledge of how male competition contributes to speciation is dominated by investigations of competition between within-species morphs or closely related species that differ in conspicuous traits expressed during the breeding season (e.g. color, song). In such studies, it is important to consider the manner in which putatively sexually selected traits influence the outcome of competitive interactions within and between types because these traits can communicate information about competitor quality and may not be utilized by homotypic and heterotypic receivers in the same way. We studied the roles of breeding color and aggressive behaviors in competition within and between two divergent threes- pine stickleback Gasterosteus aculeatus color types. Our previous work in this system showed that the switch from red to black breeding coloration is associated with changes in male competition biases. Here, we find that red and black males also use different currencies in competition. Winners of both color types performed more aggressive behaviors than losers, regardless of whether the competitor was of the same or opposite color type. But breeding color differently predicted competitive outcomes for red and black males. Males who were redder at the start of competition were more likely to win when paired with homotypic competitors and less likely to win when paired with heterotypic competi- tors. In contrast, black color, though expressed in the breeding season and condition dependent, was unrelated to competitive outcomes. Placing questions about the role of male competition in speciation in a sexual signal evolution framework may provide insight into the “why and how” of aggression biases and asymmetries in competitive ability between closely related morphs and species. Key words: male competition, speciation, sexual signal, color, threespine stickleback, Gasterosteus aculeatus Darwin (1859, 1871) reasoned that intrasexual selection would lead to role of female choice, rather than male competition, in driving diversifi- differential fitness outcomes when males varied in traits that enhance cation of sexually selected traits (McCullough et al. 2016)and generat- success in competition for mates or mating resources (Darwin 1871; ing reproductive isolation (Panhuis et al. 2001; Seehausen and Schluter Simmons 2001). Indeed, male competition is an important determinant 2004; Qvarnstro ¨m et al. 2012; Tinghitella et al. in press). This is likely of reproductive success. This is particularly true in systems in which because early sexual selection work focused on mate choice (the more males compete for access to pools of mating resources such as harems controversial of Darwin’s mechanisms of sexual selection; Anderson of females or territories (e.g., LeBouf 1974; Alatalo et al. 1986), and 1994), male ornaments are commonly correlated with female preferen- when male competition and female choice jointly determine mating ces and female sensory perception, implicating mate choice in ornament outcomes (as when the two act simultaneously or sequentially, and evolution (e.g. Boughman 2001; Mendelson and Shaw 2002; when mate choice is based on male dominance; Wong and Candolin Rodriguez et al. 2006), and because there is a direct relationship 2005; Hunt et al. 2009). Despite this, there has been a strong and per- between accepting or rejecting a mate and reproductive isolation sistent bias in the sexual selection literature toward investigating the (Qvarnstro ¨m et al. 2012). V C The Author (2017). Published by Oxford University Press. 115 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 journals.permissions@oup.com Downloaded from https://academic.oup.com/cz/article-abstract/64/1/115/4665099 by Ed 'DeepDyve' Gillespie user on 16 March 2018 116 Current Zoology, 2018, Vol. 64, No. 1 Evidence is accumulating in support of a role for male competi- outcomes. The study system is one in which male competition inter- tion in speciation, however. For instance, recent work sheds light on acts with female choice to influence reproductive success and male how competitive phenotypes and competitor recognition coevolve color functions in both male competition and female choice contexts (e.g., Martin and Mendelson 2016; Scordato 2017). Male competi- (e.g., Candolin 1999). During the breeding season, most male tive strategies (anything that leads to success in competition in a par- threespine sticklebacks Gasterosteus aculeatus, including the marine ticular environment) can come to reflect variation in the ecological ancestors of the freshwater fish studied here, express a carotenoid- and social environments (predators and parasites, competitor den- based red throat that can extend from the mouth to the pelvic sities, mating resources, habitat patchiness, and transmission prop- spines. The red throat is strongly preferred by females in mate choice erties of the environment, mate availability) with the result that because it is correlated with health, condition, and parental care well-adapted males can be socially dominant and gain access to ability (e.g., Milinksi and Bakker 1990; Frischknecht 1993; mates. When divergent types come into contact, male competition Candolin 2000; Boughman 2007; Kurtz et al. 2007) and is also used researchers often find evidence of aggression biases (e.g., Dijkstra in male competition (reviewed in Bakker and Sevenster 1983; et al. 2006, 2007; Tyers and Turner 2013; Lackey and Boughman McLennan and McPhail 1989; Rowland 1994; Rowland et al. 1995; 2013; Lehtonen 2014; Tinghitella et al. 2015) and asymmetries in Bolyard and Rowland 1996; McKinnon 1996; Baube 1997; Rick competitive ability (e.g., Owen-Ashley and Butler 2004; Dijkstra and Bakker 2008; but see McKinnon and McPhail 1996). Male et al. 2005; Pryke 2009; Winkelmann et al. 2014; Martin et al. threespine sticklebacks compete with one another for breeding terri- 2017). Each could contribute to reproductive isolation, though the tories and males must have a territory and a nest built before they consequences for hybridization and speciation are not well charac- can court potential mates (Tinbergen and van Iersel 1947; van Iersel terized. For instance, aggression biases may 1) facilitate co-existence 1953), so male competition is an important determinant of repro- of multiple morphs/species in sympatry through negative frequency ductive success. Females then search and choose amongst nesting dependent selection if aggression is directed toward homotypic males, and males subsequently provide all parental care. In some males (Seehausen and Schluter 2004) or 2) lead to competitive studies, brighter red males are more aggressive (Rowland 1984; exclusion (Hardin 1960) and habitat isolation if aggression is McLennan and McPhail 1989) and more likely to win in dominance biased toward heterotypics (e.g., Peiman and Robinson 2007). tests (Bakker and Sevenster 1983; McKinnon 1996; Baube 1997). Asymmetries in dominance can also facilitate habitat and reproduc- The dominance status of a male may also reflect his parental tive isolation if one competitor is able to displace the other, compel- ability (McKinnon 1996; Candolin 2000). Males frequently ling the latter to use less ideal habitat (Winkelmann et al. 2014)or destroy one another’s nests, and conspecifics eat and steal eggs drive local extinction of the lesser competitor. Previous work regularly (Wootton 1976), so dominance likely influences success in has nicely identified these patterns and their implications for specia- parenting. tion, but less attention has been paid to why those patterns occur In several locations across the west coast of North America, from a sexual signal evolution (signalers/receivers) perspective. male sticklebacks no longer display red nuptial coloration and Here, we illuminate the “how and why” of aggression biases and instead have full-body black coloration (McPhail 1969; Moodie asymmetries. 1972; Reimchen 1989; von Hippel 1999; Boughman 2001; Catchen Many of the study systems in which the role of male competition et al. 2013; Bolnick et al. 2016). Black color is expressed during the in speciation has been addressed are systems in which alternate color breeding season and is condition-dependent (Scott 2011), suggesting morphs are maintained within species (e.g. Kissenda Island cichlids a sexual selection function. The best-supported explanation for the (Dijkstra et al. 2005; Dijkstra et al. 2009), sticklebacks (Tinghitella switch from red to black coloration is sensory drive, which posits et al. 2015; Bolnick et al. 2016), European wall-lizards (While et al. that signals and sensory systems coevolve to maximize signal detect- 2015), or in which color is a primary phenotype that differs between ability in a given habitat (Endler 1992; Boughman 2002; Maan and closely related species [e.g., lake Victoria cichlids (Seehausen and Seehausen 2011). Black populations are typically found in locations Schluter 2004); Nicaraguan cichlids (Lehtonen et al. 2015); darters with red-shifted water color, whereas red populations are found in (Martin and Mendelson 2016; Roberts and Mendelson 2017); ruby- clear water (Reimchen 1989; Boughman 2001; Scott 2001; spot damselflies (Anderson and Grether 2010a, 2010b), limnetic- Tinghitella, unpublished data). Habitats with red-shifted and full- benthic stickleback species pairs (Lackey and Boughman 2013; Keagy spectrum light within each drainage are not separated by physical et al. 2016)]. In a handful of these systems, variation in the sensory barriers and there are documented polymorphic regions where red environment is implicated in color shifts (Reimchen 1989; Boughman and black fish interact (Hagen and Moodie 1979). The difference in 2001; Scott 2001; Maan et al. 2006; Seehausen et al. 2008). coloration between red and black males affects male competitive Environment-associated differences in color can influence male behavior in both regions where it has been investigated (Tinghitella aggressive responses to heterotypic competitors (e.g., Macedonia et al. 2015; Bolnick et al. 2016). In simulated mixed color assemb- et al. 1994; Macedonia and Stamps 2010; Macedonia et al. 2013, lages in the lab, black males from Washington state (WA) rivers 2015; Tinghitella et al. 2015; Bolnick et al. 2016), as identifying biased their aggression toward males with red nuptial coloration, appropriate competitors is a first step in competitive interactions. If whereas red males show no bias in aggressive behavior (Tinghitella agonistic color signal evolution is hypothesized to contribute to repro- et al. 2015). This pattern of aggression (bias toward heterotypic ductive isolation, it is important to understand how divergent colors males) suggests that red males are the recipients of more aggression function in male competition (the extent to which and which compo- overall which could allow black males to exclude red males from nents of putative signals dictate competitive outcomes) and whether preferred breeding sites (e.g., Adams 2004; Peiman and Robinson the relationship between signals and competitive outcomes depends 2007; Vallin and Qvarnstro ¨ m 2011; Winkelmann et al. 2014; on the competitor (receiver) type. Lehtonen et al. 2015), enhancing habitat use differences. However, We tested whether two threespine stickleback Gasterosteus acu- leatus color types have diverged in the manner in which male- our understanding of how male competition might contribute to limited breeding color is associated with male competition genetic divergence (through male competition outcomes) in this Downloaded from https://academic.oup.com/cz/article-abstract/64/1/115/4665099 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Tinghitella et al.  Currencies of competition in divergent color morphs 117 system has been limited by not knowing whether black nuptial colo- tupperware container 19 cm  12 cm  2 cm). Larger mass provides ration itself is related to male competition outcomes. a competitive advantage for red sticklebacks (Rowland 1989), so we We conducted dyadic within and between color morph male com- controlled for this by pairing males that differed in mass by no more petition trials to determine whether and how red and black breeding than 10% at the start of the trial. Males were given two days from color contribute to competition outcomes. Previous work also introduction into the tank to interact and to establish a territory supports a possible relationship between measurements of male and/or build a nest. This time period was chosen because male aggressiveness (performing more aggressive behaviors) and territory sticklebacks begin to build nests within two days of introduction to establishment in threespine stickleback males with red coloration a new nesting area when alone, and must have an established terri- (Bakker and Sevenster 1983), so we also tested for a role of aggressive tory in order to build a nest (Tinbergen and van Iersel 1947; van behaviors. We predicted that red throat coloration would predict Iersel 1953). Twenty-four hours and 48 h after introduction to the competitive outcomes, with males who have a larger proportion body competition tanks, we presented competing males with a gravid coverage of red winning more territories (Bakker and Sevenster 1983; female in a clear glass jar for 10 min near the nesting site (following Baube 1997). Given that black body coloration likely evolved as a Lackey and Boughman 2013). This reinforced that sexually mature consequence of sensory drive, is conspicuous to the stickleback visual females were present and motivated males to compete and build a system (Boughman 2001), and is condition-dependent (Scott 2011), nest. We also recorded whether males had established a territory, we also predicted that melanin-based black body coloration would and inspected the sand substrate for signs of nest-building. When a predict competitive outcomes, with more melanic males winning terri- territory has been established, the losing male becomes restricted to tories. There is reason to believe this may be the case. Melanin-based a small area of the tank, and upon moving toward the territory edge coloration is correlated with increased aggression and sexual activity is attacked by the dominant fish (Rowland 1989). Forty-eight hours in a wide variety of vertebrates through pleiotropic effects of the mela- after introduction to the competition tank and immediately after nocortin system (reviewed in Ducrest et al. 2008). However, if signals experience with the gravid female, we observed the competing males and competitor recognition have co-evolved within color types, we for 20 min and recorded all aggressive behaviors (approaches, bites, predict that body color (red or black) and/or the number of aggressive and chases) using the event recorder JWatcher (Blumstein et al. competitive behaviors will be differentially associated with competi- 2006). During approaches a male swims, sometimes rapidly, tion outcomes in homotypic versus heterotypic interactions and towards the other, often at territorial boundaries, coming within that more homotypic than heterotypic trials will end with territory one body-length. These have variously been described as “charges” establishment (assuming homotypic competitors have mutually in the literature as well. Bites can range in intensity from nips to sus- “understood” signals). tained biting. Chases occur when one male swims vigorously behind the other (within one body-length), and can involve movement across the length of the tank multiple times. Materials and Methods Competition trials Color quantification We collected sexually mature sticklebacks in regions of WA where To measure nuptial color, we photographed fish immediately before red and black fish are allopatric and transferred them to the placing them into competition tanks. For 187 of the 195 competi- University of Denver by air in summers of 2014–2017. Fish with red tion trials, we also photographed males immediately after the nuptial coloration were collected from the Chehalis River 20-min focal observation period. Red breeding color is quite plastic, 0 00  0 00  0 00 (46 56 22 N, 123 18 46 W) and Campbell Slough (47 2 40 N, including during competitive interactions between males (Candolin 0 00 124 3 33 W), and fish with black nuptial coloration were collected 1999, personal observation). Photographing males before and after 0 00  0 00 from Vance Creek (46 59 48 N, 123 24 43 W), Scatter Creek competition trials allowed us to capture the starting conditions and 0 00  0 00  0 00 (46 49 20 N, 123 3 11 W), Conner Creek (47 4 10 N, changes in body color that might occur during the course of the trial. 0 00  0 00  0 00 124 9 58 W), and Black River (46 49 45 N, 123 8 1 W). We All photographs were taken with a digital camera (Canon housed fish in visually isolated 110-L tanks (77 cm  32 cm  48 cm) PowerShot G15) under standardized lighting (four evenly spaced separated by collection location and sex at densities of no more than xenon 20 W bulbs) inside a photobox that held the camera and 30 fish per tank. Tanks were housed in a temperature and photoper- blocked ambient light. In each photo the fish was on its right side, iod controlled room set to 17 C and a 15: 9-h light: dark at the unanesthetized, against a neutral background with a millimeter ruler beginning of the season. Temperature and photoperiod conditions in view for scale. The process was brief, minimizing the potential for tracked those in the field for the remainder of the season. We fed the color change and stress. fish a mixture of frozen bloodworms (Chironomus spp.) and brine Using FIJI (Schindelin et al. 2012), we quantified red and black shrimp (Artemia spp.) daily ad libitum. coloration as a proportion of total body area from each photo We conducted dyadic competition trials between two fish with (Figure 1). To obtain red color area, we selected red coloration using the same nuptial coloration and between two fish with different the Threshold Color plugin (Y ¼ 32–255, U ¼ 0–143, V ¼ 141–255; nuptial coloration. In the field, the red populations we worked with following Wong et al. 2007). To obtain black area, we selected are typically found in full-spectrum light and the black populations black coloration using the Threshold Color plugin (Y ¼ 0–20; are found in red-shifted light environments. All trials were con- U ¼ 0–255; V ¼ 0–255). We determined total body area using the ducted under full spectrum lighting because our previous work with SIOX: Simple Interactive Object Extraction and scaled all measured these fish revealed no differences in competitive or mate choice areas using the millimeter ruler in each photo. biases under red-shifted light in the lab (Tinghitella et al. 2015). Trials followed established methods (Bakker and Sevenster 1983; Rowland 1989) wherein two size-matched sexually mature males Statistical analysis were introduced to a tank (77 cm  32 cm  48 cm) and allowed to We assessed the effects of proportion red body color and aggressive compete for a single nesting space (in this case, a sand-filled behaviors of the focal red male on competitive outcomes in dyadic Downloaded from https://academic.oup.com/cz/article-abstract/64/1/115/4665099 by Ed 'DeepDyve' Gillespie user on 16 March 2018 118 Current Zoology, 2018, Vol. 64, No. 1 Figure 1. Quantification of red and black body color using ImageJ. Panels A and B show representative red males with a high (A) and low (B) proportion coverage of red coloration. Panels (C) and (D) show representative black males with a high (C) and low (D) proportion coverage of black coloration. trials between two red competitors and between one red and one which we discuss in the Appendix. The measure of focal male black male. A focal red male was chosen randomly within each red aggressive behavior was again PC1-behaviors from the PCA red trial for analyses. To examine territory establishment by red described above and the model structure was as described above. focal males, we ran two sets of models: one using “before-compet- For all models, we followed a model comparison method in ition” red area and the other using difference in red area, calculated which the full models were compared to reduced models using v from before and after competition photos, as measures of male tests (performed using the anova function in lme4 in R) to assess color. “Before-competition” red area captures color at the start of whether the fit of the model decreased significantly when the effects competition trials and the difference in red area (after competition  of interest were systematically removed (Winter 2013). For interac- before competition) captures color change. We also ran a model tion effects, we plotted logistic curves using geom_smooth (method- using “after” red area as a predictor variable, which we provide in ¼ “glm”, family ¼ “binomial”) in the ggplot 2 package (version 2.2.1) in R (Wickham 2017) and for fixed effects, we plotted logistic the Appendix. To examine the effect of aggressive behaviors on curves using sjp.glmer in the sjPlot package (version 2.3.3) in R competitive outcomes, we combined approaches, bites, and chases (Lu ¨ decke and Schwemmer 2017). using PCA as a variable reduction technique. All factors loaded evenly onto PC1, which explained 70.2% of the variation. We tested the effects of red color and behavior on competition outcomes Results using logistic regression via a generalized linear mixed model (family ¼ binomial) in the lme4 package (version 1.212) in R (ver- When competing with homotypic competitors (other red males), sion 3.3.1; RStudio version 1.0.143; Bates et al. 2017). The “before- males with greater “before” red area were more likely to establish a competition” model included before red color area, aggressive territory, but when competing with heterotypics (black males) red behaviors (PC1-behaviors), trial type (homotypic or heterotypic), males with less “before” red body area were more successful in and the interactions between color and trial type and between establishing a territory (A in Table 1 and Figure 2A). Red males aggressive behaviors (PC1-behaviors) and trial type as independent were also more likely to establish a territory, regardless of trial type, variables. Trial outcome [whether or not the focal (red) male estab- when they performed more aggressive behaviors. We found no inter- lished a territory] was the dependent variable. The model also action between behaviors and trial type and no effect of trial type included the difference in mass between the two males in a trial as a alone or “before” red area alone on whether red focal males estab- covariate and the source populations of the red and black fish as lished a territory (A in Table 1). random effects. The difference in red area model had the same struc- The difference in red area (after competition  before competi- ture. “Before-competition” color measurements were square-root- tion) was also associated with territory establishment (B in Table 1 transformed prior to analysis to meet assumptions of normality. and Figure 3A). Males who established a territory gained more red We assessed the effects of proportion black body color and body area during competition, regardless of trial type. As above, red aggressive behaviors of the focal black male on competitive out- males that successfully established a territory performed more comes in dyadic trials between two black competitors and between aggressive behaviors (Figure 3B). We found no interaction between one red and one black male. Again, a focal black male was chosen at difference in red and trial type, no interaction between behaviors random within each black  black trial for analyses. We performed and trial type, and no effect of trial type alone on whether red focal a parallel analysis for focal black males using “before-competition” males established a territory (B in Table 1). Because the relationships black area and difference in black area (after competition  before between “before” red color and territory establishment and differ- competition) as our measures of male color in separate models. ence in red color and territory establishment were different (see Again, we also ran a model using only “after” black color area A versus B in Table 1), we also investigated the relationship between Downloaded from https://academic.oup.com/cz/article-abstract/64/1/115/4665099 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Tinghitella et al.  Currencies of competition in divergent color morphs 119 Table 1. Color measures and aggressive behaviors associated with territory establishment by focal red or black males in heterotypic and homotypic competition trials v df P Red focal males A Before red area * trial type 4.58 1 0.03 Aggressive behaviors * trial type 1.41 1 0.24 Before red area 1.28 1 0.26 Aggressive behaviors 13.7 1 <0.001 Trial type 0.12 1 0.73 B Difference in red * trial type 0.57 1 0.45 Aggressive behaviors * trial type 0.76 1 0.38 Difference in red 3.97 1 0.046 Aggressive behaviors 15.01 1 <0.001 Trial type 0.11 1 0.74 Black focal males C Before black area * trial type 0.29 1 0.59 Aggressive behaviors * trial type 0.33 1 0.57 Before black area 1.64 1 0.20 Aggressive behaviors 49.95 1 <0.0001 Trial type 0.01 1 0.94 D Difference in black * trial type 1.28 1 0.26 Aggressive behaviors * trial type 1.37 1 0.24 Difference in black 0.85 1 0.36 Aggressive behaviors 51.29 1 <0.0001 Trial type 0.12 1 0.73 types (v ¼ 3.89, P ¼ 0.14). There was a trend toward a greater pro- portion of red  red trials than black  black trials ending with ter- Figure 2. Dependence of territory establishment on the interaction between ritory establishment, but this was not significant (v ¼ 2.91, “before-competition” color area and competitor type. Panel A shows the out- comes of competition for red focal males and B shows the outcomes for black P ¼ 0.09). Males performed 135% more aggressive behaviors (PC1- focal males. In both panels, closed circles and solid lines indicate outcomes behaviors) in trials in which a territory was established (LS in homotypic competition [other red males (A) or other black males (B)] and means6 SE: territory ¼ 0.196 0.20, no territory¼0.546 0.30; open circles and dashed lines indicate outcomes in heterotypic competition v ¼ 5.13, P ¼ 0.02), but the number of aggressive behaviors per- [black males (A) and red males (B)]. formed during competition did not differ across trial types (LS means6 SE; red  red ¼ -0.336 0.48, red  black ¼ 0.256 0.23, “before” red color and difference in red color. Males that entered a black  black¼0.096 0.26; v ¼ 2.18, P ¼ 0.34). Neither color competition trial with less red body area (before) gained more red type was more likely to win in heterotypic competition (red coloration than males who entered a competition trial with more wins ¼ 42, black wins ¼ 35, v ¼ 0.46, P ¼ 0.50), and both red and red body area (v ¼65.7, P< 0.0001; mixed effects linear model black focal males performed similar numbers of aggressive behav- with red focal male population ¼ random). iors (LS means6 S.E.; red males¼0.336 0.49, black mal- In contrast to red body area, and contrary to our hypotheses, we es ¼ 0.106 0.21; v ¼ 0.861, P ¼ 0.35). found no effect of “before” black body area or the difference in black body area (after competition  before competition) on terri- tory establishment by black males, regardless of competitor type Discussion (C and D in Table 1; Figure 3C). The only measured variable signifi- In this study, we first asked whether male nuptial coloration and cantly related to the likelihood of a black male establishing a aggressive behavior function differently in the competitive interac- territory was aggressive behaviors; black males performed more tions of two diverging stickleback color types, one with red nuptial aggressive behaviors when successfully establishing a territory coloration (the ancestral type; McLennan 1996) and one with black (Figure 3D). The interactions between “before” black body area and nuptial coloration (the derived type). Overall, we found that differ- trial type and difference in black area and trial type on territory ent combinations of traits influence the outcome of dyadic competi- establishment were non-significant. Trial type alone did not affect tive interactions involving red and black males. Males of both color territory establishment in models using either black measurement types who performed more aggressive behaviors were more likely to (C and D in Table 1). establish a territory, regardless of whether the competitor was Of 195 male competition trials, 148 (76% overall) ended with homotypic or heterotypic (Figure 3B, D; Table 1). But, whereas a clear winner who had established a territory. A total of 20 of 23 competition outcomes for red males varied with “before-compet- red  red trials, 51 of 74 black  black trials, and 77 of 98 red black trials ended with territory establishment. The proportion of ition” red body area and the degree to which red body area changed trials ending with a territory did not differ among the three trial during competition (difference in red area), black body area was Downloaded from https://academic.oup.com/cz/article-abstract/64/1/115/4665099 by Ed 'DeepDyve' Gillespie user on 16 March 2018 120 Current Zoology, 2018, Vol. 64, No. 1 Figure 3. Difference in red area and aggressive behavior during competition correlate with territory establishment by red males, but only aggressive behaviors vary with territory establishment by black males. Pictured are the effects of A) difference in red area and B) PC1-behaviors of red focal males on probability of establishing a territory and the effects of C) difference in black area and D) PC1-behaviors of black focal males on the probability of establishing a territory (from the logistic regression). Increasing values of PC1-aggressive behaviors mean more behaviors were performed. Shading indicates the 95% confidence intervals, and circles represent individual focal male scores. unrelated to competitive success (Table 1; Figure 3A, C). This is con- versus heterotypic, and whether competition between homotypic trary to our original hypothesis that condition-dependent (Scott males was more likely to be resolved than competition between heter- 2011) melanism expressed during the breeding season is sexually otypic males. First, we found no evidence that homotypic trials were selected and that more melanic males would win territories, but sup- resolved more often that heterotypic trials, though there was a trend ports the idea that competitive strategies (anything that leads to suc- toward more red–red than black–black trials ending with a territory cess in competition in a particular environment) of red and black holder. In Pacific field crickets that have lost the ability to produce an males have diverged. Divergent competitive strategies may lead to aggressive song that mitigates the costs of fighting, fights involving aggression biases and asymmetries upon secondary contact. one or more silent males are more physically aggressive than those What, then, is the function of black coloration, if any? Several involving two calling males (Logue et al. 2010). Fights between two authors have suggested that black coloration was favored through threespine stickleback males who do not have the ancestral red threat sensory drive and increases male conspicuousness in red-shifted light signal did not involve more physically aggressive behaviors. However, habitats (Boughman 2001; Scott 2001; Boughman 2002). Black col- red males with greater “before-competition” red color were more oration may function in female choice, as suggested in some work likely to establish a territory in homotypic red x red trials (consistent (Scott 2004; but see Tinghitella et al. 2015 and below). Other evi- with previous work in threespine sticklebacks; Bakker and Sevenster dence suggests that black is maximally expressed during care for 1983; Baube 1997), and the same trait (extensive red color) led to eggs and fry (Scott 2004) and thus may signal parental care ability reduced success in competition with heterotypic (black) males (A in to female conspecifics (Scott 2011) and/or act as a threat to egg Table 1; Figure 2A). The relationship between black color (either predators or conspecific egg thieves (McPhail 1969; Hagen et al. measure) and competitive outcomes did not differ depending on 1980; but see Scott and Foster 2000). Alternatively, black coloration whether a male faced a homotypic or heterotypic competitor (C and Din Table 1; Figure 2B). We interpret this to mean that the ancestral may not be a signal at all, but instead have been “pulled along” by selection on another trait (Ducrest et al. 2008). Experiments in color, which does function in competition among red males, does not which black body coloration is manipulated directly to assess effects influence competitive outcomes with the derived color type in the on sexual selection or success in parental care would reveal whether same way—competitive strategies have diverged and the relationship black coloration is a signal and, if so, of what. between color and competitive outcomes differs depending on the We also asked whether color or aggressive behavior varied with reciever. This supports the hypothesis that signals and competitor rec- competitive outcomes differently when the competitor is homotypic ognition have co-evolved within color types. Downloaded from https://academic.oup.com/cz/article-abstract/64/1/115/4665099 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Tinghitella et al.  Currencies of competition in divergent color morphs 121 In a previous study, we found that in mixed sex assemblages in Acknowledgments the lab, black males biased their aggressive behaviors toward red We thank Jolysa Gallegos, Kyle Robrock, and Brian Ketterman for assistance males, while red males had no aggression bias (Tinghitella et al. with male competition trials and color quantification. Clara Jenck and Lauren 2015). The aggression bias we observed previously and difference Tyree helped maintain fish in the lab. in competition currencies (which traits are correlated with success in competition) uncovered here are likely related. For instance, less red (dull) males may be more successful in competition with black Funding males because black males bias aggression toward red competitors. This work was supported by the University of Denver, including through Said another way, if black males bias aggression toward red Partners in Scholarship Summer Research grants to Jolysa Gallegos, Brian males with greater red body area, it would be reasonable to expect Ketterman, and Kyle Robrock. Three anonymous reviewers and Maria males with greater red body area to lose in one on one competitive Servedio provided feedback that greatly improved the article. interactions with black males, relative to dull red males (as we find here). In the mixed sex assemblage experiment described above, we Author contributions found no evidence that the preferences of females from red and RMT conceived of and designed the experiment. RMT and VFL conducted black populations had diverged; all females, regardless of color type male competition trials. VFL quantified color from photos with undergradu- preferentially directed courtship interest behaviors toward red males ate assistants, using methods developed by WRL. WRL analyzed the data and (Tinghitella et al. 2015; but see Scott 2004). Ongoing no-choice made the figures. All co-authors contributed to writing and editing the courtship trials in the lab support this pattern (R.T., unpublished manuscript. data). It is relatively straightforward to imagine how male competi- tion and female choice might act in concert to drive speciation, for References instance, if both favor the same alternative male trait combinations or females choose mates on the basis of competition outcomes. If Adams DC, 2004. Character displacement via aggressive interference in females retain a preference for ancestral type males, however, there Appalachian salamanders. Ecology 85:2664–2670. Alatalo RV, Lundberg A, Glynn C, 1986. Female pied flycatchers choose terri- must be some other mechanism contributing to reproductive tory quality and not male characteristics. Nature 323:152–153. isolation. Anderson CN, Grether GF, 2010a. Character displacement in the fighting col- To illuminate whether male competition contributes to repro- ours of Hetaerina damselflies. Proc R Soc Lond B doi: 10.1098/rspb.2010. ductive isolation in this system, we suggest several lines of inquiry. It will be critical to know, for instance, whether the patterns we have Anderson CN, Grether GF, 2010b. Interspecific aggression and character dis- uncovered in the lab also exist in the field. Are red males the recipi- placement of competitor recognition in Hetaerina damselflies. Proc R Soc ents of more aggression in the field, and does this pattern lead red Lond B 277:549–555. males to nest in less preferred habitat, reinforcing isolation? Bakker TCM, Sevenster P, 1983. 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Aggressive behaviors * trial type 1.03 1 0.31 Tinghitella RM, Lackey ACR, Martin M, Dijkstra PD, Drury JP et al. After red area 1.26 1 0.26 Forthcoming. On the role of male competition in speciation: a review and Aggressive behaviors 14.20 1 <0.001 research agenda. Behav Ecol. Trial type 2.38 1 0.12 Tyers AM, Turner GF, 2013. Signal and preference divergence among popula- Black focal males tions of the non-endemic basal lake Malawi cichlid fish Astatotilapia callip- After black area* trial type 0.35 1 0.55 tera (Perciformes: Cichlidae). Biol J Linn Soc. 110:180–188. Aggressive behaviors * trial type 1.25 1 0.26 Vallin N, Qvarnstro ¨ m A, 2011. Learning the hard way: Imprinting can After black area 0.56 1 0.45 enhance enforced shifts in habitat choice. I J Ecol 2011:287532. Aggressive behavior 55.06 1 <0.0001 van Iersel JJA, 1953. An analysis of the parental behavior of the male Trial type 0.41 1 0.52 three-spined stickleback Gasterosteus aculeatus L. Behaviour Suppl 3: 1–159. 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Color and behavior differently predict competitive outcomes for divergent stickleback color morphs

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Abstract

Our knowledge of how male competition contributes to speciation is dominated by investigations of competition between within-species morphs or closely related species that differ in conspicuous traits expressed during the breeding season (e.g. color, song). In such studies, it is important to consider the manner in which putatively sexually selected traits influence the outcome of competitive interactions within and between types because these traits can communicate information about competitor quality and may not be utilized by homotypic and heterotypic receivers in the same way. We studied the roles of breeding color and aggressive behaviors in competition within and between two divergent threes- pine stickleback Gasterosteus aculeatus color types. Our previous work in this system showed that the switch from red to black breeding coloration is associated with changes in male competition biases. Here, we find that red and black males also use different currencies in competition. Winners of both color types performed more aggressive behaviors than losers, regardless of whether the competitor was of the same or opposite color type. But breeding color differently predicted competitive outcomes for red and black males. Males who were redder at the start of competition were more likely to win when paired with homotypic competitors and less likely to win when paired with heterotypic competi- tors. In contrast, black color, though expressed in the breeding season and condition dependent, was unrelated to competitive outcomes. Placing questions about the role of male competition in speciation in a sexual signal evolution framework may provide insight into the “why and how” of aggression biases and asymmetries in competitive ability between closely related morphs and species. Key words: male competition, speciation, sexual signal, color, threespine stickleback, Gasterosteus aculeatus Darwin (1859, 1871) reasoned that intrasexual selection would lead to role of female choice, rather than male competition, in driving diversifi- differential fitness outcomes when males varied in traits that enhance cation of sexually selected traits (McCullough et al. 2016)and generat- success in competition for mates or mating resources (Darwin 1871; ing reproductive isolation (Panhuis et al. 2001; Seehausen and Schluter Simmons 2001). Indeed, male competition is an important determinant 2004; Qvarnstro ¨m et al. 2012; Tinghitella et al. in press). This is likely of reproductive success. This is particularly true in systems in which because early sexual selection work focused on mate choice (the more males compete for access to pools of mating resources such as harems controversial of Darwin’s mechanisms of sexual selection; Anderson of females or territories (e.g., LeBouf 1974; Alatalo et al. 1986), and 1994), male ornaments are commonly correlated with female preferen- when male competition and female choice jointly determine mating ces and female sensory perception, implicating mate choice in ornament outcomes (as when the two act simultaneously or sequentially, and evolution (e.g. Boughman 2001; Mendelson and Shaw 2002; when mate choice is based on male dominance; Wong and Candolin Rodriguez et al. 2006), and because there is a direct relationship 2005; Hunt et al. 2009). Despite this, there has been a strong and per- between accepting or rejecting a mate and reproductive isolation sistent bias in the sexual selection literature toward investigating the (Qvarnstro ¨m et al. 2012). V C The Author (2017). Published by Oxford University Press. 115 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 journals.permissions@oup.com Downloaded from https://academic.oup.com/cz/article-abstract/64/1/115/4665099 by Ed 'DeepDyve' Gillespie user on 16 March 2018 116 Current Zoology, 2018, Vol. 64, No. 1 Evidence is accumulating in support of a role for male competi- outcomes. The study system is one in which male competition inter- tion in speciation, however. For instance, recent work sheds light on acts with female choice to influence reproductive success and male how competitive phenotypes and competitor recognition coevolve color functions in both male competition and female choice contexts (e.g., Martin and Mendelson 2016; Scordato 2017). Male competi- (e.g., Candolin 1999). During the breeding season, most male tive strategies (anything that leads to success in competition in a par- threespine sticklebacks Gasterosteus aculeatus, including the marine ticular environment) can come to reflect variation in the ecological ancestors of the freshwater fish studied here, express a carotenoid- and social environments (predators and parasites, competitor den- based red throat that can extend from the mouth to the pelvic sities, mating resources, habitat patchiness, and transmission prop- spines. The red throat is strongly preferred by females in mate choice erties of the environment, mate availability) with the result that because it is correlated with health, condition, and parental care well-adapted males can be socially dominant and gain access to ability (e.g., Milinksi and Bakker 1990; Frischknecht 1993; mates. When divergent types come into contact, male competition Candolin 2000; Boughman 2007; Kurtz et al. 2007) and is also used researchers often find evidence of aggression biases (e.g., Dijkstra in male competition (reviewed in Bakker and Sevenster 1983; et al. 2006, 2007; Tyers and Turner 2013; Lackey and Boughman McLennan and McPhail 1989; Rowland 1994; Rowland et al. 1995; 2013; Lehtonen 2014; Tinghitella et al. 2015) and asymmetries in Bolyard and Rowland 1996; McKinnon 1996; Baube 1997; Rick competitive ability (e.g., Owen-Ashley and Butler 2004; Dijkstra and Bakker 2008; but see McKinnon and McPhail 1996). Male et al. 2005; Pryke 2009; Winkelmann et al. 2014; Martin et al. threespine sticklebacks compete with one another for breeding terri- 2017). Each could contribute to reproductive isolation, though the tories and males must have a territory and a nest built before they consequences for hybridization and speciation are not well charac- can court potential mates (Tinbergen and van Iersel 1947; van Iersel terized. For instance, aggression biases may 1) facilitate co-existence 1953), so male competition is an important determinant of repro- of multiple morphs/species in sympatry through negative frequency ductive success. Females then search and choose amongst nesting dependent selection if aggression is directed toward homotypic males, and males subsequently provide all parental care. In some males (Seehausen and Schluter 2004) or 2) lead to competitive studies, brighter red males are more aggressive (Rowland 1984; exclusion (Hardin 1960) and habitat isolation if aggression is McLennan and McPhail 1989) and more likely to win in dominance biased toward heterotypics (e.g., Peiman and Robinson 2007). tests (Bakker and Sevenster 1983; McKinnon 1996; Baube 1997). Asymmetries in dominance can also facilitate habitat and reproduc- The dominance status of a male may also reflect his parental tive isolation if one competitor is able to displace the other, compel- ability (McKinnon 1996; Candolin 2000). Males frequently ling the latter to use less ideal habitat (Winkelmann et al. 2014)or destroy one another’s nests, and conspecifics eat and steal eggs drive local extinction of the lesser competitor. Previous work regularly (Wootton 1976), so dominance likely influences success in has nicely identified these patterns and their implications for specia- parenting. tion, but less attention has been paid to why those patterns occur In several locations across the west coast of North America, from a sexual signal evolution (signalers/receivers) perspective. male sticklebacks no longer display red nuptial coloration and Here, we illuminate the “how and why” of aggression biases and instead have full-body black coloration (McPhail 1969; Moodie asymmetries. 1972; Reimchen 1989; von Hippel 1999; Boughman 2001; Catchen Many of the study systems in which the role of male competition et al. 2013; Bolnick et al. 2016). Black color is expressed during the in speciation has been addressed are systems in which alternate color breeding season and is condition-dependent (Scott 2011), suggesting morphs are maintained within species (e.g. Kissenda Island cichlids a sexual selection function. The best-supported explanation for the (Dijkstra et al. 2005; Dijkstra et al. 2009), sticklebacks (Tinghitella switch from red to black coloration is sensory drive, which posits et al. 2015; Bolnick et al. 2016), European wall-lizards (While et al. that signals and sensory systems coevolve to maximize signal detect- 2015), or in which color is a primary phenotype that differs between ability in a given habitat (Endler 1992; Boughman 2002; Maan and closely related species [e.g., lake Victoria cichlids (Seehausen and Seehausen 2011). Black populations are typically found in locations Schluter 2004); Nicaraguan cichlids (Lehtonen et al. 2015); darters with red-shifted water color, whereas red populations are found in (Martin and Mendelson 2016; Roberts and Mendelson 2017); ruby- clear water (Reimchen 1989; Boughman 2001; Scott 2001; spot damselflies (Anderson and Grether 2010a, 2010b), limnetic- Tinghitella, unpublished data). Habitats with red-shifted and full- benthic stickleback species pairs (Lackey and Boughman 2013; Keagy spectrum light within each drainage are not separated by physical et al. 2016)]. In a handful of these systems, variation in the sensory barriers and there are documented polymorphic regions where red environment is implicated in color shifts (Reimchen 1989; Boughman and black fish interact (Hagen and Moodie 1979). The difference in 2001; Scott 2001; Maan et al. 2006; Seehausen et al. 2008). coloration between red and black males affects male competitive Environment-associated differences in color can influence male behavior in both regions where it has been investigated (Tinghitella aggressive responses to heterotypic competitors (e.g., Macedonia et al. 2015; Bolnick et al. 2016). In simulated mixed color assemb- et al. 1994; Macedonia and Stamps 2010; Macedonia et al. 2013, lages in the lab, black males from Washington state (WA) rivers 2015; Tinghitella et al. 2015; Bolnick et al. 2016), as identifying biased their aggression toward males with red nuptial coloration, appropriate competitors is a first step in competitive interactions. If whereas red males show no bias in aggressive behavior (Tinghitella agonistic color signal evolution is hypothesized to contribute to repro- et al. 2015). This pattern of aggression (bias toward heterotypic ductive isolation, it is important to understand how divergent colors males) suggests that red males are the recipients of more aggression function in male competition (the extent to which and which compo- overall which could allow black males to exclude red males from nents of putative signals dictate competitive outcomes) and whether preferred breeding sites (e.g., Adams 2004; Peiman and Robinson the relationship between signals and competitive outcomes depends 2007; Vallin and Qvarnstro ¨ m 2011; Winkelmann et al. 2014; on the competitor (receiver) type. Lehtonen et al. 2015), enhancing habitat use differences. However, We tested whether two threespine stickleback Gasterosteus acu- leatus color types have diverged in the manner in which male- our understanding of how male competition might contribute to limited breeding color is associated with male competition genetic divergence (through male competition outcomes) in this Downloaded from https://academic.oup.com/cz/article-abstract/64/1/115/4665099 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Tinghitella et al.  Currencies of competition in divergent color morphs 117 system has been limited by not knowing whether black nuptial colo- tupperware container 19 cm  12 cm  2 cm). Larger mass provides ration itself is related to male competition outcomes. a competitive advantage for red sticklebacks (Rowland 1989), so we We conducted dyadic within and between color morph male com- controlled for this by pairing males that differed in mass by no more petition trials to determine whether and how red and black breeding than 10% at the start of the trial. Males were given two days from color contribute to competition outcomes. Previous work also introduction into the tank to interact and to establish a territory supports a possible relationship between measurements of male and/or build a nest. This time period was chosen because male aggressiveness (performing more aggressive behaviors) and territory sticklebacks begin to build nests within two days of introduction to establishment in threespine stickleback males with red coloration a new nesting area when alone, and must have an established terri- (Bakker and Sevenster 1983), so we also tested for a role of aggressive tory in order to build a nest (Tinbergen and van Iersel 1947; van behaviors. We predicted that red throat coloration would predict Iersel 1953). Twenty-four hours and 48 h after introduction to the competitive outcomes, with males who have a larger proportion body competition tanks, we presented competing males with a gravid coverage of red winning more territories (Bakker and Sevenster 1983; female in a clear glass jar for 10 min near the nesting site (following Baube 1997). Given that black body coloration likely evolved as a Lackey and Boughman 2013). This reinforced that sexually mature consequence of sensory drive, is conspicuous to the stickleback visual females were present and motivated males to compete and build a system (Boughman 2001), and is condition-dependent (Scott 2011), nest. We also recorded whether males had established a territory, we also predicted that melanin-based black body coloration would and inspected the sand substrate for signs of nest-building. When a predict competitive outcomes, with more melanic males winning terri- territory has been established, the losing male becomes restricted to tories. There is reason to believe this may be the case. Melanin-based a small area of the tank, and upon moving toward the territory edge coloration is correlated with increased aggression and sexual activity is attacked by the dominant fish (Rowland 1989). Forty-eight hours in a wide variety of vertebrates through pleiotropic effects of the mela- after introduction to the competition tank and immediately after nocortin system (reviewed in Ducrest et al. 2008). However, if signals experience with the gravid female, we observed the competing males and competitor recognition have co-evolved within color types, we for 20 min and recorded all aggressive behaviors (approaches, bites, predict that body color (red or black) and/or the number of aggressive and chases) using the event recorder JWatcher (Blumstein et al. competitive behaviors will be differentially associated with competi- 2006). During approaches a male swims, sometimes rapidly, tion outcomes in homotypic versus heterotypic interactions and towards the other, often at territorial boundaries, coming within that more homotypic than heterotypic trials will end with territory one body-length. These have variously been described as “charges” establishment (assuming homotypic competitors have mutually in the literature as well. Bites can range in intensity from nips to sus- “understood” signals). tained biting. Chases occur when one male swims vigorously behind the other (within one body-length), and can involve movement across the length of the tank multiple times. Materials and Methods Competition trials Color quantification We collected sexually mature sticklebacks in regions of WA where To measure nuptial color, we photographed fish immediately before red and black fish are allopatric and transferred them to the placing them into competition tanks. For 187 of the 195 competi- University of Denver by air in summers of 2014–2017. Fish with red tion trials, we also photographed males immediately after the nuptial coloration were collected from the Chehalis River 20-min focal observation period. Red breeding color is quite plastic, 0 00  0 00  0 00 (46 56 22 N, 123 18 46 W) and Campbell Slough (47 2 40 N, including during competitive interactions between males (Candolin 0 00 124 3 33 W), and fish with black nuptial coloration were collected 1999, personal observation). Photographing males before and after 0 00  0 00 from Vance Creek (46 59 48 N, 123 24 43 W), Scatter Creek competition trials allowed us to capture the starting conditions and 0 00  0 00  0 00 (46 49 20 N, 123 3 11 W), Conner Creek (47 4 10 N, changes in body color that might occur during the course of the trial. 0 00  0 00  0 00 124 9 58 W), and Black River (46 49 45 N, 123 8 1 W). We All photographs were taken with a digital camera (Canon housed fish in visually isolated 110-L tanks (77 cm  32 cm  48 cm) PowerShot G15) under standardized lighting (four evenly spaced separated by collection location and sex at densities of no more than xenon 20 W bulbs) inside a photobox that held the camera and 30 fish per tank. Tanks were housed in a temperature and photoper- blocked ambient light. In each photo the fish was on its right side, iod controlled room set to 17 C and a 15: 9-h light: dark at the unanesthetized, against a neutral background with a millimeter ruler beginning of the season. Temperature and photoperiod conditions in view for scale. The process was brief, minimizing the potential for tracked those in the field for the remainder of the season. We fed the color change and stress. fish a mixture of frozen bloodworms (Chironomus spp.) and brine Using FIJI (Schindelin et al. 2012), we quantified red and black shrimp (Artemia spp.) daily ad libitum. coloration as a proportion of total body area from each photo We conducted dyadic competition trials between two fish with (Figure 1). To obtain red color area, we selected red coloration using the same nuptial coloration and between two fish with different the Threshold Color plugin (Y ¼ 32–255, U ¼ 0–143, V ¼ 141–255; nuptial coloration. In the field, the red populations we worked with following Wong et al. 2007). To obtain black area, we selected are typically found in full-spectrum light and the black populations black coloration using the Threshold Color plugin (Y ¼ 0–20; are found in red-shifted light environments. All trials were con- U ¼ 0–255; V ¼ 0–255). We determined total body area using the ducted under full spectrum lighting because our previous work with SIOX: Simple Interactive Object Extraction and scaled all measured these fish revealed no differences in competitive or mate choice areas using the millimeter ruler in each photo. biases under red-shifted light in the lab (Tinghitella et al. 2015). Trials followed established methods (Bakker and Sevenster 1983; Rowland 1989) wherein two size-matched sexually mature males Statistical analysis were introduced to a tank (77 cm  32 cm  48 cm) and allowed to We assessed the effects of proportion red body color and aggressive compete for a single nesting space (in this case, a sand-filled behaviors of the focal red male on competitive outcomes in dyadic Downloaded from https://academic.oup.com/cz/article-abstract/64/1/115/4665099 by Ed 'DeepDyve' Gillespie user on 16 March 2018 118 Current Zoology, 2018, Vol. 64, No. 1 Figure 1. Quantification of red and black body color using ImageJ. Panels A and B show representative red males with a high (A) and low (B) proportion coverage of red coloration. Panels (C) and (D) show representative black males with a high (C) and low (D) proportion coverage of black coloration. trials between two red competitors and between one red and one which we discuss in the Appendix. The measure of focal male black male. A focal red male was chosen randomly within each red aggressive behavior was again PC1-behaviors from the PCA red trial for analyses. To examine territory establishment by red described above and the model structure was as described above. focal males, we ran two sets of models: one using “before-compet- For all models, we followed a model comparison method in ition” red area and the other using difference in red area, calculated which the full models were compared to reduced models using v from before and after competition photos, as measures of male tests (performed using the anova function in lme4 in R) to assess color. “Before-competition” red area captures color at the start of whether the fit of the model decreased significantly when the effects competition trials and the difference in red area (after competition  of interest were systematically removed (Winter 2013). For interac- before competition) captures color change. We also ran a model tion effects, we plotted logistic curves using geom_smooth (method- using “after” red area as a predictor variable, which we provide in ¼ “glm”, family ¼ “binomial”) in the ggplot 2 package (version 2.2.1) in R (Wickham 2017) and for fixed effects, we plotted logistic the Appendix. To examine the effect of aggressive behaviors on curves using sjp.glmer in the sjPlot package (version 2.3.3) in R competitive outcomes, we combined approaches, bites, and chases (Lu ¨ decke and Schwemmer 2017). using PCA as a variable reduction technique. All factors loaded evenly onto PC1, which explained 70.2% of the variation. We tested the effects of red color and behavior on competition outcomes Results using logistic regression via a generalized linear mixed model (family ¼ binomial) in the lme4 package (version 1.212) in R (ver- When competing with homotypic competitors (other red males), sion 3.3.1; RStudio version 1.0.143; Bates et al. 2017). The “before- males with greater “before” red area were more likely to establish a competition” model included before red color area, aggressive territory, but when competing with heterotypics (black males) red behaviors (PC1-behaviors), trial type (homotypic or heterotypic), males with less “before” red body area were more successful in and the interactions between color and trial type and between establishing a territory (A in Table 1 and Figure 2A). Red males aggressive behaviors (PC1-behaviors) and trial type as independent were also more likely to establish a territory, regardless of trial type, variables. Trial outcome [whether or not the focal (red) male estab- when they performed more aggressive behaviors. We found no inter- lished a territory] was the dependent variable. The model also action between behaviors and trial type and no effect of trial type included the difference in mass between the two males in a trial as a alone or “before” red area alone on whether red focal males estab- covariate and the source populations of the red and black fish as lished a territory (A in Table 1). random effects. The difference in red area model had the same struc- The difference in red area (after competition  before competi- ture. “Before-competition” color measurements were square-root- tion) was also associated with territory establishment (B in Table 1 transformed prior to analysis to meet assumptions of normality. and Figure 3A). Males who established a territory gained more red We assessed the effects of proportion black body color and body area during competition, regardless of trial type. As above, red aggressive behaviors of the focal black male on competitive out- males that successfully established a territory performed more comes in dyadic trials between two black competitors and between aggressive behaviors (Figure 3B). We found no interaction between one red and one black male. Again, a focal black male was chosen at difference in red and trial type, no interaction between behaviors random within each black  black trial for analyses. We performed and trial type, and no effect of trial type alone on whether red focal a parallel analysis for focal black males using “before-competition” males established a territory (B in Table 1). Because the relationships black area and difference in black area (after competition  before between “before” red color and territory establishment and differ- competition) as our measures of male color in separate models. ence in red color and territory establishment were different (see Again, we also ran a model using only “after” black color area A versus B in Table 1), we also investigated the relationship between Downloaded from https://academic.oup.com/cz/article-abstract/64/1/115/4665099 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Tinghitella et al.  Currencies of competition in divergent color morphs 119 Table 1. Color measures and aggressive behaviors associated with territory establishment by focal red or black males in heterotypic and homotypic competition trials v df P Red focal males A Before red area * trial type 4.58 1 0.03 Aggressive behaviors * trial type 1.41 1 0.24 Before red area 1.28 1 0.26 Aggressive behaviors 13.7 1 <0.001 Trial type 0.12 1 0.73 B Difference in red * trial type 0.57 1 0.45 Aggressive behaviors * trial type 0.76 1 0.38 Difference in red 3.97 1 0.046 Aggressive behaviors 15.01 1 <0.001 Trial type 0.11 1 0.74 Black focal males C Before black area * trial type 0.29 1 0.59 Aggressive behaviors * trial type 0.33 1 0.57 Before black area 1.64 1 0.20 Aggressive behaviors 49.95 1 <0.0001 Trial type 0.01 1 0.94 D Difference in black * trial type 1.28 1 0.26 Aggressive behaviors * trial type 1.37 1 0.24 Difference in black 0.85 1 0.36 Aggressive behaviors 51.29 1 <0.0001 Trial type 0.12 1 0.73 types (v ¼ 3.89, P ¼ 0.14). There was a trend toward a greater pro- portion of red  red trials than black  black trials ending with ter- Figure 2. Dependence of territory establishment on the interaction between ritory establishment, but this was not significant (v ¼ 2.91, “before-competition” color area and competitor type. Panel A shows the out- comes of competition for red focal males and B shows the outcomes for black P ¼ 0.09). Males performed 135% more aggressive behaviors (PC1- focal males. In both panels, closed circles and solid lines indicate outcomes behaviors) in trials in which a territory was established (LS in homotypic competition [other red males (A) or other black males (B)] and means6 SE: territory ¼ 0.196 0.20, no territory¼0.546 0.30; open circles and dashed lines indicate outcomes in heterotypic competition v ¼ 5.13, P ¼ 0.02), but the number of aggressive behaviors per- [black males (A) and red males (B)]. formed during competition did not differ across trial types (LS means6 SE; red  red ¼ -0.336 0.48, red  black ¼ 0.256 0.23, “before” red color and difference in red color. Males that entered a black  black¼0.096 0.26; v ¼ 2.18, P ¼ 0.34). Neither color competition trial with less red body area (before) gained more red type was more likely to win in heterotypic competition (red coloration than males who entered a competition trial with more wins ¼ 42, black wins ¼ 35, v ¼ 0.46, P ¼ 0.50), and both red and red body area (v ¼65.7, P< 0.0001; mixed effects linear model black focal males performed similar numbers of aggressive behav- with red focal male population ¼ random). iors (LS means6 S.E.; red males¼0.336 0.49, black mal- In contrast to red body area, and contrary to our hypotheses, we es ¼ 0.106 0.21; v ¼ 0.861, P ¼ 0.35). found no effect of “before” black body area or the difference in black body area (after competition  before competition) on terri- tory establishment by black males, regardless of competitor type Discussion (C and D in Table 1; Figure 3C). The only measured variable signifi- In this study, we first asked whether male nuptial coloration and cantly related to the likelihood of a black male establishing a aggressive behavior function differently in the competitive interac- territory was aggressive behaviors; black males performed more tions of two diverging stickleback color types, one with red nuptial aggressive behaviors when successfully establishing a territory coloration (the ancestral type; McLennan 1996) and one with black (Figure 3D). The interactions between “before” black body area and nuptial coloration (the derived type). Overall, we found that differ- trial type and difference in black area and trial type on territory ent combinations of traits influence the outcome of dyadic competi- establishment were non-significant. Trial type alone did not affect tive interactions involving red and black males. Males of both color territory establishment in models using either black measurement types who performed more aggressive behaviors were more likely to (C and D in Table 1). establish a territory, regardless of whether the competitor was Of 195 male competition trials, 148 (76% overall) ended with homotypic or heterotypic (Figure 3B, D; Table 1). But, whereas a clear winner who had established a territory. A total of 20 of 23 competition outcomes for red males varied with “before-compet- red  red trials, 51 of 74 black  black trials, and 77 of 98 red black trials ended with territory establishment. The proportion of ition” red body area and the degree to which red body area changed trials ending with a territory did not differ among the three trial during competition (difference in red area), black body area was Downloaded from https://academic.oup.com/cz/article-abstract/64/1/115/4665099 by Ed 'DeepDyve' Gillespie user on 16 March 2018 120 Current Zoology, 2018, Vol. 64, No. 1 Figure 3. Difference in red area and aggressive behavior during competition correlate with territory establishment by red males, but only aggressive behaviors vary with territory establishment by black males. Pictured are the effects of A) difference in red area and B) PC1-behaviors of red focal males on probability of establishing a territory and the effects of C) difference in black area and D) PC1-behaviors of black focal males on the probability of establishing a territory (from the logistic regression). Increasing values of PC1-aggressive behaviors mean more behaviors were performed. Shading indicates the 95% confidence intervals, and circles represent individual focal male scores. unrelated to competitive success (Table 1; Figure 3A, C). This is con- versus heterotypic, and whether competition between homotypic trary to our original hypothesis that condition-dependent (Scott males was more likely to be resolved than competition between heter- 2011) melanism expressed during the breeding season is sexually otypic males. First, we found no evidence that homotypic trials were selected and that more melanic males would win territories, but sup- resolved more often that heterotypic trials, though there was a trend ports the idea that competitive strategies (anything that leads to suc- toward more red–red than black–black trials ending with a territory cess in competition in a particular environment) of red and black holder. In Pacific field crickets that have lost the ability to produce an males have diverged. Divergent competitive strategies may lead to aggressive song that mitigates the costs of fighting, fights involving aggression biases and asymmetries upon secondary contact. one or more silent males are more physically aggressive than those What, then, is the function of black coloration, if any? Several involving two calling males (Logue et al. 2010). Fights between two authors have suggested that black coloration was favored through threespine stickleback males who do not have the ancestral red threat sensory drive and increases male conspicuousness in red-shifted light signal did not involve more physically aggressive behaviors. However, habitats (Boughman 2001; Scott 2001; Boughman 2002). Black col- red males with greater “before-competition” red color were more oration may function in female choice, as suggested in some work likely to establish a territory in homotypic red x red trials (consistent (Scott 2004; but see Tinghitella et al. 2015 and below). Other evi- with previous work in threespine sticklebacks; Bakker and Sevenster dence suggests that black is maximally expressed during care for 1983; Baube 1997), and the same trait (extensive red color) led to eggs and fry (Scott 2004) and thus may signal parental care ability reduced success in competition with heterotypic (black) males (A in to female conspecifics (Scott 2011) and/or act as a threat to egg Table 1; Figure 2A). The relationship between black color (either predators or conspecific egg thieves (McPhail 1969; Hagen et al. measure) and competitive outcomes did not differ depending on 1980; but see Scott and Foster 2000). Alternatively, black coloration whether a male faced a homotypic or heterotypic competitor (C and Din Table 1; Figure 2B). We interpret this to mean that the ancestral may not be a signal at all, but instead have been “pulled along” by selection on another trait (Ducrest et al. 2008). Experiments in color, which does function in competition among red males, does not which black body coloration is manipulated directly to assess effects influence competitive outcomes with the derived color type in the on sexual selection or success in parental care would reveal whether same way—competitive strategies have diverged and the relationship black coloration is a signal and, if so, of what. between color and competitive outcomes differs depending on the We also asked whether color or aggressive behavior varied with reciever. This supports the hypothesis that signals and competitor rec- competitive outcomes differently when the competitor is homotypic ognition have co-evolved within color types. Downloaded from https://academic.oup.com/cz/article-abstract/64/1/115/4665099 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Tinghitella et al.  Currencies of competition in divergent color morphs 121 In a previous study, we found that in mixed sex assemblages in Acknowledgments the lab, black males biased their aggressive behaviors toward red We thank Jolysa Gallegos, Kyle Robrock, and Brian Ketterman for assistance males, while red males had no aggression bias (Tinghitella et al. with male competition trials and color quantification. Clara Jenck and Lauren 2015). The aggression bias we observed previously and difference Tyree helped maintain fish in the lab. in competition currencies (which traits are correlated with success in competition) uncovered here are likely related. For instance, less red (dull) males may be more successful in competition with black Funding males because black males bias aggression toward red competitors. This work was supported by the University of Denver, including through Said another way, if black males bias aggression toward red Partners in Scholarship Summer Research grants to Jolysa Gallegos, Brian males with greater red body area, it would be reasonable to expect Ketterman, and Kyle Robrock. Three anonymous reviewers and Maria males with greater red body area to lose in one on one competitive Servedio provided feedback that greatly improved the article. interactions with black males, relative to dull red males (as we find here). In the mixed sex assemblage experiment described above, we Author contributions found no evidence that the preferences of females from red and RMT conceived of and designed the experiment. RMT and VFL conducted black populations had diverged; all females, regardless of color type male competition trials. VFL quantified color from photos with undergradu- preferentially directed courtship interest behaviors toward red males ate assistants, using methods developed by WRL. WRL analyzed the data and (Tinghitella et al. 2015; but see Scott 2004). Ongoing no-choice made the figures. All co-authors contributed to writing and editing the courtship trials in the lab support this pattern (R.T., unpublished manuscript. data). It is relatively straightforward to imagine how male competi- tion and female choice might act in concert to drive speciation, for References instance, if both favor the same alternative male trait combinations or females choose mates on the basis of competition outcomes. If Adams DC, 2004. Character displacement via aggressive interference in females retain a preference for ancestral type males, however, there Appalachian salamanders. Ecology 85:2664–2670. Alatalo RV, Lundberg A, Glynn C, 1986. 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