Male and female responses to species-specific coloration in darters (Percidae: Etheostoma)

Animal Behaviour 85 (2013) 1251e1259

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Male and female responses to species-specific coloration in darters (Percidae: Etheostoma) Tory H. Williams*, Tamra C. Mendelson Department of Biological Sciences, University of Maryland, Baltimore County, Baltimore, MD, U.S.A.

a r t i c l e i n f o Article history: Received 19 December 2012 Initial acceptance 1 February 2013 Final acceptance 6 March 2013 Available online 11 April 2013 MS. number: A12-00968 Keywords: association preference behavioural isolation colour darter Etheostoma intrasexual interaction mate choice model pattern

Male secondary sexual traits often comprise multiple components and can function in different contexts. Male signals can target females for mating, males for aggression, and may also function in interspecific interactions. In darter fishes (Percidae: Etheostoma), male nuptial coloration is a multicomponent secondary sexual signal. Females are known to respond to this signal in both intra- and interspecific contexts; they prefer conspecific over heterospecific coloration and prefer a particular colour variant within species. We also have shown that two components of this complex signal (colour and pattern) are each sufficient, in isolation, to attract females. Here, we demonstrate that males also respond to variation in nuptial coloration. Males of two sympatric species showed an association preference for motorized models with conspecific over heterospecific coloration. We also sought to determine whether colour or pattern presides over the other in its influence on female and male behaviour. Using model fish representing conspecific colour (hue) with mismatched heterospecific patterning (and vice versa), we asked whether either sex preferred one element over the other. Female and male Etheostoma barrenense preferred models showing conspecific male coloration in a heterospecific pattern. In contrast, neither male nor female Etheostoma zonale showed a preference for models displaying conspecific colour or conspecific pattern. We speculate that colour may be under stronger sexual selection than pattern in E. barrenense, whereas colour and pattern may be equally important social cues for E. zonale. Ó 2013 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

Elaborate sexually dichromatic signals are typically thought to result from sexual selection, by which differential mating success leads to differential reproductive success (Darwin 1871). Different forms of sexual selection describe whether mating success is dictated by intrasexual competition or intersexual preferences (Huxley 1938a, b; Andersson 1994). Arguably, all sexual selection is competition, either direct (intrasexual) or indirect (intersexual) (Wiley & Poston 1996); however, the traditional distinction of intrasexual versus intersexual interactions is helpful in identifying the relevant target of an elaborate signal. Under intrasexual selection, signals become elaborated if males with exaggerated signalling traits are more likely to intimidate or win against competing males; in this case, the receiver of the signal is another male. In the case of intersexual selection, signals become elaborated if females prefer to mate with males that exhibit elaborate traits; in this case, the receiver of the signal is a female. Therefore, different forms of sexual selection also describe which sex represents the primary recipient of the elaborate signal, although it is certainly possible for

* Correspondence: T. H. Williams, Department of Biological Sciences, University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250, U.S.A. E-mail address: [email protected] (T. H. Williams).

sexually selected traits to target members of both sexes (Tinbergen 1953; Ord et al. 2001). Elaborate male signals can affect interactions not only within but between species. Species-specific differences in elaborate traits may signal to males whether an approaching individual represents competition for access to a reproductive female (i.e. rival conspecific male) or not (i.e. behaviourally isolated heterospecific male) (e.g. Matyjasiak 2005). Efficient signalling may save energy by discouraging unnecessary competitions between males (see Maynard Smith & Harper 2003). In haplochromine African cichlids, red male Pundamilia nyererei and blue male Pundamilia pundamilia show a disproportional bias in aggression towards males showing conspecific coloration and are more ‘tolerant’ of males showing the other (heterospecific) colour morph (Dijkstra et al. 2005, 2007; Dijkstra 2006). Similar observations have been made in male Metriaclima mbenjii, which appear to show more aggression towards males of a similar red coloration than to those of a different colour (Pauers et al. 2008). For females, species-specific differences in elaborate male signals may prevent courtship with genetically incompatible mates. Heterospecific signals of closely related species may simply be ‘ignored’ while conspecific signals draw attention; alternatively, reinforcing selection could favour active avoidance of heterospecific male signals if heterospecific mating is

0003-3472/$38.00 Ó 2013 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.anbehav.2013.03.012

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indeed costly (e.g. Dobzhansky 1937; Servedio & Kirkpatrick 1997). Thus, for both males and females, selection on elaborate signals due to interspecific interactions may contribute to developing and maintaining species boundaries between closely related sympatric species (Baker & Baker 1990; Grant & Grant 1996, 1998; Price 1998; Irwin et al. 2001). One aim of the present study was therefore to determine whether species-specific differences in an elaborate male signal that are known to affect female preferences also affect the behaviour of males. Measuring the function of elaborate signals is challenging, however, when the signal has multiple elements that may or may not convey similar information to a receiver. For example, the elaborate breeding coloration of a fish contains elements of both colour (hue) and pattern resulting from multiple colours or melanization. Either the elaborate colour itself is meaningful, or the spatial pattern it creates alongside other colours may be critical (see Marshall 2000). These hypotheses are not mutually exclusive; both aspects of the visual signal can convey information. Different elements of a complex signal might advertise multiple distinct messages to a receiver(s) or alternatively might serve as redundant information, among other possibilities (see Candolin 2003). The divergent evolution of multicomponent signals also can contribute to speciation, as independent changes within each of two populations can lead to behavioural isolation (Lande 1981). By dissecting the elements of a multicomponent signal, the relative importance of each element can be measured with respect to intra- and interspecific communication. The objective of this study was to examine the role of multiple components of elaborate male coloration in intra- and intersexual communication in darters. Darters in the genus Etheostoma comprise one of the most diverse groups of freshwater fish in North America (Page & Burr 1991). These small benthic fish are sexually dimorphic, typically expressing elaborate coloration in adult males during the breeding season, and most of the 200þ species are distinguishable by variation in species-specific nuptial colour pattern of males (Kuehne & Barbour 1983; Page 1983). The extensive diversification of elaborate male signals in the genus makes it an excellent study system to examine the targets of elaborate male signalling both within and between species. Behavioural isolation evolves more quickly than other forms of postmating isolation in numerous pairs of allopatric species (Mendelson 2003; Mendelson et al. 2007), suggesting that differences in courtship and mating behaviour serve as the primary mechanism of isolation upon secondary contact. The sympatric darter species Etheostoma barrenense and Etheostoma zonale are a case study in understanding the relationship between sexual selection and behavioural isolation (Williams & Mendelson 2010, 2011; T. H. Williams, J. M. Gumm & T. C. Mendelson, unpublished data). Males of E. barrenense are primarily orange-red with black blotches fused along the lateral line; male E. zonale are primarily green in the face and fins, with alternating green and yellow bars along the body. Females of both species are by comparison drab and cryptic, expressing the pattern but not the elaborated colour of conspecific males. The two species are approximately 6.5 million years divergent (cytochrome b, uncorrected genetic distance: T. C. Mendelson, unpublished data; molecular clock: Near & Benard 2004) and represent among the most closely related darter species to cooccur without hybridizing in nature (Hubbs 1955, 1967; Keck & Near 2009). Furthermore, these species have similar ecological preferences (T. H. Williams & T. C. Mendelson, unpublished data), spawning behaviour and roaming territoriality, and both lack parental care (Kuehne & Barbour 1983; Page 1983, 1985; Etnier & Starnes 1993).

Behavioural tests utilizing motorized model stimuli demonstrated that female E. barrenense prefer a hue of orange-red similar to the measured average of wild males over a more extreme (redshifted) hue; whereas, males show no such preference, suggesting sexual selection on male nuptial colour via female preference within species (T. H. Williams, J. M. Gumm & T. C. Mendelson, unpublished data). Females of both species also associate with models exhibiting conspecific male coloration over heterospecific coloration, suggesting that female preference for conspecific male signals contributes to behavioural isolation in nature (Williams & Mendelson 2010, 2011). Specifically, females prefer both conspecific colour (hue) and patterning elements over those of the sympatric congener. Additionally, it appears that either conspecific colour or pattern, when presented in isolation, is sufficient for eliciting a significant behavioural response from females. What remains unclear, however, is whether males discriminate between conspecific and heterospecific coloration; that is, to what extent does male nuptial colour function as a signal in intrasexual, interspecific interactions? Furthermore, it is not yet clear whether colour or pattern better explains the patterns of discrimination we observe in both sexes. The first aim of this study was to determine whether male darters distinguish conspecific from heterospecific coloration, by asking whether they show an association preference for one over the other. The second aim of the study was to tease apart colour from pattern to determine whether one trait presides over the other in its influence on female or male behaviour. To address the first aim, we used motorized model fish to test whether male darters show an association preference or an elevated activity level towards conspecific over heterospecific male coloration. To address the second aim, we also used motorized models and tested whether males and females of both species prefer one component of conspecific nuptial coloration over another (e.g. colour over pattern). METHODS Fish Collection and Care We collected adult E. barrenense and E. zonale from two confluent tributaries of the Barren River: the East Fork Barren River (Monroe County, KY, U.S.A.) and from Line Creek (Clay County, TN, U.S.A.) in MarcheApril of 2010, 2011 and 2012; the two species are syntopic in both of these locations. Permission for the collection and use of these species in behavioural experiments was granted by the Kentucky Department of Fish and Wildlife Resources and the Tennessee Wildlife Resources Agency. Live specimens were transported to the Department of Biological Sciences at the University of Maryland, Baltimore County and housed in a recirculating aquarium system reflecting natural abiotic conditions (i.e. temperature, pH, hardness and photoperiod). Behavioural testing occurred MayeJune 2010, AprileMay 2011 and AprileMay 2012, after which the darters were kept for future use in in vitro fertilization experiments. Typical breeding time for these species is April and May (Etnier & Starnes 1993). Although testing occurred in June 2010, all males were evaluated as sexually receptive on the basis of their intense body coloration and ability to produce motile sperm, examined post hoc. These males were also fully coloured and produced motile sperm during the next breeding season, after 1 year in captivity. All female subjects of both species were tested during the 2011 breeding season and were determined as sexually receptive due to gravid abdomens. Experimental procedures were approved by the Institutional Animal Care and Use Committee of the University of Maryland, Baltimore County.

T. H. Williams, T. C. Mendelson / Animal Behaviour 85 (2013) 1251e1259

Model Stimuli Darters were tested in two dichotomous choice experiments that utilized motorized urethane models as the paired stimuli. The first experiment was carried out in 2010 and tested the association preference of male E. barrenense and male E. zonale for models exhibiting conspecific versus heterospecific colour pattern. These models were previously used to test female preference for speciesspecific coloration (Williams & Mendelson 2011). That study provides detailed methods for the construction, colour analysis and motorization of the models. Twenty individual darter-shaped models were used in the present study. Ten models had orangered bodies with a black-blotched stripe along the lateral line, thus representing male E. barrenense coloration (Fig. 1a). The other 10 models had alternating green and yellow bars along the length of the body, representing male E. zonale coloration (Fig. 1a). The ‘E. barrenense’ or ‘red-stripe’ models were paired with ‘E. zonale’ or ‘green-barred’ models in such a way as to create multiple pairs of unique stimuli. The second experiment was carried out during the breeding seasons of 2011 and 2012. This experiment tested the association preference of male and female E. zonale and E. barrenense for models exhibiting conspecific colour in a heterospecific pattern versus heterospecific colour in a conspecific pattern. By ‘swapping’ colour and pattern cues in this manner, we tested whether one phenotypic component of male nuptial coloration was more attractive than the other, asking whether the fish showed an association preference in the context of conflicting visual cues. An additional 20 models were used in this experiment, with the same size and shape as the models in experiment 1, but differing in coloration. The paints used on these models were identical to those used on the models in experiment 1 (see Williams & Mendelson 2011 for paint details and spectra). Ten of the models had orange-

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red bodies with black bars, thus representing the colour of E. barrenense and the pattern of E. zonale (Fig. 1b). The remaining 10 models had green bodies with a yellow-blotched stripe along the lateral line, representing the colour of E. zonale and the pattern of E. barrenense (Fig. 1b). The ‘red-barred’ models were combined with ‘green-stripe’ models to create multiple pairs of unique stimuli. For both experiments, each focal individual was presented a single and unique pair of model stimuli that were motorized in synchrony. The use of models in lieu of live male stimuli controlled for confounding factors (stimulus size, shape, behaviour) and allowed the colour pattern difference to be the isolated, independent variable on which test fish were likely to base their behavioural response. Choice Trials Focal individuals were presented with model stimuli pairs using a standard dichotomous choice design with one motorized model displayed outside either end of a 37-litre glass ‘test’ tank (50 L  25 W  30 H cm). The models were suspended over gravel substrate in water-filled 9.5-litre tanks so that their visual surroundings matched that of the test tank. A 5 cm ‘preference zone’ was defined at both ends of the test tank to allow quantification of the time spent by males in close proximity to either motorized model stimulus (see Williams & Mendelson 2011). Models were attached to stepper motors and controlled through a computer program (GadgetMaster Script Editor v.1.2, LabVIEW Run-time Engine 8.2.1 software, and GadgetmasterÔ driver, LightMachinery Inc., Nepean, ON, Canada) so that they would pivot in synchrony. The rapid, quiver-like movements of the models approximate natural courting and spawning behaviour. Prior to each observation period, the focal fish was allowed to acclimate to the experimental environment. The acclimation standard was defined as the amount of time required for the fish to enter each of the preference zones and return to the middle of the tank; therefore, acclimation time varied with each observation period. Focal individuals were never reused within an experiment or used in both experiments within a single breeding season. Fish used in experiment 1 were caught from the wild and tested within 2 months of capture. Fish used in experiment 2 were previously used in behavioural trials 1 year preceding their use in the present study, with the exception of freshly caught male E. barrenense. Limited availability of fish prevented testing entirely freshly caught individuals; however, we justified the reuse of captive fish as they had gone through a full autumn and winter cycle since the last experiments, and the coloration of the model stimuli was novel in experiment 2. Experiment 1

Figure 1. Experiment 1 used ‘E. barrenense’ or ‘red-stripe’ models and ‘E. zonale’ or ‘green-barred’ models to test male preference for conspecific versus heterospecific colour cues (a). Experiment 2 used ‘red-barred’ and ‘green-stripe’ models to test male and female preferences for conspecific colour versus pattern (b).

For experiment 1, each male was subject to two consecutive treatments. The male was alone in the test tank during treatment 1; immediately after treatment 1, a conspecific female was added to the test tank to join the male in treatment 2. The purpose of adding a female in the second treatment was to ensure that the social environment was a sexual one, in which the model stimuli could be interpreted as a sexual rival. For both treatments, males were exposed to a red-stripe (E. barrenense) and a green-barred (E. zonale) model. Following initial acclimation in the test tank, male behaviour (see below) was recorded for 10 min for treatment 1. Then, a conspecific female was introduced into the male’s tank; after both fish showed free-swimming behaviour, male behaviour was recorded for a subsequent 10 min for treatment 2. Each male was tested again after 24 h to assess side bias. Therefore, each focal male ‘replicate’ comprised two 20 min observation periods

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separated by 24 h, where a replicate represents a unique focal fish presented with a unique pair of models. The arrangement of the two models on either side of the tank was chosen pseudorandomly for the first observation period and reversed for the second; the same female was used for both observation periods for treatment 2. The species of focal male was ordered pseudorandomly so for every pair of successive replicates, one male E. barrenense and one male E. zonale was tested but the order within the pair was random. We tested 18 unique male E. barrenense and 18 unique male E. zonale. For treatment 2, each male was paired with a unique conspecific female with whom the male had not previously been housed. Experiment 2 In experiment 2, males and females were subject to a single treatment where they were alone in the test tank and visually exposed to pairs of red-barred and green-stripe (colour-pattern ‘swapped’) models. We tested 17 unique male E. zonale, 18 female E. zonale, 20 male E. barrenense and 19 female E. barrenense. Each individual was tested twice, in immediate succession, to assess side bias. Therefore, replicates in experiment 2 were composed of two consecutive 20 min observation periods of the same focal individual. Within each replicate, the arrangement of the two models on either side of the tank was chosen pseudorandomly for the first observation period and reversed for the second. For both experiments, we used JWatcher software (http://www. jwatcher.ucla.edu) to measure time spent in the preference zone associated with each model in a stimulus pair. Time spent head jabbing at the glass (‘glass jabbing’) was measured during both experiments, and time spent erecting the anterior dorsal fin next to the model (‘fin raise’) was measured during experiment 1. Glass jabbing towards live or model males is interpreted as an effort by the test fish to approach the stimulus (Williams & Mendelson 2010, 2011) and may indicate attraction in females and possibly aggression in males. Although not previously quantified, dorsal fin raises in males are primarily observed in courtship behaviour with females and during aggressive encounters with conspecific males (T. H. Williams & T. C. Mendelson, personal observation). Both head jabbing and dorsal fin raises were measured as durations, rather than events, because the behaviour was rapid and repeated, and occurred in clear bouts. The choice test protocol mirrored that for live stimuli in Williams & Mendelson (2010). Analysis Side bias was assessed by calculating the proportion of time spent in a preference zone on a given side of the tank across both observation periods per replicate. A focal fish was assumed to be side biased if he or she spent more than 80% of the total experimental time on the same side of the tank (cutoff value selected a priori and consistent with analyses in Williams & Mendelson 2010, 2011). A fish was deemed inactive if it failed to enter either preference zone of the test tank during the total experimental time. Inactive individuals and those showing side bias were excluded from analysis. Of the 18 males per species tested in experiment 1, all individuals were included for analysis. One male E. zonale showed side bias during experiment 2, resulting in 16 males for analysis. One female E. barrenense showed side bias and two were inactive in experiment 2, resulting in 16 female E. barrenense for analysis. All 18 female E. zonale and 20 male E. barrenense were included in the analysis for experiment 2. The data for experiment 1 were not normally distributed according to the ShapiroeWilk and KolmogoroveSmirnov tests for normality, thus nonparametric statistics were implemented. Male preferences for paired model stimuli were tested using Wilcoxon signed-ranks tests. For experiment 2,

data for glass-jabbing behaviour in female and male E. zonale and female E. barrenense were not normally distributed and were analysed using Wilcoxon signed-ranks tests. Data for glass-jabbing behaviour in male E. barrenense and for all time in preference zones by both species were normally distributed and therefore analysed using paired t tests. All paired t tests and Wilcoxon signedranks tests were two tailed; all Wilcoxon signed-ranks test statistics are represented as approximated z scores. In both experiments, strength of preference (SOP) for the conspecific-like model was calculated for each individual as:

SOP ¼ ðTC  TH Þ=ðTC þ TH Þ where TC is time spent in the conspecific-like model preference zone and TH is time spent in the heterospecific-like model preference zone. For experiment 1, strength of preference values across two social categories, male (alone) versus male with conspecific female, were compared for each species using a Wilcoxon signed-ranks test. Comparisons of strength of preference between these categories were conducted separately for three behavioural response measures: (1) time spent occupying the conspecific-like versus heterospecific-like preference zone, (2) time spent glass jabbing in the conspecific-like versus heterospecific-like preference zone and (3) time spent with a fully raised first dorsal fin in the conspecificlike versus heterospecific-like preference zone (see Table 1). For experiment 2, strength of preference was calculated such that TC represented time spent with models of conspecific colour and heterospecific pattern and TH represented time spent with models of heterospecific colour and conspecific pattern. Mean strength of preference for the models as measured by time spent occupying each preference zone was compared between female and male E. barrenense and E. zonale using the KruskaleWallis test. Additionally, mean strength of preference for the models as measured by time spent glass jabbing in each preference zone was compared between each sex and species of test fish (see Table 2). JWatcher software assisted in the quantification of behaviours including total time spent occupying preference zones, time spent glass jabbing in preference zones and time spent raising the first dorsal fin in preference zones. All experimental observation periods were scored live and simultaneously documented on a Sony Handycam HDR-XR100. For experiment 2, 18 replicates initially scored live by research assistants were rescored by video analysis so that a single observer (T.H.W.) scored all trials. Statistical analyses were performed through SPSS 17.0, JWatcher 2001, and Microsoft Office Excel 2007. RESULTS Experiment 1 Treatment 1: male alone In the first treatment of experiment 1, male E. barrenense spent X  SD ¼ 41:0  28:9% of the time in the red-stripe (conspecific) preference zone and 4.6  8.8% of the time in the green-barred zone (Wilcoxon signed-ranks test: Z ¼ 3.290, N ¼ 18, P ¼ 0.001; Fig. 2a). Glass-jabbing behaviour occurred during 2.0  4.5% of the trial time towards the red-stripe stimulus and 0% of the time towards the green-barred stimulus (Z ¼ 2.201, N ¼ 18, P ¼ 0.028). Dorsal fin raises occurred in the red-stripe and green-barred zones for 17.0  20.2% and 1.0  2.6% of the trial time, respectively (Z ¼ 2.803, N ¼ 18, P ¼ 0.005). Male E. zonale occupied the green-barred (conspecific) preference zone for 53.0  27.6% of the trial time and occupied the redstripe zone for 6.0  9.5% (Z ¼ 3.549, N ¼ 18, P < 0.001; Fig. 2b).

T. H. Williams, T. C. Mendelson / Animal Behaviour 85 (2013) 1251e1259

(a) E. barrenense alone

Average proportion of time that males occupied model preference zones

1

(c) E. barrenense with female 1

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(d) E. zonale with female 1

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Model preference zone Figure 2. The average proportion of time that male E. barrenense (a) and male E. zonale (b) spent with the red-stripe and green-barred models in the absence of a conspecific female. The average proportion of time that male E. barrenense (c) and male E. zonale (d) spent with either model in the presence of a conspecific female. Boxes define the upper and lower quartiles, horizontal lines within boxes indicate medians, whiskers indicate minima and maxima, circles and triangles indicate outliers and extreme values, respectively. * Indicates a significant difference between average preferences for models within a treatment.

Glass jabbing towards the green-barred stimulus occurred during 13.0  13.8% of the trial time and towards the red-stripe stimulus during 3.0  5.7% (Z ¼ 2.726, N ¼ 18, P ¼ 0.006). Dorsal fins were raised in the green-barred zone for 34.0  27.5% of the trial time versus 5.0  9.0% in the red-stripe zone (Z ¼ 3.154, N ¼ 18, P ¼ 0.002). Treatment 2: focal male with female In the second treatment, with focal males in the presence of a conspecific female, male E. barrenense associated with red-stripe models for 27.7  28.3% of the trial and with green-barred models for 11.3  18.4% of the trial (Z ¼ 1.851, N ¼ 16, P ¼ 0.064; Fig. 2c). Trial time spent glass jabbing was similar in the red-stripe (1.0  2.7%) and green-barred (2.0  3.8%) zones (Z ¼ 0.845, N ¼ 16, P ¼ 0.398). Occurrence of raised dorsal fin behaviour was not significantly different between the red-stripe (14.0  22.0%) and green-barred (6.0  10.8%) zones (Z ¼ 1.287, N ¼ 16, P ¼ 0.198).

Male E. zonale occupied the green-barred zone for 44.0  30.1% of the trial time and occupied the red-stripe zone for 3.0  6.0% in the presence of a conspecific female (Z ¼ 3.419, N ¼ 18, P ¼ 0.001; Fig. 2d). Glass-jabbing behaviour occurred towards the greenbarred and red-stripe stimuli during 15.0  19.7% and 1.0  4.1% of the trial time, respectively (Z ¼ 2.343, N ¼ 18, P ¼ 0.019). Dorsal fins were raised in the green-barred zone during 36.0  31.7% of the trial time and raised in the red-stripe zone for 2.0  5.6% of the trial (Z ¼ 3.288, N ¼ 18, P ¼ 0.001). Treatment comparisons Strength of preference for the conspecific- versus heterospecificlike model, as measured by time spent in the preference zones, was not significantly different between the two social categories for either focal species: male (alone) versus male with conspecific female (Wilcoxon signed-ranks test: E. barrenense: Z ¼ 1.789, N ¼ 18, P ¼ 0.074; E. zonale: Z ¼ 0.000, N ¼ 18, P ¼ 1.000; Table 1). Likewise, among social categories, no difference was found in strength of

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Table 1 Treatment comparisons for experiment 1 Treatment

Time

Glass jabs

Fin raises

_ E. barrenense _þ\ E. barrenense Treatment comparison

0.6530.628 0.3630.733 Z¼1.789, P¼0.074

0.3330.485 0.0660.539 Z¼2.064, P¼0.039

0.5150.490 0.1560.791 Z¼1.419, P¼0.156

_ E. zonale _þ\ E. zonale Treatment comparison

0.6770.506 0.6700.546 Z¼0.000, P¼1.000

0.5030.656 0.5280.810 Z¼0.059, P¼0.953

0.5600.511 0.6550.672 Z¼0.400, P¼0.689

Mean  SD strength of preference values across two social categories per focal species: male (alone) versus male with conspecific female. Wilcoxon signed-ranks comparisons of strength of preference across social categories are shown for three behavioural response measures: (1) association time, (2) glass jabbing and (3) dorsal fin raises in the conspecific-like versus heterospecific-like model preference zone. N ¼ 18 for all treatments.

preference as measured by dorsal fin raises (E. barrenense: Z ¼ 1.419, N ¼ 18, P ¼ 0.156; E. zonale: Z ¼ 0.400, N ¼ 18, P ¼ 0.689). However, differences were detected in E. barrenense, but not in E. zonale, when comparing strength of preference for the

conspecific-like model as measured by glass-jabbing behaviour among social categories (E. barrenense: Z ¼ 2.064, N ¼ 18, P ¼ 0.039; E. zonale: Z ¼ 0.059, N ¼ 18, P ¼ 0.953), as strength of preference in E. barrenense for the conspecific model was weaker when in the presence of a female. Experiment 2 Male E. barrenense spent a significantly greater proportion of total trial time in the red-barred preference zone (conspecific colour, heterospecific pattern; X  SD ¼ 26:6  17:6%) compared to the green-stripe zone (14.9  9.1%; paired t test: t19 ¼ 3.039, P ¼ 0.007; Fig. 3a). There was no significant difference in proportion of time spent glass jabbing towards either model (8.1  8.4% versus 4.9  4.2%; t19 ¼ 1.874, P ¼ 0.076). Male E. zonale spent a nearly equal proportion of time in the preference zones of the red-barred model (heterospecific colour, conspecific pattern; 32.0  23.5%) and the green-stripe model (34.7  20.4%; t15 ¼ 0.653, P ¼ 0.523; Fig. 3b). These males also glass-jabbed for similar durations of time towards both the red-barred (10.7  8.3%) and green-stripe

(c) Female E. barrenense

Average proportion of time spent occupying the model preference zones

(a) Male E. barrenense 1

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Model preference zone Figure 3. Male E. barrenense (a) and E. zonale (b), and female E. barrenense (c) and E. zonale (d) preferences for models with mismatched species colour and pattern. Boxes define the upper and lower quartiles, horizontal lines within boxes indicate medians, whiskers indicate minima and maxima, and the circle represents an outlier. * Indicates a significant difference between average preferences for models within a treatment.

T. H. Williams, T. C. Mendelson / Animal Behaviour 85 (2013) 1251e1259

(13.6  12.9%) models (Wilcoxon signed-ranks test: Z ¼ 1.293, N ¼ 16, P ¼ 0.196). Female E. barrenense associated with the red-barred model (30.7  21.5%) significantly more than with the green-stripe model (13.4  14.5%; paired t test: t15 ¼ 2.242, P ¼ 0.040; Fig. 3c). Likewise, these females performed more glass-jabbing behaviour towards the red-barred stimulus compared to the green-stripe stimulus (10.0  15.8% versus 1.4  3.9%; Wilcoxon signed-ranks test: Z ¼ 2.366, N ¼ 16, P ¼ 0.018). Female E. zonale spent similar amounts of time in the preference zones adjacent to the red-barred (25.6  14.5%) and green-stripe (25.3  16.6%) models (paired t test: t17 ¼ 0.514, P ¼ 0.614; Fig. 3d). The proportion of time spent glass jabbing towards the red-barred (12.8  10.9%) or green-stripe (12.0  11.3%) model also did not differ significantly (Wilcoxon signed-ranks test: Z ¼ 0.450, N ¼ 18, P ¼ 0.653). Finally, no significant difference was detected between either sex of either species when comparing the strength of preference for conspecific coloured versus conspecific patterned models (KruskaleWallis test: H2 ¼ 5.960, P ¼ 0.114). Similarly, no significant difference was detected with respect to glass-jabbing behaviour of either sex or species (H2 ¼ 3.965, P ¼ 0.265). For these comparisons, strength of preference was calculated such that values greater than zero indicate a preference for conspecific colour and values less than zero indicate a preference for conspecific pattern (Table 2). DISCUSSION Our experiments were designed to address two questions regarding elaborate male coloration in Etheostoma. First, we asked whether males are receivers of this signal, by testing whether they discriminate between conspecific and heterospecific coloration and preferentially respond to the former. Second, of two primary phenotypic components of male nuptial coloration, colour and pattern, we asked which component better explains the behavioural responses observed in males and females. Results of experiment 1 demonstrate that male darters can distinguish between conspecific and heterospecific coloration and that they prefer to associate with models exhibiting conspecific coloration. Male nuptial coloration in darters therefore appears to function in an intrasexual, interspecific context. In darters, a conspecific male can pose a risk to a courting male either passively, by distracting the female’s attention, or actively, by aggressively disrupting the courtship. The ability of males to respond preferentially to species-specific coloration therefore could be beneficial, allowing the courting male to respond to the risk before the approaching male gains proximity to the pair (Grether et al. 2009; Martín & López 2009; Anderson & Grether 2010). In addition, recognizing and responding preferentially to conspecific coloration can allow males to locate a receptive conspecific female by her

Table 2 Treatment comparisons for experiment 2 Treatment

Time

Glass jabs

\ E. barrenense _ E. barrenense \ E. zonale _ E. zonale Treatment comparison

0.3590.635 0.2460.389 0.0570.490 0.0580.548 H2¼5.960, P¼0.114

0.3480.446 0.0770.570 0.1540.669 0.2700.699 H2¼3.965, P¼0.265

Mean  SD strength of preference values for each focal sex and species: female E. barrenense, male E. barrenense, female E. zonale, and male E. zonale. Kruskale Wallis comparisons of strength of preference across each sex/species are shown for (1) association time and (2) glass-jabbing behaviour in the conspecific colour versus conspecific pattern model preference zone.

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association with a highly colourful conspecific male. For example, breeding aggregations in frogs have been hypothesized to occur when low-quality males associate with high-quality males and attempt to spawn with females attracted by the high-quality males (Pfennig et al. 2000). Moreover, our result is consistent with previous observations that males more frequently chase away conspecific than heterospecific males in the seminatural environment of an artificial stream (Williams & Mendelson 2010). Although males of both species appeared to react preferentially towards models exhibiting conspecific male coloration, they generally did not appear to change the strength of that preference in the presence of a conspecific female. Therefore, wild male darters may respond to a conspecific rival with similar vigour in various social contexts (i.e. the presence versus absence of a conspecific female), and male colour may be a constant competitive stimulus throughout the short breeding season, regardless of whether spawning is imminent. In experiment 2, we attempted to disentangle the importance of colour versus pattern in predicting association preferences. We previously showed that females’ strength of preference for conspecific colour was not significantly different than strength of preference for conspecific pattern when colour and pattern were presented in isolation (Williams & Mendelson 2011). The current study directly measured a preference for colour versus pattern by simultaneously presenting the focal fishes with paired stimuli that exhibited conflicting visual cues and asked whether fishes preferred one phenotypic component (i.e. colour) at the expense of another (i.e. pattern). We found that both female and male E. barrenense associated with the models exhibiting conspecific colour significantly more than they did models exhibiting the conspecific pattern. However, neither female nor male E. zonale showed a significant preference for models displaying either conspecific colour or pattern. This species-specific difference could be explained in a few ways, none of which are mutually exclusive. One is that E. zonale may not prefer one phenotypic component over the other; that is, conspecific colour and conspecific pattern are equally stimulating, perhaps serving as redundant signals (see Candolin 2003). Another explanation is that individuals were confused by the novel combinations of stimuli and could not express a preference for these unfamiliar phenotypes (Pierotti & Seehausen 2006). A third possibility is that the relative importance of colour or pattern as a social signal is context dependent, and dichotomous laboratory trials are unable to replicate the social or environmental context in which different components of nuptial coloration are most informative (see Endler 1992). Finally, the green colour of E. zonale may not hold the same information content as the orange-red of E. barrenense and preferences for green may be under weaker selection. Preference for colour over pattern, shown by both sexes of E. barrenense, suggests that red colour is an informative signal in that species. A concurrent study demonstrated that female E. barrenense prefer the average orange body hue representative of wild-caught males over a red-shifted hue representative of naturally found ‘extreme’ male coloration (T. H. Williams, J. M. Gumm & T. C. Mendelson, unpublished data). Attraction towards the population mean trait value suggests that male nuptial coloration in darters is under stabilizing selection (Paterson 1985), although further experiments with a wider range of intraspecific colour variation are necessary to test that hypothesis. Nevertheless, evidence of female preference for intraspecific colour variants in E. barrenense (T. H. Williams, J. M. Gumm & T. C. Mendelson, unpublished data) combined with results of this study, showing that both female and male E. barrenense respond preferentially to conspecific colour over pattern, suggest that red coloration is subject to sexual selection in this species. Perhaps the information

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content of ‘red’ signals in E. barrenense is of greater value in the context of sexual selection than that of ‘green’ signals in E. zonale. Future studies investigating female preference for naturally occurring colour variation in E. zonale will address this hypothesis. The orange-red coloration of male E. barrenense is probably carotenoid based (Gumm et al. 2011; B. Porter, unpublished data) and therefore signal values may be influenced by diet and thus reflect individual fitness (Kodric-Brown 1989; see Garratt & Brooks 2012). As a reliable indicator of condition, carotenoid based coloration in males may lead to strong selection on preferences for orange-red colour signals in females. For example, carotenoid consumption influences how male threespine sticklebacks vary in orange-red throat coloration (McLennan 2006), and female stickleback visual sensitivity towards orange versus red stimuli appears to correlate with paternal expression of orange versus red hue (Rick et al. 2011). Therefore, one explanation for the preference in E. barrenense for conspecific colour over pattern, when forced to choose, is that orange-red nuptial coloration of male E. barrenense is influenced by carotenoid consumption and has coevolved with female preference for that trait. The predominant colour of male E. zonale is green. Green coloration in darters, including E. zonale, is produced at least in part by pigment-containing cells called cyanophores (Pearsall 2005), which have been identified as the basis of blue coloration in other fish species (Goda & Fujii 1995; Gagnon 2006; Yu et al. 2008). Some of the green hues exhibited by darters appear to be due to the overlap of both yellow and blue pigment-containing cells (Gumm et al. 2011). Little is known regarding the biochemical composition of cyanophores in darters (Pearsall 2005), nor to what degree cyanophores are endogenously synthesized or exogenously acquired. The function of blue/green coloration in darters is also unknown, but is speculated to play a role in camouflage in dense vegetation (McCormick & Aspinwall 1983; Gumm et al. 2011). Etheostoma zonale is observed at high densities in areas of attached vegetation (Etnier & Starnes 1993), providing a possible relationship between male coloration and ecology, although we find extensive ecological overlap between the two species (T. H. Williams & T. C. Mendelson, unpublished data). Given that the greenstripe models were not more stimulating to E. zonale than the red-barred models in experiment 2, we speculate that blue and green colours in darters are less reliable indicators of condition than carotenoid-based colours, which would lead to comparatively weak selection on preferences for green. Alternatively, as noted above, colour and pattern may be redundant or equally informative signals in E. zonale, such that a preference for colour would not outweigh that for pattern. Between experiments 1 and 2, we provide evidence that elaborate male nuptial coloration in darters serves as a signal to both males and females. We therefore hypothesize that both intra- and intersexual selection pressures play a role in the evolution of male nuptial coloration. Males of two sympatric species respond preferentially to conspecific over heterospecific coloration (as do females; Williams & Mendelson 2011), and both males and females of one species respond preferentially to colour over pattern when presented with conflicting visual cues. The behavioural differences found between E. barrenense and E. zonale, however, demonstrate that the attractiveness of colour per se may not be equal across all species in the genus, perhaps suggesting differences in information content across colours. That is, certain hues (e.g. ‘red’) may indicate male fitness in some species while different hues in other species may have evolved under independent pressures and cannot be assumed to convey convergent information. Future comparative studies examining the function of multiple components of darters’ complex visual communication systems would therefore provide valuable insight into the role of these elaborate signals in darter speciation.

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