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Male rainbow darters (Etheostoma caeruleum) prefer larger conspecific females
Behavioural Processes 170 (2020) 104013
Contents lists available at ScienceDirect
Behavioural Processes journal homepage: www.elsevier.com/locate/behavproc
Male rainbow darters (Etheostoma caeruleum) prefer larger conspecific females
T
Olivia Soudry, Hatib Kaira, Shabnam Parsa, Tamra Mendelson* University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, Maryland 21250 United States
A B S T R A C T
Female mate choice is a well studied mechanism of sexual selection that can affect the evolution of male secondary sexual traits. However, males also exhibit selective mate choice in some animals, which can affect the evolution of female phenotypes. We tested for evidence of intraspecific male mate choice in the Rainbow Darter (Percidae: Etheostoma caeruleum). Males of numerous darter species have been shown to prefer conspecific over heterospecific females; however, intraspecific preferences in males have not yet been demonstrated in this genus. This study investigated male preference for indicators of female fecundity, specifically female size. We found that male Rainbow Darters prefer larger females, both in dichotomous choice tests, when access between individuals was prevented, and in unrestricted behavioral tests with full access to females. Notably, however, the dichotomous trials were not repeatable; males only displayed a significant preference for larger females in the first dichotomous trial and in the unrestricted trial. We discuss our results in light of experimental methods and the role of male mate choice in sexual selection.
1. Introduction Animal behavior research strongly supports the contribution of female mating preferences to the evolution of male ornamentation (Andersson and Simmons, 2006; Ryan and Keddy-Hector, 1992). By comparison, our understanding of male mate choice and its role in the evolution of female phenotypes is less well studied. Sexual selection theory dictates that operational sex ratios (OSR) in animals generally skew toward an abundance of males, due to females having larger energy investments in reproduction (Andersson, 1994; Darwin, 1871). This skew is thought to generate conventional sex roles, defined by a polygynous mating system with competitive, promiscuous males and choosy females. However, it has long been noted that male mate choice should evolve even in populations with male-biased sex ratios if male reproductive effort, including costly ejaculates, mate guarding or intense displaying, reduces opportunities for future mating (Edward and Chapman, 2011; Dewsbury, 1982; Halliday, 1983). Indeed, male mating preferences have been documented in many species, including those with seemingly typical conventional sex roles (reviewed in AlaHonkola et al., 2010; Dewsbury, 1982; and see below). Evidence of male mate choice within species has been documented for a variety of traits (e.g., Ala-Honkola et al., 2010; Bonduriansky, 2001; MacLeod and Andrade, 2014). For example, male two-spotted gobies (Gobiusculus flavescens) prefer more colorful females both when choosing among natural variation in female color and among females that have been artificially enhanced for color (Amundsen and Forsgren, 2001). In the gift giving cabbage butterfly (Pieris rapae), males prefer
⁎
females with more colorful wings (Tigreros et al., 2014), and males of the western white butterfly (Pieris occidentalis) prefer normally pigmented over highly melanized females (Wiernasz, 1995). But the most common female trait preferred by males is size, likely because it can indicate fecundity. In the guppy (Poecilia reticulata), for example, males prefer larger females in dichotomous choice trials and in unrestricted trials (Herdman et al., 2004; Jeswiet and Godin, 2011). Male terrestrial salamanders (Plethodon shermani) spend more time performing their “foot dancing” courtship ritual in the presence of larger females than smaller ones (Eddy et al., 2016), and males of the red-backed salamander (Plethodon cinereus) prefer larger female salamanders, but only when detecting females by chemical, rather than visual cues (Jaworski et al., 2018). Species in the genus Etheostoma are freshwater fishes, commonly known as “darters,” that inhabit streams and lakes across eastern Northern America (Page and Burr, 2011). All darters exhibit classic sexual dimorphism, with males generally larger and displaying exaggerated secondary sexual traits relative to females. In most species, males have vivid nuptial coloration, and they compete physically and intensely for access to females. Etheostoma caeruleum, the Rainbow Darter, is native to the Great Lakes and Mississippi River basin. Rainbow Darters have an average lifespan of about 3 years, and adults can grow to about 6−8 cm. Male nuptial coloration consists of alternating bands of reddish orange and blue-green, and their dorsal fins are equally striking, with highly contrasting red, white, and blue horizontal bars. Females usually exhibit duller coloration, mainly brown, black, and olive green, arranged in blotches and bars that resemble the gravel
Corresponding author.
https://doi.org/10.1016/j.beproc.2019.104013 Received 11 July 2019; Received in revised form 20 October 2019; Accepted 14 November 2019 Available online 18 November 2019 0376-6357/ © 2019 Elsevier B.V. All rights reserved.
Behavioural Processes 170 (2020) 104013
O. Soudry, et al.
focal and stimulus tanks, a video camera was set to record, a female was placed into one of each of the stimulus tanks, and the focal male was placed in the focal tank. After removing the opaque partitions, an acclimation period consisted of the male visiting both sides of the focal tank, within 10-cm of the stimulus tanks, and returning to the neutral zone, to increase the likelihood that both stimulus females were observed by the male. After the acclimation period, twenty minutes of behavior were quantified from the video using JWatcher™ (Blumstein et al., 2000). Entrance into and time spent in association zones, as well as number of “jabs” against the glass adjacent to the stimulus tanks (attempted entrance into stimulus tank), were quantified. Each experimental triad (a female stimulus pair and its focal male) was tested in the dichotomous set up twice, alternating female sides to control for side bias. Trials were conducted from April 28 to May 25, 2017; thus, all fish had approximately one month to acclimate to lab conditions. After undergoing two dichotomous trials, each experimental triad underwent a third, unrestricted trial. The three fish were placed in a single 37.9 L tank and videotaped for 20 min (Fig. 1). Females were placed in the tank first and the male thereafter. Behavior of the male was quantified from video using JWatcher™. Numbers of approaches, nudges, fin displays, quivers, mounts, and spawns toward each female were recorded.
upon which they reside (Page, 1983; Kuehne and Barbour, 1983; Page and Burr, 2011). Male darters have been shown to prefer conspecific over heterospecific females in many species pairs (Ciccotto et al., 2013; Martin and Mendelson, 2016; Mendelson et al., 2018; Moran et al., 2017; Roberts and Mendelson, 2017; Williams and Mendelson, 2010); however, male preference for female phenotypes within a species has not yet been demonstrated in darters. Fuller (1998) showed that standard length is positively correlated with egg mass in females, suggesting that standard length is an indicator of fecundity in this species. The goal of this study was to examine mating preferences of male darters for conspecific females differing in size. Mate preferences can be measured using a variety of methods, including “choice” paradigms, where subjects are presented with two or more options, as well as “no choice” paradigms, where subjects are presented with a single option ( Dougherty and Shuker, 2015). Most choice paradigms do not allow the options to interact with one another, thereby minimizing potential effects of those interactions on the chooser’s preference. However, this restricted-access study design does not allow individuals to express their preference by mating, so mate choice must be inferred from proxies like time spent near option individuals. Moreover, restricted choice trials can preclude communication in some sensory modalities. We therefore allowed male darters to choose between differently sized females in both restricted dichotomous choice tests and an unrestricted test in which the male had access to both females. If males prefer larger females, we expected to see greater association times with larger females in restricted dichotomous trials and greater courtship in the unrestricted trials toward the larger female.
2.3. Data analysis Thirteen males were tested in Trial 1. For Trial 2, one male’s trial was compromised by human interference and therefore not scored. For Trial 3, a different male exhibited escape behavior during most of the trial, so the trial was discarded. Thus, 12 males were tested in Trials 2 and 3. For the dichotomous mate choice trials, to control for variation in activity levels among males, time spent in either association zone was converted to a proportion, as the time spent in one preference zone divided by the sum of time spent in both preference zones. All data were tested for normality using the Shapiro-Wilks normality test. Data from the dichotomous mate choice trials were normally distributed; thus, proportions of time spent in the large and small association zones were compared using a parametric paired t-test. Of the male behaviors quantified in the unrestricted trial, only approaches and fin displays occurred with sufficient frequency for statistical analysis (see supplementary material). Approaches were defined as a male pursuing a female; fin displays were defined as the male erecting the first dorsal fin from a resting position. Fin displays reveal the highly contrasting red, white, and blue bands that are normally concealed at rest. Approaches and fin displays were converted to proportions, as the number of approaches (or fin displays) toward one female divided by the total number of approaches (or fin displays) to both females. Proportions of approach toward large and small females were normally distributed and compared with a parametric paired ttest. Proportions of fin displays were not normally distributed; thus proportions of fin displays to large versus small females were compared using a non-parametric Wilcoxon signed rank test. To test for an effect of belly width on male preference, we used a general linear model to test for a relationship between proportion of time spent with the larger female and her absolute belly width, as well as belly width as a proportion of standard length. We also used a general linear model to test the relationship between normalized female size difference, measured as the difference in absolute size of the two females in a stimulus pair divided by their average size, and male behavior (approach, fin display) in the unrestricted trial. This test allowed us to determine if a greater difference in female size was associated with more behaviors directed toward the larger female. All data analysis was conducted in R Studio.
2. Methods 2.1. Fish collection and care Etheostoma caeruleum were collected in the breeding season (March 23rd, 2017) from the East Fork Barren River, Monroe County, KY, U.S.A. (coordinates: 36.7451434, - 85.6967475). Fish were transported to the University of Maryland Baltimore County in aerated coolers and housed in a recirculating aquarium system (Aquatic Habitats, Inc.). Each fish resided alone in a 0.8 gallon aquaria. Males and females were housed separately with no visual contact to avoid interaction prior to trials. Fish were fed a diet of live blackworms daily. After introduction to the aquarium system, all fish were lightly sedated with tricaine methanesulfonate (MS-222) and measured for standard length (snout to tip of caudal peduncle) and abdomen width to the nearest 0.10 mm using standard calipers. Fish were housed under natural lighting with supplementary fluorescent lighting that matched the natural photoperiod. Fish were euthanized humanely at the end of the study; all procedures were ethical and complied with UMBC Institutional Animal Care and Use Committee protocol number TM011061821. 2.2. Choice trials Female stimulus pairs were created using two females that differed in standard length by > 20%. Each male was randomly assigned a stimulus pair and underwent three choice trials with that same pair, each > 24 h apart. Due to unanticipated female mortality over the course of the experiment, some stimulus pairs were assigned to more than one male. The first two trials were dichotomous, restricted choice trials, in which each individual was placed in a separate tank, thus precluding all but visual communication. The dichotomous trial set up consisted of a 37.9 L “focal” tank flanked by two 9.5 L “stimulus” tanks (Fig. 1). Association zones of 5-cm and 10-cm were marked on both sides of the focal tank closest to stimulus tanks. The 10-cm association zone was used in the acclimation period, and the 5-cm zone was used to estimate preference. The zone in the focal tank between the 10-cm association zones was considered a neutral zone. For each dichotomous trial, opaque partitions were placed between
3. Results We found that males spent significantly more time on average in the 2
Behavioural Processes 170 (2020) 104013
O. Soudry, et al.
Fig. 1. Schematic front view of experimental set up for dichotomous trials 1 & 2 (left) and unrestricted trial 3 (right). Association zones of 5-cm and 10-cm were marked directly on the glass. In each trial, females were placed in their respective tanks first and the male was added after.
Fig. 2. Proportion of time male spent in association zones of large and small females in dichotomous Trial 1 (a) and dichotomous Trial 2 (b). Asterisk indicates significant difference, p < 0.002.
Fig. 3. Proportion of approaches (a) and fin displays (b) to large and small females in unrestricted Trial 3. Asterisk indicates significant difference, p < 0.007 (a) and p < 0.04 (b).
larger female association zone in the first dichotomous choice trials (mean proportion of time with large female was 68% versus 32% with small female; t = 3.997, df = 12, p = 0.0018, Fig. 2a) but not the second (mean proportion of time with large female was 58% versus 42% with small female; t = 1.25, df = 11, p = 0.237, Fig. 2b). Proportion of time spent with the larger female was not predicted by her absolute belly width or by belly width as a proportion of standard length in either Trial 1 (width: F = 1.23, df = 11, p = 0.291; proportional width: F = 3.17, d = 11, p = 0.102) or Trial 2 (width: F = 0.136, d = 10, p = 0.72; proportional width: F = 0.172, d = 10, p = 0.687). In the unrestricted trial (Trial 3), males approached the large female significantly more than the small female (t = 3.34, df = 11, p = 0.0065, Fig. 3a). Males also displayed their fins significantly more to the larger female in the unrestricted trial (V = 66.5, p = 0.034, Fig. 3b). Of the two male behaviors that occurred frequently in the unrestricted trial (i.e., approaches, fin displays), only proportion of fin
displays to the larger female was significantly predicted by normalized size difference of the stimulus female pair (R-squared = 0.42, F = 9.07, p = 0.013, Fig. 4). That is, the more the stimulus females differed in size, the more the male disproportionately directed fin displays toward the larger female. 4. Discussion Our results demonstrate a potential role for male mating preference in sexual selection and trait evolution in darters. We showed that males spent significantly more time with large females in a dichotomous mate choice trial, but this significance was not repeatable; males did not show a significant preference in the ensuing dichotomous trial. However, a significant preference appeared when males were given subsequent access to the females in unrestricted trials. These results suggest that dichotomous mate choice trials provide a reliable indication of mate choice, even if they are not repeatable in the short term. 3
Behavioural Processes 170 (2020) 104013
O. Soudry, et al.
sustain a preference over the course of multiple experiments. A third possible explanation is conditioned place preference (CPP), which occurs when an animal develops a preference for a spatial context that is associated with a reward (e.g., a mate) and is motivated to return to the space associated with the reward (Pfaus et al., 2001). In a study of CPP in fish, for example, individuals presented with an appetitive stimulus (pellets) significantly increased the time spent and distance moved in the reward side, while fish exposed to an aversive stimulus significantly decreased the time spent in the stimulation side (Millot et al., 2014). Given that males in our study preferred larger females in the unrestricted trials that followed the second dichotomous trial, males might have developed a place preference associated with the larger female in Trial 1 and returned to that place in Trial 2. CPP might explain why males spent more time with the larger female in Trial 2 but the difference was not significant; a considerable amount of time may have been spent initially inspecting the previous location of the large female. If this hypothesis is true, then dichotomous choice studies in which individuals are tested twice, with stimulus sides alternated to control for side bias (e.g., Williams and Mendelson, 2010), may need to consider potential effects of CPP. We also found that the proportion of fin displays directed to the large female in the unrestricted trial was greater overall and also greater when females differed more in size. Male darters frequently erect their dorsal fins while competing with other males (pers. obs.); this behavior reveals the striking color patterns that characterize the dorsal fins of most darter species. Demonstrating that males also fin display toward females in the absence of males, and disproportionately to the female with whom they spent more time associating in a dichotomous trial, suggests that fin displays are not only an intrasexual competitive signal but also a courtship signal. Many communication signals are known to serve this dual purpose (e.g., Berglund et al., 1996), but the relative importance of fin displays in these two communication contexts is yet unknown for darters. Previous studies of mating in E. caeruleum suggest that intrasexual competition may be more important than female preference in determining male mating success (Fuller, 2003). If this is true, then males might not be able to express a mate preference in nature, or under more realistic social conditions in the laboratory, due to interference from competing males. A future study that adds an additional trial, in which two males must compete for females, could indicate the extent to which male-male interference may mask male mate choice in nature. In sum, our study provides the first evidence that male darters do not mate randomly within species but rather base their mating decisions on criteria such as female size. Our study also suggests a caveat in experimental design, specifically questioning whether dichotomous mate choice trials conducted in close succession might obscure a true association preference. Results therefore expand our framework of sexual selection in this sexually dichromatic fish to add male choice as well as female choice to the dynamics of sexual selection in darter species.
Fig. 4. Relationship between percent difference in female length and proportion of fin displays to larger females.
Etheostoma caeruleum appears to have a typical operational sex ratio and traditional sex roles, in that females produce a limited number of large, nutritious eggs compared to millions of sperm produced by males; male E. caeruleum are highly ornamented relative to females; and, males spend considerable amount of time during the breeding season intensely competing with other males for access to females. However, neither sex provides parental care, both sexes mate multiply, and males invest considerable energy in securing a mate, both in competing and courting, suggesting suitable conditions for male choice (e.g., Edward and Chapman, 2011). Previous studies have shown that males of many darter species prefer conspecific over heterospecific females (see above). Our study suggests that males in at least one species of this sexually dimorphic lineage are choosy within species as well, preferring larger females. A previous study of mate choice in the Roanoke Darter (Percina roanoka) found that neither males nor females preferred larger conspecific members of the opposite sex in dichotomous mate choice trials (Ciccotto et al., 2014). Why neither sex of P. roanoka preferred larger mates is unclear; fish were collected and tested in the mating season, and the experimental setup was the same as in the present study. One possible explanation is that mate choice in P. roanoka is based on traits other than size, though the same study also ruled out several aspects of color variation as explanatory variables. The two species in question belong to different genera, and species of Percina generally exhibit less striking nuptial coloration and less dramatic sexual dimorphism, though P. roanoka is among the more colorful species of the genus (e.g., Page, 1983). One hypothesis is that sexual selection is less intense in Percina as compared to Etheostoma, but to conclusively explain the difference in results between the two species will require further hypothesis testing. One important result of our study is that a significant difference in association time with the larger female was not repeated in the second dichotomous mate choice trial. Bell et al. (2009) conducted a metaanalysis of repeatability in studies of animal behavior and found that mate preference was among the least repeatable classes of behavior, particularly so for ectotherm vertebrates studied in the lab. Interestingly, their study also showed that male mate preference is much less repeatable than female mate preference. One explanation for the lack of repeatability in our study is that our sample size in Trial 2 lacked the power to detect a preference. The effect size (Cohen’s d) for the second trial was 0.72, which is not large but could indicate a lack of power. Another explanation is that visual signals alone are insufficient to
Acknowledgements We are grateful for the contributions of Natalie Roberts, Courtney Mattson, Sam Hulse, Jong Park, Kevin Justus & Caitlyn McCaulley. This work was funded in part by National Science Foundation grant IOS1708543. References Ala-Honkola, O., Säilä, L., Lindström, K., 2010. Males prefer small females in a dichotomous choice test in the Poeciliid fish Heterandria formosa. Ethology 116, 736–743. Amundsen, T., Forsgren, E., 2001. Male mate choice selects for female coloration in a fish. Proc. Natl. Acad. Sci. 98, 13155–13160. Andersson, M., 1994. Sexual Selection. Princeton University Press, Princeton, NJ. Andersson, M., Simmons, L.W., 2006. Sexual selection and mate choice. Trends Ecol. Evol. 21, 296–302.
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Jeswiet, S.B., Godin, J.-G.J., 2011. Validation of a method for quantifying male mating preferences in the guppy (Poecilia reticulata). Ethology 117, 422–429. Kuehne, R.A., Barbour, R.W., 1983. The American Darters. University Press of Kentucky, Lexington, Ky. MacLeod, E.C., Andrade, M.C., 2014. Strong, convergent male mate choice along two preference axes in field populations of black widow spiders. Anim. Behav. 89, 163–169. Martin, M.D., Mendelson, T.C., 2016. Male behavior predicts trait divergence and the evolution of reproductive isolation in darters (Percidae: etheostoma). Anim. Behav. 112, 179–186. Mendelson, T.C., Gumm, J.M., Martin, M.D., Ciccotto, P.J., 2018. Preference for conspecifics evolves earlier in males than females in a sexually dimorphic radiation of fishes. Evolution 72, 337–347. Millot, S., Cerqueira, M., Castanheira, M.F., Øverli, Ø, Martins, C.I.M., Oliveira, R.F., 2014. Use of conditioned place preference/avoidance tests to assess affective states in fish. Appl. Anim. Behav. Sci. 154, 104–111. Moran, R.L., Zhou, M., Catchen, J.M., Fuller, R.C., 2017. Male and female contributions to behavioral isolation in darters as a function of genetic distance and color distance. Evolution 71, 2428–2444. Page, L.M., 1983. Handbook of Darters. Neptune City, NJ : TFH Publications. Page, L.M., Burr, B.M., 2011. Peterson Field Guide to Freshwater Fishes of North America North of Mexico. Houghton Mifflin, Harcourt. Pfaus, J.G., Kippin, T.E., Centeno, S., 2001. Conditioning and sexual behavior: a review. Horm. Behav. 40, 291–321. Roberts, N.S., Mendelson, T.C., 2017. Male mate choice contributes to behavioural isolation in sexually dimorphic fish with traditional sex roles. Anim. Behav. 130, 1–7. Ryan, M.J., Keddy-Hector, A., 1992. Directional patterns of female mate choice and the role of sensory biases. Am. Nat. 139, S4–S35. Tigreros, N., Mowery, M.A., Lewis, S.M., 2014. Male mate choice favors more colorful females in the gift-giving cabbage butterfly. Behav. Ecol. Sociobiol. (Print) 68, 1539–1547. Wiernasz, D.C., 1995. Male choice on the basis of female melanin pattern in Pieris butterflies. Anim. Behav. 49, 45–51. Williams, T.H., Mendelson, T.C., 2010. Behavioral isolation based on visual signals in a sympatric pair of darter species. Ethology 116, 1038–1049.
Bell, A.M., Hankison, S.J., Laskowski, K.L., 2009. The repeatability of behaviour: a metaanalysis. Anim. Behav. 77, 771–783. Berglund, A., Bisazza, A., Pilastro, A., 1996. Armaments and ornaments: an evolutionary explanation of traits of dual utility. Biol. J. Linn. Soc. 58, 385–399. Blumstein, D.R., Evans, C., Daniel, J.C., 2000. JWatcher TM 0.9 An Introductory User’S Guide, vol. 2005. Bonduriansky, R., 2001. The evolution of male mate choice in insects: a synthesis of ideas and evidence. Biol. Rev. 76, 305–339. Ciccotto, P.J., Gumm, J.M., Mendelson, T.C., 2013. Male association preference for conspecifics in the Redband Darter, Etheostoma luteovinctum (Teleostei: percidae) based on visual cues. Copeia 2013, 154–159. Ciccotto, P.J., Gumm, J.M., Mendelson, T.C., 2014. No evidence for color or size preference in either sex of a dichromatic stream fish, Percina roanoka. Environ. Biol. Fishes 97, 187–195. Darwin, C., 1871. The Descent of Man and Selection in Relation to Sex. J. Murray, London. Dewsbury, D.A., 1982. Ejaculate cost and male choice. Am. Nat. 119, 601–610. Dougherty, L.R., Shuker, D.M., 2015. The effect of experimental design on the measurement of mate choice: a meta-analysis. Behav. Ecol. 26, 311–319. Eddy, S.L., Wilburn, D.B., Chouinard, A.J., Doty, K.A., Kiemnec-Tyburczy, K.M., Houck, L.D., 2016. Male terrestrial salamanders demonstrate sequential mate choice based on female gravidity and size. Anim. Behav. 113, 23–29. Edward, D.A., Chapman, T., 2011. The evolution and significance of male mate choice. Trends Ecol. Evol. 26, 647–654. Fuller, R.C., 1998. ). Fecundity estimates for rainbow darters, Etheostoma caeruleum, in southwestern Michigan. Ohio J. Sci. 98, 2–5. Fuller, R.C., 2003. Disentangling female mate choice and male competition in the Rainbow Darter, Etheostoma caeruleum. Copeia 2003, 138–148. Halliday, T.R., 1983. The study of mate choice. Pp. 3-32. In: Bateson, P.P. (Ed.), Mate Choice. Cambridge University Press, Cambridge. Herdman, E., Kelly, C.D., Godin, J.-G.J., 2004. Male mate choice in the guppy (Poecilia reticulata): do males prefer larger females as mates? Ethology 110, 97–111. Jaworski, K.E., Lattanzio, M.S., Hickerson, C.M., Anthony, C.D., 2018. Male mate preference as an agent of fecundity selection in a polymorphic salamander. Ecol. Evol. 8, 8750–8760.
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Contents lists available at ScienceDirect
Behavioural Processes journal homepage: www.elsevier.com/locate/behavproc
Male rainbow darters (Etheostoma caeruleum) prefer larger conspecific females
T
Olivia Soudry, Hatib Kaira, Shabnam Parsa, Tamra Mendelson* University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, Maryland 21250 United States
A B S T R A C T
Female mate choice is a well studied mechanism of sexual selection that can affect the evolution of male secondary sexual traits. However, males also exhibit selective mate choice in some animals, which can affect the evolution of female phenotypes. We tested for evidence of intraspecific male mate choice in the Rainbow Darter (Percidae: Etheostoma caeruleum). Males of numerous darter species have been shown to prefer conspecific over heterospecific females; however, intraspecific preferences in males have not yet been demonstrated in this genus. This study investigated male preference for indicators of female fecundity, specifically female size. We found that male Rainbow Darters prefer larger females, both in dichotomous choice tests, when access between individuals was prevented, and in unrestricted behavioral tests with full access to females. Notably, however, the dichotomous trials were not repeatable; males only displayed a significant preference for larger females in the first dichotomous trial and in the unrestricted trial. We discuss our results in light of experimental methods and the role of male mate choice in sexual selection.
1. Introduction Animal behavior research strongly supports the contribution of female mating preferences to the evolution of male ornamentation (Andersson and Simmons, 2006; Ryan and Keddy-Hector, 1992). By comparison, our understanding of male mate choice and its role in the evolution of female phenotypes is less well studied. Sexual selection theory dictates that operational sex ratios (OSR) in animals generally skew toward an abundance of males, due to females having larger energy investments in reproduction (Andersson, 1994; Darwin, 1871). This skew is thought to generate conventional sex roles, defined by a polygynous mating system with competitive, promiscuous males and choosy females. However, it has long been noted that male mate choice should evolve even in populations with male-biased sex ratios if male reproductive effort, including costly ejaculates, mate guarding or intense displaying, reduces opportunities for future mating (Edward and Chapman, 2011; Dewsbury, 1982; Halliday, 1983). Indeed, male mating preferences have been documented in many species, including those with seemingly typical conventional sex roles (reviewed in AlaHonkola et al., 2010; Dewsbury, 1982; and see below). Evidence of male mate choice within species has been documented for a variety of traits (e.g., Ala-Honkola et al., 2010; Bonduriansky, 2001; MacLeod and Andrade, 2014). For example, male two-spotted gobies (Gobiusculus flavescens) prefer more colorful females both when choosing among natural variation in female color and among females that have been artificially enhanced for color (Amundsen and Forsgren, 2001). In the gift giving cabbage butterfly (Pieris rapae), males prefer
⁎
females with more colorful wings (Tigreros et al., 2014), and males of the western white butterfly (Pieris occidentalis) prefer normally pigmented over highly melanized females (Wiernasz, 1995). But the most common female trait preferred by males is size, likely because it can indicate fecundity. In the guppy (Poecilia reticulata), for example, males prefer larger females in dichotomous choice trials and in unrestricted trials (Herdman et al., 2004; Jeswiet and Godin, 2011). Male terrestrial salamanders (Plethodon shermani) spend more time performing their “foot dancing” courtship ritual in the presence of larger females than smaller ones (Eddy et al., 2016), and males of the red-backed salamander (Plethodon cinereus) prefer larger female salamanders, but only when detecting females by chemical, rather than visual cues (Jaworski et al., 2018). Species in the genus Etheostoma are freshwater fishes, commonly known as “darters,” that inhabit streams and lakes across eastern Northern America (Page and Burr, 2011). All darters exhibit classic sexual dimorphism, with males generally larger and displaying exaggerated secondary sexual traits relative to females. In most species, males have vivid nuptial coloration, and they compete physically and intensely for access to females. Etheostoma caeruleum, the Rainbow Darter, is native to the Great Lakes and Mississippi River basin. Rainbow Darters have an average lifespan of about 3 years, and adults can grow to about 6−8 cm. Male nuptial coloration consists of alternating bands of reddish orange and blue-green, and their dorsal fins are equally striking, with highly contrasting red, white, and blue horizontal bars. Females usually exhibit duller coloration, mainly brown, black, and olive green, arranged in blotches and bars that resemble the gravel
Corresponding author.
https://doi.org/10.1016/j.beproc.2019.104013 Received 11 July 2019; Received in revised form 20 October 2019; Accepted 14 November 2019 Available online 18 November 2019 0376-6357/ © 2019 Elsevier B.V. All rights reserved.
Behavioural Processes 170 (2020) 104013
O. Soudry, et al.
focal and stimulus tanks, a video camera was set to record, a female was placed into one of each of the stimulus tanks, and the focal male was placed in the focal tank. After removing the opaque partitions, an acclimation period consisted of the male visiting both sides of the focal tank, within 10-cm of the stimulus tanks, and returning to the neutral zone, to increase the likelihood that both stimulus females were observed by the male. After the acclimation period, twenty minutes of behavior were quantified from the video using JWatcher™ (Blumstein et al., 2000). Entrance into and time spent in association zones, as well as number of “jabs” against the glass adjacent to the stimulus tanks (attempted entrance into stimulus tank), were quantified. Each experimental triad (a female stimulus pair and its focal male) was tested in the dichotomous set up twice, alternating female sides to control for side bias. Trials were conducted from April 28 to May 25, 2017; thus, all fish had approximately one month to acclimate to lab conditions. After undergoing two dichotomous trials, each experimental triad underwent a third, unrestricted trial. The three fish were placed in a single 37.9 L tank and videotaped for 20 min (Fig. 1). Females were placed in the tank first and the male thereafter. Behavior of the male was quantified from video using JWatcher™. Numbers of approaches, nudges, fin displays, quivers, mounts, and spawns toward each female were recorded.
upon which they reside (Page, 1983; Kuehne and Barbour, 1983; Page and Burr, 2011). Male darters have been shown to prefer conspecific over heterospecific females in many species pairs (Ciccotto et al., 2013; Martin and Mendelson, 2016; Mendelson et al., 2018; Moran et al., 2017; Roberts and Mendelson, 2017; Williams and Mendelson, 2010); however, male preference for female phenotypes within a species has not yet been demonstrated in darters. Fuller (1998) showed that standard length is positively correlated with egg mass in females, suggesting that standard length is an indicator of fecundity in this species. The goal of this study was to examine mating preferences of male darters for conspecific females differing in size. Mate preferences can be measured using a variety of methods, including “choice” paradigms, where subjects are presented with two or more options, as well as “no choice” paradigms, where subjects are presented with a single option ( Dougherty and Shuker, 2015). Most choice paradigms do not allow the options to interact with one another, thereby minimizing potential effects of those interactions on the chooser’s preference. However, this restricted-access study design does not allow individuals to express their preference by mating, so mate choice must be inferred from proxies like time spent near option individuals. Moreover, restricted choice trials can preclude communication in some sensory modalities. We therefore allowed male darters to choose between differently sized females in both restricted dichotomous choice tests and an unrestricted test in which the male had access to both females. If males prefer larger females, we expected to see greater association times with larger females in restricted dichotomous trials and greater courtship in the unrestricted trials toward the larger female.
2.3. Data analysis Thirteen males were tested in Trial 1. For Trial 2, one male’s trial was compromised by human interference and therefore not scored. For Trial 3, a different male exhibited escape behavior during most of the trial, so the trial was discarded. Thus, 12 males were tested in Trials 2 and 3. For the dichotomous mate choice trials, to control for variation in activity levels among males, time spent in either association zone was converted to a proportion, as the time spent in one preference zone divided by the sum of time spent in both preference zones. All data were tested for normality using the Shapiro-Wilks normality test. Data from the dichotomous mate choice trials were normally distributed; thus, proportions of time spent in the large and small association zones were compared using a parametric paired t-test. Of the male behaviors quantified in the unrestricted trial, only approaches and fin displays occurred with sufficient frequency for statistical analysis (see supplementary material). Approaches were defined as a male pursuing a female; fin displays were defined as the male erecting the first dorsal fin from a resting position. Fin displays reveal the highly contrasting red, white, and blue bands that are normally concealed at rest. Approaches and fin displays were converted to proportions, as the number of approaches (or fin displays) toward one female divided by the total number of approaches (or fin displays) to both females. Proportions of approach toward large and small females were normally distributed and compared with a parametric paired ttest. Proportions of fin displays were not normally distributed; thus proportions of fin displays to large versus small females were compared using a non-parametric Wilcoxon signed rank test. To test for an effect of belly width on male preference, we used a general linear model to test for a relationship between proportion of time spent with the larger female and her absolute belly width, as well as belly width as a proportion of standard length. We also used a general linear model to test the relationship between normalized female size difference, measured as the difference in absolute size of the two females in a stimulus pair divided by their average size, and male behavior (approach, fin display) in the unrestricted trial. This test allowed us to determine if a greater difference in female size was associated with more behaviors directed toward the larger female. All data analysis was conducted in R Studio.
2. Methods 2.1. Fish collection and care Etheostoma caeruleum were collected in the breeding season (March 23rd, 2017) from the East Fork Barren River, Monroe County, KY, U.S.A. (coordinates: 36.7451434, - 85.6967475). Fish were transported to the University of Maryland Baltimore County in aerated coolers and housed in a recirculating aquarium system (Aquatic Habitats, Inc.). Each fish resided alone in a 0.8 gallon aquaria. Males and females were housed separately with no visual contact to avoid interaction prior to trials. Fish were fed a diet of live blackworms daily. After introduction to the aquarium system, all fish were lightly sedated with tricaine methanesulfonate (MS-222) and measured for standard length (snout to tip of caudal peduncle) and abdomen width to the nearest 0.10 mm using standard calipers. Fish were housed under natural lighting with supplementary fluorescent lighting that matched the natural photoperiod. Fish were euthanized humanely at the end of the study; all procedures were ethical and complied with UMBC Institutional Animal Care and Use Committee protocol number TM011061821. 2.2. Choice trials Female stimulus pairs were created using two females that differed in standard length by > 20%. Each male was randomly assigned a stimulus pair and underwent three choice trials with that same pair, each > 24 h apart. Due to unanticipated female mortality over the course of the experiment, some stimulus pairs were assigned to more than one male. The first two trials were dichotomous, restricted choice trials, in which each individual was placed in a separate tank, thus precluding all but visual communication. The dichotomous trial set up consisted of a 37.9 L “focal” tank flanked by two 9.5 L “stimulus” tanks (Fig. 1). Association zones of 5-cm and 10-cm were marked on both sides of the focal tank closest to stimulus tanks. The 10-cm association zone was used in the acclimation period, and the 5-cm zone was used to estimate preference. The zone in the focal tank between the 10-cm association zones was considered a neutral zone. For each dichotomous trial, opaque partitions were placed between
3. Results We found that males spent significantly more time on average in the 2
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Fig. 1. Schematic front view of experimental set up for dichotomous trials 1 & 2 (left) and unrestricted trial 3 (right). Association zones of 5-cm and 10-cm were marked directly on the glass. In each trial, females were placed in their respective tanks first and the male was added after.
Fig. 2. Proportion of time male spent in association zones of large and small females in dichotomous Trial 1 (a) and dichotomous Trial 2 (b). Asterisk indicates significant difference, p < 0.002.
Fig. 3. Proportion of approaches (a) and fin displays (b) to large and small females in unrestricted Trial 3. Asterisk indicates significant difference, p < 0.007 (a) and p < 0.04 (b).
larger female association zone in the first dichotomous choice trials (mean proportion of time with large female was 68% versus 32% with small female; t = 3.997, df = 12, p = 0.0018, Fig. 2a) but not the second (mean proportion of time with large female was 58% versus 42% with small female; t = 1.25, df = 11, p = 0.237, Fig. 2b). Proportion of time spent with the larger female was not predicted by her absolute belly width or by belly width as a proportion of standard length in either Trial 1 (width: F = 1.23, df = 11, p = 0.291; proportional width: F = 3.17, d = 11, p = 0.102) or Trial 2 (width: F = 0.136, d = 10, p = 0.72; proportional width: F = 0.172, d = 10, p = 0.687). In the unrestricted trial (Trial 3), males approached the large female significantly more than the small female (t = 3.34, df = 11, p = 0.0065, Fig. 3a). Males also displayed their fins significantly more to the larger female in the unrestricted trial (V = 66.5, p = 0.034, Fig. 3b). Of the two male behaviors that occurred frequently in the unrestricted trial (i.e., approaches, fin displays), only proportion of fin
displays to the larger female was significantly predicted by normalized size difference of the stimulus female pair (R-squared = 0.42, F = 9.07, p = 0.013, Fig. 4). That is, the more the stimulus females differed in size, the more the male disproportionately directed fin displays toward the larger female. 4. Discussion Our results demonstrate a potential role for male mating preference in sexual selection and trait evolution in darters. We showed that males spent significantly more time with large females in a dichotomous mate choice trial, but this significance was not repeatable; males did not show a significant preference in the ensuing dichotomous trial. However, a significant preference appeared when males were given subsequent access to the females in unrestricted trials. These results suggest that dichotomous mate choice trials provide a reliable indication of mate choice, even if they are not repeatable in the short term. 3
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sustain a preference over the course of multiple experiments. A third possible explanation is conditioned place preference (CPP), which occurs when an animal develops a preference for a spatial context that is associated with a reward (e.g., a mate) and is motivated to return to the space associated with the reward (Pfaus et al., 2001). In a study of CPP in fish, for example, individuals presented with an appetitive stimulus (pellets) significantly increased the time spent and distance moved in the reward side, while fish exposed to an aversive stimulus significantly decreased the time spent in the stimulation side (Millot et al., 2014). Given that males in our study preferred larger females in the unrestricted trials that followed the second dichotomous trial, males might have developed a place preference associated with the larger female in Trial 1 and returned to that place in Trial 2. CPP might explain why males spent more time with the larger female in Trial 2 but the difference was not significant; a considerable amount of time may have been spent initially inspecting the previous location of the large female. If this hypothesis is true, then dichotomous choice studies in which individuals are tested twice, with stimulus sides alternated to control for side bias (e.g., Williams and Mendelson, 2010), may need to consider potential effects of CPP. We also found that the proportion of fin displays directed to the large female in the unrestricted trial was greater overall and also greater when females differed more in size. Male darters frequently erect their dorsal fins while competing with other males (pers. obs.); this behavior reveals the striking color patterns that characterize the dorsal fins of most darter species. Demonstrating that males also fin display toward females in the absence of males, and disproportionately to the female with whom they spent more time associating in a dichotomous trial, suggests that fin displays are not only an intrasexual competitive signal but also a courtship signal. Many communication signals are known to serve this dual purpose (e.g., Berglund et al., 1996), but the relative importance of fin displays in these two communication contexts is yet unknown for darters. Previous studies of mating in E. caeruleum suggest that intrasexual competition may be more important than female preference in determining male mating success (Fuller, 2003). If this is true, then males might not be able to express a mate preference in nature, or under more realistic social conditions in the laboratory, due to interference from competing males. A future study that adds an additional trial, in which two males must compete for females, could indicate the extent to which male-male interference may mask male mate choice in nature. In sum, our study provides the first evidence that male darters do not mate randomly within species but rather base their mating decisions on criteria such as female size. Our study also suggests a caveat in experimental design, specifically questioning whether dichotomous mate choice trials conducted in close succession might obscure a true association preference. Results therefore expand our framework of sexual selection in this sexually dichromatic fish to add male choice as well as female choice to the dynamics of sexual selection in darter species.
Fig. 4. Relationship between percent difference in female length and proportion of fin displays to larger females.
Etheostoma caeruleum appears to have a typical operational sex ratio and traditional sex roles, in that females produce a limited number of large, nutritious eggs compared to millions of sperm produced by males; male E. caeruleum are highly ornamented relative to females; and, males spend considerable amount of time during the breeding season intensely competing with other males for access to females. However, neither sex provides parental care, both sexes mate multiply, and males invest considerable energy in securing a mate, both in competing and courting, suggesting suitable conditions for male choice (e.g., Edward and Chapman, 2011). Previous studies have shown that males of many darter species prefer conspecific over heterospecific females (see above). Our study suggests that males in at least one species of this sexually dimorphic lineage are choosy within species as well, preferring larger females. A previous study of mate choice in the Roanoke Darter (Percina roanoka) found that neither males nor females preferred larger conspecific members of the opposite sex in dichotomous mate choice trials (Ciccotto et al., 2014). Why neither sex of P. roanoka preferred larger mates is unclear; fish were collected and tested in the mating season, and the experimental setup was the same as in the present study. One possible explanation is that mate choice in P. roanoka is based on traits other than size, though the same study also ruled out several aspects of color variation as explanatory variables. The two species in question belong to different genera, and species of Percina generally exhibit less striking nuptial coloration and less dramatic sexual dimorphism, though P. roanoka is among the more colorful species of the genus (e.g., Page, 1983). One hypothesis is that sexual selection is less intense in Percina as compared to Etheostoma, but to conclusively explain the difference in results between the two species will require further hypothesis testing. One important result of our study is that a significant difference in association time with the larger female was not repeated in the second dichotomous mate choice trial. Bell et al. (2009) conducted a metaanalysis of repeatability in studies of animal behavior and found that mate preference was among the least repeatable classes of behavior, particularly so for ectotherm vertebrates studied in the lab. Interestingly, their study also showed that male mate preference is much less repeatable than female mate preference. One explanation for the lack of repeatability in our study is that our sample size in Trial 2 lacked the power to detect a preference. The effect size (Cohen’s d) for the second trial was 0.72, which is not large but could indicate a lack of power. Another explanation is that visual signals alone are insufficient to
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