Male-driven reproductive and agonistic character displacement in darters and its implications for speciation in allopatry

Male-driven reproductive and agonistic character displacement in darters and its implications for... Selection against hybridization can cause mating traits to diverge between species in sympatry via reproductive character displacement (RCD). Additionally, selection against interspecific fighting can cause aggressive traits to diverge between sympatric species via agonistic character displace- ment (ACD). By directly affecting conspecific recognition traits, RCD and ACD between species can also incidentally cause divergence in mating and fighting traits among populations within a species [termed cascade RCD (CRCD) and cascade ACD]. Here, we demonstrate patterns consistent with male-driven RCD and ACD in 2 groups of darters (orangethroat darter clade Ceasia and rainbow darter Etheostoma caeruleum). In both groups, males that occur in sympatry (between Ceasia and E. caeruleum) have higher levels of preference for mating and fighting with conspecifics over heter- ospecifics than do males from allopatry. This is consistent with RCD and ACD. We also found patterns consistent with CRCD and cascade ACD among species of Ceasia. Ceasia males that are sympatric to E. caeruleum (but allopatric to one another) also have heightened preferences for mat- ing and fighting with conspecific versus heterospecific Ceasia. In contrast, Ceasia males that are allopatric to E. caeruleum readily mate and fight with heterospecific Ceasia. We suggest that RCD and ACD between Ceasia and E. caeruleum has incidentally led to divergence in mating and fight- ing traits among Ceasia species. This study is unique in that male preferences evolve via both RCD (male preference for conspecific females) and ACD (male preference to fight conspecific males) which leads to subsequent divergence among allopatric lineages. Key words: agonistic character displacement, behavioral isolation, cascade reinforcement, reinforcement, reproductive charac- ter displacement, speciation. Reproductive interference between species can cause mating traits et al. 2003; Hoskin et al. 2005; Higgie and Blows 2007, 2008; (signals and/or preferences) to diverge via reproductive character Lemmon 2009; Porretta and Urbanelli 2012; Bewick and Dyer displacement (RCD; Howard 1993; Servedio and Noor 2003). RCD 2014; Pfennig and Rice 2014; Kozak et al. 2015). is often confirmed by a pattern of enhanced behavioral isolation be- Selection against interspecific aggression can also lead to the evo- tween 2 species in sympatry compared with allopatry. Recent re- lution of traits involved in species recognition. Maladaptive inter- search suggests that secondary effects of RCD in sympatry can also specific fighting over resources (such as mates) can cause shifts in initiate divergence between allopatric lineages (Pfennig and Pfennig aggressive signals and behavior via agonistic character displacement 2009; Hoskin and Higgie 2010). Cascade RCD (hereafter CRCD; (ACD; Grether et al. 2009; Okamoto and Grether 2013). A pattern Ortiz-Barrientos et al. 2009) occurs when behavioral isolation of divergent ACD is said to be present when 2 species are less likely evolves among populations within a species as a correlated effect of to engage in contests when they occur in sympatry compared with RCD. CRCD has been documented in a variety of taxa (e.g., Nosil allopatry. Both RCD and ACD may contribute to trait divergence V C The Author (2017). Published by Oxford University Press. 101 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/cz/article-abstract/64/1/101/4665098 by Ed 'DeepDyve' Gillespie user on 16 March 2018 102 Current Zoology, 2018, Vol. 64, No. 1 Figure 1. Ranges for Etheostoma caeruleum and 5 Ceasia species (Etheostoma spectabile, Etheostoma pulchellum, Etheostoma fragi, Etheostoma uniporum, and Etheostoma burri) used in behavioral assays in the current study and in Moran et al. (2017). Numbers on the map represent approximate collection locations for study populations (see Table 1 for details). between species that results in decreased heterospecific interactions diverged approximately 22 million years ago (Near et al. 2011). in sympatry. Although numerous studies have shown that RCD can Time calibrated gene trees indicate that Ceasia subsequently diversi- incidentally lead to divergence in mating traits among populations fied 6–7 million years ago (Bossu et al. 2013). The Ceasia clade con- within species via CRCD, whether selection against interspecific sists of 15 species, all of which are allopatric with respect to one aggression can also cause divergence in agonistic traits among popu- another (Ceas and Page 1997; Bossu and Near 2009). Phylogenetic lations within species (i.e., cascade ACD, hereafter CACD) has yet and palaeogeographical analyses support allopatric divergence of to be determined. this clade (Bossu et al. 2013). Twelve Ceasia species occur in sym- Distinguishing between RCD and ACD is essential to determin- patry with respect to E. caeruleum throughout their range, and 2 ing the underlying selective pressure (i.e., heterospecific mating or Ceasia species occur in allopatry with respect to E. caeruleum fighting) and relative contribution of male–female and male–male throughout their range (see Bossu and Near 2009; Page and Burr interactions in driving speciation. However, disentangling the im- 2011). The one remaining Ceasia species (orangethroat darter portance of RCD versus ACD to speciation can be difficult because Etheostoma spectabile) occurs in both sympatry and allopatry with many sexually selected traits are used in both female mate choice respect to E. caeruleum (Figure 1). Within Ceasia, time since diver- and male–male competition over mates (Alatalo et al. 1994; gence does not differ significantly between lineages that occur in Berglund 1996; Sætre et al. 1997; Dijkstra et al. 2007; Saether et al. sympatry versus allopatry with respect to E. caeruleum (Bossu et al. 2007; Lackey and Boughman 2013; Tinghitella et al. 2015). Here, 2013). Ceasia and E. caeruleum have similar male coloration, mat- we examine female mating preferences, male mating preferences, ing behavior, and ecology. There is little evidence that male color- and male–male aggression (MA) to test for patterns consistent with ation in either Ceasia or E. caeruleum is the target of female mate RCD, ACD, CRCD, and CACD. choice; females lack preferences for either male size or color pattern This study focuses on 2 groups of darters in the subgenus within species, and Ceasia females lack preferences for conspecific Oligocephalus: the orangethroat darter clade Ceasia and the over heterospecific Ceasia and E. caeruleum males (Pyron 1995; rainbow darter Etheostoma caeruleum. Ceasia and E. caeruleum Fuller 2003; Zhou et al. 2015; Moran et al. 2017). Instead, there is Downloaded from https://academic.oup.com/cz/article-abstract/64/1/101/4665098 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Moran and Fuller  Reproductive and agonistic character displacement in darters 103 Table 1. Collection locations for populations of each species examined in behavioral trials in the present study as well as in Moran et al. (2017) Range Geography Species Collection location Drainage information Source of behav- map popu- ioral data lation number 1 Allopatric E. caeruleum 42.426825, 85.428370 Prairieville Creek, Kalamazoo River, Barry Present study County, MI 2 Sympatric E. spectabile 40.054447, 88.089887 Unnamed tributary, Salt Fork of Vermillion Present study and River, Champaign County, IL Moran et al. (2017) 3 Sympatric E. caeruleum (Same as above) (Same as above) Present study and Moran et al. (2017) 4 Allopatric E. spectabile 40.027663, 88.577180 Unnamed tributary, Sangamon River, Piatt Present study County, IL 5 Allopatric E. pulchellum 38.952839, 95.517654 Deer Creek, Kansas River, Shawnee County, KS Present study 6 Sympatric E. fragi 36.304214, 91.927684 Rose Branch tributary of Strawberry River, Moran et al. Fulton County, AR (2017) 7 Sympatric E. uniporum 36.250560, 91.359318 Unnamed tributary of Spring River, Sharp Moran et al. County, AR (2017) 8 Sympatric E. caeruleum 36.065396, 91.610420 Mill Creek tributary of Strawberry River, Sharp Moran et al. County, AR (2017) 9 Sympatric E. burri 37.146415, 90.907459 North Fork Webb Creek, Black River Drainage, Moran et al. Wayne County, MO (2017) Notes: Sympatry and allopatry refer to the geographic relationship between Ceasia and E. caeruleum (all species of Ceasia are allopatric from one another). Range map population number refers to numbers shown in Figure 1. Etheostoma caeruleum study population used in sympatric comparisons with Ceasia species from the Ozarks regions (i.e., E. fragi, E. uniporum, and E. burri) in Moran et al. (2017). strong evidence that male coloration is under intrasexual selection also influence species diversification at a macroevolutionary scale and functions as an aggressive signal in male–male competition over (Pfennig and Pfennig 2012; Grether et al. 2017). Over time, CRCD access to females (Zhou and Fuller 2016; Moran et al. 2017). and CACD can cause isolated populations within a species to diverge Several recent studies have indicated that RCD and ACD are likely from one another to such an extent that they merit classification as occurring in this system. First, hybridization occurs between Ceasia distinct, allopatric species. The outcome of this process can result in a and E. caeruleum in nature (Bossu and Near 2009; Moran et al. complex of closely related, allopatric species that exhibit enhanced 2017), and their hybrids have reduced fitness (Zhou 2014; Moran R, mating trait divergence with one another (via CRCD/CACD), and unpublished data), providing the potential for RCD to occur via re- with a more distantly related sympatric species (via RCD/ACD). inforcement (Brown and Wilson 1956; Coyne and Orr 2004). Second, In this manner, CRCD and CACD can fuel hierarchical “speciation in pairings between 4 species of Ceasia and sympatric E. caeruleum, cascades” among allopatric lineages at multiple taxonomic levels sim- males preferentially mate and fight with conspecifics, suggesting RCD ultaneously (Pfennig and Ryan 2006). We hypothesize that this scen- and ACD (Figure 1 and Table 1; Moran et al. 2017). Third, a pattern ario is ongoing in the Ceasia–E. caeruleum system. consistent with RCD was observed in a no-choice mating experiment To test for RCD and ACD, we measured preferences for mating which found that allopatric pairings of female E. spectabile and male and fighting with conspecifics in pairings between E. spectabile and E. caeruleum yielded more eggs than sympatric pairings (Zhou and E. caeruleum that occur in sympatry versus allopatry with respect to Fuller 2014). Zhou and Fuller (2014) is the only study to date to com- one another. This allowed us to examine whether patterns consistent pare sympatric and allopatric pairings between a Ceasia species and with RCD and ACD are present at the population level within E. caeruleum, but the no-choice assay they used was not able to meas- E. spectabile and E. caeruleum. Additionally, we measured prefer- ure the contribution of each sex to behavioral isolation in sympatry. ences for mating and fighting with conspecifics in pairings between Furthermore, Zhou and Fuller (2014) did not consider male competi- Etheostoma pulchellum and E. caeruleum that occur in allopatry tion, and could not test for ACD. with respect to one another (Figure 1 and Table 1). Because E. pul- A unique aspect of this study system is that it allows us to test for chellum and E. caeruleum do not co-occur, these species should patterns consistent with RCD and ACD at 2 taxonomic levels within show a reduced level of bias against mating and fighting with one Ceasia: populations within a species, and closely related species another compared with species of Ceasia and E. caeruleum that do within a recently diverged clade. We first tested for RCD and ACD co-occur. Measuring mating and fighting biases in allopatric pair- between populations of a single species of Ceasia as a function of sym- ings of Ceasia and E. caeruleum thus serves as a critical test against patry with E. caeruleum. We next asked whether RCD and ACD are which we can compare levels of behavioral preferences in sympatric present between species of Ceasia as a function of sympatry with pairings of Ceasia and E. caeruleum that were previously reported E. caeruleum. Most studies involving RCD and ACD have considered by Moran et al. (2017). differences in mating traits between populations within a pair of spe- We also investigated whether patterns consistent with CRCD cies as a function of sympatry versus allopatry. However, RCD can and CACD are present among Ceasia species. Males within the Downloaded from https://academic.oup.com/cz/article-abstract/64/1/101/4665098 by Ed 'DeepDyve' Gillespie user on 16 March 2018 104 Current Zoology, 2018, Vol. 64, No. 1 4 Ceasia species examined by Moran et al. (2017; Figure 1 and Ceasia and sympatric E. caeruleum, and pairings between sympatric Table 1), which all occur in sympatry with respect to E. caeruleum, Ceasia species, in Moran et al. (2017; Figure 1 and Table 1). prefer conspecific over heterospecific Ceasia females and bias their Fish were collected with a kick seine in March 2016 and April aggression preferentially toward conspecific over heterospecific 2017 and transported back to the laboratory at the University of Ceasia males. This divergence in male mating and fighting traits Illinois at Urbana-Champaign in aerated coolers. Fish were sepa- among Ceasia species is not associated with differences in male color rated into stock aquaria according to population and sex, and were pattern or genetic distance. Therefore, RCD and ACD between fed daily ad libitum with frozen bloodworms. Stock aquaria were Ceasia and E. caeruleum may have incidentally contributed to spe- maintained at 19 C and fluorescent lighting was provided to mimic cies divergence within the Ceasia clade via CRCD and CACD. To the natural photoperiod. test this hypothesis, we examine preferences for mating and fighting with conspecifics (over a heterospecific member of the Ceasia clade) Testing for RCD and ACD between Ceasia and E. in pairings between E. spectabile and E. pulchellum that occur in al- caeruleum lopatry with respect to E. caeruleum. We then ask whether E. spec- Dichotomous MC assay tabile and E. pulchellum have lower levels of preference for mating We first used a dichotomous MC assay to test for RCD in male mate and fighting with conspecifics compared with that previously choice. Each trial included a focal male E. spectabile or E. pulchel- observed between pairs of Ceasia species that occur in sympatry lum with a conspecific female and a heterospecific (E. caeruleum) fe- with respect to E. caeruleum (Moran et al. 2017). male (Figure 2A). This assay allowed males to choose between (1) sympatric E. spectabile and sympatric E. caeruleum, (2) allopatric E. spectabile and allopatric E. caeruleum, and (3) allopatric E. pul- Materials and Methods chellum and allopatric E. caeruleum females (n ¼ 12 each). RCD predicts that preferences for conspecific mates should be higher in Mating system details sympatric E. spectabile focal males than both allopatric E. specta- During the spring spawning season, Ceasia and E. caeruleum travel bile and allopatric E. pulchellum focal males. to shallow gravel riffles in headwater streams (Hubbs and Strawn Behavioral trials occurred in 38 L test aquaria filled with 5 cm of 1957; Hubbs 1985). Females look for a suitable place to lay eggs by naturally colored aquarium gravel. To minimize disturbance to the performing “nosedigs” in which they jab their snout into the gravel. fish, test aquaria were covered with black opaque plastic on 3 sides. One to several males swim in tandem with a female as she searches We used unique fish in each trial, chosen haphazardly from stock for a spawning location. Males fight aggressively to ward off rival tanks. Females in each trial were size matched to within 10% of males by actively chasing them off and/or by flaring their dorsal and their total body length. Each trial began by placing the 3 fish being anal fins in a threat display. When the female is ready to spawn, she tested into a test aquarium and allowing them to acclimatize for dives into the substrate, leaving only her head and caudal fin fully 5 min. The trial then began and lasted 30 min. Each trial was broken visible. Spawning initiates when a male positions himself above the up into 60 30-s blocks (Zhou et al. 2015; Moran et al. 2017). female, and they release sperm and eggs into the substrate. We examined male mate choice by measuring focal male pursuit of Spawning often involves multiple males mating simultaneously with each female in each trial. Male pursuit of a female is highly predictive 1 female, and males sometimes exhibit sneaking behavior. Females of spawning in Ceasia and in E. caeruleum (Zhou et al. 2015; Moran will ovulate clutches of up to 200 eggs throughout the spawning sea- et al. 2017). A male was scored as having pursued a female during a son, but only release a few eggs per spawning bout (Heins et al. 30-s block if he spent a minimum consecutive time of 5-s within one 1996; Fuller 1998). Hence, the female must spawn multiple times to body length of the female. We calculated a focal male mate choice be- fertilize all the eggs from a given clutch. havioral variable from this data as described in Table 2. We performed analyses using proportional data (i.e., the behav- ioral variables described in Table 2) that varied from 0 to 1. A score Study species/populations and collection locations of 1 indicates only conspecific interactions occurred, 0.5 indicates All Ceasia species occur in allopatry with respect to one another. an equal number of interactions between conspecifics and heterospe- Throughout the rest of this paper, the terms “allopatric” and “sym- cifics, and 0 indicates only heterospecific interactions occurred. patric” refer to the geographic relationship between Ceasia and However, for ease of interpretation, we graphed the raw number of E. caeruleum (not between Ceasia species). To test for RCD and behaviors observed. ACD between E. spectabile and E. caeruleum, we examined prefer- We used analysis of variance (ANOVA) to test for RCD in male ences for mating and fighting with conspecifics over heterospecifics mating preference by asking whether focal male mate choice differed in pairings between allopatric E. spectabile and allopatric E. caeru- among the focal Ceasia study populations (i.e., sympatric E. specta- leum versus pairings between sympatric E. spectabile and sympatric bile, allopatric E. spectabile, and allopatric E. pulchellum). We E. caeruleum (Figure 1 and Table 1). We also tested for a pattern included focal male mate choice as the dependent variable, and focal consistent with RCD and ACD in pairings between allopatric E. pul- male population identity as the independent variable. We then used chellum and allopatric E. caeruleum (Figure 1 and Table 1). Finally, post hoc t-tests to directly compare populations. We also asked we tested for a pattern consistent with CRCD and CACD among whether focal male mate choice differed from a null expectation of Ceasia species by pairing allopatric E. spectabile with allopatric 0.5 (equal amounts of time spent with each female) in each popula- E. pulchellum (Figure 1 and Table 1). tion using one-sample t-tests. We used 2 types of behavioral assays [“dichotomous male choice (MC) assay” and “male competition assay,” detailed below] to com- pare preferences for engaging in mating and fighting with conspe- Male competition assay cifics versus heterospecifics. We then compared these behavioral We conducted a second type of assay in which males could compete measurements to those documented in pairings between sympatric with one another to test for RCD and ACD. This assay paired (1) Downloaded from https://academic.oup.com/cz/article-abstract/64/1/101/4665098 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Moran and Fuller  Reproductive and agonistic character displacement in darters 105 in these species is complex and varies within populations (Zhou et al. 2014), allowing us to distinguish conspecific males. Males in each trial were size matched within 10% of their total body length to control for any larger differences in color pattern and competitive ability associated with body size (Zhou et al. 2014). In each trial, we measured the behavior of the focal female, the focal male, and the rival male. Due to low collection numbers, some allopatric E. caeru- leum males were used twice, but never more than once on the same day or with the same Ceasia study population. To test for ACD, we recorded the number of aggressive behaviors (i.e., fin flares and attacks) that both males in a trial directed toward the other male. We calculated 4 behavioral variables to quantify male aggressive bias toward conspecific males: focal male fin flare bias, focal male attack bias, rival male fin flare bias,and rival male attack bias (see Table 2). We asked whether these behavioral variables dif- fered in sympatric versus allopatric pairings. To examine focal male Ceasia aggressive behavior, we conducted 2 separate ANOVAs with focal male fin flare bias and focal male attack bias as the dependent variables, and focal Ceasia male identity (sympatric E. spectabile, allopatric E. spectabile,or allopatric E. pulchellum) as the independ- ent variable in both analyses. Similarly, to examine the aggressive be- havior of E. caeruleum rival males relative to Ceasia rival males, we conducted ANOVAs with rival male fin flare bias and rival male attack bias as dependent variables, and focal Ceasia male identity as the independent variable. Additionally, we made pairwise compari- sons among groups using post hoc 2-sample t-tests. To test for RCD in male mate preference, we split each male competition trial into 60 30-s blocks (as in the dichotomous MC tri- als), and counted the number of 30-s blocks in which each male pur- sued the female. Unlike the dichotomous MC assay, the male competition assay considers the preference of male E. caeruleum for E. spectabile and E. pulchellum females. We calculated rival male Figure 2. Setup for behavioral experiments. (A–C) Trials testing for RCD and mate choice as described in Table 2. As focal males were always ACD. In these trials, sympatric E. spectabile, allopatric E. spectabile, and allo- paired with conspecific females in the male competition trials, we patric E. pulchellum served as focal Ceasia in turn. Note that in (A) and (C), did not measure focal male mate choice in these trials. The male allopatric E. caeruleum were paired with allopatric focal Ceasia, and sympat- competition assay presented males with a no-choice situation, where ric E. caeruleum were paired with sympatric focal Ceasia. (A) Experimental setup for dichotomous MC trials that tested for RCD in focal Ceasia male they could choose whether to pursue a female. This assay also exam- mate choice. (B–C) Experimental set up for male competition trials that tested ined male mate preference in the presence of a male competitor, for patterns consistent with RCD in E. caeruleum rival male mate preference, which is closer to what a male would experience in nature during RCD in focal Ceasia female mate preference, ACD in focal Ceasia male ag- the spawning season. We asked whether rival male mate choice dif- gressive behavior, and ACD in E. caeruleum rival male aggressive behavior. fered between sympatric and allopatric trial sets. We conducted an (D–E) Trials testing for CRCD and CACD. In these trials, allopatric E. spectabile ANOVA with rival male mate choice as the dependent variable and and allopatric E. pulchellum acted as focal Ceasia and as heterospecific trial set (i.e., sympatric E. spectabile, allopatric E. spectabile,or Ceasia in turn. (D) Experimental set up for dichotomous MC trials that tested for patterns consistent with CRCD in focal Ceasia male mate choice. (E) allopatric E. pulchellum as the focal pair) as the independent vari- Experimental set up for male competition trials that tested for patterns con- able, followed by pairwise post hoc 2-sample t-tests. sistent with CRCD in heterospecific Ceasia rival male mate preference, CRCD Finally, we tested for RCD in female mating preferences. The in focal Ceasia female mate preference, and CACD in focal Ceasia male and setup of the male competition assay was equivalent to a dichotom- heterospecific Ceasia rival male aggressive behavior. We did not repeat male ous female choice (FC) assay. We counted the number of nosedigs a competition trials in which a conspecific Ceasia acted as the rival male female performed towards the rival male in each trial. Females typic- (shown in B). We compared the behavior of individuals in trials with a con- specific Ceasia rival male (B) to individuals in trials with an E. caeruleum rival ally perform nosedigs directly before spawning, and this behavior is male (C). We also compared the behavior of individuals in trials with a con- often used to measure female mating preferences in darters (Fuller specific Ceasia rival male (B) to individuals in trials with a heterospecific 2003; Williams and Mendelson 2011; Zhou et al. 2015; Zhou and Ceasia rival male (E). Fuller 2016). We asked whether focal female mate choice (Table 2) differed among sympatric E. spectabile, allopatric E. spectabile, and sympatric E. spectabile and sympatric E. caeruleum, (2) allopatric allopatric E. pulchellum using ANCOVA. The model included focal E. spectabile and allopatric E. caeruleum, and (3) allopatric E. pul- female mate choice as the dependent variable and focal female iden- chellum and allopatric E. caeruleum (n ¼ 12 each). Each trial tity as the independent variable. We included the proportion of time included a focal male and a focal female pair from the same Ceasia that conspecific rival males pursued the focal female as a covariate study population. Each focal Ceasia pair was observed once with a in the analysis, as male pursuit has been shown to predict female rival male that was conspecific to them (Figure 2B), and once with a nosedigs and spawning (Zhou et al. 2015; Moran et al. 2017). rival male that was an E. caeruleum (Figure 2C). Male color pattern We also used ANCOVA to test for focal female mate preference Downloaded from https://academic.oup.com/cz/article-abstract/64/1/101/4665098 by Ed 'DeepDyve' Gillespie user on 16 March 2018 106 Current Zoology, 2018, Vol. 64, No. 1 Table 2. Definition of the behavioral variables measured in the dichotomous MC assay and the male competition assay Variable Definition RCD ACD CRCD CACD Dichotomous MC assay (2 females, 1 male) Focal Male Mate Number of time blocks spent pursuing the conspecific divided by the Yes NA Yes NA Choice total number of time blocks spent pursuing either female. Male competition assay (2 males, 1 female) Rival Male Mate Proportion of time blocks the focal female was pursued by conspecific Yes NA Yes NA Choice versus heterospecific rival males across 2 trials ¼ Number of time blocks conspecific rival male pursued the female/(sum of time blocks the conspecific and heterospecific rivals pursued the female). Focal Female Proportion of nosedigs towards conspecific versus heterospecific rival No NA No NA Mate Choice males across 2 trial ¼ Number of nosedigs toward conspecific rivals/ (sum of nosedigs toward conspecific and heterospecific rivals); the analysis of this variable was corrected for male pursuit. Focal Male Fin Proportion of fin flares toward conspecific versus heterospecific rivals NA Yes NA Yes Flare Bias across 2 trials ¼ Number of fin flares to conspecific rival/(sum of fin flares to conspecific and heterospecific rivals). Focal Male Attack Proportion of attacks toward conspecific versus heterospecific rivals NA Yes NA Yes Bias across 2 trials ¼ Number of attacks on conspecific rival/(sum of at- tacks on conspecific and heterospecific rivals). Rival Male Fin Proportion of fin flares performed by conspecific versus heterospecific NA Yes NA Yes Flare Bias rivals across 2 trials ¼ Number of fin flares by conspecific rival to- ward the focal male/(sum of fin flares by conspecific and heterospe- cific rivals toward the focal male). Rival Male Attack Proportion of attacks performed by conspecific versus heterospecific NA Mixed NA Yes Bias rivals across 2 trials ¼ Number of attacks by conspecific rival to- ward the focal male/(sum of attacks by conspecific and heterospe- cific rivals towards the focal male). Notes: We indicate whether we observed a pattern consistent with predictions for RCD, ACD, CRCD, and CACD for each behavioral variable, or whether the be- havioral variable was not applicable (NA) to testing a given prediction. Allopatric E. caeruleum males tended to attack allopatric E. spectabile males more than sympatric E. caeruleum males attacked sympatric E. spectabile males, but no other differences were found. for conspecific rival males versus E. caeruleum rival males. differed from a null expectation of 0.5 (equal amounts of time spent The number of nosedigs the focal female directed toward each rival with each female) using one-sample t-tests. male was the independent variable, the rival male’s identity (conspe- cific or E. caeruleum) was the dependent variable, and the propor- Male competition assay tion of time the rival male spent in pursuit of the female was We also conducted a male competition assay between allopatric included as a covariate. We note that although the females’ ability E. spectabile and allopatric E. pulchellum to test for patterns con- to exert mating preferences may be precluded by the outcome of sistent with CRCD and CACD. Earlier work showed that Ceasia male contests, male competition over females is pervasive in these males that are sympatric with E. caeruleum prefer to mate and fight species, so this assay reflects what females most frequently encoun- with conspecifics over heterospecific Ceasia (Moran et al. 2017). ter in nature. Here, we asked whether Ceasia males that are allopatric with re- spect to E. caeruleum lacked such preferences. We performed trials Testing for CRCD and CACD between Ceasia species in which both allopatric E. spectabile and allopatric E. pulchellum Dichotomous MC assay acted as the focal pair and as the heterospecific rival male in turn To test for patterns consistent with CRCD within Ceasia, we paired (n ¼ 12 each; Figure 2E). CRCD and CACD predict that allopatric allopatric E. spectabile with allopatric E. pulchellum in a dichotom- E. spectabile and allopatric E. pulchellum should show similarly ous MC assay. We conducted this assay in the manner described low levels of preference for mating and fighting with conspecifics above to test for RCD, but here the heterospecific female was an over heterospecifics. We measured rival male mate choice, and focal allopatric E. spectabile or allopatric E. pulchellum, in place of an female mate choice, focal male fin flare bias, focal male attack bias, E. caeruleum (Figure 2D). We performed trials in which allopatric rival male fin flare bias, and rival male attack bias as described in E. spectabile acted as the focal male and conspecific female, with Table 2. We conducted ANOVAs as described above for the male E. pulchellum as the heterospecific female, and vice versa (n ¼ 12 competition trials that tested for RCD and ACD, but with the ap- each). CRCD predicts no significant difference between allopatric propriate species (i.e., E. spectabile or E. pulchellum) in place of E. spectabile and allopatric E. pulchellum in focal male mate choice E. caeruleum as the heterospecific rival male. (Table 2). To compare focal male mate choice between these species, We used ANOVA to test for RCD, ACD, CRCD, and CACD in we conducted ANOVAs that included focal male mate choice as the both sets of dichotomous MC and male competition assays. dependent variable and focal male identity (allopatric E. spectabile Repeating all analyses using generalized linear models with a quasi- or allopatric E. pulchellum) as the independent variable. We also binomial error function and logit link function yielded qualitatively tested whether focal male mate choice for the conspecific female identical results. Downloaded from https://academic.oup.com/cz/article-abstract/64/1/101/4665098 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Moran and Fuller  Reproductive and agonistic character displacement in darters 107 Table 3. Results of ANOVA testing for RCD in focal Ceasia male mate choice between conspecific females and E. caeruleum females in di- chotomous MC male trials Focal male mate choice df Test statistic P Focal Ceasia population identity 2, 33 45.21 <0.00001 Sympatric E. spectabile versus allopatric E. spectabile 22 11.38 <0.00001 Sympatric E. spectabile versus allopatric E. pulchellum 22 8.10 <0.00001 Allopatric E. spectabile versus allopatric E. pulchellum 220 0.38 0.71 Notes: We asked focal male mate choice differed among focal Ceasia males in 3 study populations: sympatric E. spectabile, allopatric E. spectabile, and allopatric E. pulchellum. Pairwise post-hoc t-test results are also shown for the analysis. Behavioral isolation indices occur in sympatry versus allopatry. This would indicate that sym- We used the MA, male mate choice, and female mate choice data patric males bias their aggression more toward conspecifics over from both sets of male competition assays (i.e., those testing for heterospecifics. RCD and ACD, and those testing for CRCD and CACD) to calcu- Second, we tested for differences between Ceasia and Ceasia late 3 behavioral isolation indices following Moran et al. (2017). species pairs that occur in sympatry versus allopatry with respect Behavioral isolation indices were calculated individually for each to E. caeruleum. CRCD predicts higher MC and FC indices in trial and then averaged across all replicates within each species com- Ceasia–Ceasia pairings that occur in sympatry with respect to parison. These indices allowed for a comparison of levels of prefer- E. caeruleum, indicating enhanced mate preference for conspecific ence for mating and fighting with conspecifics over heterospecifics over heterospecific Ceasia. Likewise, CACD predicts higher MA in- at a macroevolutionary scale among Ceasia–E. caeruleum and dices in Ceasia–Ceasia pairings that occur in sympatry with respect Ceasia–Ceasia species pairs. Indices range from 1 (complete pref- to E. caeruleum. This would indicate that Ceasia males that occur in erence for heterospecifics) to 1 (complete preference for conspe- sympatry with respect to E. caeruleum bias their aggression more to- cifics), with 0 indicating no preference for conspecifics versus ward conspecific males versus heterospecific Ceasia males. heterospecifics (Stalker 1942; Martin and Mendelson 2016; Moran For all analyses, we used Type III sums of squares using the et al. 2017). “car” package in R (version 3.4.0). Raw data have been deposited in We calculated MA indices for each species pair as: Dryad (doi:10.5061/dryad.g8d1v). a  a c h MA ¼ ; a þ a c h Results where a and a represent the combined number of fin flares and at- c h tacks performed between conspecific males and between heterospe- RCD between Ceasia and E. caeruleum cific males, respectively. The dichotomous MC trials revealed a pattern consistent with RCD We calculated MC indices as: in focal Ceasia male mate preference. RCD predicts that MC for conspecifics should be heightened in Ceasia populations/species that m  m c h MC ¼ ; are sympatric with respect to E. caeruleum. Focal male mate choice m þ m c h was 2 higher in sympatric E. spectabile compared with allopatric where m and m represent the proportion of time in each trial that c h E. spectabile and allopatric E. pulchellum, but did not differ be- conspecific males and heterospecific males spent pursuing the Ceasia tween allopatric E. spectabile and allopatric E. pulchellum (Table 3 female. and Supplementary Figure S1a). In addition, focal male mate choice As previous studies have indicated that male pursuit of a female was much greater than the null expectation of 0.5 in trials with is highly correlated with female nosedigs (a measure of female mat- sympatric E. spectabile serving as the focal male (mean6 SE: ing preference), FC indices controlled for male pursuit of the female. 0.976 0.01; one-sample t-test: t ¼ 51.58, P < 0.00001). We calculated the FC indices as: Conversely, focal male mate choice did not differ from 0.5 in trials where allopatric E. spectabile and allopatric E. pulchellum served f f c h FC ¼  ; as the focal males (Supplementary Figure S1b, c; allopatric E. spec- p p c h tabile mean6 SE: 0.516 0.04; one-sample t-test: t ¼ 0.17, where f and f represent the number of nosedigs females performed c h P ¼ 0.87; E. pulchellum mean6 SE: 0.536 0.05; one-sample t-test: toward conspecific males and toward heterospecific males, respect- t ¼ 0.60, P ¼ 0.56). ively. p and p represent the number of 30-s blocks in which con- c h RCD in male mate preference was also indicated in the male specific males and heterospecific males were scored as having competition trials, which compared E. caeruleum rival male prefer- pursued the female during a trial, respectively. ence for the focal Ceasia female with that of the conspecific Ceasia We used ANOVA to make 2 sets of comparisons among the rival male. RCD predicts that sympatric E. caeruleum males should 3 types of behavioral isolation indices (i.e., MA, MC, and FC). First, be less likely to pursue Ceasia females than allopatric E. caeruleum we tested for differences between Ceasia and E. caeruleum pairs that males. Rival male mate choice differed significantly between sym- occur in sympatry versus allopatry with respect to one another. patric and allopatric E. caeruleum (Supplementary Table S1). In tri- RCD predicts higher MC and FC indices in Ceasia–E. caeruleum als where sympatric E. spectabile served as the focal Ceasia pair, pairings that occur in sympatry versus allopatry, indicating conspecific rival males were much more likely to pursue the focal enhanced mate preference for conspecifics. Similarly, divergent female compared with the sympatric E. caeruleum rival males ACD predicts higher MA indices in Ceasia–E. caeruleum pairs that (Supplementary Figure S2a). In both trials where allopatric Downloaded from https://academic.oup.com/cz/article-abstract/64/1/101/4665098 by Ed 'DeepDyve' Gillespie user on 16 March 2018 108 Current Zoology, 2018, Vol. 64, No. 1 E. spectabile and E. pulchellum served as the focal Ceasia pair, con- fin flares toward conspecific (versus E. caeruleum) rival males specific rival males and allopatric E. caeruleum rival males spent (Supplementary Figure S1d). Similarly, sympatric E. spectabile focal roughly the same amount of time pursuing the focal female males attacked conspecific rival males 6 more than they attacked (Supplementary Figure S2b, c). Hence, allopatric E. caeruleum males sympatric E. caeruleum rival males (Supplementary Figure S1g). On chose to pursue allopatric E. spectabile and allopatric E. pulchellum average, both allopatric E. spectabile and allopatric E. pulchellum females. Sympatric E. caeruleum males largely ignored sympatric focal males directed an equal number of fin flares (Supplementary E. spectabile females. Figure S1e, f) and attacks (Supplementary Figure S1h, i) toward con- We did not find support for RCD in female mating preferences specific rival males and allopatric E. caeruleum rival males. in the male competition trials. When male pursuit was included as a We also found a pattern consistent with divergent ACD in covariate in the analysis, focal female mate choice did not differ E. caeruleum male aggressive behavior. Divergent ACD predicts among the sympatric E. spectabile, allopatric E. spectabile, and allo- that sympatric E. caeruleum rival males should show higher levels of patric E. pulchellum trials (Table 4). Females did not exert prefer- aggression toward focal male Ceasia compared with allopatric ence for conspecific males over E. caeruleum males, regardless of E. caeruleum rival males. Rival male fin flare bias showed a pattern sympatry with respect to E. caeruleum (Supplementary Table S2). like that found with focal Ceasia males (Supplementary Table S3). Sympatric E. caeruleum rival males were much less likely to flare their fins toward E. spectabile focal males compared with allopatric ACD between Ceasia and E. caeruleum E. caeruleum rival males (Supplementary Figure S2d, f). The aggressive behavior of focal Ceasia males in the male competi- Conversely, rival male attack bias did not differ between sympat- tion trials was consistent with divergent ACD. Divergent ACD ric and allopatric E. caeruleum (Supplementary Table S3). Both predicts that Ceasia males that are sympatric with respect to sympatric and allopatric E. caeruleum rival males directed a low E. caeruleum should bias their aggression toward conspecific rival number of attacks toward the focal Ceasia males (Supplementary males over E. caeruleum rival males. Focal male fin flare bias and Figure S2g, i). Thus, while allopatric E. spectabile and allopatric focal male attack bias were higher for sympatric E. spectabile com- E. pulchellum focal males did not bias their aggression more toward pared with allopatric E. spectabile and allopatric E. pulchellum conspecific rival males (versus allopatric E. caeruleum rival males; (Table 5). Sympatric E. spectabile focal males directed 9 more see previous paragraph), allopatric E. caeruleum rival males typic- ally preferred not to attack allopatric E. spectabile and allopatric Table 4. Results ANCOVA testing for RCD in focal Ceasia female E. pulchellum focal males. mate choice between conspecific rival males and E. caeruleum rival males in male competition trials CRCD between Ceasia species Focal female mate choice df Test statistic P CRCD predicts that males from Ceasia species that are sympatric with respect to E. caeruleum should show higher levels of male mate Focal Ceasia population identity 2, 32 0.09 0.92 preference for conspecific females over heterospecific Ceasia fe- Male pursuit 1, 32 0.74 0.40 males, despite the fact that the 2 Ceasia species are allopatric with Notes: We asked whether focal female mate choice differed among focal respect to one another. Moran et al. (2017) showed that in Ceasia Ceasia females in 3 study populations: sympatric E. spectabile, allopatric species that are sympatric with respect to E. caeruleum, male mate E. spectabile, and allopatric E. pulchellum. Male pursuit of the female was preference for conspecific over heterospecific Ceasia females was sur- included as a covariate in the analysis. prisingly high. This study shows that male Ceasia (i.e., E. spectabile Figure 3. Patterns of RCD and ACD between Ceasia and E. caeruleum. Behavioral isolation indices (with 95% confidence intervals) for (A) MA, (B) MC, and (C)FC for comparisons between Ceasia species and E. caeruleum. Allopatric comparisons (i.e., those including Ceasia and E. caeruleum that occur in allopatry with re- spect to one another) are shown in black. Sympatric comparisons (i.e., those including Ceasia and E. caeruleum that occur in sympatry with respect to one an- other) are shown in white. Grouping bars are also used to indicate allopatric species pairs (left) versus sympatric species pairs (right). Significance levels from ANOVAs comparing allopatric and sympatric species pairs are shown. Downloaded from https://academic.oup.com/cz/article-abstract/64/1/101/4665098 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Moran and Fuller  Reproductive and agonistic character displacement in darters 109 Table 5. Results of ANOVA testing for ACD in focal Ceasia MA bias in male competition trials Focal male fin flare bias df Test statistic P Focal Ceasia population identity 2, 33 8.34 0.0012 Sympatric E. spectabile versus allopatric E. spectabile 22 5.28 <0.0001 Sympatric E. spectabile versus allopatric E. pulchellum 22 2.85 0.0093 Allopatric E. spectabile versus allopatric E. pulchellum 22 0.84 0.41 Focal male attack bias df Test statistic P Focal Ceasia population identity 2, 33 9.12 <0.001 Sympatric E. spectabile versus allopatric E. spectabile 22 4.53 0.0002 Sympatric E. spectabile versus allopatric E. pulchellum 22 3.82 <0.001 Allopatric E. spectabile versus allopatric E. pulchellum 22 0.65 0.52 Notes: We asked whether focal male fin flare bias and focal male attack bias differed among focal Ceasia males in 3 study populations: sympatric E. spectabile, allopatric E. spectabile, and allopatric E. pulchellum. Pairwise post-hoc t-test results are also shown for both analyses. and E. pulchellum) that are allopatric with respect to E. caeruleum males compared with the conspecific E. spectabile rival males do not prefer conspecific over heterospecific Ceasia females. In di- (Supplementary Figure S3c). This resulted in a significant difference chotomous MC trials, focal male mate choice did not differ between in rival male fin flare bias between allopatric E. spectabile and allo- allopatric E. spectabile and allopatric E. pulchellum (F ¼ 0.29; patric E. pulchellum (F ¼ 5.79; P ¼ 0.025; Supplementary Figure 1,22 1,22 P ¼ 0.60; Supplementary Figure S3a, b). Additionally, focal male S4), despite the pattern being consistent with the prediction for mate choice did not differ from a null expectation of 0.5 in allopat- CACD. Rival male attack bias did not differ between trials with ric E. spectabile (mean6 SE: 0.426 0.04; one-sample t-test: allopatric E. spectabile versus allopatric E. pulchellum serving as the t ¼1.94, P ¼ 0.08) or in allopatric E. pulchellum (mean6 SE: focal male (F ¼ 0.10; P ¼ 0.75; Supplementary Figure S4). 11 1,22 0.456 0.04; one-sample t-test: t ¼1.28, P ¼ 0.23). Similarly, in the male competition trials rival male mate choice did not differ between allopatric E. spectabile and allopatric E. pulchellum Behavioral isolation indices (F ¼ 0.12; P ¼ 0.73; Supplementary Figure S4). To examine macroevolutionary patterns of RCD and ACD among 1,22 In contrast, there was no evidence for CRCD in female mating Ceasia–E. caeruleum species pairs, and CRCD and CACD among preference. Focal female mate choice did not differ between allopat- Ceasia–Ceasia species pairs, we compared the behavioral isolation ric E. spectabile and allopatric E. pulchellum, and these preferences indices calculated in this study with behavioral isolation indices cal- did not differ from 0.5 (Supplementary Table S4). There was no sig- culated by Moran et al. (2017; Table 6 and Figures 3 and 4). The nificant difference in the proportion of female nosedigs toward rival pattern in male mating preference was consistent with RCD between males as function of their identity (conspecific or heterospecific) Ceasia and E. caeruleum species pairs and CRCD between Ceasia when we controlled for the proportion of time each male pursued and Ceasia species pairs. MC indices were consistently higher be- the female (Supplementary Table S5). tween sympatric species pairs compared with allopatric species pairs, signifying enhanced preference for mating with conspecifics in sympatry. RCD was indicated in the Ceasia–E. caeruleum compari- CACD between Ceasia species sons as MC was higher for sympatric compared with allopatric spe- CACD predicts that Ceasia males that are sympatric with respect to cies pairs (F ¼ 56.35, P < 0.0001; Figure 3). CRCD was 1,82 E. caeruleum should bias their aggression toward conspecific over indicated in the Ceasia–Ceasia comparisons as male Ceasia that are heterospecific Ceasia males, despite the fact that the 2 Ceasia species sympatric with respect to E. caeruleum had heightened MC indices, are allopatric with respect to one another. CACD also predicts that despite the fact that all Ceasia are allopatric to one another Ceasia males that are allopatric with respect to E. caeruleum should (F ¼ 6.64, P ¼ 0.01; Figure 4). The difference in MC indices in 1,70 not bias their aggression more toward conspecific versus heterospe- sympatry versus allopatry was greater in Ceasia–E. caeruleum pair- cific males. Moran et al. (2017) paired Ceasia species that occur in ings than in Ceasia–Ceasia pairings (Table 6). sympatry with respect to E. caeruleum and found high levels of male Conversely, we did not observe a pattern consistent with RCD preference for fighting with conspecific over heterospecific Ceasia or CRCD in female mating preferences. FC indices did not differ as males. Here, we show that Ceasia species (i.e., E. spectabile and a function of sympatry with respect to E. caeruleum in Ceasia–E. E. pulchellum) that are allopatric with respect to E. caeruleum show caeruleum (F ¼ 0.96, P ¼ 0.33) or Ceasia–Ceasia comparisons 1,82 no such male bias in aggressive behavior. Focal male fin flare bias (F ¼ 0.18, P ¼ 0.67; Table 6 and Figures 3 and 4). This was due 1,70 did not differ between allopatric E. spectabile and allopatric E. pul- to females not exerting any detectable mating preferences for con- chellum (F ¼ 1.79; P ¼ 0.19; Supplementary Figure S3c, d), nor specific males. 1,22 did focal male attack bias (F ¼ 0.84; P ¼ 0.37; Supplementary We observed a pattern consistent with divergent ACD between 1,22 Figure S3e,f). Ceasia and E. caeruleum species pairs and CACD between Ceasia and Rival male behavior showed a similar pattern consistent with Ceasia species pairs. MA indices were consistently higher between CACD. In the trials where allopatric E. pulchellum served as focal sympatric species pairs compared with allopatric species pairs, indi- males, both conspecific E. pulchellum rival males and the allopatric cating increased male preference for fighting with conspecific over E. spectabile rival males directed a similar number of fin flares to- heterospecific males in sympatry. This pattern was present both ward focal males (Supplementary Figure S4d). However, in trials within the Ceasia–E. caeruleum comparisons (F ¼ 136.30, 1,166 where allopatric E. spectabile served as focal males, the allopatric P < 0.0001; Figure 3; indicating ACD) and within the Ceasia–Ceasia E. pulchellum rival males directed more fin flares toward the focal comparisons (F ¼ 34.17, P < 0.0001; Figure 4; indicating CACD). 1,142 Downloaded from https://academic.oup.com/cz/article-abstract/64/1/101/4665098 by Ed 'DeepDyve' Gillespie user on 16 March 2018 110 Current Zoology, 2018, Vol. 64, No. 1 Figure 4. Patterns of CRCD and CACD between Ceasia species. Behavioral isolation indices (with 95% confidence intervals) for (A) MA, (B) MC, and (C) FC be- tween pairs of Ceasia species. Allopatric comparisons (i.e., comparisons including Ceasia species that both occur in allopatry with respect to E. caeruleum) are shown in black. Sympatric comparisons (i.e., comparisons including Ceasia species that both occur in sympatry with respect to E. caeruleum) are shown in white. Grouping bars are also used to indicate allopatric species pairs (left) versus sympatric species pairs (right). Significance levels from ANOVAs comparing allopat- ric and sympatric species pairs are shown. Table 6. Behavioral isolation indices (mean6 standard error) for MA, MC, and FC, calculated from male competition assays that paired 2 Ceasia species or paired Ceasia with E. caeruleum Geography Pairing Species Hypotheses tested n MA MC FC Allopatric Ceasia–Ceasia E. spectabile–E. pulchellum CRCD/CACD 24 0.0160.07 0.1160.07 0.0160.02 Sympatric Ceasia–Ceasia E. fragi–E. uniporum CRCD/CACD 16 0.3860.08 0.3160.07 0.0160.01 Sympatric Ceasia–Ceasia E. fragi–E. burri CRCD/CACD 16 0.5060.06 0.3060.07 0.0260.01 Sympatric Ceasia–Ceasia E. fragi–E. spectabile CRCD/CACD 16 0.3560.06 0.3460.10 0.0160.02 Allopatric Ceasia–E. caeruleum E. spectabile–E. caeruleum RCD/ACD 24 0.0960.09 0.2260.12 0.1660.16 Allopatric Ceasia–E. caeruleum E. pulchellum–E. caeruleum RCD/ACD 24 0.3060.12 0.2560.12 0.0160.02 Sympatric Ceasia–E. caeruleum E. fragi–E. caeruleum RCD/ACD 48 0.8060.05 0.7660.06 0.0160.04 Sympatric Ceasia–E. caeruleum E. uniporum–E. caeruleum RCD/ACD 16 0.8260.06 0.7060.09 0.1160.13 Sympatric Ceasia–E. caeruleum E. burri–E. caeruleum RCD/ACD 16 0.9260.03 0.6660.08 0.0560.05 Sympatric Ceasia–E. caeruleum E. spectabile–E. caeruleum RCD/ACD 32 0.8560.05 0.8460.06 0.0360.02 Notes: As all species of Ceasia occur allopatrically with respect to one another, here geography for a given pairing refers to the relationship between Ceasia and E. caeruleum. For each species pairing, the Ceasia species that acted as the focal Ceasia in behavioral trials is listed first, followed by the species that it was observed with (a heterospecific Ceasia or E. caeruleum). Sample size (n) and hypotheses tested (CRCD/CACD in pairings between 2 Ceasia species, or RCD/ACD in pairings between Ceasia and E. caeruleum) are listed. Data from Moran et al. (2017). Calculated using data from the present study combined with data from Moran et al. (2017). MA was higher between sympatric Ceasia and E. caeruleum pairs (Table 5). Second, we found evidence for ACD and RCD among than it was in sympatric Ceasia and Ceasia pairs (Table 6). closely related species in the Ceasia species complex. Males showed no preference for mating (Table 3) or fighting (Table 5) with conspe- cifics over heterospecifics in pairings of allopatric E. pulchellum and Discussion allopatric E. caeruleum. This stands in contrast to the results of Moran et al. (2017), which found high levels of male preference for Striking patterns of RCD and ACD driven by male behavior are pre- mating and fighting with conspecifics over heterospecifics in sympat- sent at 2 taxonomic levels within Ceasia. First, we found evidence for ric pairings of Ceasia species and E. caeruleum. We discuss how the both RCD and ACD among populations within species (Figure 3 and data from the present study and Moran et al. (2017) reveal a pattern Supplementary Figure S1; Table 2). We observed RCD in male mate consistent with RCD and ACD at a macroevolutionary scale between choice among populations of E. spectabile and E. caeruleum. Male Ceasia species and E. caeruleum (see below). (but not female) preference for conspecific mates was enhanced in Most of our efforts were directed at testing for RCD and ACD in sympatric (versus allopatric) population pairings of these species Ceasia. However, we also found evidence for RCD in male mate (Tables 3, 4, and Supplementary Table S2). We also found evidence choice (Supplementary Figure S2 and Table S1) and ACD in MA of divergent ACD among populations within E. spectabile and bias in E. caeruleum (Supplementary Figure S2 and Tables S3), but E. caeruleum. Males preferentially biased their aggression toward the pattern of divergent ACD observed in male E. caeruleum conspecific males to a greater extent in sympatric population pairings Downloaded from https://academic.oup.com/cz/article-abstract/64/1/101/4665098 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Moran and Fuller  Reproductive and agonistic character displacement in darters 111 behavior was not as extreme as that observed in Ceasia. ACD was preferences (Panhuis et al. 2001), male coloration in darters appears indicated in E. caeruleum in that sympatric male E. caeruleum were to be under intrasexual selection due to intense male–male competi- less likely to flare their fins at sympatric male E. spectabile, but tion. RCD and ACD can lead to shifts in behavioral response to het- E. caeruleum males from both sympatric and allopatric populations erospecifics and in the signals used in species recognition (Brown did not perform many attacks toward E. spectabile or E. pulchellum and Wilson 1956; Grether et al. 2009). Thus, examining whether males. We hypothesize that this difference may be related to the level character displacement in male color pattern corresponds to the of gene flow present between populations of Ceasia species versus observed ACD and CACD in male aggressive response to heterospe- E. caeruleum. RCD and ACD are more likely to be maintained over cifics would be of interest. time (and to lead to CRCD and CACD) when gene flow is low Our results also uphold previous examinations of female mate among populations within species (Yukilevich and Aoki 2016). choice in this system, which have consistently failed to detect female Ceasia and E. caeruleum both occur in small headwater streams, but preferences for conspecific males in sympatric or allopatric pairings E. caeruleum can also inhabit larger order streams and rivers (Page of Ceasia and E. caeruleum (Pyron 1995; Fuller 2003; Zhou et al. 1983), leading to more opportunities for gene flow among popula- 2015; Moran et al. 2017). FC may be prevented by the presence of tions (Echelle et al. 1975, 1976). Gene flow from sympatric to allo- intense male competition in these species. Further study is needed to patric populations of E. caeruleum may result in the loci for MA determine whether females exhibit any cryptic forms of mate choice bias spreading beyond the zone of sympatry. Indeed, population (Eberhard 1996), such as adjusting the number of eggs laid when genetic analyses of 4 species of Ceasia and E. caeruleum found mating with conspecific versus heterospecific males. increased heterozygosity and higher levels of nucleotide diversity present in E. caeruleum compared with Ceasia (Moran et al. 2017), Selection underlying RCD and ACD indicating lower levels of gene flow in species of Ceasia. The presence of hybridization in conjunction with high levels of We also tested for patterns consistent with CRCD and CACD postzygotic isolation between Ceasia and E. caeruleum (Zhou 2014; between species of Ceasia (Table 2 and Figure 4). We observed that Moran R, unpublished data) suggests that RCD in these species may allopatric E. spectabile and allopatric E. pulchellum males showed occur via reinforcement. Selection for males to prefer conspecific no preference for conspecific over heterospecific Ceasia females, nor mates (to avoid maladaptive hybridization) would establish females did they bias their aggression more toward conspecific over hetero- as an unshared resource between species, making interspecific fight- specific Ceasia males (Supplementary Figures S3 and S4). Our previ- ing over females costly. Theoretical treatments of ACD predict that ous work indicated that sympatric Ceasia species have a clear selection may favor divergence in male aggressive traits between spe- preference to mate and fight with conspecific over heterospecific cies when males compete for separate resources (i.e., females), which Ceasia (Moran et al. 2017). Together, these data reveal a clear pat- decreases the prevalence of interspecific aggression in sympatry tern of CRCD in male mate choice and CACD in MA among Ceasia (Okamoto and Grether 2013). In the case of Ceasia and E. caeru- species (see below). leum, a lowered aggressive response to heterospecific males may also facilitate their co-occurrence within the same habitat in sympat- Relationship to previous studies in darters ric drainages. The fact that the 2 species can co-occur in sympatry Considering our results together with those of a recent study by provides further opportunities for interspecific encounters and hy- Moran et al. (2017) reveals 2 macroevolutionary patters: (1) RCD bridization, further strengthening selection for divergence in mating and ACD are present between species of Ceasia and E. caeruleum traits and behavioral isolation via RCD. In this manner, RCD and and (2) cascading effects of RCD and ACD between Ceasia and ACD may strengthen one another in a positive feedback loop. There E. caeruleum have incidentally contributed to allopatric divergence is evidence for such a feedback loop scenario between types of char- among closely related lineages within the Ceasia clade (i.e., CRCD acter displacement acting in Ficedula flycatchers (Qvarnstro ¨ m et al. and CACD). RCD and ACD are indicated in that Ceasia species that 2012; Vallin et al. 2012). occur in sympatry with E. caeruleum consistently show almost com- plete preference for mating and fighting with conspecifics over E. caeruleum, but no such preferences exist in Ceasia species that Selection underlying CRCD and CACD occur in allopatry with E. caeruleum (this study; Zhou and Fuller Theory predicts that CRCD or CACD can occur when populations 2014). Similarly, CRCD and CACD are indicated in that Ceasia spe- stochastically respond to selection on mating and fighting traits in cies that occur in sympatry with E. caeruleum (but allopatry with re- unique ways during RCD and ACD (i.e., mutation-order selection; spect to one another) show surprisingly high levels of male Abbott et al. 2013; Mendelson et al. 2014; Comeault and Matute preference for mating with and fighting with conspecifics over heter- 2016). Under mutation-order selection, trait divergence may occur ospecific Ceasia, but these preferences are absent in pairings of despite the presence of similar types of ecological and sexual selec- Ceasia that occur in allopatry with respect to E. caeruleum (this tion. In this way, stochastic variation in response to the same select- study; Moran et al. 2017). Future studies should determine whether ive pressures (i.e., maladaptive heterospecific interactions in patterns of CRCD and CACD are also present among populations sympatry) can potentially lead to allopatric divergence among popu- within individual species of Ceasia (as is the case with RCD and lations within species. ACD within E. spectabile). Although theory predicts that CRCD and CACD can lead to This study corroborates the results of several recent studies allopatric speciation (McPeek and Gavrilets 2006; Pfennig and Ryan which have shown that male mate choice and male competition play 2006), the majority of empirical studies that have examined CRCD an important role in driving sympatric and allopatric trait diver- and CACD to date have only tested for differences in behavioral gence in darters (Ciccotto et al. 2013; Zhou et al. 2015; Zhou and preferences among populations within species. In addition, many Fuller 2016; Martin and Mendelson 2016; Moran et al. 2017). studies have tested for CRCD by comparing levels of behavioral iso- Furthermore, although the presence of elaborate male coloration lation between populations within species that are allopatric versus is typically attributed to intersexual selection via female mate sympatric with respect to another species (Nosil et al. 2003; Downloaded from https://academic.oup.com/cz/article-abstract/64/1/101/4665098 by Ed 'DeepDyve' Gillespie user on 16 March 2018 112 Current Zoology, 2018, Vol. 64, No. 1 Lemmon 2009; Hopkins et al. 2014; Kozak et al. 2015; Comeault References et al. 2016). The implication with these studies is that RCD changes Abbott R, Albach D, Ansell S, Arntzen JW, Baird SJE et al., 2013. mating traits in such a way that increases behavioral isolation be- Hybridization and speciation. 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Male-driven reproductive and agonistic character displacement in darters and its implications for speciation in allopatry

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Abstract

Selection against hybridization can cause mating traits to diverge between species in sympatry via reproductive character displacement (RCD). Additionally, selection against interspecific fighting can cause aggressive traits to diverge between sympatric species via agonistic character displace- ment (ACD). By directly affecting conspecific recognition traits, RCD and ACD between species can also incidentally cause divergence in mating and fighting traits among populations within a species [termed cascade RCD (CRCD) and cascade ACD]. Here, we demonstrate patterns consistent with male-driven RCD and ACD in 2 groups of darters (orangethroat darter clade Ceasia and rainbow darter Etheostoma caeruleum). In both groups, males that occur in sympatry (between Ceasia and E. caeruleum) have higher levels of preference for mating and fighting with conspecifics over heter- ospecifics than do males from allopatry. This is consistent with RCD and ACD. We also found patterns consistent with CRCD and cascade ACD among species of Ceasia. Ceasia males that are sympatric to E. caeruleum (but allopatric to one another) also have heightened preferences for mat- ing and fighting with conspecific versus heterospecific Ceasia. In contrast, Ceasia males that are allopatric to E. caeruleum readily mate and fight with heterospecific Ceasia. We suggest that RCD and ACD between Ceasia and E. caeruleum has incidentally led to divergence in mating and fight- ing traits among Ceasia species. This study is unique in that male preferences evolve via both RCD (male preference for conspecific females) and ACD (male preference to fight conspecific males) which leads to subsequent divergence among allopatric lineages. Key words: agonistic character displacement, behavioral isolation, cascade reinforcement, reinforcement, reproductive charac- ter displacement, speciation. Reproductive interference between species can cause mating traits et al. 2003; Hoskin et al. 2005; Higgie and Blows 2007, 2008; (signals and/or preferences) to diverge via reproductive character Lemmon 2009; Porretta and Urbanelli 2012; Bewick and Dyer displacement (RCD; Howard 1993; Servedio and Noor 2003). RCD 2014; Pfennig and Rice 2014; Kozak et al. 2015). is often confirmed by a pattern of enhanced behavioral isolation be- Selection against interspecific aggression can also lead to the evo- tween 2 species in sympatry compared with allopatry. Recent re- lution of traits involved in species recognition. Maladaptive inter- search suggests that secondary effects of RCD in sympatry can also specific fighting over resources (such as mates) can cause shifts in initiate divergence between allopatric lineages (Pfennig and Pfennig aggressive signals and behavior via agonistic character displacement 2009; Hoskin and Higgie 2010). Cascade RCD (hereafter CRCD; (ACD; Grether et al. 2009; Okamoto and Grether 2013). A pattern Ortiz-Barrientos et al. 2009) occurs when behavioral isolation of divergent ACD is said to be present when 2 species are less likely evolves among populations within a species as a correlated effect of to engage in contests when they occur in sympatry compared with RCD. CRCD has been documented in a variety of taxa (e.g., Nosil allopatry. Both RCD and ACD may contribute to trait divergence V C The Author (2017). Published by Oxford University Press. 101 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/cz/article-abstract/64/1/101/4665098 by Ed 'DeepDyve' Gillespie user on 16 March 2018 102 Current Zoology, 2018, Vol. 64, No. 1 Figure 1. Ranges for Etheostoma caeruleum and 5 Ceasia species (Etheostoma spectabile, Etheostoma pulchellum, Etheostoma fragi, Etheostoma uniporum, and Etheostoma burri) used in behavioral assays in the current study and in Moran et al. (2017). Numbers on the map represent approximate collection locations for study populations (see Table 1 for details). between species that results in decreased heterospecific interactions diverged approximately 22 million years ago (Near et al. 2011). in sympatry. Although numerous studies have shown that RCD can Time calibrated gene trees indicate that Ceasia subsequently diversi- incidentally lead to divergence in mating traits among populations fied 6–7 million years ago (Bossu et al. 2013). The Ceasia clade con- within species via CRCD, whether selection against interspecific sists of 15 species, all of which are allopatric with respect to one aggression can also cause divergence in agonistic traits among popu- another (Ceas and Page 1997; Bossu and Near 2009). Phylogenetic lations within species (i.e., cascade ACD, hereafter CACD) has yet and palaeogeographical analyses support allopatric divergence of to be determined. this clade (Bossu et al. 2013). Twelve Ceasia species occur in sym- Distinguishing between RCD and ACD is essential to determin- patry with respect to E. caeruleum throughout their range, and 2 ing the underlying selective pressure (i.e., heterospecific mating or Ceasia species occur in allopatry with respect to E. caeruleum fighting) and relative contribution of male–female and male–male throughout their range (see Bossu and Near 2009; Page and Burr interactions in driving speciation. However, disentangling the im- 2011). The one remaining Ceasia species (orangethroat darter portance of RCD versus ACD to speciation can be difficult because Etheostoma spectabile) occurs in both sympatry and allopatry with many sexually selected traits are used in both female mate choice respect to E. caeruleum (Figure 1). Within Ceasia, time since diver- and male–male competition over mates (Alatalo et al. 1994; gence does not differ significantly between lineages that occur in Berglund 1996; Sætre et al. 1997; Dijkstra et al. 2007; Saether et al. sympatry versus allopatry with respect to E. caeruleum (Bossu et al. 2007; Lackey and Boughman 2013; Tinghitella et al. 2015). Here, 2013). Ceasia and E. caeruleum have similar male coloration, mat- we examine female mating preferences, male mating preferences, ing behavior, and ecology. There is little evidence that male color- and male–male aggression (MA) to test for patterns consistent with ation in either Ceasia or E. caeruleum is the target of female mate RCD, ACD, CRCD, and CACD. choice; females lack preferences for either male size or color pattern This study focuses on 2 groups of darters in the subgenus within species, and Ceasia females lack preferences for conspecific Oligocephalus: the orangethroat darter clade Ceasia and the over heterospecific Ceasia and E. caeruleum males (Pyron 1995; rainbow darter Etheostoma caeruleum. Ceasia and E. caeruleum Fuller 2003; Zhou et al. 2015; Moran et al. 2017). Instead, there is Downloaded from https://academic.oup.com/cz/article-abstract/64/1/101/4665098 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Moran and Fuller  Reproductive and agonistic character displacement in darters 103 Table 1. Collection locations for populations of each species examined in behavioral trials in the present study as well as in Moran et al. (2017) Range Geography Species Collection location Drainage information Source of behav- map popu- ioral data lation number 1 Allopatric E. caeruleum 42.426825, 85.428370 Prairieville Creek, Kalamazoo River, Barry Present study County, MI 2 Sympatric E. spectabile 40.054447, 88.089887 Unnamed tributary, Salt Fork of Vermillion Present study and River, Champaign County, IL Moran et al. (2017) 3 Sympatric E. caeruleum (Same as above) (Same as above) Present study and Moran et al. (2017) 4 Allopatric E. spectabile 40.027663, 88.577180 Unnamed tributary, Sangamon River, Piatt Present study County, IL 5 Allopatric E. pulchellum 38.952839, 95.517654 Deer Creek, Kansas River, Shawnee County, KS Present study 6 Sympatric E. fragi 36.304214, 91.927684 Rose Branch tributary of Strawberry River, Moran et al. Fulton County, AR (2017) 7 Sympatric E. uniporum 36.250560, 91.359318 Unnamed tributary of Spring River, Sharp Moran et al. County, AR (2017) 8 Sympatric E. caeruleum 36.065396, 91.610420 Mill Creek tributary of Strawberry River, Sharp Moran et al. County, AR (2017) 9 Sympatric E. burri 37.146415, 90.907459 North Fork Webb Creek, Black River Drainage, Moran et al. Wayne County, MO (2017) Notes: Sympatry and allopatry refer to the geographic relationship between Ceasia and E. caeruleum (all species of Ceasia are allopatric from one another). Range map population number refers to numbers shown in Figure 1. Etheostoma caeruleum study population used in sympatric comparisons with Ceasia species from the Ozarks regions (i.e., E. fragi, E. uniporum, and E. burri) in Moran et al. (2017). strong evidence that male coloration is under intrasexual selection also influence species diversification at a macroevolutionary scale and functions as an aggressive signal in male–male competition over (Pfennig and Pfennig 2012; Grether et al. 2017). Over time, CRCD access to females (Zhou and Fuller 2016; Moran et al. 2017). and CACD can cause isolated populations within a species to diverge Several recent studies have indicated that RCD and ACD are likely from one another to such an extent that they merit classification as occurring in this system. First, hybridization occurs between Ceasia distinct, allopatric species. The outcome of this process can result in a and E. caeruleum in nature (Bossu and Near 2009; Moran et al. complex of closely related, allopatric species that exhibit enhanced 2017), and their hybrids have reduced fitness (Zhou 2014; Moran R, mating trait divergence with one another (via CRCD/CACD), and unpublished data), providing the potential for RCD to occur via re- with a more distantly related sympatric species (via RCD/ACD). inforcement (Brown and Wilson 1956; Coyne and Orr 2004). Second, In this manner, CRCD and CACD can fuel hierarchical “speciation in pairings between 4 species of Ceasia and sympatric E. caeruleum, cascades” among allopatric lineages at multiple taxonomic levels sim- males preferentially mate and fight with conspecifics, suggesting RCD ultaneously (Pfennig and Ryan 2006). We hypothesize that this scen- and ACD (Figure 1 and Table 1; Moran et al. 2017). Third, a pattern ario is ongoing in the Ceasia–E. caeruleum system. consistent with RCD was observed in a no-choice mating experiment To test for RCD and ACD, we measured preferences for mating which found that allopatric pairings of female E. spectabile and male and fighting with conspecifics in pairings between E. spectabile and E. caeruleum yielded more eggs than sympatric pairings (Zhou and E. caeruleum that occur in sympatry versus allopatry with respect to Fuller 2014). Zhou and Fuller (2014) is the only study to date to com- one another. This allowed us to examine whether patterns consistent pare sympatric and allopatric pairings between a Ceasia species and with RCD and ACD are present at the population level within E. caeruleum, but the no-choice assay they used was not able to meas- E. spectabile and E. caeruleum. Additionally, we measured prefer- ure the contribution of each sex to behavioral isolation in sympatry. ences for mating and fighting with conspecifics in pairings between Furthermore, Zhou and Fuller (2014) did not consider male competi- Etheostoma pulchellum and E. caeruleum that occur in allopatry tion, and could not test for ACD. with respect to one another (Figure 1 and Table 1). Because E. pul- A unique aspect of this study system is that it allows us to test for chellum and E. caeruleum do not co-occur, these species should patterns consistent with RCD and ACD at 2 taxonomic levels within show a reduced level of bias against mating and fighting with one Ceasia: populations within a species, and closely related species another compared with species of Ceasia and E. caeruleum that do within a recently diverged clade. We first tested for RCD and ACD co-occur. Measuring mating and fighting biases in allopatric pair- between populations of a single species of Ceasia as a function of sym- ings of Ceasia and E. caeruleum thus serves as a critical test against patry with E. caeruleum. We next asked whether RCD and ACD are which we can compare levels of behavioral preferences in sympatric present between species of Ceasia as a function of sympatry with pairings of Ceasia and E. caeruleum that were previously reported E. caeruleum. Most studies involving RCD and ACD have considered by Moran et al. (2017). differences in mating traits between populations within a pair of spe- We also investigated whether patterns consistent with CRCD cies as a function of sympatry versus allopatry. However, RCD can and CACD are present among Ceasia species. Males within the Downloaded from https://academic.oup.com/cz/article-abstract/64/1/101/4665098 by Ed 'DeepDyve' Gillespie user on 16 March 2018 104 Current Zoology, 2018, Vol. 64, No. 1 4 Ceasia species examined by Moran et al. (2017; Figure 1 and Ceasia and sympatric E. caeruleum, and pairings between sympatric Table 1), which all occur in sympatry with respect to E. caeruleum, Ceasia species, in Moran et al. (2017; Figure 1 and Table 1). prefer conspecific over heterospecific Ceasia females and bias their Fish were collected with a kick seine in March 2016 and April aggression preferentially toward conspecific over heterospecific 2017 and transported back to the laboratory at the University of Ceasia males. This divergence in male mating and fighting traits Illinois at Urbana-Champaign in aerated coolers. Fish were sepa- among Ceasia species is not associated with differences in male color rated into stock aquaria according to population and sex, and were pattern or genetic distance. Therefore, RCD and ACD between fed daily ad libitum with frozen bloodworms. Stock aquaria were Ceasia and E. caeruleum may have incidentally contributed to spe- maintained at 19 C and fluorescent lighting was provided to mimic cies divergence within the Ceasia clade via CRCD and CACD. To the natural photoperiod. test this hypothesis, we examine preferences for mating and fighting with conspecifics (over a heterospecific member of the Ceasia clade) Testing for RCD and ACD between Ceasia and E. in pairings between E. spectabile and E. pulchellum that occur in al- caeruleum lopatry with respect to E. caeruleum. We then ask whether E. spec- Dichotomous MC assay tabile and E. pulchellum have lower levels of preference for mating We first used a dichotomous MC assay to test for RCD in male mate and fighting with conspecifics compared with that previously choice. Each trial included a focal male E. spectabile or E. pulchel- observed between pairs of Ceasia species that occur in sympatry lum with a conspecific female and a heterospecific (E. caeruleum) fe- with respect to E. caeruleum (Moran et al. 2017). male (Figure 2A). This assay allowed males to choose between (1) sympatric E. spectabile and sympatric E. caeruleum, (2) allopatric E. spectabile and allopatric E. caeruleum, and (3) allopatric E. pul- Materials and Methods chellum and allopatric E. caeruleum females (n ¼ 12 each). RCD predicts that preferences for conspecific mates should be higher in Mating system details sympatric E. spectabile focal males than both allopatric E. specta- During the spring spawning season, Ceasia and E. caeruleum travel bile and allopatric E. pulchellum focal males. to shallow gravel riffles in headwater streams (Hubbs and Strawn Behavioral trials occurred in 38 L test aquaria filled with 5 cm of 1957; Hubbs 1985). Females look for a suitable place to lay eggs by naturally colored aquarium gravel. To minimize disturbance to the performing “nosedigs” in which they jab their snout into the gravel. fish, test aquaria were covered with black opaque plastic on 3 sides. One to several males swim in tandem with a female as she searches We used unique fish in each trial, chosen haphazardly from stock for a spawning location. Males fight aggressively to ward off rival tanks. Females in each trial were size matched to within 10% of males by actively chasing them off and/or by flaring their dorsal and their total body length. Each trial began by placing the 3 fish being anal fins in a threat display. When the female is ready to spawn, she tested into a test aquarium and allowing them to acclimatize for dives into the substrate, leaving only her head and caudal fin fully 5 min. The trial then began and lasted 30 min. Each trial was broken visible. Spawning initiates when a male positions himself above the up into 60 30-s blocks (Zhou et al. 2015; Moran et al. 2017). female, and they release sperm and eggs into the substrate. We examined male mate choice by measuring focal male pursuit of Spawning often involves multiple males mating simultaneously with each female in each trial. Male pursuit of a female is highly predictive 1 female, and males sometimes exhibit sneaking behavior. Females of spawning in Ceasia and in E. caeruleum (Zhou et al. 2015; Moran will ovulate clutches of up to 200 eggs throughout the spawning sea- et al. 2017). A male was scored as having pursued a female during a son, but only release a few eggs per spawning bout (Heins et al. 30-s block if he spent a minimum consecutive time of 5-s within one 1996; Fuller 1998). Hence, the female must spawn multiple times to body length of the female. We calculated a focal male mate choice be- fertilize all the eggs from a given clutch. havioral variable from this data as described in Table 2. We performed analyses using proportional data (i.e., the behav- ioral variables described in Table 2) that varied from 0 to 1. A score Study species/populations and collection locations of 1 indicates only conspecific interactions occurred, 0.5 indicates All Ceasia species occur in allopatry with respect to one another. an equal number of interactions between conspecifics and heterospe- Throughout the rest of this paper, the terms “allopatric” and “sym- cifics, and 0 indicates only heterospecific interactions occurred. patric” refer to the geographic relationship between Ceasia and However, for ease of interpretation, we graphed the raw number of E. caeruleum (not between Ceasia species). To test for RCD and behaviors observed. ACD between E. spectabile and E. caeruleum, we examined prefer- We used analysis of variance (ANOVA) to test for RCD in male ences for mating and fighting with conspecifics over heterospecifics mating preference by asking whether focal male mate choice differed in pairings between allopatric E. spectabile and allopatric E. caeru- among the focal Ceasia study populations (i.e., sympatric E. specta- leum versus pairings between sympatric E. spectabile and sympatric bile, allopatric E. spectabile, and allopatric E. pulchellum). We E. caeruleum (Figure 1 and Table 1). We also tested for a pattern included focal male mate choice as the dependent variable, and focal consistent with RCD and ACD in pairings between allopatric E. pul- male population identity as the independent variable. We then used chellum and allopatric E. caeruleum (Figure 1 and Table 1). Finally, post hoc t-tests to directly compare populations. We also asked we tested for a pattern consistent with CRCD and CACD among whether focal male mate choice differed from a null expectation of Ceasia species by pairing allopatric E. spectabile with allopatric 0.5 (equal amounts of time spent with each female) in each popula- E. pulchellum (Figure 1 and Table 1). tion using one-sample t-tests. We used 2 types of behavioral assays [“dichotomous male choice (MC) assay” and “male competition assay,” detailed below] to com- pare preferences for engaging in mating and fighting with conspe- Male competition assay cifics versus heterospecifics. We then compared these behavioral We conducted a second type of assay in which males could compete measurements to those documented in pairings between sympatric with one another to test for RCD and ACD. This assay paired (1) Downloaded from https://academic.oup.com/cz/article-abstract/64/1/101/4665098 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Moran and Fuller  Reproductive and agonistic character displacement in darters 105 in these species is complex and varies within populations (Zhou et al. 2014), allowing us to distinguish conspecific males. Males in each trial were size matched within 10% of their total body length to control for any larger differences in color pattern and competitive ability associated with body size (Zhou et al. 2014). In each trial, we measured the behavior of the focal female, the focal male, and the rival male. Due to low collection numbers, some allopatric E. caeru- leum males were used twice, but never more than once on the same day or with the same Ceasia study population. To test for ACD, we recorded the number of aggressive behaviors (i.e., fin flares and attacks) that both males in a trial directed toward the other male. We calculated 4 behavioral variables to quantify male aggressive bias toward conspecific males: focal male fin flare bias, focal male attack bias, rival male fin flare bias,and rival male attack bias (see Table 2). We asked whether these behavioral variables dif- fered in sympatric versus allopatric pairings. To examine focal male Ceasia aggressive behavior, we conducted 2 separate ANOVAs with focal male fin flare bias and focal male attack bias as the dependent variables, and focal Ceasia male identity (sympatric E. spectabile, allopatric E. spectabile,or allopatric E. pulchellum) as the independ- ent variable in both analyses. Similarly, to examine the aggressive be- havior of E. caeruleum rival males relative to Ceasia rival males, we conducted ANOVAs with rival male fin flare bias and rival male attack bias as dependent variables, and focal Ceasia male identity as the independent variable. Additionally, we made pairwise compari- sons among groups using post hoc 2-sample t-tests. To test for RCD in male mate preference, we split each male competition trial into 60 30-s blocks (as in the dichotomous MC tri- als), and counted the number of 30-s blocks in which each male pur- sued the female. Unlike the dichotomous MC assay, the male competition assay considers the preference of male E. caeruleum for E. spectabile and E. pulchellum females. We calculated rival male Figure 2. Setup for behavioral experiments. (A–C) Trials testing for RCD and mate choice as described in Table 2. As focal males were always ACD. In these trials, sympatric E. spectabile, allopatric E. spectabile, and allo- paired with conspecific females in the male competition trials, we patric E. pulchellum served as focal Ceasia in turn. Note that in (A) and (C), did not measure focal male mate choice in these trials. The male allopatric E. caeruleum were paired with allopatric focal Ceasia, and sympat- competition assay presented males with a no-choice situation, where ric E. caeruleum were paired with sympatric focal Ceasia. (A) Experimental setup for dichotomous MC trials that tested for RCD in focal Ceasia male they could choose whether to pursue a female. This assay also exam- mate choice. (B–C) Experimental set up for male competition trials that tested ined male mate preference in the presence of a male competitor, for patterns consistent with RCD in E. caeruleum rival male mate preference, which is closer to what a male would experience in nature during RCD in focal Ceasia female mate preference, ACD in focal Ceasia male ag- the spawning season. We asked whether rival male mate choice dif- gressive behavior, and ACD in E. caeruleum rival male aggressive behavior. fered between sympatric and allopatric trial sets. We conducted an (D–E) Trials testing for CRCD and CACD. In these trials, allopatric E. spectabile ANOVA with rival male mate choice as the dependent variable and and allopatric E. pulchellum acted as focal Ceasia and as heterospecific trial set (i.e., sympatric E. spectabile, allopatric E. spectabile,or Ceasia in turn. (D) Experimental set up for dichotomous MC trials that tested for patterns consistent with CRCD in focal Ceasia male mate choice. (E) allopatric E. pulchellum as the focal pair) as the independent vari- Experimental set up for male competition trials that tested for patterns con- able, followed by pairwise post hoc 2-sample t-tests. sistent with CRCD in heterospecific Ceasia rival male mate preference, CRCD Finally, we tested for RCD in female mating preferences. The in focal Ceasia female mate preference, and CACD in focal Ceasia male and setup of the male competition assay was equivalent to a dichotom- heterospecific Ceasia rival male aggressive behavior. We did not repeat male ous female choice (FC) assay. We counted the number of nosedigs a competition trials in which a conspecific Ceasia acted as the rival male female performed towards the rival male in each trial. Females typic- (shown in B). We compared the behavior of individuals in trials with a con- specific Ceasia rival male (B) to individuals in trials with an E. caeruleum rival ally perform nosedigs directly before spawning, and this behavior is male (C). We also compared the behavior of individuals in trials with a con- often used to measure female mating preferences in darters (Fuller specific Ceasia rival male (B) to individuals in trials with a heterospecific 2003; Williams and Mendelson 2011; Zhou et al. 2015; Zhou and Ceasia rival male (E). Fuller 2016). We asked whether focal female mate choice (Table 2) differed among sympatric E. spectabile, allopatric E. spectabile, and sympatric E. spectabile and sympatric E. caeruleum, (2) allopatric allopatric E. pulchellum using ANCOVA. The model included focal E. spectabile and allopatric E. caeruleum, and (3) allopatric E. pul- female mate choice as the dependent variable and focal female iden- chellum and allopatric E. caeruleum (n ¼ 12 each). Each trial tity as the independent variable. We included the proportion of time included a focal male and a focal female pair from the same Ceasia that conspecific rival males pursued the focal female as a covariate study population. Each focal Ceasia pair was observed once with a in the analysis, as male pursuit has been shown to predict female rival male that was conspecific to them (Figure 2B), and once with a nosedigs and spawning (Zhou et al. 2015; Moran et al. 2017). rival male that was an E. caeruleum (Figure 2C). Male color pattern We also used ANCOVA to test for focal female mate preference Downloaded from https://academic.oup.com/cz/article-abstract/64/1/101/4665098 by Ed 'DeepDyve' Gillespie user on 16 March 2018 106 Current Zoology, 2018, Vol. 64, No. 1 Table 2. Definition of the behavioral variables measured in the dichotomous MC assay and the male competition assay Variable Definition RCD ACD CRCD CACD Dichotomous MC assay (2 females, 1 male) Focal Male Mate Number of time blocks spent pursuing the conspecific divided by the Yes NA Yes NA Choice total number of time blocks spent pursuing either female. Male competition assay (2 males, 1 female) Rival Male Mate Proportion of time blocks the focal female was pursued by conspecific Yes NA Yes NA Choice versus heterospecific rival males across 2 trials ¼ Number of time blocks conspecific rival male pursued the female/(sum of time blocks the conspecific and heterospecific rivals pursued the female). Focal Female Proportion of nosedigs towards conspecific versus heterospecific rival No NA No NA Mate Choice males across 2 trial ¼ Number of nosedigs toward conspecific rivals/ (sum of nosedigs toward conspecific and heterospecific rivals); the analysis of this variable was corrected for male pursuit. Focal Male Fin Proportion of fin flares toward conspecific versus heterospecific rivals NA Yes NA Yes Flare Bias across 2 trials ¼ Number of fin flares to conspecific rival/(sum of fin flares to conspecific and heterospecific rivals). Focal Male Attack Proportion of attacks toward conspecific versus heterospecific rivals NA Yes NA Yes Bias across 2 trials ¼ Number of attacks on conspecific rival/(sum of at- tacks on conspecific and heterospecific rivals). Rival Male Fin Proportion of fin flares performed by conspecific versus heterospecific NA Yes NA Yes Flare Bias rivals across 2 trials ¼ Number of fin flares by conspecific rival to- ward the focal male/(sum of fin flares by conspecific and heterospe- cific rivals toward the focal male). Rival Male Attack Proportion of attacks performed by conspecific versus heterospecific NA Mixed NA Yes Bias rivals across 2 trials ¼ Number of attacks by conspecific rival to- ward the focal male/(sum of attacks by conspecific and heterospe- cific rivals towards the focal male). Notes: We indicate whether we observed a pattern consistent with predictions for RCD, ACD, CRCD, and CACD for each behavioral variable, or whether the be- havioral variable was not applicable (NA) to testing a given prediction. Allopatric E. caeruleum males tended to attack allopatric E. spectabile males more than sympatric E. caeruleum males attacked sympatric E. spectabile males, but no other differences were found. for conspecific rival males versus E. caeruleum rival males. differed from a null expectation of 0.5 (equal amounts of time spent The number of nosedigs the focal female directed toward each rival with each female) using one-sample t-tests. male was the independent variable, the rival male’s identity (conspe- cific or E. caeruleum) was the dependent variable, and the propor- Male competition assay tion of time the rival male spent in pursuit of the female was We also conducted a male competition assay between allopatric included as a covariate. We note that although the females’ ability E. spectabile and allopatric E. pulchellum to test for patterns con- to exert mating preferences may be precluded by the outcome of sistent with CRCD and CACD. Earlier work showed that Ceasia male contests, male competition over females is pervasive in these males that are sympatric with E. caeruleum prefer to mate and fight species, so this assay reflects what females most frequently encoun- with conspecifics over heterospecific Ceasia (Moran et al. 2017). ter in nature. Here, we asked whether Ceasia males that are allopatric with re- spect to E. caeruleum lacked such preferences. We performed trials Testing for CRCD and CACD between Ceasia species in which both allopatric E. spectabile and allopatric E. pulchellum Dichotomous MC assay acted as the focal pair and as the heterospecific rival male in turn To test for patterns consistent with CRCD within Ceasia, we paired (n ¼ 12 each; Figure 2E). CRCD and CACD predict that allopatric allopatric E. spectabile with allopatric E. pulchellum in a dichotom- E. spectabile and allopatric E. pulchellum should show similarly ous MC assay. We conducted this assay in the manner described low levels of preference for mating and fighting with conspecifics above to test for RCD, but here the heterospecific female was an over heterospecifics. We measured rival male mate choice, and focal allopatric E. spectabile or allopatric E. pulchellum, in place of an female mate choice, focal male fin flare bias, focal male attack bias, E. caeruleum (Figure 2D). We performed trials in which allopatric rival male fin flare bias, and rival male attack bias as described in E. spectabile acted as the focal male and conspecific female, with Table 2. We conducted ANOVAs as described above for the male E. pulchellum as the heterospecific female, and vice versa (n ¼ 12 competition trials that tested for RCD and ACD, but with the ap- each). CRCD predicts no significant difference between allopatric propriate species (i.e., E. spectabile or E. pulchellum) in place of E. spectabile and allopatric E. pulchellum in focal male mate choice E. caeruleum as the heterospecific rival male. (Table 2). To compare focal male mate choice between these species, We used ANOVA to test for RCD, ACD, CRCD, and CACD in we conducted ANOVAs that included focal male mate choice as the both sets of dichotomous MC and male competition assays. dependent variable and focal male identity (allopatric E. spectabile Repeating all analyses using generalized linear models with a quasi- or allopatric E. pulchellum) as the independent variable. We also binomial error function and logit link function yielded qualitatively tested whether focal male mate choice for the conspecific female identical results. Downloaded from https://academic.oup.com/cz/article-abstract/64/1/101/4665098 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Moran and Fuller  Reproductive and agonistic character displacement in darters 107 Table 3. Results of ANOVA testing for RCD in focal Ceasia male mate choice between conspecific females and E. caeruleum females in di- chotomous MC male trials Focal male mate choice df Test statistic P Focal Ceasia population identity 2, 33 45.21 <0.00001 Sympatric E. spectabile versus allopatric E. spectabile 22 11.38 <0.00001 Sympatric E. spectabile versus allopatric E. pulchellum 22 8.10 <0.00001 Allopatric E. spectabile versus allopatric E. pulchellum 220 0.38 0.71 Notes: We asked focal male mate choice differed among focal Ceasia males in 3 study populations: sympatric E. spectabile, allopatric E. spectabile, and allopatric E. pulchellum. Pairwise post-hoc t-test results are also shown for the analysis. Behavioral isolation indices occur in sympatry versus allopatry. This would indicate that sym- We used the MA, male mate choice, and female mate choice data patric males bias their aggression more toward conspecifics over from both sets of male competition assays (i.e., those testing for heterospecifics. RCD and ACD, and those testing for CRCD and CACD) to calcu- Second, we tested for differences between Ceasia and Ceasia late 3 behavioral isolation indices following Moran et al. (2017). species pairs that occur in sympatry versus allopatry with respect Behavioral isolation indices were calculated individually for each to E. caeruleum. CRCD predicts higher MC and FC indices in trial and then averaged across all replicates within each species com- Ceasia–Ceasia pairings that occur in sympatry with respect to parison. These indices allowed for a comparison of levels of prefer- E. caeruleum, indicating enhanced mate preference for conspecific ence for mating and fighting with conspecifics over heterospecifics over heterospecific Ceasia. Likewise, CACD predicts higher MA in- at a macroevolutionary scale among Ceasia–E. caeruleum and dices in Ceasia–Ceasia pairings that occur in sympatry with respect Ceasia–Ceasia species pairs. Indices range from 1 (complete pref- to E. caeruleum. This would indicate that Ceasia males that occur in erence for heterospecifics) to 1 (complete preference for conspe- sympatry with respect to E. caeruleum bias their aggression more to- cifics), with 0 indicating no preference for conspecifics versus ward conspecific males versus heterospecific Ceasia males. heterospecifics (Stalker 1942; Martin and Mendelson 2016; Moran For all analyses, we used Type III sums of squares using the et al. 2017). “car” package in R (version 3.4.0). Raw data have been deposited in We calculated MA indices for each species pair as: Dryad (doi:10.5061/dryad.g8d1v). a  a c h MA ¼ ; a þ a c h Results where a and a represent the combined number of fin flares and at- c h tacks performed between conspecific males and between heterospe- RCD between Ceasia and E. caeruleum cific males, respectively. The dichotomous MC trials revealed a pattern consistent with RCD We calculated MC indices as: in focal Ceasia male mate preference. RCD predicts that MC for conspecifics should be heightened in Ceasia populations/species that m  m c h MC ¼ ; are sympatric with respect to E. caeruleum. Focal male mate choice m þ m c h was 2 higher in sympatric E. spectabile compared with allopatric where m and m represent the proportion of time in each trial that c h E. spectabile and allopatric E. pulchellum, but did not differ be- conspecific males and heterospecific males spent pursuing the Ceasia tween allopatric E. spectabile and allopatric E. pulchellum (Table 3 female. and Supplementary Figure S1a). In addition, focal male mate choice As previous studies have indicated that male pursuit of a female was much greater than the null expectation of 0.5 in trials with is highly correlated with female nosedigs (a measure of female mat- sympatric E. spectabile serving as the focal male (mean6 SE: ing preference), FC indices controlled for male pursuit of the female. 0.976 0.01; one-sample t-test: t ¼ 51.58, P < 0.00001). We calculated the FC indices as: Conversely, focal male mate choice did not differ from 0.5 in trials where allopatric E. spectabile and allopatric E. pulchellum served f f c h FC ¼  ; as the focal males (Supplementary Figure S1b, c; allopatric E. spec- p p c h tabile mean6 SE: 0.516 0.04; one-sample t-test: t ¼ 0.17, where f and f represent the number of nosedigs females performed c h P ¼ 0.87; E. pulchellum mean6 SE: 0.536 0.05; one-sample t-test: toward conspecific males and toward heterospecific males, respect- t ¼ 0.60, P ¼ 0.56). ively. p and p represent the number of 30-s blocks in which con- c h RCD in male mate preference was also indicated in the male specific males and heterospecific males were scored as having competition trials, which compared E. caeruleum rival male prefer- pursued the female during a trial, respectively. ence for the focal Ceasia female with that of the conspecific Ceasia We used ANOVA to make 2 sets of comparisons among the rival male. RCD predicts that sympatric E. caeruleum males should 3 types of behavioral isolation indices (i.e., MA, MC, and FC). First, be less likely to pursue Ceasia females than allopatric E. caeruleum we tested for differences between Ceasia and E. caeruleum pairs that males. Rival male mate choice differed significantly between sym- occur in sympatry versus allopatry with respect to one another. patric and allopatric E. caeruleum (Supplementary Table S1). In tri- RCD predicts higher MC and FC indices in Ceasia–E. caeruleum als where sympatric E. spectabile served as the focal Ceasia pair, pairings that occur in sympatry versus allopatry, indicating conspecific rival males were much more likely to pursue the focal enhanced mate preference for conspecifics. Similarly, divergent female compared with the sympatric E. caeruleum rival males ACD predicts higher MA indices in Ceasia–E. caeruleum pairs that (Supplementary Figure S2a). In both trials where allopatric Downloaded from https://academic.oup.com/cz/article-abstract/64/1/101/4665098 by Ed 'DeepDyve' Gillespie user on 16 March 2018 108 Current Zoology, 2018, Vol. 64, No. 1 E. spectabile and E. pulchellum served as the focal Ceasia pair, con- fin flares toward conspecific (versus E. caeruleum) rival males specific rival males and allopatric E. caeruleum rival males spent (Supplementary Figure S1d). Similarly, sympatric E. spectabile focal roughly the same amount of time pursuing the focal female males attacked conspecific rival males 6 more than they attacked (Supplementary Figure S2b, c). Hence, allopatric E. caeruleum males sympatric E. caeruleum rival males (Supplementary Figure S1g). On chose to pursue allopatric E. spectabile and allopatric E. pulchellum average, both allopatric E. spectabile and allopatric E. pulchellum females. Sympatric E. caeruleum males largely ignored sympatric focal males directed an equal number of fin flares (Supplementary E. spectabile females. Figure S1e, f) and attacks (Supplementary Figure S1h, i) toward con- We did not find support for RCD in female mating preferences specific rival males and allopatric E. caeruleum rival males. in the male competition trials. When male pursuit was included as a We also found a pattern consistent with divergent ACD in covariate in the analysis, focal female mate choice did not differ E. caeruleum male aggressive behavior. Divergent ACD predicts among the sympatric E. spectabile, allopatric E. spectabile, and allo- that sympatric E. caeruleum rival males should show higher levels of patric E. pulchellum trials (Table 4). Females did not exert prefer- aggression toward focal male Ceasia compared with allopatric ence for conspecific males over E. caeruleum males, regardless of E. caeruleum rival males. Rival male fin flare bias showed a pattern sympatry with respect to E. caeruleum (Supplementary Table S2). like that found with focal Ceasia males (Supplementary Table S3). Sympatric E. caeruleum rival males were much less likely to flare their fins toward E. spectabile focal males compared with allopatric ACD between Ceasia and E. caeruleum E. caeruleum rival males (Supplementary Figure S2d, f). The aggressive behavior of focal Ceasia males in the male competi- Conversely, rival male attack bias did not differ between sympat- tion trials was consistent with divergent ACD. Divergent ACD ric and allopatric E. caeruleum (Supplementary Table S3). Both predicts that Ceasia males that are sympatric with respect to sympatric and allopatric E. caeruleum rival males directed a low E. caeruleum should bias their aggression toward conspecific rival number of attacks toward the focal Ceasia males (Supplementary males over E. caeruleum rival males. Focal male fin flare bias and Figure S2g, i). Thus, while allopatric E. spectabile and allopatric focal male attack bias were higher for sympatric E. spectabile com- E. pulchellum focal males did not bias their aggression more toward pared with allopatric E. spectabile and allopatric E. pulchellum conspecific rival males (versus allopatric E. caeruleum rival males; (Table 5). Sympatric E. spectabile focal males directed 9 more see previous paragraph), allopatric E. caeruleum rival males typic- ally preferred not to attack allopatric E. spectabile and allopatric Table 4. Results ANCOVA testing for RCD in focal Ceasia female E. pulchellum focal males. mate choice between conspecific rival males and E. caeruleum rival males in male competition trials CRCD between Ceasia species Focal female mate choice df Test statistic P CRCD predicts that males from Ceasia species that are sympatric with respect to E. caeruleum should show higher levels of male mate Focal Ceasia population identity 2, 32 0.09 0.92 preference for conspecific females over heterospecific Ceasia fe- Male pursuit 1, 32 0.74 0.40 males, despite the fact that the 2 Ceasia species are allopatric with Notes: We asked whether focal female mate choice differed among focal respect to one another. Moran et al. (2017) showed that in Ceasia Ceasia females in 3 study populations: sympatric E. spectabile, allopatric species that are sympatric with respect to E. caeruleum, male mate E. spectabile, and allopatric E. pulchellum. Male pursuit of the female was preference for conspecific over heterospecific Ceasia females was sur- included as a covariate in the analysis. prisingly high. This study shows that male Ceasia (i.e., E. spectabile Figure 3. Patterns of RCD and ACD between Ceasia and E. caeruleum. Behavioral isolation indices (with 95% confidence intervals) for (A) MA, (B) MC, and (C)FC for comparisons between Ceasia species and E. caeruleum. Allopatric comparisons (i.e., those including Ceasia and E. caeruleum that occur in allopatry with re- spect to one another) are shown in black. Sympatric comparisons (i.e., those including Ceasia and E. caeruleum that occur in sympatry with respect to one an- other) are shown in white. Grouping bars are also used to indicate allopatric species pairs (left) versus sympatric species pairs (right). Significance levels from ANOVAs comparing allopatric and sympatric species pairs are shown. Downloaded from https://academic.oup.com/cz/article-abstract/64/1/101/4665098 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Moran and Fuller  Reproductive and agonistic character displacement in darters 109 Table 5. Results of ANOVA testing for ACD in focal Ceasia MA bias in male competition trials Focal male fin flare bias df Test statistic P Focal Ceasia population identity 2, 33 8.34 0.0012 Sympatric E. spectabile versus allopatric E. spectabile 22 5.28 <0.0001 Sympatric E. spectabile versus allopatric E. pulchellum 22 2.85 0.0093 Allopatric E. spectabile versus allopatric E. pulchellum 22 0.84 0.41 Focal male attack bias df Test statistic P Focal Ceasia population identity 2, 33 9.12 <0.001 Sympatric E. spectabile versus allopatric E. spectabile 22 4.53 0.0002 Sympatric E. spectabile versus allopatric E. pulchellum 22 3.82 <0.001 Allopatric E. spectabile versus allopatric E. pulchellum 22 0.65 0.52 Notes: We asked whether focal male fin flare bias and focal male attack bias differed among focal Ceasia males in 3 study populations: sympatric E. spectabile, allopatric E. spectabile, and allopatric E. pulchellum. Pairwise post-hoc t-test results are also shown for both analyses. and E. pulchellum) that are allopatric with respect to E. caeruleum males compared with the conspecific E. spectabile rival males do not prefer conspecific over heterospecific Ceasia females. In di- (Supplementary Figure S3c). This resulted in a significant difference chotomous MC trials, focal male mate choice did not differ between in rival male fin flare bias between allopatric E. spectabile and allo- allopatric E. spectabile and allopatric E. pulchellum (F ¼ 0.29; patric E. pulchellum (F ¼ 5.79; P ¼ 0.025; Supplementary Figure 1,22 1,22 P ¼ 0.60; Supplementary Figure S3a, b). Additionally, focal male S4), despite the pattern being consistent with the prediction for mate choice did not differ from a null expectation of 0.5 in allopat- CACD. Rival male attack bias did not differ between trials with ric E. spectabile (mean6 SE: 0.426 0.04; one-sample t-test: allopatric E. spectabile versus allopatric E. pulchellum serving as the t ¼1.94, P ¼ 0.08) or in allopatric E. pulchellum (mean6 SE: focal male (F ¼ 0.10; P ¼ 0.75; Supplementary Figure S4). 11 1,22 0.456 0.04; one-sample t-test: t ¼1.28, P ¼ 0.23). Similarly, in the male competition trials rival male mate choice did not differ between allopatric E. spectabile and allopatric E. pulchellum Behavioral isolation indices (F ¼ 0.12; P ¼ 0.73; Supplementary Figure S4). To examine macroevolutionary patterns of RCD and ACD among 1,22 In contrast, there was no evidence for CRCD in female mating Ceasia–E. caeruleum species pairs, and CRCD and CACD among preference. Focal female mate choice did not differ between allopat- Ceasia–Ceasia species pairs, we compared the behavioral isolation ric E. spectabile and allopatric E. pulchellum, and these preferences indices calculated in this study with behavioral isolation indices cal- did not differ from 0.5 (Supplementary Table S4). There was no sig- culated by Moran et al. (2017; Table 6 and Figures 3 and 4). The nificant difference in the proportion of female nosedigs toward rival pattern in male mating preference was consistent with RCD between males as function of their identity (conspecific or heterospecific) Ceasia and E. caeruleum species pairs and CRCD between Ceasia when we controlled for the proportion of time each male pursued and Ceasia species pairs. MC indices were consistently higher be- the female (Supplementary Table S5). tween sympatric species pairs compared with allopatric species pairs, signifying enhanced preference for mating with conspecifics in sympatry. RCD was indicated in the Ceasia–E. caeruleum compari- CACD between Ceasia species sons as MC was higher for sympatric compared with allopatric spe- CACD predicts that Ceasia males that are sympatric with respect to cies pairs (F ¼ 56.35, P < 0.0001; Figure 3). CRCD was 1,82 E. caeruleum should bias their aggression toward conspecific over indicated in the Ceasia–Ceasia comparisons as male Ceasia that are heterospecific Ceasia males, despite the fact that the 2 Ceasia species sympatric with respect to E. caeruleum had heightened MC indices, are allopatric with respect to one another. CACD also predicts that despite the fact that all Ceasia are allopatric to one another Ceasia males that are allopatric with respect to E. caeruleum should (F ¼ 6.64, P ¼ 0.01; Figure 4). The difference in MC indices in 1,70 not bias their aggression more toward conspecific versus heterospe- sympatry versus allopatry was greater in Ceasia–E. caeruleum pair- cific males. Moran et al. (2017) paired Ceasia species that occur in ings than in Ceasia–Ceasia pairings (Table 6). sympatry with respect to E. caeruleum and found high levels of male Conversely, we did not observe a pattern consistent with RCD preference for fighting with conspecific over heterospecific Ceasia or CRCD in female mating preferences. FC indices did not differ as males. Here, we show that Ceasia species (i.e., E. spectabile and a function of sympatry with respect to E. caeruleum in Ceasia–E. E. pulchellum) that are allopatric with respect to E. caeruleum show caeruleum (F ¼ 0.96, P ¼ 0.33) or Ceasia–Ceasia comparisons 1,82 no such male bias in aggressive behavior. Focal male fin flare bias (F ¼ 0.18, P ¼ 0.67; Table 6 and Figures 3 and 4). This was due 1,70 did not differ between allopatric E. spectabile and allopatric E. pul- to females not exerting any detectable mating preferences for con- chellum (F ¼ 1.79; P ¼ 0.19; Supplementary Figure S3c, d), nor specific males. 1,22 did focal male attack bias (F ¼ 0.84; P ¼ 0.37; Supplementary We observed a pattern consistent with divergent ACD between 1,22 Figure S3e,f). Ceasia and E. caeruleum species pairs and CACD between Ceasia and Rival male behavior showed a similar pattern consistent with Ceasia species pairs. MA indices were consistently higher between CACD. In the trials where allopatric E. pulchellum served as focal sympatric species pairs compared with allopatric species pairs, indi- males, both conspecific E. pulchellum rival males and the allopatric cating increased male preference for fighting with conspecific over E. spectabile rival males directed a similar number of fin flares to- heterospecific males in sympatry. This pattern was present both ward focal males (Supplementary Figure S4d). However, in trials within the Ceasia–E. caeruleum comparisons (F ¼ 136.30, 1,166 where allopatric E. spectabile served as focal males, the allopatric P < 0.0001; Figure 3; indicating ACD) and within the Ceasia–Ceasia E. pulchellum rival males directed more fin flares toward the focal comparisons (F ¼ 34.17, P < 0.0001; Figure 4; indicating CACD). 1,142 Downloaded from https://academic.oup.com/cz/article-abstract/64/1/101/4665098 by Ed 'DeepDyve' Gillespie user on 16 March 2018 110 Current Zoology, 2018, Vol. 64, No. 1 Figure 4. Patterns of CRCD and CACD between Ceasia species. Behavioral isolation indices (with 95% confidence intervals) for (A) MA, (B) MC, and (C) FC be- tween pairs of Ceasia species. Allopatric comparisons (i.e., comparisons including Ceasia species that both occur in allopatry with respect to E. caeruleum) are shown in black. Sympatric comparisons (i.e., comparisons including Ceasia species that both occur in sympatry with respect to E. caeruleum) are shown in white. Grouping bars are also used to indicate allopatric species pairs (left) versus sympatric species pairs (right). Significance levels from ANOVAs comparing allopat- ric and sympatric species pairs are shown. Table 6. Behavioral isolation indices (mean6 standard error) for MA, MC, and FC, calculated from male competition assays that paired 2 Ceasia species or paired Ceasia with E. caeruleum Geography Pairing Species Hypotheses tested n MA MC FC Allopatric Ceasia–Ceasia E. spectabile–E. pulchellum CRCD/CACD 24 0.0160.07 0.1160.07 0.0160.02 Sympatric Ceasia–Ceasia E. fragi–E. uniporum CRCD/CACD 16 0.3860.08 0.3160.07 0.0160.01 Sympatric Ceasia–Ceasia E. fragi–E. burri CRCD/CACD 16 0.5060.06 0.3060.07 0.0260.01 Sympatric Ceasia–Ceasia E. fragi–E. spectabile CRCD/CACD 16 0.3560.06 0.3460.10 0.0160.02 Allopatric Ceasia–E. caeruleum E. spectabile–E. caeruleum RCD/ACD 24 0.0960.09 0.2260.12 0.1660.16 Allopatric Ceasia–E. caeruleum E. pulchellum–E. caeruleum RCD/ACD 24 0.3060.12 0.2560.12 0.0160.02 Sympatric Ceasia–E. caeruleum E. fragi–E. caeruleum RCD/ACD 48 0.8060.05 0.7660.06 0.0160.04 Sympatric Ceasia–E. caeruleum E. uniporum–E. caeruleum RCD/ACD 16 0.8260.06 0.7060.09 0.1160.13 Sympatric Ceasia–E. caeruleum E. burri–E. caeruleum RCD/ACD 16 0.9260.03 0.6660.08 0.0560.05 Sympatric Ceasia–E. caeruleum E. spectabile–E. caeruleum RCD/ACD 32 0.8560.05 0.8460.06 0.0360.02 Notes: As all species of Ceasia occur allopatrically with respect to one another, here geography for a given pairing refers to the relationship between Ceasia and E. caeruleum. For each species pairing, the Ceasia species that acted as the focal Ceasia in behavioral trials is listed first, followed by the species that it was observed with (a heterospecific Ceasia or E. caeruleum). Sample size (n) and hypotheses tested (CRCD/CACD in pairings between 2 Ceasia species, or RCD/ACD in pairings between Ceasia and E. caeruleum) are listed. Data from Moran et al. (2017). Calculated using data from the present study combined with data from Moran et al. (2017). MA was higher between sympatric Ceasia and E. caeruleum pairs (Table 5). Second, we found evidence for ACD and RCD among than it was in sympatric Ceasia and Ceasia pairs (Table 6). closely related species in the Ceasia species complex. Males showed no preference for mating (Table 3) or fighting (Table 5) with conspe- cifics over heterospecifics in pairings of allopatric E. pulchellum and Discussion allopatric E. caeruleum. This stands in contrast to the results of Moran et al. (2017), which found high levels of male preference for Striking patterns of RCD and ACD driven by male behavior are pre- mating and fighting with conspecifics over heterospecifics in sympat- sent at 2 taxonomic levels within Ceasia. First, we found evidence for ric pairings of Ceasia species and E. caeruleum. We discuss how the both RCD and ACD among populations within species (Figure 3 and data from the present study and Moran et al. (2017) reveal a pattern Supplementary Figure S1; Table 2). We observed RCD in male mate consistent with RCD and ACD at a macroevolutionary scale between choice among populations of E. spectabile and E. caeruleum. Male Ceasia species and E. caeruleum (see below). (but not female) preference for conspecific mates was enhanced in Most of our efforts were directed at testing for RCD and ACD in sympatric (versus allopatric) population pairings of these species Ceasia. However, we also found evidence for RCD in male mate (Tables 3, 4, and Supplementary Table S2). We also found evidence choice (Supplementary Figure S2 and Table S1) and ACD in MA of divergent ACD among populations within E. spectabile and bias in E. caeruleum (Supplementary Figure S2 and Tables S3), but E. caeruleum. Males preferentially biased their aggression toward the pattern of divergent ACD observed in male E. caeruleum conspecific males to a greater extent in sympatric population pairings Downloaded from https://academic.oup.com/cz/article-abstract/64/1/101/4665098 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Moran and Fuller  Reproductive and agonistic character displacement in darters 111 behavior was not as extreme as that observed in Ceasia. ACD was preferences (Panhuis et al. 2001), male coloration in darters appears indicated in E. caeruleum in that sympatric male E. caeruleum were to be under intrasexual selection due to intense male–male competi- less likely to flare their fins at sympatric male E. spectabile, but tion. RCD and ACD can lead to shifts in behavioral response to het- E. caeruleum males from both sympatric and allopatric populations erospecifics and in the signals used in species recognition (Brown did not perform many attacks toward E. spectabile or E. pulchellum and Wilson 1956; Grether et al. 2009). Thus, examining whether males. We hypothesize that this difference may be related to the level character displacement in male color pattern corresponds to the of gene flow present between populations of Ceasia species versus observed ACD and CACD in male aggressive response to heterospe- E. caeruleum. RCD and ACD are more likely to be maintained over cifics would be of interest. time (and to lead to CRCD and CACD) when gene flow is low Our results also uphold previous examinations of female mate among populations within species (Yukilevich and Aoki 2016). choice in this system, which have consistently failed to detect female Ceasia and E. caeruleum both occur in small headwater streams, but preferences for conspecific males in sympatric or allopatric pairings E. caeruleum can also inhabit larger order streams and rivers (Page of Ceasia and E. caeruleum (Pyron 1995; Fuller 2003; Zhou et al. 1983), leading to more opportunities for gene flow among popula- 2015; Moran et al. 2017). FC may be prevented by the presence of tions (Echelle et al. 1975, 1976). Gene flow from sympatric to allo- intense male competition in these species. Further study is needed to patric populations of E. caeruleum may result in the loci for MA determine whether females exhibit any cryptic forms of mate choice bias spreading beyond the zone of sympatry. Indeed, population (Eberhard 1996), such as adjusting the number of eggs laid when genetic analyses of 4 species of Ceasia and E. caeruleum found mating with conspecific versus heterospecific males. increased heterozygosity and higher levels of nucleotide diversity present in E. caeruleum compared with Ceasia (Moran et al. 2017), Selection underlying RCD and ACD indicating lower levels of gene flow in species of Ceasia. The presence of hybridization in conjunction with high levels of We also tested for patterns consistent with CRCD and CACD postzygotic isolation between Ceasia and E. caeruleum (Zhou 2014; between species of Ceasia (Table 2 and Figure 4). We observed that Moran R, unpublished data) suggests that RCD in these species may allopatric E. spectabile and allopatric E. pulchellum males showed occur via reinforcement. Selection for males to prefer conspecific no preference for conspecific over heterospecific Ceasia females, nor mates (to avoid maladaptive hybridization) would establish females did they bias their aggression more toward conspecific over hetero- as an unshared resource between species, making interspecific fight- specific Ceasia males (Supplementary Figures S3 and S4). Our previ- ing over females costly. Theoretical treatments of ACD predict that ous work indicated that sympatric Ceasia species have a clear selection may favor divergence in male aggressive traits between spe- preference to mate and fight with conspecific over heterospecific cies when males compete for separate resources (i.e., females), which Ceasia (Moran et al. 2017). Together, these data reveal a clear pat- decreases the prevalence of interspecific aggression in sympatry tern of CRCD in male mate choice and CACD in MA among Ceasia (Okamoto and Grether 2013). In the case of Ceasia and E. caeru- species (see below). leum, a lowered aggressive response to heterospecific males may also facilitate their co-occurrence within the same habitat in sympat- Relationship to previous studies in darters ric drainages. The fact that the 2 species can co-occur in sympatry Considering our results together with those of a recent study by provides further opportunities for interspecific encounters and hy- Moran et al. (2017) reveals 2 macroevolutionary patters: (1) RCD bridization, further strengthening selection for divergence in mating and ACD are present between species of Ceasia and E. caeruleum traits and behavioral isolation via RCD. In this manner, RCD and and (2) cascading effects of RCD and ACD between Ceasia and ACD may strengthen one another in a positive feedback loop. There E. caeruleum have incidentally contributed to allopatric divergence is evidence for such a feedback loop scenario between types of char- among closely related lineages within the Ceasia clade (i.e., CRCD acter displacement acting in Ficedula flycatchers (Qvarnstro ¨ m et al. and CACD). RCD and ACD are indicated in that Ceasia species that 2012; Vallin et al. 2012). occur in sympatry with E. caeruleum consistently show almost com- plete preference for mating and fighting with conspecifics over E. caeruleum, but no such preferences exist in Ceasia species that Selection underlying CRCD and CACD occur in allopatry with E. caeruleum (this study; Zhou and Fuller Theory predicts that CRCD or CACD can occur when populations 2014). Similarly, CRCD and CACD are indicated in that Ceasia spe- stochastically respond to selection on mating and fighting traits in cies that occur in sympatry with E. caeruleum (but allopatry with re- unique ways during RCD and ACD (i.e., mutation-order selection; spect to one another) show surprisingly high levels of male Abbott et al. 2013; Mendelson et al. 2014; Comeault and Matute preference for mating with and fighting with conspecifics over heter- 2016). Under mutation-order selection, trait divergence may occur ospecific Ceasia, but these preferences are absent in pairings of despite the presence of similar types of ecological and sexual selec- Ceasia that occur in allopatry with respect to E. caeruleum (this tion. In this way, stochastic variation in response to the same select- study; Moran et al. 2017). Future studies should determine whether ive pressures (i.e., maladaptive heterospecific interactions in patterns of CRCD and CACD are also present among populations sympatry) can potentially lead to allopatric divergence among popu- within individual species of Ceasia (as is the case with RCD and lations within species. ACD within E. spectabile). Although theory predicts that CRCD and CACD can lead to This study corroborates the results of several recent studies allopatric speciation (McPeek and Gavrilets 2006; Pfennig and Ryan which have shown that male mate choice and male competition play 2006), the majority of empirical studies that have examined CRCD an important role in driving sympatric and allopatric trait diver- and CACD to date have only tested for differences in behavioral gence in darters (Ciccotto et al. 2013; Zhou et al. 2015; Zhou and preferences among populations within species. In addition, many Fuller 2016; Martin and Mendelson 2016; Moran et al. 2017). studies have tested for CRCD by comparing levels of behavioral iso- Furthermore, although the presence of elaborate male coloration lation between populations within species that are allopatric versus is typically attributed to intersexual selection via female mate sympatric with respect to another species (Nosil et al. 2003; Downloaded from https://academic.oup.com/cz/article-abstract/64/1/101/4665098 by Ed 'DeepDyve' Gillespie user on 16 March 2018 112 Current Zoology, 2018, Vol. 64, No. 1 Lemmon 2009; Hopkins et al. 2014; Kozak et al. 2015; Comeault References et al. 2016). The implication with these studies is that RCD changes Abbott R, Albach D, Ansell S, Arntzen JW, Baird SJE et al., 2013. mating traits in such a way that increases behavioral isolation be- Hybridization and speciation. 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Current ZoologyOxford University Press

Published: Feb 1, 2018

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