Eggspot Number And Sexual Selection In The Cichlid Fish .
Eggspot Number and Sexual Selection in the Cichlid FishAstatotilapia burtoniFrederico Henning, Axel Meyer*Lehrstuhl für Zoologie und Evolutionsbiologie, Department of Biology, University of Konstanz, Konstanz, GermanyAbstractSexual selection on male coloration is one of the main mechanisms proposed to explain the explosive speciation rates inEast African cichlid fish. True eggspots are color patterns characteristic of the most species-rich lineage of cichlids, theHaplochromini, and have been suggested to be causally related to the speciation processes. Eggspots are thought to haveoriginated by sensory exploitation and subsequently gained several roles in sexual advertisement. However, for most ofthese functions the evidence is equivocal. In addition, the genetic architecture of this trait still is largely unknown. Weconducted bidirectional selective breeding experiments for eggspot numbers in the model cichlid, Astatotilapia burtoni.After two generations, low lines responded significantly, whereas the high lines did not. Body size was both phenotypicallyand genotypically correlated with eggspot number and showed correlated response to selection. Males with highernumbers of eggspots were found to sire larger offspring. Despite the potential to act as honest indicators of fitness, thebehavioral experiments showed no evidence of a role in either intra- or inter-sexual selection. Visual-based femalepreference was instead explained by courtship intensity. The evolution of this trait has been interpreted in light of adaptivetheories of sexual selection, however the present and published results suggest the influence of non-adaptive factors suchas sensory exploitation, environmental constraints and sexual antagonism.Citation: Henning F, Meyer A (2012) Eggspot Number and Sexual Selection in the Cichlid Fish Astatotilapia burtoni. PLoS ONE 7(8): e43695. doi:10.1371/journal.pone.0043695Editor: Dirk Steinke, Biodiversity Insitute of Ontario - University of Guelph, CanadaReceived May 2, 2012; Accepted July 23, 2012; Published August 24, 2012Copyright: ß 2012 Henning, Meyer. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Funding: FH is supported by the Brazilian National Counsel of Technological and Scientific Development (CNPq)/German Academic Exchange Program (DAAD)cooperation program (GDE 290049/2007-5). AM is funded by the Deutsche Forschungsgemeinschaft and the University of Konstanz. The funders had no role instudy design, data collection and analysis, decision to publish, or preparation of the manuscript.Competing Interests: The authors have declared that no competing interests exist.* E-mail: axel.meyer@uni-konstanz.despawning and release of semen (see Video S1). Femalehaplochromines collect the eggs in their mouth immediately afterthey are spawned, presumably as an adaptive response to theheavy rates of predation on eggs. It was originally proposed thateggspots deceive females to mouth at the males anal fin to ensurefertilization [4]. However, the experimental removal of eggspotshad no effect on fertilization success [7,8]. Some mate choicestudies suggest that advantages to males can instead derive fromincreased mating frequency [7]. It was reported that in severalhaplochromines, females discriminate among males based onpresence [7], particular number and size [9] and high numbers[7,8,10]. Most haplochromines have a variable numbers ofeggspots. In others, such as Pseudotropheus (Maylandia) lombardoi, itwas reported that males have a single spot and that female matechoice is the likely source of selection [9].By mimicking eggs eggspots might constitute one of the fewclear examples of sensory exploitation and pre-existing bias [11]and might have evolved by exploitation of an ancestral sensorybias [12]. Empirical and theoretical studies suggest that sensoryexploitation might be important for the initial evolution ofpreference and signal [13]. The costs of being exploited by malescan subsequently favor the evolution correlations with fitnessrelated traits and sexual advertisement functions leading toadaptive female choice [13]. A role in sexual advertisement wassuggested to account for female preference for high numbers ofeggspots. It was proposed that high numbers indicate geneticIntroductionThe haplochromine lineage of cichlid fishes has the fastestknown speciation rate [1]. With little overall genetic differentiation, cichlids have achieved an extraordinary diversity includingecological types and coloration polymorphisms [2]. Eggspots(Figure 1), also referred to as egg-dummies or –mimics, areconsidered by some authors to be key-evolutionary innovations ofhaplochromines that might influence speciation rates [3,4,5,6]. Adirect role of eggspots in speciation was suggested by Goldschmidtand Visser [5], who reasoned that divergent selection regimes onegg morphology could lead to divergence of eggspots and femalepreference, thus facilitating speciation. Furthermore, the origin ofthis trait coincides with the origin of the modern haplochrominesleading to the perception that eggspots might be causally involvedin the explosive speciation rate of this lineage [3]. In the presentmanuscript, we analyze the short-term response of number ofeggspots to artificial selection and test the hypotheses that thenumber of eggspots is an honest indicator of survival and socialstatus and therefore a target for adaptive female choice.Eggspots are color traits usually located on the anal fin and areconsidered to mimic eggs. They play many important roles in themating behavior of haplochromines. In order to attract mates,territorial males approach and quiver their anal fin therebydisplaying it and their eggspots to females. During courtship andmating displays, both sexes perform this quivering behavior andthis induces the partner to mouth at their anal fin, elicitingPLOS ONE www.plosone.org1August 2012 Volume 7 Issue 8 e43695
Sexual Selection and Eggspots in a Cichlid Fishquality [8] and are correlated with male fitness traits such assurvival and social dominance [14].Little is known about the factors responsible for the variation innumbers of eggspots. Even the simplest explanation for thevariation in numbers (positive phenotypic correlation with bodysize) has received mixed support [14,15]. Furthermore, the sourcesof sexual selection (both inter- and intra-sexual) are based on alimited number of studies and species and modest sample sizes[7,8,9,10,14].In spite the enthusiasm for eggspots and their role in cichlidspeciation, the genetic architecture and transmission genetics ofthis trait still is largely unknown. Artificial selection experimentsare invaluable tools for directly investigating the response toselection, general evolvability (sustainability of the response) andthe existence of genetic correlations [16,17]. Few studies haveapplied these methods to teleosts models of evolutionary and notaquacultural research [18,19,20].The focal species of the present study, the model haplochromineAstatotilapia burtoni is polymorphic for eggspot numbers and exhibitslek-like mating behavior [21]. A recent study on the variation ofeggspot numbers in this species shows that numbers of eggspots arehighly heritable. On the basis of a correlation with age and successin male-male competition, the authors proposed that highnumbers of eggspots function as sexual advertisers [14].The present study aims to analyze a) the response of eggspotnumbers to bidirectional selection b) the phenotypic and geneticcorrelations with body size; c) the strength of female matingpreference on this trait and; d) test the sexual advertisementfunctions. In addition, ontogenetic series were analyzed todisentangle of effects of aging and growth on eggspot numbersand assess the proximal mechanisms of eggspot gain.Figure 1. Eggspot number variation in Astatotilapia burtoni. Adominant male is shown on the top. A histogram showing thefrequency of eggspot numbers in a sample of 167 males of 6 months ofage (bottom-left). An anal fin with four conspicuous eggspots is shownon the Materials and MethodsExperiments were approved by the German authorities(Regierungspräsdium Freiburg, Abteilung Landwirtschaft, Ländlicher Raum, Veterinär- und Lebensmittelwesen).reproductively active and most females spawn in regular intervals(20–30 days). Ripe females have a distinct morphology (swollenabdomen and genital papilla) and respond actively to malecourtship. The sample sizes are shown in Table S1. In one case(control line, replicate one) a brood consisted exclusively offemales. Although females normally spawn in regular intervals, insome cases the selected females failed to spawn or the pair failed tomate successfully. These factors caused the second generation toconsist of one (low 1, control 1 and high 2) or two families (high 1and low 2) in some lines. This does not affect our analysis sinceinbreeding is not biased towards a particular selection line orphenotypic value.Because the eggspots of females are sometimes difficult to countunambiguously, only the male values were used in the analysis ofselection response. The number of eggspots was counted based onstandard photographs and scaled to the mean and S.D of eachsex/replicate (z score). The significance of the response to selectionof eggspots and correlated response of body size was analyzed bycomparing selection to control lines using Mann-Whitney testsbecause normality of the data in all replicates and lines wasrejected (Shapiro-Wilks test, P,0.05).Heritability was estimated using the animal model (mixedmodel) approach according to Wilson et al. [22] using theMCMCGlmm R package [23]. This estimate was based on thetotal pedigree information and all the phenotypic records. Themixed model approach including females and was chosen insteadof the traditional midparent-offspring correlation because it isResponse to Artificial SelectionBidirectional artificial selection for eggspot number was carriedout for two generations using a stock of Astatotilapia burtonimaintained and randomly mated in the laboratory for over10 years (generation time is about six months). Three selectionlines (low, high and control), with two replicates each, wereestablished from a base population of 82 individuals (58 males and22 females) of approximately one year of age. The distribution ofeggspot number of the base population is shown in Figure S1.First, six individuals were randomly selected to constitute each ofthe control lines then the three males and females with the highestand lowest numbers of eggspots were selected to establish the firstgeneration of the high and low lines, respectively. To avoid sibmatings, the second generation was generated by selecting onemale and one female from each family (within-family truncationselection). Each replicate was housed in a single tank, and thefamilies kept separate using mesh dividers. To avoid effects ofdensity resulting from different brood sizes, families were reducedthree months prior to selection to make their sizes comparable tothe smallest family. The tanks were randomized in each generationof selection. At six months of age, pairs were selected, isolated andkept separate using plexi-glass (to avoid aggression from the maleand ensure visual contact and maintenance of an activereproductive state). The pairs were inspected daily and allowedto interact without the dividers after showing signs of sexualbehaviors. In these conditions, all males become territorial andPLOS ONE www.plosone.org2August 2012 Volume 7 Issue 8 e43695
Sexual Selection and Eggspots in a Cichlid Fishmore robust to the violations of assumptions of absence of selectionand normality. This method can also account for differentunderlying error distributions, as is expected to be the case withsex in the present study (female values are uncertain, particularlyat lower phenotypic values) [16,24]. Models were compared usingthe Akaike and deviance information criteria (AIC and DIC). Thefinal model had sex as a fixed effect and tank, selection line, ageand animal (pedigree matrix) as random effects. To test for geneticcorrelations between eggspot number and body size, offspringeggspot number and body length were regressed on midparentbody length and eggspot number, respectively.Phenotypic Correlation with Body Size and OntogenyTo test whether eggspot numbers can act as an honest indicatorof survival, we investigated the correlation of eggspot number(response) with standard length and age (predictor variables) on asample of 542 males. Colinearity of the predictors was assessed bycalculating correlation coefficients and the variance inflation factor(VIF) using the R package MASS. The analysis was initiallycarried out using a generalized linear model (GLM) with Poissonerrors. However the residuals were not normally distributed, therewas evidence of heteroscedasticity and very high VIF values. Thedata was further analyzed in a linear model (LM) with robustestimates of the standard error and P values obtained using asandwich covariance matrix estimator in the R package sandwich[28]. In a second LM, the response variable was Box-Coxtransformed based on the likelihoods of the l values using the Rpackage MASS and analyzed in a LM. The transformation led toa more reliable model in which errors were normally distributedand homoscedastic. To disentangle the unique contributions of ageand size, a residual regression was performed. In this approach,the residuals of the regression of weakest predictor on the strongestare used instead of the observed values [29].The mechanisms of eggspot addition were investigated throughthe analysis of ontogenetic series from 14 individuals. Allspecimens were raised in individual tanks to avoid potential effectsof social environments and photographed at approximately6 months of age and in intervals of 2–6 months until the age of24 months. Ontogeny of eggspots in earlier stages has alreadybeen investigated [30].Female Mating PreferenceAssociation time, a widely used predictor of mating preference[10,25,26], was measured as a proxy for female preference. Thetest tank (500 l, 190670640 cm) was divided into threeenclosures using glass dividers. The two most extreme quartilesof the center compartment were considered choice zones. Thetime spent in each of the three zones was recorded for 20 min.All trials were video-taped for later quantification of malecourtship. One male from each group was introduced randomlyto each of the lateral compartments (125 l, 50 cm length). Maleswere acclimated for at least two hours in the presence of conspecifics before the trial.37 mature males (13–18 months of age) were selected andpaired to minimize size difference and maximize the difference ineggspot numbers. Two groups consisting of males with high (9–13,n 8) and low (6–7, n 8) numbers of eggspots were used in thevalid trials. To achieve a good match of size, weight and eggspotnumber some males had to be used more than once, but werepaired to different males. The mean difference in eggspot numberbetween the two males in each trial was 5.09 (S.D. 1.87). Allmales were kept in individual tanks for at least four days prior tothe experiment. A ripe female (identified visually from a stock of26 kept in sexual isolation) was transferred to the centercompartment immediately before each trial. Females took lesstime to acclimate than males, and were actively exploring the testtank one or two minutes after being introduced.Courtship intensity was measured by summing the counts of thefollowing male behaviors: approach, quiver and lead swim. The sumwas normalized by the time spent in proximity to the female toavoid obtaining a spurious correlation. Males only court in thepresence of females, so the male with more association timeautomatically courts more. After the trial, the test female wastransferred to a tank containing a sexually active, territorial maleto determine whether the female was ripe. Ripe A. burtoni femalesmate immediately with the resident male in these conditions. Somecan take up to 48 h depending on the degree of maturation of theeggs.Mate choice trials were considered valid when a) both malescourted; b) the female had visual contact with both males withinthe first 10 min and c) the female spawned within 48 h of the trial[10,25]. Multiple-regression was carried out using female association time as the dependent variable and factors known toinfluence mate choice in haplochromines were independentvariables. Those were pelvic fin length, standard length, eggspotnumber and courtship intensity. The values included in theanalysis were calculated as the difference between both males.Model simplification proceeded through sequential dropping ofthe least significant terms. Nested models were compared using Ftests. All statistical analyses were performed using R version 2.10.1[27].PLOS ONE www.plosone.orgMale-male CompetitionThe correlation of eggspot number with dominance wasassessed by allowing two males to directly compete in a tankmeasuring 90670640 cm (250 l) in the presence of 10 females(added to improve acclimation). Under these conditions, malesengage in intense territorial battles within 10 min. A total of 112males were photographed and paired to minimize size differenceand distribute other factors (such as age and social context)uniformly between the two groups of males with high and lownumbers of eggspots. 33 pairs (total number of males 66) wereselected. The difference between both males in relevant traits isshown in supporting Figure S2.Subordinate males were immediately removed after thehierarchy had been established, which usually took place within5–20 min. The probability of success of the individuals with moreeggspots in the 33 trials was tested against the null hypothesis of0.5 using a binomial test.ResultsResponse to Artificial SelectionThe standardized selection differential and responses are givenin Table 1. The response to selection was asymmetric, the low lineresponded significantly whereas the high line did not (Table 2,Figure 2A). The response trajectories are shown in Figure 2B. Thenumber of eggspots of the low line differed significantly from thecontrol line in both the first generations. Due to biased sex ratiosand failure to spawn in one family (see Materials and Methods),replicate 2 of the low line (generation 2) consisted of only 2 males.In this case, the small sample size did not allow for statisticalsignificance although the trend is clear (Figure 2B). The high linewas not significantly different in any of the generations. Thenarrow sense heritability estimated with the animal model was of0.3860.2.3August 2012 Volume 7 Issue 8 e43695
Sexual Selection and Eggspots in a Cichlid FishTable 1. Standardized selection differentials (S) and response(R) for each of the selection trolHighLowS0.1220.36Table 2. Results of two-sample Wilcox test of response toselection.RLineGenerationReplicateWP 0.81313221.5220.48Combined4150.67230The test statistic (W), P values and number of males in each replicate are given(N). Significant P values are shown in bold.doi:10.1371/journal.pone.0043695.t002Values are presented as z scores (as described in the Materials and 001The formation of 32 eggspots in 14 individuals was observed inthe ontogenetic series. All individuals gained eggspots at the tip ofthe anal fin (mean number of new eggspots per individual 1.57)(Figure 4A), three individuals (21%) gained eggspots at theproximal region (Figure 4B) and two individuals (14%) gainedeggspots at the center of the anal fin (Figure 4C). The ontogenetictrajectories of all 14 individuals are shown in Figure 4D.The correlated response of body size is shown in Figure 2C.Body size showed significant response in the first generation ofselection for high numbers of eggspots (W 1062.5, P 0.012) andfor low numbers in the second generation (W 2773, P,0.001)(Figure 2D).Female Mating Preference24 trials were conducted, out of which 10 were considered validaccording to the criteria outlined in the Materials and Methodssection. Seven trials were eliminated due to criteria a, two due to band five due to c. Females that were not ripe spent more time inthe neutral zone (48%) than females from valid trials (16.7%). Thisindicates that the difference in association time between malesreflects sexual choice, rather than schooling behavior. Someexamples of the recorded mating behaviors are shown in the VideoS1.Female association time was normally distributed (ShapiroWilks test, W 0.975, P 0.932) and was not significantlycorrelated with pelvic fin length, body size or eggspot number(Figure 3A). Courtship intensity was significantly correlated withfemale association (adjusted r2 0.435, F 7.941, 8 df, P 0.023)(Figure 3B).DiscussionHere we show that a) eggspot number responds significantlyand asymmetrically to bidirectional selection with correlatedresponse of body size; b) the phenotypic and genetic correlationswith body size are strong, and likely due to the ontogeneticmechanism by which eggspots are formed and; c) the main visualcue for mate choice in this species might be courting intensity.Our results, based on larger samples, broader distribution of thepredictor variables (age and size) and more appropriateexperimental designs, question the strength and universality ofthe previously proposed sources of sexual selection and thenotion of a paradox of standing genetic variation in this trait.Instead, we propose that the current levels of standing additivegenetic variation are consistent with the existence of weakselection pressures and possibly of selection on a geneticallycorrelated and condition-dependent trait.Male-male CompetitionAll pairs of males engaged in aggressive territorial disputes andthe dominance hierarchy was clearly established in all trials. Themales with higher numbers of eggspots were dominant in 19 out of33 trials. The estimated probability of success of 0.58 (95% C.I.,0.39–0.75) does not differ significantly from 0.5 (binomial test,P 0.49) (Figure 3C).Response to Artificial SelectionWe found an asymmetric response to selection, the lower halfof the distribution of eggspot numbers (z ,0.5) had nearlydouble the realized heritability of the upper half (Figure 5).Asymmetric responses to selection are a common finding inselection experiments. The reasons given by Falconer andMackay [31] involve experimental artifacts (random drift,inbreeding depression and unmeasured natural or sexual selectionacting during the experiment) as well as a multitude of potentialgenetic causes (genetic asymmetry, presence of major genes,scalar asymmetry). Random drift is unlikely to explain thepresent results because of the consistency of response betweenboth replicates (Figure 2B). Inbreeding depression is alsounlikely because the mean of the unselected control line didPhenotypic Correlation with Body Size and OntogenyStandard length had a significant positive effect on eggspotnumbers in all analyses. Those were a GLM (b 0.17,S.E. 0.013, Z 13.77, P,0.001), a LM with robust estimates(b 1.08, S.E. 0.055, T 19.72, P,0.001) and a LM withtransformation of the response variable (b 0.03, S.E. 0.001,T 28.84, P,0.001). The effect of age was not significant, anddropped from the models.PLOS ONE www.plosone.org14August 2012 Volume 7 Issue 8 e43695
Sexual Selection and Eggspots in a Cichlid FishFigure 2. Response and correlated response to selection. Response of eggspot numbers (A,B) and correlated response of body size (C,D) totwo generations of artificial selection. (A) Boxplots of eggspot numbers of each selection line. (B) Selection response trajectories. Eggspot numberswere scaled (z score) to eliminate the difference in range between the generations. Plotted values are the means of each replicate. (C) Boxplots ofstandard length of each line. (D) Correlated response trajectories of standard length values scaled (z score) to eliminate the difference in rangebetween the generations and sexes. Values plotted in the line plots (B and D) are the means of each replicate.doi:10.1371/journal.pone.0043695.g002not decline. Post-hatching mortality was negligible in the presentexperiment, thus indicating that differences in viability alsocannot account for the asymmetric response. This suggests thatPLOS ONE www.plosone.orgthe observed asymmetry is likely to have a genetic cause. Wildcaught A. burtoni specimens hardly develop more than a singlerow of eggspots (G. Fryer, personal communication). But wild5August 2012 Volume 7 Issue 8 e43695
Sexual Selection and Eggspots in a Cichlid FishFigure 3. Results of behavioral trials. (A) Association times of the two groups of males with high and low numbers of eggspots. Boxes representthe interquartile range, the median is shown by the dark bar and outliers are displayed as circles; (B) Regression of association time on the differencein courtship intensity of the two males in each trial; (C) Number of trials won by males with lower (light grey) and higher (dark grey) numbers ofeggspots is shown on the stacked barplot. The left axis shows the total number of trials (33) and the height of each stack represents the number oftrials won. The right axis shows the associated significance values for a binomial test with 33 events. Values more extreme than the two full lines (.22or ,11) are significantly different from a 50% chance of t broods reared in the laboratory for a period of one yearalso show high range of variation and multiple rows (F.Henning, personal observation). Although we can not at thispoint rule out the other genetic causes (major genes, directionaldominance or genetic asymmetry), it is possible that theasymmetric response is explained by scalar asymmetry: highphenotypic values might be particularly subject to environmental influence (e.g condition-dependency, rearing conditions) andthe extreme values an artifact of laboratory rearing. Theabsence of individuals with phenotypic values above 0.35 (seeTable 1) in the first generation of the high line also suggeststhat the more extreme high values observed in the basepopulation have non-genetic causes and supports the scalarasymmetry hypothesis. The intensity of selection that could beapplied on the second generation in the high line (Table 1) wasconstrained by two factors: The lack of response of the firstround of selection and the smaller variance of eggspot numbersin the first and second generations (6 months of age) ascompared to the base population (one year of age).One interesting possibility is that the asymmetry results from theprevious action of selection in the base population. Favorablealleles are expected to have frequencies above their symmetricalpoints [31]. This would suggest that the low numbers of eggspots(,6–7) or small body size were previously selected against.Body size responded in the direction of selection for eggspotnumbers in two cases (high line in generation one and low line ingeneration 2). Although this pattern is not as clear as the directselection response (presumably due to environmental variance),cross-heritability is high (0.6060.26) and significant whenestimated by the regression of mean male offspring body size onsire eggspot number (r2 0.17, F1,24 6.021, df, P 0.02). Due tothe advantages that body size confers to males in the competitionfor social dominance, it would be expected that females useeggspot number as a mate choice cue.PLOS ONE www.plosone.orgDo Females have Strong Preferences for EggspotNumbers?Female association in A. burtoni was predicted by courtshipintensity with little or no effect of eggspot number. Preference formales that court in high frequency is widespread, but notnecessarily open-ended [32]. Activity level is a potential sexualadvertiser, as it can be an indicator of condition and viability [33].Despite the limited sample size (in part due to the stringentvalidation criteria that were used), the significant association withcourtship intensity and the difference in association time betweenmature and immature females shows that our experiment wasvalid and had enough power to detect the variable that bestexplained female preference.Sexual selection might instead act on eggspot numbers byinfluencing the size of clutches that are spawned. The number ofclutches spawned with males more eggspots was larger in othercichlids, A. elegans [7] and in P. aurora [8]).A recent study on A. burtoni [34] could not demonstrate astatistically significant effect of the number of eggspots on themale’s mating success. Only one replicate (out of three) wassignificant in one experiment. Furthermore, all females in each ofthree replicates chose among the same four males. The finding,that one single male with no eggspots was more successful mightwell have been due to other causes (perhaps activity level) [33]. Forthese reasons, we have to regard the evidence for the effect ofeggspot numbers on female choice in A. burtoni as tentative only.Does Eggspot Number Signal Social Status?Eggspots have also been seen as a signal for social dominanceand, in this way, are related to spawning success [14,34]. But evenwith our larger sample, we did not detect a significant relationshipbetween eggspot number and intra-sexual competition. We believethat the discrepan
East African cichlid fish. True eggspots are color patterns characteristic of the most species-rich lineage of
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Key words: Organization, Classical theory, Taylor, Fayol and Weber. Introduction The society we belong is an organizational society. Modern society has retained high morale value of rationality, efficiency and effectiveness in contrast to previous society (Etzioni, 1964). There are relationships between individuals and organizations. It is
