Male courtship preferences demonstrate discrimination against allopatric colour morphs in a cichlid fish.

0.37 MB·English

by  Zoppoth, P

doi:10.1111/jeb.12074
Male courtship preferences demonstrate discrimination against
allopatric colour morphs in a cichlid fish
€
P. ZOPPOTH, S. KOBLMULLER & K. M. SEFC
DepartmentofZoology,UniversityofGraz,Graz,Austria
Keywords: Abstract
allopatricdivergence; Whether premating isolation is achieved by male-specific, female-specific or
Cichlidae; sex-independent assortative preferences often depends on the underlying
LakeTanganyika; evolutionary processes. Here we test mate preferences of males presented
malepreference; withfemalesofdifferentallopatriccolourvariantsofthecichlidfishTropheus
matechoice; sp., a Lake Tanganyika endemic with rich geographical colour pattern varia-
reproductiveisolation; tion,inwhichthestrengthofsexualisolationvariesbetweenpopulations.We
Tropheus. conductedtwo-waymatechoiceexperimentstocomparebehaviourofmales
of a red-bodied morph (population Moliro) towards females from their own
population with behaviour towards females from four allopatric populations
at different stages of phylogenetic and phenotypic divergence. Males courted
same-population females significantly more intensely than females of other
populations, and reduced their heteromorphic courtship efforts both with
increasing genetic and increasing phenotypic distinctness of the females. In
particular,femalesofacloselyrelatedred-bodiedpopulationreceivedsignifi-
cantly more courtship than either genetically distinct, similarly coloured
females (‘Kirschfleck’ morph) or genetically related, differently coloured
females (‘yellow-blotch’ morph), both of which were courted similarly.
Genetically and phenotypically distinct females (Tropheus polli) were not
courted at all. Consistent with previous female-choice experiments, female
courtship activity also decreased with increasing genetic distance from the
males’ population. Given successful experimental and natural introgression
between colour morphs and the pervasive allopatry of related variants, we
consider itunlikelythat assortative preferencesof bothsexes weredrivenby
direct selection during periods of secondary contact or, in turn, drove colour
pattern differentiation in allopatry. Rather, we suggest that sexual isolation
evolvedasby-productofallopatricdivergence.
assortative preferences support this hypothesis (Saetre
Introduction
et al., 1997; Russell et al., 2006; Hochkirch et al., 2007;
Assortative and conspecific mate preferences play an Kozak & Boughman, 2009; Verzijden et al., 2009;
important role in maintaining reproductive isolation O’Rourke & Mendelson, 2010). Nonetheless, a fairly
between species or among morphs within a species large proportion of studies comparing male and female
(Ritchie, 2007). It has been argued that the pressure to mate preferences between sympatric species and in
avoid hybridization should be stronger on the sex pay- hybrid zones found that male mate choice contributes
ing the higher costs for mating mistakes, which is fre- equally or even more than female choice to sexual iso-
quently assumed to be the female (Parker & Partridge, lation (Knight & Turner, 1999; Seehausen et al., 1999;
1998; Wirtz, 1999; but see Servedio, 2007). Several Jiggins et al., 2001; Shine et al., 2002, 2004; Peterson
empirical examples of female biases in the strengths of et al., 2005; Svensson et al., 2007; Pryke & Griffith,
2007; Pierotti et al., 2008; Espinedo et al., 2010), either
because males incur high costs by courtship and by
Correspondence:KristinaM.Sefc,DepartmentofZoology,Universityof
Graz,Universit€atsplatz2,8010Graz,Austria.Tel.:[phone]; mating mistakes (Albert & Schluter, 2004; Peterson
fax:[phone];e-mail:[email protected] et al., 2005; Svensson et al., 2007) or because of
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578 P. ZOPPOTH ET AL.
processes other than reinforcement and direct selection or may have been promoted during periods of second-
on mating preferences (Jiggins et al., 2004; Nosil et al., arycontactwithothermorphsprecipitatedbylakelevel
2007; Pierotti et al., 2008; Espinedo et al., 2010; Men- fluctuations (Sturmbauer et al., 2005; Egger et al.,
delson & Shaw, 2012). For example, sexual isolation 2007). With uniparental maternal brood care and a
can arise as a by-product of allopatric divergence (Coy- mating system leading to a male-biased operational sex
ne & Orr, 2004), in which case a sex bias in the ratio (Sefc, 2008), avoidance of potentially disadvanta-
strength of assortative preferences is not necessarily geous inter-morph matings would be expected to evolve
expected. Accordingly, assortative preferences were primarily in females. However, inter-morph fertility in
strong in both males and females of a Lake Malawi experimental crosses (K.M. Sefc, personal observation)
cichlid fish species when given the choice between as well as natural introgression between morphs and
individuals from their own and from an allopatric pop- the putative hybrid origin of some colour morphs
ulation(Pauerset al.,2010). (Egger et al., 2007, 2012) cast doubt on whether het-
The endemic Lake Tanganyika genus Tropheus eromorphic matings necessarily incur severe costs on
comprises another cichlid species complex with rich eitherof thesexes.
allopatric colour pattern variation (Konings, 1998; In the previous mate choice experiments, females
Schupke, 2003). Most Tropheus populations are sexually were offered a two-way choice between a homomor-
monomorphic or display only subtle sexual differences phic and a heteromorphic male, and interactions of
in size and colour pattern, and males and females each females with the males were scored in categories repre-
defend their individual territories. Both sexes employ senting aggression and mate preference. Although
colour signals to communicate in social and sexual designed as female choice experiments, both male and
interactions (Nelissen, 1976). For mating, females give female preferences could have contributed to the
up their territories and move into a chosen male’s terri- observed patterns, for instance because the intensity of
tory for several days to weeks until spawning, upon male courtship apparently influences female prefer-
which they leave the male and mouthbrood their off- ences in within-population interactions (Steinwender
spring alone for about 4 weeks (Yanagisawa & Nishida, et al., 2012). In this study, we quantified behaviour in
1991). Genetic analyses indicated that broods are fertil- homomorphic and heteromorphic intersexual interac-
ized by a single male each, that is presumably by the tions to examine whether males discriminate against
females’ temporary social mates (Egger et al., 2006). heteromorphic females and modify their behaviour
Dispersal of the stenotopic rock-dwellers is restricted by according to theirphenotypic and phylogenetic distance
habitat barriers, and significant genetic differentiation from the heteromorphic stimulus females. A two-way
as well as slight colour pattern differentiation exists male choice design was selected to assess male mate
among many adjacent populations, whereas large and preferences in situations when both homomorphic and
longstanding barriers such as river estuaries often sepa- heteromorphic choices are available. Behaviours of
rate highly distinct colour variants (Egger et al., 2007; females, for which the experiment presented a
Koblmu€ller et al., 2011). The classification of the geo- no-choice situation, were scored to assess congruency
graphically separated, phenotypically diverse but closely with malebehaviour.
related colour variants into the nominal species is not The tested males belong to a red-bodied morph
completely resolved, and populations are typically spec- occurring in southern Lake Tanganyika (population
ified by cheironyms indicating their geographical origin Moliro, Fig. 1), proposed alternatively to represent its
or colour pattern. The most recent common ancestor of own species Tropheus sp. ‘red’ (Konings, 1998) or
the species complex (the genus Tropheus excepting the belong to Tropheus moorii (Egger et al., 2007). Females
clearly divergent Tropheus duboisi) dates back to approx- of the red-bodied morph displayed strictly assortative
imately 1.5 million years ago (Ma; Koblmu€ller et al., mating preferences vis-(cid:2)a-vis several distinct morphs in
2010). Divergence times between individual popula- previous studies (Egger et al., 2008; Hermann, 2011; H.
tions range between this onset of diversification and Brindl & K. M. Sefc unpublished), but did not discrim-
the population fragmentation associated with the latest inate against a closely related and similarly coloured
major lake level rise between 10 000 and 20 000 years population (Egger et al., 2010). For this study, the
ago (Egger et al., 2007; Koblmu€ller et al., 2011), provid- heteromorphic stimulus females were chosen from
ing opportunities to examine reproductive isolation at populations with different degrees of genetic and col-
differentlevels ofphenotypic andgeneticdistance. our pattern differentiation from the Moliro population,
Studies of mate choice in laboratory experiments and namely a genetically closely related and similarly
in an artificially admixed field population suggested coloured population of the same red-bodied lineage
that reproductive isolation among allopatric morphs (Ndole), a related population with a distinct yellow-
depends (with exceptions) on the extent of phenotypic blotch colouration (population Chiseketi, T. moorii),
and genetic differentiation (Salzburger et al., 2006; a genetically distinct population with a red ‘Kirsch-
Egger et al., 2008, 2010, 2012). Where present, assorta- fleck’ (cherry-blotch) colour pattern (assigned to Tro-
tive preferences may either have evolved in allopatry pheus sp. ‘black’ by Konings, 1998) and a genetically
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Fig.1 Phylogeneticrelationships,colourpatternvariationandgeographicaldistributionoftheTropheuspopulationsusedinthisstudy.
ThephylogenetictreeofTropheuspopulationsisbasedonAFLPdataandanalysesbyEggeretal.(2007).Thecolouredbarsnexttothetree
indicatethecladesinwhichthetestedpopulationsareplaced.Colouredlinesalongthelakeshorerepresentthedistributionofthe
Tropheusmorphs(greylines:stripedTropheuspolli;blacklines:‘Kirschfleck’morph;yellowlines:theyellow-blotchmorph;redlines:the
red-bodiedmorph).PhotographscourtesyofWolfgangGessl(www.pisces.at).
and phenotypically distinct population assigned to Tro- were divided into three compartments for two stimulus
pheus polli (Fig. 1). females (outer compartments, 45 cm) and the test male
(centre compartment, 60 cm) by mesh partitions (mesh
size 13 mm). The tanks were equipped with internal
Materials and methods
box filters, heaters and overhead lights, and each com-
The fish were wild caught adults imported by an orna- partment was provided with identical hollow bricks
mental fish trader. Prior to the experiments, the fish serving as hiding place and territory focus. The fish
were weighed, measured and transferred into individ- were allowed an acclimatization period of 2 days after
ual aquaria (60 9 30 9 30 cm) equipped with internal introduction into the experimental tank, during which
box filters, heaters and hollow bricks and illuminated males and females remained separated by the partitions
with overhead white light in a 12:12 h light:dark but were able to communicate by visual, olfactory and
cycle. acoustic signals. This part of the trial corresponds to the
Experimental setup was analogous to the female- ‘decision phase’ of the design by Egger et al. (2008),
choice design developed for Tropheus by Egger et al. during which the test fish can assess the two stimulus
(2008, 2010). Experimental tanks (150 9 70 9 50 cm) fish under standardized conditions. Next, males were
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580 P. ZOPPOTH ET AL.
allowed to interact freely with each of the two stimulus number of times the female was involved in a ‘T-
females at a time in ‘sequential access’ test sessions position’ display, that is, either presented herself to the
(Egger et al., 2008), for which the partition between maleorlethimnuzzlehergenitalpapilla,or,withroles
the male and one of the females was removed, whereas reversed, nuzzled the papilla of her courtship partner.
a nontransparent panel was positioned between the Tallies were summed across the three access sessions to
male and the other female to prevent distraction of the obtainonedatapoint foreach male-femalepair.
tested individuals and manipulation of the by-standing To account for the repeated use of individual males
female. In the next session, the male was given access and females, we employed generalized linear mixed
to the other female. Access sessions lasted for 20 min models (GLMM) with male and female identities as
after the first interaction between the pair. Intervals crossed random factors. As male behaviour towards
between access sessions were at least 1 h. Each trial homomorphic females did not differ between experi-
consisted of three access sessions per stimulus female, ments (see Results), data from all four experiments
and the order of access (beginning with either the were combined to build models testing for morph-
homomorphic or the heteromorphic female) was alter- dependent differences in male and female behaviour.
nated between trials. The scoring of unobstructed inter- As the response variables were tallies of events, models
actions between pairs had proven necessary in previous were fitted with negative binomial error distributions
experiments, which had shown that association time (NB1 or NB2, dependent on which model achieved the
and interactions through a partition did not predict higher AIC value) and log link functions, and included
mating preferences, but could represent territorial total observation time as offset. Analyses were con-
behaviour as well (Egger et al., 2008). Furthermore, the ducted in R v. 2.15.0 (R Development Core Team,
repetitions of the access sessions take into account that http://www.R-project.org) withpackage glmmADMB.
males and females interact over a longer period of time
during theirpre-spawning pair-bondingphase.
Results
Males of the Moliro population (n = 15 males) were
given a choice of one female from the Moliro popula- Behaviour of males towards homomorphic females did
tion (the ‘homomorphic’ female, n = 19 females) and notdifferbetweenexperiments,andtherewasnoeffect
one female from one of the four other populations (the of body size asymmetry between males and females on
‘heteromorphic’ female), resulting in four experiments the amounts of male aggression displays and male
with the four different heteromorphic populations. Het- courtship quivers towards homomorphic females (Table
eromorphic stimulus females were from Ndole (n = 13 S2). Courtship quivers towards homomorphic females
trials, five females), Chiseketi (n = 20 trials, eight occurred in all trials (minimum rate of 0.1 quivers per
females), a location north of Mabilibili (‘Kirschfleck’; minute). In five trials (7.5% of all trials), homomorphic
n = 20 trials, eight females) and a location south of Iso- females receivednomaleaggression.
nga (T. polli; n = 14 trials, eight females). Logistic con- Homomorphic male behaviours were compared with
straints limited the numbers of stimulus females that behaviours towards females of the four different
could be used in the experiments. Figure 1 illustrates heteromorphic populations. The intensity of male
the geographical distribution, genetic relatedness and courtship, scored as number of courtship quivers,
colour patterns of the different morphs. Individuals towards homomorphic females was significantly stron-
were used in one to three trials (once, in four trials) ger than towards heteromorphic females (Table 1).
per experiment (Table S1). Body sizes (standard length, Moreover, the courtship intensities towards the differ-
SL) of individuals are reported as supplementary infor- ent heteromorphic populations differed significantly
mation (Table S1). Relative body size differences from each other except for the similar quiver rates
between males and females were calculated as towards the Chiseketi and the ‘Kirschfleck’ females
RSD = (male SL (cid:1) female SL)/(male SL + female SL). (Table 1). In particular, males courted the phenotypi-
Fish were observed continuously during the access cally and genetically similar Ndole females more inten-
sessions and components of their behaviour were sely than both, the phenotypically similar, genetically
scored with EthoLog 2.2 (Ottoni, 2000). Males and distinct ‘Kirschfleck’ females and the phenotypically
females court (among other behaviours) by quiver dis- distinct, genetically related Chiseketi females. Males did
plays, and mutual courtship involves reciprocal T-posi- not court females of the phenotypically and genetically
tions, in which one of the pair quivers and the other distinctT. pollipopulation (Fig. 2).
one nuzzles the genital papilla of the partner (Nelissen, Female courtship was observed only in encounters
1976). Courtship quivers of males and aggressive dis- between the Moliro males and females from Moliro,
plays (charges and displays with spread fins) of males Ndole and Chiseketi, and there was a positive correla-
and females were tallied to provide measures of male tionbetweenmaleandfemalecourtshipactivity(Fig. 2,
courtship and male and female aggression respectively. Table S3). The genetically and phenotypically similar
Female sexual behaviour was quantified as the sum of Ndole females courted Moliro males as frequently as
the number of courtship quivers by the female and the did the homomorphic Moliro females, and the rate, at
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Table1 Generalizedlinearmixedmodels(GLMM)estimates(interceptsandeffectestimatesbwithstandarderrorsSE)oftheeffectsof
femalepopulationontheratesofmalecourtshipquivers.Themodelwasfittedusinganegativebinomialerrordistribution(NB2)witha
loglinkfunction,andmaleandfemaleidentityascrossedrandomfactors.Pairwisecomparisonsbetweenfemalepopulationswerecarried
outbyre-runningmodelsandalternatingthepopulationusedasreference.Negativesignsofthebvaluesindicatethatcourtshiptowards
thefocuspopulationwaslessvigorousthantowardsthereferencepopulation.
Referencepopulation(interceptestimate(cid:3)SE)
Focuspopulation Moliro(0.58(cid:3)0.25) Ndole((cid:1)0.65(cid:3)0.41) Chiseketi((cid:1)2.04(cid:3)0.36) ‘Kirschfleck’((cid:1)2.39(cid:3)0.37)
Ndole b=(cid:1)1.22(cid:3)0.39
P=0.0015
Chiseketi b=(cid:1)2.62(cid:3)0.33 b=(cid:1)1.39(cid:3)0.44
P=3.8910(cid:1)15 P=0.0015
‘Kirschfleck’ b=(cid:1)2.94(cid:3)0.33 b=(cid:1)1.72(cid:3)0.44 b=(cid:1)0.33(cid:3)0.39
P=<2910(cid:1)16 P=9.0910(cid:1)5 P=0.40
Tropheuspolli b=(cid:1)7.60(cid:3)1.07 b=(cid:1)6.38(cid:3)1.11 b=(cid:1)4.98(cid:3)1.09 b=(cid:1)4.65(cid:3)1.09
P=1.3910(cid:1)12 P=9.2910(cid:1)9 P=5.1910(cid:1)6 P=2.1910(cid:1)5
which female courtship vigour increased with increas-
ing male quiver rates, did not differ between the two
morphs. In contrast, this rate was significantly higher
in females from the genetically related but phenotypi-
cally distinct Chiseketi population (Fig. 2, Table S3).
Females of the genetically and phenotypically distinct
T. polli and the genetically different but similarly col-
oured ‘Kirschfleck’ did not court the males at all
(Fig. 2).
Males directed significantly more aggressive behav-
iour against heteromorphic females than against
females of their own population, but did not vary their
levels of aggression between the different heteromor-
phic populations (Fig. 3; Table S4). Female aggression
was displayed almost exclusively by Moliro females
(Fig. 3; TableS5).
Fig.2 Ratesofmaleandfemalecourtshipandbodysize Fig.3 RatesofaggressivedisplaysbyMoliromalesagainstfemales
asymmetry(RSD)betweenmalesandfemales.Theregressionlines ofdifferentpopulations(upperpanel),andbyfemalesofthe
aredrawnusingtheinterceptsandslopesestimatedbythe differentpopulationsagainstMoliromales(lowerpanel).Different
generalizedlinearmixedmodels(GLMM)reportedinTableS3. lettersaboveboxplotsindicatesignificantpairwisecontrastswithin
Greytrianglesmarktheaveragecourtshipratesofmalesand panels[generalizedlinearmixedmodels(GLMM)inTablesS4
females(omittedinthepanelforTropheuspolli). andS5].
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582 P. ZOPPOTH ET AL.
not necessarily reflections of female behaviour and
Discussion
preferences (Takahashi et al., 2008), but may rather
We found that (i) aggressive and courtship behaviour result from male mating preferences and the ability of
of males towards homomorphic females was indepen- malesto discriminate between populations.
dent of the morph of the alternative stimulus female, The importance of male mate choice for species isola-
(ii) males behaved differently towards homomorphic tioningeneralisdifficulttoassess.Thenumberoftaxa,
and heteromorphic females in the contexts of both in which male preferences have been examined, is still
courtship and aggression, and addressed significantly rathersmallandthestudiedtaxadifferdistinctlyincon-
more courtship and significantly less aggression to ditions known or assumed to influence the evolution of
homomorphic females, and (iii) the intensity of male mating preferences (Parker & Partridge, 1998; Servedio,
heteromorphic courtship, but not of aggression, 2007),suchas matingandbreedingsystems,patternsof
depended on the females’ population, as genetically current and past geographical distributions, natural and
and phenotypically similar females received signifi- sexual selection regimes and hybrid fitness. In several
cantly more courtship than females of genetically and/ taxa, only the females displayed strong assortative pref-
or phenotypically distant populations. Females from erences (Tetrix ground hoppers, Hochkirch et al., 2007;
genetically distant populations also showed reduced darters, O’Rourke & Mendelson, 2010; guppies, Russell
courtship activity towards Moliro males, but an oppo- et al., 2006; Ficedula flycatchers, Saetre et al., 1997),
site trend in the allocation of aggression, which they whereas in others, males contributed significantly or
only displayed in homomorphic encounters. Aggression exclusively to the maintenance of assortative mating
between males and females reflects competition for (damselflies, Svensson et al., 2007; Heliconius butterflies,
feeding territories (Yanagisawa & Nishida, 1991), and Jiggins et al., 2001, 2004; mosquito fish, Espinedo et al.,
may have been determined by the interplay of body 2010; garter snakes, Shine et al., 2004; cichlid fishes,
size asymmetries, which were in favour of the Moliro Pauers et al., 2010; Egger et al., 2010; present study).
males especially in the heteromorphic encounters Frequently, assortative male preferences were found in
(Fig. 2), and sexual attraction between the pair, which studies of sympatric taxa, where costs of nonassortative
interferes withagonistic behaviour. mating could explain the evolution of mate choice in
Observations of different courtship intensities males, for example to avoid egg predation by allomor-
between different morphs are consistent with previous phic females led to a stickleback male’s nest (Albert &
studyonreproductiveisolationinTropheus,whichdem- Schluter, 2004). Moreover, low hybrid fitness favours
onstrated that discrimination against foreign popula- assortative mate choice in males when mating opportu-
tions is not universal and amongst other possible nities are limited (Peterson et al., 2005) or when court-
factors dependent on the similarity between the shipisassociatedwithmortalityrisks,asforexampledue
involved morphs (Salzburger et al., 2006; Egger et al., to predation on conspicuous, courting male damselflies
2008, 2010, 2012). In particular, in female choice (Svenssonet al.,2007).
experiments with the Moliro population and another During allopatric divergence, assortative preferences
red-colouredpopulation (Chimba; closelyrelated toand can arise in both sexes for example when the social
resembling the Ndole population tested in this study), systempromotessexualimprintingandlearning(Verzij-
courtship was equally likely to occur within and den et al., 2012) or as a consequence of the correlated
between populations (Egger et al., 2010). In contrast, divergence of sensory systems and signals (Boughman,
Moliro females exhibited strictly assortative mate choice 2002). Indeed, assortative mate preferences of cichlids
in two-way laboratory trials and in mixed-morph have been shown to be influenced by sexual imprinting
breeding populations, when the alternative populations (Verzijden & ten Cate, 2007; Verzijden et al., 2008) and
had distinctcolour patterns (bluish and yellow-blotched by associations between sensory systems, signals and
populations), whereas Moliro males occasionally environmental variation (Maan & Seehausen, 2010).
courted or mated with the heteromorphic females Both processes could theoretically be at work in Tro-
(Egger et al., 2008; Hermann, 2011; H. Brindl & K. M. pheus, as their territorial system, involving social inter-
Sefc, unpublished). The present data suggest that male actions with numerous conspecifics, may be conducive
courtship behaviour can contribute to sexual isolation to learning and individual recognition, and as divergent
between Moliro and other populations, provided that selection implied in colour pattern differentiation (Mat-
male courtship affects the probability of mating (Fors- tersdorfer et al., 2012) could also have acted on the
gren, 1997; Oliveira et al., 2000; Shamble et al., 2009; sensory system. Mate discrimination by both sexes and
Lehtonen, 2012). Asymmetric courtship between Mol- the weak isolation between similar phenotypes are con-
iro males and ‘Kirschfleck’ females and between sistent with both sexual imprinting on and a sensory
Chiseketi females and Moliro males further suggest that bias for homomorphic phenotypes, but discrimination
courtship can be displayed by one sex independent of betweenthesimilarlycolouredbutdifferentlypatterned
the behaviour of the other sex. Hence, the population- Moliro and ‘Kirschfleck’ might be expected to be
dependent courtship intensities on the part of males are weaker if preferences were based on biased colour
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Malematechoiceamongcichlidcolourmorphs 583
perception. Selection could also have targeted mate Although males behaved similarly to Chiseketi and
preferences directly or by reinforcement in periods ‘Kirschfleck’ females, the females of the two popula-
when population displacements associated with lake tions differed in their courtship behaviour towards
levelfluctuationsresultedinsecondarycontactofdiffer- Moliro males. ‘Kirschfleck’ females did not respond to
entiated morphs (Egger et al., 2007). It is certainly unli- male courtship at all, whereas Chiseketi females
kely that each of the different pairs of morphs in this showed a higher courtship rate relative to male court-
study have been interacting with each other in their ship activity than forexample the homomorphic Moliro
past, but it has been proposed that single bouts of rein- females. Although the exaggerated heteromorphic
forcementmaygiverisetogeneralchoosiness(Coyne& courtship of Chiseketi females could be a consequence
Orr, 2004). Nonetheless, offspring viability and fertility of a lack of choice offered to the females in our experi-
in experimental crosses between some morphs (K.M. ment, it is also consistent with previous observations
Sefc, personal observation), introgression and the made with females pertaining to the same yellow-
potential hybrid origin of morphs (Sturmbauer et al., blotched morph (a population from Mbita Island),
2005; Egger et al., 2007), and the link between sexual which did not discriminate between homo- and hetero-
isolation and genetic divergence provide no support for morphic males in laboratory two-way female choice
selection against heteromorphic mating. Furthermore, tests against a bluish morph (Nakaku; Egger et al.,
while genetically distinct lineages of Tropheus co-occur 2010), and spawned preferentially with the bluish
atsomelocations,itisstrikingthatthereisnosympatry males in experimental ponds stocked with yellow-
of related colour variants (Konings, 1998; Egger et al., blotch andbluish Tropheus(Hermann,2011).
2007), which would be expected if premating isolation Overall,matechoicedataofTropheussuggestthatsex-
was a product of secondary contact. Finally, a Fisherian ual isolation among populations increases with increas-
run-away process driven by female preferences has ing genetic distance. Positive correlations between
been implied in the allopatric colour differentiation in a premating isolation and genetic distance have also been
cichlid species complex from Lake Malawi, consistent observed in other allopatric species pairs, including
with assortative mating in experimental situations Drosophilaflies(Coyne&Orr,1997)andhaplochromine
(Knight&Turner,2004).Ifmatepreferencesweredriv- cichlids (Stelkens et al., 2009). In the haplochromines,
ing colour pattern differentiation in Tropheus, we would whichisthecichlidcladecomprisingthegenusTropheus
expectstrongdiscriminationalreadyintheearlieststage (Koblmu€ller et al., 2008), premating isolation was esti-
of divergence, which is, however, not the case between matedtoaccumulatefastestduringinitialdivergenceand
the genetically related morphs studied here (Moliro and reach completion after 4.8/10/22 million years (Myr),
Ndole)and previously(Moliro andChimba, Eggeret al., depending on the underlying molecular clock (Stelkens
2010). et al.,2009).Inourexperiment,thedeficiencyinrepro-
In previous studies of mate choice between Tropheus ductively motivated interactions between males of the
populations, the extent of colour pattern differentiation MoliropopulationandfemalesofT. polliandthe‘Kirsch-
between populations covaried with their genetic dis- fleck’ population is consistent with the proposed species
tance, and it was not possible to discern whether body delineations (Konings, 1998; Egger et al., 2007),
colour differences affected sexual isolation indepen- provided that Moliro females do not successfully solicit
dently of divergence time (Egger et al., 2010). In this matings from the foreign males, which has not been
study, we compared sexual isolation in four different testedhere.Sofar,however,red-morphfemalesexerted
combinations of genetic and colour pattern differentia- strictlyassortativematechoiceintwo-wayandmultiple
tion, and presented Moliro males with heteromorphic choice experiments with twoother distinct morphs (Eg-
choices of genetically and phenotypically similar ger et al., 2008; Hermann, 2011; H. Brindl & K. M. Sefc
females (Ndole), genetically and phenotypically distinct unpublished).Using amolecularclockcalibrationcorre-
females (T. polli), genetically related females with sponding to the one yielding the 4.8 Myr estimate for
distinct colour pattern (Chiseketi) and genetically dis- the completion of premating isolation in the above ha-
tinct but similarly coloured females (‘Kirschfleck’). plochrominestudy,themostrecentcommonancestorof
Males did not differentiate in their courtship rates the Tropheus species complex (excluding T. duboisi), and
between the latter two heteromorphic populations hence the maximum divergence between populations,
(Chiseketi and ‘Kirschfleck’), whereas they displayed datestoapproximately1.5 Ma (Koblmu€lleret al.,2010).
significantly more courtship quivers to the genetically This does not necessarily imply that isolation evolved
and phenotypically similar population (Ndole), and sig- exceptionally fast in Tropheus, as premating isolation
nificantly less courtship towards the genetically and among the haplochromine species was estimated in
phenotypically distinct T. polli. Apparently, male dis- no-choicetestsanddiscriminationagainstheterospecifics
crimination was influenced not only by colour pattern in the presence of conspecific females, as in our experi-
differences but also by divergence in other, for example ments, may be achieved earlier during divergence.
olfactory or acoustic, cues (Plenderleith et al., 2005; Accounting further for the imprecision of molecular
Verzijdenet al.,2010). clock dating and the different measures of premating
ª2012THEAUTHORS.J.EVOL. BIOL.26(2013)577–586
JOURNAL OF EVOLUTIONARYBIOLOGY ª2012EUROPEANSOCIETY FOREVOLUTIONARY BIOLOGY
584 P. ZOPPOTH ET AL.
isolation(spawning inStelkenset al.,2009vs. courtship morphs of the endemic Lake Tanganyika cichlid genus
intheTropheusstudies),thetimespanwithinwhichsex- Tropheus.Hydrobiologia615:37–48.
ual isolation evolved among Tropheus populations fits Egger, B., Mattersdorfer, K. & Sefc, K.M. 2010. Variable dis-
wellwiththehaplochrominemodel. crimination and asymmetric preferences in laboratory tests
of reproductive isolation between cichlid colour morphs. J.
In conclusion, males of the Moliro population
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Egger, B., Sefc, K.M., Makasa, L., Sturmbauer, C. & Salzburg-
and even vary their courtship behaviour between
er, W. 2012. Introgressive hybridization between color mor-
females of the same population and a phenotypically
phs in a population of cichlid fishes twelve years after
and genetically similar population. Discrimination human-inducedsecondaryadmixis.J.Hered.103:515–522.
among populations appears to increase with both, Espinedo,C.M.,Gabor,C.R.& Aspbury,A.S. 2010.Males,but
genetic distance and colour pattern dissimilarity. On notfemales,contributetosexualisolationbetweentwosym-
the basis of current evidence, we consider it likely that patricspeciesofGambusia.Evol.Ecol.24:865–878.
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by-product of allopatric divergence under natural or gobies.Anim.Behav.53:267–276.
Hermann,C.2011.Matechoiceinacichlidfish.PhDthesis.Uni-
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versityofGraz,Graz.
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ranted to address the mechanisms underlying sexual
the devil: reproductive interference could hamper species
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assortative mate preferences among Tropheus popula- Jiggins, C.D., Naisbit, R.E., coe, R.L. & Mallet, J. 2001. Repro-
tions at their various stages of phylogenetic and pheno- ductive isolation caused by colour pattern mimicry. Nature
typic divergence has the potential to improve our 411:302–305.
general understanding of how allopatric populations Jiggins, C.D., Estrada, C. & Rodrigues, A. 2004. Mimicry and
evolve prematingisolation ontheirway to speciation. the evolution of premating isolation in Heliconius melpomene
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Knight, M.E. & Turner, G.F. 1999. Reproductive isolation
Acknowledgments among closely related Lake Malawi cichlids: can males rec-
ognize conspecific females by visual cues? Anim. Behav. 58:
We thank Wolfgang Gessl for allowing us to use his 761–768.
photographs of fish used in this study, and Caroline
Knight, M.E. & Turner, G.F. 2004. Laboratory mating trials
Hermann for help with the experiments. Many thanks indicate incipient speciation by sexual selection among pop-
to Karen Grace-Martin for her accessible statistical ulations of the cichlid fish Pseudotropheus zebra from Lake
tutoring made available through ‘The Analysis Factor’ Malawi.Proc.R.Soc.Lond.B271:675–680.
website. Three anonymous reviewers provided valuable Koblmu€ller, S., Schliewen, U.K., Duftner, N., Sefc, K.M.,
comments. This work was supported by the Austrian Katongo, C. & Sturmbauer, C. 2008. Age and spread of the
Science Fund (FWF; grant P20883-B16 to KMS). Data haplochromine cichlid fishes in Africa. Mol. Phylogenet. Evol.
49:153–169.
deposited in the Dryad repository: doi:10.5061/dryad.
Koblmu€ller, S., Egger, B., Sturmbauer, C. & Sefc, K.M. 2010.
b78g9.
Rapid radiation, ancient incomplete lineage sorting and
ancient hybridization in the endemic Lake Tanganyika cich-
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586 P. ZOPPOTH ET AL.
effects (b (cid:3) SE) of female population (Moliro, Ndole,
Supporting information
Chiseketi) and male courtship intensity (quiver rate) on
Additional Supporting Information may be found in the female courtship.
onlineversion ofthisarticle: Table S4 Generalized linear mixed model estimating
Table S1 Body size(standard length SL) of females and effects (b (cid:3) SE) of female population on male aggres-
males, and the number of times each individual was sion.
usedin theexperiments. Table S5 Generalized linear mixed model estimating
Table S2 Generalized linear mixed model estimating effects (b (cid:3) SE) of female population on female aggres-
effects of experiment (heteromorphic female popula- sion.
tion) and RSD (relative body size difference between
the male and the homomorphic female) on aggressive DatadepositedatDryad:doi:10.5061/dryad.b78g9
and courtship behaviour of Moliro males towards the
homomorphic Molirofemales. Received 25 July 2012; revised 15 October 2012; accepted 12
Table S3 Generalized linear mixed model estimating November2012
ª2012THEAUTHORS. J.EVOL. BIOL.26(2013)577–586
JOURNAL OF EVOLUTIONARYBIOLOGYª2012EUROPEANSOCIETY FOREVOLUTIONARYBIOLOGY