University of Groningen The role of male courtship ... · Aitana Peire §and Saleta Perez Vila,...

University of Groningen

The role of male courtship behaviour in prezygotic isolation in NasoniaPeire Morais, Aitana

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7. Male courtship

behaviour and female preference in Nasonia

Aitana Peire§ and Saleta Perez Vila§, Rense Veenstra, Louis van de Zande,

Leo W. Beukeboom

§ both authors contributed equally

07-Male courtship 19/2/07 11:19 Página 129

Abstract

Recombinant inbred lines (RIL) between N. vitripennis and N.longicornis were used to dissect the structure of sexual behaviour atthe phenotypic and genotypic level. Different male behavioural traitswere found to significantly affect female’s receptivity in each of thetwo species: longer cycles, characteristic of N. longicornis males, arepreferred by N. longicornis females whereas N. vitripennis femalestend to prefer males with second number of headnods lower thanfirst, as is typical for N. vitripennis males. We confirmed the presenceof prezygotic isolation between these two species, reflected by thelower female receptivity rates in inter- than intraspecific crosses (1 vs0.45 in N. vitripennis and 0.95 vs 0 in N. longicornis). Hybrid lineswith one backcrossing to N. longicornis have a high composition ofN. vitripennis markers and traits while lines with more backcrosseshave a high composition of N. longicornis markers and traits. Thisdichotomy is unexpected as we would expect a gradient from onespecies to the other. These results indicate that sexual selection formale behaviour may be important in the speciation process inNasonia.

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Introduction

Sexual selection via female preference of male behaviour constitutesan important force driving evolution of populations. Females might usebehaviour of conspecific males as a cue to identify best fit mates, whiledivergence in male behaviour between closely related species allowsfemales to discriminate against heterospecific males, preventing themfrom wasting reproductive effort on (partial) incompatible hybrid matings(Maynard Smith 1991). The possibility of hybrid matings for populationsof different species living in sympatry might lead to sexual selectiondriving divergence between species (prezygotic isolation; MaynardSmith 1991, Coyne 1992; Turelli et al. 2001). To discriminate betweenintra- and interspecific sexual selection it is necessary to identify maletraits that induce female preference. Furthermore, to understand theevolution of sexual behaviours it is important to determine the geneticbasis of male traits under female preference. The main difficulty insexual selection studies is that species constitute “blocks” of traits, thusmaking it difficult to separate which trait or traits are the particular targetof female preference. Hybrids between closely related species, wherethe traits can be genetically segregated from each other are a precioustool available in our model organism, the parasitic wasp Nasonia.

The species group of Nasonia consists of three closely relatedspecies that present distinct variations to a cyclical stereotypedcourtship (Jachmann and van den Assem 1993; see chapter 1 for acomplete description). Although both sexes take part in the courtshipperformance, the male’s sexual behaviour is the most conspicuous(Jachmann and van den Assem 1996). Its structure is well known andextensively studied as it can easily be scored. Male courtship iscomposed of different traits that can be measured qualitatively(presence/absence) or quantitatively (duration or repetitions) and isbasically similar for all three species. When a female is presented to amale, there is a “latency” time until the male mounts onto the back ofthe female and adopts a stereotyped position (forelegs on female’s

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The role of male courtship behaviour in prezygotic isolation in Nasonia...


head, the other legs on female’s wings). After a certain time “displaystarts”: the male begins consecutive series of “headnods” accompaniedby the release of male pheromones (van den Assem et al. 1980;Jachmann and van den Assem 1993) and punctuated by pauses. A“cycle” includes a series of headnods and a pause. After a certainnumber of cycles the female becomes receptive, the male backs up andcopulates. If a female does not become receptive, the male gives upand dismounts (van den Assem and Werren 1994; van den Assem andBeukeboom 2004). Interestingly, length and number of repetitions ofquantitative courtship components differ markedly between the threeNasonia species. In general, females show a preference for the malesof their own species and reject males of, at least, one of the other twospecies, which might cause prezygotic isolation in nature (Bordensteinet al. 2000). It remains a challenge to determine the causes of femalespecies discrimination in Nasonia.

Until now, male pheromones have been implied as the onlyfactor inducing receptivity in females (van den Assem et al. 1980;Jachmann and van den Assem 1993). Male courtship seems to beinternally regulated and does not respond to external cues since malesbehave normally with dummy females (Jachmann and van den Assem1993). Female behaviour during courtship is basically remainingimmobile and signalling overt “receptivity” raising the abdomen toexpose the genital orifice, and lowering the antenna. It is this lastmovement that induces the male to go to the rear of the female andcopulate (Jachmann and van den Assem 1993). However in interspecificcrosses a female of each species typically shows reduced receptivitytowards another species’ male (van den Assem and Werren 1994;Bordenstein et al. 2000; Velthuis et al. 2005). This supports the view thatdifferences in courtship behaviour between the Nasonia species mayplay a role in reproductive isolation (Bordenstein et al. 2000).

The three sibling species of Nasonia: N. longicornis, N.vitripennis and N. giraulti, are reproductively isolated as the result ofinfection with different (species specific) strains of Wolbachia bacteriathat cause cytoplasmic incompatibility in the interspecific crosses

7. Male courtship behaviour and female preference in Nasonia

133


(Breeuwer and Werren 1995; Werren et al. 1995; Bordenstein et al.2001). However, after antibiotic treatment to remove the infection, thespecies can be mated and viable and fertile hybrids are produced(Breeuwer and Werren 1995; Beukeboom and van den Assem 2001).Hybrids between closely related species allow genetical segregation oftraits, which is a precious tool in investigating traits involved in the(sympatric) speciation process.

In this project we investigate whether there are components ofmale courtship behaviour in Nasonia that serve as cues for femalepreference. To this end, we established hybrid recombinant inbredlines (RILs) formed between two species, N. longicornis and N.vitripennis. Hybrid RILs are highly inbred lines that are expected tohave different combinations of genes and traits of the parental species.In different RILs the different components of male behaviour areindependently segregated, enabling us to analyse their influence onfemale preference. RILs constitute a more powerful tool than simplehybrids because they enable replicate testing of genetically identicalhybrid individuals.

For the genomes of N. vitripennis, and N. longicornis amedium resolution map of AFLP and microsatellites has beenestablished (Pietsch et al. in prep.; Velthuis et al. 2005), allowing toassess the relative representation of the vitripennis and longicornisgenomes in any particular RIL genome. This makes it possible to linkphenotypic characteristics of the RILs and their effect on femalepreference, to their genetic make-up.

Material & Methods

Recombinant Inbred Lines (RILs)In the Evolutionary Genetics group of the University of Groningen,recombinant inbred lines were created to analyse the genetic basis aswell as the structure of various phenotypes such as courtship

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behaviour in Nasonia. Two cured lab lines were used: N. longicornis(L) IV7R2 and N. vitripennis (V) AsymC. Since L females are choosieragainst V males than viceversa, only the easy cross direction was usedto make the RILs (hL x mV). The parental lines were highly inbred lablines, so all males and all females can be considered as replicas. Theannotation used is LV[V]: paternal nuclear genome, maternal nucleargenome, and cytoplasm origin respectively. Females constitute thefirst generation of hybrids in haplodiploids. F1 females of the crosshL x mV were labelled L1V[V] or L1V, as were haploid males derivedfrom these females. Further backcrosses were made with L males, sothat with each backcross a new RIL is formed with a 50% increase inL genes. The annotation in that case is L2V for offspring producedafter one backcross, L3V after two backcrosses, etc (fig. 1). L1V hybridswere expected to have 50% genetic content of each parental species,but a pilot molecular screen showed a strong bias towards N.vitripennis, probably due to nucleo-cytoplasmic incompatibilities(Beukeboom unpublished data). To compensate for this bias L1Vhybrid males were backcrossed towards L2V females to increasecontent in L genes. Males derived from that cross, intermediatebetween one and two backcrosses (L1.5V) were used instead of L1Vmales. For simplicity, however, we kept the L1V label for L1.5V males.We sometimes refer to RILs with more than one backcross as “LxV”where x stands for 2, 3, 6 and 10 generations of backcrosses. Afterbackcrossing, all RILs were inbred by several generations of brother-sister matings and by using only one mated female at each generation tocontinue the line. This strong inbreeding homogenizes the lines, but thelevel of homogenisation varies with the number of generations ofinbreeding. At the beginning of this project, L1V lines were inbred for 16generations, L2V lines for 15, L3V lines for 12, L6V lines for about 10, andL10V lines for about 5. Thus L1V lines are expected to be the mosthomogeneous and L10V lines the least. In summary RILs are hybridisofemale lines, each line representing a stock of a particularcombination of genes due to genetic recombination between theparental species genomes and with a progressive increase in L genes


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and behaviour with the increase of backcrosses. After one year ofcrosses and inbreeding, there were 100 different lines created. For thisproject we analysed nine L1V lines, two L2V, two L3V, two L6V and oneL10V (table 1). As representative of the pure species we used the samelines as were used to generate the RILs (N. vitripennis AsymC and N.longicornis IV7R2).

Table 1.RIL names and codes used in this chapter. The first letter of the codecorresponds to the grandfather, L: N. longicornis; the second to thegrandmother, V: N. vitripennis, the number in between indicatesbackcrosses towards N. longicornis. Last is the line number, e.g.there are 9 L1V lines (L1V-1 to 9). %L: expected percentage of

N. longicornis genes.

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Original name code %L

LV6.1 A6.2 L1V-1 62.5

LV6.1 A7.2 L1V-2 62.5

LV62.4 A6.2 L1V-3 62.5

LV63.1 A1.2 L1V-4 62.5

LV63.1 A 7.3 L1V-5 62.5

LV63.1 A9.2 L1V-6 62.5

LV68.4 A1.1 L1V-7 62.5

LV68.5 A8.2 L1V-8 62.5

LV68/64.4 A8.1 L1V-9 62.5

L2V6 B8.1 L2V-1 75

L2V7 C6.1.1 L2V-2 75

L3V1 B7.1 L3V-1 87.5

L3V10 B2.1 L3V-2 87.5

L6V1 A8.1 L6V-1 94

L6V6 B9.2 L6V-2 94

L10V66.4.3 C21 L10V 97


Figure 1. Construction of Recombinant Inbred Lines. A cross between N. longicornis (L) males and N. vitripennis (V)females yielded F1 LV hybrids. F1 LV virgin females produced F2 LVmales that were each a particular combination of genes from thetwo species. Those males crossed with new LV females (nephew-aunt crosses) produced N. vitripennis biased offspring which wasthe reason to cross those males with L2V females. This yielded L1Vlines (in fact L1.5V) with expected 62.5% L alleles. Subsequentbackcrosses to L males increased the expected proportion of Lgenome in L2V, L3V, L6V and L10V RILs. After completion ofbackcrosses, brother-sister crosses were used to homogenise eachline. Doing this with different females (isofemale lines) generatedgenetically different lines due to genetic recombination.Percentages indicate expected amount of N. longicornis genome.Boxes contain the types of lines used in this study.


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Genotypic analysisRIL females were hosted as virgins to produce only haploid maleoffspring, which were used for the molecular analysis. In this way,problems associated with diploid samples (females), such asdominance/recessive effects in lines where homogenisation is notcomplete, were avoided.

To characterise the genetic composition of the lines fourmicrosatellite markers specifically designed for N. vitripennis andwith different alleles for N. longicornis (Nv-18, Nv-39, Nv-40 and Nv-46) were used. In addition, four AFLP primer pairs (CG, FA, DD andAD, see appendix in chapter 6 for the composition of the primerpairs) were used to amplify discriminating fragments of the DNA thatare diagnostic for N. vitripennis or N. longicornis.

The number of individuals screened with molecular markersdiffered per line. The degree of homogeneity within a RIL depends onthe number of generations the line was inbred. Lines with fewbackcrosses should be more homogeneous since they haveundergone more generations of inbreeding and fewer individuals willbe required to characterise those lines than lines when morebackcross (and hence fewer inbreeding) generations have takenplace. We analysed 4 males to characterise RILs with more generationsof backcrossing (L3V, L6V and L10V) and between 1 and 4 individualsto characterise L1V and L2V RILs. The choice of the male was based onthe receptivity of its female partner(s). We chose our samples in sucha way that half of the males analysed in a RIL induced receptivity toboth pure females (L and V), and half induced receptivity to only onepure species’ female. For example, for L1V lines, 2 males that inducedreceptivity to both L and V females, and 2 males that inducedreceptivity to V females but not L females, were chosen for the geneticanalysis. When a male did not provoke receptivity to any kind offemale (see below phenotypic analysis) we discarded it. Notprovoking receptivity in any kind of female is probably associatedwith male behavioural dysfunction.

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Phenotypic analysisBehavioural observations were done with couples (1h + 1m) that wereplaced in a 12 x 75-mm glass tube under the binocular microscope (10 x)for a maximum of 10 minutes. Both male and female behaviour wasscored. Since age and temperature influence the behaviour of Nasonia(Peire, unpublished data) we standardised conditions. Both males andfemales were virgin, inexperienced and one day old. Observationswere done at constant temperature in a climate room at 25˚C.

Traits. Since RILs are a combination of N. longicornis and N.vitripennis, all components that differ between the two speciescourtship behaviours were observed and scored (table 2). There aretwo kinds of differences between the species, quantitative andqualitative. Quantitative differences are found in latency, display start,duration display, duration of the cycles, number of headnods in thecycles and total number of cycles. Qualitative differences are in maletraits present in N. longicornis and absent in N. vitripennis: antennalsweep, feet rubbing, and minus nods. Number of headnods in theconsecutive series are highly correlated, as are duration of consecutivecycles, justifying the calculation of a principal component (PC) ofnumber of headnods and cycle durations (chapter 4). A principalcomponent was calculated for the first four series of headnods andanother for the first four cycle durations. The number of cycles and theduration of the display are determined by the moment at which femalesbecome receptive. Duration of the display was calculated only whenthe female did not become receptive and can be interpreted as malegiving up time. One-way ANOVA (univariate analysis and post-hocTukey test of honestly significant difference (HSD) for homogeneousgroups) was performed to characterise the RILs according to theirphenotypic composition. In this way, in our study each trait of a RIL canbe compared to the pure species and be allocated to N. vitripennis, N.longicornis or neither. Receptivity of the females was analysed withTukey HSD tests as a characteristic of the cross, representing aninteraction between male and female. To quantify receptivity, we used


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the fraction of females that became receptive in a particular cross andthe average number of cycles required for females to become receptive.

Table 2. Courtship traits that are different between N. longicornis and N. vitripennis males.

Latency: Time from female introduction to the male, until malemounts on top of the female. Display start: time from mounted maleuntil first head-nod. Duration cycle: time from first nod of a seriesto the first nod of the next series. Duration display: time from firsthead-nod until female receptivity or male dismounting. Headnodsnumber: number of headnods in a series. 2 minus 1: the number ofnods in the second cycle minus the number of nods in the firstcycle. Number cycles: number of cycles till receptivity. Antennalsweep: large-amplitude antennal movement before the first nod ofeach cycle. Feet rubbing: forefeet circular movement of the maleover the eyes of the female. Minus nods: nods with small amplitudewithout mandible extrusion performed between the series of nods.

Trait N. vitripennis N. longicornis

Latency Short LongDisplay start Short LongCycle duration Short LongDuration display Short LongHeadnod numbers1 5-6 1-22 minus 1 negative positiveNumber cycles2 2.5 3.5Antennal sweep3 Absent PresentFeet rubbing Absent PresentMinus nods Absent Present

1 Number of headnods in the first series (first cycle only).2 Average number of cycles till receptivity.3 The antennal sweep is absent in N. vitripennis only in the first cycle

and is present in the rest of the cycles.

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C rosses. A total of 1584 couples were observed. The behaviourof 30 males per RIL was scored. RIL males were observed against bothpure species females and their own RIL females and pure species maleswere observed against both pure species and RIL females (table 3). Ineach of these crosses female differences in mate discrimination made itpossible to form two groups of males: one with males that made morethan 80% of females become receptive (“non-choosy” group) and onewhere males provoked receptivity in less than 80% of the females(“choosy” group). Comparison between those two groups showswhether female preference correlates with particular components ofmale courtship. It is to be expected that the RILs rejected by N.longicornis females are those that have N. vitripennis traits responsiblefor species discrimination. To a lesser extent, because N. vitripennis isless choosy, the same will apply to RILs rejected by N. vitripennisfemales. Because distribution of traits did not generally follow a normaldistribution (data non shown) we performed non-parametric MannWhitney U-tests to find the traits that were significantly differentbetween males that provoked female receptivity and males that did not.These traits are considered as candidate cues for species discrimination.

Table 3. Crosses scheme. RIL males with pure species females and RIL females, and purespecies males with conspecific, heterospecific and both types of RIL

females (L1V and LxV).

RIL males Pure species males

10 h RIL x m L then m V 10 h L (or V) x m L then m V10 h RIL x m V then m L 10 h L (or V) x m V then m L

10 h RIL x m RIL 10 h L (or V) x m RIL


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Results

Genotypic analysisAnalysis with molecular markers that discriminate between N.longicornis and N. vitripennis allowed to determine the proportion ofthe genome of either species in the RILs. A maximum of fourindividuals were analysed per RIL with four microsatellites and fourAFLP primer pairs which yielded 91 discriminating fragments(appendix 1). Two groups can clearly be distinguished, lines with onebackcross (L1V) had a majority of N. vitripennis markers and lineswith further backcrosses (L2,3,6,10V = LxV) had a majority of N.longicornis markers. These data can be used to obtain the percentageof N. longicornis markers (genetic composition) in each RIL. First, wecan calculate for every analysed male the proportion of L markers(number of grey cells divided by the total number of cells, seeappendix 1) and then we can estimate an average of the proportionsof the different analysed males in each RIL (fig. 2). The two groups donot correspond with the expected values of figure 1. L1V lines (in factL1.5V) have a very low percentage of N. longicornis markers and arefar below the 62.5% expected (table 1), and the LxV lines have a highpercentage of N. longicornis markers. All LxV lines show a percentagearound the 87.5% expected for L3V. Thus, we found the RILs dividedin two groups depending on the level of backcrossing.

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Figure 2. RIL genotypes. Expected (see figure 1) and observed (see appendix 1) percentage

of N. longicornis marker alleles in each RIL.

Phenotypic analysisTraits. Each of 16 RILs was analysed for sexual behaviour. As

a first categorization at the behavioural level RILs were classifiedaccording to their similarity to either or none of the parental speciesbased on Tukey homogeneous groups ANOVA1 (table 4). The testdivides all lines into homogeneous groups for each trait. Each groupcan contain some RILs and a pure species, or some RILs and both purespecies, or only RILs. The general outcome of these results isconsistent with the genotypic split of RILs in two groups. All nine L1Vlines are mostly composed of traits grouping with N. vitripennis,while the LxV lines are mostly composed of traits grouping with N.longicornis. Only latency, 2 minus 1 and duration display show somesimilarity in the two groups of RILs. Latency strongly depends on the


143


experimental conditions (place where the male is in the tube when thefemale is introduced). N. vitripennis males have a lower latency than N.longicornis males (table 2) and hybrids showed a tendency towardslonger latencies than pure species males (appendix 2). In hybridslatency serves as an indication of fitness, with higher latenciesassociated with longer reaction time and lower fitness. PC headnodswas the only trait that showed a progressive increase towards N.longicornis with increasing backcrosses (appendix 3). L1V-1, L1V-4,L1V-7, and L1V–8 lines for the 2 minus 1, and L1V-4 for the durationdisplay where grouped with N. longicornis while for all the other traitsthey were grouped with N. vitripennis. The duration display for all theLxV lines was grouped with N. vitripennis while all other traits weregrouped with N. longicornis. Some traits proved more difficult to groupwith any of the pure species, such as number of cycles and feetrubbing. The average number of cycles performed by N. vitripennis islower than N. longicornis, but the lowest numbers correspond to N.longicornis (appendix 4). While L1V lines generally grouped with N.vitripennis, we found a trend in LxV lines to perform fewer cycles thanpure species males, though in the range of N. longicornis (appendix 4).Low number of cycles could also be an indication of male fitnessassociated with feebleness in courtship. Scoring of antennal sweep, feetrubbing and minus nods in the RILs is somewhat subjective since itdepends on the locomotor ability of the male. Hybrids tend to havesome difficulties staying motionless, so that subtle movements are moredifficult to distinguish. For these reasons we let these traits out of furtheranalysis, but we present a general overview in table 4. In furtheranalyses we grouped the lines in four categories: V, L, L1V or LxV.

With the phenotypic data from table 4 we calculated theproportion of N. longicornis traits present in each RIL (number of greycells divided by the total number of cells) and compared the results tothe expected values calculated from the level of backcrossing asshown in table 1 (fig. 3). We confirmed that while theoretical valuesincreased progressively with the backcrossing level, observed valueswere in two distinct groups (table 4; fig. 3). L1V RILs have a low

144



percentage of L traits, much lower than the predicted 62.5%. LxV RILshave a high percentage of L traits but do not follow a clear increasingpattern. L6V have the highest percentage, followed by L10V, then L2V,and finally L3V. Overall, there are two groups of RILs, L1V with mostlyN. vitripennis traits and LxV with a majority of N. longicornis traits.

Figure 3. RIL phenotypes. Expected (see figure 1) and observed (see table 4) percentage of N.longicornis traits in each RIL. Observed values were calculated with

all scored traits (not shown).


145


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146



Receptivity.According to the proportion of females that became receptive

we divided the crosses in two groups: non-choosy, where more than80% of the females became receptive, and choosy, where receptivefemales were less than 80% (table 5). In receptive females, readiness tomate is represented by the number of male courtship cycles thatfemales required to become receptive. N. vitripennis males provokedcomplete receptivity (100%) in their conspecific females and noreceptivity (0%) in N. longicornis females. N. longicornis malesprovoked high but not complete receptivity (95%) in their conspecificfemales, and low but not zero receptivity (45%) in heterospecificfemales. In general, groups with high receptivity proportion involvedintraspecific crosses or crosses between N. vitripennis and L1V orbetween N. longicornis and LxV. As within N. vitripennis, crosseswithin L1V resulted in complete receptivity. The lowest receptivitywas shown by N. longicornis females towards either N. vitripennis orL1V males (15%). Consistent with these results, the lowest number ofcycles till receptivity was found in conspecific crosses or crosses withmales and females from the same RIL. The lowest average number ofcycles till receptivity was between N. vitripennis males and L1Vfemales (1.95±0.43 S.D.) and the highest in L1V males crossed with N.longicornis females (7.10±0.97) which was also the cross with theleast receptive females. In general, L1V females behaved more like N.vitripennis and accepted N. longicornis and N. vitripennis males. LxVbehaved more like N. longicornis and accepted N. longicornis malesmore than N. vitripennis males, as do pure N. longicornis females.Thus, choosy crosses generally involved heterospecific crosses andreciprocal crosses of V with LxV and of L with L1V, while non-choosycrosses involved males and females from either the same species orRIL or reciprocal crosses of V with L1V and of L with LxV. The onlyexception are the LxV-LxV crosses in which there are very few malesprovoking receptivity, probably due to low male fitness (see below).

In order to find particular traits that females may use asdiscriminating cues, we compared the behaviour of males in choosy


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versus non-choosy crosses. Overall, we found significant effects ofheadnod numbers (2 minus 1 and PC headnods, L-L1V; table 6), cycleduration (L1V-L and LxV-V; table 7), latency (LxV-V; table 7) anddisplay start (LxV-LxV; table 7) on receptivity induction, although afterBonferroni corrections only PC cycles in the cross between L1V malesand L females and display start in the cross between LxV females andmales remained significant. Despite the lack of significance we candistinguish some trends in figures 4 and 5. N. vitripennis and L1Vfemales preferred males with lower 2 minus 1 and higher headnodnumbers, which are N. vitripennis traits, while N. longicornis and LxVfemales preferred males with higher 2 minus 1 and cycle durations,which are N. longicornis traits. Interestingly, N. vitripennis and L1Vfemales accepted more readily males with higher number ofheadnods (fig. 5) except when crossed to LxV males. LxV RILs have ahigh percentage of N. longicornis traits (table 4) but they also showedsome transgressive traits usually associated with lower hybrid fitness,such as exaggerated latency and display start. LxV males accepted bythe females, including their own RIL’s females, were in general theones more similar to N. longicornis with less transgressive traits andlower values of display start and latency (fig. 4).

It should be noted that high values of display start and latencycan be an indication of low male fitness, since less fit males areweaker and slower. The fact that fitness of LxV males might influencefemale receptivity was confirmed by the fact that N. vitripennisfemales became receptive at higher proportion to LxV males whenthey were one day old than when they were two days old (64%receptivity towards 1-day-old males, 34% towards 2-days-old) andtheir fitness was also affected by age (Pérez Vila pers. obs.). Fitnessalso seemed to affect choice of N. longicornis females towards LxVmales. In general N. longicornis females prefer LxV males with non-transgressive cycle durations of values similar to pure N. longicornismales.

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Table 5. Proportion of females becoming receptive inthe different types of crosses and average number of

cycles till receptivity.V: N. vitripennis; L: N. longicornis; L1V: RILs with one backcross;LxV: RILs with two or more backcrosses towards N. longicornis.

Groups: n.c.: non-choosy; c: choosy.


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Table 6. Receptivity of females towards pure species males.

Male courtship traits in pure species males provoking (Yes) or not(No) receptivity (rec.) in females. V: N. vitripennis; L: N.longicornis; L1V: RILs with one backcross; LxV: RILs with two ormore backcrosses towards N. longicornis. Results from MannWitney U test * p<0.05; there are no significant values after

Bonferroni corrections.

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Table 7. Receptivity of females towards RIL males. Male courtship traits in RIL males provoking (Yes) or not (No)receptivity (rec.) in females. V: N. vitripennis; L: N. longicornis; L1V:RILs with one backcross; LxV: RILs with two more backcrossestowards N. longicornis. Results from Mann Witney U test * p<0.05;

** p<0.01; after Bonferroni corrections only ** are significant.


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Figure 4. Latency, display start and headnods 2 minus 1 in the differentcrosses (male-female). Average and standard errors are given. Onthe left N. vitripennis and L1V females, on the right N. longicornisand LxV females. In each graph the first four are “non-choosy”crosses and the last three “choosy”. Solid circles are for males thatprovoked receptivity and open circles for males that did not.

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Figure 5. Duration of the cycles (D; left of each graph) and number of headnods (N;right of each graph) in the 1st four cycles. Average and standard errors aregiven. On the left crosses with N. vitripennis and L1V females, on the rightcrosses with N. longicornis and LxV females. Top graphs represent “non-choosy” crosses and lower graphs “choosy”. Solid circles are for males that

provoked receptivity and open circles for males that did not.


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Discussion

We used recombinant inbred lines (RILs) generated from crossesbetween N. vitripennis females and N. longicornis males with the aimof segregating the different components of male courtship behaviourand determine which particular traits were subject to femalepreference causing prezygotic isolation between N. vitripennis and N.longicornis. Different RILs were created by repeatedly backcrossinghybrid females with N. longicornis males to increase the proportionof N. longicornis genes. Instead of the expected gradual increase in N.longicornis alleles we found a strong bias towards N. vitripennis inthe lines with one backcross to N. longicornis both at the genotypicand phenotypic level, while lines with more backcrosses were biasedtowards N. longicornis, perhaps reflecting postzygotic geneticincompatibility between the parental species. From the phenotypicstudy, we found indications for duration of the cycles playing a majorrole in mate discrimination for N. vitripennis and N. longicornisfemales when crossed with the other species males. Our data alsoseem consistent with headnod numbers, in particular the differencebetween the first two series of nods (2 minus 1) representing a cue formating in N. vitripennis. This is the first study to show that Nasoniafemales might use particular components of male courtship behaviouras cue for becoming receptive.

GenotypeWe found RILs split into two groups, lines resulting from a singlebackcross (L1V) composed mainly of N. vitripennis traits and markers,and lines with two or more backcrosses (LxV) composed of N.longicornis traits and markers. That finding differs from what wasexpected, since the aim of constructing these RILs was to have agradient of different combinations of genes from both species.

Nucleo-cytoplasmic interactions can explain a lowerrepresentation of N. longicornis genes in L1V lines. The fact that all

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RILs have a N. vitripennis cytoplasm can result in a bias in markercomposition towards N. vitripennis in the once-backcrossed RILs, sincegenes and cytoplasm have coevolved and result in fitter individuals iftogether. On the other hand, nuclear-nuclear incompatibilities might beresponsible for the sudden transition towards a majority of N.longicornis genes from lines with two backcrosses or more to N.longicornis (LxV). Accumulation of different mutations in each speciescan cause genetic incompatibility in hybrids and favour parentalcombinations of alleles. Since LxV lines have per se more N. longicornisgenes, the most viable individuals may be those with mostly N.longicornis alleles. This form of postzygotic isolation is known asDobzhansky-Muller incompatibilities (Orr and Turelli 2001). Theexperimental set up itself might have enhanced the lack of intermediateforms. At each generation a single individual female was chosen toproduce offspring and continue the line. If the first females to emergewere always chosen, only the fittest individuals would contribute to thenext generation. The fittest individuals in the first backcross would bethose with majority of N. vitripennis genes, since they would not sufferof nucleo-cytoplasmic incompatible interactions. In the consecutivebackcrosses the proportion of genes is forced to an increase of N.longicornis, since that is the species to which the hybrids are crossedback. Thus, because of Dobzhansky-Muller incompatibilities, thoseindividuals with a majority of N. longicornis genes would be fitter. It isknown that both kind of hybrid incompatibilities are responsible forpostzygotic isolation between two of the Nasonia species, N.vitripennis and N. giraulti (Breeuwer and Werren 1995; Gadau et al.1999), and we now found indication of their presence in hybrids of N.longicornis and N. vitripennis.

PhenotypeWe analysed the male courtship behaviour of several RILs withdifferent levels of backcrossing towards N. longicornis expecting tofind a progressive decrease of N. vitripennis traits in favour of N.longicornis traits. Instead, the observations showed a clear split in the


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RILs’ behaviour. Lines with one backcross (L1V) behaved in general asN. vitripennis and in lines with two or more backcrosses (LxV) malebehaviour resembled in general that of N. longicornis. This somewhathampered our analysis because courtship components inherited asblocks rather than independently. We could nevertheless use the RILsto identify courtship components that served as mating cues forfemales using the pure species as controls.

To identify cues for mating we first analysed mating preferenceof the females. We scored a female accepting a male when sheshowed receptivity and took the number of cycles before femalesbecame receptive as a measure of choosiness. Receptivity of thefemales depended on the type of cross. N. vitripennis males alwaysprovoked receptivity in conspecific N. vitripennis females and in themajority of the L1V females, whereas only about 40% of LxV femalesbecame receptive and none of the N. longicornis females. N.longicornis males provoked receptivity in almost all conspecific andLxV females, in roughly 75% of L1V females and in less than half of N.vitripennis females. L1V males provoked receptivity in N. vitripennisfemales whereas they got mostly rejected by N. longicornis. On theother hand, LxV males were widely accepted by N. longicornisfemales, but by less than 50% of N. vitripennis. These patterns of matediscrimination are broadly consistent with the presence of prezygoticisolation between N. vitripennis and N. longicornis. The fact that N.vitripennis and L1V females showed a higher level of receptivitytowards N. longicornis and LxV males, than did N. longicornis andLxV females towards N. vitripennis and L1V males is consistent withthe observation that N. longicornis is more choosy against N.vitripennis than the other way around. This might be a consequenceof the distribution of the species in nature. N. vitripennis iscosmopolitan and can be found in sympatry with either N. longicornisin western North America or N. giraulti in eastern North America andalso in allopatry, for example in Europe. N. longicornis shares all itsdistribution range with N. vitripennis and can even be found sharingthe same fly pupae (Darling and Werren 1990). The selection pressure

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is therefore much higher for N. longicornis to discriminate, as thisspecies is never free of competition with N. vitripennis. Moreover, theN. vitripennis line used to produce the RILs originated in Leiden (TheNetherlands) and was therefore allopatric to the other two Nasoniaspecies.

Receptivity in both species seems to depend on at leastpartially different cues. N. vitripennis and L1V females’ receptivityvaried with differences in headnod numbers, whereas receptivity ofN. longicornis and LxV females was much more influenced by theduration of cycles. Cycle durations in L1V lines were similar to N.vitripennis, and headnod numbers in LxV similar to N. longicornis.However the reciprocal is not true, headnod numbers varied acrossthe L1V lines as did cycle durations in the LxV lines. This could beexplained by coevolution of male trait and female preference withinthe RILs, with headnod numbers constituting the trait selected by L1Vfemales and cycle duration by LxV females. N. vitripennis receptivitywas only significantly related to duration of the cycles when crossedto LxV males. In that particular cross females preferred males thatperformed more N. longicornis-like. This seemingly contradictoryresult might be due to the fact that LxV males were much weaker thanall others, and possibly the fittest ones were those with a betterintegration of all N. longicornis characters. This was confirmed by theobservation that LxV males that made any of the females, N.longicornis, N. vitripennis or LxV, receptive showed a lower displaystart and latency than LxV males that did not provoke femalereceptivity. High values of these traits are associated with N.longicornis, but they can also be indicative of a reduction in fitness.

If male behaviour played a role in the speciation of theNasonia genus causing prezygotic isolation we would expect femalesto discriminate among mates following differences in courtshipbehaviour in sympatric but not in allopatric populations (Coyne andOrr 1998). The N. vitripennis line used to construct the RILs is ofEuropean origin and it shows, nevertheless, the capacity ofdiscriminating against N. longicornis. Perhaps N. vitripennis are


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competing for mates with yet undetected sibling European species.Small chalcidoids resembling Nasonia have been found in Europeannest boxes (Koevoets, Burton and Sykes, pers. comm.) but so far thereare no records of other species capable of mating with N. vitripennisin Europe. Another explanation could come from intraspecific sexualselection causing N. vitripennis females to choose among conspecificmales according to headnod numbers. Although this possibility wouldbe consistent with the observed effect of headnod numbers onreceptivity in L1V lines, so far we have detected no evidence ofdiscrimination between N. vitripennis lines (Peire pers. obs.).

Our data also confirmed the partial discrimination of N.longicornis females against their own males (Bordenstein et al. 2000;Beukeboom and van den Assem 2001). N. longicornis are, inprinciple, subject to higher selection pressure against interspecificmatings in the field, since their distribution overlaps with that of N.vitripennis (Darling and Werren 1990). The partial rejection ofconspecific males might be the consequence of a trade off in N.longicornis between avoiding incompatible matings and acceptingtheir own males. The third possibility is that male behaviour hasdiverged between the three Nasonia species as a consequence of driftand be the outcome of neutral evolution with no significance in thespeciation process and that pheromones are the only cause of femalemate discrimination (Van den Assem et al. 1980, Van den Assem 1986;Van den Assem and Werren 1994). For this last point, analysis ofpheromones composition of the RILs could be of particular help. Ifpheromone composition varied between RILs with differentbehaviours it would be crucial to perform experiments in whichpheromone composition and male behaviour were uncoupled todetermine whether one alone or both provoke female receptivity.Pheromones and male behaviour may also be intertwined. Forexample, females might be susceptible to the pace at whichpheromones are released at the beginning of each series, controlledby the duration of the cycles. Another possibility is that femalesrespond to the amount of pheromone discharged in each cycle,

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controlled by the number of headnods. So far, only the first headnodof a series seems to be accompanied by the release of pheromones(Van den Assem et al. 1980, Van den Assem 1986; Van den Assem andWerren 1994), but future experiments could be aimed at unravellingvariability for that performance and a possible relation with thenumber of headnods in the series.

We have found evidence that some male courtship traits,mainly headnod numbers and cycle durations influence femalereceptivity in Nasonia. We also found that N. longicornis and N.vitripennis appear to have different recognition cues. Until now, itwas assumed that pheromones were the only responsible factorinducing receptivity in Nasonia. However, experiments at that levelwere done only within the N. vitripennis species (van den Assem etal. 1980). It is not known whether pheromones inducing femalereceptivity are the same in the different Nasonia species, and to whatextent they are used as cues for discrimination. This study points atthe complementary importance that male courtship behaviour mighthave, particularly on the discrimination between species, thereforeconstituting an important means of prezygotic isolation in Nasonia.


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Appendix 1 [In the next page]

Genetic analysis of the RILs with 4 microsatellites and fourcombinations of AFLP primers. Seven L1V lines (L1V-2 to 9) and sixLxV lines (L2V 1 and 2; L3V 1 and 2; L6V-2 and L10V) were analysed.Each column corresponds to one individual. One to four individualswere analysed per line (see Material and Methods). Dashed cellsrepresent N. vitripennis alleles and solid grey cells N. longicornisalleles. Blank cells represent alleles that did not correspond to eitherpure species. Codes for AFLP primer pairs correspond to those in theappendix of chapter 6.

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Appendix 2 Mean and standard deviation of latency for all males within a RILcompared to the pure species (V and L). Symbols correspond to

the results with the Tukey test shown in table 4.

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Appendix 3 Mean and standard deviation of PC headnods for all males within aRIL compared to the pure species (V and L). PC headnods is thefactor resulting from a PCA done on the number of headnods in thefirst four cycles. Symbols correspond to the results with the Tukey

test shown in table 4.


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Appendix 4 Mean and standard deviation of the total number of cycles for allmales within a RIL compared to the pure species. Only unreceptivefemales are taken into account. Symbols correspond to the results

with the Tukey test shown in table 4.

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