University of Groningen The role of male courtship ... · importance of male courtship behaviour in the speciation process. Material & Methods Lines Nasonia are parasitic wasps of

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

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2.Natural variation of male

courtship behaviour inNasonia species

Aitana Peire,Louis van de Zande and

Leo W. Beukeboom

02-Nat Variations 16/2/07 16:18 Página 25


Abstract

To determine the standing genetic variation of male courtshipbehaviour we analysed both field and lab lines of the three Nasoniaspecies: N. vitripennis, N. longicornis and N. giraulti. Our resultsshow that while the highest differences in behaviour are foundbetween species, there is also significant variation within species.Crosses between species and lines allowed calculation of theminimum number of effective factors (nef) underlying the phenotypicdifferences. Nef was low both within and between species. Althougha low nef might indicate an oligogenic structure it can also reflect theaction of selection. Low nef between species is consistent with strongselection for divergence acting on few factors of large effect. Low nefwithin species is consistent with stabilizing selection.

27


IntroductionPrezygotic isolation mechanisms prevent maladaptive hybrid matings.Divergence of male courtship behaviour and associated femalepreference are the most studied mechanisms of premating prezygoticisolation between closely related species in sympatric populations(e.g. Noor 1999). However, one of the biggest challenges is todistinguish premating mechanisms from simple phenotypicdifferences, since divergence in courtship behaviour might also be theconsequence of intraspecific sexual selection or of random drift(Coyne and Orr 2004, table 6.1). Traits that diverge causing prezygoticisolation between species usually fulfil a common role within species.For example, male courtship behaviour is typically necessary toinduce female receptivity and while variations of behaviour withinspecies might constitute a cue for females to choose a conspecificmate, variations between species might serve as a cue for speciesrecognition (Maynard Smith 1991). Determining which traits aredifferent between closely related species might be useful as a firstapproach to find the traits causing reproductive isolation.Furthermore, phenotypic species differences are interesting per se tounderstand how evolutionary forces produce different outcomes fromthe same ancestral state.

Selection for differences on a polygenic trait might act onmany loci or affect one or few loci that have major effects on thedivergence (Orr 2001). Changes in a few factors are possibly theconsequence of strong selection forces provoking a major shift inphenotypic balance, while changes invoking a high number of factorsare likely either neutral or the consequence of gradual selection, suchas intraspecific sexual selection with gradual changes in malebehaviour and associated female preference (Saldamando et al. 2005).Crosses between species or different behavioural lines within aspecies allow estimation of the minimum number of effective factors(nef) responsible for the differences (Lynch and Walsh 1998).

An amenable system for studying the genetics of courtship

28

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


behaviour divergence is the three sibling species complex of theparasitic wasp Nasonia: N. giraulti, N. vitripennis and N. longicornis.Nasonia species are reproductively isolated by infection withdifferent strains of Wolbachia bacteria that cause cytoplasmicincompatibility in the interspecific crosses (Werren 1997; Bordensteinand Werren 1998). In nature N. longicornis and N. giraulti live inallopatry and are endemic of western respectively eastern NorthAmerica whereas N. vitripennis is cosmopolitan and lives both insympatry and allopatry with the other two species (Darling andWerren 1990). The three species show variations to a general patternof male courtship behaviour (van den Assem et al. 1980; van denAssem 1986; van den Assem and Werren 1994; chapter 1). Two of thecomponents of this behaviour, headnod numbers and cycle durationsare particularly interesting, as they show different phenotypic meansbetween the three species. Headnod numbers is the number of headmovements a male makes to accompany the release of pheromones,whereas cycle duration is the lapse of time between a series ofheadnods and the next one that are punctuated by pauses. Differencesin behaviour between the three Nasonia species probably came aboutafter the infection with Wolbachia (Bordenstein et al. 2001), and mayplay a role in reinforcing the speciation process, or be simply aconsequence of random drift (van den Assem and Werren 1994).

In this study we first determined the standing natural geneticvariation in headnod numbers and cycle durations in the threeNasonia species. To this end, we scored field and lab lines of N.vitripennis, N. longicornis and N. giraulti at constant conditions sothat phenotypic differences were not caused by environmental factorsand therefore could serve as an indication of genotypic variation(Falconer and Mackay 1996). To determine the number of effectivefactors (nef) responsible for intraspecific differences in male courtshipwe analysed F2 males from lines that showed significant differencesin at least one behavioural component. Hybrid males of N. giraultiand N. longicornis and from a cross previously performed between N.longicornis and N. vitripennis (Pietsch et al. in prep.) were analysed

2. Natural variation of male courtship beaviour in Nasonia species

29


to determine the nef for interspecific differences. Comparison of intra-and interspecific nef may yield valuable information as to theimportance of male courtship behaviour in the speciation process.

Material & Methods

LinesNasonia are parasitic wasps of blowfly pupae (Protocalliphora andCalliphora). They are easily cultured in the laboratory and have ageneration time of approximately two weeks at 25°C. Thehaplodiploid sex determining system makes it simple to obtain males,since virgin females produce all male families. Virgins can becollected by opening fly pupae before wasps emerge. A total oftwenty two lines of the three Nasonia species (11 N. vitripennis; 9 N.longicornis; 2 N. giraulti) were scored for male behaviour (table 1).While the N. longicornis lines were kept in the lab as mass culturesfor several years, all the N. vitripennis and one of the N. giraulti lines(Ng PA) were scored shortly after collection from the field. Eight N.vitripennis lines were from Europe and three from North America, thelatter being sympatric with N. longicornis.

Table 1. Lines scored for male courtship behaviour fromthe three Nasonia species with the labels used in this

study.[In next page]

h (m): total number of observed males that were sons of femaleshosted as virgin (number of mothers); h: total number of malesobserved that were collected from the mass culture. Comments: a:collected from a nestbox; b: collected from a bait; c: derived from“Lab II” line (van den Assem and Werren 1994); d: Wolbachia-cured;e: isofemale line; f: data are from Pietsch et al. (in prep.), part wereobtained in this study; g: used by van den Assem and Werren (1994).

30




31

CollectionMales

observedThis study

labelOriginal label

Location Date massculture

Comments

N. vitripennis (Nv)

EuropeanNv HV-A HV 202.1 25 (1)Nv HV-B HV 202.2 25 (1)Nv HV-C HV 236.2 25 (5)Nv HV-D HV 271.2

HogeVeluwe,

TheNetherlands 25 (1)

a

Nv Mosc BaitMoscow,Russia

52(52)

b

Nv Zven 277

ZvenigorodBiologicalStation,

60km west ofMoscow,Russia

52(52)

a

Nv St. G St. Germaine

SaintGermaine au

Mont d’Or,22km north

of Lyon,France

2002

25 (5)

Nv Asym AsymCLeiden, TheNetherlands

1971 46 (1) c, d

North AmericanNv UT-A E5.2 25 (6) aNv UT-B 10x.2 25 (5) a, eNv UT-C 13.1

Monastery inHuntsville,

Utah2005

25 (7) a

N. longicornis (Nl)

Nl CA-A STCA-JB 25 (5)

Nl CA-B09 314.2.86III

CA25

Nl CA-C CA 00 3271 25 (1)Nl CA-D CA 9304

California

20 (1)Nl UT-A IV7 R2 1986 20 (1) d, fNl UT-B UT 218 1989 25Nl UT-C 36w strain 2

Utah

1999 25 aNl-ID ID Idaho 1991 10 (1)

Nl-MN MN 8510 Minnesota 25 (1)

N. giraulti (Ng)

Ng VA RV2 R2 Virginia 1987 20 (1) d, e, gNg PA PA 233 F2 Pennsylvania 20 (6)

h h(m)


Courtship observationsNasonia courtship behaviour is stereotypic and can easily beobserved and quantified (Barrass 1960, van den Assem 1986, van denAssem and Werren 1994, Beukeboom and van den Assem 2001, 2002)Typically, Nasonia males mount on top of the females, and start thecourtship, performing series of strong movements with the headinterrupted by pauses; the so-called “headnods”. The first headnod ina series is accompanied by the release of pheromones. After a fewseries of headnods a female may become receptive, and the malebacks up and copulate. Females typically mate only once. If femalesdo not become receptive, males eventually dismount. The number ofheadnods in the first four series were scored, as well as the cycle time(time period between the first headnod of two consecutive series) inthe first three series. We used a stereo binocular microscope at 10xmagnification for all observations. Individual males were placed in 60mm glass tubes, diameter 10mm, closed off with a plug of cottonwool, and mated females were then introduced. Mated femalestypically do not mate again, allowing observation of longer courtshipbouts. All males were inexperienced and one day old, since male ageand previous experience may have an effect on the courtshipperformance (Beukeboom and van den Assem 2001).

CrossesMated females were collected from a mass culture after emergenceand hosted. In the next generation females were collected from the flypupae before emergence and hosted as virgin to produce all-maleoffspring to be scored (Fig. 1, GF). In some cases, males werecollected from the mass culture before emergence (table 1). Afterscoring, males were mated to virgin females (Fig. 1, GM) to producefemales in the first generation (Fig. 1, M) and males in the second (Fig.1, F2 males).

32



Figure 1. Crossing scheme for haplodiploids. Viewed from the F2 generation, P is the grandparental generation(GM: grandmother; GF: grandfather); F1 females are the mothers (M),and F2 males are the sons (S). Courtship behaviour of grandfatherswas scored, and they were then mated to females from a differentspecies or line. One female (F1) per couple resulting from this cross

was hosted as virgin. Emerging F2 males were then scored.

Interspecific crosses. We analysed the cross between N.vitripennis and N. longicornis first published by Beukeboom and vanden Assem (2001) with new data from Pietsch et al. (in prep.). Weperformed the cross between N. longicornis and N. giraulti with a N.longicornis line from Utah (Nl UT-A), which is the same line (IV7 R2) asused by Beukeboom and van den Assem (2001, 2002) and Pietsch et al.(in prep.), and a N. giraulti line from Virginia (Ng VA). Both lines had


33


been maintained in the lab for almost 20 years (table 1). Offspring wasanalysed from five couples between N. giraulti males and N. longicornisfemales and four couples from the reciprocal cross. One hundred F2males were scored for their behaviour for each reciprocal cross.

Intraspecific crosses. We performed crosses between differentlines within N. vitripennis and within N. longicornis. All lines scoredwere tested for differences in headnod numbers and cycle durationswith Tukey Honest Significant Differences (HSD) tests. Whendifferences were found for at least one of the courtship componentsbetween two lines we crossed them. The number of couples andscored F2 males varied between the lines and ranged from one to fiftycouples, and from one to thirteen F2 males per couple (table 2).

Table 2. Intraspecific crosses.Number of couples per cross, number of F2 males scored percouple and total number of F2 males scored in the cross betweenbrackets. First data for male from the first line x female from thesecond line followed by data for the reciprocal cross. Line labels are

as in table 1.

34


CrossCouples

male x female/reciprocal

F2 males per couplemale x female/reciprocal

(total)

N. vitripennisEurope

Nv Zven x Nv Mosc 50/50 1 (50)/1 (50)Nv Zven x Nv HV-C 9/9 5 (45)/5 (45)Nv Mosc x Nv HV-A 9/9 5 (45)/5 (45)

North AmericaNv UT-B x Nv UT-A 16/16 4 (64)/4 (64)Nv UT-B x Nv UT-C 4/4 15 (60)/15 (60)

N. longicornisNl CA-B x Nl MT 5/1 10 (50)/10 (10)

Nl Nl UT-B x Nl MT 5/4 10 (50)/13 (52)Nl UT-B x Nl UT-C 3/3 10 (30)/12 (36)


Data analysisSince number of headnods in the consecutive series are highlycorrelated as are cycle durations, principal component (PC) analyseswere used to reduce the variables to two (chapter 5). The firstprincipal component is the one that accounts for most variation andthe one we used in our analysis, which we will refer to as PC.Homogeneity of variances, the main condition for parametric tests,was tested with Brown-Forsythe tests. The hybrid crosses did not fulfilthis requirement whereas all the intraspecific crosses did (data nonshown). Therefore, in the hybrid crosses the PC of headnod numbersand cycle durations are represented with median, percentiles andoutliers. In the intraspecific crosses differences were tested withTukey HSD and the PC of headnod numbers and cycle durationsrepresented with average and standard errors.

In different behavioural lines or species, the Castle-Wrightestimators compare the segregation variance of the offspring with theparental phenotypic means to get an estimate of the minimumnumber of factors responsible for the differences (Lynch and Walsh1998). We used an adaptation of the formula for haplodiploids (Jones2001) that was previously used for Nasonia wing size difference byWeston et al. (1999):

nef = (P–

1 – P–

2) /8s2s

where P1 and P2 are the mean parental phenotypes and ss is the se-gregation variance, calculated as the variance of the pooled F2 males.The variance of the estimator is

var (nef) = (R 2 / 8s2s)2 [4 var (R) / R2 + (var (s2

s ) / (s2s )2],

where R = P–

1 – P–

2, and the var (s2s) = 2 (s2

s) / N +2, where N is thesample size (Lynch and Walsh 1998). The most critical assumption ofthe Castle-Wright estimator is that all the phenotypic variationbetween lines or species is due to additive effects of the genes. Whenthis assumption is not met it tends to underestimate the number of


35


factors (Jones 2001). We were able to check this assumption with theanalysis of the inter- and intraspecific crosses (chapter 3).

Results

Phenotypic variationMale courtship behaviour differed greatly between the three Nasoniaspecies, with a general structure consistent with previous reports (Fig.2; appendices 1 and 2). In general, N. giraulti has the highest averageheadnod numbers and cycle durations, with a high peak in headnodnumbers in the second cycle and a gradual decrease in the third andfourth cycles. N. longicornis has the lowest values for headnodnumbers, with the lowest number in the first cycle and a gradualincrease in the consecutive cycles, and intermediate cycle durations.N. vitripennis has headnod numbers intermediate to the other twospecies and the lowest cycle durations. In contrast to the other twospecies, N. vitripennis has a high peak in the headnods of the firstcycle and a low peak in the second with a gradual increase in the thirdand fourth cycles. The grouped populations of N. vitripennissympatric with N. longicornis differ more in headnod numbers withthe latter than the allopatric populations (the only non significantdifference after Bonferroni corrections is in headnod numbers ofthe fourth series between N. longicornis and allopatric N. vitripennis:t = -1.03; d.f. = 404; p = 0.3).

The lowest average number of headnods (±variance) in thefirst cycles were found in N. longicornis and ranged from 1.90±1.88(UT-A) to 4.24±2.52 (UT-B), with an average increase between theheadnods of the first and second cycles of 0.56 headnods. The highestheadnods numbers were found in the two N. giraulti lines (VA9.05±9.42; PA 8.05±5.52) with an average increase of 1.6 headnodsbetween the first and second cycles. In the N. vitripennis lines the

36



number of headnods in the first cycle ranged from 6.04±1.92 (UT-A)to 6.80±1.42 (UT-B) with an average decrease between the first twocycles of 1.55 in the sympatric lines and from 4.72±1.38 (Zven) to6.20±1.92 (Mosc) and 6.20±0.83 (HV-D) with an average decrease of1.23 headnods between the first and second cycles.

Figure 2. Average numbers and 95% confidence intervals of headnodnumbers in the first four cycles (a) and durations of the first three

cycles

37

2. NATURAL VARIATION OF MALE COURTSHIP BEAVIOUR IN NASONIA SPECIES


(b) in the three Nasonia species.

Shortest average (±variance) cycle durations were found in theN. vitripennis lines and ranged from 7.68±1.06 (UT-A, third cycle) to9.00±2.33 (UT-B, second cycle) in the North American, and from6.62±1.05 (HV-A third cycle) to 9.67±1.45 (HV-C first cycle) in theEuropean lines. Highest cycle durations were in the N. giraulti linesand ranged from 13.42±11.26 (PA first cycle) to 19.70±53.06 (VA firstcycle). Cycle durations in the N. longicornis lines were intermediateto the other two species and ranged from 7.88±26.36 (MT, first cycle)to 15.58±3.04 (CA-D, third cycle).

These results are broadly consistent with previous reports(Barras 1960; van den Assem and Werren 1994). The higher differencein headnod numbers between N. longicornis and N. vitripennissympatric than allopatric might be the consequence of selection fordivergence in sympatry (see discussion).

Number of effective factors (nef)The minimum number of factors responsible for differences inphenotype between species and lines was calculated for all thecrosses using the Castle-Wright estimator corrected for haplodiploids(Jones 2001; Figs. 3 and 4; appendix 3). Violations of the assumptionof additivity underestimate nef between species. By comparing inter-and intraspecific crosses we can estimate differences in the number offactors responsible for differences between species and the numberof factors responsible for intraspecific variation.

Interspecific crosses. We estimated a low number ofeffective factors responsible for differences in the PC of headnodnumbers between N. longicornis and the other two species (with N.vitripennis: 0.60±0.75 variance; with N. giraulti: 1.49±3.76). BetweenN. vitripennis and N. longicornis the number of factors responsiblefor differences in duration of the first cycle was 0.31±0.22 and for thePC of cycle durations between N. longicornis and N. giraulti0.72±1.28. Both values were roughly half the nef for headnod

38



numbers. Hybrids between N. longicornis males and N. giraultifemales show complete additivity in inheritance of headnod numbers(chapter 3), so the Castle-Wright will less likely be underestimated.When we estimated nef with this cross only the variance differed(1.49±0.50) which suggests that the estimates in the other crossesmight also not be significantly underestimated. The low number offactors suggests that the differences are due to mutation in one or afew loci of large effect, consistent with a bout of strong selection.

Figure 3. Castle-Wright estimator of the number of effective factors (nef)underlying interspecific differences in headnod numbers (left) andcycle durations (right) adapted for haplodiploids. Nv: N. vitripennis;Nl: N. longicornis; Ng: N. giraulti; F2 males of both reciprocal

crosses are pooled.


39


Figure 4. Castle-Wright estimator of the number of effective factors (nef)underlying differences between lines within a species in headnodnumbers (left) and cycle durations (right) adapted forhaplodiploids. Nv: N. vitripennis; Nl: N. longicornis; Ng: N. giraulti;

line codes correspond to table 1.

40



Intraspecific crosses. As in the hybrid crosses, the estimatednef responsible for intraspecific differences in headnod numbers andcycle duration was low, particularly for cycle durations. The greatestdifferences were between the Russian lines (Nv Mosc and Nv Zven)and the Dutch lines (HV-A and HV-C) probably due to drift provokedby isolation by distance. This is consistent with the fact that betweenthe Russian lines differences in cycle duration were estimated to bedue to virtually zero factors. The fact that headnod numbers hadlargely similar estimated nef between geographically distant and closelines indicates a higher intraspecific variability for this trait. The resultsof the N. vitripennis lines sympatric with N. longicornis were similarto the allopatric lines. In N. longicornis the estimated nef for headnodnumbers and cycle duration was consistently lower than in N.vitripennis even though some of the populations were from distantplaces. The lowest nef was estimated in cycle durations, where it wasalmost zero in the three crosses. This might indicate lower variationwithin N. longicornis, perhaps as a consequence of higher selectionpressures.

Discussion

We have analysed male courtship behaviour in field and lab lines ofthe three Nasonia species, N. vitripennis, N. longicornis and N.giraulti. The crosses allowed us to calculate the minimum number ofeffective factors (nef) responsible for behavioural differencesbetween and within species. Our results show that, while the highestdifferences in behaviour are found between species there is alsosignificant variation within species. Furthermore, the nef was low andsimilar for intra- and interspecific differences. Our results areconsistent with strong disrupting selection provoking divergencebetween species and stabilizing selection within species.


41


Phenotypic variationSome of the features of male courtship behaviour in the Nasonia spp.are characteristic of a single species and absent in the others, allowingspecies classification. However, there are also common elements thatvary only in phenotypic average (Darling and Werren 1990; van denAssem 1986; Beukeboom and van den Assem 2001, 2002), such asheadnods numbers and cycle durations. After a screening of 22different lines we confirmed that the differences in average number ofheadnods in the first four series and the cycle durations in the firstthree series are characteristic of the species. The feature that showsthe highest species fidelity is the structure of the courtship. Thedifference in headnod number between the first and second series,increases for N. longicornis and N. giraulti, and decreases for N.vitripennis (van den Assem 1986). After the second series, headnodnumbers increase gradually in N. longicornis and N. vitripenniswhereas they go down in N. giraulti. Cycle durations were alsodistinct between the species, N. giraulti showed the highest values, N.vitripennis the lowest, and N. longicornis intermediate. In nature, N.vitripennis lives allopatric (Europe) and sympatric (North America)with the other two species. Interestingly, headnod numbers of N.vitripennis lines that came from areas sympatric with N. longicornisdiffered from the latter species more than allopatric populations. Thisis consistent with reinforcement acting in the field to enhance pre-zygotic isolation between these two species, although furtherexperiments are needed to confirm the role of courtship in pre-zygotic isolation.

Number of effective factors (nef)From the hybrid and line crosses we calculated the minimum numberof factors responsible for the differences in headnod numbers andcycle durations between species or lines with the Castle-Wrightestimator adapted for haplodiploids (Jones 2001, Weston et al. 1999).Although maybe underestimated by the presence of non-additive(e.g. epistatic) effects, we found a very low number of effective

42



factors responsible for phenotypic differences in headnod numbersand cycle durations. In general, the number of factors was lower forcycle durations, except between European (allopatric) lines of N.vitripennis. Between species, the number of factors for differences inheadnod numbers was highest between N. longicornis and N.giraulti, while nef between N. longicornis and N. vitripennis wassimilar to nef for intraspecific differences. The number of factorsresponsible for differences in cycle durations was similar betweenand within species. The species with lowest factors for intraspecificdifferences was N. longicornis and the highest were the allopatricpopulations of N. vitripennis. In the latter, differences in number offactors in cycle durations are consistent with differences accumulateddue to isolation by distance that prevents gene flow between distantpopulations. Thus, differences between the Russian lines aregoverned by fewer factors than differences between the Russian andthe Hoge Veluwe lines. Isolation by distance does not explain the nefbetween N. longicornis populations in cycle duration that was lowirrespective of the distance between the compared populations. Theresults might reflect the nature of selection and the geneticarchitecture of each trait. Allopatric European populations are inprinciple not subject to interspecific sexual selection on malecourtship, whereas sympatric North American populations are.Selection on courtship in allopatric populations would therefore beintraspecific. Traits under sexual selection within species are expectedto evolve gradually (Orr and Coyne 1992) via runaway selection ofmating behaviour and preference (Maynard Smith 1991) and are moreprone to have a polygenic basis, whereas a single bout of strongselection is more likely to affect one or few genes of major effect (Orr2001). Such a strong selection might, even if they had not such drasticeffect before, transform a complex trait into a simpler one (Tregenzaet al. 2000).

We found a reduced number of factors explaining variationwithin N. longicornis. Inbreeding within the lab lines might explainthis result. Genetic variation has likely been depleted within the lab


43


lines, since all used N. longicornis lines had long been kept in thelaboratory. But the low number of underlying factors in the traitswithin and between species could also indicate an oligogenicarchitecture and/or that they are subject to strong selection, as theywould if they served as cue to species recognition.

Evolutionary implicationsDifferences in behaviour might be a consequence of lack of gene flowbetween distant populations, or populations isolated by differentWolbachia infections. If the behavioural differences were related toWolbachia-induced cytoplasmic incompatibility they would representan indication of incipient speciation (Telschow et al. 2002, 2005).Natural variation in behaviour is common in N. vitripennis but so farit has not been found to be related with any degree of incompatibility(Bordenstein et al. 2000).

Our results are consistent with few loci of large effect in thephenotype of headnod numbers and cycle duration both within andbetween species. N. longicornis is the species that appeared to haveleast factors underlying intraspecific phenotypic differences, perhapsindicating a strong selection pressure in this species responsible fordepletion of variation. N. longicornis is sympatric in all its distributionarea to N. vitripennis, while N. vitripennis also occurs in allopatrywithout the pressure of selection against hybrid matings. Interestingly,we found headnod numbers of sympatric N. vitripennis lines to differmore from N. longicornis than allopatric lines, consistent withselection for divergence in sympatric, but not in allopatric areas.Although the number of factors is similar within and between speciesit is possible that the causes are different kinds of selection. Whileselection for divergence might have acted on few factors of largeeffect, stabilizing selection within species keeps genetic variationwithin species low. To ascertain the role of courtship behaviour inNasonia it is necessary to determine which components are subjectto female preference, and whether female preference exists forintraspecific or interspecific variation (chapter 7).

44



Appendix 1Phenotypic variation in headnod numbers in the first four series forseveral lines of all three species. Average ± variance and sample size

are given. Line labels are as in table 1.


45

1st headnods 2nd headnods 3rd headnods 4th headnodsN. vitripennis European

HV-A 4.96±0.71 (25) 3.72±0.46 (25) 3.92±0.33 (25) 4.60±1.08 (25)HV-B 5.00±1.75 (25) 4.00±1.25 (25) 4.12±0.69 (25) 4.48±1.01 (25)

HV-C 5.72±1.21 (25) 4.40±0.58 (25) 4.52±1.09 (25) 4.76±2.69 (25)HV-D 6.20±0.83 (25) 4.08±0.66 (25) 4.17±1.54 (24) 4.92±1.73 (24)Mosc 6.20±1.92 (25) 5.16±0.89 (25) 5.52±1.18 (25) 5.96±1.46 (25)Zven 4.72±1.38 (25) 3.72±0.71 (25) 3.84±0.64 (25) 4.08±0.66 (25)StG 5.04±2.04 (25) 4.16±1.72 (25) 4.40±1.00 (25) 4.52±1.59 (25)

Asym 4.97±1.71 (70) 3.84±0.97 (68) 4.20±1.18 (54) 4.66±1.42 (53)overall 5.31±1.79 (221) 4.10±1.02 (221) 4.35±1.16 (220) 4.72±1.51 (216)

North AmericanUT-A 6.04±1.21 (25) 4.24±0.77 (25) 4.32±0.48 (25) 4.72±0.63 (25)UT-B 6.80±1.42 (25) 5.52±1.68 (25) 5.68±1.39 (25) 6.52±2.34 (25)UT-C 6.24±2.11 (25) 4.68±1.39 (25) 5.40±2.08 (25) 5.96±1.71 (25)

overall 6.36±1.64 (75) 4.81±1.53 (75) 5.13±1.63 (75) 5.73±2.09 (75)

N. longicornisCA-A 2.04±0.46 (25) 2.44±0.59 (25) 2.84±0.97 (25) 3.56±5.01 (25)CA-B 2.56±1.59 (25) 3.20±1.42 (25) 3.44±1.92 (25) 3.68±1.81 (25)CA-C 2.28±1.29 (25) 3.48±2.51 (25) 3.72±1.88 (25) 4.67±3.10 (24)CA-D 2.60± 0.57 (20) 3.65±1.50 (20) 4.45±2.26 (20) 5.37±1.25 (19)UT-A 1.90±1.88 (20) 2.25 ±1.88 (20) 3.00 ±2.74 (20) 3.75±2.47 (19)UT-B 4.24±2.52 (25) 4.12±1.61 (25) 4.64±2.91 (25) 5.40±4.42 (25)UT-C 2.56±1.59 (25) 3.20±1.42 (25) 4.24±1.19 (25) 4.52±1.68 (25)

ID 2.20±1.29 (10) 2.50±0.72 (10) 3.90±1.21 (10) 3.70±0.90 (10)MT 2.08±1.66 (25) 3.04±1.29 (25) 3.72±1.63 (25) 4.36±3.24 (25)

overall 2.73±2.04 (194) 3.24±1.69 (193) 3.92±2.16 (193) 4.57±2.78 (190)

N. giraultiVA 9.05±9.42 (20) 10.75±7.25 (20) 9.40±3.73 (20) 9.60±14.88 (20)PA 8.05±5.52 (20) 9.55±7.42 (20) 10.15±4.45 (20) 8.42±4.26 (19)

overall 8.55±7.54 (40) 10.15±7.52 (40) 9.78±4.13 (40) 9.03±9.82 (39)


Appendix 2 Phenotypic variation in cycle durations (seconds) in the first threeseries for several lines of all three species. Average ± variance andsample size are given. -: no data available. Line labels are as in table 1.

46


1st cycle 2nd cycle 3rd cycleN. vitripennis

EuropeanHV-A 6.68±1.06 (25) 6.79±0.43 (24) 6.62±1.05 (21)HV-B 7.20±1.25 (25) 7.48±1.84 (25) 7.56±3.01 (25)

HV-C 9.67±1.45 (24) 9.18±1.39 (22) 9.27±1.06 (22)HV-D 7.88±0.98 (24) 7.71±1.01 (21) 7.53±0.82 (19)Mosc 8.52±2.01 (25) 8.20±2.00 (25) 8.08±1.16 (25)Zven 7.48±1.01 (25) 7.56±0.76 (25) 7.44±0.84 (25)St.G 7.29±4.04 (24) 7.25±3.50 (24) 6.79±1.65 (24)

Asym 7.37±2.99 (70) - -overall 7.81±2.47 (172) 7.72±2.01 (166) 7.61±2.00 (161)

North AmericanUT-A 8.20±0.92 (25) 7.88±0.94 (25) 7.68±1.06 (25)UT-B 8.80±2.08 (25) 9.00±2.33 (25) 8.68±1.39 (25)UT-C 8.79±0.95 (24) 8.00±1.39 (24) 8.38±1.03 (24)

overall 8.59±1.37 (74) 8.30±1.77 (74) 8.24±1.31 (74)

N. longicornis CA-A 13.20±2.33 (25) 13.00±1.67 (25) 13.08±1.64 (24) CA-B 11.20±3.00 (25) 12.48±2.18 (25) 12.60±2.33 (25) CA-C 10.17±37.01 (24) 14.88±7.51 (24) 14.21±8.73 (19) CA-D 11.42±24.70 (19) 15.37±4.36 (19) 15.58±3.04 (19)UT-A 11.05±3.00 (20) 10.79±2.06 (19) 10.56±2.61 (18) UT-B 12.08±5.58 (25) 12.32±3.14 (25) 12.60±5.42 (25) UT-C 11.20±3.00 (25) 12.48±2.18 (25) 12.60±2.33 (25)

ID 9.60±18.27 (10) 13.70±36.68 (10) 12.50±6.94 (10) MT 7.88±26.36 (25) 13.75±3.93 (24) 14.04±2.04 (24)

overall 11.43±10.71 (188) 12.91±6.50 (192) 13.03±5.57 (190)

N. giraulti VA 19.70±53.06 (20) 16.75±25.57 (20) 16.76±19.82 (17) PA 13.42±11.26 (19) 14.63±24.25 (19) 15.05±9.27 (19)

overall 16.64±41.97 (39) 15.72±25.42 (39) 15.86±14.58 (36)


Appendix 3Minimum number of effective factors (nef) underlying phenotypicdifferences between species or lines within species calculated

usingthe pooled F2 males. Nef ± variance.


47

BETWEEN SPECIES

N. vitripennis x N. longicornis N. giraulti x N. longicornis

headnods 0.60±0.75 1.49±3.76cycles 0.31±0.22 0.72±1.28

WITHIN SPECIES

N. vitripennisoverall

mean±var

EuropeanZven

xHV-C

Zvenx

Mosc

Moscx

HV-Aheadnods 0.35±1.28 0.54±1.30 0.50±1.05 0.46±1.21

cycles 0.85±2.03 0.03±0.07 0.57±1.70 0.48±1.27

NorthAmerican

UT-Bx

UT-A

UT-Cx

UT-Aheadnods 0.55±1.52 0.14±0.25 0.34±0.89

cycles 0.23±0.60 0.06±0.10 0.14±0.35

N. longicornis

MT x CA-B MT x UT-B UT-B x UT-Cheadnods 0.23±0.60 0.24±0.47 0.13±0.24 0.20±0.43

cycles 0.002±0.003 0.01±0.02 0.002±0.003 0.01±0.01



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