Genetics: Published Articles Ahead of Print, published on October 11, 2005 as 10.1534/genetics.105.044743

No patrigenes required for femaleness in the haplodiploid Nasonia

vitripennis

Leo W. Beukeboom & Albert Kamping

Evolutionary Genetics, Centre for Ecological and Evolutionary Studies, University of

Groningen,

P.O. Box 14, NL-9750 AA Haren, The Netherlands

1 Running head: Females from unfertilized diploid eggs in Nasonia

Key words: diploid male, polyploidy, gynandromorph, Nasonia, determination

Corresponding author:

Leo W. Beukeboom Evolutionary Genetics Centre for Ecological and Evolutionary Studies Biological Centre University of Groningen, P.O. Box 14, NL-9750 AA Haren The Netherlands Tel. (31) 50-363 4884 Fax. (31) 71-363 2348 E-mail. [email protected]

The parasitoid wasp is an emerging model organisms for developmental and behavioural genetics. It reproduces by ; males typically develop parthenogenetically from haploid eggs and females from fertilized diploid eggs. A polyploid mutant strain is available in which females are triploid and lay haploid and diploid eggs that normally develop into males when unfertilized. In contrast to previous reports, about 2% of triploid females were found to occasionally produce daughters as well as gynandromorphs from diploid unfertilized eggs. Daughter production increased with age and differed between familial lineages. This is the first report of parthenogenetic female development in Nasonia. The results show that a paternally provided genome is not required for femaleness and call for modifications of existing models of sex determination in Nasonia.

2

All (, and ) have haplodiploid sex determination. The most common mode of reproduction is arrhenotoky, i.e. males develop from unfertilized eggs and are haploid, whereas females develop from fertilized eggs and are diploid. As a consequence, males inherit genes from their mother only and have no genetic father.

Thelytoky also occurs widespread among Hymenoptera. This reproductive mode refers to species that produce females parthenogenetically in the absence of males.

Parthenogenesis is automictic, i.e. haploid eggs are produced meiotically but undergo diploidy restoration by a variety of mechanisms (SUOMALAINEN et al. 1987). Hence, these diploid eggs develop in to uniparental females.

In haplodiploids there are no heteromorphic sex chromosomes; the only genetic difference between males and females is the number of chromosome sets. The question of how this difference in copy number of the genome can lead to the development of either males or females can still not be answered satisfactorily. A number of models have been proposed for sex determination in Hymenoptera and available data indicate that at least two different mechanisms exist (reviewed in COOK 1993a; DOBSON and

TANOUYE 1998).

WHITING (1943) introduced single locus Complementary Sex Determination (sl-

CSD) based on his studies of the parasitoid Bracon hebetor. Sex is determined by a single locus; heterozygotes develop into females and hemizygotes and homozygotes into males. This mode of sex determination is typically detected with inbreeding crosses which lead to diploid homozygous males (WHITING 1943; STOUTHAMER et al. 1992;

COOK 1993a,b). A sl-CSD mechanism of sex determination has now been shown for

3 over 40 species of Hymenoptera (reviewed in STOUTHAMER et al. 1992; PERIQUET et al.

1993; COOK 1993a; BUTCHER et al. 2000) covering all major suborders. However, the precise phylogenetic distribution of this mode of sex determination is still unclear

(BEUKEBOOM et al. 2000). Moreover, although the sex locus has recently been characterized from the honey (BEYE et al. 2003) the precise molecular genetic mechanism of sl-CSD remains to be elucidated.

In a number of hymenopteran species inbreeding does not result in diploid males and indicates the absence of a sl-CSD mechanism (SKINNER and WERREN 1980; COOK

1993b; BEUKEBOOM et al. 2000). Sl-CSD is also incompatible with most forms of . Peri-meiotic mechanisms that include union of meiotic products, such as central and terminal fusion, cause an increase of homozygosity. Homozygosity of the csd gene leads to diploid males rather than females. Therefore, CSD mechanisms can only be reconciled with thelytoky if the csd gene resides in a region that remains permanently heterozygous (BEUKEBOOM and PIJNACKER 2000). Wolbachia-induced thelytoky (STOUTHAMER et al. 1990; BRAIG et al. 2002) causes gamete duplication leading to complete homozygosity and can therefore not occur in species with CSD.

The parasitoid wasp Nasonia vitripennis is one of the best studied hymenopterans genetically. It is an emerging model organism for studying complex traits (BEUKEBOOM and DESPLAN 2003; PULTZ and LEAF 2003; SHUKER et al. 2003) because genetical analysis is greatly facilitated by haploidy of males. It has been firmly established that

Nasonia does not have sl-CSD (WHITING 1967; SKINNER and WERREN 1980; KAMPING and BEUKEBOOM, unpublished). Several alternative models for sex determination in

Nasonia have been proposed. They include Fertilization Sex Determination (FSD,

4 following notation of DOBSON and TANOUYE 1998), Maternal Effect Sex Determination

(MESD) and Sex Determination (GISD). DOBSON and TANOUYE

(1998) tested these alternative models of sex determination using a polyploid mutant strain of N. vitripennis. Polyploid mutants of N. vitripennis have been known for a long time (WHITING 1960; MACY and WHITING 1969). They appeared several times spontaneously in Whiting’s stock cultures and at least one strain has been maintained ever since. Triploid females produce, in addition to unviable aneuploid eggs, haploid and diploid eggs that typically develop into males. Diploid males are fertile and produce diploid sperm. Use of the polyploid strain enables one to vary the number of chromosome sets contributed by either parent to the embryo and test their effect on offspring sex.

Here, we present additional results of breeding experiments with the polyploid mutant of N. vitripennis. We show for the first time that unfertilized diploid eggs of triploid females do not always develop into males, but can yield fertile females or gynandromorphs, i.e. male-female mosaics. Our results differ from previous studies that used this polyploid strain. Moreover, they have important implications for the mode of sex determination in N. vitripennis and the validity of different models.

MATERIALS AND METHODS

Nasonia strains and culturing: Nasonia vitripennis is a pupal parasitoid of cyclorraphous flies and is easily cultured in the laboratory on Protocalliphora. It has a generation time of 14 days at 25ºC. Virgin wasps are obtained by breaking open the host puparia a few days prior to emergence and separating male and female wasp pupae.

5 Both can easily be distinguished because males have small wing buds (short wings as adults) and females large wing buds (long wings as adults). Mated females typically produce female-biased progenies when provided with hosts individually. Virgin wasps only lay unfertilized eggs that develop into all-male broods.

The polyploid mutant strain originates from Whiting’s cultures (WHITING 1960) and has been maintained ever since by the late G. Saul (University of Vermont). It is the same strain as used by DOBSON and TANOUYE (1998). It carries two recessive eye-color mutations; oyster (oy) and scarlet (st) (SAUL 1990). Both mutations occur at a single segregation unit (“R-locus”) in which no recombination occurs (WHITING 1965).

Homozygous and hemizygous oyster individuals have pink-grayish eyes and scarlet individuals bright red eyes. Diploid heterozygous oy +/ + st individuals have dark purple eyes similar to wildtype. The females of this polyploid strain are triploid and lay haploid, diploid and aneuploid eggs. Aneuploid eggs do not develop into adult wasps which greatly reduces the number of offspring of triploid females (WHITING 1960). Brood size of females can therefore be diagnostic for level: triploid females produce 0-3 offspring per fly host, whereas normal diploid females produce 20-30 offspring.

The maintenance scheme of the polyploid mutant strain is shown in Figure 1.

Triploid oy + / + st / + st females reproduce as virgins and lay haploid oy + and + st eggs as well as diploid oy + / + st and + st / + st eggs, all of which typically develop into males. Diploid oy + / + st males are crossed to standard diploid + st / + st females giving rise to triploid females again. Thus, triploid females receive the full diploid complement from their father (due to mitotic spermatogenesis) in addition to a haploid complement of their mother. Note that haploid and diploid scarlet males cannot be distinguished even

6 though diploid males are typically larger than haploid males, but this difference is not always conclusive.

Experimental design: Previous culturing of the polyploid mutant strain (BEUKEBOOM

1992) suggested that unmated triploid females very rarely produce daughters. After confirming this finding in a pilot experiment, we systematically tested the sex of all offspring in progenies of 1200 virgin triploid females. Three diploid males each were collected from 8 broods of the stock culture (designated the grand paternal generation).

Each male was individually mated to five scarlet females (see Figure 1) yielding 120 progenies containing triploid females. Next, ten virgin triploid females (designated the parental generation) were collected from each progeny and individually given four hosts at three consecutive ages, 0-1, 7-8 and 20-21 days, respectively. All experimental procedures, including matings, culturing and testing, were carried out at 25°C to minimize variation in phenotypic and epigenetic effects. The number, sex and eye-color of the F1 offspring were scored.

Females and males can easily be recognized in N. vitripennis (Figure 2A and 2B).

Females have dark brown antennae and legs, large wings and an ovipositor. Males have yellow antennae and legs, short wings and no ovipositor. Close observation of the offspring was performed to identify gynandromorphs, defined here as having both female and male body parts (Figure 2C and 2D). In gynandromorphic individuals the distribution of male and female tissues was also determined. The design of this experiment additionally allowed us to track rare female production back to the grand paternal generation, and to measure the effects of female age on daughter production.

7 If daughters were found among the F1 progeny they were bred further. Note that due to the low fecundity of triploid females leading to incomplete digestion of the fly hosts, the usual separation of male and female offspring a few days prior to emergence was not feasible. Moreover, developmental times among progenies of triploid females are slightly longer and more variable than standard progenies. Therefore, it was not always possible to obtain virgin daughters from broods of triploid females. If F1 females had emerged in the absence of brothers, they were hosted as virgins (three hosts for life).

If they had emerged simultaneously with one or more of their haploid or diploid brothers, they were likely to already be mated. Their ploidy level (diploid or triploid) and genotype was inferred from the number, eye-color and sex of their offspring.

RESULTS

Segregation of eye-color markers

A random sample of 3805 offspring of 245 triploid virgin females were screened for eye- color and ploidy level (haploid or diploid). The observed numbers of oyster, scarlet and wildtype were 903 (24%), 2136 (56%) and 766 (20%), respectively (Table 1). These breeding data are broadly consistent with previously reported ones (CONNER 1966;

DOBSON and TANOUYE 1998). Note that haploid and diploid scarlet males cannot be distinguished unequivocally. The oyster and scarlet mutant eye color markers can be treated as a single segregation unit because recombination does not occur between these loci. If one assumes Mendelian inheritance and equal numbers of haploid and diploid males, the expected ratios of oyster, scarlet and wildtype are 1: 3 : 2 (scenario 1 in Table

1, see also Figure 1). The observed numbers deviate significantly from this ratio; there is

8 a great shortage of wildtype males. A previous study (CONNER 1966) showed that triploid females produce on average 40% diploid sons. If the expected ratios are adjusted for 40% diploids and 60% haploids, the observed numbers still deviate significantly

(scenario 2 in Table 1). CONNER (1966) also reported preferential transmission of the maternal allele at a frequency of 40% versus 30% for each of the paternal alleles. If the expected ratios are also adjusted for preferential transmission, the observed numbers do not fit better (scenario 3 in Table 1). However, an alternative scenario based on 70% haploids and 30% diploids without preferential segregation fit the data well (scenario 4 in Table 1).

A decrease by age of the triploid female in the proportion of diploids in the offspring was observed in a random sample of 166 families and ranges from 0.225 (0-1 days), 0.195 (7-8 days) to 0.161 (20-21 days). Paired t-tests on the diploid frequencies of the different paternal lineages show that this decrease is significant for 0-1 days versus 7-

8 days (P < 0.01) and for 0-1 days versus 20-21 days (P < 0.05). The frequency of purple diploids in the offspring is highly different for the eight paternal lineages and ranges from 0.151 to 0.267 (χ2 test Yates corrected, P < 0.001).

Daughter and gynandromorph production

Three subsequent broods of 1200 triploid virgin females derived from 24 families (eight times 3 diploid males crossed with 5 scarlet females each) were scored for female and gynandromorph production. A total of 13 females (from 11 triploid mothers = 0.92%,

N=1200) and 16 gynandromorphs (from 16 triploid mothers = 1.33%) were found among the offspring. Table 2 lists all progenies of triploid females that contained daughters or gynandromorphs. Two progenies (4a3#7 and 7b3#3) contained more than one daughter.

9 All progenies with females or gynandromorphs also contained one or more diploid males that could unequivocally be determined by their wildtype eye-color, with two exceptions.

These two progenies contained only oyster and scarlet individuals, of which the latter could be haploid or diploid.

The frequency of females and gynandromorphs differed greatly between consecutive hostings (Table 3). In the first broods (0-1 day old females), no females and gynandromorphs were found. In the second broods (7-8 days), 9 females and 16 gynandromorphs were found. In the third broods (20-21 days) 4 females were found, of which two had a mother that also produced a female in the second brood. Note that the number of third broods is lower due to premature death or ageing of females. When the number of females and gynandromorphs in each age class are corrected for the number of offspring per triploid female at different ages (see Table 2), the expected numbers are significantly different from observed (χ2 Yates corrected = 10.00 df = 2, P <0.01). These data show that later eggs in the oviposition sequence have a higher probability to become female.

The production of females and gynandromorphs is not random. Paternal lineage

#3 (see Table 3) has a significantly higher number of gynandromorphs than the other lineages (χ2 test, P < 0.001). Paternal lineage #7 has a significantly higher number of females than the other lineages (P < 0.01). These two lineages produce a significantly higher (P < 0.001) number of females and gynandromorphs than the others, probably indicating a genetic basis for the production of females and gynandromorphs from unfertilized eggs.

10 Table 4 compares the composition of broods (all three consecutive broods pooled) containing at least one female or gynandromorph with broods lacking any aberrant individuals. Most females and gynandromorphs had either scarlet or wildtype eyes.

These ratios are significantly different from the distribution among their male brothers

(χ2 = 25.97, P < 0.001, broods with females and gynandromorphs pooled). The overall frequencies (males, gynandromorphs and females pooled) of the different eye-colors in the families containing females or gynandromorphs do not differ significantly from those in the families with only males (χ2 = 0.59, P = 0.74). These results indicate that females and gynandromorphs develop predominantly from diploid eggs. However, there were also two gynandromorphs with oyster eyes reminiscent of haploidy. Note again that the ploidy level of the scarlet individuals cannot be unequivocally determined (see Figure 1).

Twelve out of 13 uniparentally produced females were bred. Eight produced normal sized broods with Mendelian segregation of the eye-color markers among sons and daughters, indicating that they were diploid and that they had mated with one of their brothers. Two females produced normal sized broods consisting entirely of males, indicating that these females were unmated. Two other females did not produce progeny which may be due to gynandromorphism that was not apparent from the external morphology and one female was dead prior to the test.

None of the 16 individuals scored as gynandromorphs produced offspring indicating that they were sterile. This is not surprising since 15 out of 16 appeared to have a male abdomen (Table 5). The phenotype of gynandromorphs showed a clear pattern. They shifted from being female in the anterior body parts to being

11 predominantly male in the posterior parts. All individuals had female antennae, but only one had a partial female abdomen.

These results document for the first time development of females from unfertilized diploid eggs in Nasonia vitripennis and confirm our previous loose observations during many years of culturing of this polyploid strain. Furthermore, these parthenogenetically produced females appear diploid and fully fertile, but they themselves did in turn not produce daughters parthenogenetically.

DISCUSSION

Segregation in the triploid strain

Like all Hymenoptera, Nasonia vitripennis has haplodiploid sex determination; unfertilized haploid eggs develop into males and fertilized diploid eggs develop into females. Mutations to polyploidy have arisen spontaneously in laboratory cultures several times (WHITING 1960). Triploid females produce haploid and diploid eggs, in addition to a large proportion of aneuploid eggs resulting in low offspring number.

Results of this study are consistent with previous reports that the proportion of diploids among the adult offspring is lower than that of haploids, but we find 30% diploids instead of 40% as previously reported (CONNER 1966; DOBSON and TANOUYE 1998).

Moreover, preferential transmission or segregation (CONNER 1966) needs not be invoked to explain our results. Molecular marker studies indicate that recombination rates in triploid females are comparable to diploid females (Beukeboom, unpublished results).

12 Female and gynandromorph production

Until now unfertilized haploid and diploid eggs of N. vitripennis were reported to always develop into males (CONNER 1966; BEUKEBOOM 1992; DOBSON and TANOUYE 1998).

Using one of Whiting’s original strains, we now have shown that unfertilized diploid eggs occasionally develop into females or gynandromorphs. These uniparentally produced females are diploid and have normal fertility, and appear in no way different from normally produced females from fertilized eggs.

A number of important observations require explanation: (1) about 2% of all females produced one or more daughters or a gynandromorph, (2) all females and almost all gynandromorphs have the wildtype or scarlet phenotype indicating that they are diploid, (3) the frequency of diploid eggs decreases with female age, (4) the frequency of females and gynandromorphs increases with age, (5) the frequency of females and gynandromorphs differs significantly between lineages, (6) gynandromorphs show an anterior-posterior gradient in femaleness, and (7) progenies with females or gynandromorphs also contained one or more diploid males The rare production of daughters is discussed below in relation to the mode of sex determination. Daughter production increases with the mother’s age, but the frequency of diploid eggs decreases with age. Therefore, daughter production is not simply the result of an increase in the production of diploid eggs by age, and implies that these two phenomena are at least partially independent. It suggests that daughter production is partially under maternal control and/or under control of genes which have age-dependent expression. The low frequency of virgin triploid females that produce females can be increased by changing culture conditions. In a pilot experiment we found a strong increase for female and

13 gynandromorph production at high temperature, providing further evidence for a maternal effect.

Only diploid eggs appear to undergo a sex switch. This indicates that the ploidy level of the egg plays an important role in sex determination. In this respect it would be informative to know the ploidy level of the gynandromorphs. They could be ordered haplodiploid mosaics (i.e. consisting of haploid male and diploid female tissues) as described for other hymenopterans (WHITING et al. 1934; HALSTEAD 1988; GROSCH

1988) or intersexes (i.e. with incompletely differentiated gender). The fact that our gynandromorphs descend from uninseminated females rules out any mechanism involving fertilization, such as polyspermy or fertilization of binucleate eggs (STERN

1968, PETTERS and GROSCH 1976). Our results suggest that most gynandromorphs are diploid. We found only two gynandromorphs with oyster eyes, which is a recessive mutation, indicating that (at least) their eye tissue was haploid. Moreover, our breeding results show that all fertile uniparental females are diploid (not haploid). Haploidy of females has never been described in the Hymenoptera although WHITING et al. (1934) reported a type of sex-intergrade consisting of two types of genetically male tissue that interact to produce feminization. The higher frequency of feminization in anterior versus posterior body parts in our gynandromorphs suggests that some sort of gradient exists in female induction. We are currently investigating ploidy levels of the gynandromorphs in order to reveal the mechanism of their origin.

Another finding requiring explanation are the significantly higher proportions of females and gynandromorphs in some maternal and grand paternal lineages as well as the fact that some females produce more than one daughter in addition to diploid sons.

14 Uniparentally produced females do however not produce females or diploid males parthenogenetically themselves. This rules out a dominant mutation that is transmitted over generations. The data rather suggest the occurrence of a rare event in the germ line of some males that is partially manifested (e.g. semi-dominant) in some of their daughters. The expression of the trait varies within the triploid females as well, given that not all of their diploid offspring were transformed into females. Our data therefore appear most easily explained by invoking a dosage effect and/or an epigenetic mutation.

Epigenetic inheritance of traits has been invoked for Nasonia before (POIRIÉ et al. 1992;

BEUKEBOOM 1995). Epigenetic control over inheritance is now well established although much of the underlying mechanisms remain to be uncovered (CONSTANCIA et al. 1998;

LYKO and PARO 1999; VU and HOFFMAN 2000). Genomic imprints usually undergo cyclic application and erasure within single generations, but MORGAN et al. (1999) showed that the epigenetically inherited agouti locus in mice can retain its imprint for several generations through incomplete erasure of the modification in the female germ line. Although purely speculative at this moment, it is not inconceivable that a similar process is responsible for the results found in this study. Such a scenario would implicate that one may be able to artificially select for the trait “occasional parthenogenetic female production”. This is what we are currently attempting.

Implications for sex determination

Our recovery of diploid daughters and gynandromorphs from unfertilized triploid females indicates that the sex determining mechanism in Nasonia is most likely more complicated than the existing models predict. Different models have been reviewed by

COOK (1993a) and DOBSON and TANOUYE (1998). According to the Fertilization Sex

15 Determination (FSD) model (WHITING 1960) sex is determined by fertilization, not by ploidy level, i.e. fertilized eggs develop into females and unfertilized eggs into males.

Our results now show that female development can also occur without fertilization in N. vitripennis. Together with female development under thelytokous reproduction, which is known from many Hymenoptera, these data discard the FSD model. This model had previously also been rejected based on studies with the Paternal Sex Ratio (PSR) chromosome which showed that fertilized eggs can develop into males (WERREN et al.

1987; NUR et al. 1988; BEUKEBOOM and WERREN 1993; DOBSON and TANOUYE 1998).

The Genomic Imprinting Sex Determination (GISD) model (POIRIÉ et al. 1992;

BEUKEBOOM 1995) proposes that paternally and maternally derived genes function differently in sex determination and that at least one paternally inherited set of chromosomes is required for female development. This model was prompted by breeding data of the polyploid strain used in this study. Diploid eggs containing two maternally derived chromosome sets were found to develop into males, whereas diploid eggs with one maternal and one paternal set (derived from fertilization of haploid eggs of triploid females) resulted in females. This led DOBSON and TANOUYE (1998) to consider

GISD as the only model consistent with all available data. However, we now have shown that diploid eggs without patrigenes can also develop into females which cannot easily be accounted for by GISD. We therefore conclude that sex determination in Nasonia is more complicated than any of the existing models predict.

STOUTHAMER and KAZMER (1994) proposed that triploid females carry a non- functional variant (mutation) of a locus involved in sex determination, i.e. triploid females are functionally diploid and diploid males are functionally haploid. Assuming

16 Mendelian segregation of this mutation in the polyploid strain, 2/3 of diploid eggs are expected to carry one mutated and one intact copy of the sex locus and develop into diploid males. The other 1/3 are expected to receive two intact copies of the sex locus, resulting in female development. Our data do not support these predictions because only a very small fraction (<1%) of triploid daughters of a particular diploid father produces females or gynandromorphs. Preferential segregation (CONNER 1966) of the mutation to diploid eggs can also be excluded since the ratio of haploid and diploid individuals in the progeny of these “exceptional” triploid females is similar to that of a random sample of virgin triploid females.

Although the proportion of virgin triploid females that produce daughters is very low, the presence of a diploid daughter in the offspring of an “affected” mother is not a sporadic event. The frequency of daughters among their diploid offspring does not significantly deviate from 1/3 (Table 2). This could simply be explained by a single dominant mutation that occurred in the germ line of these females. However, our data also show a difference between paternal lineages in production of diploid females and gynandromorphs through their virgin triploid daughters. Moreover, the data show that female and gynandromorph production are largely independent events. These observations indicate that sex determining factors are polymorphic in the polyploid strain and suggest that at least two segregating genes are involved, e.g. a sex determining gene and a gene that modifies its expression. Recombination in triploid females appears to be completely random (unpublished results). The absence of females and gynandromorphs in the offspring of virgin diploid F2 daughters in turn indicates that the ploidy level of the mother also plays a role in the production of daughters from unfertilized eggs.

17 The mode of reproduction of N. vitripennis is called arrhenotoky, which is the common form among haplodiploid Hymenoptera. Some species are thelytokous and produce only daughters from haploid eggs that have undergone a diploidy restoration process. Although we have now shown uniparental production of females in N. vitripennis, the mechanism by which these females arise is different from thelytokous species. They develop from unfertilized diploid eggs that result from a reduction division without any subsequent ploidy increase. This is still the consequence of arrhenotokous reproduction, where it are typically the males that arise by automictic . Polyploid Hymenoptera with this mode of reproduction are not known from nature. The mechanism is most reminiscent of pre-meiotic doubling thelytoky, whereby the diploid chromosome complement is doubled before meiosis and the meiotic products are diploid (SUOMALAINEN et al. 1987). This has been reported for a few hymenopterans (DONCASTER 1916; PEACOCK and SANDERSON 1939; DODDS 1939;

LEDOUX 1954).

In conclusion, we showed for the first time uniparental development of females in

N. vitripennis. The used polyploid mutant strain proofs very useful for manipulating the number of paternally and maternally transmitted chromosome sets. Occasional daughter development from eggs without a paternal chromosome complement poses a new challenge for the existing models of sex determination and additional modifications seem to be required. Our results will contribute to the long term goal of elucidating the genetic regulation of haplodiploid sex determination. Further study of gynandromorphic individuals, such as obtaining data on ploidy level and dosage compensation, appears particularly promising.

18

We thank MARY ANNE PULTZ, RICHARD STOUTHAMER, CAROL TRENT and JOHN WERREN for stimulating discussions on Nasonia sex determination, GEORGE SAUL and JOHN WERREN for providing the polyploid mutant, REYHAN ÖZKAN, BIANCA HOLLEBEEK, ANDY RUMAHLOINE and

JOOP PRIJS for technical assistance, and LOUIS VAN DE ZANDE, CAROL TRENT and PETER

MAYHEW for their valuable comments on the manuscript. This work has been made possible by a fellowship of the Royal Netherlands Academy of Arts and Sciences and a Pionier grant of the

Dutch Scientific Organization to LWB.

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25 26 Table 1. Observed and expected genotypes and phenotypes (eye-color) among progeny of 245 virgin triploid females. Four scenarios are used for the expected numbers in each class. Scenario 1 is based on equal ratios of haploid and diploid males and Mendelian segregation of the eye-color markers. Scenario 2 is based on 40% diploid males versus

60% haploid males (CONNOR, 1966) and Mendelian segregation. Scenario 3 is based on

40% diploid males versus 60% haploid males and preferential transmission of the maternal versus both paternal alleles in the ratio of 4:3:3 (CONNOR, 1966). Scenario 4 is based on 30% diploid males versus 70% haploid males and Mendelian segregation.

Scenario 4 fits the data by far the best.

Phenotype oyster scarlet wildtype Total

Genotype haploid haploid + st or diploid oy + diploid + st / + st oy + / + st

Observed 903 2136 766 3805 number

Expected ratio 1 3 2 χ2 = 341.57, df = 2, under scenario P < 0.001 1

Expected ratio 1.2 3.2 1.6 χ2 = 93.05, df = 2, under scenario P < 0.001 2

Expected ratio 1.07 3.42 1.51 χ2 = 113.07, df = 2, under scenario P < 0.001 3

Expected ratio 1.40 3.40 1.20 χ2 = 0.48, df = 2, under scenario not significant 4

27

28 Table 2. Progeny numbers of individual virgin triploid females that produce at least one female (A) or one gynandromorph (B) during life (three hostings). Eye-colour is informative for ploidy level: oyster is haploid, scarlet is haploid or diploid and wildtype is diploid (see Figure 1).

A Triploid Males Females Total mother oyster scarlet wildtype oyster scarlet wildtype

2a5#61 1 7 1 - - 1 10 2c3#1 2 5 3 - - 1 11 3c3#3 3 8 - - - 1 12 4a3#7 1 7 3 - - 2 13 5b3#2 3 4 3 - - 1 11 7a2#6 - 5 3 - 1 - 9 7a5#5 6 7 1 - 1 - 15 7b3#3 9 15 2 - 1 1 28 7c4#3 4 4 3 - - 1 12 Total2 29 62 19 - 3 8 121

29

B Triploid Males Gynandromorphs Total mother oyster scarlet wildtype oyster scarlet wildtype

1c4#7 6 21 6 - 1 - 34 2a1#1 4 2 4 - - 1 11 3a1#4 3 7 - - - 1 11 3a2#3 3 4 5 - - 1 13 3a3#2 1 3 2 - 1 - 7 3b2#5 4 9 1 - 1 - 15 3b5#5 2 11 5 1 - - 19 3c1#3 6 18 4 - 1 - 29 3c2#7 7 19 4 - - 1 31 3c3#8 7 9 3 - - 1 20 4a2#10 1 10 1 - - 1 13 4b1#3 3 6 1 - - 1 11 4b4#1 4 10 3 - 1 - 18 5b2#6 6 10 3 - - 1 20 7a2#5 1 8 2 1 - - 12 7c2#8 1 9 5 - 1 - 16 Total 59 156 49 2 6 8 280

1 Female code 2a5#6 indicates: paternal line 2 (#1-8), father a (a, b or c), mother 5 (#1-

5), female number 6 (#1-10).

2 Two additional females, a wildtype and a scarlet respectively, were found in the progeny of mothers 3a3#7 and 3a4#7. These progenies were not scored for numbers and phenotypes of males.

30 Table 3. Number of females (F) and gynandromorphs (G) in the offspring of 1200 virgin triploid females at different age in eight paternal lines (150 females per line).

Paternal line Age of female 0-1 day 7-8 days 20-21 days Total F G F G F G F G 1 0 0 0 1 0 0 0 1 2 0 0 2 1 0 0 2 1 3 0 0 1 8 2 0 3 8 4 0 0 1 3 1 0 2 3 5 0 0 1 1 0 0 1 1 6 0 0 0 0 0 0 0 0 7 0 0 4 2 1 0 5 2 8 0 0 0 0 0 0 0 0 Total 0 0 9 16 4 0 13 16 Average progeny number1 (number 4.67 (n=166) 1.01 (n=166) 3.0 (n=48)2 females) 1 based on a subsample of the 1200 females 2 based on 48 of 166 females that were still alive

31 Table 4. Offspring of virgin triploid females per family type of only males, males and females, or males and gynandromorphs.

Proportions refer to the relative frequencies of each eye-color within a sex (row wise)

Family type Offspring type oyster scarlet purple total Only males1 Number males (proportion) 664 (0.242) 1528 (0.556) 555 (0.202) 2747 Females and Number females (proportion) 0 4 (0.308) 9 (0.692) 13 males Number males (proportion) 29 (0.264) 62 (0.563) 19 (0.173) 110 Total number offspring 29 (0.236) 66 (0.536) 28 (0.228) 123 (proportion) Gynandromorphs Number gynandromorphs 2 (0.125) 6 (0.375) 8 (0.5) 16 and males (proportion) Number males (porportion) 59 (0.223) 156 (0.591) 49 (0.186) 264 Total number offspring 61 (0.217) 162 (0.579) 57 (0.203) 280 (proportion)

1 based on a random sample of 166 triploid females

32

Table 5. Phenotypes of gynandromorphs. Double symbols refer to the left respectively right side of the body, F=female and M=male.

Body part Eye-color oyster scarlet purple

Antennae F F F F F F F F F F F F F F F F F F F F F F F F

Fore F F F F F F F F F F F F F F F F/M F F F F F F F F Wings Hind F F F F F F F F M M F F F F F/M F/M F F F F F F M M wings Front M M F F F F F F F F M M M M F/M F/M F F F F F F F M legs Middle M M M M F F F M M F/M M M M M M M F F F F F M M M legs Hind M M M M F F/M M F/M M F/M M M M M M M F F F M F M M M legs Abdomen M M M M M F M M M M F/M M

Number 1 1 1 1 1 1 1 1 5 1 1 1 individuals

33

Figure 1. The polyploid mutant strain of Nasonia. Oyster (pink eyes) and scarlet (red eyes) are recessive complementing eye-color mutations that occur at a single segregation unit (“R” locus) in which no recombination occurs. Virgin triploid oy + / + st / + st females are designated as the parental generation in the experiments. They lay haploid oy + and + st eggs as well as diploid oy + / + st and + st / + st eggs which typically develop into males. Diploid oy + / + st males can be distinguished by their purple

(wildtype) eyes and produce fertile diploid sperm. They are crossed with + st / + st females of a standard stock resulting in triploid females again. Genotypes are indicated inside and phenotypes and expected ratios (scenario 1, see Table 1) below the male and female symbols.

Figure 2. The distinction between females and males in N. vitripennis. Females (A) have dark brown antennae and legs, large wings and an ovipositor. Males (B) have yellow antennae and legs, short wings and no ovipositor. Gynandromorphs have features of both sexes (examples C and D). Black arrows indicate femaleness, red arrows maleness.

34

oy + + st + st

haploid diploid

+ st oy + + st oy + + st + st pink red red purple

+ st oy + + st X + st

oy + + st + st

Beukeboom & Kamping, figure 1

35