A neo-W chromosome in a tropical butterfly links colour pattern, male-killing, and speciation

Abstract

Sexually antagonistic selection can drive both the evolution of sex chromosomes and speciation itself. The tropical butterfly the African Queen, Danaus chrysippus, shows two such sexually antagonistic phenotypes, the first being sex-linked colour pattern, the second, susceptibility to a male-killing, maternally inherited mollicute, Spiroplasma ixodeti, which causes approximately 100% mortality in male eggs and first instar larvae. Importantly, this mortality is not affected by the infection status of the male parent and the horizontal transmission of Spiroplasma is unknown. In East Africa, male-killing of the Queen is prevalent in a narrow hybrid zone centred on Nairobi. This hybrid zone separates otherwise allopatric subspecies with different colour patterns. Here we show that a neo-W chromosome, a fusion between the W (female) chromosome and an autosome that controls both colour pattern and male-killing, links the two phenotypes thereby driving speciation across the hybrid zone. Studies of the population genetics of the neo-W around Nairobi show that the interaction between colour pattern and male-killer susceptibility restricts gene flow between two subspecies of D. chrysippus Our results demonstrate how a complex interplay between sex, colour pattern, male-killing, and a neo-W chromosome, has set up a genetic 'sink' that keeps the two subspecies apart. The association between the neo-W and male-killing thus provides a 'smoking gun' for an ongoing speciation process.

Keywords: Danaus chrysippus; Spiroplasma; colour pattern; male-killing; neo-W chromosome; speciation.

Figure 1.

Map of the hybrid zone and the location of the different Danaus subspecies studied. (a) Map of East Africa showing the location of samples, the approximate position of the hybrid zone (b) and the distribution of Danaus chrysippus subspecies chrysippus (c) and dorippus (d). The hybrid form D. c. transiens (underside shown in e) is confined to the hybrid zone, as are the endosymbiont Spiroplasma ixodetis (f) and the ‘fused’ neo-W karyotype (g). Outside the hybrid zone the wild-type ‘unfused’ (Wu) karyotypes for chrysippus (h) and dorippus (i) are fixed. Sampling sites mentioned in the text are shown in the insets, southern Kenya (j) and the environs of Nairobi (k). The symbol A refers to the autosome carrying the C locus.

Figure 2.

Colour phenotypes, genotypes, and karyotypes of subspecies D. c. chrysippus (cc), D. c. dorippus (CC), and the hybrid form transiens (Cc) in the Nairobi region. (a–d) Meiosis prophase I pachytene chromosome pairs from female ovaries (a,c) and metaphase I pairs from male testes (b,d): Danaus chrysippus dorippus, a female, b male, 30 bivalents (2n = 60) in both sexes; D. c. chrysippus, c female, 28 bivalents, 1 trivalent (2n = 59); d male, (2n = 60) as b; the white dots in the female micrographs a and c are telomere signals. Scale bars represent 10 µm. (e,f) F₁ progeny, female (e) and male (f) from a cross between a Spiroplasma-positive D. c. chrysippus female (c) and a Spiroplasma-negative D. c. dorippus male (d). The provenance of chromosomes is shown as red, dorippus C-autosome; pink, chrysippus c-autosome; dark blue, dorippus Z chromosome; light blue, chrysippus Z chromosome; yellow, W chromosome unfused; green, W chromosome fused. Wild-type bivalents (in a,b,d) comprise, respectively, two sex chromosomes (Z/Z or Z/W) with two autosomes carrying the C locus, whereas the trivalent is found only in the mutant females c and e. Chromosomes in square brackets in females c, e, and male f are lost in successive generations of dead sons (marked with a cross in f). A few f males escape death by either immunity (MK suppression) or failed transmission of Spiroplasma [8]; most male survivors have the transiens (Cc) phenotype detectable by the white spots (arrowed in e,f) on the underside and/or scattered black scales on the upper side of the forewing apex.

Figure 3.

(a) The array of karyotypes in the F₂ from founder crosses between D. c. dorippus and D. c. chrysippus populations in the Nairobi hybrid zone, displayed by sex and the generation in which they first appear. F₂ karyotype frequencies are calculated on the following assumptions: (1) the sex ratio in the founder crosses is 1 : 1 in dorippus and 90% female in chrysippus, (2) the numbers of dorippus and chrysippus females in the founder population is equal, (3) all females are mated, (4) all male offspring of neo-W females die, and (5) there is neutrality and no migration. The upper pair of chromosomes comprises the C-autosomes, red for dorippus and pink for chrysippus. The lower pair shows the Z chromosomes of dorippus (dark blue) and chrysippus (light blue); the yellow W chromosome is always unfused (Wu) in dorippus, whereas in chrysippus the W chromosome shown in green may be unfused or fused to the C-autosome (neo-W).