JournaloftheLepidopterists' Society 44(4), 1990, 229-244 FEMALE COLOR AND SEX RATIO IN HYBRIDS BETWEEN PAPILIO GLAUCUS GLAUCUS AND P. EURYMEDON, P. RUTULUS, AND P. MULTICAUDATUS (PAPILIONIDAE) Mark Scriber,1 Robert V. Dowell,2 Robert C. Lederhouse1 J. and Robert H. Hagen1 1 Department of Entomology, Michigan State University, East Lansing, Michigan 48824 21681 Pebblewood Dr., Sacramento, California 95833 ABSTRACT. Female offspring of blackPapilio glaucus glaucus females handpaired to P. eurymedon, P. rutulus, or P. multicaudatus males show variable expression of the black phenotype. Hybridization with P. rutulus yielded black, yellow, and intermediate females, in agreement with previous observations. Hybridization with P. multicaudatus also yielded black and intermediate females, which has not been reported previously. Hybridization with P. eurymedon yielded only one yellow female. Suppression of the blackphenotypeininterspecieshybridsisnotcompleteandmaynothaveasimplegenetic basis. The sex ratio among progeny of these hybridizations was skewed drastically towards males, with most females dying prior to adult eclosion. This "Haldane effect" appears to bemuchlesssevereamongprogeny ofP. glaucusglaucus males paired toP. eurymedon orP. rutulus females than in the case of reciprocal pairings using P. g. glaucus females. Additional keywords: Haldane effect, mimetic coloration, suppressor genes, enabler genes, pupal diapause. Papilio glaucus glaucus (Papilionidae) females show a striking color dimorphism that has attracted considerable study for over 100 years (Edwards 1884, Clarke & Sheppard 1959, 1962, Brower 1958, Brower 1959a, 1959b, Brower & Brower 1962, Scriber et al. 1987, Lederhouse & Scriber 1987). Female P. g. glaucus may have a yellow ground color and resemble the monomorphic males, or they may have a dark or black ground color and act as Batesian mimics of Rattus philenor (Brower 1958). Papiliog.glaucusappearstobeuniqueamongtaxawithinthePapilio glaucus species group in exhibiting this dimorphism. Only monomor- phic females occur in P. glaucuscanadensis, P. rutulus, P. eurymedon, P. multicaudatus, and P. alexiares alexiares. Only black females are known in P. alexiares garcia (Beutelspacher & Howe 1984). Valuable insight into the evolution of mimicry in P. g. glaucus can be obtained through comparative study of the genetic basis for female color in these closely related species and subspecies. Female color in P. g. glaucus is almost always maternally inherited, implying that it is determined primarily by a Y-linked gene (Clarke & Sheppard 1959, 1962, Clarke & Clarke 1983). Exceptions to the usual rule of maternal inheritance (cases of black females producing yellow daughters and the reverse) have been noted repeatedly, however (Ed- 230 Journal of the Lepidopterists' Society wards 1884, Clarke & Sheppard 1959, 1962, Scriber & Evans 1986, Scriber et al. 1987). Chromosome abnormalities have been invoked as explanations for some of these cases (Clarke & Sheppard 1959, Clarke & Clarke 1983, Scriber & Evans 1987, West & Clarke 1987). Cases of non-maternal inheritance may also result from effects of autosomal or X-linked "suppressor" alleles that inhibit the production or deposition of black ground color (Clarke & Willig 1977, Clarke & Clarke 1983, Scriber et al. 1987, West & Clarke 1987, Hagen & Scriber 1989). An X-linked suppressor of this type has been identified and mapped in P. g. canadensis (Hagen & Scriber 1989). Presence of the canadensis suppressor allele is probably responsible for reported cases of non-maternal inheritance involving females collected near the P. g. glaucus/P.g. canadensishybrid zone (Scriberetal. 1987, Scriber 1988). In the case of P. g. canadensis, laboratory hybridizations with P. g. glaucus have revealed that the absence of black females is due both to lack of the black-determining Y-linked allele and to high frequency of the X-linked suppressor allele in natural populations M. Scriber, R. (J. Hagen & R. C. Lederhouse, unpublished). Do homologous suppressors occur in other taxa also lacking a black female form? Interspecies hybrids within the glaucus species group may be ob- tained through hand-pairing (Clarke & Sheppard 1955, 1957, West & Clarke 1987, Scriber et al. 1988, 1991). Based on such crosses, West and Clarke (1987) presented evidence for suppressors in P. eurymedon, P. rutulus, and P. multicaudatus. Here we report results of additional laboratory hybridizations between P. g. glaucus and P. eurymedon, P. rutulus,andP. multicaudatus. ResultsofhybridizationwithP.alexiares garcia have been presented elsewhere (Scriber et al. 1988). Reduced viability of hybrid females, the heterogametic sex, may result from genetic differentiation after speciation (the "Haldane ef- fect": Haldane 1922, Ae 1979, Oliver 1979,Coyne & Orr 1989a). Recent studies have implicated sex chromosome interactions as primary factors in sex-biased hybrid viability and fertility (Coyne 1985, Coyne & Orr 1989b). Imperfect integration of the genome of hybrid Lepidoptera can result in a syndrome of developmental and diapause abnormalities, possibly from hormonal imbalances between regulatory and secretory positions of sex-linked co-adapted gene complexes (Oliver 1983). Pro- longed post-diapause development of pupae may result in delays of adult females, and diapause may extend for one or more years (Oliver 1983, Scriber et al. 1987). More extreme cases may result in death of female hybrids at the egg, larval, or pupal stage, and therefore skewed sex-ratios may serve as indicators of the negative effects of the X- and Y-chromosome interactions. Sex ratios for interspecific crosses within the glaucus species group are presented here. Volume 44, Number 4 231 Methods Male and female P. rutulus and P. eurymedon were collected from Orange, Placer, Solano, Sacramento, and Mono counties, California and the Blue Mountains (Columbia County) of Washington during 1982- 90 and mailed in envelopes or carried on ice to our laboratory. Papilio multicaudatus were collected from Placer and Solano counties in Cal- ifornia and Columbia County, Washington and also from Nuevo Leon, Mexico. Male P. rutulus, P. eurymedon, and P. multicaudatus were hand- paired to lab-reared virgin P. glaucus females. Field collected and laboratory-mated females were set up in plastic oviposition boxes (10 cm x 20 cm x 27 cm) with a sprig of foodplant under saturated m humidity. The boxes were placed 0.7-1.0 from continuously lighted 100 watt incandescent bulbs. From 1987-90 a repeating 4:4 h photo: scotophase was used. Females were fed a mixture of 1 part honey to 4 parts water at least once daily. Most females were allowed to oviposit until death. Eggs were collected and counted at 2-day intervals. Larvae were removed as they eclosed, and the remaining eggs were monitored for 10 days after the first larva hatched. First instar (neonate) larvae were gently placed on fresh leaves of various host plants. Leaf moisture was maintained using Aquapics® and fresh leaves were provided three times a week throughout larval development. Pupae were held at room temperature (23°C) for a minimum of three months after pupation. Those that had not emerged were then refrigerated six months at 5- 7°C and then held at room temperature the following summer. This procedure was repeated for those apparently alive, healthy pupae that did not emerge by the end of the second summer. Some progeny of thefield-collectedfemaleswere used inthesubsequentmatings. Hybrid crosses are given with the female parent listed first. Results Hybridization with Papilio eurymedon A total of 25 pairings of P. g. glaucus females and P. eurymedon males was successful as judged by production of offspring reaching at least the pupal stage (Table 1). Only one female eclosed successfully from these broods whereas 223 males eclosed. The number of dead or developmentally delayed pupae (250) was not greatly different from the total number of males eclosing, suggesting that the majority may have been females (sex of pupae was not determined for this portion of our study). Extremely low viability of female hybrids was indepen- dent of maternal color phenotype. Black P. g. glaucus females were 232 Journal of the Lepidopterists' Society Table 1. Hybrids between Papilio glaucus and P. eurymedon. Offspring Dead Broodno. Mother(source) Father(source) Males Females pupae 1083 black Pgg(OH) eurymedon (CA) 13 5 1170 black Pgg(OH) eurymedon (CA) 1 3 1196 black Pgg(WI) eurymedon (CA) 6 9 2309 black Pgg(WVA) eurymedon (CA) 1 2311 black Pgg(IL) eurymedon (CA) 11 11 2312 black Pgg(IL) eurymedon (CA) 1 4 2313 black Pgg(IL) eurymedon (CA) 21 27 2314 black Pgg(WVA) eurymedon (CA) 1 2315 black Pgg(WVA) eurymedon (CA) 6 9 2321 black Pgg(WVA) eurymedon (CA) 24 21 2322 black Pgg(WVA) eurymedon (CA) 4 2327 black Pgg(IL) eurymedon (CA) 14 1* 29 2328 black Pgg(IL) eurymedon (CA) 33 41 2518 black Pgg(GA) eurymedon (CA) 4 9 2547 black Pgg(WI) eurymedon (WA) 1 1 2671 black Pgg(WI) eurymedon (CA) 16 16 Subtotal (148) (1) (190) 544 yellow Pgg(PA) eurymedon (CA) 22 14 1084 yellow Pgg(OH) eurymedon (CA) 3 5 1117 yellow Pgg'OH) eurymedon (CA) 2 1119 yellow Pgg(OH) eurymedon (CA) 21 23 1168 yellow Pgg(FL) eurymedon (CA) 5 3 1187 yellow Pgg(FL) eurymedon (CA) 1 1198 yellow Pgg(FL) eurymedon (CA) 1 2269 yellow Pgg(WVA) eurymedon (CA) 15 11 2318 yellow Pgg(WVA) eurymedon (CA) 6 3 Subtotal (75) (0) (60) 4465 eurymedon (WA) Fgg(FL) 6 1* Pgg=Papilioglaucusglaucus,*=yellow used in 16 of the pairings and yellow females in the remaining nine; the only daughter produced was from a black mother (brood 2327). Female viability appears to be higher in the reciprocal cross (P. eurymedon female x P. g. glaucus male). Few crosses in this direction were attempted in our study and only one was successful (Table 1: brood 4465). However, one of two successful crosses in the same di- rection reported by West and Clarke (1987) produced 13 females and 13 males; the other produced two males only. The overall sex ratiofrom these three crosses was 1.5 male: 1.0 female (n = 35 offspring). Thecolor of thesingle hybrid (P. g. glaucus x P. eurymedon) female was yellow, which could indicate that her phenotype resulted from a suppressor contributed by her father. Additional evidence of a P. eu- rymedon suppressor is provided by the yellow daughters from 2 back- Volume 44, Number 4 233 Table 2. Backcrosses involving P. eurymedon.1 Offspring Brood Dead no. Mother(source) Father(source) Males Females pupae 1278 black Pgg(TX) Fi (yellow Pgg x pe 2 1* 4 ) 1544 black Pgg(TX) Fi (black Pgg x Pe) 3 5* 3 1TheP.g.glaucusfemalenumbersfortheg x ehybridmalesare544and1083,respectively,forpairings1278and 1544. Pgg=Papilioglaucusglaucus,Pe=Papilioeurymedon,*=yellow. crosses of hybrid (P. g. glaucus x P. eurymedon) males to black P. g. glaucusfemales (Table2). Toofew offspring (6females) were produced to determine whether yellow and black phenotypes depart significantly from the 1:1 ratio expected of a single suppressor allele contributed from the P. eurymedon grandparent. The combined sex ratio among the backcross progeny (0.83 male: 1.0 female, n = 11) was similar tothe combined ratio from three similar backcross families obtained by West and Clarke (1987) (1.33:1, n = 21) and neither differed significantly from a 1:1 ratio (x2, both P's > 0.50). Fertility of the hybrid males did not appear to be greatly reduced relative to that of other laboratory-reared males (Lederhouse et al. 1990). Hybridization with Papilio rutulus There were 26 successful pairings of P. g. glaucus females with P. rutulus males using 13 black and 13 yellow females (Table 3). As in the case of pairings with P. eurymedon males, most of the progeny that eclosed were male: 362 males versus 12 females (28:1 ratio). Also similar to crosses with P. eurymedon, a large number of pupae (407) failed to develop. Live pupae of 1987 crosses (brood #4562, 4564, and 4664) that had not emerged by August 1988 were sexed. Only 2 of the 34 pupae were male and all died subsequently without emerging. No effect of maternal color on sex ratio was apparent (Table 3). West and Clarke (1987) reported a total of 19 males and two females from two crosses of this type; two additional females were obtained by ecdysone injection of pupae (Clarke & Willig 1977). Far fewer reciprocal crosses (P. rutulusfemale x P. g. glaucus male) were attempted, but the one that was successful (#4447) yielded an equal number of males and females. A similar, nearly equal, ratio of sexes (10 males, 8 females) was obtained by Clarkeand Sheppard (1955) in an earlier cross of this type. Fertility of hybrid males, backcrossed to P. g. glaucus females did not appear to be substantially impaired (Table 4; also West & Clarke 1987). The sex ratio among backcross 234 Journal of the Lepidopterists' Society Table 3. Hybrids between Papilio glaucus and P. rutulus. Offspring Dead Broodno. Mother(source) Father(source) Males Females pupae 1115 black Pgg(WI) rutulus (CA) 2 1152 black Pgg(FL) rutulus (CA) 1 1153 black Pgg(FL) rutulus (CA) 1 1+ 1155 black Pgg(WI) rutulus (CA) 15 2+ 18 1156 black Pgg(OH) rutulus (CA) 16 16 1181 black Pgg(FL) rutulus (CA) 3 4 1183 black Pgg(OH) rutulus (CA) 5 1* 7 2517 black Pgg(GA) rutulus (WA) 43 1* 33 2553 black Pgg(GA) rutulus (WA) 2 1 2830 black Pgg(WVA) rutulus (CA) 40 15 4562 black Pgg(FL) rutulus (CA) 47 2* 41 4564 black Pgg(FL) rutulus (CA) 2 1 4664 black Pgg(OH) rutulus (CA) 24 12 Subtotal (198) (7) (151) 2 yellow Pgg(PA) rutulus (CA) 18 30 277 yellow Pgg(PA) rutulus (CA) 15 2* 25 433 yellow Pgg(PA) rutulus (CA) 12 17 546 yellow Pgg(WI) rutulus (CA) 6 4 547 yellow Pgg(PA) rutulus (WA) 30 1* 68 548 yellow Pgg(WI) rutulus (WA) 9 11 1179 yellow Pgg(WI) rutulus (CA) 33 23 334 yellow Pgg(FL) rutulus (CA) 7 19 335 yellow Pgg(FL) rutulus (CA) 2 8 336 yellow Pgg(FL) rutulus (CA) 6 29 343 yellow Pgg(FL) rutulus (CA) 2 1178 yellow Pgg(FL) rutulus (CA) 9 2* 2 1180 yellow Pgg(FL) rutulus (CA) 17 18 Subtotal (164) (5) (256) 4465 rutulus (CA) Pgg(FL) 5 5 2 Pgg=Papilioglaucusglaucus,*=yellow, + =black. progeny was 2.4 male: 1.0 female (n = 150 offspring), similar to the ratio 2:1 (n = 71) reported by West and Clarke (1987). Five pairings with black P. g. glaucus females produced a total of 3 black and 4 yellow hybrid (Fl) daughters (Table 3, Fig. 1). Mixed phenotypes (two yellow, two intermediate) were also reported by West and Clarke (1987) from crosses of this type. (Hybrid intermediates are figured in Clarke & Willig [1977] and Clarke & Clarke [1983].) Three successful backcrosses of Fl males to black P. g. glaucus females also produced a range of color phenotypes among daughters (Table 4). The majority of backcross females were intermediate in color, with varying proportions of black and yellow scales intermixed (Fig. lc-f). Four backcrossesofthistype reported by Westand Clarke (1987) alsoyielded Volume 44, Number 4 235 lHOC0O5 MI> S£ bSfi _"|3> > c Pr Pr)Pr) PQ x x x Pgg PggPgg (yellow (black(black fa ti-i fa 3 3 CiO 60 txo &0 C>0 b0 ^CXh Rh Bh o_o*^o! 3^ O lO CD CO l^ t- CD CO CO Journal of the Lepidopterists' Society 236 Fig. 1. Ventral (a) anddorsal (b) viewsofablackfemale hybridfromablackfemale P.g.glaucus x maleP.rutulus(brood#1153). Backcrossoffspringexhibitingfourfemale color forms (yellow [c], "intermediate" mostly yellow [d], "intermediate" mostly black [ej, and black [fl). These 4 females are from a single backcross (brood #1876) between a dark P. g. glaucus female and a hybrid male (from a black female glaucus x male rutulus). . Volume 44, Number 4 237 Table 5. Hybrids between Papilio glaucus and P. multicaudatus Offspring Inter- Brood Black mediate Yellow Dead no. Mother(source) Father(source) Males females females females pupae 2265 black Pgg(OH) multicaudatus (CA) 10 2 3 10 3619 black Pgg(GA) multicaudatus (WA) 4 1 3660 black Pgg(OH) multicaudatus (Mex) 1 3 4473 black Pgg(OH) multicaudatus (Mex) 2 4 4475 black Pgg(IN) multicaudatus (Mex) 1 1 4498 black Pgg(FL) multicaudatus (Mex) 10 9 4512 black Pgg(IN) multicaudatus (Mex) 3 4516 black Pgg(OH) multicaudatus (Mex) 2 1 Subtotal (30) (3) (3) (0) (31) Pgg=Papilioglaucusglaucus. a mixture of phenotypes (16 black; 5 intermediate; 3 yellow). Differ- ences in the proportions of black and intermediate females between studies may reflect differences in criteria used for phenotype classifi- cation. Hybridization with Papilio multicaudatus We obtained eight successful pairings of black P. g. glaucus females withP. multicaudatus males (Table 5). Nopairingswithyellow females weresuccessfulandwedidnothavesufficientP. multicaudatusfemales to attempt reciprocal pairings using P. g. glaucus males. Two pairings yielded a total of six female offspring, for an overall sex ratio of 5:1 male female (n = 36 offspring). Pupae from broods #2265, 3660, 4473, : 4498, and 4516 that were alive, but had not eclosed after one year were sexed. All ten were female and all died without eclosing. West and Clarke (1987) report only male offspring in crosses in this type. The hybrid females consisted of three black and three intermediate individuals (Fig. 2). The intermediate phenotype may indicate partial suppression of the black color in these hybrids. West and Clarke (1987) postulate the occurrence of a P. multicaudatus suppressor on the basis of 2 yellow daughters obtained from a backcross of a hybrid male to a black P. g. glaucus female. Miscellaneous Interspecific Crosses Five pairings of P. rutulus females with P. eurymedon males and five of the reciprocal pairings were successful (Table 6, Fig. 3). Despite overall low numbers of emerging adults, hybrid females were obtained from both types. The one successful hybridization between a P. eury- medon female and a P. multicaudatus male also yielded both male 238 Journal of the Lepidopterists' Society Fig. 2. Hybrid offspring (brood #2265) of a black female of P. g. glaucus x male P. multicaudatus. Ventral (a) and dorsal (b) views of a black female with some yellow scaling and the dorsal view (c) of an intermediate female areshown. The lower right (d) is a sibling hybrid male.