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Journal of the Lepidopterists' Society 36(2), 1982, 121-131

EXPERIMENTAL HYBRIDIZATION BETWEEN PHYCIODES THAROS AND P. PHAON (NYMPHALIDAE)

Charles G. Oliver

R. D. 1, Box 78, Scottdale, Pennsylvania 15683

ABSTRACT. Phyciodes tharos and P. phaon are common species that occur sym-patrically over much of the southern United States. The species differ in larval, pupal, and adult phenotypic appearance, habitat preference, and larval foodplant. Fx hybrids and backcrosses between the species showed an unusual pattern of incompatibility, with relatively slight hybrid breakdown in one direction of the cross and total invia-bility in the reciprocal cross. The results differ strongly from those obtained in crosses between P. tharos and other Phyciodes species.

In the southern part of the southestern United States Phyciodes tharos Drury and P. phaon Edwards are the dominant Phyciodes species. They are closely related taxonomically, but while P. tharos ranges northward into southern Canada, P. phaon is confined to the South and Southwest and ranges into Central America. Except for P. phaon, all of the half-dozen or so members of the P. tharos species group feed on asters in the larval stage. The larval foodplant of P. phaon is Lippia (Verbenaceae), an herbaceous perennial found commonly along sandy roadsides in the Deep South.

In northern Florida (e.g., Alachua, Bradford, and Levy Cos.) P. tharos and P. phaon are often sympatric along roadsides and in other open, sandy areas, but while P. phaon seems fairly closely restricted to this habitat, P. tharos is common also in moist grassy fields, lawns, and pine-palmetto savannah. Laboratory experiments indicate that P. tharos larvae feed readily on a wide array of Aster species, and that each habitat contains suitable foodplants.

Both species are multivoltine, with the spring emergence beginning in mid-March in northern Florida. First generation adults of both species are of the extreme "spring" phenotype ("marcia" in P. tharos, "hiemalis" in P. phaon) with much more extensive dark markings on the ventral hindwing than in the "summer" phenotype ("morpheus" and "phaon," respectively). Although fairly similar in appearance, adults of the two species are easily distinguished by differences in color pattern (Fig. 1, Table 1). The larvae also differ in appearance (Table 1), although in general that of P. phaon is more similar in appearance to P. tharos than are those of P. campestris Behr (Oliver, 1978) or P. batesii Reakirt (Oliver, 1979a). In addition first instar larvae of both P. campestris and P. batesii form rudimentary communal webs, whereas P. tharos and P. phaon do not. The haploid chromosome numbers of both latter species is 31 (Maeki and Remington, 1960). There are apparently no records of natural hybrids. The rela-

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Fig. 1. Parental-type, F2 hybrid, and backcross adults: Row A, P. phaon; B, P. tharos; C, F2 hybrids P. phaon 9 x P. tharos 6; D, backcrosses (P. phaon 9 x P. tharos 6) 9 x P. tharos 6. Specimens show, left to right: male dorsal, female dorsal, male ventral, female ventral.

tively close taxonomic relationship of P. tharos and P. phaon, together with the unusual foodplant of P. phaon, made it seem especially desirable to me to investigate the relationship between these two species as part of my ongoing study of the evolutionary genetics of the P. tharos group.

Methods and Materials

Laboratory stock was derived from one female of P. tharos taken 17 March 1979 in Gainesville, Alachua Co., Florida; two females of P. tharos and one male and two females of P. phaon taken 16 and 21 March 1979 four mi. west of Otter Creek, Levy Co., Florida; and from one male and four females of P. tharos and three females of P. phaon taken 17, 18, and 24 March 1979 three mi. north of Waldo, Bradford Co., Florida. Cultures at the University of Florida were maintained at 25°C and under approximately natural photoperiod conditions (for April at latitude 29°30'N). On 19-21 April 1979 the cultures were transferred to my laboratory in Pennsylvania, where they were maintained at 27°C days, 24°C nights, and given 16 h light/24 h using rows of fluorescent tubes.

Table 1. Differences in phenotypic appearance of mature larva, pupa, and adult of P. tharo phaon 9 x P. tharos 6).

f. Color, head capsule

II. Pupa

a.   Color

b.  Overall shape

III. Adult.

a.   Dorsal body vestiture

b.   Color, ventral antennal club

c.   Intensity, black pattern elements

d.   Color, dorsal and ventral forewing

Brown w. dorsal white patch, very small lateral-ventral patch

Light tan to dark wood brown

More angular, projections more pronounced

W. large proportion of tawny hairs

Almost always very dark

Relatively light

Tawny areas nearly even; median black band often broken into sep. patches

Dorsally like tharos but w large ventral white pat

Usually light tan

More rounded, projection less pronounced

Very few tawny hairs

Light tawny gray

Relatively heavy

Median tawny band, mar al lunule, submedian s contrastingly pale; blac band heavy and contin

e. Color, ventral hindwing

Ground even straw yellow; discal and basal dark markings tan, outer marginal patch chocolate brown; dark markings less crisp

Ground cream w. a large contrasting central taw spot; dark markings all chocolate brown, usual very crisp

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Matings were made by the hand-pairing method (Clarke, 1952) or in small cages. Mated females were housed in 10 x 20 cm glass cylinders and given potted plants or cut sprigs of various asters or Lippia nodiflora (L.) Michx. for oviposition. Eggs were removed daily and counted, and the substrate leaf kept fresh until the eggs hatched. Larvae of P. tharos were reared on cut sprigs of Aster in 10 x 20 cm glass cylinders with screening over the tops; whereas, P. phaon were reared on cut sprigs of Lippia in 7 X 10 cm closed plastic boxes, since the Lippia plants tended to desiccate if exposed to the open air. After transferral of the cultures to Pennsylvania all hybrid larvae were reared on Aster because of the unavailability of Lippia. For this same reason no control broods of P. phaon were reared in Pennsylvania after early May.

Fx progeny of wild-collected wild-mated females or (in two cases) unmated, wild-collected females were used for the hybrid pairings and as parental-type stock for backcrosses. No stock used was inbred. Observations were made on parental population, Fx hybrid, and back-cross phenotypic appearance and larval foodplant acceptance, interspecific courtship behavior, development times and adult eclosion patterns, fertility, adult sex ratios, and embryonic, pupal, and eclosing adult viability. Single species controls were reared simultaneously for comparison with hybrid and backcross broods.

Data on egg fertility, viability, and sex ratios were treated statistically using the Wilcoxon Two-Sample Test. Adult fertility was measured by a count of the number of visibly developing eggs divided by the total number of eggs laid after a single mating. Development times from hatching of the egg to eclosion of the adult were estimated by calculating the 99% confidence intervals for the medians of the distributions (Owen, 1962). Distributions of development times within broods or series of broods have been represented by adult eclosion graphs showing the number of adults emerging from pupae each day.

Results

Interspecific Courtship Behavior

Males of both species show vigorous courtship behavior when caged with females of their own or of the other species. Females apparently rarely accept males of other species, however, and only one interspecific pairing, P. phaon 2 x P. tharos 6, was made without forced pairing.

Phenotypic Appearance

Differences in phenotypic appearance of the fifth instar larvae, pupae, and adults of the parental species and their Fx hybrid (P. phaon

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2 X P. tharos 8) are summarized in Table 1. Adults are shown in Fig. 1. The backcross (P. phaon 2 x P. tharos 6) 2 x P. tharos 6 generally resembled P. tharos except for the following characters: 1) larva— width of lateral light stripes slightly wider than on P. tharos; 2) adult— color of ventral antennal club dark, sometimes with a light tip; 3) color of dorsal and ventral forewing intermediate between P. tharos and P. phaon; 4) color of ventral hindwing with ground lighter than P. tharos, often with a slight, diffused central tawny spot; discal and ventral markings more like P. tharos but with some P. phaon influence present; dark markings more sharply defined than on P. tharos. The larva and pupa of P. phaon and the larva of P. tharos have been figured by Emmel and Emmel (1973) and the pupa of P. tharos by Holland (1931). In nature both P. tharos and P. phaon show seasonal polyphenism regulated by photoperiod (Oliver, 1976, unpubl. data). In both species the short-day forms, which fly in fall and spring, have the underside of the hindwings suffused with dark brown or violet-brown, which obscures the other dark markings. The character of this suffusion is closely similar in both species. Under the laboratory conditions described above only light-colored long-day forms were present in the parental cultures. The males in the Fx hybrid broods were only of the long-day form; varying proportions of the females were of the short-day form (Brood No. 79-6: 23.3%; 79-17: 0%; 79-18: 13.1%; 79-34: 0%; 79-35: 17.6%; 79-36: 71.1% [see Table 4 for numbers of females involved]). A few backcross females showed moderate expression of the short-day phenotype.

Foodplant Acceptance

In laboratory oviposition tests P. phaon accepted only Lippia, P. tharos only Aster. Fx hybrid females (P. phaon 2 x P. tharos 6) accepted either plant readily.

Newly hatched larvae without feeding experience given the food-plant of the other species fed to a very limited extent and died without increase in size. Fx hybrid larvae of the cross P. phaon 2 x P. tharos 6 fed readily on either foodplant. One early brood of this cross was split into Lippia- and Aster-feeding groups. Growth was markedly slower on Lippia, and the Lippia-feeding group was changed to Aster when the cultures were moved to Pennsylvania. Part of one brood of the backcross (P. phaon 2 x P. tharos 6) 2 x P. tharos 6 was started on Lippia and grew until the second instar, when all of the larvae (N = 40) died.

Viability, Fertility, and Sex Ratio

Embryonic viability in the parental control broods was extremely high. In the Fx hybrid (P. phaon 2 X P. tharos 6) there was significant

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reduction in viability (Table 2). In the reciprocal hybrid there was either complete very early embryonic inviability or a complete lack of egg fertilization following insemination. Females mated in this cross oviposited normally (in large egg patches), rather than in the pattern demonstrated by uninseminated females (very few eggs laid, widely scattered over the foodplant). Since in other Phyciodes crosses this has proved to be an infallible indicator of mating success, these mat-ings were assumed to be successful even though the presence of sper-matophores was not verified. The backcross (P. phaon 9 x P. tharos S) 9 x P. tharos 6 showed a significant reduction in visible egg fertility and reduced embryonic viability. The reciprocal backcross P. tharos 9 x (P. phaon 9 x P. tharos S) S showed massive reductions in egg fertility and complete embryonic inviability. The reduction in visible egg fertility in these crosses may have been due to very early embryonic inviability or to reduced parental fertility. No stock of P. phaon could be maintained in Pennsylvania for backcrosses because of a lack of Lippia for foodplant.

Post-larval (i.e. prepupal, pupal, and ecdysing adult) viability was significantly lower for the P. phaon parental broods than for P. tharos (P = .005) (Table 3). This may have been due to the different larval rearing containers for P. phaon, since humidity in these containers was very high. Fx hybrid and backcross broods were reared in the same manner as P. tharos; post-larval viabilities of these two series of broods were significantly lower than for P. tharos (P = .005 for both values) but not lower than both P. tharos and P. phaon considered together. It is possible that neither parental foodplant was ideal for the hybrid larvae, and that some reduction in viability was due to this.

Adult sex ratios in the Fx hybrid broods and in the backcross broods showed no change from those of the parental control broods (Table 3).

Development Times and Eclosion Graphs

Development times from hatching of the egg to eclosion of the adult were about the same for the two parental species, although some broods of P. phaon averaged a day or so faster than P. tharos (Table 4). Development times of the P. phaon broods varied more than did those of P. tharos.

In Fx hybrid broods of the cross (P. phaon 9 x P. tharos 6) reared on Aster, males showed development times similar to those of the control broods, but those of the Fx females averaged at least several days longer than those of the controls. Development times of both males and females of this F1 hybrid tended to vary more than those of the controls. In addition, the eclosion graphs of both sexes showed

Table 2. Mean egg fertility and embryonic viability (with standard deviation) of P and backcross broods. P values refer to comparison for differences of hybrid or backc

Species/cross

No. of broods

No. of eggs

Fertile/laid

H

P. tharos P. phaon

th 9 ph 9

x ph 8 x th 8

(ph 9 x th 8) 9 x th 8 th 9 x (ph 9 x th 8) 8

Parental Controls

0.995 ± 0 1.000 ± 0

0.000 ± 0 0.945 ± 0

0.747 ± 0 0.000 ± 0

Table 3. Mean incidence (percentages) of prepupal through eclosing adult invia P. phaon (ph), Fj hybrids, and backcrosses. Tests of significance refer to comparison tharos alone (viability).

Species/cross

No. of broods

Dead prepupae

Dead pupae

Total no. eclosing

S (me

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Table 4. Development times in days from hatching of egg until eclosion of adult for P. tharos and P. phaon control broods, Fx hybrid (P. phaon 9 x P. tharos 8) and backcrosses ([P. phaon 9 x P. tharos 8] 9 x P. tharos 8). "Medians" show 99% confidence limits. See text for rearing conditions. Mr = March, A = April, My = May, J = June.

Males                                                        Females

a tailing-off effect. This was more marked in the females (Fig. 2). Because of slightly different rearing conditions, broods hatched during March and early April cannot be compared with those hatching during May and June. Development times of the backcrosses to P. tharos were significantly shorter than those of P. tharos or of the F1 hybrids.

Discussion

At first glance the results seem to give a somewhat contradictory picture of incompatibility between Phyciodes tharos and P. phaon. In the F2 hybrid (P. phaon 2 x P. tharos 6) and its backcross to P. tharos S, hybrid sex ratios are normal and development times only slightly affected; whereas, the reciprocal hybrid is totally inviable,

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30                                  30                     40                     50

Days until Eclosion

Fig. 2. Distributions of times required for development of typical P. tharos, P. phaon, F2 hybrid, and backcross broods from hatching of eggs until eclosion of adults. A, P. tharos, Brood 79-9; B, P. phaon, Brood 79-7; C, F! hybrid P. phaon ?xP, tharos 6, Brood 79-6; D, backcross (P. phaon ?xP. tharos 6) 9 x P. tharos 6, Brood 79-55. (See Table 4 and text for rearing conditions and dates.)

and the backcross P. tharos 9 x (P. phaon 2 x P. tharos 6) 6 almost so. It would appear from these results that, while the nuclear materials of P. tharos and P. phaon are quite compatible (i.e. can cooperate to direct harmonious growth and development) and while P. tharos nuclear material is relatively compatible with P. phaon or hybrid cytoplasm, P. phaon nuclear material is highly incompatible with P. tharos cytoplasm. This incompatibility may involve crucial differences in one or more of the many factors that determine the composition of the cytoplasmic environment in which foreign nuclear material must

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function to produce a viable hybrid individual organism. An incompatibility of this sort may be ultimately attributable to a relatively slight degree of differentiation in gene regulation (see Oliver, 1979b, for a fuller discussion) and does not contradict the other results presented here. The conclusion is, then, that P. phaon and P. tharos show relatively slight overall genetic differentiation. Hybridization in nature is probably prevented by barriers involving courtship behavior.

The pattern of hybrid incompatibility in the present series of crosses is quite different from that shown in hybridization experiments using P. tharos and P. campestris (Oliver, 1978), P. batesii (Oliver, 1979a), or the entity I have referred to as P. "tharos Type B" (Oliver, 1979a, 1980). These combinations show a homogeneous pattern of effects, which involves mainly slight to moderate (but not total) reduction in viability in the Fj hybrids and backcrosses, skewed Fj adult sex ratios, and abnormal Fj hybrid development times. In the Fj hybrid females eclosion occurs before that of the males when P. tharos is the male parent. In the reciprocal crosses female eclosion is delayed, and male and female curves usually do not overlap. In the (P. phaon 9 x P. tharos 6) Fj hybrid broods, however, females show slightly delayed rather than speeded up eclosion times. These abnormalities in development time probably are in some way related to parental species differences in larval diapause induction thresholds (Oliver, unpubl. data).

Expression of the "marcia"-"hiemalis" phenotypes in the female Fj hybrids may indicate that this form results unless the "morpheus"-"phaon" form is induced. One possible explanation for failure of this induction in female hybrids is that the "switch" gene and/or modifiers are linked with both a diapause induction-development rate gene complex and with sex. Since it is the females that are heterogametic in Lepidoptera, in this cross the female "morpheus"-"phaon" form must be induced in P. phaon cytoplasm using regulation by P. tharos genetic material, this induction fails in a high percentage of individuals. This case seems to be analogous to that in the cricket genus Pteronemobius (Masaki, 1978), where the Fj hybrid males (the heterogametic sex in Orthoptera) show abnormal growth rates and photoperiodic responses.

The pattern of hybrid incompatibility between P. tharos and P. phaon differs also from that in butterfly hybrids outside the genus (reviewed in Lorkovic, 1978, and Oliver, 1979b). In general these latter show incompatibility similar to that in the other Phyciodes crosses discussed above. I know of no case in which the reciprocal Fj hybrids differ so drastically in viability as do those between P. tharos and P. phaon, although recent crosses between Pieris callidice

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Hiibner and P. occidentalis Reakirt (Pieridae) (Shapiro, 1980) show a basic similarity that may be due to the same genetic effects.

Acknowledgment

I am grateful to the Department of Zoology, University of Florida, Gainesville, Florida, for the use of laboratory facilities during my visit there in March and April 1979.

Literature Cited

Emmel, T. C. & J. F. Emmel. 1973. The butterflies of Southern California. Nat. Hist.

Mus. L. A. Co., Sci. Ser. 26:1-148. Holland, W. J. 1931. The Butterfly Book, revised ed. Doubleday & Co., New York.

424 pp. L0RKOV16, Z. 1978. Types of hybrid sterility in diurnal Lepidoptera speciation and

taxonomy. Acta Entomol. Jugoslavica 14:13-24. MAEKI, K. & C. L. REMINGTON. 1960. Chromosomes of North American Lepidoptera.

Part 4. J. Lepid. Soc. 14:179-201. Masaki, S. 1978. Seasonal and latitudinal adaptations in the life cycles of crickets.

In Evolution of Insect Migration and Diapause (H. Dingle, ed.). Springer-Verlag,

New York. 284 pp. Oliver, C. G. 1978. Experimental hybridization between the nymphalid butterflies

Phyciodes tharos and P. campestris montana. Evolution 32:594-601.

--------- 1979a. Experimental hybridization between Phyciodes tharos and P. batesii

(Nymphalidae). J. Lepid. Soc. 33:6-20.

--------- 1979b. Genetic differentiation and hybrid viability within and between some

Lepidoptera species. Amer. Natur. 114:681-694.

--------- 1980. Phenotypic differentiation and hybrid breakdown within Phyciodes

"tharos" (Lepidoptera: Nymphalidae) in the northeastern United States. Ann. Entomol. Soc. Amer. 73:715-721.

Owen, D. B. 1962. Handbook of Statistical Tables. Addison-Wesley, Reading, Mass. 580 pp.

Shapiro, A. M. 1980. Genetic incompatibility between Pieris callidice and Pieris occidentalis nelsoni: differentiation within a periglacial relict complex (Lepidoptera: Pieridae). Canad. Entomol. 112:463-468.