Copyright 0 1987 by the Genetics Society of America

High Fitness of Heterokaryotypic Individuals Segregating Naturally Within a Long-standing Laboratory Population of Drosophila silvestris

Hampton L. Carson Department of Genetics, University of Hawaii, Honolulu, Hawaii 96822 Manuscript received September 8, 1986 Revised copy accepted March 23, 1987

ABSTRACT Natural populations of Drosophila szlvestris are polymorphic for inversions in one or more of four of the five major chromosome arms; laboratory stocks tend to retain this heterozygosity. A laboratory stock, U28T2, was started from a single naturally inseminated wild female caught at Kilauea Forest Reserve, Hawaii, in January 1977. Polytene analysis in 1980 showed the presence of three natural inversions in chromosome 4: k2 is distal, t is central and 1’ is proximal. The inversions are short but only short uncovered euchromatic sections exist at the distal and proximal ends. Periodic examinations through 1986 showed all three inversions to be persistent at moderately high frequencies. In 1984, a series of tests of mating performance of caged, mature males, taken at random as they eclosed from the stock, were followed by cytological testcrosses to females from a homokaryotypic stock. Only three of the eight possible haplotypes, k2/t/+ (A), +/+/12 (a)and +/+/+ (a’) were present. Tests of crossing over show none in males; in females, there is about 1% in each of the two regions between the inversions. Only one such apparent crossover haplotype was found among 1084 examined in samples from this stock. Thus, chromosome arrangements A, a and a’ virtually behave as whole- chromosome alleles in both sexes. Of 146 males marked and tested in cages, 61 produced progeny; the others failed to reproduce. Of 58 males and 80 females producing progeny and analyzed cytologically, there were, respectively, 49 and 59 heterokaryotypes. On the basis of frequencies calculated for fertilized eggs, 33.6 males and 46.3 females are expected. The facts suggest that individual males with the Aa karyotype are particularly successful in production of offspring. Adult females show an excess of Aa‘ as well as Aa. Such high fitness of heterokaryotypes in the effective breeding adults could be a major factor in the maintenance of stable chromosomal polymorphisms both in laboratory stocks and in nature. Although some of this heterosis is clearly ascribable to differential survival, the facts suggest that there is a substantial opportunity, indeed a likelihood, for a contribution from differential mating among surviving adults.

F the various components of Darwinian fitness, to, the deme under examination. Since fitness char- 0 simple survivability is fairly easy to measure and acters have a polygenic basis, the use of single-gene has tended to dominate thinking about selective proc- markers is insufficient. Polymorphisms consisting of esses. New data on sexual selection, however, indicate inversions, however, may cover some considerable that many individual organisms, especially males, may part of the genome; as markers, these are much more survive well and engage in courtship yet fail to partic- desirable. This set of conditions has almost never been ipate significantly in reproduction (e.g., LANDE198 1 ; realized experimentally. For example, BOSIGER THORNHILLand ALCOCK1983). The sexual activity (1962), and those who followed him using similar of the individual is surely of paramount theoretical techniques, have tended to rely on the use of geno- importance in fitness yet has been little studied (but types marked experimentally by initially making inter- see ANDERSONet al. 1979). The reason for the lack of strain crosses. Individuals carrying the markers are data appears to stem from the double difficulty of then introduced into an experimental population, per- measuring this trait phenotypically and then relating mitting them to be distinguished from the resident the measure directly to a genetic basis. The individual members of the deme. As a result, the differential not only must be marked so that sexual performance reproduction that may be observed does not represent can be monitored but the relative contribution of this that which occurs naturally within the deme. Rather, individual to the next generation must also be esti- any effect is the result of artificial interdeme hybridi- mated. zation. Striking heterozygote advantage has often As fitness of an individual must be determined been demonstrated in this manner, yet the relevance relative to other competing members of the same local of the results is questionable. The goal of such exper- population or deme, proper genetic control requires iments, namely, to understand the genetics of the the use of markers that are intrinsic in, and not foreign mating patterns that have evolved and exist in a

Genetics 116 415-422 (July, 1987) 416 H. L. Carson natural, complex balanced gene pool is not well served a series of such samples from a single locality, the frequencies by such a procedure. of the various inversions carried in the source wild popula- tion can be deduced from orcein smears of the salivary When a field of related surviving males compete gland chromosomes of the F, larvae. It may sometimes be for the favors of females in natural populations, a inferred that the female was carrying sperm from more than crucial question is: to what extent does relevant seg- one wild male (CRADDOCKand JOHNSON 1978). regating genetic variation occur among the members Laboratory stocks: In order to produce a vigorous iso- female stock of D. silvestris, F, of such a field? If such genetic variation can indeed an from a single wild female of no less than 30 flies of each sex is desirable. This should be shown to exist, then is it possible to demonstrate assure that about 10 high-fitness males are available for that differential reproductive success is served by any getting the stock started (CARSON1986). If possible, how- of the blocks of polygenic genetic variants that are ever, 72 flies matured in vials are used. These imagoes are segregating naturally within it? placed together in a large (4 liter) glass jar, the bottom of The experiments to be described here employ a which contains a layer of about 8 cm of sterile sand mois- tened with distilled water. As a source of food and as an single laboratory population of Drosophila silvestris, a oviposition site, about six shell vials (95 X 30 mm) containing species endemic to the Hawaiian islands. This species a small amount of food high in plant proteins are placed in has been subjected to a number of genetic and behav- the jar. The formula of WHEELERand CLAYTON(1965) is ioral investigations (for reviews, see CARSON1978, used but the yeast is omitted. These vials are left open at 1982, 1986). Like many Hawaiian Drosophilas, the end and are laid horizontally on the sand. Each vial also D. contains a piece of paper tissue that has been saturated with silvestris displays a number of secondary sexual char- an aqueous extract of the leaves of a host plant. The flies acters of males that are used in the courtship proce- are free to enter and leave the vials for feeding and ovipo- dure (SPIETH 1978). The species has extensive genetic sition. A cloth screen covers the tap. The large open area variability in inversions and allozymes both within and within the jar serves as an arena for courtship, mating and between populations (SENEand CARSON1977; CRAD- reproduction much as in an ordinary population cage. The life cycle is completed in the following manner. Vials DOCK and JOHNSON 1979). More recently, intra- and containing eggs and small larvae are harvested from the jars interpopulation genetic variance in certain secondary and at the same time are replaced with fresh ones. During sexual characters has also been demonstrated (CARSON this procedure, the standing adult population is allowed to and BRYANT1979; CARSONet al. 1982; CARSONand remain undisturbed within the jar. Vials containing larvae LANDE1984; CARSON1985). are given a heavy feeding with cornmeal medium, plugged with stoppers, slanted and stored separately until mature Experiments with marked males in experimental third-instar larvae start leaving the food. At this time, the cage situations strongly suggest that both epigamic vials are placed on sterile sand in a fresh 4-literjar. Pupation and intrasexual sexual selection is operative in popu- occurs in the sand. lations of this species (SPIESSand CARSON198 1 ; CAR- After the larvae have evacuated the food vials, the latter are removed and replaced with open yeastless vials that SON 1986). In cages, approximately one third of ma- serve as a source of nutrition for the emerging imagoes. ture, healthy, actively courting males surviving to four Occasionally, young maturing adults are added to the stand- weeks of age were found to be unsuccessful in achiev- ing population in the oviposition jars in order to gradually ing copulation whereas another third accomplish replace the older flies, create a distribution of ages and to about two-thirds of the observed matings. maintain a census population size of between 40 and 60 flies The present study extends the differential analysis per jar. Two such jars are normally maintained for each stock. These cultures are maintained at 18" and yield 3-4 of marked males to include assaying the reproduc- generations per year; they are maintained without outcross- tively successful ones for genetic inversion markers ing. carried naturally by the experimental population. The Experimental population: The experiments to be de- results indicate (1) that there is ample genetic varia- scribed here were done with a single chromosomally poly- bility carried naturally in males of this population, (2) morphic laboratory population (U28T2). It is descended from a single wild female collected at Kilauea Forest Re- that this variation can be related to the reproductive serve, Island of Hawaii, in January 1977. It is a vigorous success or failure of these males and (3) that success- stock that has been maintained approximately as described fully reproducing individuals of both sexes show a above without bottlenecks. The local population in this area high representation of heterokaryotypes for a series has been well characterized for both allozymes and inver- of naturally segregating chromosomal inversion hap- sions (CRADDOCKand JOHNSON 1978). The method for recording male copulatory performance lotypes that cover virtually the entire length of one of used in the present experiments is slightly modified from the five major chromosome arms. that described in the preliminary work (CARSONand TERA- MOTO 1980; SPIESSand CARSON198 1). A group of 25 male MATERIALS AND METHODS specimens eclosing at approximately the same time from the sand jars are aged for 4 weeks in groups of five in unyeasted The species: Drosophila silvestris is wholly confined to the food vials. In addition, virgin females from both the Kilauea island of Hawaii, the newest in the archipelago. It exists in stock (U28T2) and a homokaryotypic test stock from the disjunct populations of small size in the moderate-altitude nearby Olaa area (T94X) were also aged to 4 weeks, in a rainforests. Most samples of these populations have been similar manner. All flies were handled individually by gentle taken by capture of individual wild female specimens from aspiration; no anesthetic was used at any time. which F, progeny are then reared in the laboratory. From Analysis of breeding capacity reported here concentrates Heterokaryotypes and Fitness 417 on the male sex. Experiments were begun by dividing 4- week-old surviving males from the group of 25 into two groups of between 9 and 1 1 each and introducing them into two Plexiglas cages (20 X 20 X IO cm). Before introduction, each male was marked with one or two small dots of nontoxic enamel paint (The Testor Corporation, Rockford, Illinois) so that each fly in each cage could be individually recog- nized. Flies to be marked were held in the dark for about 1 hr. When brought again into the light, there is about a 20- niin period before light-adaptation is complete. During this time, the flies may be approached without startling them, allowing a sharpened toothpick to be used to dab the paint onto the mesonotum. Over the next 6 days, the flies were observed for approx- imately 2.5 hr each morning under fluorescent light in a constant-temperature room (20 f 1 "). Following each ob- servation period, any remaining females were removed and the cages were covered with an opaque cloth. Under such FIGURE I.-Polytene chromosome 4 of D. n'luestris showing circumstances, activity is greatly reduced. Ordinarily, the three inversions in the heterokaryotypic state. The distal end is to cages were uncovered for about 2 hr in the afternoon in the left. From left to right the inversions are ha, t and I*. The triply order to allow the males to move around and to feed on heterokaryotypic state shown has the zygotic formula 'Aa" being shallow dishes of unyeasted food that were replaced daily. formed by the pairing of an 'A" haplotype (hz/f/+) with an -a" On each day of testing, the cages containing the males haplotype (+/+/Iz). were uncovered at about 8 am and left for a light-adjustment period of 20 min. Females, which were held in vials in the peza) all have no variants in chromosome 4, that is, have the dark, were brought into the light at the same time as the standard sequence (+/+/+). This arrangement of genes is males. Routinely, two virgin females were released simulta- identical to D. silvestric haplotype a' and is clearly the neously into each cage. As soon as a copulation occurred, ancestral arrangement. the pair was covered with an empty vial, the time until The exceptional outlier mentioned above was detected in separation was recorded and a new virgin female intro- an adult female that, when testcrossed, showed a unique duced. Females not mating in 30 min were removed and haplotype, k2/+/1', in heterozygous combination with hap returned to vials; they were used again on a later day. The lotype A. Preliminary data on crossing over in heterokary- identities of the copulating male and female were recorded, otypic females show about 1% recombination in each of the the female was then isolated in a fresh vial for the rearing two regions between the inversion sites. Accordingly, the of larvae. After each copulation, the male was left in the unique haplotype carried by this female has been interpreted cage and thus given opportunity to engage in further cop as a rare recombinant. Crossing over has not been detected ulations on that day and the rest of the week. in males. Further analysis of haplotypes and crossing over Chromosomal polymorphisms: Approximately 1 1 poly- will be presented elsewhere. morphic inversions are known in this species; a given wild Polytene chromosome samples: Most of the females used population may carry from 5 to 10of these. Each population in the cages were from the homokaryotypic T94X stock. tends to be characterized by a unique combination of inver- Karyotyping of copulating males was done by examining the sions and/or frequencies. polytene chromosomes of seven larvae from such a mating. The Kilauea stock displays three inversion polymorphisms This affords a probability of 63 out of 64 that both homologs in chromosome 4 but is homokaryotypic in the other major of each paternal autosome have been observed. In each chromosomes. Spatially, these three inversions are separate experimental run, U28T2 females were also used in the from one another along the length of the chromosome cages; if used together with T94X females, one of the two (Figure 1). The distal inversion is known as 4k2, the central types was marked with paint dots. The data indicate that one as 41 and the proximal one as 412 (read this last formula the males are accepted equally well by both types of females. as "four-ell-two"). These inversions are unique to D. dvestris Larval salivary gland preparations from copulations in the and were originally described and the break points mapped cages between U28 males and females were also prepared. by CARSONand STALKER(1 968). They are widespread geo- These data cannot be used to determine either of the graphically in the species (CARSONand BRYANT1979; CRAD parental karyotypes but they serve as a source of data for DOCK and JOHNSON 1979; CARSON1982). larval karyotypes from the stock. Although eight chromosome 4 haplotypes would theoret- Karyotype samples from stock adult females were ob- ically be expected, the data show that, with one rare excep tained by crossing a series of mature adults to T94X males tion, only three are found segregating in the stock. This and determining their karyotypes from seven FI smears in type of linkage disequilibrium was first discovered in natural the same manner as was done for the males. populations of D. silvestris by CRADWK and JOHNSON Samples of third instar larvae from the stock were cap (1 979). These same three inversions were reported by them tured at random as they wandered in the pupation jar after in the same Kilauea Forest Reserve site where the U28T2 having emerged from food vials placed therein. No more stock was collected. Testcrosses of U28T2 individuals to a than seven larvae were taken in any week in order to homokaryotypic laboratory st&k (T94X: chromosome 4 is minimize sibling effects. Although small samples were taken k'/t/+) reveal that the three haplotypes present in U28T2 from the stock as early as 1980, the samples on which both are k'/t/+ (designated haplotype A), +/+/12 (haplotype a) the gametic and zygotic frequencies to be reported were and +/+/+ (haplotype a'). Each "+" refers to the relevant based were taken during 1984- 1986, closer to the time that standard banding sequence for this subgroup of flies. It is the cage experiments were done. The frequencies of these worth noting that the four species most closely related to D. various samples are homogeneous and these random larval silvestris (D. heteroneura, D. phitibia, D. differens, D. hemi- samples were pooled with those of like sex from the U28 x 418 H. L. Carson TABLE 1 cohorts by sex (female larvae and adults and male

Frequencies of haplotypes A, a and a’ observed in chromosome larvae and adults) were used to assess egg to adult 4 of Drosophila silvestris frequency changes. The gametic frequencies in adults have been used to calculate the frequencies expected A a a’ in fertilized eggs, by uniting female gametes with male

Sample No. Percent No. Percent No. Percent Total gametes. This gives the zygotic frequencies given in - ~~~ the top line in the body of Table 2. For example: Female larvae 253 0.588 141 0.328 36 0.084 430 Femaleadults 91 0.569 46 0.287 23 0.144 160 f(Aa) = female A (0.569) X male a (0.404) Male larvae 229 0.603 130 0.342 21 0.055 380 male A (0.517) X female a (0.287) = 0.378, etc. Maleadults 59 0.517 46 0.404 9 0.079 114 +

~~ Samples from a laboratory population originating from isofemale When the zygotic distributions of each of the four U28T2, Kilauea Forest Reserve, Hawaii, January 1977. Zygotic samples is compared on the hypothesis that they are frequencies are shown in Table 2. maintained at the same frequencies found in fertilized U28 larval samples mentioned above. Due to the faint- eggs, it will be seen that they do not conform (Table staining condition of the unpaired X chromosome of males, 2). This suggests the presence of selection both be- it is routinely possible to diagnose each individual larval tween eggs and larvae and between larvae and adults. preparation as to whether it was from a male or a female larva. To give an indication of the contribution of the indi- In summary, four types of chromosomal samples from vidual karyotypes to these differences, Table 2 gives this population have been obtained: larval females, larval the percent above (+) or below (-) the expected males, adult females and adult males. Only the latter, how- frequency for each genotype. These calculations are ever, were assayed for copulatory performance. presented in Table 2 and are shown graphically in Figure 2. OBSERVATIONS These changes may also be viewed by computing Table 1 gives the observed gametic frequencies of the fitness for each genotype that would be required the three haplotypes inferred from the diploid kary- in order to transform each relevant egg frequency otypes (zygotes) for each of the four samples. Ob- into that observed in the other two life-cycle stages served zygotic frequencies for these same samples are (Table 4). Larvae to adult transformations are also given in the lower part of Table 2. Table 3 provides given. Without exception, all fitnesses of 1 and above a homogeneity analysis for both gametic and zygotic involve the three heterokaryotypes. To express this frequencies. The haplotype frequencies (above diag- apparent heterosis, the fitnesses of the heterokary- onal) are homogeneous in larvae and adults within the otypes have been renormalized to the mean of the same sex but two of the comparisons between the two more frequent homokaryotypes (Table 5). The sexes appear to be different from one another. Except rare a’a’ homozygote is omitted from these calcula- in one instance (male larvae ‘us. female larvae) the tions. The very high fitness of the aa’ karyotype distribution of the zygotic frequencies are widely dif- between male larvae and adults in Table 4 is partly an ferent among the samples. artifact of the small numbers involved, i.e., 2 in larvae To investigate the situation further, the two age and 5 in adults. Disregarding this, it is noteworthy

TABLE 2 Observed zygotic frequencies in samples of larvae and adults compared to those expected on the basis of calculated frequencies for fertilized eggs

Aa Aa’ ea ‘ AA aa ala’ N xp d.f. P Expected (Exp.) frequency in 0.378 0.120 0.081 0.294 0.116 0.01 1 fertilized eggs FEMALE LARVAE Observed (Obs.) frequency 0.461 (99) 0.073 (16) 0.074(16) 0.321 (69) 0.061 (13) 0.009(2) 215 14.0 5 0.02 (N) Obs. - Exp. (%) +8.3 -4.6 -0.7 +2.7 -5.5 -0.2 FEMALE ADULTS Observed frequency (N) 0.450 (36) 0.238 (19) 0.050 (4) 0.225 (18) 0.038 (3) 0 (0) 80 17.7 4 0.002 Obs. - Exp. (%) +7.2 +11.8 -3.1 -6.9 -7.8 -1.1 MALE LARVAE Observed frequency (N) 0.453 (86) 0.068 (1 3) 0.021 (4) 0.342(65) 0.105(20) 0.011 (2) 190 17.1 5 0.002 Obs. - Exp. (%) +7.5 -5.2 -6.0 +4.8 -1.1 0 MALE ADULTS Observed frequency (N) 0.684 (39) 0.070 (4) 0.088 (5) 0.140 (8) 0.018 (1) 0 (0) 57 25.4 4 0.0001 Obs. - Exp. (%) +30.6 -5.0 +0.7 -15.4 -9.8 -1.1 Heterokaryotypes and Fitness 419 TABLE 3 Homogeneity chi-square tests on the frequency distributions of three haplotypes (above diagonal) and six zygotes (below diagonal) among the four samples

Female larvae Male larvae Female adults Male adults Female larvae - 2.5 2 0.30 4.8 2 0.18 2.3 2 0.30 Male larvae 8.5 5 0.12 - 12.1 2 0.003 2.8 2 0.25 Female adults 16.7 5 0.005 21.6 5 0.001 - 5.4 2 0.06 Male adults 11.9 5 0.04 20.6 5 0.001 11.3 4 0.02 - Each entry, from left to right, gives chi-square value, degrees of freedom and probability.

I TABLE 5 Fitnesses renormalized to the mean of AA and aa (ignoring a’a’) +w i FEMALES Females +20 Mean of AA Value Aa Aa’ aa’ andaa of mean + 15 i Eggs to larvae 1.50 0.77 1.13 1 0.665 Eggstoadults 2.17 3.65 1.13 1 0.460 Larvae to adults 1.47 4.84 1.01 1 0.680

Males

Eggs to larvae 1.16 0.54 0.25 1 0.865 Eggs to adults 5.71 1.83 3.43 1 0.175 Larvae to adults 5.26 3.58 14.58 1 0.190

TABLE 6 Reproductive performance of the 98 males that survived to 4 \ weeks and were tested in cages i i i. No. of Percent t Category males of males -‘*i AWLTS FIGURE 2.-Changes in karyotype frequencies from fertilized Did not copulate 18 0.184 eggs to larvae and adults in a stock of D. silvestris. Copulated but no progeny 19 0.194 Gave progeny 61 0.622 TABLE 4 Total 98 Fitnesses necessary to transform egg and larval frequencies into TABLE 7 adult frequencies (normalized to Aa) Reproductive failure of some copulating males of D. Eilvesttis Females No. of No. of Transformation Aa Aa’ aa‘ AA aa a‘a’ No. of CO ulations copulations Category males otserved per male Eggs to larvae 1 0.51 0.75 0.90 0.43 0.67 ~~ ~ Eggs to adults 1 1.66 0.52 0.64 0.28 0 Gave no progeny 19 44 2.32 Larvae toadults 1 3.29 0.69 0.72 0.64 0 Gave Droszenv 53 132 2.49

Males ports of male reproductive performance (e.g. CARSON Eggs to larvae 1 0.47 0.22 0.97 0.76 0.84 and TERAMOTO1980; SPIESSand CARSON198 1 ; sum- Eggs to adults 1 0.32 0.60 0.26 0.09 0 marized in CARSON1986), were based on males that Larvae toadults 1 0.68 2.77 0.27 0.11 0 similarly had survived to 4 weeks of age. In these cases, about one-third of the males did not copulate. that the highest fitness among adult males is ascribable In the present case (Table 6), if only the survivors are to the triple heterozygote (Aa), whereas in females, considered, there are 18.4% that do not copulate. this is true for the double heterozygote Aa’ (Table 5). These, together with those that copulated but gave Among the 146 individual males followed in this no progeny again document the fact that a consider- study, 48 died during the aging process, leaving 98 able percent (37.8% in this case) of healthy, surviving that survived and were tested in cages. Previous re- adult males again are shown to be nonreproductive. 420 H. L. Carson A curious category in Table 5 is the group of 19 alleles. The six zygotes produced are viable both as males that were observed to copulate but nevertheless larvae and adults. gave no progeny. This was studied further by calcu- During the life cycle in the laboratory, the zygotic lating the mean number of observed copulations by frequencies apparently formed in the fertilized eggs such males and comparing this with the mean number are not maintained in the third instar larvae or in the of copulations engaged in by males that did produce effective population of breeding adults. Instead, there progeny. There are 53 rather than 61 males in the is a preponderance of heterokaryotypes, especially latter category since some copulations recorded in among the adult males. The distribution of the inver- Table 6 were not actually observed. There is no sions along the chromosome shows that most of the difference between the two means, as shown in Table length of the chromosomal homologues is segregating 7 (chi-square with one degree of freedom = 0.13; P as a unit, or haplotype. This appears to reflect the = 0.9). This point will be the object of further study. participation of the entire length of the chromosome, Dissections of males have not given any evidence of about one-fourth of the diploid male genome, in a sterility manifested by lack of motile sperm. balanced system. Current cytological techniques permit the recogni- tion of the inversion karyotypes only in the salivary DISCUSSION gland cells of third instar larvae or by testcrossing adults. Individuals that die without progeny later than Balanced polymorphism is characteristic of many the third instar larval stage remain unknown cytolog- sexual populations (DOBZHANSKY1955). Genetic var- ically, but it may be inferred that they represent some iability in the form of inversions or translocations of the zygotic classes that are underrepresented commonly display such balance. The populations of among the breeding adults. many species also retain high frequencies of recessive The data suggest that the heterotic effect already lethals, semilethal and subvital genes as well as genes begins to express itself in viability differences express- that produce various kinds of sterility. An enormous ing themselves in the period between the fertilized amount of biochemical polymorphism in the form of eggs and the third instar larvae, an effect that is allozyme variation is also carried in both natural and observed about equally in females and males; the triple artificial populations. Much of the allozyme variation heterozygote apparently leads the way (Table Fur- appears to be neutral to selection and this condition 4). thermore, as the cohorts approach the breeding stage, can explain its presence. Where such alleles appear further opportunity for the assertion of viability dif- balanced at high frequencies, they may not have this ferences between the zygotic types exists, since it may property themselves but may be hitchhiking on larger sections of the genome that do have the property of be noted that 48 males, or 32.9% of the full cohort balance. of 146 need to be accounted for. A useful aspect of some of these balances is that It does not appear, however, that inviability can they are often tenaciously retained in the laboratory account for all of the reciprocal increase in heterozy- as well as in wild populations, even though the lab gotes and decrease in homozygotes. In the perform- populations are started from single gravid females and ance tests mentioned earlier, it has been observed that considerable inbreeding continues to be present in one third of the mature healthy males are not ob- subsequent generations. served to engage in copulation at all. This category DOBZHANSKYsuggested that balanced polymor- appears to be represented in the current experiments phism is due to a widespread tendency for genetic by the 18.4% of males that did not copulate during adjustment to the environment to be mediated by the the test period. Viability also apparently does not formation of superior heterozygotes or heterokary- affect another group of 19.4% that copulate but do otypes. These are thought to arise through the coad- not produce progeny. Thus 37.8% of healthy viable aptation of large sections of the chromosomes that adult males are not included in the effective breeding DOBZHANSKYcalled “supergenes.” Rather than fit the population. This suggests that the fitness of the males organism through directional selection and fixation may have a component due to sexual activity as well of alleles at many loci individually, a process that as viability. Further experiments will have to be done, exhausts genetic variability, balancing selection ap- however, to settle this matter. One may also notice pears to serve fitness by favoring multigenic states. that the karyotype displaying the highest fitness in In the present case, the D. silvestris stock from females (Aa’) is different from that in males (Aa). Kilauea Forest Reserve is polymorphic for three in- Whatever the proximate cause, the observed selec- versions in chromosome 4 that have remained in tion results in a narrowed reproductive class in which equilibrium for 9 yr in the laboratory. These inver- some zygotes come to represent a genetic elite. This sions exist as three haplotype sequences; these appear results in a stable equilibrium of chromosomal types to be supergenes that segregate in the manner of that expresses itself strongly under the environmental Heterokaryotypes and Fitness 42 1 conditions obtaining in the laboratory. Stable multi- material. 1 am also extremely grateful to Professor TIMOTHY ple-allele polymorphisms such as the one studied here PROUT, who not only made an extensive review of the manuscript but suggested the valuable cohort method of devising expectations. are difficult to model theoretically (CROWand KI- This work was supported by National Science Foundation grant MURA 1970). This may be invoked as another reason BSR 84-15633, to the University of Hawaii. why it seems unlikely that simple survivability of het- erozygous types can be used to mount a sufficient explanation of their maintenance. A differential mat- LITERATURE CITED ing pattern such as sexual selection may be involved ANDERSON,W. W., L. LEVINE,0. OLVERA,J. R. POWELL,M. E. DE but no theoretical construction will be attempted here. LA ROSA, V. M. SALCEDA,M. I. G~soand J. GUZMAN, Such reproductive genetic elitism may be wide- 1979 Evidence for selection by male mating success in natural spread in animal and plant populations but at the same populations of Drosophila pseudoobscura. Proc. Natl. Acad. Sci. time can remain hidden from easy view. In order to USA 76 1519-1523. ~IGER,E., 1962 Sur le degri d’heterozygositie des populations recognize a genetic basis, one needs genetic markers naturelles de Drosophila melanogaster et son maintien par la that are intrinsic to the gene pool and which cover a silection sexuelle. Bull. Biol. Fr. Belg. 96: 3-122. considerable length of chromosome; as pointed out CARSON,H. L., 1978 Speciation and Sexual Selection in Hawaiian earlier, this is a difficult assignment. Further, the Drosophila. pp. 93-107. In: Ecological Genetics: The Interface, differential reproduction of certain individuals re- Edited by P. F. BRUSSARD.Springer-Verlag, New York. CARSON,H. L., 1982 Evolution of Drosophila on the newer Ha- quires a special demographic scrutiny of the perform- waiian volcanoes. Heredity 48: 3-25. ance of the individual members of the population. CARSON,H. L., 1985 Genetic variation in a courtship-related male These two parameters, brought together in the pres- character in Drosophila silvestris from a single Hawaiian locality. ent study, have rarely been juxtaposed in intrapopu- Evolution 39 678-686. lational studies. An exception is the evidence for dif- CARSON,H. L., 1986 Sexual selection and speciation. pp. 391- 409. In: Evolutionary Processes and Theory, Edited by S. KARLIN ferential male mating success in several populations and E. NEVO.Academic Press, London. of Drosophila pseudoobscura (ANDERSONet al. 1979). CARSON,H. L. and P. J. BRYANT,1979 Change in a secondary The role of inversions in such cases as the present sexual character as evidence of incipient speciation in Drosoph- one may not be a primary effect. Thus, the relevant ila. Proc. Natl. Acad. Sci. USA 76 1929-1932. balances may preexist as a result of natural or sexual CARSON,H. L. and R. LANDE,1984 lnheritance of a secondary sexual character in Drosophila silvestris. Proc. Natl. Acad. Sci. selection, having arisen in a chromosomally mono- USA 81: 6904-6907. morphic population in the absence of inversions. In CARSON,H. L. and H. D. STALKER,1968 Polytene chromosome this view, the inversion serves as a chance chromo- relationships in Hawaiian species of Drosophila. 11. The D. somal mutation that essentially captures a balance that planitibia subgroup. Univ. Tex. Publ. 6818: 355-365. is already present and renders it less prone to destruc- CARSON,H. L. and L. T. TERAMOTO,1980 Differences in copu- latory success among laboratory males of Drosophila silvestris. tion by recombination. This idea would take some of Genetics 94: S14. the mystery out of the occurrence of multiple inver- CARSON,H. L., F. C. VAL, C. M. SIMONand J. W. ARCHIE, sions in a single chromosome, such as in D. pseudoob- 1982 Morphometric evidence for incipient speciation in Dro- scura. Almost any inversion would be perpetuated if sophila silvestris from the island of Hawaii. Evolution 36 132- it can “parasitize” a prebalanced condition. 140. CRADDOCK,E. M. and W. E. JOHNSON,1978 Multiple insemination A demonstration of this is possibly afforded by the in natural populations of Drosophila silvestris. Drosophila In- experiments of PAGET(1 954). Examining an irradi- form. Serv. 53: 138. ated population of Drosophila melanogaster, he found CRADDOCK,E. M. and W. E. JOHNSON,1979 Genetic variation in that a number of new inversions were induced and Hawaiian Drosophila. V. Chromosomal and allozymic diversity that at least one increased in frequency and apparently in Drosophila silvestris and its homosequential species. Evolution 33: 137-155. became balanced. The results of VANN(1 966) suggest CROW,J. R. and M. KIMURA,1970 An Introduction to Population a similar result. Genetics Theory. Harper & Row, New York. Heterotic effects involving inversions such as those DOBZHANSKY,TH., 1955 A review of some fundamental concepts described in this paper are widespread in nature and and problems of population genetics. Cold Spring Harbor certainly come as no surprise. What is unique in the Symp. Quant. Biol. 20 1-15. LANDE,R., 1981 Models of speciation by sexual selection on present situation, however, is that marking and mon- polygenic traits. Proc. Natl. Acad. Sci. USA 78: 3721-3725. itoring of individuals and their behavior was purely PAGET,0. E., 1954 A cytological analysis of irradiated popula- intrapopulational; the heterosis found in this case is tions. Am. Nat. 88: 105-107. closely proximate to the final reproductive act itself. SENE,F. M. and H. L. CARSON,1977 Genetic variation in Ha- waiian Drosophila. 11. Allozymic similarity between D. silvestris I am grateful for the intense hard work of Ms. LINDENTERA- and D. heteroneura from the island of Hawaii. Genetics 86 187- MOTO in caring for the specimens and preparing most of the 198. chromosome smears. Drs. TERRENCELYTTLE and DAVIDHAYMER SPIESS,E. B. and H. L. CARSON,198 1 Evidence for sexual selection gave advice on statistics. I also thank Professor HUKAIof Hainan in Drosophila silvestris of Hawaii. Proc. Natl. Acad. Sci. USA University, People’s Republic of China, for her assistance in some 78 3088-3092. of the experimental runs. Dr. A. T. OHTA collected the wild SPIETH,H., 1978 Courtship patterns and evolution of the Dro- 422 H. L. Carson sophila adiastola and planitibia species subgroups. Evolution 32 rearrangements introduced into laboratory populations of Dro- 435-451. sophila melanogaster. Am. Nat. 100 425-449. THORNHILL,R. and J. ALCOCK,1983 TheEuolution ofInsectMahng WHEELER,M. R. and F. E. CLAYTON,1965 A new Drosophila Systems. Harvard University Press, Cambridge, Mass. culture technique. Drosophila Inform. Serv. 40: 98. VANN, E. G., 1966 The fate of X-ray induced chromosomal Communicating editor: J. R. POWELL