DANE'S rule: In all of the crosses of T. castaneum males ..... WHITLOCK, CHARLES GOODNIGHT, M O W E D NOOR, ANDY CLARK and at least two ...
Copyright Q 0000 by the Genetics Society of America
Genetic Variation Segregatingin Natural Populations of Tribolium castuneum Affecting Traits Observed in Hybrids With T. fiemu& Michael J. Wade,* Norman A. Johnson: Rachel Jones,* Vera Siguel* and Michael McNaughton* *Department of Ecology and Evolution, University of Chicago, Chicago, Illinois 60637 and tUniversity of Texas at Arlington, Arlington, Texas 76019 Manuscript received April 5, 1997 Accepted for publication August 14, 1997
ABSTRACT We investigatedpatternsofwithin-speciesgeneticvariationfor traits observedinhybrids(hybrid numbers, hybrid sex ratios, and hybrid male deformities) between two species of flour beetles,Tribolium castaneum and T. Peemani. We found genetic variation segregating among four natural populations of T. castaneum as well as within these populations. For some hybrid traits,we observed as much variation among populations 750 km apart as between populations on different continents, suggesting genetic differentiation at a local scale. Within natural populations, the variation segregating among sires is greater than that found in an earlier study for an outbred laboratory population and comparable to that observed between inbred lines derived from the outbred stock by eight generations of brothersister mating. When sires from T. castaneum are mated to conspecific and heterospecific females,we do not observe a significant correlation at the level of the family mean between the intraspecific and interspecific phenotypes, suggesting the independence of the hybrid traits from comparable traits within species. We discuss our findingsin relation to the evolutionary geneticsof speciation and the expression of epistatic genetic variance in interspecific crosses
I
WADE and JOHNSON 1994; ROBINSONet
Corresponding authur: Michael J. Wade, Departmentof Ecology and Evolution, Universityof Chicago, 1101 E. 57th St., Chicago, IL 60637.
castaneum affecting several hybrid traits, including two traits, male hybrid inviability and male hybrid deformity, that conform to HALDANE’S (1922) rule. When T. castaneum males are crossed with T. freemani females, the interspecific hybrids express the following traits: (1) the sex ratio of hybrid families is significantly female biased owing, at least in part, to hybrid male inviability (WADE and JOHNSON 1994; WADE et al. 1994a); (2)male hybrids, more frequentlythanfemales, display deformed antennaeand limbs (WADE and JOHNSON 1994; WADEet al. 1994a); and (3) there is a reversal of the typical sex dimorphism for body size within the genus so that hybrid males are substantially larger than hybrid females (WADEandJOHNSON 1994). In therecip rocal cross ( T. castaneum females by T. fremani males), hybrid sex ratios do not greatly deviate from 50% females and few deformities are observed (WADE and JOHNSON 1994). Because males are the heterogametic sex in this genus, observations l and 2 (above) are expressions of HALDANE’S(1922) rule, which states that, in interspecific hybridizations, the F, hybrids of the heterogametic sex are more adversely affected than hybrids of the homogametic sex. Several different evolutionary explanations for HALDANE’Srule have been proposed (COYNE and ORR1989; FRANK1991; HURSTand POMIANKOWSKI
N speciation genetic research, it is common practice
to introgress specific regions of chromosomes from one species into another to identify and characterize genetic factors affecting interspecific hybrids, especially hybrid sterility and inviability (c$ COYNE1992; WU and PALOPOLI1994; FORJET1996; TRUEet al. 1996). These studies emphasize the fixed genetic differences between species both in method and in interpretation. In contrast, intraspecific studies in evolutionary genetics and evolutionary ecology attempt to characterize genetic variation segregating within and among naturalpopulations. Once segregating variation is identified, subsequent research focuses on the role of evolutionary forces, like mutation, natural selection, and random genetic drift, in the origin and maintenance of this variation. Ultimately, wewish to understand how the standard evolutionary forces operating within populations give riseto geneticdifferences at thespecific level (LEWONTIN 1974). Thus, the two approaches are complementary. In this article, we combine thespeciation genetic and evolutionary genetic approaches. We used four natural populations of the flourbeetle, Tribolium castaneum, and created hybrids by crossing these populations to T. freemani, a closely related congener. Adults of T. castaneum and T. Peemani mate readily in the absence of conspecific mates and hybrids are produced in large numbers
Genetics 147: 1235-1247 (November, 1997)
(NAKAKITA et al. 1981; BROWNLEEand SOKOLOFF 1988; at 1994; WADE et al. 1994a,b).We investigated patterns of genetic variation segregating within and among populations of T.
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M. J. Wade et al.
1991; JABLONKA and LAMB 1991, 1997; Wu and DAVIS 1993; TURELLI and ORR1995; WU et aZ. 1996). We expand our previous study of a laboratory population (WADE et U L 1994a) toexamine intraspecific genetic variation for the expression of HALDANE’S rule within and among four wild populations. We previously showed that there was significant autosomal genetic variation segregating within the c-SM laboratory strain of T. castaneum for factors affecting hybrid sex ratio and male hybrid deformities (WADEet aL 1994a). Furthermore, inbred lines, derived by eight generations of brother-sister mating from this same cSM population, exhibited an increase in the variance of both hybrid traits without a substantial change in the mean,suggesting that interspecific hybrid inviability and intraspecific inbreeding depression appear to have different genetic bases (WADEet aL 1994a). We report our study of four natural populations of T. castaneum, two from the United States and two from Spain. We show in this article that beetles from these populations are phenotypically identical and that crosses between the populations produce abundant,fertile, and intact hybrids when crossed inter se. We established full-sib and half-sib hybrid families using 20-25 T. castaneum sires from each population and several T. freemani dams per sire. The among-sire component of variation for hybrid family size, hybrid sex ratio, and the frequency of hybrid males with morphological a b normalities is an estimate of the genetic variation segregating within the T. castaneum populations. This amongsire variation does not reflect additive genetic variance in the standard sense. The traits in hybrids tend to be deleterious (broken antennae, limbs, and inviability), which is evidence of epistatic gene action since these genes cause abnormalities only in the heterospecific and not in the conspecific crosses (for similar interpretations see COYNE 1992;Wu and PALOPOLI 1994; ORR 1995). Furthermore, the segregating genes in T. castaneum, which interact epistatically with the fixed genetic differences in T.freemani, will appear as among-sire variation. This is the interspecific manifestation of the conversion of nonadditive variance to additive variance (GOODNIGHT 1988; WHITLOCK et al. 1993; WHITLOCK 1995). In addition to the incidence of hybrid male deformities, we also counted the numbers of missing antennal segments on the right and left sides of the head, the numbers of missing limb segments, and the sex dimorphism in hybrid dry weight.From the difference in numbers of antennal segments between the right and left sides we can compute an estimate of the degree of “fluctuating asymmetry,”which is believed to be inversely related to the degree of developmental homeostasis (LERNER1954;WADDINGTON 1957; VAN VALEN 1962; ZAKHAROV 1981; LEAMY 1984; PALMER and STROBECK 1986). Fluctuating asymmetryhas been investi-
gated in interspecific crosses as a means of testing for the disruption of coadapted gene complexes in interspecific hybrids (FELLEY 1980; GRAHAMandFELLEY 1985; LEARYet al. 1985;COYNE1985; MARKOW and F W K E R 1991). Previous studies have found fluctuating asymmetry in hybrid traits but have not reported genetic variation segregating within either of the parent species for this developmental phenomenon. Inthe discussion, we argue thatevolutionary forces within species affect this developmental genetic variation and, thus, it may be important to the origin of postzygotic isolation between the species. The genetic variation that we see in the hybrids in the present study is for traits that do not appear in crosses within or among populations of T. castaneum. In this study, we are using the interspecific hybrids to see genetic variation segregating within T. castaneum that would otherwise be hiddenby homeostasis in much the same way that WADDINGTON (1953,1957) used high temperatures in his artificial selection experiments on genetic assimilation. (see also GIBSONand HOGNESS 1996). MATERIALS AND METHODS
Biological materials T. castaneum is a human-commensual beetle found worldwide in a variety of stored products (SOKOLOFF 1974). T. fieemani shares morphological, karyotypical, and molecular genetic similaritywith T. castaneum but is nearly three times as massive (HINTON1948; SOKOLOFF 1974; NAKAKITAet al. 1981; BROWNLEE and SOKOLOFF 1988;JUAN et al. 1993; WADEand JOHNSON 1994). Crosses between these species usually yield many viable but sterile or quasi-sterile offspring of both sexes. We used four wild-caught populations of T. castaneum (US: cArkansas and c-Texas; Spain: c-Jerez and c-Campanario) in the experiments described below. The c-Arkansas population was founded from 100 adult beetles collected by ORALuW, in Little Rock, Arkansas (WADE 1990) and the c-Texaswas founded from using 70 adults collected in Austin, Texas, by ROBERTSRYGLEY. The distance between Austin and Little Rock is -750 km. Both Spanishpopulations were established from groups of >50 adults collected in Jerez (c-Jerez) and in Campanario (c-campanario), at least 750 km apart (WADE 1991). We used the standard laboratory strain of T. f i m a n i (NAKAKITA et al. 1981; BROWNLEE and SOKOLOFF 1988;WADE and JOHNSON 1994). Hybridhalf-sibbreeding design: From each of the four populations of T. castaneum, we chose at random 20-25 males (sires) and mated them individually to three to five virgin T. freemani females for 7 days. The females were then separated and each was placed in an 8dram shell vial containing 8 g standard media (WADEand GOODNIGHT 1991; WADE and JOHNSON 1994). The beetles were cultured in a darkened incubator at 29” and 50-7096 relative humidity for 7 days. After that time, females were transferred to a fresh vial for a second 7day period before being discarded. Approximately 40 days after a female was cleared from a vial, her offspring were censused and placed at 4-5” to allow for later sexing and scoring for morphological abnormalities. For the family size data (number of adult offspring), we analyzed the offspring only from mothers still living at the end of the second 7day period.
GeneticsQuantitative Hybrids Males, but not females, of the T. castamurn clade possess femoral pits on the first pair of legs (~OKOLOFF1974).We assayed sex via the presence of these pits, using a dissecting microscope. On rare occasions, when both legs of the first pair weremissing the femur, we sexed the individual by everting the genitalia. For each offspring, we counted the numbers of segments on the right and left antennae where 22 is the normal number for both parent species. We also examined each leg and recorded whether any of the major segments, tarsus, tibia, or femur, were missing or deformed. In every case of a basal deformity of either antenna or leg, all distal segments were also deformed or missing. We separated the males and females from each dam and dried them for 224 hr in an oven at 58".We then weighed the beetles of each sex as a group on a MettlerAE-163balance, to a precision of 0.01 mg. Dry weights are -60% of the live weights. Paternal correlations of intraspecific and hybrid traits: To compare traits within and between species, we mated males the c-Texas and T. of T. castamurn tovirginfemalesfrom fiemani stocks. All adults were collected and separated by sex at the pupal stage. For each of 20 T. castaneum males, we observed 2 wkof reproduction with a heterospecific female and 2 wk of reproduction with a conspecific female(20males X 2 wk per female per species X 2 species of female = 80 vials total). Each of the 20 heterospecific pairswas permitted to reproduce for 7 days in an 8dramvial with 8 g of standard medium husbanded under the conditions described above. After 1 wk, each pair was transferred to a new vial with fresh medium and permitted to reproduce for an additional 7 days. After a total of 14 daysof heterospecific reproduction, we removed the adults, separated the males from the females, and repaired each male with a conspecific female. These 20 conspecific pairs were placed in fresh medium and permitted to reproduce for 7 days. A second 7day period of reproduction was obtained with an additional transfer. With this protocol, we can compare the traits of conspecific and hybrid offspring and their correlation across sires. The genetic covariance across species can provide a formalmethod to describe how the indirect effects of selection within a species affect the evolution of traits seen in hybrids between species(JOHNSON and WADE 1996). Thirty-five days after removal of the adults, we censused each vial for l a m e and pupae. Those vialswithlarvaeremaining were recensused every 3-4 days until all larvae had matured to pupae. After an additional 3 wk, pupae were censused for adults and at least five adults of each sexwere weighed. All adults were examined with a dissecting scope for antennal and limb deformities. Becausewe found only 50:50 sex ratios and no limb or antennal deformities in the conspecific progeny, we could investigate only the correlations for the numbers and weights of conspecific and heterospecific offspring. Statistical anahpis: For the hybrid half-sib design, we conducted a general multivariate analysis ofvariance investigating the differences in trait means among continents and among states nested within continents. In this analysis, the family size data from the two 7day periods were averaged and those families that averaged fewer than seven progeny per 7day period were excluded (Table 1, see below).Thus, our discussion of trait means focuseson the larger interspecificfamilies, producing 214 progeny. For binomial traits, such as the frequency of deformed males, data from the two 7day periods were pooled. All frequency data were arcsin-square root transformed to meet the criteria of nested analysisof variance (Statistica Software, 4.0). Although we report the untrans-
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TABLE 1 Geographic variationin unproductive interspecific matings Progeny number
Total Population Texas
Sires Dams matings
All matings Sires"
0
1-6
0
1-6
0 0
0 0 1 0
80
7
2
125
7
8
3
75
5
125
32 5
9
5
13
0
Arkansas
20 25
4 5
Jerez Campanario
25 25
a Number of sires that produced no progeny or fewer than seven offspring per two "day periods with all dams.
formed means of these traits, allstatisticaltestswereconducted on the transformed data. RESULTS
Geographicvariation in unproductive interspecific matings: In Table 1, we list the proportion of unproductive matings, defined as those producingan average of less than seven progeny per 7day period. We find significant variationin theproportion of failuresamong the four populations(G test of2 X 4 contingency table, G = 56.30, 3 d.f., P Q 0.0001). This difference is owing to Jerez males being unproductive 55% of the time, more than three times greater than that of any of the other three populations. If the Jerez population is removed from the analysis, there are no significant differences in the rate of unproductive matings among the other populations(G = 0.53,2d.f., P > 0.50). Although five Jerez sires failed to produce any offspring with all three heterospecific mates, the expected number,given 75 matings and an absolute failurerate of 0.43,is 5.82. Thus, the mating failuresare not clustered in a way that suggests genetic differences among Jerez males in the tendency to produce offspring with T.Jkemni females. Geographicvariation in hybrid traits: The means over sires for eight hybrid traits are reported in Table 2 with the standard error of the mean in parentheses. I n a MANOVA, we find significant geographic variation for all traits except the mean number of limb segments lost. For three traits, family size, sex ratio, and number of antennal segments lostor deformed in males, there is as much variation among populations 750 km apart as there is between continents. The local geographic variation in sex ratio is significant even whenthe effects of family size are statistically removed by treating family size as a covariate in the analysis. T. castaneum males from different continents produce different numbers of hybrids, different hybrid sex ratios, frequencies of deformed male hybrids, and different kinds of male hybrid deformities. [Female hybrids did not exhibit morphological deformities to any sig-
M.J. Wade et al.
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TABLE 2 Ceagraphic variation between states and between continents in hybrid traits Significant differences Arkansas Texas
21.07 Family
Trait
20.66 29.01 (1.53)(0.91)(0.99) Sex ratio (% males) 0.401 0.399 0.367 0.436 (0.017) (0.024) (0.020) (0.015) males deformed Frequency of 0.4940.4790.4600.360 (0.039) Frequency ofdefects antennal 0.4510.4260.4150.320 (0.040) Frequency defect of limb 0.2310.2240.1660.143 (0.109) lost segments Antennal 6.35 7.15 7.21 7.25 (0.46) of Asymmetry losssegment 3.06 3.27 3.58 3.64 (0.27) lost segments Limb 2.47 2.46 2.51 2.42 (0.18) dry wt. (mg x 1000) 1.81 1.97 Male (0.06) 1.75 1.76 Female (0.07) size
Population Jerez
21.28
Campanario
BS"
***
***
(1.OO)
(0.032)
(0.055)
(0.030)
(0.030)
(0.050)
(0.030)
(0.025)
(0.034)
(0.021)
(0.33)
(0.38)
(0.33)
(0.17)
(0.19)
(0.13)
(0.19)
(0.15)
(0.11)
1.98 (0.02)
1.67 (0.03) 1.66 (0.30)
(0.03) 1.81 (0.02)
(0.02)
BCb
***
*c
(*)
***
(*)
***
NS
***
*
NS
NS
*
NS
NS
***
NS
***
NS
BS, between states; BC, between continents. Results of a general MANOVA contrasting states within continents. All significant differences are between traits of the Texas and Arkansas populations with the exception of antennal segments lost, whichis between thetwoSpanish cities. One, two, or three asterisks indicate P < 0.050, 0.010, and 0.005, respectively. The symbol (*) indicates borderline significance with 0.050 < P < 0.070; NS indicates P > 0.070. Results of general MANOVA with state nested within continent. Results of general MANOVA with state nested within continent and family size as a covariate.
nificant extent. Forexample, the incidence of deformed hybrid females in the Campanario population is 0.30; Figure 1). Figure 1 illustrates that the Spanish population from the city of Infantes studied earlier (WADE andJOHNSON 1994) produces, on average, 30% larger familysizes (27.0) than beetles from either Jerez and Campanario as well as hybrid sex ratios much more biased toward females (70.4% ws. an average. of 60.0%, respectively). Also shown in Figure 1 are the results from the c-BS strainfromNaperville,Illinois (WADE and JOHNSON 1994), which is less productive than either the Texas or Arkansas populations but intermediate in sex ratio. Although the pure bred beetles from thesenatural and laboratory populations of T. castaneumappear similar in
Genetics Quantitative Hybrids
I _I Ru'
IS
17
19
21
23
n
n
29
31
M a n Hybrid Family Size
FIGURE 1.-The regression of mean sex ratio on mean hybrid familysize for two laboratory (c-SM and c-pearl) and seven natural T. castamurn populations. The two points for the Jerez and Campanario populations are so similar that they are coincident to the resolution of this graph.
phenotype, they clearly differ from one another when viewed through the lens of hybridization to a standard population of T. Peemani. Genetic variation segregating within populations: We found significant among-sire variation segregating within each of our four natural populations for most of the hybrid traits studied (Tables 3 and 4). In every population, we found among-sirevariance for both traits expressingHALDANE'S rule, male hybrid inviability (reflected in the hybrid sex ratios) (Figure 2) and the
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frequency of male hybrid deformities (Figure 3). Histog r a m s of the half-sib mean hybrid sex ratios (Figure 2) reveal that the among-sirevariationinevery natural population is greater than that observed among sires for an outbred laboratory population c-SM (compare our Figure 2 with Figure 1, top, in WADEet al. 1994a). Indeed, it is as great as that observed previouslyamong c-SM inbred lineages derived independently by eight generations of sib mating (compare our Figure 2 with Figure 1, bottom, of WADE et al. 1994a). This genetic variationsegregatingwithinall T. castaneum populations is autosomal; it cannot be X-linked because none of the interspecific male hybrids receives an Xchromosome from its T. castaneum sire. In both U.S. populations but in neither Spanish p o p ulation, there was significant among-sire variation in the degree of asymmetry of antennal loss (Table 3 and 4). Thus, there is genetic variance for the degree of fluctuating asymmetry ofthe hybrids segregating within some populations of T. castaneum. Correlations among half-sib family means for hybrid traits: The correlations among half-sibfamilymeans are reported in Tables 5 and 6 for all populations.We are not using these hybriddata to estimate genetic correlations for purposes like an animal breeder: the hybrid population cannot be selectedon since the hybrids are all sterile. Instead, we use the correlation of the half-sib family means across sires to that showthe hybrid
TABLE 3 Among-sire variation for hybrid traits of U.S.
Trait Texas Family size Sex ratiob Frequency of deformed males Frequency of antennal defects Frequency of limb defects Asymmetry of antennal loss Antennal segments lost Limb segments lost dry wt. (mg X 1000) Male Female Arkansas Family size sex ratio" Frequency of deformed males Frequency of antennal defects Frequency of limb defects Asymmetry of antennal loss Antennal segments lost Limb segments lost DryWt. (mg X 1000) Male Female
Populations
SS error
d.f."
MS error
1807.3 0.590 1.761 1.716 1.613 44.51 148.94 58.41
19, 51 19, 51 19, 51 19, 51 19, 51 19, 45 19, 45 19, 40
35.44 0.012 0.035 0.034 0.032 0.989 3.310 1.460
1.88 1.76 4.99 6.65 2.34 2.31 4.17 1.17
0.0385 0.0552 0.0000 0.0000 0.0083 0.0110 0.0000 0.3300
19, 51 19, 51
0.035 0.018
7.41 17.88
0.0000 0.0000
3330.9 0.960 6.383 7.264 4.552 123.12 11.23 2.190
24, 85 24, 85 24,84 24, 85 24, 85 24, 79 24,80 23,62
39.19 0.011 0.076 0.086 0.054 1.559 5.517 1.584
2.25 4.87 2.03 1.92 3.44 1.68 2.04 1.38
0.0035 0.0000 0.0097 0.0153 0.0000 0.0460 0.0098 0.1571
0.889 1.172
24, 80 24, 81
0.011 0.015
5.67 3.00
0.0000 0.0001
1.76 0.90
F
P
M. J. Wade et al.
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TABLE 4 Amongsire variation for hybrid traits of Spanish populations
Trait
SS error
d.f."
MS error
F
P
Jerez Family size Sex ratio'
deformed Frequency of defects antennal Frequency of defects limb Frequency of Asymmetry of loss antennal lost lost Dry Wt. (mg X 1000) Male
segments Antennal segmentsLimb
Female Campanairo Family size Sex ratio" males deformedof Frequency Frequency ofdefect antennal Frequency defects of limb Asymmetry of loss antennal lost segments Antennal segments Limb lost Dry wt. (mg X 1000) Male
Female
males
396.33 0.097 0.603 0.797 0.303 22.06 55.19 11.79
19, 14 19,14 19, 14 19, 14 19, 14 19, 14 19, 14 19, 13
28.31 0.007 0.043 0.057 0.022 1.576 3.942 0.73 0.907
2.22 2.72 3.06 1.97 4.04 0.76 0.85
0.0668 0.0309 0.0190 0.1007 0.0053 0.7110 0.6313 0.7308
0.11 0.10
19, 14 19, 14
0.008 0.007
4.43 4.57
0.0034 0.0029
24,84 24,84 24,81 24,81 24,81 24,80 2480 24,68
44.42 0.013 0.098 0.098 0.066 1.168 4.302 1.703
2.57 2.94 1.74 1.61 1.37 1.37 2.36 0.62
0.0008 0.0001 0.0348 0.0585 0.1488 0.1498 0.0023 0.9061
24,83 24, 82
0.047 0.017
1.92 2.26
0.0158 0.0034
3731.1 1.081 7.930 7.936 5.352 93.42 344.19 115.83 3.90 1.41
traits covary and to test the significance of the correlations and the heterogeneity among them. This is different from estimatinga genetic varianceor genetic covariance component for use in formal theory or applied breeding. (Because ofthe large number of correlations, we report significance at the more conservative 0.025 level or less as is indicated in bold in Tables 5 and 6.) Despite the significant variation among half-sib family means forboth sex ratio (Tables 3 and 4 and Figure 2) and family size (Tables 3 and 4), these two traits are not correlated significantly within any population (cf: Tables 5 and 6 and Figure 4). In contrast, in every population, there is a large negative correlation across sires between the frequency ofmaleswith deformed antennae and the degree of sex ratio bias (cf: Tables 5 and 6 and Figures 3 and 5). Furthermore, the correlation is heterogeneous across populations, ranging from -0.35 to -0.73 (test of heterogeneity, x ' = 9.09, d.f. = 3, P < 0.028), with a pooledestimate of -0.524 (inverse ofaverage z = -0.581). The frequencies of antennal and limb deformitiesare highly positively correlated in every population and homogeneous across populations ($ Tables 5 and 6). The mean number of limb segments lost varies neither between populations nor among sires within any population. It is, however, correlated with other traits and these correlations are also heterogeneous among populations (Tables 5 and 6). The two U.S. populations
are heterogeneous in the correlation between the mean number of limb segments lostand the following traits: family size( P = 0.016), sex ratio ( P= 0.031), frequency of limb deformities ( P = 0.028), and mean number of antennal segments lost ( P = 0.057). The only other traits exhibitingheterogeneous among-sire correlations are male and female dry weight, which are highly correlated in the Texas population but onlyweakly correlated in the Arkansas population. The only heterogeneous correlation in the Spanish populations is also between the mean number of limb segments lost and the frequency of limb segments lost ( P = 0.026). The correlations are significantly heterogeneous among all four populations for these two traits (x' = 23.43, d.f. = 3, P < 0.0001; Figure 6). The matrix correlation between the two U.S. populations is 0.62 ( n = 36) and between the Spanish populations it is 0.71 ( n = 36). Although both are significantly different from zero, it is more important to note that the average withincontinent matrix correlation is significantly different from one (SOW and ROHLF1981, p. 779; combined probability test, x ' = 12.045, d.f. = 4, P = 0.017). This indicatesthat local populations differ in the genetic relationships among hybrid traits. The average of the four matrix correlations between pairs of populations on different continents is very similar to that betweenpopulationswithin continents, 0.690. This average correlation isalso different from
GeneticsQuantitative 8,
o
of Hybrids
1241
I
0.05
0.1
0.15
0.2
11.25
0.3
(1.35
0.4
11.45
11.5
11.55 0.6
Halfsib Mean Hybrid Sex Ratio
Halfsib Mean Frequency o f Hybrid Male Deformities
'I Campanarlo
I
I
€!Si J e w
0.05
0.1
I
0.15
0.2
0.25
11.5
11.35
0.4
11.45
0.5
0.5?
0.6
Halfsib Mean Hybrid Sex Ratio
o
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Halfsib Mean Frequency o f Hybrid Male Deformities
FIGURE2.-Histograms illustrating the among-sire variation in the hybrid sex ratio for the two U.S. (top) and two Spanish (bottom) populations.
FIGURE3.-Histograms illustrating the among-sire variation in the frequency of hybrid maleantennal deformities for the two U.S. (top) and two Spanish (bottom) populations.
one (combined probability test, x* = 23.413, d.f. = 8, P = 0.0029), indicating heterogeneityof the correlation matrix among populations on different continents. Paternal correlations of conspecific and hybrid traits: One quarterof the heterospecific crosses (5/20) between c-Texas males and T.fremuni females failed to reproduce. In contrast,none of the c-Texas conspecific crosses and only two of the conspecific T. fremani crosses failed. That is, all five c-Texas males that failed to reproducewith a T.frmnani female were fertile when subsequently paired with a conspecific female. The mean productivity of the heterospecific cross was 16.5 (SE = 2.85) in the first week and 17.2 (SE = 3.00) in the second week while the conspecific crosses averaged 55.6 (SE = 5.58) offspring in the third week and 42.0 (SE = 4.93) in the fourth. The correlation in hybrid productivity between weeks 1 and 2 was high and positive, +0.81 ( P < O.OOOl), as was the correlation in conspecific productivity between weeks 3 and 4, +0.84 ( P < 0.0001). However, the correlationbetween heterospecific and conspecific offspring numbers between weeks 2 and 3 was negative, -0.19, but not significantly so ( P = 0.48). For the heterospecific offspring, the family sex ratio was female biased inboth weeks 1 and 2, withan average frequency of hybrid males of 0.404 (SE = 0.030) and
0.364 (SE = 0.037), respectively. In contrast, for the conspecific offspring, family sex ratio did not differ significantly from 0.50in either weeks 3 (0.480, SE = 0.013) or 4 (0.493, SE = 0.035). Furthermore, thecorrelation across sires between heterospecific sex ratio in week 2 and conspecific sex ratio in week 3 was essentially zero (-0.01). Although 27.3% of the hybrid sons exhibited antennal or limb deformities, none of the hundreds of conspecific offspring did. Within c-Texas families, the correlation in mean weight of sons and daughters across sires was significantly positive, 0.412 ( P < 0.04), whereas the average cross correlation between conspecific and heterospecific offspringwas much lower, 0.160, and not significant [mean of four cross correlations: ( 1 ) conspecific and heterospecific sons, 0.166; (2) conspecific sons and heterospecific daughters, 0.093; (3) conspecific daughters and heterospecific sons, 0.170; and, (4) conspecific and heterospecific daughters, 0.2121. DISCUSSION
The nature of the hybrid traits: In this study and in previous studies (M'ADE andJOHNSON 1994; WADE et al. 1994), we observe female-biased hybrid sex ratios and morphological deformities in the male hybrids between
M. J. Wade
1242
et
al.
TABLE 5 Halfkb meau correlations between hybrid traits in the A m r and the Texas populations Half-sib mean correlations Hybrid traits [ 11 Family size [2] Sex ratio [3] Frequency of antennal defects [4] Frequency of limb deformaties [5] Asymmetrical of antennal loss [6] Antennal segments lost [7] Limb segments lost [8] Male wt. [9] Female wt
1 -0.19 -0.15 +0.15 +0.05 +0.08 +0.30
-0.15 1 -0.73 -0.72 +0.28 -0.68
-0.35 +0.49 +0.02 +0.41 -0.18
-0.05
-0.02
-0.64
-0.47 +0.53
1 -0.34
1 -0.33
+0.57
+0.65
+0.74
+0.23 +0.62 -0.14 -0.17 +0.01 -0.06
+0.16 -0.01 +0.36 -0.60
-0.47"
+0.35 -0.28 t-0.74 -0.14 -0.46 +0.34 -0.02 1 -0.17 -0.04 1 +0.06 -0.05 1 -0.20 +0.53 +0.04 -0.05 -0.09 -0.16 -0.03 -0.06
+0.28 +0.37 -0.30 -0.26 +0.01
+0.13 -0.54 +0.36 +0.36 -0.42 -0.47 +0.17 +0.01 +0.09 1 +0.18 +0.93 1
Significant correlations ( P < 0.025) in bold type. For the Arkansas and Texas populations, sample sizes are 24 and 20, respectively, so that the levels of significance are as follows: (1) P < 0.025 for (0.46, 0.50); (2) P < 0.010 for (0.52, 0.56); (3) P < 0.005 for (0.55, 0.60); and (4) P < 0.001 for (0.63, 0.68). Correlations in the Arkansas and Texas populations are above and below the diagonal, respectively.
T. castaneum males and T. jivemani females. As we have shown above, thesetraits are not evident in conspecific crosses ofT. castamurn and thus are clearly hybrid traits. The hybrid phenotypes are not due to the endosymbiont Wolbachia, as no known populations (including the ones usedinthisstudy)of T. castaneum harbor this microbe (CHANGand WADE 1996) and the hybrid phenotypes do not appear altered upon antibiotic treatment (WADEandJoHNsoN 1994). The hybrid traits are probably not due totransposableelements: we find moresevereexpression of thesehybridtraits upon exposuretohigh temperature (N. A. JOHNSON, Y. TOQUENACA and M. J. WADE,unpublished results) whereas the hybrid dysgenesis causedby the Woot transposable element is mostsevere at low temperatures (THOMSON et al. 1995; BEEMAN et al. 1996). Among-population variation for expression of HAL DANE'S rule: In all of the crosses of T. castaneum males
to T. freemani females, the hybrid progeny have a female-biased sex ratio but the degree of female bias differs significantly among populations on a local scale (c.& TABLES 1 and 2; see alsoWADEand JOHNSON 1994; WADEet al. 1994a). Populationsexhibiting the most female-biased sex ratios also have the highest incidence of morphological abnormalities inthe surviving hybrid males(Figure 1). However, the correlation issmall, -0.157, suggesting that these two hybrid male traits do not share a similar genetic basis. Populations of T. castaneum differ in hybrid phenotypes on alocalgeographicscale.Forexample, the two southcentral U.S. populations differmore from one another in hybrid sex ratios than they do from the cJerez population on a differentcontinent (Figure 1 and Table 2). The within-population genetic correlations betweenhybridtraits are alsosignificantlyheterogeneous among populations, especiallythoseinvolving
TABLE 6
The half-sib mean correlations betweenhybrid traits in the Campanario and Jerez p ~ p u l a t i ~ ~ ~~~~~~~~
Half-sib mean correlations [91 [l] [2] [3] [4] [5] [6]
[81traits [71 [61 Hybrid
[51
[41 [I1 [31
Familysize Sex ratio Frequency of antennal deform Frequency of limb deformaties Asymmetry of antennal loss Antennal segments lost [7] Limb segments lost [8] Male wt. [9] Female wt.
1 -0.09 -0.26 -0.10 -0.11 -0.34 -0.07
[21 +0.15 1 -0.57" -0.63
-0.21 -0.04 -0.44 +0.66 +0.09 +0.42 -0.47
+0.18 -0.12 +0.73 -0.25 -0.17 +0.70 1 1 -0.18 -0.24 +0.52 +0.46 -0.52 +0.50 +0.74 -0.17 -0.50 -0.38 +0.06 -0.11 +0.36 0.00 -0.26 -0.35 1
-0.18 -0.33
-0.17 -0.39
-0.30 -0.05 +0.76 +0.17 +0.57 +0.15 -0.05 -0.13 1 +0.43 1 +0.52 -0.39 -0.21 -0.30 +0.28
+0.16 +0.16 -0.18 -0.26 -0.05
+0.10 -0.05
+0.15 -0.12 -0.06 +0.32 +0.18
-0.19 -0.19 1 +0.45 +0.38 1
Correlations in the Arkansas and Texas populations are above and below the diagonal, respectively. a Significant correlations ( P < 0.025) in bold type. For the Campanario and Jerez populations, sample sizes are 25 and 17, respectively, so that the levels of significance are as follows: (1) P < 0.025 for (0.45, 0.54); (2) P < 0.010 for (0.51, 0.61); (3) P < 0.005 for (0.54, 0.65); and (4) P < 0.001 for (0.62, 0.73).
1243 Genetics Quantitative Hybrids
of a6
0.7 0.6
0.1
I
Toxn Popuhtlon
a2 I
i
i
e 1
0'
5
a
10
B.ILib M
So
25
20
1s
35
40
m Family SLze
a7 I
I
0.5
.
0.4
8 '
I
s
1
0
u
m
s
~
I o
SI a
0.3
absence of a half-sib mean correlationbetween family size and sex ratio for the two U.S. populations (top: 0,Texas; 0,Arkansas) and the two Spanish populations (bottom: 0, Campanario; W, Jerez).
0
0.1
.
. rn
oo 0
0.2
"mnh4lsh.
FIGURE 4.-The
0
Carnp.rmri0 PopukUon
1
I
1
c
00
0.1
0.1
0.3
a4
as
0.6
0.7
Edfslb Mean Frequeacy of Antccuul Momttla FIGURE
5.-The half-sib
mean correlation between the fre-
quency of hybrid maleantennal deformities and sex ratio for male limb deformities (Tables 5 and 6). The overall the two U.S. populations (top: 0, Texas; 0, Arkansas) and similarity of the genetic correlation matrices, estimated the two Spanishpopulations(bottom: 0, Carmpanario; W, as the correlation among matrix elements, were signifi- Jerez). In each population, the fewer the number of hybrid cantly greater than zero but also significantly lessthan males, the greater the likelihood that the existinghybrid males will be deformed. one between populations on the same continent. This also indicates localgeographic differentiation of populations. population genetic variation in fitness has been found Within-population variation for expression of HAL in previous studies of both the c-Arkansas and cCampaDANE'S rule: We found significant genetic variationfor nario populations (WADE1991). two hybrid traits related to HALDANE'S rule within wildAveraged across all populations, the correlation becaught populations of T. cmtaneum.WADEet al. (1994a), tween hybrid sexratio and male hybrid deformitieswas using a similarbreeding design with anoutbred labora-0.524, although it varied significantlyamong populatory strain (c-SM),also found significantvariation tions (see above). WADEet al. (1994a) observed a negaamong sires for both traits. Upon inbreeding the ctive correlation (-0.284, correlation among half-sib SM strain, the variation among lineages for both traits means)betweenthesesame two hybridtraitsin the markedly increased,but the mean values werenot sigc-SM strain and across the means ofthe inbred lineages nificantly altered (Figure 1 of WADEet al. 1994a). The derived from it (-0.289). among-sire variation observed in our four wildcaught We observed significant correlations across sires for populations is greater than that found in the laboratory a number of other hybrid traitsas well, including correstrain and comparable to that observed among the lations betweenthe frequency of antennal segment loss highly inbred lineagesderived from it (WADEet al. and the number or severity of the losses and between 1994a).Because some genetic drift has occurred during loss ofantennal and limb segments.The day weights of the maintenance of these populations in the laboratory, male and female hybrids tended to be positively correour measurementsprobably underestimate the true lated with one another and withfamilysize, so that amount of genetic variation capable of affecting hybrids larger families alsohad larger individuals of both sexes. in natural populations. Largeamounts of heritable variMale hybrid weight was weakly but consistently negaation for competitive abilityand sensitivity to temperatively correlated with the frequency of antennal deforture and humidity have been reported in earlier studies mities and with the severity ofantennal and limb deforof the c-Arkansas population (WADE 1990) and withinmities. However, onlytwo of these 12 correlations were
M. J. Wade
1244
et
al.
PALMER and STROBECK 1986). In the interspecific hybrids, this developmental homeostasis is apparently lost or diminished.Fluctuatingasymmetry,owingtoa breakdowninhomeostasis,has been frequently o h served in interspecific hybrids in other taxa (FELLEY 1980; GRAHAMand FELLEY1985;COYNE1985; LEARY et al. 1985; MARKOW and RICKER 1991). Because, by definition, there is no additivegeneticvariation for fluctuating asymmetry, these data provide further evidence of epistatic gene action affectingthe hybrid traits. On a finer genetic scale,we found evidence of genes E8 O O 0.1 03 0.3 a4 0.5 0.6 1 segregating within T. castaneum that cause variations in W i b M m Fraqprq ofDeformedLimbs the degree of developmental stability of the interspecifichybrids. We observed fluctuating asymmetryof e male hybrid antennal deformities among populations on different continents (Table 2) and among-sires within both U.S. populations (Tables 3 and 4). Correlations between conspecificand heterospecific traits Productivity,sex ratio, maledeformities, and adult dry weights exhibit no correlations between conspecific and heterospecific half-sib families (ie., across sires mated to females of both species). Productivity between weeks, within either type of cross, has significant positive correlations but not between crosses. E OO 0.1 0.2 0.3 a4 0.5 0.6 A negative correlation between heterospecific and W i b Meam Freqoanq of Deformed Limba conspecific productivities would be indicativeof adap FIGURE 6.-The half-sib mean correlation between thefretive divergence leading to hybrid incompatibility(JOHNquency of hybrid male limb deformities and severity of the SON and WADE1996).We found a weak nonsignificant, limb deformities for thetwo U.S. populations (top:0,Texas; but negative, correlation between conspecific and het0 , Arkansas) and the two Spanish populations (bottom: 0, erospecific productivities. Given the relatively few ( n = Campanario; a,Jerez) In the Texas and Jerez populations, the most severely deformed hybrid males occur in those hy20) sires used in this experiment, our power to detect brid families with the greatest proportion of limb-defomed correlations was limited. Further research is warranted. males. With respect to dry weights,the correlation in weight between conspecificbrothers and sisters is almost three times greater than the averagecross correlation besignificant. Our results suggestthat larger males are less tween conspecific and heterospecific half-sibs. Allikely to be deformed than smaller males but further though adult weight isa highlyheritable and apparently study of this is warranted. additive trait within T. castaneum (c.f review in WADE et In both WADE et ul. (1994a)and the present study, the al. 1996), the genes affecting weight segregating within variation segregating within populations of T. castaneum this speciesare expressed differentlyor not at all in the must be autosomal because the sires ( i e . , males) do not heterospecific background. transmit their X chromosomes to their sons. Hence, Our findings in relation to the genetics of speciaour results showthat autosomal variation can be respontion: In the neo-Darwinian viewof speciation, reprosible for large differences in the expression of HAL ductive barriers between geographically separated pop DANE’S rule. ulations are believed to arise from the accumulation of Fluctuating asymmeq The segregatinggenes we myriad small adaptive changes over time(DOBZHANSKY detected within T, castaneum are not associatedwith 1937; MULLER 1942; MAYR 1963; TEMPLETON 1981; phenotypicdifferencesbetween the sires that carry COYNE 1992; PALOPOLI and Wu 1994;Wu and PALOPOLI them or among their conspecificprogeny. Further1994). more, the paternal correlationsbetweenconspecific Unfortunately the relationship between the genetic and heterospecific traits (for those traits that do vary basis of reproductive isolationand origin of the genetic among sires within species) are near zero. The convariation between speciesfor those phenotypesthat are stancy of the phenotype in the conspecific background “the real stuff of evolution” (LEWONTIN 1974, p. 19) despite pervasive among-sire variation in the hybrids has been difficult to unravel, especially when only fixed suggests intraspecific homeostasis of the developmental effectsofthese genes (LERNER 1954;WADDINGTON differences are investigated. Not all fixed differences between species are involved in reproductive isolation 1957; VAN VALEN1962; ZAKHAROV 1981; LEAMY1984;
I
i
0
GeneticsQuantitative and some of those that currently affect interspecific fertility may have accumulated after isolation was established. One important difference between postzygotic reproductive isolation and other species differences is that the genes involvedin postzygotic reproductive isolation (hybrid sterility/inviability)do not cause manifest problems in the genetic background of their own species but only in the hybrid background (Wuand PALOPOLI 1994; ORR1995). The converse can also be true: for example, the alleles responsiblefor inbreeding depression withinT. castuneunaappear not to be those involved in either aspect of HALDANE’S rule shown by hybrids with T. fieemani (WADE et al. 1994a). Thus, as was pointed out by DOBZHANSKY (1937) and MULLER (1942), epistasis between heterospecific loci must be involved in hybridsterility and inviability(seealso COYNE1992 and Wu and PALOPOLI 1994 for recent reviews). Because the expression of such “speciation genes” is believed to be background specific, it is difficult to establish a correlation between a trait deleterious in hybrids and any conspecific phenotype. According to theory recently developed by ORR(1995), derived alleles should cause hybrid problems far more often than ancestral alleles, making within-species genetic variation even more difficult to analyze. To the extent that this scenario is accurate, it potentially hinders our ability to understand the possible adaptivefunction of these genes within species and the evolutionary processesthat affect them. An important initial step for determining the adaptive function of speciationgenes in the conspecific background is the characterization of intraspecific genetic variation for hybrid traits. If variation of this kind is segregating within species (aswe have shown in this case), then the evolutionary forces currently affecting it should be relevant to the origin of species differences. The parameter r in the model of JoHNsoN and WADE (1996) explicitlymodels the effectofanalleleina hybrid genetic background in terms of its selective effect in a conspecific background (see also ORR1995). Unfortunately, few studies of the genetic bases ofreproductive isolationor interspecific differenceshave examined genetic variation within species for traits expressed upon hybridization withother species (INOUE and KITAGAWA 1990; WADE and JOHNSON 1994; WADE et al. 1994a). None have examined the correlation across heterospecific and conspecific backgrounds. When visiblemutants have been studied in both conspecific and hybrid genetic backgrounds, they tend to act similarlyin both (e.&, BROWNLEEand SOKOLOFF 1988; SPICER 1991; COYNE 1992; PALOPOLI and Wu 1994). Assuming purely additive gene action, allelic effects will change with gene frequency but the sign of the effects (positive or negative) will remain the same
of Hybrids
1245
irrespectiveofgeneticbackground (LANDE 1981). Thus, whenthe genetic variationis additive and individual sires are mated to conspecific and heterospecific dams, the sign ofthe among-sire variance within species should be the same as that observed among the hybrid half-sib progenies, because the genetic background is irrelevant. Furthermore, the genetic variancein the hybrid progeny should equal the genetic correlation between the intraspecific and interspecificphenotypes and, hence, always bepositive UOHNSON and WADE 1996). Deviations from this expectation are evidence of nonadditive gene action. With strictly additive gene effects within species, it is difficult to see how the negative genetic correlation between intraspecificand interspecific phenotypes, necessary for speciation by adap tive divergence, could arise. Without epistasis, the additive effect of a gene cannot change sign from positive withinspeciestonegativebetweenspecies (WADE 1992). Our observation of among-sire variation in T. castaneum for traits of interspecific hybridsis consistent with the predictions of theoretical modelsthat examine the relationship between epistatic genetic varianceand genetic variation segregating within and among populations (GOODNIGHT1987, 1988; COCKERHAM and TACHIDA 1988; WADE 1992; WHITLOCK et al. 1993; WHITLOCK 1995). The amounts ofadditive and epistatic variance within a populationchange as allelic frequencies change and fixation occurs owing to random genetic drift and natural selection. In particular, the epistatic varianceis “converted” (sensu GOODNIGHT 1988) into additive genetic variance (WADE 1992; WHITLOCK et al. 1993). We wouldexpecttoobservethiseffect as variance among half-sib hybrid progenies whenever epistatically acting genes segregating within one species are expressed on the interspecific background of another. Because ofthe fixed genetic differences between two species, epistatic variance within one species will be manifest as among-sire variance in half-sib hybrid families. Thus, our experimental method of establishing half-sib hybrid families is exceptionally well suited for detecting the genetic variation segregating within one species that interacts with the fixed genetic difference between itselfand a congener. These genes might be especially relevant understanding to the evolutionary genetics of speciation. The sample sizes that we used in creating our halfsib hybrid progenies, 20-25 sires, are relatively small for quantitative genetic studiesof traits within species. Only when heritabilities are exceptionally high should we be able to detect significant sires effects with only two dozen sires. With more typical heritabilities, “many dozens or scores of sires” are recommended (ARNOLD 1994, p. 33-34). The fact that we can observe significant sire effects in the half-sib hybrid analysis with relatively modest degrees of freedom indicates that many genes
M. J. Wacde et al.
1246
Hybrid Background
FORJET,J., 1996 Hybrid sterility in the mouse. Trends Genet. 1 3
sire Genotypes
Backgound
P1 Pz P3
P
P 4
Ps PC RGURE 7.-A schematic illustrationof the differential phenotypic (P)expression of genes ( G )on conspecificand hybrid backgrounds. The genotypic differences among conspecifics are masked in a unitaryphenotype owing to canalization and homeostasis during development. In contrast, these same genotypic differences are revealed in the hybrid background.
still segregating within species interact epistatically with the fixed gene differences between species. In other words, the lens of hybridization magnifies segregating genetic differences within species (see Figure7). We thank ORALEE L u w and NANCY C W G for assistance in the MICHAEL laboratory. We also thank MIKE PALOPOLI, CHUNGI WU, WHITLOCK, CHARLES GOODNIGHT, M O W E D NOOR,ANDY CLARK and at least two anonymous reviewers for comments during this work. This research was supported by National Institutes of Health grant GM-22523to M.J.W.
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