Bicyclus Anynana

Genet. Res., Camb. (2001), 77, pp. 167–181. With 5 ﬁgures. Printed in the United Kingdom # 2001 Cambridge University Press 167

Eﬀects of bottlenecks on quantitative genetic variation in the butterﬂy Bicyclus anynana

" $ # " ILIK J. SACCHERI , *, RICHARD A. NICHOLS  PAUL M. BRAKEFIELD " Research Group in Eolutionary Biology, Institute of Eolutionary and Ecological Sciences, Uniersity of Leiden, Kaiserstraat 63, PO Box 9516, 2300 RA Leiden, The Netherlands # School of Biological Sciences, Queen Mary & Westﬁeld College, Uniersity of London, Mile End Road, London E1 4NS, UK $ Conseration Genetics Group, Institute of Zoology, The Zoological Society of London, Regent’s Park, London NW1 4RY, UK (Receied 19Noember 1997 and in reised form 7 August 2000)

Summary The effects of a single population bottleneck of differing severity on heritability and additive genetic variance was investigated experimentally using a butterfly. An outbred laboratory stock was used to found replicate lines with one pair, three pairs and 10 pairs of adults, as well as control lines with approximately 75 effective pairs. Heritability and additive genetic variance of eight wing pattern characters and wing size were estimated using parent–offspring covariances in the base population and in all daughter lines. Individual morphological characters and principal components of the nine characters showed a consistent pattern of treatment effects in which average heritability and additive genetic variance was lower in one pair and three pair lines than in 10 pair and control lines. Observed losses in heritability and additive genetic variance were significantly greater than predicted by the neutral additive model when calculated with coefficients of inbreeding estimated from demographic parameters alone. However, use of molecular markers revealed substantially more inbreeding, generated by increased variance in family size and background selection. Conservative interpretation of a statistical analysis incorporating this previously undetected inbreeding led to the conclusion that the response to inbreeding of the morphological traits studied showed no significant departure from the neutral additive model. This result is consistent with the evidence for minimal directional dominance for these traits. In contrast, egg hatching rate in the same experimental lines showed strong inbreeding depression, increased phenotypic variance and rapid response to selection, highly indicative of an increase in additive genetic variance due to dominance variance conversion.

Mackay, 1996). In reality, many polygenic characters 1. Introduction deviate from additivity to some degree (reviewed in The role of population bottlenecks in adaptive Moreno, 1994). In light of this, several theoretical evolution rests crucially on how genetic drift and studies have found that, under certain model assump- inbreeding aﬀect heritable variation of quantitative tions, inbreeding can lead to a net increase (or characters (Lande, 1980). In a purely additive model, ! 1\2Ne reduction) in VA through the ‘conversion’ of with no selection, a bottleneck of Ne individuals non-additive genetic variance. This non-additive vari- reduces additive genetic variance VA by a factor of ance was hitherto unavailable to selection because it 1\2Ne, lowering the ability of the population to was in the form of dominance variance (Robertson, respond to selection (Wright, 1951; Falconer & 1952; Willis & Orr, 1993), epistatic variance (Good- night, 1987, 1988; Cheverud & Routman, 1996; * Corresponding author. Present address: Population and Evol- Lo! pez-Fanjul et al., 1999) or both (Tachida & utionary Biology Research Group, School of Biological Sciences, Cockerham, 1989; Whitlock et al., 1993). However, University of Liverpool, Nicholson Building, Brownlow Street, the validity of these models for a given trait is highly Liverpool L69 3GS, UK. Tel: j44 (0)151 7945116. Fax: j44 (0)151 7945094. e-mail: saccheri!liverpool.ac.uk dependent on its underlying genetic architecture (i.e.

the relative magnitudes of additive, dominance and for by variation in dominance variance for the traits epistatic variances, as well as the distribution of allelic under study (Willis & Orr, 1993). Increases in VA are effects), which is, in general, unknown (Barton & usually associated with characters showing inbreeding Turelli, 1989). Moreover, it is difficult to predict depression (Bryant et al., 1986; Lo! pez-Fanjul & whether selection will act unconditionally against the Villaverde, 1989; Garcı!a et al., 1994; Ferna! ndez et al., novel genotypes produced by inbreeding, such that 1995; Ruano et al., 1996; Wade et al., 1996), implying any increase in VA is counterbalanced by a reduction that dominance variance (as opposed to epistatic in mean fitness, hence compromising its adaptive variance) is the major contributor to the increase. In value. contrast, studies which conform to the neutral additive The endurance of the debate on the likelihood of model measured either VA of bristle number in bottleneck-induced increases in VA (e.g. Coyne et al., Drosophila (Da Silva, 1961; James, 1971; Frankham, 2000; Goodnight & Wade, 2000) stems from the 1980; Franklin, 1980), heritability of pupal weight in fundamental implications of such effects for founder T. castaneum (Wade et al., 1996) or VA of wing size event speciation (e.g. Barton & Charlesworth, 1984; and shape in D. melanogaster (Whitlock & Fowler, Carson & Templeton, 1984; Barton, 1989; Carson, 1999). These characters show minimal inbreeding 1990; Templeton, 1996), Wright’s shifting balance depression (e.g. Latter & Robertson, 1962; Kidwell & (e.g. Wright, 1977; Whitlock, 1995) and the evol- Kidwell, 1966; Goodwill, 1975), indicating no sub- utionary consequences of metapopulation dynamics stantial directional dominance for these characters. (Lande, 1992; Harrison & Hastings, 1996). A fuller A notable exception to this trend is the absence of understanding of the effect of drift on polygenic any significant reduction in VA for adult body weight variation is also of applied importance in the in mice inbred to an average coefficient of inbreeding conservation of biodiversity (Frankham, 1999). of 0n39 (Cheverud et al., 1999). Given the minimal The debate has been fuelled by the results of inbreeding depression for this trait, direct evidence for laboratory experiments: some demonstrate close epistasis (from QTL data) and high level of ex- concordance to the neutral additive model, while perimental replication this is the most convincing others have found significant deviations from this example to date of inbreeding-induced epistatic to classical model. The most influential of these empirical additive variance conversion. Loss of VA may also be studies was that of Bryant et al.(1986), followed up in retarded by selection against homozygotes in com- Bryant & Meffert (1993), showing that VA for some bination with linkage disequilibrium (see Latter et al., morphological characters of Musca domestica can 1995), as reported by Tantawy & Reeve (1956) who increase substantially after bottlenecks. Bottleneck- found that VA for wing size in D. melanogaster induced increases in VA have also been shown for showed no significant decline up to 50–60% in- sternopleural bristle number (Lints & Bourgois, 1984), breeding and remained higher than expected up to viability (Lo! pez-Fanjul & Villaverde, 1989; Garcı!a et nearly 90% inbreeding. al., 1994) and courtship song (Ritchie & Kyriacou, The present experiment was conducted to inves- 1994) in Drosophila melanogaster; and for viability tigate the effects of single generation bottlenecks (Ferna! ndez et al., 1995) and fitness (Ruano et al., (founder events) of different size on quantitative 1996; Wade et al., 1996) in Tribolium castaneum.In genetic variation of morphological characters – wing contrast, some earlier experiments (Da Silva, 1961; size and pattern – in the satyrine butterfly Bicyclus James, 1971; Frankham, 1980; Franklin, 1980) found anynana (Butler). This butterfly has a wing pattern that bottlenecked lines of Drosophila exhibited a which is both highly variable and easily measured. reduced short-term response to artificial selection on Moreover, the wing pattern is known to be under sternopleural or abdominal bristle number. Moreover, strong visual selection in relation to attacks by for a bottleneck of one breeding pair, the magnitude vertebrate predators (Brakefield, 1997). Wing pattern of the response was close to the expected three diversity is also a feature of the speciose genus quarters of the response of non-bottlenecked lines, in Bicyclus, consisting of about 80 species (Condamin, all four experiments. More recently, Wade et al. 1973). In nature, B. anynana populations are probably (1996) found that the reduction in the realized more stable and also more spatially substructured heritability of pupal weight in differentially inbred (Brakefield & Reitsma, 1991) than Musca or Droso- lines of T. castaneum was of the order predicted by the phila populations, which are typically very large, yet neutral additive model. Similarly, Whitlock & Fowler prone to crashes, partly as a result of more ephemeral (1999), on the basis of a highly replicated isofemale food resources and more rapid turnover of generations treatment, concluded that the change in VA for D. (e.g. Sevenster & van Alphen, 1993). The influence of melanogaster wing size and shape was consistent with demographic history and ecology on genetic archi- neutral additive expectations. tecture is complex (Roff, 2000), but is likely to have In most of the cases presented above the observed implications for the response to bottlenecks (see pattern of response to inbreeding can be accounted Saccheri et al., 1996).

It is evident from the foregoing overview that, in the number of individuals used to start the ex- order to critically assess the influence of genetic perimental populations or lines. The founder numbers architecture on the response of VA to bottlenecks, one used were two, six, 20 and c. 300 (control treatment), should ideally (pre)select a suite of traits which vary replicated six times for the smallest bottleneck and substantially with respect to additive, dominance and four times for the rest. epistatic variances. For practical reasons it was not Husbandry techniques are detailed in Saccheri et al. possible to study the non-additive components of (1996). With the exception of the control lines (see variance directly, though the magnitude of directional below), all lines were established from clutches of dominance was assessed. Furthermore, while the known parentage in the following way. One hundred change in VA was measured for morphological traits and twenty-five clutches were collected over 7 days only, we also report on the likely genetic basis of the from females isolated in copula. From those clutches selection response of a major component of fitness with more than 40 fertile eggs (representing 80% of all (egg-larva viability) in the same experimental lines fertile clutches and 57% of all clutches, including non- (Saccheri et al., 1996). fertile ones) the required number of clutches (i.e. one, The aim of this experiment was to provide results three or 10) were randomly chosen to found the which could be compared with previous empirical bottlenecked populations of one pair, three pairs and studies, particularly those of Bryant et al.(1986), and 10 pairs. These populations were then allowed to with the neutral additive model, using a novel model freely increase in size to a maximum adult population organism. A specific strength of this study is that the size of about 300, controlled by random culling of degree of inbreeding in the experimental lines was larvae. Three generations elapsed before data were estimated directly with molecular markers (Saccheri et collected (population sizes from P to F3 are given in al., 1999), overcoming the difficulty of uncertain Ne Saccheri et al., 1999). encountered by some other bottleneck experiments. It was impractical to establish control lines with This paper describes the effects of the bottlenecks on clutches obtained from mating pairs collected in heritability and additive genetic variance within lines copula. However, to control for the small chance that for wing size and a series of wing pattern characters. the procedure for choosing clutches to found bottle- Given the paucity of empirical data on this important neck lines was biased with respect to genotype, two issue, primarily on a few traits in four species (D. types of control were used. Initially, four populations melanogaster, M. domestica, T. castaneum and Mus each consisting of 150 virgin females and 150 virgin musculus), fresh data describing the response of very males were established and random mating allowed different traits in an unrelated species are of substantial for 2 weeks. For two of these populations we collected interest for understanding the response of polygenic eggs from the entire population; these eggs were used traits to inbreeding. to found two control lines. For the remaining two populations we collected clutches from all females 2. Materials and methods individually, but only used those clutches with more than 40 fertile eggs (85 and 60 clutches from each (i) Experimental populations population respectively) to found the other two control A laboratory population of B. anynana was established lines. In subsequent generations, all females were from approximately 80 gravid females collected from given equal opportunity to lay eggs on potted maize the wild at a single locality (Nkhata Bay) in Malawi in plants placed in the cages for a fixed period of time, all August 1988. Prior to the experiment the stock was control lines being maintained at an adult population maintained for about 20 generations at a population size of about 300. Clutches used to found the 10 pair size of 400–600 adults with some overlap of gener- and control lines were culled randomly to keep larval ations. High levels of allelic diversity and hetero- densities equivalent in all lines. No differences in zygosity detected by single-locus DNA fingerprinting inbreeding were detected between the two types of and allozyme electrophoresis (Saccheri et al., 1999) control (see Saccheri et al., 1999). suggest that the stock population at the start of the experiment had not undergone any substantial bottle- (ii) Morphological measurements neck and that the husbandry technique was effective in maintaining genetic variability. Butterflies to be measured were frozen and dissected Previous experimental results (e.g. Bryant et al., to separate the wings from the body, which was 1986) and theoretical predictions suggest that the required for molecular analysis (Saccheri et al., 1999). largest changes in additive genetic variance are most Nine morphological characters were measured on the likely to occur for population bottleneck sizes (founder ventral surface of the hind wing (Fig. 1): the areas of events) of 20 or fewer individuals. Consequently, three concentric rings of colour constituting the fifth daughter populations were derived from the base and largest of the eyespots occuring in the distal part population according to four treatments, differing in of the wing (referred to as white area, black area and

5, 8

9 4 6, 7 1 2 3

Fig. 1. Morphometric measurements of Bicyclus anynana hind wing. No. Object Measurement Character name # 1 White centre spot Area (mm ) White area # 2 Black ring Area (mm ) Black area # 3 Gold ring Area (mm ) Gold area 4 Extra ring Standard deviation of grey values Extra ring 5 Whole wing Coarse grey value diﬀerences Contrast 6 Mid-cell Mean grey value (small l dark) Colour 7 Mid-cell Fine grey value diﬀerences Marbling # 8 Whole wing Area (mm ) Wing area 9 Mid-cell Distance from crossing point of cubital and Band index median veins to edge of white band (mm); inversely proportional to band area

gold area); four indices of colour and contrast over 2000). Principal component analysis was used to different parts of the wing surface, measured on a summarize the variation contained in the nine mor- scale of grey values ranging from 0 to 255 (extra ring, phological characters, while at the same time providing colour, contrast and marbling); the area of the entire principal components which could be treated as wing (wing area); and an index of the area of the white statistically independent. The raw character values transverse band (band index). The left hind wing was were transformed into standardized normal deviates measured unless damage necessitated measurements as part of the analysis, which was performed in from the right hind wing. All traits were measured Genstat (Payne et al., 1988). A correlation matrix, as with a computerized image analyser developed specifi- opposed to a covariance matrix, was used because of cally for this purpose (Windig, 1993). Repeated the widely differing scales of measurement and units. measurements of the nine morphological traits for a subsample of 100 males yielded estimates of repeat- (iii) Heritability tests ability from 0n697 to 0n936 with a mean of 0n852. The two traits associated more with wing colour and Parent–offspring heritability tests were carried out for variation in grey values (colour and marbling) showed the base population at the start of the experiment (test lower repeatability than the size and pattern traits. 1) and all 18 lines in the F3 generation (test 2). Single The nine morphological traits which were measured male–female pairs were isolated and their offspring are not all developmentally independent. This is most reared under the same conditions of temperature and obvious for the three eyespot components (Brakefield, humidity. Clutches were culled to standardize larval

(a) PC1 PC2 and 49 families for one pair, three pair, 10 pair and control treatments respectively; both parents, five Control daughters and five sons were measured for each # family. Heritability, h , was estimated from the slope of the regression of offspring on parental values Treatment 1 within each line. Additive genetic variance, VA, was estimated as the product of the midparent heritability and twice the phenotypic variance, VMP, of 30 mid- Treatment 2

Offspring value Offspring pair values (parents plus randomly chosen pairs from the same line) for each line. The environmental variance, VE, was calculated as 2VMPkVA. Treatment 3

Midparent value (iv) Inbreeding depression The extent of any reduction in the mean value of a (b) trait following inbreeding (i.e. inbreeding depression) is a measure of the magnitude of directional dominance for that trait. ANOVA was used to test for equality of treatment (bottleneck size) means for all nine morphological traits in each sex of the parental F3 generation sample (30 individuals of each sex per

Offspring line). Statistical signiﬁcance was assessed by com- paring the ratio of treatment to replicate (nested within treatment) mean squares with the appropriate critical value of the F-distribution.

Parents (c) (v) Analysis The experiment was designed to evaluate the effect of # bottleneck treatments on h , which can be divided into effects on VA and on VE. The neutral additive model makes specific predictions which can be compared with the observed results. The rationale for

Offspring the analysis was as follows. The slope of the regression # for the trait in offspring on parents is an estimate of h . Under the null hypothesis that treatment had no effect, the parent–offspring regressions should not differ across treatments for each principal component Parents (Fig. 2a). Given a normal distribution, the likelihood Fig. 2. Diagrams to clarify our analysis and estimation for a particular regression line is a function of procedures. (a) Under the null hypothesis we expect no deviation of each offspring value from the regression significant difference in the slopes of parent–offspring line, d, and the variance, V: regressions (i.e. heritability) among treatments (only one # replicate with six families is shown per treatment), though Log likelihood lk0n5Σ ln(V)jd \V, slopes may differ among principal components. (b) The heritability for a particular treatment can be estimated by where the sum is over all offspring. The likelihood can fitting parallel regression lines, with a different intercept therefore be maximized by minimizing the sum of for each of the four (or more) replicates. Failure to fit squares of deviations, and likelihoods can be combined different intercepts would result in the upwardly biased over the principal components if each regression is estimate shown by the dashed line. (c) The full model fits weighted by its residual deviance. different slopes for each replicate within a treatment (i.e. different heritabilities). Although principal components are constructed so that they are uncorrelated (Manly, 1986), they may not be independent within a line after the bottleneck. density in the sleeve cages, which were kept abundantly This is because linkage disequilibrium will have been supplied with maize. In test 1, 57 families were reared; generated by the bottlenecks (Lynch, 1988), with the # both parents, 10 daughters and 10 sons were measured result that changes in h and VA for different principal for each family. In test 2, an average of 12 families per components within a line may be associated. This line were reared giving treatment totals of 63, 55, 51 would have the effect of inflating the variation in slope

Table 1. Estimates of the coeﬃcient of inbreeding at F3, used to calculate the expected heritability and additie genetic ariance for each bottleneck size, according to three series of estimates (see text)

Bottleneck size E(null) E(demographic) E(molecular)

One pair 0 0n27 0n32 Three pairs 0 0n100n12 Ten pairs 0 0n03 0n07 Control 0 0n01 0n02

between replicates (measured by the Expectationi combined across principal components by weighting bottleneck sizeireplicate term in Tables 4–6) and each regression by its residual deviance. reducing it between principal components (pc) of the If one assumes that the morphological characters same replicate (Expectationireplicateipc). In fact have no non-additive variance, as is reasonable based the mean squares for these two terms were very on our observations implying small dominance vari- similar, but it might be considered prudent to interpret ance, and that environmental variance is unaffected the data using the former term as the denominator in by inbreeding, the expected heritability after inbreed- F-tests. Correlated changes for different principal ing is given by components within lines may also reflect variation in # inbreeding among replicate lines within treatments, # h!(1kFt) # E(ht ) l # but the precision of our estimates for h is insufficient (1kh!Ft) to study the behaviour of individual replicates. # where E(ht ) and Ft are the heritability and inbreeding Each treatment (bottleneck size) slope (heritability) # was estimated by fitting a common slope to all coefficient at time t, and h! is the original heritability replicate lines of the treatment, while allowing for in the base population (Falconer & Mackay, 1996). different intercepts for each replicate. In effect, this For the analysis of heritability, the expected deviation procedure fitted a set of parallel straight lines (four or of offspring values from the mean was calculated as six per treatment) through the data (Fig. 2b). This # (midparent value–line mean):E(h ), estimation procedure is preferable to taking the mean t # slope because the precision of the estimates differs for each principal component. E(ht ) is the ratio of the between replicates. Fitting parallel lines takes this into expected additive variance, E(VA), and the expected account automatically. Notice that this approach is midparental phenotypic variance, E(VMP). A second not equivalent to pooling the data across replicates, # approach attempted to account for differences in which would lead to upwardly biased h estimates due environmental variance between replicates in order to to differentiation among replicate line means (Fig. focus on the response of VA. In this analysis the 2b). expected deviation of offspring values from the mean A test for deviation from parallel regression lines was calculated as (Fig. 2a) was conducted by fitting a different slope to each treatment and testing for a significant improve- # E(VMP) (midparent value–line mean):E(ht ): , ment in fit (Expectationibottleneck size in Tables VMP 4–6). Any improvement in fit produced by allowing a different slope for each replicate (Fig. 2c) quantifies where E(VMP) l VA!(1kFt)jVE!, VE! being the ad- the different response of the replicates of each ditive and environmental variances in the base treatment (Expectationibottleneck sizeireplicate in population, respectively. The observed heritabilities Tables 4–6). Terms were also introduced to quantify and phenotypic variances for the base population the different response of principal components within were not substantially different from the control replicates (Expectationireplicateipc in Tables 4–6). averages, but as the base population was reared under A more stringent null hypothesis is that the ratio of slightly different conditions, the control estimates # slopes for each principal component is predicted by were used for h! and VMP!. the neutral additive model and the effective population Three different expectations, referred to as E(null), size Ne of each bottleneck. In that case the regressions E(demographic) and E(molecular), were specified by were transformed to be parallel using the procedure different estimates of Ft for each bottleneck size described below. Deviation from the null expectation (Table 1). E(null) is equivalent to the null hypothesis was tested in the same way by fitting a separate slope that the bottlenecks have no effect; E(demographic) for each treatment, and again information was are the null expectations from the calculation that

includes inbreeding due to observed population sizes indicating that the characters are normally distributed. and variances in family size in the F1 and F2 (see A principal component analysis was carried out on all Saccheri et al., 1996); and E(molecular) uses the the test 2 offspring (1042 females and 985 males), these estimates obtained from the analysis of allozyme and being in a better preserved state than the parent minisatellite data (Saccheri et al., 1999). The control generation sample (Table 2). The first three principal data were included in each analysis, with Ft set to zero. components can each be interpreted in a straight- The estimated slope for control values against their forward way: PC1 (which explains 31% of the total expectation need not be zero. This scheme allowed for variation) is essentially an index of eyespot size, large # error in the estimation of h! and VMP! which could eyespots being associated with high values of extra otherwise lead to a systematic deviation from expecta- ring and contrast; PC2 (21%) is essentially an index tions across all the treatments. of darkness and grain in the mid-cell; and PC3 (13%) contrasts wing area and band area. PC4, PC5 and 3. Results PC6 are more difficult to interpret, but are consistent with other data sets and each explains between 5% (i) Principal component analysis and 10% of the total variation. Plots of the cumulative frequency distribution of each character against the cumulative normal were linear,

Table 2. Principal component character loadings

Principal component

Character loadings 1 23456

White area k0n445 k0.198 k0n035 0n044 0n358 k0n225 Black area k0n437 k0n298 0n097 0n098 0n281 k0n064 Gold area k0n480 k0n2130n056 0n123 0n086 0n188 Extra ring k0n374 0n340 k0n323 k0n400 k0.136 k0n110 Contrast k0n428 0n227 k0n043 k0n312 k0n475 0n090 Colour k0n148 0n507 0n371 0n198 0n222 0n664 Marbling k0n107 0n507 0n387 0n335 k0n013 k0n664 Wing area k0n107 k0n375 0n507 0n134 k0n642 0n033 Band index 0n111 k0n069 0n580 k0n739 0n284 k0n087 Eigenvalue (%) 32 2113 10 105

The eigenvalues (as percentages) and eigenvectors of the correlation matrix for the nine wing characters measured in B. anynana. The ﬁrst six principal components are shown; the largest loadings for each principal component are underlined. Note that band index is inversely proportional to band area.

Table 3. Analysis of coariance from the GLM of oﬀspring alues with the midparental alues as the explanatory ariate (control only)

Source of variation d.f. s.s. m.s. v.r. Pa

Oﬀspring sexireplicatebipc 47 281n055 5n980 7n67 * Tryc parent sexioﬀspring sex 4 428n032 107n008 137n33 *** Midparent 1 425n013 425n013 545n45 *** Midparentipc 5 15n358 3n072 3n94 ns Midparentireplicate 3 2n761 0n920 1n18ns Midparentireplicateipc 1555n394 3n693 4n74 ns Replicateipcifamily 246 757n457 3n079 3n95 * Residual 2574 2005n671 0n779 Total 2891 3542n708 1n225

*0n01 ! P % 0n05; ** 0n001 ! P % 0n01; *** P % 0n001; ns, non-significant. a Levels of significance are calculated in relation to the m.s. for replicateipcifamily as the denominator (with the exception of the P value for replicateipcifamily, where the residual m.s. is the denominator). P value for offspring sexireplicateipc refers to the separate effects of replicate and pc, which were the only factors in this group that differed significantly in their means. b Replicate stands for replicate within treatment. c Try is an option in generalized linear modelling which adds terms then drops them.

0.8 0.9 White area Contrast 0.7 0.8 0.7 0.6 0.6 0.5 0.5 0.4 0.4 0.3 0.3 0.2 0.2 0.1 0.1 0.0 0.0 0.1 0.2 0.3 0.0 0.1 0.2 0.3

1.0 0.6 0.9 Black area Colour 0.5 0.8

0.7 0.4 0.6

0.5 0.3

0.4 0.2 0.3

0.2 0.1 0.0 0.1 0.2 0.3 0.0 0.1 0.2 0.3

0.7 0.7 Heritability Gold area Wing area 0.6 0.6 0.5 0.5 0.4 0.4 0.3 0.3 0.2

0.2 0.1

0.1 0.0 0.0 0.1 0.2 0.3 0.0 0.1 0.2 0.3

0.7 0.7 Extra ring Band index 0.6 0.6

0.5 0.5

0.4 0.4

0.3 0.3

0.2 0.2 0.0 0.1 0.2 0.3 0.0 0.1 0.2 0.3 Coefficient of inbreeding Fig. 3. Narrow-sense heritabilities for eight morphometric traits determined by oﬀspring–midparent regressions for bottleneck lines of one, three and 10 founding pairs and control lines (continuous line). Average heritabilities for each bottleneck size and their standard errors were calculated by ﬁtting the best set of parallel regressions to replicate lines within each bottleneck size (six for the one pair and four for the other bottleneck sizes). These average heritabilities are

Differences between base population and F3 control (ii) Inbreeding depression # h are most likely to be due to differences in the There was no detectable inbreeding depression for any rearing environment resulting from higher larval of the morphological traits and, by extension, for the densities in the test 1 parental generation. The closer principal components. The one significant result (P l correspondence between base population and control # 0n04) was for male wing area. However, the pattern of F3 h for area measures (white area, black area, gold line and treatment means shows that this result, which area and wing area), as opposed to grey value measures is primarily due to two low three pair lines, cannot be (extra ring, contrast and colour), suggest that the accounted for by inbreeding depression. Such a lack former characters are the most reliable, at least in part of evidence for directional dominance also suggests because they have higher repeatabilities and are less little dominance variance in general for these traits. affected by wing wear. # The analyses of effects on h and VA in relation to the null hypothesis are presented in Tables 4 and 5, (iii) Heritability, additie genetic ariance and respectively. The variation between replicates is not enironmental ariance significantly greater than the variation between prin- An initial exploratory analysis of principal component cipal components within replicates. It is not clear, heritabilities was performed on the four control lines therefore, whether the treatment effect should be (Table 3). There was a significant common family compared with the mean square of summing all the effect (replicateipcifamily), so mean squares of terms of Eibsireplicate not already included in the previous terms were compared with the ‘family’ mean model, or with the Eireplicateipc mean square (i.e. square to assess their level of statistical significance. the pooled sums of squares). Sokal & Rohlf (1981,p. Separate intercepts were first fitted for sons and 284) state that ‘statisticians do not agree about the daughters (offspring sex), replicate lines and each of conditions under which mean squares should be the six principal components. Only replicates and pooled and about the desirability of pooling’ in such principal components differed significantly in mean nested models. They propose criteria under which (remembering that female and male values were pooling should be performed. According to these, the standardized), as might be expected due to environ- sums of squares should be pooled in this analysis if the mental variance between cages and the differing prior belief was that the variance among replicates character composition of principal components. The was less than twice that among principal components. pooled sums of squares obtained by fitting a different This might be applicable in this case where the slope for each parent sex and their interaction with principal components are considered to reflect differ- each offspring sex is not significantly larger than the ent traits. The increased number of degrees of freedom sum of squares for the regression of all offspring on changes the significance level of the results as indicated # midparent values. This result indicates that h do not in parentheses in Tables 4 and 6, but relies on the differ significantly between parent or offspring sex and assumption of independence between principal com- # validates the use of midparent h in subsequent ponents. # analyses. Somewhat surprisingly, principal compo- Bottleneck size changed h and VA significantly nents do not appear to differ significantly with respect (E(null)ibs in Tables 4 and 5). This result holds # to their h (midparentipc). There were no significant whether the treatment effect is compared with the # differences in h among the four replicates (mid- mean square of Eibsireplicate or Eireplicateipc, parentireplicate), or any significant interaction be- but is more convincing in the latter case. The pooled tween replicates and principal components with sums of squares for E(null) and E(null)ibs is larger # # respect to h (midparentireplicateipc). (though not significantly) in Table 4 (h analysis) than Character heritabilities are not independent and in Table 5 (VA analysis). This suggests that the second cannot therefore be combined directly, but are shown approach, using VMP, is a less efficient predictor of to illustrate the results in their simplest form (Fig. 3). offspring values, implying either that the error on VMP # In general, there is a clear tendency for the one pair estimates is too large for them to be used to adjust h , # and three pairs bottleneck size lines to have lower h or that the assumption that variation in VMP was than the 10 pairs and control bottleneck lines. The predominantly due to environmental variation was raw character VA graphs (Fig. 4) show a pattern of inappropriate. # treatment effects similar to that observed for h :an The analyses investigating whether the observed # increasing trend from the one pair and three pair changes in h and VA deviated significantly from the bottlenecks to the 10 pair bottleneck and control. changes expected under the neutral additive model,

plotted against the treatment level coeﬃcient of inbreeding estimated from demographic and molecular data combined (Saccheri et al., 1999). Base population values (dashed line) are shown for comparison. The graph for marbling is similar to that for colour with which it is correlated, and is not shown.

0.0009 4.5 0.0008 White area 4.0 Contrast 0.0007 3.5 0.0006 3.0 0.0005 2.5 0.0004 2.0

0.0003 1.5 0.0002 1.0 0.0001 0.5 0.0 0.1 0.2 0.3 0.0 0.1 0.2 0.3

0.19 55 Black area Colour 0.17 50

0.15 45

0.13 40

0.11 35

0.09 30

0.07 25 0.0 0.1 0.2 0.3 0.0 0.1 0.2 0.3

0.12 90 Gold area Wing area 0.11 80 0.10 Additive genetic variance Additive 70 0.09 60 0.08 50 0.07 40 0.06 0.05 30

0.04 20 0.0 0.1 0.2 0.3 0.0 0.1 0.2 0.3

20 0.014 Extra ring Band index 18 0.013

16 0.012

14 0.011

12 0.010

10 0.009

8 0.008

6 0.007 0.0 0.1 0.2 0.3 0.0 0.1 0.2 0.3 Coefficient of inbreeding Fig. 4. Additive genetic variances for eight morphometric traits averaged across replicate lines within each bottleneck size, plotted against the treatment level coeﬃcient of inbreeding.

Table 4. Analysis of coariance from the GLM of oﬀspring alues with the midparental alues as the explanatory ariate (all treatments)

Source of variation d.f. s.s. m.s. v.r.a Pb

pcioﬀspring sexibsireplicate 170 2520n839 14n829 – E(null) 11123n6411123n641 446n91 *** E(null)ibs 3 37n002 12n334 4n58 * (**) E(null)ibsireplicate 1437n674 2n691 – E(null)ireplicateipc 45 110n575 2n457 – Residual 11928 11144n486 0n934 Total 1216114974n217 1n231

a Variance ratios are calculated using the mean square for E(null)ibsireplicate as the denominator. b The asterisks in parentheses indicate a comparison with the pooled m.s. for E(null)ibsireplicate and E(null)ireplicateipc. bs, bottleneck size treatment. This notation also applies to Tables 5 and 6.

Table 5. Analysis of coariance from the GLM of oﬀspring alues with the midparental alues adjusted by VMP as the explanatory ariate (all treatments)

Source of variation d.f. s.s. m.s. v.r. P

pcioﬀspring sexibsireplicate 170 2520n839 14n829 – E(null) 1 986n728 986n728 172n72 *** E(null)ibs 3 63n123 21n041 3n68 * E(null)ibsireplicate 1479n988 5n713– E(null)ireplicateipc 45 149n446 3n321 – Residual 11928 11174n093 0n937 Total 1216114974n217 1n231

Notation as for Table 4.

Table 6. Analysis of coariance from the GLM of oﬀspring alues with E(molecular) as the explanatory ariate (all treatments)

Source of variation d.f. s.s. m.s. v.r. P

pcioﬀspring sexibsireplicate 170 2520n839 14n829 – E(molecular) 11136n689 1136n689 428n78 *** E(molecular)ibs 3 21n525 7n175 2n71 ns (*) E(molecular)ibsireplicate 1437n1122n651 – E(null)ireplicateipc 45 113n1162n514– Residual 11928 11144n936 0n934 – Total 1216114974n217 1n231

Notation as for Table 4.

using either E(demographic) or E(molecular), gave among principal components. This was also apparent # essentially the same result. The analysis of h for from a visual comparison of the separate graphs for E(molecular), which predicts the greatest change, is each principal component. Therefore, a useful sum- # shown in Table 6. The outcome is ambiguous: the mary is provided by the average h and VA for each # change in h does not differ significantly from the bottleneck size across PC1–6 (Fig. 5). Standard errors expected change when the treatment effect is compared were calculated from the variation among replicates, with the Eibsireplicate mean square, but it does rather than principal components. The VA values were differ significantly (P ! 0n05) when compared with the ln-transformed as their distributions were skewed and pooled mean square. the treatment effects were expected to be multi- # It was found that the behaviour of h and VA with plicative. The values expected from the neutral respect to bottleneck size did not differ significantly additive model, using the molecular estimates of Ft

Downloaded from https://www.cambridge.org/core. IP address: 170.106.33.42, on 30 Sep 2021 at 16:09:05, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0016672301004906 I. J. Saccheri et al. 178 # 0.60 significantly different, from the control average h . # The expected h is within 2 standard errors of the # 0.55 observed h for all bottleneck sizes. Similarly, Fig. 5 shows large reductions in VA for the one pair and three 0.50 pair bottlenecks, but no change in VA for 10 pairs relative to control lines. As was indicated by the 0.45 analysis of variance, there is some evidence that average losses in one pair lines, and to a lesser extent Heritability 0.40 in three pair lines, were greater than predicted by the neutral additive model. The close concordance of # the 10 pair and control V values suggests that h 0.35 A differences between these two treatments (see Fig. 5) were due to environmental variation. 0.30 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 Table 7 indicates that inbreeding did not alter VE, Coefficient of inbreeding and, not surprisingly, that principal components have different VE. There was also no evidence for VE –0.30 differing among replicate lines. –0.40 –0.50 4. Discussion –0.60 Our experiment indicates that bottlenecks of one pair –0.70 and three pairs of individuals result in large and –0.80 # significant losses in h and VA for wing size and pattern –0.90 (as analysed using principal components) in Bicyclus –1.00 anynana. Furthermore, conservative interpretation of –1.10 our statistical analysis indicates that these changes are Additive genetic variance Additive –1.20 in quantitative agreement with the neutral additive –1.30 model of quantitative genetic variation. There is some evidence to suggest that the losses –1.40 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 were greater than expected under the neutral additive Coefficient of inbreeding model, but it has not been possible to demonstrate this unequivocally (Table 6). Greater than expected losses Fig. 5. Observed (continuous line) and expected (dashed # in h and VA imply undetected inbreeding. For the line) heritability and ln-transformed additive genetic # variance for each bottleneck size (coefficient of observed and expected values of h and VA to coincide, inbreeding) averaged across PC1–6. Standard errors the average level of inbreeding of the one pair and describe the variation among replicates. three pairs lines (relative to the control level of inbreeding) would need to be close to 0n48 and 0n32, (relative to control Ft), are superimposed on both respectively. Analysis of molecular data (Saccheri et graphs. al., 1999) revealed that the experimental lines had Fig. 5 illustrates the general pattern of treatment indeed undergone more inbreeding than was pre- # effects on h shown by the individual principal viously thought (Table 1), probably as a result of components and characters, confirming that the increased variance in reproductive viability and smaller bottlenecks caused heritability to decline; the background selection (hitch-hiking). This additional biggest reduction occurred for the one pair bottleneck, inbreeding, which is incorporated in the analysis with # followed by the three pair bottleneck. The average h the E(molecular) expectation, may explain much of for the 10 pair bottleneck is marginally higher, but not the loss in additive genetic variation (Table 6). Given

Table 7. ANOVA of enironmental ariance

Source of variation d.f. s.s. m.s. v.r. P

pc 5 2n285 0n457 9n33 *** bs 3 0n198 0n066 1n35 ns pcibs 150n320 0n021 0n44 ns Replicate 140n497 0n036 0n73 ns Residual 70 3n428 0n049 Total 107 6n729 0n063

the low level of replication imposed by the system, the for the view that increases in VA following inbreeding remaining discrepancy could plausibly be accounted will most often be due to large dominance variance in for by the variance of the realization of the drift traits which are closely related to fitness. process and error on parameter estimates (Lynch, Existing empirical evidence does not rule out the 1988). Alternative mechanistic explanations based on possibility that epistasis may also contribute sub- extreme distributions of allelic effects (conceivably stantially to increases in VA of some characters leading to greater than expected losses in VA) are less following inbreeding. Because of the difficulties in- plausible, both on theoretical grounds (Maynard herent in accurately estimating even the simplest form Smith, 1998, p. 120) and because they are incom- of epistatic variance (additiveiadditive) few studies patible with the evidence for several genes, each of of inbreeding effects, including our own, have at- small phenotypic effect, controlling eyespot size tempted to measure it. In one inbreeding experiment (Wijngaarden, 2000). where additive dominance (VD), epistatic (VAA), and A strength of our analysis is the use of principal environmental variances were measured (Bryant & components, rather than raw characters, because it Meffert, 1996) the results were somewhat equivocal, allows a rigorous examination of the treatment effects but did show a positive association between increases for the measured set of characters as a whole and in VA and VAA in the base population, among four overcomes the problem of correlations faced by morphological characters. The results of Cheverud et analyses of raw characters (e.g. Bryant et al., 1986). In al.(1999), while clearly demonstrating a potentially this respect, an important result is that the effect of major role for epistatic to additive variance con- # bottleneck size on both h and VA is consistent for the version, were obtained in a highly derived population first six principal components. For several of these (F2 intercross of inbred high and low selected lines), specific traits (viz. eyespot elements) strong evidence leading us to question the relevance of this result for of a common genetic and developmental determi- natural populations. In future, the application of nation system (Brakefield et al., 1996) further justifies molecular quantitative genetics (QTLs) to describe the the use of principal components. Moreover, from an genetic architecture of traits in natural populations evolutionary perspective, the most likely target of should provide greater insights into the role of non- selection in nature is the wing pattern as a whole additive variance components for adaptation in the (Brakefield, 2000), which is described more effectively presence of drift. by the principal components than the individual Even when non-additive variance is negligible, VA characters. may increase above control levels in a small fraction The additive behaviour of the wing pattern and size of cases, simply because of the variance about the traits in response to inbreeding is consistent with the expectation of the drift process, which is likely to be lack of any detectable inbreeding depression for these particularly large for polygenic traits. This has been traits. The implication is that there is relatively little demonstrated by the highly replicated Drosophila (directional) dominance variance for these traits experiment of Whitlock & Fowler (1999). The same which, as mentioned in Section 1, has been found to study found highly variable but significant overall be the most common source of non-additive to additive increases in VE for four of six wing area and angle variance conversion. The apparent lack of directional measurements, paralleling the results of Bryant & dominance for the wing traits in B. anynana conforms Meffert (1996). Such increases in VE are traditionally to the now well supported hypothesis (Kearsey & attributed to lowered developmental homeostasis, Kojima, 1967; Crnokrak & Roff, 1995; DeRose & though the genetic basis and developmental mech- Roff, 1999) predicting low directional dominance for anism for this hypothesis are unclear. We were unable traits under weak stabilizing selection, such as are to detect any significant effect of inbreeding on VE for most morphological traits. We have also detected very the B. anynana traits under investigation. This may be large directional dominance for a major component of because of substantial differences in developmental fitness, egg hatching rate, in the same experimental canalization (Gibson & Wagner, 2000) or less stressful lines (Saccheri et al., 1996), as expected for fitness- environmental conditions (e.g. less crowding). related traits subject to strong directional selection Another factor that should be taken into con- (DeRose & Roff, 1999). The rapid and large response sideration is that in natural (as opposed to laboratory) to natural selection for increased egg hatching rate in environments, founding colonists or population crash some of the one pair lines (Saccheri et al., 1996), survivors are unlikely to be a random set from the coupled with the observed increase in phenotypic base population. With the exception of the statistically variance, implies substantial increases in VA for egg problematic experiment of Lints & Bourgois (1984) hatching rate, as a result of inbreeding. Thus, our who measured an increase in VA for sternopleural results, which demonstrate losses of VA for mor- bristle number following an extended bottleneck phological traits and a probable increase in VA for a induced by high temperature, this possibility has not major component of fitness, provide strong support been explored by any of the empirical studies aiming

to assess the consequences of bottlenecks for VA. If, Bicyclus butterflies (Satyridae) in Malawi. Ecological for example, the more heterozygous genomes are Entomology 16,291–303. favoured, for which there is increasing evidence (Keller Brakefield, P. M., Gates, J., Keys, D., Kesbeke, F., Wijngaarden, P. J., Monteiro, A., French, V. & Carroll, et al., 1994; Saccheri et al., 1998), VA for the selected S. B. (1996). Development, plasticity and evolution of traits themselves or for those traits in linkage butterfly eyespot patterns. Nature 384, 236–242. disequilibrium with the selected regions may increase, Bryant, E. H. & Meffert, L. M. (1993). The effect of serial or decrease less than predicted by the neutral additive founder flush cycles on quantitative genetic variation in model. the housefly. Heredity 70, 122–129. Bryant, E. H. & Meffert, L. M. (1996). Nonadditive genetic There now exists an accumulated body of work structuring of morphometric variation in relation to a indicating that bottlenecks are likely to cause losses in population bottleneck. Heredity 77, 168–176. VA of most characters most of the time, but increases Bryant, E. H., McCommas, S. A. & Combs, L. M. (1986). in others. The evolutionary consequences of bottle- The effect of an experimental bottleneck upon quantitative necks are therefore less predictable than implied by genetic variation in the housefly. Genetics 114, 1191–1211. Carson, H. L. (1990). Increased genetic variance after a the neutral additive model. An important consider- population bottleneck. Trends in Ecology and Eolution 5, ation is the relative fitness contribution of characters 228–230. which gain VA and those that lose VA. Future studies Carson, H. L. & Templeton, A. R. (1984). Genetic revolu- aiming to make a general assessment of the effect of tions in relation to speciation phenomena: the founding bottlenecks on quantitative characters should choose of new populations. Annual Reiew of Ecology and Systematics 15, 97–131. characters carefully, including a range of contrasting Cheverud, J. M. & Routman, E. J. (1996). Epistasis as a genetic architectures (i.e. varying relative magnitudes source of increased additive genetic variance at population of additive, dominance and epistatic variances) and bottlenecks. Eolution 50, 1042–1051. presumed selective regimes (major determinants of Cheverud, J. M., Vaughn, T. T., Pletscher, L. S., King- fitness versus weakly selected traits). The present Ellison, K., Bailiff, J., Adams, E., Erickson, C. & 1 study has also emphasized the need to obtain accurate Bonislawski, A. ( 999). Epistasis and the evolution of addititve genetic variance in populations that pass through estimates of the level of inbreeding when interpreting a bottleneck. Eolution 53, 1009–1018. the effects of bottlenecks on quantitative genetic Condamin, M. (1973). Monographie du genre Bicyclus variation, and suggests that cryptic selection (Saccheri (Lepidoptera, Satyridae). Memoires de l’Institut Fonda- et al., 1999) can accelerate the loss of VA. mental d’Afrique Noire 88, Ifan-Dakar. Estimates of heritabilities, phenotypic variances Coyne, J. A., Barton, N. H. & Turelli, M. (2000). Is Wright’s shifting balance process important in evolution? Eolution and means are available on request from I.J.S. 54, 306–317. Crnokrak, P. & Roff, D. A. (1995). Dominance variance: We thank R. Heywood, E. Schlatmann and B. de Winter for associations with selection and fitness. Heredity 75, their assistance during the experimental work. Bas Zwan 530–540. and three anonymous reviewers provided valuable com- Da Silva, J. M. P. (1961). Limits of response to selection. ments. This research was funded by the Institute of Zoology PhD thesis, University of Edinburgh. and the University of Leiden. The final period of writing was DeRose, M. A. & Roff, D. A. (1999). A comparison of supported by the European Union TMR Network ‘Frag- inbreeding depression in life-history and morphological land’. traits in animals. Eolution 53, 1288–1292. Falconer, D. S. & Mackay, T. F. C. (1996). Introduction to References Quantitatie Genetics, 4th edn. Harlow, Essex: Longman. Ferna! ndez, A., Toro, M. A. & Lo! pez-Fanjul, C. (1995). The Barton, N. H. (1989). Founder effect speciation. In effect of inbreeding on the redistribution of genetic Speciation and its Consequences (ed. D. Otte & J. A. variance of fecundity and viability in Tribolium castaneum. Endler), pp. 229–256. Sunderland, MA: Sinauer Asso- Heredity 75, 376–381. ciates. Frankham, R. (1980). The founder effect and response to Barton, N. H. & Charlesworth, B. (1984). Genetic revolu- artificial selection in Drosophila. In Selection Experiments tions, founder effects, and speciation. Annual Reiew of in Laboratory and Domestic Animals (ed. A. Robertson), Ecology and Systematics 15, 133–164. pp. 87–90. Wallingford: Commonwealth Agricultural Barton, N. H. & Turelli, M. (1989). Evolutionary quan- Bureau. titative genetics: How little do we know? Annual Reiew Frankham, R. (1999). Quantitative genetics in conservation of Genetics 23, 337–370. biology. Genetical Research 74, 237–244. Brakefield, P. M. (1997). Phenotypic plasticity and fluctuat- Franklin, I. R. (1980). Evolutionary change in small ing asymmetry as responses to environmental stress populations. In Conseration Biology: An Eolutionary- in the butterfly Bicyclus anynana. In Enironmental Ecological Perspectie. (ed. M. E. Soule! & B. A. Wilcox), Stress, Adaptation and Eolution. (ed. R. Bijlsma & V. pp. 135–149. Sunderland, MA: Sinauer Associates. Loeschcke), pp. 65–78. Basel: Birkha$ user. Garcı!a, N., Lo! pez-Fanjul, C. & Garcı!a-Dorado, A. (1994). Brakefield, P. M. (2000). The structure of a character and The genetics of viability in Drosophila melanogaster: the evolution of patterns. In Eolutionary Biology of effects of inbreeding and artificial selection. Eolution 48, Characters (ed. G. P. Wagner). New York: Academic 1277–1285. Press (in press). Gibson, G. & Wagner, G. (2000). Canalization in evol- Brakefield, P. M. & Reitsma, N. (1991). Phenotypic plas- utionary genetics: a stabilizing theory? BioEssays 22, ticity, seasonal climate and the population biology of 372–380.

Goodnight, C. J. (1987). On the effect of founder events on Ritchie, M. G. & Kyriacou, C. P. (1994). Genetic variability epistatic genetic variance. Eolution 41, 80–91. in courtship song in a population of Drosophila melano- Goodnight, C. J. (1988). Epistasis and the effect of founder gaster. Animal Behaiour 48, 425–434. events on the additive genetic variance. Eolution 42, Robertson, A. (1952). The effect of inbreeding on the 441–454. variation due to recessive genes. Genetics 37, 189–207. Goodnight, C. J. & Wade, M. J. (2000). The ongoing Roff, D. (2000). The evolution of the G matrix: selection or synthesis: a reply to Coyne, Barton, and Turelli. Eolution drift? Heredity 84, 135–142. 54,317–324. Ruano, R. G., Silvela, L. S., Lo! pez-Fanjul, C. & Toro, Goodwill, R. (1975). An analysis of the mode of gene action M. A. (1996). Changes in the additive variance of a fitness- affecting pupa weight in Tribolium castaneum. Genetics related trait with inbreeding in Tribolium castaneum. 79,219–229. Journal of Animal Breeding and Genetics 113, 93–97. Harrison, S. & Hastings, A. (1996). Genetic and evolutionary Saccheri, I. J., Brakefield, P. M. & Nichols, R. A. (1996). consequences of metapopulation structure. Trends in Severe inbreeding depression and rapid fitness rebound in Ecology and Eolution 11, 180–183. the butterfly Bicyclus anynana (Satyridae). Eolution 50, James, J. W. (1971). The founder effect and response to 2000–2013. artificial selection. Genetical Research 16,241–250. Saccheri, I., Kuussaari, M., Kankare, M., Vikman, P., Kearsey, M. J. & Kojima, K. (1967). The genetic architecture Fortelius, W. & Hanski, I. (1998). Inbreeding and of body weight and egg hatchability in Drosophila extinction in a butterfly metapopulation. Nature 392, melanogaster. Genetics 56, 23–37. 491–494. Keller, L. F., Arcese, P., Smith, J. N. M., Hochachka, Saccheri, I. J., Wilson, I. J., Nichols, R. A., Bruford, M. W. W. M. & Stearns, S. C. (1994). Selection against inbred & Brakefield, P. M. (1999). Inbreeding of bottlenecked song sparrows during a natural population bottleneck. butterfly populations: estimation using the likelihood of Nature 372, 356–357. changes in marker allele frequencies. Genetics 151, Kidwell, J. F. & Kidwell, M. M. (1966). The effects of 1053–1063. inbreeding on body weight and abdominal chaeta number Sevenster, J. G. & van Alphen, J. M. (1993). A life history in Drosophila melanogaster. Canadian Journal of Genetics trade-off in Drosophila species and community structure and Cytology 8, 207–215. in variable environments. Journal of Animal Ecology 62, Lande, R. (1980). Genetic variation and phenotypic evol- 720–736. ution during allopatric speciation. American Naturalist Sokal, R. R. & Rohlf, F. J. (1981). Biometry. New York: 116, 463–479. W. H. Freeman. Lande, R. (1992). Neutral theory of quantitative genetic Tachida, H. & Cockerham, C. C. (1989). A building block variance in an island model with local extinction and model for quantitative genetics. Genetics 121, 839–844. colonization. Eolution 46,381–389. Tantawy, A. O. & Reeve, E. C. R. (1956). Studies in Latter, B. D. H., Mulley, J. C., Reid, D. & Pascoe, L. quantitative inheritance. IX. The effects of inbreeding at (1995). Reduced genetic load revealed by slow inbreeding different rates in Drosophila melanogaster. Zeitschrift fuW r in Drosophila melanogaster. Genetics 139, 287–297. indukt. Abstammungs- und Vererbungslehre 87, 648–667. Latter, B. D. H. & Robertson, A. (1962). The effects of Templeton, A. R. (1996). Experimental evidence for the inbreeding and artificial selection on reproductive fitness. genetic-transilience model of speciation. Eolution 50, Genetical Research 3, 110–138. 909–915. Lints, F. A. & Bourgois, M. (1984). Population crash, Wade, M. J., Shuster, S. M. & Stevens, L. (1996). In- population flush and genetic variability in cage popula- breeding: its effect on response to selection for pupal tions of Drosophila melanogaster. Genetics, Selection, weight and the heritable variance in fitness in the flour Eolution 16, 45–56. beetle, Tribolium castaneum. Eolution 50, 723–733. Lo! pez-Fanjul, C. & Villaverde, A. (1989). Inbreeding Whitlock, C. M., Phillips, P. C. & Wade, M. J. (1993). Gene increases genetic variance for viability in Drosophila interaction affects the additive genetic variance in sub- melanogaster. Eolution 43, 1800–1804. divided populations with migration and extinction. Lo! pez-Fanjul, C., Ferna! ndez, A. & Toro, M. A. (1999). The Eolution 47, 1758–1769. role of epistasis in the increase in the additive genetic Whitlock, M. C. (1995). Variance-induced peak shifts. variance after population bottlenecks. Genetical Research Eolution 49, 252–259. 73, 45–59. Whitlock, M. C. & Fowler, K. (1999). The changes in Lynch, M. (1988). Design and analysis of experiments on genetic and environmental variance with inbreeding in random drift and inbreeding depression. Genetics 120, Drosophila melanogaster. Genetics 152, 345–353. 791–807. Wijngaarden, P. J. (2000). The genetic basis of eyespot size: Manly, B. F. J. (1986). Multiariate Statistical Methods: A an analysis of line crosses. Heredity (in press). Primer. London: Chapman and Hall. Willis, J. H. & Orr, H. A. (1993). Increased heritable Maynard Smith, J. (1998). Eolutionary Genetics, 2nd edn. variation following population bottlenecks: the role of Oxford: Oxford University Press. dominance. Eolution 47, 949–957. Moreno, G. (1994). Genetic architecture, genetic behaviour, Windig, J. J. (1993). The genetic background of plasticity in and character evolution. Annual Reiew of Ecology and wing pattern of Bicyclus butterflies. PhD thesis, University Systematics 25,31–44. of Leiden. Payne, R. W., Lane, P. W., Ainsley, A. E., Bicknell, K. E., Wright, S. (1951). The genetical structure of populations. Digby, P. G. N., Harding, S. A., Leech, P. K., Simpson, Annals of Eugenics 15, 323–354. H. R., Todd, A. D., Verrier, P. J. & White, R. P. (1988). Wright, S. (1977). Eolution and the Genetics of Populations. Genstat 5 Reference Manual. New York: Oxford Uni- Experimental Results and Eolutionary Deductions, vol. 3. versity Press. Chicago: University of Chicago Press.