INBREEDING, DEVELOPMENTAL STABILITY, AND CANALIZATION IN THE SAND CRICKET GRYLLUS FIRMUS

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1 Evolution, 57(), 00, pp INBREEDING, DEVELOPMENTAL STABILITY, AND CANALIZATION IN THE SAND CRICKET GRYLLUS FIRMUS DENIS RÉALE 1, AND DEREK A. ROFF,4 1 Department of Biology, McGill University, Montreal, Quebec HA 1B1, Canada dreale@po-box.mcgill.ca Department of Biology, University of California, Riverside, California Derek.Roff@ucr.edu Abstract. Inbreeding, the mating of close relatives, is known to have deleterious effects on fitness traits in organisms. Developmental stability (DS) and canalization may represent two processes that allow an organism to maintain a stable development that will produce the fittest phenotype. Inbreeding is thus expected to affect either DS or canalization. We tested if inbreeding affects DS and canalization using an inbreeding experiment on the cricket Gryllus firmus. We compared mean length, fluctuating asymmetry (as an index of DS), and morphological variation (as an index of canalization) of four limb traits between seven highly inbred lines, their F 1 crosses, and outbred lines originated from the same stock population and maintained in the same environmental conditions. We show evidence for moderate inbreeding depression on the four measures of leg length. The nonsystematic difference in fluctuating asymmetry indices between breed types indicates that inbreeding or heterozygosity did not affect DS, or that fluctuating asymmetry is not a reliable index of DS. In contrast, inbreeding appears to affect canalization, as shown by the significantly higher variation in inbred lines compared to other lines. Identical low variation values in the crossbred and outbred lines indicate that heterozygosity could affect canalization. High variation in morphological variation and fluctuating asymmetry within crossbred or inbred lines, however, suggest the effect of recessive deleterious alleles on both canalization and DS. Although the strong correlation in morphological variation among traits suggests that identical genetic mechanisms govern canalization for all the limb traits, the absence of significant correlation in fluctuating asymmetry among traits causes us to reject this hypothesis for DS. For most of the traits, morphological variation and fluctuating asymmetry were not significantly correlated, which support the hypothesis that canalization and DS consist in two distinct mechanisms. Key words. Fluctuating asymmetry, Gryllus firmus, inbreeding depression, limb length, morphological variation. The phenotype of an organism depends in part on its genetically determined developmental trajectory. Developmental homeostasis is the ability of an organism to maintain a stable development that will produce the fittest phenotype, either in different environmental conditions (canalization) or in specific environmental conditions (developmental stability; Waddington 194; Lerner 1954). Canalization and developmental stability (DS) are assumed to correspond to two different processes, although this has been rarely tested (Hoffmann and Woods 001), and these two processes have often been confounded (Debat and David 001). Recently, Debat and David (001, p. 50) proposed defining canalization as a set of processes historically selected to keep the phenotype constant in spite of genetic or environmental variation (see also Hoffmann and Woods 001), whereas DS is a set of mechanisms historically selected to keep the phenotype constant in spite of small, random irregularities potentially inducing slight differences among homologous parts within individuals (Debat and David 001, p. 50 and references therein; see also Lerner 1954). In this paper, we analyze the relation between inbreeding depression and developmental homeostasis in the cricket Gryllus firmus. Because of its known deleterious effects on fitness traits in organisms, inbreeding would be expected to affect either DS or canalization. We first tested for the presence of inbreeding depression on four morphological traits (forefemur, midfemur, hindfemur, and hindtibia lengths) by comparing seven highly inbred lines with their F 1 crosses (crossbred lines) and with outbred lines. Inbred and outbred lines originated from the same stock population and were 00 The Society for the Study of Evolution. All rights reserved. Received March 8, 00. Accepted November 11, maintained in the same environmental conditions. Inbreeding depression on weight, fecundity, and muscle mass has been found for these cricket lines (Roff 1998, 00; Roff and DeRose 001). Shorter limbs are thus expected in inbred lines compared to crossbred or outbred lines. We then tested if inbreeding affects DS and canalization in the four traits under study among these lines. The most common measure of DS is fluctuating asymmetry (FA), the nondirectional deviation from bilateral symmetry (Van Valen 19). Several studies have found evidence for increasing FA with inbreeding, using individuals or populations varying in heterozygosity (Leary et al. 198, 1984; Alibert et al. 1994) or comparisons of inbred lines with their F 1 hybrids (Mather 195; Beardmore 190; Reeve 190), whereas others have failed to find such a relationship (heterozygosity: Clarke et al. 199; Gilligan et al. 000; Carchini et al. 001; controlled inbreeding experiments: Fowler and Whitlock 1994; Sheridan and Pomiankowski 1997; Hosken et al. 000). This inconsistency in the results has raised doubts about the generality of FA as an index of DS and about the reliability of FA to detect inbreeding effects (see reviews in Palmer and Strobeck 198; Møller and Swaddle 1997; Vøllestad et al. 1999; Gilligan et al. 000). Phenotypic variance has been used as an index of canalization or DS (Robertson and Reeve 195; Lerner 1954; Whitlock and Fowler 199; Woods et al. 1999). However, it seems that phenotypic variance is more appropriate as an index of canalization (Debat and David 001; Hoffmann and Woods 001). Inbreeding has been shown to affect phenotypic variance (Robertson and Reeve 195; Lerner 1954; Mitton and Grant 1989; Falconer and Mackay 199;

2 598 D. RÉALE AND D. A. ROFF Whitlock and Fowler 199; see table. in Lynch and Walsh 1998 for a review). Inbreeding may affect DS and canalization because homozygotes lack the enzymatic diversity that allows the heterozygotes to buffer their development from perturbations during their development (Lerner 1954). Alternatively, DS and canalization would decrease because of the effect of specific deleterious recessive alleles whose effects are revealed with inbreeding or as a result of the breakup of coadapted gene complexes with inbreeding or extreme outbreeding (Mather 195; Thoday 1958; Clarke 199). We tested these two hypotheses by comparing four indices of fluctuating asymmetry (FA1, FA, and FA10 both standardized and unstandardized for trait size; Palmer 1994) on limb length between inbred, crossbred, and outbred lines. We also compared two indices of canalization (VAR, among-individual variance within a line, standardized and unstandardized for trait size; Klingenberg and McIntyre 1998; Debat et al. 000) between breed types. FA presents the advantage that either genetic or environmental effects act in the same way on both sides of an individual (Palmer 1994). In contrast, VAR indices may differ between breed types because of either genetic or environmental reasons. According to quantitative genetic models the phenotypic variance of a trait V P V G V I V E, where V G corresponds to the genetic variance (additive, dominance, and epistasis components), V I is the variance due to genotype-environment interaction, and V E is the variance due to environmental effects (Falconer and Mackay 199; Lynch and Walsh 1998). V E can be decomposed in general and specific environmental effects (Lynch and Walsh 1998). The former corresponds to environmental conditions experienced by a unit group (family, group, or population), whereas the latter corresponds to the effects on one particular individual. Within-line V G for a trait is assumed to decrease in proportion to the coefficient of inbreeding F, following the equation (1 F)V Go, where V Go is the genetic variance in the outbred population (Falconer and Mackay 199). Within highly inbred lines (i.e., F 1), V G V I 0, and thus V P V E. All the phenotypic variance among inbred lines is thus attributed to between-line genetic variance (Falconer and Mackay 199). F 1 hybrids (crossbred) between two strongly inbred lines are all genetically identical but are heterozygous at the loci where their parent lines differ (Falconer and Mackay 199; Lynch and Walsh 1998). This means that on average the level of heterozygosity will be identical among crossbred lines but that it may differ at a particular locus. In a constant controlled environment, as in a laboratory experiment, general environmental effects can be considered constant for all individuals and all the lines, and therefore variation in VAR among lines would reflect differences among those lines in their ability to maintain a constant phenotype in spite of small environmental variation (i.e., environmental canalization; Gibson and Wagner 1999; Debat and David 001). In contrast, VAR within an outbred line results from both genetic and environmental variance, and variation in VAR among lines would be due to variation in genetic or environmental variance within each line and to difference between the line in their ability to canalize their development. Comparison of breed types allows one to test for the hypotheses on the genetic mechanisms of DS and canalization. Heterozygosity is restored in crossbred lines. Therefore, higher FA and VAR in inbred compared to the two other breed types and equal FA and VAR between crossbred and outbred lines would indicate that heterozygosity is the main effect responsible for genetic stress on DS and canalization, respectively. In contrast, higher FA and VAR in crossbred than in outbred lines would indicate effects of recessive alleles on DS and canalization, respectively. This should be confirmed by the presence of significant among-line variance in FA1 and FA (indices of DS measured at the individual level) within inbred and crossbred lines, because heterozygosity is equal among the lines within those breed types. We looked at the association in DS and canalization indices between traits. A positive association would indicate that the same mechanisms are responsible for variation in canalization (VAR) or DS (FAs) among lines. The absence of association would indicate that the traits may be differently canalized or stable. Canalization and DS have been assumed to correspond to two distinct processes (Waddington 1957), and some studies having found no association between morphological variability and DS support this hypothesis (Debat et al. 000; Hoffmann and Woods 001). Alternatively, because of a positive association between indices of canalization and those of DS found in some studies (Clarke 1998b; Klingenberg and McIntyre 1998; Woods et al. 1999), Clarke (1998b) has suggested that both canalization and DS may rely on the same or on interrelated genetic mechanisms, particularly when the trait under study is subject to strong stabilizing selection (Clarke 1998b). To test this hypothesis we looked at the association between VAR and FA for each trait. Assuming that locomotory traits in our study are of critical morphofunctional importance, we should observe a positive correlation between trait variability and FA (Debat et al. 000). MATERIALS AND METHODS Experimental Protocol We used females from seven inbred lines, crosses between these lines, and seven outbred lines. These lines were derived from a stock originated from about 40 individuals (sex ratio 1:1) collected in northern Florida in This stock was maintained in the laboratory (about individuals per generation) at 5 C and fed with Purina (Alberta, Canada) rabbit chow. Inbred lines were obtained by brother-sister matings over 14 generations. Outbred lines were made by mating females within each line with males randomly chosen from the stock population. Full details of the breeding procedure used in this experiment are given in Roff (00). Thirty-four inbred and 0 outbred lines were produced by brother-sister mating at each generation for inbred lines and by mating a single female from the outbred line with a male taken at random for the stock for outbred lines, respectively (Roff 00). Roff (00) could not show any significant differential extinction rate of inbred and outbred lines, suggesting the absence of purging of inbreeding depression in that experiment. At generation 14 inbred lines are assumed to have reached a high level of inbreeding (F 0.951). A complete diallel-cross was made among the seven inbred lines. Some

3 INBREEDING AND DEVELOPMENTAL HOMEOSTASIS 599 crosses between inbred lines could not be made for technical reasons (primarily generations too far out of phase), and 8 of the 49 (7 7) potential crosses were used in this study. To maintain an approximately balanced design we used individuals from the combined reciprocal crosses, which gave 19 crosses (i.e., two of the 1 combinations were missing). Our results are thus based on comparisons between seven inbred, 19 crossbred, and seven outbred lines. Offspring of between- and within-line crosses were maintained in batches of 40 with food and water ad libitum in 4- L buckets placed in growth chambers at 8 C and a photoperiod of 15:9 L:D. Each cross was distributed among five separate cages to account for potential common environmental effects. For practical reasons we used only females in these analyses. One-week posteclosion females were sacrificed and preserved in alcohol. Measurement and Estimates of Developmental Stability and Canalization Females from inbred, crossbred, and outbred lines were randomly chosen for measurements. We attempted to obtain at least five females per line evenly distributed among the cages (range in the number of females per line 1). For each female, left and right forefemurs, midfemurs, hindfemurs, and hindtibias were gently removed with forceps and placed under a microscope connected to a camera and an image analyser. Forefemur (FFL), midfemur (MFL), hindfemur (HFL), and hindtibia (HTL) length were measured twice by the same person (DR). The plate containing legs was rotated between the two series of measurements on the same individual, so that replicates were obtained from different digitized views. We excluded individuals with damaged legs. We assessed the significance of FA relative to measurement error with a mixed-model analysis of variance (ANOVA; Palmer and Strobeck 198; Palmer 1994). For each individual, we calculated trait mean value (mean [R L]; four measurements), FA1 ( L R /), and FA ({ L R /[(R L)/]}/), as indices of FA (Palmer 1994). We also calculated line values of FA5 [ (L R) /N; Palmer 1994] and of FA9a (1 R L ; Windig and Nylin 000). Because we have shown difference in within-line variance among breed types (see Results) FA9a ( R L / R L TS, where TS is trait size) was biased in our case and was not used. Results for FA1 and FA5 were similar to those for FA. For each line, we ran a mixed-model ANOVA, with trait length as the dependent variable, individual as a random factor, side as a fixed effect, and their interaction (Palmer 1994; Debat et al. 000; Palmer and Strobeck 00). This method permits to test for the significance of variance among individuals (i.e., individual effect), directional asymmetry (i.e., side effects), and nondirectional asymmetry (i.e., individual side interaction). Moreover, it allows one to obtain estimates of FA (FA10) and variability (VAR) unbiased by measurement errors (Palmer 1994; Debat et al. 000; Palmer and Strobeck 00). FA10 was calculated following the formula FA i proposed by Palmer and Strobeck (00), where i is the variance caused by the interaction between individual and side. Calculated this way, FA10 can be directly compared to FA1 (Palmer and Strobeck 00). We used as an index of canalization the variance among individuals (VAR j ) estimated from the mixed-model ANOVA (Debat et al. 000). We calculated skewness and kurtosis of signed FA, for each line and each trait, and tested their significance following Palmer (1994). We tested for the independence between FA1 and trait size ([R L]/) by running Spearman s correlation rank tests (Palmer 1994). Descriptive statistics of FA for each line are not presented here but can be supplied by the authors on request. Because of small sample size for some lines and the large number of comparisons ( lines 4 traits 1 comparisons), results have to be considered with caution. These conditions may increase both Type I or Type II error chance. However, some interesting patterns emerged from these analyses. In very few occasions, we could detect a significant association between FA1 and trait size (5/1, 4%), or a significant skewness or kurtosis (9/1, 7%) that would indicate departure from normality. Only four cases (4/1, %) side effects indicating directional asymmetry were significant. In 119 cases (90%), the variance among individuals ( j ) was significantly higher than the variance caused by measurement errors ( m ). In 54 cases (41%), the individual side interaction variance ( i ) was significantly higher than m. Finally, in 1 cases (1%), the estimate of i was negative and thus replaced by zero for subsequent analyses. Several results from preliminary analyses suggest that low and nonsignificant i for some lines and traits reflects the absence of FA rather than sampling bias; contrary to what we could expect in the case of sampling bias, m did not decline significantly (all P 0.1), and i did not increase significantly (all P 0.47) with the number of individuals per line. Moreover, except for FFL (r 0.8, P 0.05), did not decrease significantly with increasing i m (MFL: r 0.19, P 0.0; HFL: r 0.9, P 0.10; HTL: r 0.1, P 0.5). Although we did not detect strong evidence for an association between trait size and FA within each line, trait size differed among breed types and among lines within breed type, and FA may depend on mean trait size. We therefore corrected both FA and VAR indices with trait size. FA10 size is equivalent to FA10 standardized by trait size (or to i from the mixed-model ANOVA on the natural logarithm of trait size; Palmer and Strobeck 00). VAR size equals VAR divided by mean trait length. Evidence for Inbreeding Depression To test if trait length varied among breed types (inbred, crossbred, and outbred), we used a nested ANOVA on individual trait length with trait and breed type as fixed effects and with line number as a random factor nested within breed type and trait. Inbreeding depression would be revealed by smaller size of traits in inbred lines relative to outbred lines. Inbreeding depression ( ) was estimated as 100(1 X i /X o ), where X i and X o are the average values for inbred and outbred lines, respectively. Inbreeding, Developmental Stability, and Canalization We tested if FA varied among breed types by using the nested ANOVA with trait and breed type as fixed effects and

4 00 D. RÉALE AND D. A. ROFF TABLE 1. Nested analyses of variance comparing variation in mean trait length on breed type (inbred, crossbred, and outbred lines), for each trait separately (FFL, forefemur length; MFL, midfemur length; HFL, hindfemur length; HTL, hindtibia length). Line number (nested within breed type) was included as a random effect. Source df F P FFL MFL HFL HTL breed type line no. (breed type) breed type line no. (breed type) breed type line no. (breed type) breed type line no. (breed type), 0 0, 7, 0 0, 7, 0 0, 7, 0 0, with line number as a random factor nested within breed type and trait. We ran a fully factorial ANOVA using VAR, VAR size, and FA10, and FA10 size indices as a dependent variable with breed type and trait as factors (Palmer 1994; Palmer and Strobeck 00). Following the hypothesis that DS and canalization decrease with inbreeding, we expect to find higher FA (i.e., FA1, FA, FA10, and FA10 size ) and VAR (VAR and VAR size ) in inbred lines compared to crossbred and outbred lines, for both mixed-model and factorial ANOVAs. Significantly lower FA or VAR in crossbred compared to outbred would confirm the effect of recessive alleles on DS or canalization. Significant among-line variance within a breed type in FA (mixed-model ANOVA) would indicate some effects of recessive alleles on DS. Correlations among s and Relationship between Developmental Stability and Canalization We tested if similar genetic mechanisms are involved in DS or canalization of different traits by correlating line-mean values of VAR and FA10 between traits. We only considered inbred and crossbred lines, because all the variation in VAR between two of those lines can be attributed to their relative genetic ability to undergo canalized or stable development. In contrast, additive genetic and environmental influences both affect VAR and thus variation in VAR between lines may not represent only variation in canalization or DS (see introduction). We analyzed the relation between canalization and DS by calculating line-mean correlations between VAR and FA10 for each trait. As for correlation among traits, we only considered inbred and crossbred lines. RESULTS Comparing FA10 with mean-line FA1 (or FA10 size with mean-line FA) allows one to determine the effects of measurement errors on FA indices (Palmer and Strobeck 00). For each trait, the slope of the regression of the average line value of FA1 on FA10 indicates that the bias on FA estimates caused by measurement errors was stronger for small values of FA (HFL: b , t 7.4, P 0.001; MFL: b , t 7.88, P 0.001; FFL: b , t 9.7, P 0.001; HTL: b , t 9.4, P 0.001; N ). The same patterns were observed when comparing FA10 size with FA (results not shown). There was a strong relationship between VAR and V P (HFL: b 1.00 FIG. 1. Mean length and standard errors for inbred, crossbred, and outbred lines in Gryllus firmus. 0.01, t 98.9, P 0.001; MFL: b , t 5.7, P 0.001; FFL: b , t 57.15, P 0.001; HTL: b , t 5.9, P 0.001; N ), indicating that, notsurprisingly, V P is not biased by measurement errors. Evidence for Inbreeding Depression Because of the significant interaction between trait and breed type (F,.4, P 0.0) in the nested ANOVA (i.e., trait length trait breed type [trait breed type] line nested within trait and breed type), we ran ANOVAs lines nested in breed type for each trait separately (Table 1). This interaction appears to be caused by the absence of significant variation among breed types for HTL, whereas variation was moderately significant for FFL and highly significant for both MFL and HFL (Fig. 1). There was significant variation among lines within breed types and trait (Table 1). Inbreeding depression estimates ( 100%) for limb lengths were.8 1.1% (FFL), % (MFL), % (HFL), and % (HTL), respectively. Inbreeding, Developmental Stability, and Canalization FA1 did not vary significantly with breed type, but varied significantly with trait (Table A), HTL having the highest FA (Fig. A). FA varied significantly among traits (Table B), HFL having the lowest and HTL having the highest FA (Fig. B). There was a significant variation in FA among breed types (Table B), outbred having lower FA than inbred and crossbred (outbred: ; inbred: ; crossbred: ). Standardizing for trait length led to an increase in FA for FFL and MFL relative to HTL and particularly HFL (Fig. A, B). Standardized and unstandardized indices of FA10 showed significant difference among traits, but no significant differences were found among breed types and on the interaction between trait and breed

5 INBREEDING AND DEVELOPMENTAL HOMEOSTASIS 01 TABLE. Nested analysis of variance of FA1 and FA on breed type (inbred, crossbred, and outbred lines) and trait (FFL, forefemur length; MFL, midfemur length; HFL, hindfemur length; HTL, hindtibia length). Line number (nested within breed type and trait) was included as a random effect. (A) FA1 trait Line no. (breed type type) (B) FA trait Line no. (breed type trait) MS df 1 df F P FIG.. Mean values of FA1 (A), FA (B), FA10 (C), FA10 size (D), VAR (E), and VAR size (F) and their standard errors for different traits in inbred, crossbred, and outbred lines lines in Gryllus firmus. FFL, forefemur length; MFL, midfemur length; HFL, hindfemur length; HTL, hindtibia length.

6 0 D. RÉALE AND D. A. ROFF TABLE. Two-way analyses of variance of FA10 and VAR indices (both standardized and nonstandardized by mean trait length) on breed type (inbred, crossbred, and outbred lines) and trait (FFL, forefemur length; MFL, midfemur length; HFL, hindfemur length; HTL, hindtibia length). Line number (nested within breed type and trait) was included as a random effect. (A) FA10 trait (B) FA10 size trait (C) VAR trait (D) VAR size trait MS df F P type (Table A, B). Patterns of FA10 and FA10 size are similar to FA1 and FA, respectively (Fig. A D), suggesting that partition of measurement errors did not change estimates of FA in our study. Among-individual within-line variation (standardized and unstandardized VAR) varied significantly among breed types and among traits, but the interaction between trait and breed type was not significant (Table C, D). For both indices, inbred lines had a significantly higher VAR than both outbred and crossbred lines (Student-Newman-Keuls test: P 0.05), but no significant difference was found between crossbred and outbred lines. Hindfemur and hindtibia, the longest limbs, showed the highest variability, although this did not change after standardizing VAR for limb length (Fig. E, F). Correlations among s in Canalization and Developmental Stability Correlations among traits were estimated with inbred and crossbred lines only (see Materials and Methods). All the correlations in line-mean lengths or in VAR among traits were positive, strong, and significant (Table 4A, B). In contrast, no particular pattern emerges from the correlations in FA10 (Table 4B) or in FA (not shown) among traits. Relationship between Developmental Stability and Canalization Correlations were estimated with inbred and crossbred lines only and on standardized values of FA10 and VAR (see Materials and Methods). The correlation between VAR and FA10 was positive and significant only in HFL (FFL: r 0., P 0.8; MFL: r 0.0, P 0.14; HFL: r 0.5, P 0.00; HTL: r 0.1, P 0.5; N ). TABLE 4. Line-mean correlations between traits (see Table for abbreviations) in Gryllus firmus. (A) Mean length (above the diagonal). (B) VAR (above) and FA10 (below). A indicates correlations significant ( 0.05) after sequential Bonferroni corrections. Number of lines (inbred 7; crossbred 19). (A) FFL MFL HFL HTL (B) FFL MFL HFL HTL FFL MFL HFL HTL *** 0.84*** 0.79*** 0.88*** DISCUSSION 0.8*** 0.89*** *** 0.79*** 0.84*** 0.84*** 0.79*** 0.88*** Evidence for Inbreeding Depression The existence of inbreeding depression in G. firmus is shown by a decrease in limb lengths in inbred compared to outbred lines. Evidence for inbreeding depression in various life-history and morphological traits has been found in this population of G. firmus at both moderate (F 0.5; Roff 1998) and strong inbreeding coefficients (F 0.951; data from the same experiment, Roff 00). As expected, our inbreeding depression indices were less than 5%, which is equivalent to estimates in morphological traits and is low compared to what is usually found for life-history traits (Roff 1997; DeRose and Roff 1999). This low inbreeding depression can be explained by the low directional dominance of morphological traits relative to life-history traits (Falconer and Mackay 199; DeRose and Roff 1999). Limb lengths of crossbred lines were not different from those of inbred lines or were intermediate between inbred and outbred lines, a consistent patterns shown over the four traits (Fig. 1). This suggests some but not complete restoration to the outbred state. This, however, is at variance with the life-history results that showed crossbred to be higher than outbred (Roff 00). If the inbred lines were overall genetically depauperate with respect to additive component compared to the outbred, then crossing the lines would produce the intermediate results. Although Roff (00) found similar extinction rates in inbred and in outbred lines, we can not reject the possibility for selection in inbred lines during the experiment. Therefore, the seven inbred lines used in our diallel cross may be characterized by properties that differ from those expected under neutrality. The purge of deleterious alleles, however, has not been strong enough, and we were still able to find some inbreeding depression in life-history (Roff 00) and morphological traits (this paper). Variability or Fluctuating Asymmetry as Bioindicators of Genetic Stress? Although our experiment does not represent a natural situation (strong inbreeding coefficient, controlled environment) it has the advantage of controlling for potential effects that may dampen the relationship between inbreeding and

7 INBREEDING AND DEVELOPMENTAL HOMEOSTASIS 0 either DS or canalization. Evolutionary ecologists and conservation biologists are interested in finding reliable bioindicators of genetic or environmental stresses in natural populations. The high sensitivity of FA to stress is assumed to allow detection of stress on an organism before any effects on fitness leading the population to extinction (Clarke 1995). FA has often been used as an index of genetic stress within or between populations (Clarke 1995; Møller and Swaddle 1997). Considering that DS should increase with heterozygosity (Lerner 1954), a good index of DS should be able to detect difference in stability between homozygous and heterozygous individuals, before the occurrence of inbreeding depression. FA has the advantage that there is little genetic variance for unsigned difference between the sides of a trait, and it has been considered as the most reliable indicator of DS (Palmer 1994). The absence of difference in FA indices (excepted FA) between breed types indicates that FA does not appear to be a good index of DS or that DS is not affected by inbreeding in the crickets. Although our study shows that small values of FA may be subject to relatively larger measurement errors than high values of FA, comparison between FA1 and FA10 indicates that measurement errors did not affect the relative differences in FA among traits or among breed types. Partitioning for measurement errors did not lead to significant difference in FA among breed types. Standardizing FA1 for trait length (i.e., FA) led to significant difference in FA index among breed types; FA was lower for outbred than crossbred and inbred lines but crossbred and inbred lines showed similar FA, suggesting that heterozygosity is not responsible for variation in DS among breed types. This result, however, should be considered with caution, because FA10 size (the equivalent to FA after partitioning for measurement errors) did not show any significant difference among breed types. Variation in FA among breed types may thus be enhanced by measurement errors (Palmer 1994). The nonsignificant difference in FA indices among breed types cannot be explained by the lack of power of our analysis because we could detect a significant effect of the trait and significant differences between lines for the same data. An alternative explanation on the absence of association between FA and inbreeding is that there may be a threshold relationship between genetic diversity and FA (Fowler and Whitlock 1994). The high inbreeding coefficient (F 0.951) for inbred lines in this experiment, however, prevents such an explanation for absence of variation in FA among breed types. Vøllestad et al. (1999) found a stronger relationship between FA and heterozygosity for ectotherms, and thus crickets or insects are a good model for the study of DS. Leung et al. (000) have proposed to use CFA indices (FA of multiple traits) to increase the reliability of FA to detect stress. Our analyses on CFA did not show any differences among breed types (not reported here), suggesting that the absence of FA difference between inbred, crossbred, and outbred lines did not result from the low reliability of FA indices. Our results highlight the fact that the lack of difference in FA between two populations or between individuals within a population should not be interpreted systematically as evidence for an absence of genetic stress (see also Sheffer et al. 1999; Hosken et al. 000). Variability indices (VAR) appears to be convenient in estimating canalization under genetic stress, inbred lines having higher VAR values than crossbred and outbred lines. However, its use as a bioindicator of stress in natural populations is not straightforward because of the multiple determinants acting on phenotypic variation of a morphological trait; part of VAR in outbred lines may be caused by genetic variance, whereas V A is assumed to be null in inbred and crossbred lines. Differences among s in Developmental Stability and Canalization Significant differences among traits in FA was found for the four FA indices, confirming results from many studies (Palmer and Strobeck 198; Leung et al. 000). Shorter limbs (forefemur and midfemur) had lower FA than longer limbs (hindfemur and hindtibia), supporting the idea that DS could be related to trait size (Palmer and Strobeck 198). However, this difference should not be considered as a purely sizedependent FA, because we were not able to detect any size effect on FA within lines and traits. Moreover, hindfemur, the longest limb, showed the lowest FA relative to its size when compared to other traits (see Fig. B, D). Hindtibia showed a higher FA (both standardized and nonstandardized) than femurs. This may be caused by differential DS for traits that have developed at different phases or by differences in the strength of selection on both types of traits (Clarke 1998b). However, we have no evidence that tibia length may be under stronger directional selection than femur length. Hindfemur and hindtibia showed high values of VAR compared to fore- and midfemurs (Fig. E, F). This may indicate that developmental interactions dominate within a module (e.g., hind limb) and are less strong within modules (Klingenberg et al. 001). We found a high consistency in VAR between traits, indicating lines more canalized for one trait also having higher canalization for other traits. Our results indicates that withinline variability in one trait can be used as a good predictor of within-line variability for the whole organism. Such a strong consistency in VAR between traits suggests genetic differences among lines in their ability to canalize traits. In contrast, correlations in FA (FA10) between traits were weak and nonsignificant, suggesting that DS varies among traits or that FA is not a reliable index of DS. This pattern has been found in many other studies on FA (see Clarke 1998a) and has been interpreted as a consequence of the low repeatability of FA (Whitlock 199; Van Dongen 1998; Houle 000; Van Dongen and Lens 000) or of the random errors occurring during development of separate modules (Clarke 1998a,b; Klingenberg et al. 001). Canalization and Developmental Stability Same or Different Processes? We found an absence of significant correlations between VAR and FA10 for most of the traits. This, along with the among-trait correlations, suggests that canalization and DS are characterized by different genetic mechanisms. Recent studies have shown relation between canalization and DS (Clarke 1998b; Klingenberg and McIntyre 1998; Woods et al. 1999), whereas others have found independence between

8 04 D. RÉALE AND D. A. ROFF these two mechanisms (Debat et al. 000; Hoffmann and Woods 001). These contrasted results show that morphological variation and FA should not be used indifferently as indices of canalization and DS (see Debat et al. 000; Debat and David 001). Clarke (1998b) suggested that although canalization and DS consist in two different genetic and functional mechanisms, both may be similarly affected by the same genetic condition. This may hold particularly when traits are directly related to fitness (Clarke 1998b; Debat et al. 000). Limbs length in crickets can be assumed to be of strong functional importance; thus, our findings do not support this last hypothesis. Under the conditions of our experiment, the higher VAR for inbred lines compared to crossbred and outbred lines supports the idea that heterozygosity is beneficial for canalization or that the deleterious effects of recessive alleles are hidden by the heterozygote state. In contrast, the absence of difference in FA indices between inbred and both crossbred and outbred lines suggests that heterozygosity is not related to DS. There is still important variation in FA1 and in VAR among lines within each breed type (Table 1B). Some inbred lines showed DS identical to outbred or crossbred lines, and the largest range of VAR size was observed among crossbred lines ( ) relative to inbred ( ) and outbred ( ) lines. Therefore, heterozygosity alone cannot explain variation in canalization among the lines and genetic effects other than heterozygosity may also affect either canalization or DS; for instance, the existence of recessive deleterious alleles ( poor-homeostasis genes, Thoday 1958) for DS (Clarke 199). Inbred lines would differ in the number of such recessive alleles that were fixed by genetic drift during the inbreeding experiment, and heterozygosity would be restored for a different proportion of these loci in the F 1 progeny of each crossing, depending on the genetic composition of both parental lines at these loci. This helps explain why results from studies on the relationship between inbreeding and DS, comparing a low number of inbred lines and their F 1, would diverge (Fowler and Whitlock 1994). In conclusion, despite evidence for inbreeding depression shown on morphological (this study) and life-history traits (Roff 00) and for a lower canalization in inbred individuals shown by VAR, no relationship between inbreeding and DS could be detected from FA. Moreover, VAR showed higher consistency among traits than FA. Our results suggest that both canalization and DS do not result from heterozygous advantage alone and may also be caused by the effects of recessive deleterious alleles. Comparison among traits suggests that traits within an organism differ in their stability, but that limbs appeared to share the same canalization mechanisms. The low relation between FA and VAR found for all the traits does not support the hypothesis that canalization and DS are characterized by the same genetic mechanisms. The absence of relationship between FA and inbreeding does not automatically imply the absence of relationship between developmental homeostasis and inbreeding. Studies on inbreeding and development homeostasis should thus not only use FA as an index of DS but should also add comparisons in trait means and in phenotypic variances, when possible. ACKNOWLEDGMENTS We thank G. Stirling, P. O. Cheptou, and two anonymous reviewers for their valuable comments. 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