Journal of Heredity Advance Access published online on May 13, 2008
Journal of Heredity, doi:10.1093/jhered/esn026
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Effect of Mutations on Developmental Stability and Canalization in Morphological Traits in Drosophila ananassae
From the Genetics Laboratory, Department of Zoology, Banaras Hindu University, Varanasi 221 005, India
Address correspondence to B. N. Singh at the address above, or e-mail: bnsingh{at}bhu.ac.in
The present study was designed to determine the effects of visible mutations of large effect on developmental stability and canalization in different morphological traits, namely, sternopleural bristle number, wing length, wing to thorax ratio, ovariole number, and sex comb tooth number (SCTN) in Drosophila ananassae. We have compared the mean trait size, fluctuating asymmetry (FA) (as an index of developmental stability), and morphological variation (as an index of canalization) of different mutant strains (yellow body color, y; claret eye color, ca; plexus wing, px; spread wing, spr; ebony body and sepia eye color, e se; yellow body and claret eye color, y ca; and cardinal eye color, curled wing, and ebony body color, cd cu e) with wild-type strain. The mean trait size of all morphological traits differs significantly among the wild-type and mutant strains. The wild-type and mutant strains vary significantly for the morphological variation and also for the levels of the FA in different morphological traits. However, we have found no increase in either the variance or in the degree of FA with the increase of the mutations (except in SCTN in y mutant). The plausible reasons for the variation in wild-type and mutant strains with particular reference to developmental stability and canalization have been discussed.
Phenotypic variations provide the raw material for evolution on which natural selection acts, but understanding its nature and generation is an important challenge for evolutionary biologists. Variability, defined as ability to vary (Wagner and Altenberg 1996) often results from 2 antagonistic trends: sources of variation (mutations, environmental effect, and developmental errors) and a set of regulatory processes, including buffering and enhancing mechanisms (Debat and David 2001). The 2 major buffering mechanisms, which control the phenotypic variability, are canalization and developmental stability. Former is a set of processes historically selected to keep the phenotype constant in spite of genetic or environmental variation whereas the latter is defined as 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 individual (Debat and David 2001; Reale and Roff 2003). In the populations, canalization is estimated by the interindividual variance, whereas developmental stability is measured by fluctuating asymmetry (FA), that is, the intraindividual variation due to small random differences between left and right sides (Santos et al. 2005). Buffering mechanisms could be impaired by the environmental stress or by mutations and thus create a situation where the hidden reserve of genetic variations is exposed to the natural selection (Hallgrimsson et al. 2002).
Mutations provide biological diversity by generating variations that fuels evolution (Rifkin et al. 2005), and, therefore, it is considered that the mutant phenotypes are more variable than wild type (Willkins 2002), which may be explained as the significant mutant phenotypes represent the developmental configurations that have not undergone selection for canalization and are more sensitive to the environmental perturbations also (Hallgrimsson et al. 2002). Previous studies have mainly focused on the changes in the trait means and the variability (canalization and developmental stability) in mouse, Drosophila, and sheep blowfly (McKenzie and Clarke 1988; Bourguet 2000; Debat et al. 2000; Indrasamy et al. 2000; Willmore et al. 2006).
The aim of the present study was to determine whether the visible mutations of large effect (described in Table 1) have any effect on canalization and developmental stability of different morphological traits (wing length [WL], wing to thorax [W/T] ratio, sternopleural bristle number (SBN), sex comb tooth number [SCTN], and ovariole number [ON]) using a drosophilid fly as a model organism. Further, we have tried to endeavor the following questions: 1) Does variability of mutant strains is higher than that of wild type? 2) Whether the buffering mechanisms are more robust in wild-type than the mutant strains? 3) Is there any increase in the magnitude of FA and variance with the increase of mutations (single, double, and triple) in mutant strains?, and finally 4) Does the effect of mutations is trait and sex specific.
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Our study is different from the previous studies in Drosophila (e.g., Stearns et al. 1995; Bourguet 2000; Milton et al. 2003, 2006; Dworkin 2005; Santos et al. 2005) in different respects: first, we have investigated the effect of mutations on FA and canalization without any introgression of mutations in any line; second, the model system that we have used is Drosophila ananassae Doleschall 1858, which occupies a unique status among the Drosophila species due to the presence of certain unusual genetic features (Singh 2000). It is a cosmopolitan and domestic species belonging to the ananassae subgroup of the melanogaster species group and is one of the most common species in tropical and subtropical regions of the world (Singh 1996, 2000), and the third, we have measured canalization and developmental stability in different morphological traits, namely, SBN, WL, W/T ratio, SCTN, and ON.
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Drosophila Strains and Rearing Protocol
The stocks used in the present study are shown in Table 1. The wild-type strain (Skt) was established from naturally impregnated females collected from Shaktinagar, Uttar Pradesh, India. All the mutant strains except spr and yca were kindly provided by Professor M. Matsuda, Kyorin University, Japan. Spread (spr) is a spontaneous mutation from a wild-type stock of D. ananassae (Yadav and Singh 2003) whereas the yellow body and claret eye (y ca) mutant stock was generated in the laboratory by crossing virgin flies of claret eye and yellow body color. All the strains were maintained in the laboratory on simple yeast agar culture medium at 25 °C in an incubator.
Trait Measurement
Except, thorax length (TL), different morphological traits, namely WL, W/T ratio, SBN, SCTN, and ON, were scored on both left and right sides in 50 males and 50 females from wild-type and mutant strains. The traits were measured as described in Vishalakshi and Singh (2006): WL (absolute length between the anterior crossvein to the distal tip of the third longitudinal vein), TL (from anterior end of the thorax to the posterior end of the scutellum), W/T ratio (calculated from the data of wing and TLs), SCTN (total number of teeth present on the first, second, and third tarsal segments), SBN, and ON were scored. However, WL and W/T ratio were not measured in the cd cu e mutants because the wings are curled, so it is not possible to measure these traits correctly.
Data Analysis
The mean trait size of different morphological traits was calculated as the average value of right and left side (R + L)/2 for different morphological traits in wild-type and mutants strains. Then, the paired t-test was employed for comparing the mean value of different morphological traits between mutant strain and wild-type strain.
Measuring Canalization
For a given trait, the phenotypic variance can be partitioned into genetic and environmental component (Falconer and Mackay 1996):
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VE = VEm + VEg + VEs where VEm is microenvironment variance, that is the environment shared by the individuals such as nutrition and temperature. VEg is general within variance between individuals (residual) or micro environmental variance and calculated by coefficient of variation (CV), and finally VEm is specific within individual usually measured by FA (Falconer and Mackay 1996). In the present case, the macro environmental component was kept constant; therefore, VE = VEg + VEs. We have calculated both variance and CV in wild-type and different mutant strains. Further, fluctuating asymmetry was calculated following the framework laid out by Palmer and Strobeck (1986, 2003).
Asymmetry Determination
Measurement error can cause biased estimates of FA, and repeat measurements should be undertaken to ensure that FA is detectable and there is no confounding effect associated with directional asymmetry (Palmer 1994; Woods et al. 1999). A repeatability analysis on all the morphological traits using 32 flies selected at random from a laboratory culture at the onset of the study. Counts were made on different days and in different order. The magnitude of ME was assessed (following the recommendations of Palmer 1994) by 2-way mixed model analysis of variance, where sides were entered as fixed factor and individuals as a random factor. The resultant variance estimates due to ME, as a percentage of the between-sides variance representing FA (side x individual interaction mean square) were 0%, 0.147%, 0.0022%, and 0.23% for FA in SBN, WL, SCTN, and ON, respectively. This suggests that the variability between sides due to ME was negligible relative to actual trait asymmetry.
Individual asymmetry was measured as D = R – L, where R is the value of the trait on the right side and L is the value of the trait on the left side. For directional asymmetry, one-sample t-test on the signed differences (R – L) for each trait was performed to determine whether the mean values differ from zero (Palmer 1994). For antisymmetry, we checked departures from normality of the distribution of the signed differences (R – L) using Kolmogorov–Smirov test. We also examined the normality of distributions determining whether skewness and kurtosis coefficients of all the traits deviated from zero, which is the expected value for normal distribution by one-sample t-test (Palmer 1994; Llorens et al. 2002). FA has been calculated for a given trait, as the relative mean of the difference between the right and left side of the body, {|(R – L)|/(R + L)/2} for SBN, WL, W/T ratio, ON, and SCTN. To know whether trait FA covaries with the trait size, we obtained nonparametric Spearman's correlation coefficient for all the traits between absolute trait asymmetry |(R – L)| and trait size (R + L)/2. To test the effect of wild-type and mutant strains and sexes on the relative FA values for different morphological traits, 2-way ANVOA was performed. Further, paired t-test was performed in order to test the levels of FA between each mutant and wild type for each trait in both males and females.
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Results |
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Table 2 presents the mean trait size of different morphological traits, namely, SBN, WL, W/T ratio, SCTN, and ON in different mutant and wild-type strains of D. ananassae. There is significant difference among the wild-type and mutant strains for SBN (males: F7, 392 = 20.43, P < 0.001; females: F7, 392 = 77.77, P < 0.001), WL (males: F6, 363 = 47.19, P < 0.001; females: F6, 363 = 112.31, P < 0.001), W/T (males: F6, 363 = 5.103, P < 0.001; females: F6, 363 = 15.73, P < 0.001), and sexual traits (males: F7, 392 = 3.206, P < 0.001; females: F7, 392 = 160.09, P < 0.001). When the mean trait size was compared individually between the wild-type and mutant strains, significant differences were found for SBN, WL, W/T, SCTN, and ON (Table 2).
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The variance and CV of all the traits in wild-type and different mutant strains are shown in the Table 3. To compare the variability of different strains, test of homogeneity for CV was employed (Zar 2005, p. 204). There is significant strain variability for SBN (males
2 = 241.89, degrees of freedom [df] = 7, P < 0.001; females 2 = 225.84, df = 7, P < 0.001), WL (males 2 = 36.42, df = 6, P < 0.001; females 2 = 53.85, df = 6, P < 0.001), W/T ratio (males 2 = 11.38, df = 6, P > 0.05; females 2 = 67.49, df = 6, P < 0.001), and sexual traits (males 2 = 12.15, df = 7, P > 0.05; females 2 = 714.4, df = 7, P < 0.001). To compare phenotypic variability of traits, we considered trait CVs separately in males and females. Traits differed significantly in their CVs by Kruskal–Wallis test in males (H = 20.19, df = 3, P < 0.001) but not in females (H = 7.384, df = 3, P > 0.05). Similarly, there are significant differences for all the morphological traits, when the variance of wild type is compared with different mutant strains (data not shown).
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One-sample t-test reveals that mean values of each trait did not differ significantly from zero (Table 4) indicating the absence of directional asymmetry. The distribution of the signed differences (R – L) showed normal distribution in the Kolmogorov–Smirov test from normality. Moreover, none of skewness and kurtosis values differed from zero (P > 0.05) for all the traits. This indicates that we are observing true FA rather than directional asymmetry and antisymmetry in our data. FA that has been calculated as relative mean of trait asymmetry {|R – L|/(R + L/2)} was correlated with trait size (R + L)/2, for SBN (r = –0.053, P = 0.134), WL (r = 0.001, P = 0.971), W/T ratio (r = 0.022, P = 0.564), ON (r = –0.63, P = 0.208), and SCTN (r = –0.031, P = 0.539), and shows no dependence. It is evident from Figure 1 that there are differences in the levels of FA of different traits between wild-type and mutant strains. To determine this difference statistically, paired t-test was conducted between wild-type and mutant strains in both sexes (Table 5). Moreover, the levels of FA differ significantly (P < 0.001) between the males and females in wild-type and different mutant strains for different morphological traits.
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Discussion |
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The aim of the present study was to focus on the effect of mutations on canalization and developmental stability in D. ananassae. The trait size of different morphological traits, namely, SBN, WL, W/T ratio, SCTN, and ON, differs significantly when mutant strains are compared with wild-type strain in both males and females (Table 2). There is general reduction of trait size in mutants for all the morphological traits but more pronounced in SCTN as compared with the wild-type strain. This reduction may be due to pleiotropic effect of the mutant alleles and correlated response of inbreeding depression.
There is significant variation among wild-type and mutant strains (see Results). However, the wild-type strain is more variable as compared with the mutant lines suggesting that mutant lines are more canalized than the wild type. In the present study, the mutant lines that we have used are highly inbred; therefore, the genetic component of variation is zero and so the variance is only due to the environmental component, whereas in the wild-type strain, an outbred strain having both genetic and environmental component of variance that may result into an increase in the variability in wild-type strain. However, previous studies have shown that environmental variable (stressful temperatures) and mutations have been found to increase the phenotypic variance of SBN in D. melanogaster (Beadmore 1960; Bubliy et al. 2000; Indrasamy et al. 2000; Dworkin 2005).
The levels of FA also vary significantly among the wild-type and mutant strains for the morphological traits (SBN, WL, W/T, and sexual traits) indicating that there is heterogeneity in the levels of FA, which is due to the mechanisms operating at the individual and population levels. At individual level, stress is expected to disrupt the developmental stability of a trait and elevate the asymmetry levels, but it can vary in their sensitivity to specific stress factors. On the other hand, at the population level, in the presence of environment x genotype interactions attenuation of FA signal occurs, if genotypes differ in their sensitivity or direction of response to stress (Polak et al. 2002). The magnitude of FA is similar in males and females for nonsexual traits but not for the sexual traits (see Table 5) suggesting that difference is due to the SCTN in males and ON in females. There is no evidence for the increase of FA in mutant strains except few, for example, for W/T ratio in males of y ca mutant and SCTN in y mutant males and for SBN in spr mutant females (see Figure 1). This can be explained, as the mutations used in the present study are recessive, so their effect on the developmental buffering mechanisms might be less as compared with any dominant mutation, despite of the fact that they produces strong phenotypic effects on body color, eye color, and wing venations of adult flies. Willmore et al. (2006) has shown that a semidominant Brachyrrhine (Br) mutation has significant effect on the underlying developmental architecture of the skull, which results in an increase in variance and also in FA in Brachyrrhine mouse. Moreover, certain developmental genes and pathways also influence the symmetry of particular traits (Milton et al. 2003). The best-known example is the Notch gene of Drosophila, which regulates bristle development and specifically buffers bristle-trait symmetry (Milton et al. 2003). In a study, Indrasamy et al. (2000) have shown that the mutants have significant asymmetry response relative to wild type for 6 different bristle characters in 10 Notch mutants.
The strongest candidate known till date is Hsp90 chaperone, which buffers the development against a wide range of morphological changes in Drosophila and other organisms and also masks the effect of both additive and epistatic sources of genetic and genotype–environmental interaction variations (Rutherford and Lindquist 1998). But when the effect of Hsp90 was tested on developmental stability, neither trait nor global asymmetry of several bilateral bristle and wing traits was found to be affected in outbred flies treated by Hsp90 inhibitor or across a series of inbred genetic backgrounds from a wild population suggesting that genetic canalization and developmental stability are not controlled by a single Hsp90-dependent process (Milton et al. 2003). Therefore, it is difficult to imagine how genes would directly regulate the stochastic source of variation in development such as left–right sources of small numbers of critical regulatory molecules (Milton et al. 2003).
In the present study, there is no evidence for increase of FA with the increase of mutations (see Figure 1); moreover, the effects of mutations on FA in D. ananassae seem to be sex and trait specific. In future, more experimental analyses are needed to understand the underlying developmental mechanisms affecting the phenotypic changes that will help us to unravel the complex relationship between development and evolution as suggested by Willmore et al. (2006).
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Senior Research Fellowship of Centre of Advanced Study, Department of Zoology to C.V.
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Acknowledgments |
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The authors thank Professor R. MacIntyre and the 2 anonymous referees for their helpful comments on the original draft of the manuscript. We are thankful to Professor M. Matsuda, Kyorin University, Japan, for providing us the mutant strains of Drosophila ananassae.
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Footnotes |
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Corresponding Editor: Ross Maclntyre
Received August 7, 2007
Accepted March 10, 2008
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