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Journal of Heredity Advance Access published online on October 30, 2008
Journal of Heredity, doi:10.1093/jhered/esn094
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© The American Genetic Association. 2008. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org.

Fluctuating Asymmetry in Hybrids of Sibling Species, Drosophila ananassae and Drosophila pallidosa, Is Trait and Sex Specific

Chavali Vishalakshi, and Bashisth N. Singh

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.

Due to inconsistent results of the empirical studies, the relationship between fluctuating asymmetry (FA, a measure of developmental stability) and interspecific hybridization has been the subject of intense debates. In the present study, we have assessed the impact of interspecific hybridization between 2 sibling species of Drosophila: Drosophila ananassae and Drosophila pallidosa on the levels of FA over 3 generations. Trait size of different morphological traits, namely, sternopleural bristle number, wing length (WL), wing to thorax (W/T) ratio, sex comb tooth number (SCTN), and ovariole number differed significantly among parental species and their hybrids of different generations in both the sexes. However, the levels of FA of different morphological traits were similar in parental species and their hybrids of different generations in males (except SCTN) and in females (except for WL and W/T ratio). These results are interpreted in terms of developmental stability as a function of a balance between the level of heterozygosity and the disruption of coadapted gene complexes.

Key Words: developmental instability • Drosophila ananassae • Drosophila pallidosa • interspecific hybrids • morphological traits

Understanding the mechanisms of speciation remains a mystery for evolutionary biologists (Sawamura and Tomaru 2002; Coyne and Orr 2004; Wu and Ting 2004; Noor and Feder 2006). Species maintain their entity through reproductive isolating mechanisms, which restrict the intermingling of genomes from different species (Biological Species Concept, Mayr 1942). Due to these reproductive barriers, species in vicinity are unable to mate and if they do so, they are unable to produce fertile and viable progeny (Coyne and Orr 2004; Mishra and Singh 2005). Contrary to the biological species concept, there are several examples of interspecific hybridization among closely related species of lepidoptera and birds (Naisbit et al. 2001; Presgraves 2002; Price and Bouvier 2002). However, majority of species do not hybridize in nature, but they do so in laboratory, for instance, in Drosophila (Bock 1984). Interestingly, interspecific hybridization often leads to embryonic or adult lethality and reduced viability or fertility of hybrids (Sperlich 1962; Llopart et al. 2005; Mishra and Singh 2005), which is due to accumulation of genes which cause recessive incompatible epistatic interactions between the parental species (Dobzhansky–Muller model, Presgraves 2003 and references therein). Each species exists with its own history of accumulated genetic modifications during the course of its evolution. Therefore, in interspecific hybrids, the coexistence of genomes from 2 different species may destabilize the normal development by decreasing the developmental stability of hybrids (Andersen et al. 2006; Carreira et al. 2008). Developmental stability refers to the ability of organisms to buffer their developmental processes against environmental and genetic perturbations (Leamy and Klingenberg 2005). Although the genetic basis of developmental stability is not fully understood, but it is thought that genomic coadaptation and heterozygosity are the 2 main causal genetic conditions associated with sustaining developmental stability in organisms (Alibert and Auffray 2003; Pertoldi, Kristensen, et al. 2006; Pertoldi, Sorensen, et al. 2006). In hybrids, one may expect that the developmental stability may decrease due to breakdown of coadapted gene complexes, and alternatively, there may be an increase in the developmental stability levels because of the increased heterozygosity, but this depends on the degree of divergence in the genetic systems controlling development in hybridizing taxa (Alibert and Auffray 2003; Carreira et al. 2008). The most widely used parameter of developmental stability is fluctuating asymmetry (FA), which refers to subtle random deviations from ideal bilateral symmetry (Van Valen 1962) that may result due to the inability of self-regulatory mechanisms to stabilize development in the face of environmental perturbations (Van Valen 1962; Carreira et al. 2008). Several studies have demonstrated that interspecific hybrids of different genera or species have either decreased (see, e.g., Lamb et al. 1990; Blows and Sokolowaki 1995; Hutchinson and Cheverud 1995; Alibert et al. 1997) or increased developmental stability as compared with the parents (see, e.g., Leary et al. 1985; Ross and Robertson 1990; Markow and Ricker 1991), which provides indirect evidence that epistatic effects may be important in controlling the levels of FA (Leamy and Klingenberg 2005).

Other probable causes of departure from bilateral symmetry are directional asymmetry (DA) and antisymmetry (AS). Former occurs when there is normally a greater development of a character on one side of the planes of symmetry than on the other, whereas latter occurs when one side of the plane is consistently larger than the other, but the larger side may be either right or left at random, resulting in a bimodal distribution of R – L differences about a mean of zero (Van Valen 1962). However, there is evidence that DA is less associated with developmental stability, but it has been found to differ between parents and their hybrids (e.g., Klingenberg et al. 1998; Schneider et al. 2003), and also the transitions between FA and DA have been suggested as an indicator of stress in populations (Graham et al. 1993; Carreira et al. 2008). The effect of interspecific hybridization on the levels of FA has been investigated in different species pairs of Drosophila (Markow and Ricker 1991; Rego et al. 2006; Carreira et al. 2008), and the results of these studies except of Markow and Ricker (1991) do not support the suggestion that hybridization may cause a reduction in developmental stability of hybrids (Rego et al. 2006; Carreira et al. 2008).

Drosophila ananassae and Drosophila pallidosa are 2 sibling species of the ananassae complex of the ananassae subgroup of the melanogaster species group (Bock and Wheeler 1972; Lemeunier et al. 1986, but see Da Lage et al. 2007). Drosophila pallidosa is endemic to the Islands of South Pacific Ocean, where it coexists with its sibling species D. ananassae, a cosmopolitan and circumtropical species (Tobari 1993), and also exists in highly structured populations in Asia and South Pacific (Tomimura et al. 1993; Vogl et al. 2003; Das et al. 2004; Schug et al. 2007, 2008; Singh P and Singh BN 2008). Both the species are genetically distinct in nature, and strong sexual isolation between them has been considered to be crucial in maintaining the integrity of the gene pool of these 2 species (Yamada et al. 2002a), but interspecific hybrids of both sexes produced in the laboratory are viable and fertile, that is, absence of postmating isolation (Speith 1966; Futch 1973; Oguma 1993, but see Sawamura et al. 2008). These sibling species are difficult to distinguish as the only diagnostic traits in sympatric populations are body color and sex comb tooth number (SCTN) (Bock and Wheeler 1972, but see Vishalakshi and Singh 2008a). Also, female sex pheromones (Nemoto et al. 1994; Doi et al. 1997) and male courtship songs (Yamada et al. 2002a, 2002b) differ between these 2 species, which are thought to be used for species recognition (Sawamura et al. 2008). Thus, mate discrimination is the only known mechanism that prevents gene flow between these 2 sibling species, and the loci that might have played crucial role in the evolution of reproductive isolation were mapped to distinct positions near the delta locus in the middle of the left arm of the second chromosome (Doi et al. 2001). Moreover, D. pallidosa has specific inversions on XL, 2L, 2R, and 3R that are not found in sympatric strains of D. ananassae (Futch 1966; Tobari 1993).

In the present communication, we have endeavored to investigate whether interspecific hybridization contributes to reduce the developmental stability level in hybrids of D. pallidosa and D. ananassae. Moreover, the absence of postmating isolation such as hybrid inviability or sterility in F1 and F2 generations between these 2 species allows us to disassociate the effect of 2 hybridization events, that is, heterozygosity (which may result in decreased levels of FA in F1 hybrids) and breaking of coadapted gene complexes in subsequent generations (which may result in increased levels of FA in F2 or F3 generation) of interspecific hybrids. These facts have tempted us to study the effect of interspecific hybridization on the levels of FA in different morphological traits, namely, sternopleural bristle number (SBN), wing length (WL), wing to thorax (W/T) ratio, SCTN, and ovariole number (ON) in D. ananassae and D. pallidosa species pair and their hybrids. In addition to this, there are no studies in Drosophila that have examined in detail the developmental stability of hybrids for over 3 generations after interspecific hybridization, that is, up to F3 generation in different morphological traits (mentioned above). We also examined DA in morphological traits because hybridization has been found to affect DA in bees and other insects (Smith et al. 1997; Klingenberg et al. 1998; Schneider et al. 2003). Moreover, previous studies in D. ananassae have shown that FA exists in controlled laboratory conditions (Vishalakshi and Singh 2006), and there is negative relationship between FA and sexual selection (Vishalakshi and Singh 2008b). Also, FA is affected by different environmental stressors (Vishalakshi and Singh 2008c, 2008d) and mutations in D. ananassae (Vishalakshi and Singh 2008e).


   Materials and Methods
Top
 Materials and Methods
Results
 Discussion
 Funding
 Supplementary Material
 References
 
Drosophila Stocks
The mass culture stock of D. ananassae (Mysore, India) was established in the laboratory from the naturally impregnated females (n = 6) collected from Mysore, India, in 2001. The stock of D. pallidosa (NAN 57), provided by M. Matsuda Japan, is an isofemale line collected at Lautoka, Fiji (1981). FA is not affected by inbreeding in Drosophila (Fowler and Whitlock 1994; Vishalakshi and Singh 2006, 2008f; Vishalakshi C. and Singh B.N., unpublished data); therefore, these 2 stocks were used. Both these stocks of Drosophila have been maintained on 25 days generation time in the laboratory on simple yeast culture medium at approximately 24 °C temperature.

Experimental Design
Flies of both species were kept for 2 generations in an incubator maintained at 25 °C with continuous light to avoid environmental effects (Mueller and Joshi 2000). After 2 generations, 25 pairs of 7-day-old virgin females and males from both stocks were transferred in culture bottles. Flies were kept for 2 days for oviposition and were then discarded. Culture bottles were kept in the incubator at 25 °C and were positioned at random and rotated daily in order to avoid any systematic macroenvironmental effects. Virgin females and bachelor males from both stocks were separated under anesthesia within 2–4 h of eclosion and were kept in separate food vials of 3'' (height) x 1'' (diameter) for 7 days of aging. The 2 reciprocal crosses (throughout this paper the maternal species is always indicated first) were made: 1) D. ananassae femalefemale x D. pallidosa malemale (hereafter referred as AP) and 2) D. pallidosa femalefemale x D. ananassae malemale (hereafter referred as PA). For F1 hybrids, 25 virgin females of one species were crossed with 25 bachelor males of other species. Because there is strong sexual isolation between 2 sibling species, the flies in both the reciprocal crosses were kept for 2 days in food vials and then transferred to culture bottles. Flies were allowed to lay eggs for 2 days and then discarded. To obtain F2 and F3 generations, 25 pairs of flies were taken randomly from F1 and F2 generations as parents, respectively. Throughout the experiment, the rearing conditions were kept same (as mentioned above) for both parental species as well as for hybrids of different generations. In each group (parental and hybrids) when the flies emerged, virgin females and bachelor males were collected and aged for 4 days in separate food vials before scoring of different traits. All fly handling was done at room temperature (ca., 24 °C), using ether anesthesia when necessary.

Morphological Trait Measurements
Except thorax length (TL), all the other morphological traits (WL, W/T ratio, SBN, SCTN, and ON) were scored on both left and right sides in 50 males and 50 females from each parental species and their hybrids of different generations in AP and PA crosses. TL was measured from anterior end of the thorax to the posterior end of the scutellum under a microscope at magnification of 50x using ocular micrometer (1 unit = 16.67 µm). Two sets of bristles are present on the sternopleuron of males and females were counted under a stereo binocular with a magnification of 25.2x. Anterior bristles (A) occur in an oblique row from the forecoxa toward the midline, whereas the transverse bristles (T) running in a thin line toward the center of fly just anterior to middle leg. Total number of sternopleural bristles (SBN) is taken as the sum of the anterior and transverse bristles. Then wings were removed with the fine forceps and mounted on a glass slide in a drop of insect's saline (0.67% NaCl), and coverslip was placed over them. WL was measured as the absolute length between the anterior cross vein to the distal tip of the third longitudinal vein under a microscope at 50x magnification using ocular micrometer (1 unit = 16.67 µm). The W/T ratio was calculated from the data of WL and TL. In females, ovaries were dissected in insect's saline stained with 2% acetocarmine stain, mounted in 45% acetic acid, and counted ON under a microscope at 50x magnification. Sex comb in males of the ananassae subgroup are characterized by several transverse rows of stout blackish bristles on the ventral surface of first, second, and third tarsal segments of prothoracic legs (Bock and Wheeler 1972). Forelegs of males of both species and their hybrids were dissected and mounted in insect saline, and the total number of the teeth (SCTN) on the first (C1), second (C2), and third tarsal segments were counted under a microscope at 50x magnification.

Trait Size Analysis
Trait size for each trait was measured by the average value of right and left side [(R + L)/2]. In order to investigate whether the trait size of different morphological traits differs among parental species and their hybrids, 1-way analysis of variance (ANOVA) was performed in both males and females. When this difference was significant, we conducted "post hoc" comparisons by means of Bonferroni test. Further, the variability of each group (i.e., parental species and their hybrids) was estimated using the coefficient of variation (CV), and phenotypic variability of parental species and hybrids was compared by test of homogeneity for CV in both males and females. To know how different morphological traits were correlated to each other, Pearson correlation tests were performed for males and females separately.

Trait Asymmetry Analyses
For the analyses of FA, the framework laid out by Palmer (1994) and Palmer and Strobeck (1986, 2003) was followed. Measurement error (ME) can cause extreme bias in the studies of FA; therefore, accurate estimates of ME are essential (Palmer 1994). It is important to have the confidence that there are differences in R – L among individuals and not simply an artifact of ME. When sample size is large, repeat measurements of trait may not be practical, and in such cases, the effect of ME should be calculated from repeat measure of a sample of at least 30 individuals (Palmer 1994). In the present study, a subsample of 32 flies randomly collected from the cultures and 2 replicate counts were made for different traits per fly for ME. The second set of measurements was made on different days and without the reference to the first set. A mixed model 2-way ANOVA (Palmer 1994) was used to assess asymmetry in trait size in the samples of 32 flies that were measured twice. In this model, "individual" is a random factor that assesses variation among individuals, "sides" is a fixed factor that assesses DA, the "individual x sides interaction" assesses FA, and the "error" assesses variation in the replicate measurements (Palmer 1994). The significant mean squares for the interaction indicates that the amount of FA is greater than that of ME, and the asymmetry analysis may be proceeded (Palmer 1994). The mixed model ANOVA also allowed us to estimate the precision of the replicate measurements (Schneider et al. 2003).

To obtain the measure of FA and DA, individual asymmetry was calculated as the value of a given trait on the right side of the body minus its value on the left side, that is, signed difference (R – L). Then, we have tested for other departures from bilateral asymmetry, that is, DA and AS. For DA, 1-sample t-test on the signed difference (R – L) for each trait, sex and generation (parental and F1, F2, and F3), was performed to determine whether the mean (R – L) values differ from zero (Palmer 1994). Data of the hybrids of the reciprocal crosses were pooled in each generation. If these signed differences for different traits were significantly different from zero, this indicates that DA was present in the morphological traits (Van Valen 1962). AS was tested by departures of (R – L) frequency distribution from normality by Kolmogorov–Smirov test as suggested by Palmer (1994) and also 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) was examined by 1-sample t-test (Palmer 1994). However, significant platykurtosis indicates the signal of AS (Palmer and Strobeck 2003). Tests for DA and AS should be conducted in the studies of FA for 2 reasons: first they may inflate the FA indices and second if a trait exhibits either DA or AS some portion of the between-sides variation may have a genetic basis; hence, the between-sides variance may not be purely a product of developmental noise (Palmer and Strobeck 1986; Palmer 1994).

FA (FA1 of Palmer 1994, which is the FA measure reported in most of studies) has been calculated for a given trait as the mean of absolute value of the difference in trait size between the right and the left sides of the body, |(R – L)|. Positional FA (PFA; Polak 1997) is a measure of the difference between the 2 sides of the body in the way in which components of a meristic trait are arranged or positioned was calculated for both SBN (PFAB) as |(Right A/Right T) – (Left A/Left T)| (Polak and Starmer 2001) and SCTN (PFAS) as |(Right C1/Right C2) – (Left C1/Left C2)| (Polak et al. 2004). Thus, FA for SBN, WL, W/T ratio, ON, and SCTN was calculated. To know whether trait FA covaries with the trait size, we obtained nonparametric Spearman's correlation coefficients for all the traits between absolute trait asymmetry |(R – L)| and trait size (R + L)/2, for different morphological traits in parental species and different generations, and also in each sex separately. This test was performed because dependence of asymmetry on trait size can influence the inferences made in FA studies (Palmer 1994).

For all the traits (SBN, WL, W/T ratio, ON, and SCTN), we have calculated a size-corrected FA index (FA2), that is, relative FA, (|R – L|)/(R + L)/2 (for the pros and cons of the indices, FA1 and FA2, see Palmer 1994). To be sure that the correction was successful, we have again correlated the size-corrected index (i.e., [|R – L|]/[R + L]/2) with trait size (i.e., [R + L]/2). Then, to compare the degree of FA between parental species and their hybrids of different generations, 1-way ANOVA followed by Bonferroni tests for males and females separately was performed. The rationale behind this comparison was as follows: 1) Are there differences in FA between parental species and their hybrids for different morphological traits? and 2) Do the levels of FA remain the same in hybrids of different generations or there is some breakdown? In order to investigate whether the levels of FA vary between sexual (SCTN and ON) and nonsexual (SBN, WL, and W/T ratio) traits, the levels of FA were compared by 1-way ANOVA in both males and females of parental species and their hybrids.


   Results
Top
 Materials and Methods
 Results
Discussion
 Funding
 Supplementary Material
 References
 
Trait Size Analyses
The trait size [(R + L)/2] of SBN, WL, W/T ratio, SCTN, and ON varies significantly among the parental species, D. ananassae and D. pallidosa, and their hybrids of different generations in both males and females (Tables 1 and 2). Further, when Bonferroni test for pair wise analysis was employed, significant differences were found for all the traits in males and females (Tables 1 and 2). It is also evident from Tables 1 and 2 that between the 2 parental species, D. ananassae flies have larger trait size for different morphological traits than D. pallidosa flies. But when they were compared with the hybrids of F1 generation in both the reciprocal crosses, the trait size of parental species was lower than the F1 hybrids for different morphological traits, which provides evidence for increase of heterozygosity (see Tables 1 and 2). To test whether the CV differed among generations (parental species and F1, F2, and F3 hybrids), we have employed test of homogeneity of CV (Zar 2005, p. 204). The groups (parental and F1, F2, and F3 hybrids) differ significantly (P < 0.05) in trait variability both in sexes and in reciprocal crosses. This difference arises due to higher values of CV in the parental species in comparison to that of hybrids, suggesting that hybridization does not increase the variability in hybrids.


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  		Table 1. Comparison of trait size mean and standard errors among the parental Drosophila species (Drosophila ananassae and Drosophila pallidosa) and their hybrids of different generations (F1, F2, and F3) in males

 


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  		Table 2. Comparison of mean (±standard error) trait size of different morphological traits among the parental Drosophila species (Drosophila ananassae and Drosophila pallidosa) and their hybrids of different generations (F1, F2, and F3) in females

 
It is evident from Supplementary Material 1 that WL is positively correlated with SBN, W/T, and SCTN in males and in females and WL positively correlated with SBN, W/T, and ON, suggesting that WL, an index of body size, is related with adult fitness such as mating success, fecundity, and dispersal potential supporting previous findings (Partridge et al. 1987; Partridge 1988; Santos et al. 1992 ; Barker and Krebs 1995; Norry et al. 1995; Sisodia and Singh 2001).

Trait Asymmetry Analyses
Descriptive statistics for signed (R – L) asymmetry values for each trait, generation (parental and hybrids), and sexes are given in Supplementary Material 2. For all the traits, mean signed (R – L) values did not differ significantly from zero, suggesting that there is absence of DA in data (see Supplementary Material 2) in parental species as well as in hybrids of different generations (the data of both the reciprocal crosses were pooled in each generation). For AS, none of the (R – L) frequency distributions showed bimodal distribution and signed differences also showed normal distribution in Kolmogorov–Smirov test for normality but significant leptokurtosis was also detected in some traits (Supplementary Material 2), which might be due to lack of power (as in the present study, n = 50) as suggested by Babbitt et al. (2006). Although, significant platykurtosis may be the signal of presence of AS (for details, see Palmer and Strobeck 2003). Further, the mean values of (R – L) of PFA of SBN and SCTN were tested for the presence of DA and AS. The mean values of (R – L) differ significantly from a mean of zero (P < 0.001), which suggests the presence of DA in the data of PFA in parental species and their hybrids (data not shown). Therefore, the data of PFAS and PFAB have been removed from further analysis of FA, but the levels of DA were compared in parental species and different hybrid generations by 1-way ANOVA. The levels of PFA for the SBN were similar in parental species and different hybrid generations in both AP (males: F4,245 = 0.962, P = 0.429; females: F4,245 = 1.555, P = 0.187) and PA crosses (males: F4,245 = 0.565, P = 0.689; females: F4,245 = 1.413, P = 0.230). The levels of PFA in SCTN were similar in parental species and different hybrid generations in males of AP (F4,245 = 0.0094, P = 1.000) and PA (F4,245 = 0.0551, P = 0.994) crosses.

Correlation analyses conducted with the absolute value of the difference between sides for each trait and mean trait size as an estimator of total size for different traits, sex, and generations showed that there was significant correlation (data not shown). Therefore, the absolute FA data have been corrected as relative index of FA or size-corrected FA, [(|R – L|)/(R + L)/2]. No significant correlation is found between relative index of FA and trait size in any of the traits studied after correction (data not shown). Tables 3 and 4 present the levels of mean relative FA of different morphological traits in males and females, respectively. It is evident from the Table 3 that in males, the levels of relative FA are higher in the F3 generation than in the parental species and hybrids of F1 and F2 generations. Although, the levels of relative FA are similar in males of both parental species and interspecific hybrids of different generations for SBN, WL, and W/T ratio except SCTN (P < 0.001) in AP and PA crosses (Table 3). Similarly, in females, the degree of FA is similar in both parental species and hybrids for SBN and ON but not for WL and W/T ratio in both the reciprocal crosses (see Table 4). Further, when Bonferroni test for pairwise analysis was employed, significant differences were found for SCTN in males (Table 3) and WL and W/T ratio in females (Table 4). The levels of FA were significantly higher in sexual traits (SCTN and ON) than the levels of FA in nonsexual traits (SBN, WL, and W/T ratio; data not shown) in parental species, and their hybrids of different generations in both males and females (P < 0.05).


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  		Table 3. ANOVA for the mean ± standard error relative FA (mean [|R – L|/(R + L/2)] for different traits among parental species and hybrids of different generations in males

 


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  		Table 4. ANOVA for the mean ± standard error relative FA (mean [|R – L|/(R + L/2)] for different traits among parental species and hybrids of different generations in females

 

   Discussion
Top
 Materials and Methods
 Results
 Discussion
Funding
 Supplementary Material
 References
 
The present study tests whether interspecific hybridization between 2 sibling species, D. ananassae and D. pallidosa, influences the levels of developmental stability of different morphological traits. There are 2 ways by which effect of hybridization could be expressed: lesser FA in hybrids as compared with both parental species (an indication of heterozygosity) and greater FA in hybrids in comparison to the parental species (an indication of breakdown of coadapted gene complexes). Trait size of different traits increases in the hybrids of F1 generation than the parental species in both the reciprocal crosses providing evidence for increase of heterozygosity. Following Carreira et al. (2008), the genetic factors with dominant and/or epistatic effects appear to be involved in the genetic architecture of trait morphology, when the parental genomes are combined in the hybrids. Also unlike F1 hybrids of Drosophila koepferae–Drosophila buzzatii (Carreira et al. 2008), we have not found phenotypic plasticity in any trait in hybrid males and females of D. ananassae–D. pallidosa, and also the degree of differences were similar in both the reciprocal crosses. This could be explained as in the present study, hybrids are the progeny of single cross/replicate of an isofemale line of D. pallidosa and a mass culture stock of D. ananassae; therefore, the hybrid flies do not show plasticity. In contrast to our study, Carreira et al (2008) have found that the differences in wing morphology were not homogeneous among crosses, suggesting that wing morphology in hybrids depended strongly on the particular isofemale lines employed as parents, which provides evidence that the factors involved in wing morphology do not cooperate in an additive way in interspecific hybrids.

The variability (estimated as CV) of D. ananassae flies was higher than D. pallidosa flies suggesting the cosmopolitan nature of D. ananassae. Hybridization between D. ananassae and D. pallidosa does not increase the phenotypic variability in hybrids as the variability of hybrids of both sexes was more or less similar to the flies of D. pallidosa.

Further, the levels of FA were also similar in the hybrids and parental species except in few traits, for example, SCTN in males and WL and W/T ratio in females (Tables 3 and 4), indicating that hybridization does not increase the levels of FA in hybrids. Interestingly, we have found that the levels of FA were increased in the males of F3 generation for all the traits, but this increase was significant only in SCTN (Table 3), suggesting that there may be breakdown of coadapted gene complexes in F3 generation. The interbreeding of species results in the breakdown of coadapted gene complexes (which are established over evolutionary time and maintained through stabilizing selection) as a result of which developmental processes get disrupted resulting in higher FA (Møller and Swaddle 1997). Furthermore, the levels of FA are similar in both the reciprocal crosses for SBN, SCTN, and ON but not in WL and W/T ratio (see Tables 3 and 4). The degree of FA in WL and W/T ratio is lower in the hybrid males of PA cross than the hybrid males of AP cross, demonstrating the role of maternally expressed genes (Turelli and Orr 2000). Thus, our results suggest that the interspecific hybrids of D. ananassae and D. pallidosa are developmentally as stable as their parents. Also, the effect of hybridization on FA appears to be trait and sex specific.

The degree of FA is more in sexual traits (SCTN and ON) in comparison to the nonsexual traits (SBN, WL, and W/T ratio), which suggests that sexual traits are more prone to stress caused by hybridization. This could be explained as different traits have different developmental windows, in which the developmental stability of a trait is more vulnerable to stress as their development is controlled by distinct gene complexes (Parsons 1990; Andersen et al. 2002). Also, different traits are exposed to different degree of stabilizing selection and canalization depending on their functional significance. For example, greater functional significance to the organism is subject to stronger selection as in the present study, W/T ratio (related to flight capacity) and WL are having less FA in comparison to SBN, SCTN, and ON. In other words, those traits, which are more closely related to fitness, are expected to buffer, better against environmental effects (Lerner 1954; Woods et al. 1999; Vishalakshi and Singh 2006, 2008b, 2008c, 2008d, 2008e, 2008f).

The levels of developmental instability also depend on the degree of divergence, that is, greater the divergence time higher will be chance of hybrids to be developmentally unstable (Garnier et al. 2006). It has been reported that the phylogenetic separation of D. ananassae and D. pallidosa must have been a recent event in speciation of the melanogaster group (Bock and Wheeler 1972), but the estimates of divergence time (in million years) between these 2 sibling species are not known till date (Prof. T. A. Markow, Arizona, and Prof. K, Sawamura, Japan, personal communication). Because of this reason, the levels of FA of hybrids are similar to the parental species. In literature, it has been reported that the 3 species pairs differ greatly in their time of divergence, for example, Drosophila melanogaster–Drosophila simulans (divergence time: 2.3 ± 0.65 Ma; Russo et al. 1995), Drosophila madeirensis–Drosophila subobscura (divergence time: 0.6–1 Ma; Rego et al. 2006), and D. buzzatii–D. koepferae (divergence time: 3–5 Ma; Carreira et al. 2008) species pairs. The presence of morphological abnormalities in interspecific hybrids of these species pairs suggests that divergence between species in regulatory pathways may contribute to hybrid disruptions. But the levels of FA in hybrids were not higher than the parental species, thereby do not support the idea that hybridization may cause a reduction in developmental stability of hybrids (Rego et al. 2006; Carreira et al. 2008).

Moreover, the amount of out breeding depression in hybrids depends on the degree of genetic divergence between hybridizing taxa (Alibert and Auffray 2003). Most of the evidence in favor of such a mechanism comes from studies reporting increased FA in hybrids (Alibert and Auffray 2003), whereas other studies have reported reduced FA as levels of heterozygosity in populations increase (Alibert and Auffray 2003 and references therein). Thus, developmental stability may be a function of the balance between the effects of both genetic mechanisms, degree of divergence, and level of heterozygosity, suggesting 2 different but not mutually exclusive genetic causes for developmental instability in F1 hybrids: underdominace and/or direct negative epistasis (Alibert and Auffray 2003).

Although DA is less associated with developmental stability than FA (Palmer 1994), we have examined DA but have found DA only in the PFA in SBN and SCTN. The levels of PFA were similar in parental species and their hybrids for SBN in both males and females and for PFA in SCTN in males. Although none of the comparisons for the levels of DA reached significance, our results nevertheless suggest a slight tendency for hybridization to increase DA (Klingenberg et al. 1998; Schneider et al. 2003).

However, D. ananassae and D. pallidosa are 2 well-defined species reproductively isolated by strong premating barriers (Yamada et al. 2002a, 2002b). In another study, we have tested the degree of crossability, productivity, and sex ratio in D. ananassae and D. pallidosa and their F1 hybrids (Vishalakshi and Singh 2008b). The degree of crossability and productivity is more in conspecific matings than in heterospecific matings. The number of progeny produced in the parental species was higher than the hybrids produced in the AP and PA crosses (Vishalakshi and Singh 2008a), which may reflect genetic incompatibilities between the parental genomes. Moreover, we have found no sex ratio distortion in the interspecific hybrids showing that there are less genetic incompatibilities between these 2 sibling species (Vishalakshi and Singh 2008a). Therefore, it may be proposed that hybrids that managed to reach the adult stage may have conceived as compatible combinations of parental genomes, which might be sufficiently different at loci involved in trait development, as to generate the differences observed between the 2 parental species and among parental species and their hybrids, and this may be correlated with the similar levels of developmental stability of hybrids with the parental species. Further analysis using backcross progeny could disentangle the possible role of hybrid breakdown of coadapted gene complexes from potentially beneficial heterozygosity as suggested by Carreira et al. (2008).


   Funding
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 Materials and Methods
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 Funding
Supplementary Material
 References
 
Senior Research Fellowship of the Center of Advanced Study, Department of Zoology, Banaras Hindu University to C.V.


   Supplementary Material
Top
 Materials and Methods
 Results
 Discussion
 Funding
 Supplementary Material
References
 
Supplementary materials can be found at http://www.jhered.oxfordjournals.org/.


   Acknowledgments
 
We are grateful to the 2 anonymous reviewers for their helpful and constructive comments on the original draft of the manuscript, which have improved the manuscript a lot. We are thankful to Prof. M. Matsuda, Kyorin University, Tokyo, Japan, for providing Drosophila pallidosa stock.


   Footnotes
 
Corresponding Editor: James Thompson

Received May 12, 2008
Revised September 30, 2008
Accepted September 30, 2008


   References
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 Supplementary Material
 References
 

    Alibert P, Auffray J-C. Genomic coadaptation, outbreeding depression, and developmental instability. In: Developmental instability: causes and consequences—Polak M, ed. (2003) New York: Oxford University Press. 116–134.

    Alibert P, Fel-Clair K, Manolakou F, Britton Davidian J, Auffray JC. Developmental stability fitness and trait size in laboratory hybrids between European subspecies of house mice. Evolution (1997) 51:1284–1295.[CrossRef][Web of Science]

    Andersen DH, Pertoldi C, Loeschcke V, Scali V. Developmental instability, hybridization and heterozygosity in stick insects of the genus Bacillus (Insecta; Phasmatodea) with different modes of reproduction. Biol J Linn Soc (2006) 87:249–259.[CrossRef][Web of Science]

    Andersen DH, Pertoldi C, Scali V, Loeschcke V. Intraspecific hybridization, developmental stability and fitness in Drosophila mercatorum. Evol Ecol Res (2002) 4:603–621.

    Babbitt GA, Kiltie R, Bolker B. Are fluctuating asymmetry studies adequately sampled? Implications of a new model for size distribution. Am Nat (2006) 167:230–245.[CrossRef][Web of Science][Medline]

    Barker JSF, Krebs RA. Genetic variation and plasticity of thorax length and wing length in Drosophila aldrichi and D. buzzatii. J Evol Biol (1995) 8:689–709.[CrossRef][Web of Science]

    Blows MW, Sokolowaki MB. The expression of additive and non-additive genetic variation under stress. Genetics (1995) 140:1149–1159.[Abstract]

    Bock IR. Interspecific hybridization in the genus Drosophila. In: Evolutionary biology—Hecht MK, Wallace B, eds. (1984) New York: Plenum Publishing Corporation. 41–70.

    Bock IR, Wheeler MR. The Drosophila melanogaster species group. Univ Tex Publ (1972) 7:1–102.

    Carreira VP, Soto IM, Fanara JJ, Hasson E. A study of wing morphology and fluctuating asymmetry in interspecific hybrids between Drosophila buzzatii and D. koepferae. Genetica (2008) 133:1–11.[CrossRef][Web of Science][Medline]

    Coyne JA, Orr HA. Speciation (2004) Sunderland (MA): Sinauer Associates, Inc.

    Da Lage JL, Kergoat GJ, Maczkowiak F, Silvain JF, Cariou ML, Lachaise D. A phylogeny of Drosophilidae using Amyrel gene: questioning the Drosophila species group boundaries. J Zool Sys Evol Res (2007) 45:47–63.[CrossRef]

    Das A, Mohanty S, Stephan W. Inferring the population structure and demography of Drosophila ananassae from multilocus data. Genetics (2004) 168:31–41.

    Doi M, Matsuda M, Tomaru M, Matsubayashi H, Oguma Y. A locus for female discrimination behaviour causing sexual isolation in Drosophila. Proc Natl Acad Sci USA (2001) 98:6714–6719.[Abstract/Free Full Text]

    Doi M, Nemoto T, Nakanishi H, Kuwahara Y, Oguma Y. Behavioural response of males to major sex pheromone component, (Z, Z)-5,25-hentricontadiene of Drosophila ananassae females. J Chem Ecol (1997) 23:2067–2078.[CrossRef][Web of Science]

    Fowler K, Whitlock MC. Fluctuating asymmetry does not increase with moderate inbreeding in Drosophila melanogaster. Heredity (1994) 73:373–376.[CrossRef][Web of Science][Medline]

    Futch DG. A study of speciation in south Pacific populations of Drosophila ananassae. Univ Tex Publ (1966) 6615:79–120.

    Futch DG. On the ethological differentiation of Drosophila ananassae and D. pallidosa in Samoa. Evolution (1973) 27:456–467.[CrossRef][Web of Science]

    Garnier S, Gidaszewski N, Charlot M, Rasplus JY, Alibert P. Hybridization, developmental stability and functionality of morphological traits in the ground beetle Carabus solieri (Coleoptera, Carabidae). Biol J Linn Soc (2006) 89:151–158.[CrossRef][Web of Science]

    Graham JH, Freeman DC, Emlen JM. Antisymmetry, directional asymmetry and dynamic morphogenesis. Genetica (1993) 89:121–137.[CrossRef][Web of Science]

    Hutchinson DW, Cheverud JM. Fluctuating asymmetry in tamarin (Saguinus) cranial morphology: intra- and interspecific comparison between taxa with verifying levels of genetic heterozygosity. J Hered (1995) 86:280–288.[Abstract/Free Full Text]

    Klingenberg CP, McIntyre GS, Zaklan SD. Left-right asymmetry of fly wings and the evolution of body axes. Proc R Soc Lond B Biol Sci (1998) 265:1255–1259.[Medline]

    Lamb T, Novak JM, Mahoney DL. Morphological asymmetry and interspecific hybridization: a case study using hybrid frogs. J Evol Biol (1990) 3:295–309.[CrossRef][Web of Science]

    Leamy L, Klingenberg CP. The genetics and evolution of fluctuating asymmetry. Ann Rev Ecol Evol Syst (2005) 36:1–21.[CrossRef]

    Leary RF, Allendorf FW, Knudson KL. Developmental instability and high meristic counts in interspecific hybrids in Salmonoid fishes. Evolution (1985) 39:1318–1326.[CrossRef][Web of Science]

    Lemeunier F, David J, Tsacas L, Ashburner M. The melanogaster species group. In: The genetics and biology of Drosophila—Ashburner M, Carson HL, Thompson JN Jr, eds. (1986) London: Academic Press. 147–256.

    Lerner IM. Genetic homeostasis (1954) New York: John Wiley.

    Llopart A, Lachaise D, Coyne JA. An anomalous hybrid zone in Drosophila. Evolution (2005) 59:2602–2607.[CrossRef][Web of Science][Medline]

    Markow TA, Ricker JP. Developmental stability in hybrids between the sibling species pair, D. melanogaster and D. simulans. Genetica (1991) 84:115–121.[CrossRef][Web of Science][Medline]

    Mayr E. Systematics and the origin of species (1942) New York: Columbia University Press.

    Mishra PK, Singh BN. Why hybrid males are sterile in Drosophila? Curr Sci (2005) 89:1813–1819.[Web of Science]

    Møller AP, Swaddle JP. Asymmetry, developmental stability and evolution (1997) Oxford: Oxford University Press.

    Mueller LD, Joshi A. Stability in model populations (2000) Princeton and Oxford: Princeton University Press.

    Naisbit RE, Jiggins CD, Mallet JLB. Disruptive sexual selection against hybrids contributes to speciation between Heliconius cydno and H. melanpomene. Proc R Soc Lond B Biol Sci (2001) 268:1849–1854.[Medline]

    Nemoto T, Doi M, Oshio K, Matsubayashi H, Oguma Y, Suzuki T, Kuwahara Y. (Z, Z)-5,27- tritriacontadiene: major sex pheromone of Drosophila pallidosa (Diptera: Drosophilidae). J Chem Ecol (1994) 20:3029–3037.[CrossRef][Web of Science]

    Norry FM, Vilardi JC, Fanara JJ, Hasson E. Courtship success and multivariate analysis of sexual selection on morphometric traits in Drosophila buzzatii. J Insect Behav (1995) 8:219–229.[CrossRef]

    Noor MAF, Feder JL. Speciation genetics: evolving approaches. Nat Rev Genet (2006) 7:851–861.[CrossRef][Web of Science][Medline]

    Oguma Y. Sexual behaviour. In: Drosophila ananassae: genetical and biological aspects—Tobari YN, ed. (1993) Tokyo, Japan: Japan Scientific Societies Press. 199–205.

    Palmer AR. Fluctuating asymmetry analysis: a primer. In: Developmental instability: its origins and evolutionary implications—Markow TA, ed. (1994) Dordrecht (The Netherlands): Kluwer Academic Publishers. 335–364.

    Palmer AR, Strobeck C. Fluctuating asymmetry: measurement, analysis, patterns. Ann Rev Ecol Syst (1986) 17:391–421.[CrossRef][Web of Science]

    Palmer AR, Strobeck C. Fluctuating asymmetry analysis revisited. In: Developmental instability: causes and consequences—Polak M, ed. (2003) New York: Oxford University Press. 279–319.

    Parsons PA. Fluctuating asymmetry: an epigenetic measure of stress. Biol Rev (1990) 65:131–145.[Medline]

    Partridge L. Lifetime reproductive success in Drosophila. In: Reproductive success—Clutton-Brock TH, ed. (1988) Chicago: University of Chicago Press. 11–23.

    Partridge L, Ewing A, Chandler A. Male size and mating success in Drosophila melanogaster: the roles of male and female behaviour. Anim Behav (1987) 35:555–562.[CrossRef][Web of Science]

    Pertoldi C, Kristensen TN, Andersen DH, Loeschcke V. Developmental instability as an estimator of genetic stress. Heredity (2006) 96:122–127.[CrossRef][Web of Science][Medline]

    Pertoldi C, Sorensen JG, David JR, Loeschcke V. Lerner's theory on the genetic relationship between heterozygosity, genomic co-adaptation and developmental instability revisited. Evol Ecol Res (2006) 8:1487–1498.

    Polak M. Ectoparasitism in mothers causes higher positional fluctuating asymmetry in their sons: implications for sexual selection. Am Nat (1997) 149:955–974.[CrossRef][Web of Science][Medline]

    Polak M, Starmer WT. The quantitative genetics of fluctuating asymmetry. Evolution (2001) 55:498–511.[CrossRef][Web of Science][Medline]

    Polak M, Starmer WT, Wolf LL. Sexual selection for size and symmetry in a diversifying secondary sexual character in Drosophila bipectinata Duda (Diptera: Drosophilidae). Evolution (2004) 58:597–607.[CrossRef][Web of Science][Medline]

    Presgraves DC. Patterns of postzygotic isolation in Lepidopetra. Evolution (2002) 56:1168–1183.[CrossRef][Web of Science][Medline]

    Presgraves DC. A fine scale genetic analysis of hybrid incompatibilities in Drosophila. Genetics (2003) 163:955–972.[Abstract/Free Full Text]

    Price TD, Bouvier MM. The evolution of F1 post-zygotic incompatibilities in birds. Evolution (2002) 56:2083–2089.[CrossRef][Web of Science][Medline]

    Rego C, Matos M, Santos M. Symmetry breaking in interspecific Drosophila hybrids is not due to developmental noise. Evolution (2006) 60:746–761.[Web of Science][Medline]

    Ross KG, Robertson JL. Developmental stability, heterozygosity and fitness in two introduced fire ants (Solenopsis invicta and S. richteri) and their hybrid. Heredity (1990) 64:93–103.[CrossRef][Web of Science]

    Russo CA, Takezaki MN, Nei M. Molecular phylogeny and divergence of the Drosophilid species. Mol Biol Evol (1995) 12:391–404.[Abstract]

    Santos M, Fowler K, Partridge L. On the use of tester stocks to predict the competitive ability of genotypes. Heredity (1992) 69:489–495.[Web of Science][Medline]

    Sawamura K, Tomaru M. Biology of reproductive isolation in Drosophila: toward a better understanding of speciation. Pop Ecol (2002) 44:209–219.[CrossRef]

    Sawamura K, Zhi H, Setoguchi K, Yamada H, Miyo T, Matsuda M, Oguma Y. Genetic analysis of female mating recognition between Drosophila ananassae and D. pallidosa: application of interspecific mosaic genome lines. Genetica (2008) 133:179–185.[CrossRef][Web of Science][Medline]

    Schneider SS, Leamy LJ, Lewis LA, De Grandi-Hoffman G. The influence of hybridization between African and European honeybees, Apis mellifera, on asymmetries in wing size and shape. Evolution (2003) 57:2350–2364.[CrossRef][Web of Science][Medline]

    Schug MD, Baines JF, Amanda K, Mohanty S, Das A, Grath S, Smith SG, Zhargam S, McEvey SF, Stephan W. Evolution of mating isolation between populations of Drosophila ananassae. Mol Ecol (2008) 17:2706–2721.[CrossRef]

    Schug MD, Smith SG, Tozier-Pearce A, McEvey SF. The genetic structure of Drosophila ananassae populations from Asia, Australia and Samoa. Genetics (2007) 175:1429–1440.[Abstract/Free Full Text]

    Singh P, Singh BN. Population genetics of Drosophila ananassae. Gene Res (Forthcoming 2008).

    Sisodia S, Singh BN. Mating success and morphometric traits in Drosophila ananassae. Curr Sci (2001) 80:1444–1447.[Web of Science]

    Smith DR, Crespi BJ, Bookstein FL. Fluctuating asymmetry in the honeybee, Apis mellifera: effects of ploidy and hybridization. J Evol Biol (1997) 10:551–574.[CrossRef][Web of Science]

    Speith HT. Mating behaviour of Drosophila ananassae and ananassae like flies from the Pacific. Univ Texas Pub (1966) 3:133–146.

    Sperlich D. Hybrids between Drosophila melanogaster and D. simulans in nature. Drosoph Inf Serv (1962) 36:118.

    Tobari YN. Drosophila ananassae: genetical and biological aspects (1993) Tokyo, Japan: Japanese Scientific Society Press.

    Tomimura Y, Matsuda M, Tobari YN, Cariou ML, Da Lage JL, Stephan W, Langley CH. Population genetics. In: Drosophila ananassae: genetical and biological aspects—Tobari YN, ed. (1993) Tokyo (Japan): Japanese Scientific Society Press. 139–198.

    Turelli M, Orr HA. Dominance, epistasis and the genetics of post zygotic isolation. Genetics (2000) 154:1663–1679.[Abstract/Free Full Text]

    Van Valen L. A study of fluctuating asymmetry. Evolution (1962) 16:125–142.[CrossRef][Web of Science]

    Vishalakshi C, Singh BN. Fluctuating asymmetry in certain morphological traits in laboratory populations of D. ananassae. Genome (2006) 49:777–785.[Medline]

    Vishalakshi C, Singh BN. Differences in morphological traits between two sibling species, Drosophila ananassae and D. pallidosa. Zool Stud (2008a) 47(3):352–359.

    Vishalakshi C, Singh BN. Mating success is not correlated with fluctuating asymmetry in Drosophila ananassae. Curr Sci (2008b) 94:377–381.

    Vishalakshi C, Singh BN. Effect of developmental temperature stress on fluctuating asymmetry in certain morphological traits in Drosophila ananassae. J Therm Biol (2008c) 33:201–208.[CrossRef][Web of Science]

    Vishalakshi C, Singh BN. Effect of environmental stress on fluctuating asymmetry in certain morphological traits in Drosophila ananassae: nutrition and larval crowding. Can J Zool (2008d) 86:427–437.[CrossRef]

    Vishalakshi C, Singh BN. Effect of mutations on developmental stability and canalization of morphological traits in Drosophila ananassae. J Hered (2008e) 99:539–545.[Abstract/Free Full Text]

    Vishalakshi C, Singh BN. Can fluctuating asymmetry in morphological traits be used to detect inbreeding in Drosophila ananassae? Drosoph Inf Serv (Forthcoming 2008f).

    Vogl C, Das A, Beaumont M, Mohanty S, Stephan W. Populations subdivision and molecular sequences variation: theory and analysis of Drosophila ananassae data. Genetics (2003) 165:1385–1395.[Abstract/Free Full Text]

    Woods RE, Srgo CM, Hercus MJ, Hoffmann AA. The association between fluctuating asymmetry, trait variability, trait heritability and stress: a multiply replicated experiment on combined stresses in Drosophila melanogaster. Evolution (1999) 53:493–505.[CrossRef][Web of Science]

    Wu CI, Ting CT. Genes and speciation. Nat Rev Genet (2004) 5:114–122.[CrossRef][Web of Science][Medline]

    Yamada H, Matsuda M, Oguma Y. Genetics of sexual isolation based on courtship song between two sympatric species: Drosophila ananassae and D. pallidosa. Genetica (2002a) 116:225–237.[CrossRef][Web of Science][Medline]

    Yamada H, Sakai T, Tomaru M, Doi M, Matsuda M, Oguma Y. Search for species-specific mating signal in courtship songs of sympatric sibling species, Drosophila ananassae and D. pallidosa. Genes Genet Syst (2002b) 77:97–106.[CrossRef][Web of Science][Medline]

    Zar JH. Biostatistical analysis (2005) 4th ed. Delhi (India): Pearson education.


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