Evol Ecol DOI 10.1007/s10682-009-9342-0 ORIGINAL PAPER
Outbreeding causes developmental instability in Drosophila subobscura Zorana Kurbalija • Marina Stamenkovic-Radak Cino Pertoldi • Marko Andjelkovic
•
Received: 25 June 2009 / Accepted: 29 November 2009 Ó Springer Science+Business Media B.V. 2010
Abstract A possible effect of interpopulation hybridization is either outbreeding depression, as a consequence of breakdown of coadapted gene complexes which can increase developmental instability (DI) of the traits, or increased heterozygosity, which can reduce DI. One of the principal methods commonly used to estimate DI is the variability of fluctuating asymmetry (FA). We analysed the effect of interpopulation hybridization in Drosophila subobscura through the variability in the wing size and the FA of wing length and width for both sexes in parental, F1 and F2 generations. The results of the wing size per se in intra- and interpopulation hybrids of D. subobscura do not explicitly reveal the significance of either of the two hypotheses. However, the results of the FA of the wing traits give a different insight. The FA of wing length and width generally increases in interpopulation crosses in F1 with respect to the FA in the parental generation, which suggests the possibility that outbreeding depression occurred in the first generation after the hybridization event. We generally observed that the FA values for the wing length and width of interpopulation hybrids were higher in F1 and F2 generations, compared to intrapopulation hybrids in same generations. These results suggest that the association between coadaptive genes with the same evolutionary history are the most probable mechanism that maintains the developmental homeostasis in Drosophila subobscura populations. Keywords Wing size
Coadapted genome Fluctuating asymmetry Outbreeding depression
Z. Kurbalija (&) M. Stamenkovic-Radak M. Andjelkovic Institute of Biological Research, University of Belgrade, Despot Stefan Blvd. 142, 11000 Belgrade, Serbia e-mail:
[email protected] M. Stamenkovic-Radak M. Andjelkovic Faculty of Biology, University of Belgrade, Studentski trg 3, 11000 Belgrade, Serbia C. Pertoldi Department of Ecology and Genetics, Institute of Biological Science, University of Aarhus, ˚ rhus C, Denmark Ny Munkegade, Building 540, 8000 A
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Introduction The anthropogenic activities on natural ecosystems increase the risk of stochastic fluctuations in population size and cause changes in the population genetic structure, which potentially result in inbreeding or outbreeding depressions (Edmands and Timmerman 2003; Frankham 2005). In disturbed habitats, previously isolated populations may come in contact and, if individuals from two such populations mate, hybridization of the two different gene pools will occur (Ross and Robertson 1990; Edmands 1999). Mating between individuals from genetically different populations which are not taxonomically distinguishable is called intraspecific or interpopulation hybridization (Barton and Hewitt 1985). Hybridization between different populations can lead to heterosis in the first generation, followed by outbreeding depression in the consecutive generation (Dobzhansky 1950; Andersen et al. 2002; Edmands 2007). Hybridization can cause outbreeding depression within the affected population due to breakdown of coadaptive gene complexes (Dobzhansky 1950). A breakdown of coadaptation might be displayed by an individual as a decreased ability to develop an optimal phenotype due to an increased DI (Leary and Allendorf 1989). Developmental instability is the product of developmental noise or stress, which affects on individuals capacity to buffer the processes that otherwise result in the development of the specific phenotype (Zakharov 1981; Palmer 1996). This can be reflected in decreasing fitness components and an increase in phenotypic variability (Barton and Hewitt 1985). This effect might be displayed after hybridization in the F1 generation if the hybridization event occurs between very distinct genomes (Markow and Ricker 1991). In other cases, disruption of coadaptive gene complexes might not be observed before the F2 generation. The disruption of coadapted gene complexes in F2 is a result of a recombination of the F1 genomes (Graham 1992; Goldberg et al. 2004). Therefore, F2 genomes consist of genes which have evolved under different selection pressures (Felley 1980). There is growing evidence that environmental and genomic stress can induce significant levels of developmental instability (DI) (Palmer and Strobeck 1986; Palmer 1994, 1996; Møller and Swaddle 1997; Pertoldi et al. 2006a). Two principal methods are commonly used to estimate DI. Some studies used phenotypic variance of different morphological traits, where estimate can be blurred by the presence of genetic and/or environmental variability (Andersen et al. 2002; Pertoldi et al. 2006a, b). Other studies used fluctuating asymmetry (FA), defined as small deviations from the perfect bilateral symmetry in morphological traits. This dissimilarity in expression of a given character on the left and right side cannot be explained by either genotypic or environmental differences, since the development of bilateral characters in an individual is ensured by the same genotype under identical environmental conditions (Palmer and Strobeck 1986). The increase or decrease of DI as a consequence of the genomic stress has been explained by two hypotheses: heterozigosity (Lerner 1954) and the genomic coadaptation (Dobzhansky 1950). The heterozygosity theory predicts that levels of heterozygosity will be inversely correlated with the level of DI (Lerner 1954; Livshits and Kobyliansky 1985; Pertoldi et al. 2006a). It has been suggested that heterozygosity has a buffering role through increased biochemical diversity, which enables a dynamic and stable developmental pathway in changing conditions (Livshits and Smouse 1993). Lerner (1954) suggested that heterozygosity in complex multi-genetics systems provides a mechanism for maintaining potential plasticity and promoting canalization.
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The genomic coadaptation hypothesis predicts that more balanced coadapted gene complexes, established over the evolutionary history of the populations via natural selection, will show higher stability in development over time (Markow 1995). Whether genomic coadaptation or heterozygosity have influence on DI is still unclear. Although no clear patterns have been found, several trends emerged. Available data indicate a tendency of FA to increase with inbreeding and population hybridization (Palmer and Strobeck 1986; Waldmann 1999; Lens et al. 2000; Garnier et al. 2006; Andersen et al. 2008). There is evidence which suggests a positive correlation between FA and genomic stress (Leary and Allendorf 1989). However, there are several studies that report exceptions to these patterns (Clarke et al. 1992; Sheridan and Pomiankowski 1997; Pelabon et al. 2005). On the other hand, the relationships of DI (measured by FA) with a breakdown of coadapted gene complexes and heterozygosity are still unclear (Vøllestad et al. 1999; Alibert and Auffray 2003). Inversion polymorphism of Drosophila was used as a model system for studying processes involved in adaptation and genetic diversity. As crossing-over is suppressed within the inversion loops of heterokaryotypes, all genes within the inverted segments segregate as a linked group, representing one physical and functional unit, called the ‘supergene’, so the different arrangements can be regarded as ‘allelic’ complexes (Krimbas 1993). Assuming a relatively long-time of selection on the linked genes within inverted regions, Dobzhansky (1948) developed the coadaptation hypothesis, which proposed that the selective value of inversions depends on the combinations of alleles, genes and their interaction. The important aspect of this hypothesis is the effects of heterosis and fitness epistasis, causing the evolution of the genes evolve after their origin (Hoffmann et al. 2004). The coadaptation hypothesis presumes that different alleles of genes will be presented in different gene arrangements, and that interpopulation differences exist for the allelic combinations of the same arrangement (Hoffmann et al. 2004). Drosophila subobscura is a Palearctic species which displays rich inversion polymorphism on all five acrocentric chromosomes of the set (Krimbas and Loukas 1980; Krimbas 1993) which makes that species a good candidate for studying the above mentioned hypotheses. In the present paper we focused on coadaptive aspect of inversion polymorphism in Drosophila subobscura populations from three ecologically and topologically distinct habitats, knowing that they possess a certain degree of genetic differentiation due to their different evolutionary histories. The aim of this study is to detect variability of the wing size and FA of wing length and wing width between inter-population and intra-population hybrids of D. subobscura. The analysis performed over two generations after hybridization was aimed at comparing the level of fluctuating asymmetry as measure of DI between intrapopulation and interpopulation hybrids through generations. However, the most important aim of the study was to discover if there was association of coadaptive gene complexes and/or higher heterozygosity maintaining developmental homeostasis in populations.
Materials and methods For the present study, D. subobscura flies were sampled in Serbia simultaneously at the end of June 2006 using fermented fruit traps. The flies were collected from three localities (beech-B, oak-O and Botanical Garden-BG). The beech (B) and the oak (O) woods are situated in different expositions on mountain Goc in central Serbia. These two woods have distinct microclimates. Beech wood features
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higher humidity with dense vegetation coverage, whereas the oak has sparser trees and is slightly warmer. The third locality is the Botanical Garden (BG) situated in the central, urban part of Belgrade, with a specific microclimate and surrounded by high anthropogenic activity. The flies collected in these three localities were used to obtain isofemale lines (IF) and they were reared on the common cornmeal-sugar-yeast-agar medium for Drosophila. All cultures were maintained and all experiments performed under constant laboratory conditions, at 19°C, approx. 60% relative humidity, light of 300 lux and 12/12 h light/dark cycles. We used 63 IF lines from oak, 38 IF lines from beech and 64 IF lines from the Botanical Garden population. The progeny of these IF lines formed from the field samples were used as the parental (P) generation in the experiment. Virgin males and females were separated within each IF line upon emerging and intra- and interpopulation crosses were made 4 days after eclosion. The intra- (B 9 B, O 9 O and BG 9 BG) and interpopulation crosses (B 9 O, BG 9 O and BG 9 B) were made among IF lines of the three D. subobscura populations. Both direct and reciprocal crosses were made in order to take into account the potential maternal effect (i.e., direct cross: male from IF line No1 with female from IF No2, and reciprocal cross: male from IF No2 crossed with female from IF No1 etc.) (Table 1). The progeny (6 males and 6 females) from each cross was transferred to fresh vials to obtain F1 and F2 generation, respectively. The flies from P, F1, and F2 generations, from intra- and interpopulation crosses (B 9 B, O 9 O, BG 9 BG, B 9 O, BG 9 O, BG 9 B) were frozen (-20°C) and used for further wing measurements.
Wing length and width analyses The left and right wings from each fly were cut and mounted on a microscope slide using double sided scotch (12.7–22.8 mm) and cover slip was placed over them. Each wing was photographed with a Canon Power Shot camera attached to a Leica stereomicroscope under 409 magnification. The measurements were performed on photographs, with Image Tool Software 3.0 (Wilcox et al. 2002). (http://ddsdx.uthscsa.edu/dig/download.html). The wing length (L) was taken as the distance from the intersection of the third longitudinal vein (L3) with the anterior cross vein (A1) to the wing tip where the third vein ends.
Table 1 Number and type of crosses for direct and reciprocal crosses
Type of cross
Intrapopulation
Direct cross
Reciprocal cross
No. of crosses
Inter line crosses
O 9 O (63 IF lines)
O$ 9 O#
O# 9 O$
63
B 9 B (38 IF lines)
B$ 9 B#
B# 9 B$
71
BG 9 BG (64 IF lines)
BG$ 9 BG#
BG# 9 BG$
89
Interpopulation O Oak population, B Beech population, BG botanical garden population
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B9O
B$ 9 O#
B# 9 O$
82
BG 9 O
BG$ 9 O#
BG# 9 O$
81
BG 9 B
BG$ 9 B#
BG# 9 B$
73
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Fig. 1 Wings landmarks used for measuring the two wing traits: wing length distance from the intersection of the third longitudinal vein (L3) with the anterior cross vein (A1) to the wing tip where the third vein ends; wing width as the distance between the ends of the second (L2) and the fifth longitudinal vein (L5)
The wing width (W) was taken as the distance between the ends of the second (L2) and the fifth longitudinal vein (L5) (Fig. 1). Statistical analyses Before interpreting FA estimates, several statistical procedures were completed. The measurement error was estimated for all samples by the two-way ANOVA on a sample of 30 individuals measured twice (Palmer 1994). There were significant interactions between wing size and individual FA for both the length (MS = 76.013, p \ 0.01) and width (MS = 45.918, p \ 0.05), which means that FA has a grater value than the measurement error. The non-parametric tests, Shapiro–Wilk (W) and Chi-squared (v2), were used to test (R–L) for departures from normality. There are several avaliable tests for normal distribution, and Shapiro–Wilk is high power test which is optimized for small sample sizes (N \ 50), and for large sample size we used Chi-squared test. The one-sample t-test was done to test a departure of the mean of (R–L) from the expected mean of zero. Test for presence of directional asymmetry (DA) should be conducted in FA studies because, the presence of DA artificialy inflates the values of certan FA indices (Palmer 1994). To test size dependence on the absolute FA, linear regression analyses of ((R ? L)/2) on |R–L| were done for all samples. The FA1 index (Palmer 1994) of each trait was measured as the absolute (unsigned) |R–L| difference between sides in all samples of intrapopulation and interpopulation hybrids (both, direct and reciprocal crosses), separately for males and females through P, F1 and F2 generations. The FA1 index is the one of the most frequently used index to describe a level of FA in sample. It is also an unbiased estimator of the sample standard deviation, and recommended for testing FA differences between 3 or more samples (Palmer and Strobeck 1992). The F-test and t-test the are very commonly used tests which assume normal distributions. The F-test is for equal variance, while the t-test is for the equality of the means. These tests were conducted in order to test significant differences in the mean and variances of the wing length and width between sexes, populations, generations, and type of cross. All these tests were done using sex, population, generation, and types of cross as
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separate variables. The conservative F-test and t-test were used to reduce the possibility of a Type 1 error. Type 1 error is typically associated with investigations dealing with a large amount of data, as in the present study. All the statistical analyses were performed using PAST software (Hammer et al. 2001). Corrections for multiple comparisons were performed using overall Bonferroni correction (Rice 1989).
Results None of the samples manifest significant deviations from normality (Tables 2, 3) and the signed right-left (R–L) size analysis show that directional asymmetry (DA) is absent in all samples. In less than 1% of the samples a positive correlation between |R–L| and the (R ? L)/2 is found. After sequential Bonferroni correction none of the regressions were significant, indicating that FA is not correlated with the trait size. Intrapopulation hybridization Changes of the mean and variance across generations in males Analysis of the difference in the mean and variance of the wing length in males is given in Table 4a. Generally, a significant decrease of the mean is observed in males from P to F2 in all direct and reciprocal crosses. The variance, in general, significantly decreases from P to F1, and increases in F2, both in direct and reciprocal crosses. Analysis of the difference in the mean and variance of the wing width is given in Table 4b. Significant decrease of the mean is obtained in all crosses (both direct and reciprocal), except in the BG 9 BG direct cross, where different trends through generations (P [ F1 \ F2) were found. The variance significantly decreases from P to F1, and towards F2, except in the B 9 B reciprocal cross. Changes of the mean and variance across generations in females Analysis of the difference in the mean and variance of the wing length in females is given in Table 5a. A significant decrease of the mean is observed from P toward F2 in all direct and reciprocal crosses. The variance generally increases through generations (P \ F1 \ F2) in both direct and reciprocal crosses. Analyses of the difference in the mean and variance of the wing width are shown in Table 5b. A significant decrease of the mean toward F2 is observed both in direct and reciprocal crosses. The variance change shows no general trend, with a significant difference between generations, found only in direct crosses. Changes of the FA across generations in males Analysis of the FA1 index between generations for wing length in males shows no significant differences between generations either in the direct and reciprocal crosses (Table 6a). Analysis of FA1 between generations for wing width, shows no significant difference between generations, except in the hybrids from BG 9 BG reciprocal cross (tP,F1 = -2.17, p \ 0.05; tP,F2 = -2.30, p \ 0.05) (Table 6b).
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97 W = 0.98, p = 0.59
F2
188 W = 0.99, p = 0.67
464 W = 0.98, p = 0.08
F1
F2
48 W = 0.97, p = 0.19
45 W = 0.97, p = 0.58
F1
117 W = 0.96, p = 0.06
F2
13 W = 0.93, p = 0.44
62 W = 0.98, p = 0.61
F1
P
16 W = 0.96, p = 0.65
P
BG 9 BG P
B9B
O9O 16 W = 0.96, p = 0.71 50 W = 0.98, p = 0.78 110 W = 0.99, p = 0.90 13 W = 0.90, p = 0.16 45 W = 0.97, p = 0.42 98 W = 0.96, p = 0.09 48 W = 0.99, p = 0.96 148 W = 0.98, p = 0.08 491 W = 0.98, p = 0.60
v2 = 2.00, p = 0.65
v2 = 1.35, p = 0.61
v2 = 1.13, p = 0.61
v2 = 0.84, p = 0.44
v2 = 2.34, p = 0.58
v2 = 0.36, p = 0.60
v2 = 1.16, p = 0.19
v2 = 0.85, p = 0.67
v2 = 1.27, p = 0.08
Shapiro–Wilk
N
Chi-square
N
Shapiro–Wilk
Reciprocal
Direct
Gen. Males
Intrapopulation
Cross
v2 = 1.26, p = 0.06
v2 = 5.13, p = 0.08
v2 = 0.17, p = 0.96
v2 = 2.00, p = 0.09
v2 = 2.55, p = 0.42
v2 = 2.69, p = 0.16
v2 = 1.78, p = 0.90
v2 = 0.88, p = 0.78
v2 = 0.50, p = 0.70
Chi-square
Shapiro–Wilk
593 W = 0.98, p = 0.17
158 W = 0.99, p = 0.97
48 W = 0.98, p = 0.83
120 W = 0.97, p = 0.05
46 W = 0.97, p = 0.28
13 W = 0.91, p = 0.20
146 W = 0.99, p = 0.49
47 W = 0.97, p = 0.29
16 W = 0.88, p = 0.05
N
Direct
Females
v2 = 4.17, p = 0.17
v2 = 0.28, p = 0.97
v2 = 1.16, p = 0.83
v2 = 2.20, p = 0.06
v2 = 3.04, p = 0.29
v2 = 0.84, p = 0.20
v2 = 1.78, p = 0.49
v2 = 0.23, p = 0.28
v2 = 1.50, p = 0.05
Chi-square
Shapiro–Wilk
572 W = 0.99, p = 0.06
184 W = 0.99, p = 0.88
48 W = 0.98, p = 0.86
120 W = 2.60, p = 0.06
44 W = 0.96, p = 0.18
13 W = 0.82, p = 0.05
105 W = 0.97, p = 0.06
46 W = 0.97, p = 0.45
16 W = 0.92, p = 0.24
N
Reciprocal
v2 = 1.46, p = 0.07
v2 = 2.04, p = 0.88
v2 = 0.83, p = 0.86
v2 = 2.60, p = 0.06
v2 = 4.54, p = 0.18
v2 = 2.69, p = 0.05
v2 = 1.62, p = 0.60
v2 = 1.82, p = 0.45
v2 = 8.5, p = 0.24
Chi-square
Table 2 Results of Shapiro–Wilk (W) and Chi-square (v2) tests for normal distribution of wing length in samples of intra- and interpopulation crosses in P, F1 and F2 generations in males and females (direct and reciprocal crosses)
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155 W = 0.95, p = 0.05
263 W = 0.98, p = 0.06
F1
F2
426 W = 0.98, p = 0.05
F2
38 W = 0.98, p = 0.79 145 W = 0.98, p = 0.11 285 W = 0.93, p = 0.05 45 W = 0.98, p = 0.74 135 W = 0.98, p = 0.10 265 W = 0.98, p = 0.05 32 W = 0.98, p = 0.96 154 W = 0.97, p = 0.05 440 W = 0.97, p = 0.07
v2 = 0.10, p = 0.97
v2 = 1.42, p = 0.09
v2 = 1.78, p = 0.49
v2 = 4.33, p = 0.17
v2 = 2.91, p = 0.07
v2 = 2.05, p = 0.42
v2 = 2.75, p = 0.06
v2 = 1.62, p = 0.05
v2 = 4.69, p = 0.08
v2 = 2.41, p = 0.10
v2 = 4.83, p = 0.75
v2 = 0.25, p = 0.76
v2 = 6.09, p = 0.06
v2 = 3.47, p = 0.10
v2 = 4.33, p = 0.75
v2 = 1.15, p = 0.51
v2 = 3.05, p = 0.11
v2 = 1.78, p = 0.79
Chi-square
Shapiro–Wilk
548 W = 0.98, p = 0.06
150 W = 0.99, p = 0.49
32 W = 0.97, p = 0.51
364 W = 0.99, p = 0.05
169 W = 0.98, p = 0.05
45 W = 0.97, p = 0.35
365 W = 0.96, p = 0.06
149 W = 0.94, p = 0.05
38 W = 0.98, p = 0.91
N
Direct
Females
v2 = 5.78, p = 0.07
v2 = 1.62, p = 0.49
v2 = 0.25, p = 0.51
v2 = 6.34, p = 0.05
v2 = 1.62, p = 0.05
v2 = 1.67, p = 0.35
v2 = 2.30, p = 0.05
v2 = 1.69, p = 0.08
v2 = 0.52, p = 0.91
Chi-square
Shapiro–Wilk
365 W = 0.98, p = 0.06
143 W = 0.98, p = 0.09
32 W = 0.95, p = 0.19
337 W = 0.98, p = 0.05
135 W = 0.98, p = 0.16
45 W = 0.97, p = 0.37
286 W = 0.94, p = 0.05
132 W = 0.97, p = 0.05
38 W = 0.97, p = 0.49
N
Reciprocal
v2 = 1.70, p = 0.06
v2 = 4.15, p = 0.06
v2 = 3.00, p = 0.19
v2 = 8.64, p = 0.05
v2 = 1.14, p = 0.16
v2 = 0.95, p = 0.37
v2 = 1.16, p = 0.60
v2 = 1.51, p = 0.05
v2 = 0.52, p = 0.49
Chi-square
* p \ 0.05, ** p \ 0.01, *** p \ 0.001
O Oak population, B Beech population, BG botanical garden population, gen generation, R right side, L left side. Note: the pixel was used as measurement unit
169 W = 0.98, p = 0.05
F1
32 W = 0.96, p = 0.06
306 W = 0.99, p = 0.42
F2
BG 9 B P
169 W = 0.97, p = 0.05
F1
45 W = 0.96, p = 0.17
38 W = 0.99, p = 0.97
P
BG 9 O P
B9O
Shapiro–Wilk
N
Chi-square
N
Shapiro–Wilk
Reciprocal
Direct
Gen. Males
Interpopulation
Cross
Table 2 continued
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97 W = 0.99, p = 0.96
F2
150 W = 0.99, p = 0.71
447 W = 0.99, p = 0.81
F1
F2
48 W = 0.96, p = 0.14
46 W = 0.94, p = 0.06
F1
160 W = 0.99, p = 0.64
F2
13 W = 0.91, p = 0.20
62 W = 0.98, p = 0.74
F1
P
16 W = 0.93, p = 0.34
P
BG 9 BG P
B9B
O9O 16 W = 0.94, p = 0.43 50 W = 0.97, p = 0.39 110 W = 0.99, p = 0.68 13 W = 0.93, p = 0.34 45 W = 0.98, p = 0.78 97 W = 0.99, p = 0.90 48 W = 0.96, p = 0.20 149 W = 0.98, p = 0.12 485 W = 0.99, p = 0.64
v2 = 2.00, p = 0.34
v2 = 0.58, p = 0.74
v2 = 2.14, p = 0.64
v2 = 8.23, p = 0.20
v2 = 1.13, p = 0.06
v2 = 0.36, p = 0.96
v2 = 2.83, p = 0.14
v2 = 0.51, p = 0.71
v2 = 0.88, p = 0.81
Shapiro–Wilk
N
Chi-square
N
Shapiro–Wilk
Reciprocal
Direct
Gen. Males
Intrapopulation
Cross
v2 = 1.01, p = 0.63
v2 = 3.15, p = 0.13
v2 = 0.83, p = 0.20
v2 = 1.10, p = 0.90
v2 = 0.24, p = 0.78
v2 = 4.53, p = 0.34
v2 = 0.98, p = 0.68
v2 = 2.32, p = 0.39
v2 = 2.50, p = 0.43
Chi-square
Shapiro–Wilk
564 W = 0.99, p = 0.39
158 W = 0.98, p = 0.07
48 W = 0.97, p = 0.15
120 W = 0.98, p = 0.46
46 W = 0.95, p = 0.08
13 W = 0.96, p = 0.87
146 W = 0.99, p = 0.94
47 W = 0.97, p = 0.35
16 W = 0.96, p = 0.75
N
Direct
Females
v2 = 0.42, p = 0.39
v2 = 3.87, p = 0.47
v2 = 1.67, p = 0.15
v2 = 4.00, p = 0.45
v2 = 0.95, p = 0.08
v2 = 0.23, p = 0.87
v2 = 0.03, p = 0.94
v2 = 1.77, p = 0.36
v2 = 0.5, p = 0.75
Chi-square
Shapiro–Wilk
557 W = 0.99, p = 0.38
174 W = 0.98, p = 0.12
48 W = 0.98, p = 0.75
120 W = 0.99, p = 0.32
44 W = 0.97, p = 0.32
13 W = 0.87, p = 0.06
139 W = 0.99, p = 0.83
46 W = 0.97, p = 0.38
16 W = 0.87, p = 0.05
N
Reciprocal
v2 = 0.43, p = 0.38
v2 = 1.17, p = 0.12
v2 = 2,50, p = 0.75
v2 = 8.46, p = 0.32
v2 = 0.73, p = 0.32
v2 = 0.84, p = 0.06
v2 = 1.80, p = 0.83
v2 = 3.56, p = 0.38
v2 = 2.50, p = 0.05
Chi-square
Table 3 Results of Shapiro–Wilk (W) and Chi-square (v2) tests for normal distribution of wing width in samples of intra- and interpopulation crosses in P, F1 and F2 generations in males and females (direct and reciprocal crosses)
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123
155 W = 0.98, p = 0.06
250 W = 0.99, p = 0.60
45 W = 0.96, p = 0.87
165 W = 0.98, p = 0.13
298 W = 0.99, p = 0.81
32 W = 0.96, p = 0.29
160 W = 0.98, p = 0.25
426 W = 0.99, p = 0.06
F1
F2
BG 9 O P
F1
F2
BG 9 B P
F1
F2
38 W = 0.98, p = 0.82 155 W = 0.98, p = 0.66 284 W = 0.99, p = 0.37 45 W = 0.97, p = 0.54 142 W = 0.98, p = 0.06 261 W = 0.99, p = 0.48 45 W = 0.96, p = 0.51 142 W = 0.98, p = 0.08 431 W = 0.99, p = 0.53
v2 = 0.52, p = 0.65
v2 = 5.23, p = 0.07
v2 = 2.48, p = 0.60
v2 = 0.23, p = 0.87
v2 = 1.71, p = 0.13
v2 = 3.61, p = 0.80
v2 = 0.05, p = 0.29
v2 = 4.85, p = 0.25
v2 = 6.34, p = 0.06
v2 = 2.05, p = 0.53
v2 = 1.94, p = 0.08
v2 = 2.93, p = 0.51
v2 = 0.99, p = 0.08
v2 = 6.22, p = 0.05
v2 = 1.49, p = 0.54
v2 = 0.31, p = 0.37
v2 = 5.23, p = 0.06
v2 = 0.52, p = 0.83
Chi-square
Shapiro–Wilk
548 W = 0.99, p = 0.10
150 W = 0.98, p = 0.22
32 W = 0.96, p = 0.28
346 W = 0.99, p = 0.06
149 W = 0.99, p = 0.13
45 W = 0.97, p = 0.31
344 W = 0.99, p = 0.44
149 W = 0.98, p = 0.14
38 W = 0.97, p = 0.41
N
Direct
Females
v2 = 5.45, p = 0.11
v2 = 1.94, p = 0.32
v2 = 0.50, p = 0.28
v2 = 6.34, p = 0.05
v2 = 1.04, p = 0.14
v2 = 0.24, p = 0.86
v2 = 2.77, p = 0.44
v2 = 1.34, p = 0.14
v2 = 2.00, p = 0.41
Chi-square
Shapiro–Wilk
511 W = 0.99, p = 0.18
142 W = 0.99, p = 0.79
31 W = 0.98, p = 0.84
332 W = 0.99, p = 0.90
135 W = 0.98, p = 0.34
45 W = 0.98, p = 0.65
321 W = 0.99, p = 0.77
149 W = 0.98, p = 0.14
38 W = 0.98, p = 0.61
N
Reciprocal
v2 = 2.73, p = 0.18
v2 = 1.15, p = 0.79
v2 = 0.38, p = 0.84
v2 = 1.80, p = 0.91
v2 = 1.94, p = 0.08
v2 = 2.02, p = 0.65
v2 = 0.27, p = 0.77
v2 = 1.74, p = 0.14
v2 = 1.16, p = 0.61
Chi-square
* p \ 0.05, ** p \ 0.01, *** p \ 0.001
O Oak population, B Beech population, BG Botanical garden population, gen generation, R right side, L left side. Note: the pixel was used as measurement unit
38 W = 0.98, p = 0.66
P
B9O
Shapiro–Wilk
N
Chi-square
N
Shapiro–Wilk
Reciprocal
Direct
Gen. Males
Interpopulation
Cross
Table 3 continued
Evol Ecol
97 541.93 ± 3.03
F2
463 569.81 ± 1.59
F2
BG 9 B
BG 9 O
B9O
32 580.96 ± 4.60
169 580.77 ± 1.65
426 570.66 ± 1.47
P
F2
306 567.21 ± 1.80
F2
F1
45 562.10 ± 5.62
165 585.62 ± 1.62
P
263 562.37 ± 1.90
F2
F1
38 586.30 ± 5.29
155 580.71 ± 2.24
P
F1
Interpopulation
188 587.85 ± 1.70
F1
48 598.21 ± 3.37
46 582.34 ± 3.30
F1
*** 1,450.01
F1 [ F2, t = 4.20
13 598.21 ± 7.76
117 561.06 ± 3.52
P
F2
*** ** *** *** 1,176.20
F1 [ F2, t = 8.15
P [ F1, t = 2.74
P [ F2, t = 5.60
F1 [ F2, t = 6.62
781.41
***
F1 [ F2, t = 6.08
P [ F1, F = 2.71
987.97
F1 [ F2, t = 6.76
922.86
F1 [ F2, t = 3.95 ***
677.13 458.85
P [ F1, t = 0.04
P [ F2, t = 1.86
Mean ± SE
t-test
50 587.15 ± 4.58 P [ F2, t = 4.78
16 590.30 ± 9.07 P [ F1, t = 0.32
N
97 549.93 ± 3.47 F1 [ F2, t = 7.42 48 602.47 ± 3.52 P [ F1, t = 4.92
***
153 584.87 ± 1.94 P [ F2, t = 0.37
45 578.00 ± 3.60 P \ F1, t = -1.69
284 561.01 ± 2.05 F1 [ F2, t = 5.92
145 579.94 ± 2.15 P [ F2, t = 4.97
38 590.30 ± 4.99 P [ F1, t = 2.11
*** 490 571.00 ± 1.45 F1 [ F2, t = 5.71
** 194 585.22 ± 1.51 P [ F2, t = 6.62
*
45 594.80 ± 4.78 P [ F2, t = 4.64
13 595.98 ± 7.94 P [ F1, t = 0.12
** 109 543.16 ± 3.53 F1 [ F2, t = 7.25
**
p
Reciprocal
1,315.60
443.35
596.56
669.40
944.90
***
959.67
*** 1,372.26
587.44
1,540.95
574.47
581.86
*** 1,191.28
***
*
*** 1,027.09
***
***
*** 1,168.39
*** 1,030.45
820.50
*** 1,357.19
p
*
***
F1 [ F2, F = 1.43
P \ F2, F = 1.63
P \ F1, F = 2.36
**
**
F1 \ F2, F = 2.65 ***
P \ F2, F = 2.65
P [ F1, F = 1.01
F1 \ F2, F = 1.78 ***
P \ F2, F = 1.26
P [ F1, F = 1.41
F1 \ F2, F = 2.32 ***
P \ F2, F = 1.72
P [ F1, F = 1.34
F1 \ F2, F = 1.14
P \ F2, F = 1.42
P \ F1, F = 1.26
F1 \ F2, F = 1.29
P \ F2, F = 1.03
P [ F1, F = 1.26
Variance F-test
*** 1,047.45
p
45 578.00 ± 3.60 P \ F1, t = -2.66 *** 141 604.29 ± 3.12 P [ F2, t = 1.88
F1 \ F2, F = 2.01 *** 440 575.42 ± 1.47 F1 [ F2, t = 9.16
P \ F2, F = 1.36
P [ F1, F = 1.47
F1 \ F2, F = 2.28 *** 265 575.78 ± 2.41 F1 [ F2, t = 0.37
432.78 ***
P [ F1, F = 3.28 P [ F2, F = 1.44
P \ F1, t = -5.52 *** 1,422.06
F1 \ F2, F = 1.21
P [ F2, F = 1.12
P [ F1, F = 1.36
P \ F2, F = 2.17
P \ F2, F = 2.16
P [ F1, F = 1.00
F1 \ F2, F = 1.78
P \ F1, F = 1.14
P [ F1, F = 1.57
F1 \ F2, F = 2.08
P [ F2, F = 1.31
P \ F2, t = -0.99
949.07
1,064.14 ***
P [ F1, t = 1.07
P [ F2, t = 4.44
543.53
544.19
892.31
499.99
***
P [ F2, t = 6.42
783.62
*
P [ F1, t = 2.14
697.60
***
P [ F2, t = 4.16
* 1,894.46
Variance F-test
16 603.98 ± 10.88 P [ F1, t = 2.27
p
59 584.28 ± 3.44
t-test
P
Mean ± SE
F1
BG 9 BG P
B9B
O9O
Intrapopulation
N
Gen. Direct
(a) Wing length
Cross
Mean(R ? L)/2
Table 4 The mean and variance for (a) wing length and (b) wing width in males of intra- and interpopulation crosses across generations (direct and reciprocal crosses)
Evol Ecol
123
123 399.95 238.46 * 368.91
32 367.26 ± 3.53 P \ F1, t = -1.71
169 372.60 ± 1.19 P [ F2, t = -0.21
426 367.99 ± 0.93 F1 [ F2, t = 2.79
F2
165 371.07 ± 1.18 P \ F2, t = -1.80
306 361.92 ± 1.26 F1 [ F2, t = 4.76
F1
F2
P
230.12 *** 485.75
45 355.52 ± 3.58 P \ F1, t = -5.31
F1
*** 465.39 *** 576.26
263 358.86 ± 1.33 F1 [ F2, t = 5.71
F2
635.61 * 380.20
P
38 368.10 ± 4.10 P \ F1, t = -0.75
155 370.91 ± 1.57 P [ F2, t = 2.41
P
F1
521 383.76 ± 1.16 F1 \ F2, t = -4.48 *** 704.83
246.18
P [ F1, F = 1.56
Mean ± SE
t-test
*** 194 373.56 ± 1.08 P [ F2, t = 4.35
48 382.15 ± 2.68 P [ F1, t = 3.36
97 353.16 ± 2.08 F1 [ F2, t = 7.27
45 388.39 ± 5.54 P [ F2, t = 3.98
13 377.30 ± 5.90 P \ F1, t = -1.02
109 350.13 ± 2.15 F1 \ F2, t = -0.49
50 377.10 ± 3.14 P [ F2, t = 3.97
16 373.96 ± 5.60 P [ F1, t = -0.49
N
***
*
38 373.91 ± 3.58 P [ F1, t = 1.76
153 370.03 ± 1.22 P [ F2, t = 0.58
45 368.60 ± 2.40 P \ F1, t = -0.55
284 358.55 ± 1.37 F1 [ F2, t = 4.59
145 368.34 ± 1.32 P [ F2, t = 3.88
F1 \ F2,F = 1.55
P [ F2, F = 1.08
P [ F1, F = 1.68
32 374.35 ± 3.10 P \ F1, t = -0.06 154 374.57 ± 1.39 P [ F2, t = 1.1 ** 440 370.25 ± 0.98 F1 [ F2, t = 2.34
*
F1 \ F2, F = 2.11 *** 265 366.33 ± 1.56 F1 [ F2, t = 1.64
P [ F2, F = 1.19
P [ F1, F = 2.50
F1 \ F2, F = 1.22
P [ F2, F = 1.36
P [ F1, F = 1.67
F1 \ F2, F = 2.86 *** 491 367.49 ± 1.02 F1 [ F2, t = 3.44
P \ F2, F = 2.47
P [ F1, F = 1.16
F1 \ F2, F = 1.09
P [ F2, F = 1.22
P [ F1, F = 1.33
F1 \ F2, F = 1.54
P [ F2, F = 1.02
p
Reciprocal
452.67 P \ F1, F = 3.05
502.36 F1 \ F2, F = 1.02
492.34 P \ F2, F = 1.01
501.82 P [ F1, F = 1.02
Variance F-test
*
***
***
***
***
***
***
* p \ 0.05, ** p \ 0.01, *** p \ 0.001
p
*
**
***
422.12 F1 \ F2, F = 1.42
296.16 P \ F2, F = 1.37
307.33 P [ F1, F = 1.04
645.40 F1 \ F2, F = 2.81 ***
229.45 P \ F2, F = 2.49
259.45 P [ F1, F = 1.13
529.86 F1 \ F2, F = 2.09 ***
252.57 P \ F2, F = 1.08
488.28 P [ F1, F = 1.93
511.95 F1 \ F2, F = 2.25 ***
227.88 P \ F2, F = 1.48
345.65 P [ F1, F = 1.52
418.05 F1 [ F2, F = 3.31 ***
*** 1,383.27 P [ F2, F = 1.08
***
p
O Oak population, B Beech population, BG botanical garden population, gen generation, R right side, L left side. Note: the pixel was used as measurement unit
BG 9 B
BG 9 O
B9O
Interpopulation
F2
F1
188 374.55 ± 1.44 P \ F2, t = -1.21
*** 353.37
97 351.31 ± 1.91 F1 [ F2, t = 6.89
F2 285.76
*** 324.58
46 374.21 ± 2.66 P [ F2, t = 4.99
F1
48 379.05 ± 2.44 P [ F1, t = 1.74
432.52
* 583.30
13 379.40 ± 5.77 P [ F1, t = 0.89
117 361.78 ± 2.23 F1 [ F2, t = 2.58
F2
P
* 594.21 ** 379.62
Variance F-test
16 383.19 ± 6.09 P [ F1, t = 2.12
p
62 370.99 ± 2.47 P [ F2, t = 3.32
t-test
P
Mean ± SE
F1
BG 9 BG P
B9B
O9O
Intarpopulation
N
Gen. Direct
(b) Wing width
Cross
Mean(R ? L)/2
Table 4 continued
Evol Ecol
BG 9 B
BG 9 O
B9O
*** ***
158 634.37 ± 2.12 P [ F2, t = 8.02
592 615.35 ± 1.29 F1 [ F2, t = 6.96
1,188.03 663.62
*** ***
32 635.46 ± 6.09 P [ F1, t = 0.57
150 632.42 ± 2.10 P [ F2, t = 3.63
548 615.93 ± 1.25 F1 [ F2, t = 6.26
F1
F2
858.87
*** 1,148.13
346 612.62 ± 1.82 F1 [ F2, t = 7.38
F2
602.32
1,149.13
P
***
45 634.22 ± 5.05 P \ F1, t = -0.24
149 635.31 ± 2.01 P [ F2, t = 4.02
P
F1
***
365 609.35 ± 1.59 F1 [ F2, t = 7.48
F2
920.83
734.75 550.68
***
38 628.15 ± 4.40 P \ F1, t = -0.45
149 630.11 ± 1.92 P [ F2, t = 3.67
P
989.77
710.84
F1
Interpopulation
F2
F1
688.47
*** 1,444.42
F2 ***
701.80
***
46 624.31 ± 3.91 P [ F2, t = 4.02
F1
48 652.80 ± 3.77 P [ F1, t = 4.21
390.78
13 638.98 ± 5.48 P [ F1, t = 1.85
120 595.87 ± 3.47 F1 [ F2, t = 4.66
*** 1,266.56
146 596.34 ± 2.94 F1 [ F2, t = 4.26
F2
P
1,019.74 *** 1,556.47
p
t-test
45 623.15 ± 4.77 P [ F2, t = 4.40
13 642.13 ± 6.23 P [ F1, t = 1.99
139 590.99 ± 3.05 F1 [ F2, t = 4.88
46 619.94 ± 4.65 P [ F2, t = 7.65
130 624.92 ± 2.61 P [ F2, t = 5.40
38 628.20 ± 4.42 P [ F1, t = 0.61
* 571 617.98 ± 1.16 F1 [ F2, t = 5.09
174 629.80 ± 1.81 P [ F2, t = 5.90
48 642.28 ± 3.49 P [ F1, t = 3.20
** 120 598.07 ± 3.21 F1 [ F2, t = 4.17
*
Mean ± SE
16 662.36 ± 7.38 P [ F1, t = 4.70
N
Reciprocal
**
39 598.06 ± 5.95 P \ F1, t = -6.00 135 632.46 ± 2.56 P \ F2, t = -1.68
F1 \ F2, F = 1.29
P [ F2, F = 1.38
P [ F1, F = 1.79
*
520 618.63 ± 1.35 F1 [ F2, t = 5.95
143 635.70 ± 2.39 P [ F2, t = 0.93
32 623.87 ± 5.69 P \ F1, t = - 2.07
F1 \ F2, F = 1.91 *** 337 608.82 ± 2.07 F1 [ F2, t = 6.47
P [ F2, F = 1.01
P [ F1, F = 1.91
F1 \ F2, F = 1.67 *** 335 598.74 ± 1.77 F1 [ F2, t = 6.08
P \ F2, F = 1.25
P [ F1, F = 1.33
F1 \ F2, F = 1.39
P \ F2, F = 1.44
P \ F1, F = 1.03
F1 \ F2, F = 2.06
P \ F2, F = 3.70
P \ F1, F = 1.79
F1 [ F2, F = 1.23
P \ F2, F = 1.24
P \ F1, F = 1.53
Variance F-test
16 642.40 ± 7.98 P [ F1, t = 1.82
p
47 622.47 ± 5.75 P [ F2, t = 4.96
t-test
P
Mean ± SE
F1
BG 9 BG P
B9B
O9O
Intrapopulation
N
Gen. Direct
(a) Wing length
Cross
Mean(R ? L)/2
996.44
872.50
887.31
741.83
765.99
569.99
586.46
***
953.96
814.79
* 1,037.51
*** 1,444.02
883.06
*** 1,381.89
*** 1,047.27
***
***
***
**
*** 1,240.58
*** 1,025.99
505.00
p
F1 \ F2, F = 1.17
P [ F2, F = 1.09
P [ F1, F = 1.27
F1 \ F2, F = 1.63
P \ F2, F = 1.04
P [ F1, F = 1.56
F1 \ F2, F = 1.05
P \ F2, F = 1.41
P \ F1, F = 1.20
F1 \ F2, F = 1.34
P \ F2, F = 1.31
P [ F1, F = 1.03
**
*
F1 \ F2, F = 1.21 ***
P \ F2, F = 2.45
P \ F1, F = 2.03
F1 \ F2, F = 1.29
P \ F2, F = 1.48
P \ F1, F = 1.14
Variance F-test
*** 1,290.10
***
***
p
Table 5 The mean and variance for (a) wing length and (b) wing width in females of intra- and interpopulation crosses across generations (direct and reciprocal crosses)
Evol Ecol
123
123 * 356.23 ** 789.63 * 490.81 *** 369.61 *** 492.00
46 400.32 ± 2.78 P [ F2, t = 2.38
120 387.64 ± 2.56 F1 [ F2, t = 2.82
48 414.31 ± 3.18 P [ F1, t = 2.57
158 405.85 ± 1.53 P [ F2, t = 4.97
592 397.75 ± 0.91 F1 [ F2, t = 4.19
F1
F2
F1
F2
*** 421.74 622.50
346 391.14 ± 1.10 F1 [ F2, t = 7.75
45 399.91 ± 3.72 P \ F1, t = -0.75
F2
*** 515.14 *** 434.27
346 392.63 ± 1.22 F1 [ F2, t = 4.68
32 407.22 ± 3.68 P [ F1, t = 4.61
F2
P
* 324.67
149 402.44 ± 1.48 P [ F2, t = 2.00
F1
P
308.62
149 406.09 ± 1.44 P [ F2, t = 1.48
P
F1
P \ F1, F = 1.38
P \ F1, F = 1.13
F1 \ F2, F = 1.59
P [ F2, F = 1.21
P [ F1, F = 1.92
F1 \ F2, F = 1.37
P [ F2, F = 1.85
P [ F1, F = 2.53
F1 \ F2, F = 1.33
P \ F2, F = 1.00
P [ F1, F = 1.33
F1 \ F2, F = 2.22
P \ F2, F = 2.86
P \ F1, F = 1.29
F1 [ F2, F = 1.74
P [ F2, F = 1.26
Mean ± SE
t-test
*
44 401.08 ± 2.99 P [ F2, t = 2.52
13 403.64 ± 7.05 P [ F1, t = 0.38
* 139 380.68 ± 1.83 F1 [ F2, t = 4.55
46 397.22 ± 3.03 P [ F2, t = 7.32
16 421.94 ± 4.61 P [ F1, t = 4.25
N
38 401.14 ± 2.78 P [ F1, t = 0.92
*** 449.59
*** 425.86
293.51
393.88
*** 333.06
* 262.26
*** 552.26
* 394.45
647.02
*** 468.31
*** 421.90
32 397.77 ± 3.29 P \ F1, t = -2.22
* 346.45
** 657.88
** 441.95
* p \ 0.05, ** p \ 0.01, *** p \ 0.001
p
P \ F1, F = 1.13
F1 \ F2, F = 1.49 **
P \ F2, F = 1.09
P [ F1, F = 1.36
F1 \ F2, F = 1.05
P \ F2, F = 1.53
P \ F1, F = 1.45
F1 \ F2, F = 1.18
P \ F2, F = 1.50
P \ F1, F = 1.27
F1 \ F2, F = 1.40
P [ F2, F = 1.17
P [ F1, F = 1.64
F1 \ F2, F = 1.11
P \ F2, F = 1.37
P \ F1, F = 1.24
Variance F-test
*** 340.46
p
45 379.02 ± 3.66 P \ F1, t = -5.08 *** 602.09 135 398.21 ± 1.81 P \ F2, t = -2.85
** 337 390.56 ± 1.40 F1 [ F2, t = 3.07
**
* 335 384.53 ± 1.16 F1 [ F2, t = 6.08
** 130 397.76 ± 1.81 P [ F2, t = 4.65
***
* 572 399.48 ± 0.83 F1 [ F2, t = 1.72
174 402.38 ± 1.38 P [ F2, t = 3.52
48 409.85 ± 2.34 P [ F1, t = 2.57
** 120 386.20 ± 2.14 F1 [ F2, t = 3.74
p
Reciprocal
O Oak population, B Beech population, BG botanical garden population, gen generation, R right side, L left side. Note: the pixel was used as measurement unit
BG 9 B
BG 9 O
B9O
Interpopulation ** 780.56
276.18
13 406.57 ± 4.61 P [ F1, t = 1.08
38 396.54 ± 4.53 P \ F1, t = -2.62
*** 447.93
146 386.08 ± 1.75 F1 [ F2, t = 3.34
F2
P
565.34 *** 781.24
Variance F-test
16 412.66 ± 5.94 P [ F1, t = 1.75
p
47 398.97 ± 4.08 P [ F2, t = 4.71
t-test
P
Mean ± SE
F1
BG 9 BG P
B9B
O9O
Intrapopulation
N
Gen. Direct
(b) Wing width
Cross
Mean(R ? L)/2
Table 5 continued
Evol Ecol
13 2.73 ± 0.48 P \ F1, t = -0.77 45 3.22 ± 0.31 P \ F2, t = -1.00 97 3.50 ± 0.27 F1 \ F2, t = -0.62 48 4.73 ± 0.48 P [ F1, t = 0.36 194 4.53 ± 0.25 P [ F2, t = 0.20 490 4.62 ± 0.16 F1 \ F2, t = -0.31
13 2.26 ± 0.33 P \ F1, t = -1.46
46 3.38 ± 0.39 P \ F2, t = -1.78
97 3.52 ± 0.25 F1 \ F2, t = -0.31
48 4.42 ± 0.42 P [ F1, t = 0.16
188 4.34 ± 0.23 P \ F2, t = -0.09
463 4.47 ± 0.15 F1 \ F2, t = -0.45
F1
F2
F1
F2
109 2.71 ± 0.18 F1 [ F2, t = 0.84
117 3.12 ± 0.24 F1 [ F2, t = 0.80
F2
P
50 2.99 ± 0.30 P [ F2, t = 2.17
59 3.48 ± 0.39 P [ F2, t = 0.57
F1
Mean ± SE t-test
16 3.90 ± 0.75 P [ F1, t = 1.35
p N
16 3.51 ± 0.59 P [ F1, t = 0.05
Mean ± SE t-test
Reciprocal
P
BG 9 BG P
B9B
O9O
Intrapopulation
N
Direct
Gen. Males
(a) Wing length
Cross
FA1 = |R - L| wing length
*
Mean ± SE t-test
592 4.75 ± 0.14 F1 \ F2, t = -0.57
158 4.58 ± 0.26 P \ F2, t = -2.38
48 3.55 ± 0.38 P \ F1, t = -1.20
120 3.30 ± 0.26 F1 \ F2, t = -0.75
46 2.95 ± 0.31 P \ F2, t = -0.19
13 3.14 ± 0.55 P [ F1, t = 0.30
146 2.89 ± 0.18 F1 \ F2, t = -0.44
47 2.73 ± 0.33 P [ F2, t = 0.91
16 3.42 ± 0.63 P [ F1, t = 1.03
p N
Direct
Females
Mean ± SE t-test
48 3.68 ± 0.42 P \ F1, t = -1.13
120 3.48 ± 0,21 F1 \ F2, t = -0.46
45 3.28 ± 0.38 P \ F2, t = -0.56
13 3.11 ± 0.36 P \ F1, t = -0.24
139 2.56 ± 0.16 F1 [ F2, t = 1.13
46 2.96 ± 0.38 P [ F2, t = 0.61
16 2.86 ± 0.40 P \ F1, t = -0.14
N
571 4.67 ± 0.16 F1 \ F2, t = -1.44
** 174 4.22 ± 0.22 P \ F2, t = -1.78
*
p
Reciprocal p
Table 6 The FA1 index differences between generations and type of crosses for (a) wing length and (b) wing width in males and females in direct and reciprocal crosses
Evol Ecol
123
123 141 5.17 ± 0.32 P \ F2, t = -1.50
*
**
169 4.33 ± 0.29 P \ F1, t = -2.12
426 5.25 ± 0.19 F1 \ F2, t = -2.45
F1
F2
440 5.41 ± 0.20 F1 \ F2, t = -0.60
32 4.29 ± 0.49 P \ F1, t = -1.23
32 3.73 ± 0.33 P \ F1, t = -0.95
265 4.31 ± 0.18 F1 [ F2, t = 2.49
306 4.78 ± 0.20 F1 [ F2, t = 0.03
F2
BG 9 B P
153 5.24 ± 0.36 P \ F2, t = -1.03
165 4.79 ± 0.26 P \ F2, t = -1.25
F1
*** 284 4.21 ± 0.15 F1 [ F2, t = 1.56 45 3.78 ± 0.39 P \ F1, t = -2.1
263 4.35 ± 0.18 F1 [ F2, t = 4.17
F2
145 4.66 ± 0.26 P \ F2, t = -0.24
38 4.10 ± 0.47 P \ F1, t = -0.99
45 4.10 ± 0.37 P \ F1, t = -1.29
155 5.94 ± 0.39 P [ F2, t = 0.12
38 4.42 ± 0.81 P \ F1, t = -1.71
F1
P
BG 9 O P
B9O
Mean ± SE t-test
N
p
N
Mean ± SE t-test
Reciprocal
Direct
Gen. Males
Interpopulation
Cross
FA1 = |R - L| wing length
Table 6 continued
Mean ± SE t-test
149 4.84 ± 0.32 P \ F2, t = -2.07
45 4.00 ± 0.39 P \ F1, t = -1.42
365 4.60 ± 0.15 F1 [ F2, t = 0.91
149 4.88 ± 0.29 P \ F2, t = -0.54
38 4.33 ± 0.55 P \ F1, t = -0.85
N
548 5.70 ± 0.19 F1 \ F2, t = -1.45
150 5.11 ± 0.33 P \ F2, t = -2.45
31 3.69 ± 0.48 P \ F1, t = -1.84
** 346 5.22 ± 0.21 F1 \ F2, t = -1.02
*
p
Direct
Females
Mean ± SE t-test
48 3.68 ± 0.43 P \ F1, t = -1.82
337 4.90 ± 0.19 F1 \ F2, t = -0.02
135 4.89 ± 0.33 P \ F2, t = -1.97
39 3.74 ± 0.42 P \ F1, t = -1.75
335 5.17 ± 0.26 F1 \ F2, t = -1.93
130 4.32 ± 0.42 P \ F2, t = -0.12
38 5.07 ± 0.63 P [ F1, t = 1.33
N
520 5.42 ± 0.18 F1 \ F2, t = -0.43
** 143 5.23 ± 0.47 P \ F2, t = -2.88
*
p
Reciprocal
**
*
p
Evol Ecol
50 2.52 ± 0.27 P [ F2, t = 1.23 109 2.27 ± 0.17 F1 [ F2, t = 0.80 13 2.37 ± 0.29 P [ F1, t = -0.18 45 2.47 ± 0.29 P [ F2, t = -0.82 97 2.91 ± 0.24 F1 \ F2, t = -1.09 48 4.22 ± 0.44 P \ F1, t = -0.17
* 194 4.32 ± 0.25 P \ F2, t = -0.59 491 4.53 ± 0.16 F1 \ F2, t = -0.73
*
62 3.17 ± 0.31 P \ F2, t = -1.92
117 3.03 ± 0.21 F1 [ F2, t = 0.38
13 2.01 ± 0.33 P \ F1, t = -1.44
46 2.76 ± 0.26 P [ F2, t = 0.84
97 3.10 ± 0.25 F1 [ F2, t = -0.84
48 3.10 ± 0.40 P \ F1, t = -2.17
188 4.15 ± 0.22 P \ F2, t = -2.30
521 4.37 ± 0.16 F1 \ F2, t = -0.71
F2
P
F1
F2
BG 9 BG P
F1
F2
B9B
O9O
F1
Mean ± SE t-test
16 2.90 ± 0.59 P [ F1, t = 0.64
p N
16 1.87 ± 0.47 P \ F1, t = -1.98
Mean ± SE t-test
Reciprocal
P
Intrapopulation
N
Direct
Gen. Males
(b) Wing width
Cross
FA1 = |R - L| wing length
Table 6 continued
Mean ± SE t-test
592 4.32 ± 0.15 F1 [ F2, t = 1.54
158 4.82 ± 0.26 P [ F2, t = 0.05
48 4.35 ± 0.46 P \ F1, t = -0.89
120 2.63 ± 0.20 F1 \ F2, t = -0.77
46 2.34 ± 0.32 P \ F2, t = -0.30
13 2.44 ± 0.47 P [ F1, t = 0.15
146 2.65 ± 0.16 F1 [ F2, t = 2.46
47 3.53 ± 0.39 P \ F2, t = -0.38
16 2.46 ± 0.51 P \ F1, t = -1.47
p N
Direct
Females
Mean ± SE t-test
572 4.64 ± 0.17 F1 [ F2, t = 0.04
174 4.65 ± 0.24 P \ F2, t = -2.00
48 3.43 ± 0.35 P \ F1, t = -2.46
120 3.18 ± 0.20 F1 \ F2, t = -1.18
44 2.73 ± 0.30 P \ F2, t = -0.36
13 2.94 ± 0.67 P [ F1, t = 0.33
* 139 2.82 ± 0.17 F1 \ F2, t = -1.19
46 2.41 ± 0.27 P \ F2, t = -1.21
16 2.15 ± 0.63 P \ F1, t = -0.45
p N
Reciprocal
*
*
p
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123
306 4.55 ± 0.21 F1 [ F2, t = 0.34
F2
154 4.77 ± 0.34 P \ F2, t = -1.21 440 4.85 ± 0.20 F1 \ F2, t = -0.21
169 5.07 ± 0.39 P \ F2, t = -0.53
426 4.34 ± 0.17 F1 [ F2, t = 2.00
F1
F2
Mean ± SE t-test
548 4.58 ± 0.16 F1 [ F2, t = 0.41
157 4.73 ± 0.35 P \ F2, t = -0.56
32 4.20 ± 0.42 P \ F1, t = -0.67
346 4.72 ± 0.18 F1 [ F2, t = 1.18
149 5.19 ± 0.42 P \ F2, t = -1.57
45 3.87 ± 0.51 P \ F1, t = -1.61
346 4.82 ± 0.21 F1 [ F2, t = 1.17
* 149 5.29 ± 0.35 P \ F2, t = -1.13
38 4.08 ± 0.40 P \ F1, t = -1.69
p N
Direct
Females
Mean ± SE t-test
520 4.79 ± 0.18 F1 [ F2, t = 1.22
143 5.27 ± 0.37 P [ F2, t = 1.54
32 5.98 ± 1.51 P [ F1, t = 0.75
337 4.35 ± 0.19 F1 [ F2, t = 3.09
135 5.46 ± 0.31 P \ F2, t = -0.87
45 3.88 ± 0.40 P \ F1, t = -2.71
335 5.54 ± 0.27 F1 \ F2, t = -0.70
130 5.19 ± 0.43 P \ F2, t = -2.05
38 3.88 ± 0.47 P \ F1, t = -1.58
p N
Reciprocal
* p \ 0.05, **p \ 0.01, ***p \ 0.001
O Oak population, B Beech population, BG botanical garden population, gen generation, R right side, L left side. Note: the pixel was used as measurement unit
32 3.94 ± 0.59 P \ F1, t = -1.05
32 3.40 ± 0.66 P \ F1, t = -1.14
265 4.38 ± 0.22 F1 [ F2, t = 1.29
153 4.86 ± 0.30 P \ F2, t = -0.30
45 4.21 ± 0.47 P \ F1, t = -1.06
284 5.08 ± 0.24 F1 \ F2, t = -1.50
BG 9 B P
*
*
165 4.67 ± 0.26 P \ F2, t = -2.19
F1
263 4.67 ± 0.26 F1 [ F2, t = 1.37
F2
38 3.65 ± 0.43 P \ F1, t = -1.32
** 145 4.48 ± 0.30 P \ F2, t = -2.14
*
155 5.28 ± 0.37 P \ F2, t = -2.61
F1
**
45 3.32 ± 0.38 P \ F1, t = -2.53
38 2.80 ± 0.42 P \ F1, t = -3.22
P
BG 9 O P
B9O
Mean ± SE t-test
N
p
N
Mean ± SE t-test
Reciprocal
Direct
Gen. Males
Interpopulation
Cross
FA1 = |R - L| wing length
Table 6 continued
**
**
*
p
Evol Ecol
Evol Ecol
Changes of the FA across generations in females Analysis of FA1 between generations for wing length in females shows a significant increase of FA through generations (P \ F1 \ F2) only in direct cross BG 9 BG (tP,F1 = -1.20, p \ 0.05; tP,F2 = -2.38, p \ 0.001) (Table 6a). The FA1 analysis between generations for wing width shows no significant differences between generations (Table 6b), except in the hybrids from the direct cross O 9 O (tF1,F2 = 2.46, p \ 0.05). In the hybrids of the reciprocal crosses within the BG population, a significant difference is obtained between generations (tP,F1 = -2.46, p \ 0.05; tP,F2 = -2.00, p \ 0.05). Interpopulation hybridization Changes of the mean and variance across generations in males The analysis of the mean and variance of the wing length in males is given in Table 4a. A significant decrease of the mean (as the one observed for intrapopulation hybrids) is found in the hybrids from B 9 O, both in direct and reciprocal crosses. But, in the hybrids from BG 9 O and BG 9 B, a different general pattern is obtained. In direct crosses, the mean significantly increases in F1 and significantly decreases in the F2 generation (P \ F1 [ F2). The same tendency is found in hybrids of the BG 9 B reciprocal crosses. The variance, in general, significantly decreases in F1 and increases in F2, except in the hybrids of BG 9 B, where the opposite was obtained in the reciprocal crosses. Analysis of the mean and variance of the wing width in males is given in Table 4b. The same general pattern as for the wing length is obtained, with a significant decrease of the mean through generations, in B 9 O, both in direct and reciprocal crosses. In hybrids from direct crosses of BG 9 O and BG 9 B a significant increase of the mean in F1 is followed by a significant decrease in F2. The variance, in general, significantly decreases in F1 and an increase in F2, both in direct and reciprocal crosses (Table 4b.). Changes of the mean and variance across generations in females Analysis of the mean and variance of the wing length in females is shown in Table 5a. The mean significantly decreases in hybrids from both direct and reciprocal B 9 O crosses. In the hybrids from the BG 9 O cross, the mean increases in F1 and significantly decreases in the F2 generation. A similar response is found in the reciprocal crosses of BG 9 O. However, in the hybrids from BG 9 B, the mean significantly increases in consecutive generations of direct crosses, and a different trend is obtained in hybrids from the reciprocal crosses (P \ F1 [ F2). The variance, in general, significantly decreases in F1 and increases in F2, in both direct and reciprocal crosses. Analysis of the mean and variance of the wing width in females shows a significant increase of the mean in F1 and a significant decrease in the F2 generation in B 9 O hybrids from direct crosses (Table 5b). The mean significantly decreases through generations in reciprocal crosses (P [ F1 [ F2). In BG 9 O hybrids the mean increases in the F1 generation and significantly decreases in F2, in both direct and reciprocal crosses. However, in the hybrids from the BG 9 B direct cross, a significant decrease of the mean in F1 and a significant increase in F2 is found. In the reciprocal crosses, a different result is obtained (P \ F1 [ F2).
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The variance, in general, significantly decreases in F1 and increases in F2 in the hybrids from both direct and reciprocal crosses, with the exception of BG 9 B (P \ F1 [ F2) in direct crosses (Table 5b). Changes of FA across generations in males Analysis of the FA1 between generations for wing length in males is shown in Table 6a. A significant increase of FA in F1 and its significant decrease in F2 is observed for B 9 O hybrids both from direct and reciprocal crosses. In the hybrids from the BG 9 B direct cross, FA1 increases across generations (P \ F1 \ F2). The FA1 index analysis between generations for wing width in males is shown in Table 6b. In the hybrids from B 9 O (direct crosses) FA significantly increases in F1 and decreases in F2. However, a different trend (P \ F1 \ F2) is found in the reciprocal crosses. In direct crosses, BG 9 O and BG 9 B, a trend (P \ F1 [ F2) is obtained, without significant differences in reciprocal crosses. Changes of the FA across generations in females The results of the FA1 analysis between generations for wing length in females are shown in Table 6a. The FA1 significantly increases across generations in the hybrids both from direct and reciprocal BG 9 O crosses. Also, in the hybrids from BG 9 B direct crosses, FA1 increases through generations (P \ F1 \ F2) and the same significant trend is found in the hybrids from the reciprocal crosses. Analysis of FA1 between generations for wing width in females is shown in Table 6b. No significant difference between generations in the hybrids from the B 9 O direct crosses is obtained, but in the reciprocal crosses, a significant increase of FA between generations (P \ F1 \ F2) is observed. In the hybrids from the BG 9 O reciprocal crosses, FA significantly increases in F1 and decreases in F2. No significant difference of FA is obtained between generations of BG 9 B both in direct and reciprocal crosses.
Discussion In the present paper we focused on the coadaptive aspect of genetic variability at population level, and its relation to genomic stress, such as interpopulation hybridization. Populations of D. subobscura from three ecologically and topologically distinct habitats were analyzed, presuming that they possess a certain degree of genetic differences due to their different evolutionary histories. Previous analyses of the inversion polymorphism show that these populations differ in the frequencies of some gene arrangements (Andjelkovic et al. 2003; Stamenkovic-Radak et al. 2008; Jelic et al. 2009). The different gene arrangements are carriers of various alleles that are differently favoured in diverse environmental conditions and prove in most cases to be the major factor determining the gene arrangement frequencies in natural populations of D. subobscura (Andjelkovic et al. 2003). Assuming that all genes within inversion segregate as a linked group, and that they have existed together for relatively long-time under selection, we could considered each inversion as a coadapted gene complex (Krimbas 1993). The coadaptation hypothesis presumes that different alleles of genes will be presented in different gene arrangements, and that interpopulation differences exist for the allelic combinations of the same
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arrangement (Hoffmann et al. 2004), which makes these three populations suitable for testing the coadaptation versus heterozygosity hypothesis. In most studies with different Drosophila species and with various traits (Anderson 1968; David 1979), heterosis is found in F1 hybrids, and lost in F2, as a consequence of disruption of the balanced polygene complexes. This supports the coadaptation hypothesis. Our results show that the means of the wing length and width for intrapopulation hybrids significantly decrease in F1 and F2 generations with respect to parental values in both sexes, in direct and reciprocal crosses. As opposed to that, hybridization between ecologically different and distant populations causes an increase of the wing size in F1 and a decrease in F2. This confirms that distance is important in hybridization between the closely situated beech and oak populations. Their hybrids show a similar decrease of the mean wing size through generations as hybrids from intrapopulation crosses of each population, suggesting that a higher gene flow is probably involved between these two populations. Some studies showed evidence of coadaptation in this species when different experimental design, populations, and traits were considered (Orengo and Prevosti 1996; Banerjee and Singh 1998). The results concerning wing size and variance per se obtained in intra- and interpopulation hybrids of D. subobscura in our paper cannot explicitly reveal the significance of either of the two hypotheses. However, the observed results of the FA of the wing traits, give a different insight. It is generally believed that the degree of DI in hybrids is related to the genetic distance between hybridizing populations, as a result of the balance between the stabilizing effect due to the increased heterozygosity and the disruption caused by the breakdown of the coadapted gene complexes (Markow and Ricker 1991). Investigations of the genetic basis of the DI, using several approaches, gave inconclusive results, for several reasons. Both genomic coadaptation theory and the heterozygosity theory lead to similar predictions of the DI decrease in hybrids with respect to their parental strains (Andersen et al. 2002). A problem associated with the hybrid approach is that parental strains are not completely homozygous, especially in the lines from the field. Furthermore, the negative correlation between heterozygosity and DI may only become apparent in certain ecological and population contexts, possibly because DI is related to various exogenic and endogenic factors (Pertoldi et al. 2006b). Also, to obtain the highest fitness level, sexual reproducing individuals have to aim for optimal outbreeding. Optimal outbreeding is where the crosses between two individuals can raise offspring with highest fitness (fitness optimum) without expression of any detrimental effects. The optimal outbreeding can be described as a continuous variable that is under influence by other variables like population size and is flanked by the two extremes of inbreeding and outbreeding (Sagvik et al. 2005). In the interpopulation hybrids we generally observed an increase of the FA in F1, which suggests that outbreeding depression occurred in the first generation after hybridization. Theoretically, outbreeding depression occurs as a consequence of disruption of the coadapted genome phenomenon which can occur after the second generation upon hybridization (Graham 1992). The Dobzhansky-Muller model (Orr and Turelli 2001) suggested that outbreeding depression is considered to be more likely expressed in the F2, because different populations can accumulate genetic incompatibilities more easily (i.e., alleles with negative epistatic interactions). It has become widely accepted that the evolution of epistatic incompatibility is explained by the observation that isolated populations gradually accumulate neutral or advantageous mutations over time. Furthermore, selection for positive epistasis may result in development of unique coadapted gene complexes within each isolated population (Whitlock et al. 1995; Fenster et al. 1997).
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The occurrence of significant outbreeding depression in F1 generation of populations which are not too distant from each other is more surprising. Outbreeding depression is usually considered an important issue when divergent populations, often recognized as distinct subspecies, are brought into contact, rather than two demes in a local metapopulation. But, there is some evidence that outbreeding depression may also occur in the F1, due to factors such as underdominance, epistatic interactions (heterozygote-heterozygote interactions or interaction involving sex chromosomes) or disruption of local adaptations (i.e., extrinsic isolation) (Edmands 2007; Escobar et al. 2008). Furthermore, if an individual have parents originating from different populations its fitness depends on the type of selection acting on the parental populations. For example, if recessive deleterious alleles are present in the parental populations in high frequency, hybridization will mask such recessive variation and heterosis is expected. In contrast, under some scenarios of overdominant selection, outbreeding depression might be expected, particularly if different pairs of alleles are most fit in different populations or if only one of the populations is subject to overdominant selection at the locus (Charlesworth and Hughes 1999; Garnier et al. 2006; Andersen et al. 2008). We generally observed that the FA value for wing length and width, of interpopulation hybrids is higher in F2 generation compared to the FA value in intrapopulation hybrids, for both sexes. This was expected if we take into account that the intrapopulation crosses, in fact represents the simulation of random mating in parental natural populations. In each parental population, new mutations may arise and in increase of FA but, if these mutations are either neutral or advantageous for the populations they are predicted to accumulate and become fixed in the genome. Here they may be incorporate in specific gene complexes on which selection for local adaptation may invoke. In the other words, they become coadapted and increase the fitness of populations which suggest that the specific association of coadaptive gene complexes are the most probable mechanism that maintains the developmental homeostasis in populations. In the case of hybridisation between two different populations (interpopulation crosses), coadaptive gene complexes were disrupted because the new recombination due to divergence in selection for local adaptation between the two populations So, the new gene complexes will be expected to decrease overall fitness and individuals and populations will experience outbreeding depression (Dobzhansky 1936; Orr 1995; Burke et al. 1998; Andersen et al. 2002, 2008; Edmands 2007). The impact of outbreeding depression on populations differs and depends on the amount of genes involved in the coadapted gene complexes. If only few genes are involved a coadapted gene complex, outbreeding depression may be temporary, because selection may efficiently re-establish fitness. But, if the most genes within genome are involved in coadaptation the chances of re-establish fitness are very low. We expected the breakdown of coadapted gene complexes to be equal for males and females in interpopulation crosses. However, our results show that effects of the breakdown differ between sexes which are in accordance to previous studies (see Andersen et al. 2002 and references therein). The results of the FA of wing length and width in interpopulation hybrids, suggest a different response in males and a general increase of FA in F1, followed by a reduction of FA in the F2 generation. We found a different response in females, which was a constant increase of FA through the generations. The results show a greater trend of FA change in males than in females both for wing length and width. The observed difference in FA between direct and reciprocal crosses suggests the presence of the maternal effect. A large part of FA variation is non-additive and is influenced by cytoplasimic maternal effect, reflecting unpredictable interactions between genetic backgrounds that have diverged to stochastic processes. Its importance is not
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questionable in literature, and there are experiments indicating that a maternal effect can account for the variation of the individual size in offspring, survival and behavioural differences and may have a huge impact on the fitness (Cheverud and Moore 1994; McAdam et al. 2002; Andersen et al. 2005). Maternal effects arise due to a lot of different causes, both intrinsic and extrinsic. There are investigations showing that extrinsic changes in parental environment, such as temperature (Gilchrist and Huey 2001), pollution, nutrition (Gliwich and Guisande 1992; Rossiter 1996) and oviposition site (Mousseau and Fox 1998) can influence the offspring. Similarly, various more direct intrinsic effects as maternal size (Emlet and Hoegh-Guldberg 1997; Hunt and Simmons 2000), parental care (Rauter and Moore 2002) and maternal age (Kern et al. 2001) influence offspring across one or more generations. There is also growing evidence that some maternal effects are not simply the accidental transmission of environmental information from one generation to the next. Rather, type and function of maternal effects appear often to have been shaped by natural selection (Hunter 2002). The results of our study suggest that D. subobscura populations are genetically diverse even at small geographic scale with frequently strong and unpredictable consequences of outbreeding. That pattern is likely to become increasingly pronounced as a result of ongoing habitat fragmentation and destruction by the increased anthropogenic activity. In such a situation, it seems that one preventive action to increase survival may be to increase the gene flow by translocation of individuals between populations, and in that way hinder inbreeding and loss of genetic variation. Our results indicate that care should be taken with such translocations because that kind of activity could have strong consequences on the population fitness, and could potentially increase the risk of outbreeding depression. Climate change is therefore also altering the immigration-emigration dynamics which in turn will have profound consequences for the population genetic structure as increased geneflow typically increase the genetic variability within of populations simultaneously reducing their local adaptation (Pertoldi and Topping 2004). In a population, the actual degree of adaptation is the residual effect of the dynamic interaction between the selective pressure and gene flow. Hence, the high levels of gene flow can reduce or impede the capacity of adaptation to a stressor (Roush and McKenzie 1987) or may introduce essential new genes for future adaptation or increase in tolerance (Slatkin 1995). The importance of gene flow as a force for the maintenance of genetic diversity and avoidance of inbreeding depression is therefore quite evident. However, as the results of this study are showing high levels of gene flow also have the potential to introduce poorly adapted genes (outbreeding depression) that can reduce viability of the population, even if the consequences of outbreeding have been shown to not being predictable. Furthermore, different outcomes could be expected depending on the origin of the populations, microhabitat adaptation and evolutionary history of each population. Clearly, we know too little about long-term consequences of outbreeding. Overall, among-population heterogeneity in genetic architecture makes it difficult to assess short/long time results of different population crossings. However, our results confirm that the studies dealing with DI seem to provide valuable insights into the importance of the genetic factors in the long term survival of isolated populations. Acknowledgments We are grateful to anonymous reviewers for helpful and valuable suggestions on the manuscript. This work was supported by the Ministry of Science and Technology of the Republic of Serbia, Grant No. 143014.
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