Blackwell Publishing LtdOxford, UKBIJBiological Journal of the Linnean Society0024-4066The Linnean Society of London, 2006? 2006 881 3343 Original Article DEVELOPMENTAL STABILITY IN MOUSE HYBRIDS E. GAZAVE ET AL .
Biological Journal of the Linnean Society, 2006, 88, 33–43. With 3 figures
Do chromosomal hybrids necessarily suffer from developmental instability? ELODIE GAZAVE1*, JOSETTE CATALAN1, MARIA DA GRACA RAMALHINHO2, MARIA DA LUZ MATHIAS3, ANA CLAUDIA NUNES3, JANICE BRITTON-DAVIDIAN1 and JEAN-CHRISTOPHE AUFFRAY1 1
Institut des Sciences de l’Evolution, UMR CNRS, CC 064, Université de Montpellier II, 34095 Montpellier, Cedex 5, France 2 Centro de Biologia Ambiental, Museu Nacional de Historia Natural, Rua de Escola Politecnica, 1268102 Lisboa, Portugal 3 Centro de Biologia Ambiental, Departamento de Zoologia e Antropologia da Faculdade de Ciências da Universidade de Lisboa, Edificio C2, 3° piso, Campo Grande, 1749-016, Lisboa, Portugal
Received 15 September 2004; accepted for publication 4 May 2005
The role of chromosomal rearrangements in disturbing reproduction in hybrids between karyotypically differentiated groups is fairly well documented. However, the effect of chromosomal changes at other phenotypic levels is rarely considered. In Tunisia, natural chromosomal hybrids of the house mouse exhibit developmental instability, suggesting that a high karyotypic heterozygosity might also affect developmental processes. If this is true, we predict that, in this species, developmental instability should arise in hybrids between any populations with a high chromosomal differentiation. To test this hypothesis, we compare the results obtained in Tunisian mice with those obtained in the present analysis on Madeiran mice. Both systems of races have similar levels of chromosomal differentiation (nine Robertsonian fusions). Unlike Tunisian mice, hybrids in Madeira display a similar level of developmental instability as parental groups. This indicates that structural heterozygosity per se does not necessarily impair developmental stability. It further suggests that chromosomal fusions are not all equivalent in their phenotypic effects, and that the identity of each fusion should be taken into account. © 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 88, 33–43.
ADDITIONAL KEYWORDS: chromosomal differentiation – developmental stability – house mouse – Robertsonian fusion – structural heterozygosity.
INTRODUCTION Chromosomal differentiation is thought to be a key process among those leading to speciation. In hybrid zones between different karyomorphs, theoretical models postulate that heterozygote disadvantage is the main mechanism leading to the emergence of an isolation between structurally homozygous groups (White, 1978). Hybrid dysgenesis in chromosomal heterozygotes has been documented in many species (for a review, see Searle, 1993). In most cases, it leads to a dysfunction of fertility components, such as abnormal
*Corresponding author. E-mail:
[email protected]
chromosome pairing at the pachytene stage of meiosis, leading to germ cell death, or to nondisjunction of homologous chromosomes at anaphase 1, leading to aneuploidy. In the house mouse, Mus musculus domesticus Rutty, it has been suggested that chromosomal heterozygotes suffer from perturbations not only at the meiotic level, but also at a premeiotic stage (Redi et al., 1985). This raises the question of the developmental level at which chromosomal differentiation can act. In particular, the role of chromosomal rearrangements in generating phenotypic changes, by affecting development, has rarely been addressed (Qumsiyeh, 1995; for a review, see Alibert & Auffray, 2003). The house mouse is a suitable model to explore this question. This species presents a standard karyotype
© 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 88, 33–43
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E. GAZAVE ET AL.
of 2n = 40 acrocentric chromosomes. In Western Europe, North Africa, and the Middle East, numerous populations have fixed Robertsonian (Rb) fusions, reducing the diploid number to 2 n = 22 when nine Rb fusions are fixed (Nachman & Searle, 1995; Gündüz, Coskun & Searle, 2000). In addition to variation in chromosomal number, these populations also differ by the chromosomes involved in the fusions. Their unique combinations of Rb fusions (in number and identity) define different Rb races. These races have kept the ability to hybridize with the standard one, when they come into contact. As in other cases of chromosomal hybridization, meiotic perturbations have been reported in the house mouse. A positive correlation between the number of heterozygous fusions and aneuploidy (Capanna et al., 1976; Rizzoni & Spirito, 1998; Castiglia & Capanna, 2000), germ cell death (Wallace, Searle & Everett, 1992), or litter size reduction (Viroux & Bauchau, 1992; Saïd et al., 1993; Castiglia & Capanna, 2000), is well documented. Interestingly, an effect of karyomorphic heterozygosity was also found on other components of fertility which are not directly related to the meiotic process, such as relative testis weight and a decrease of tubular diameter in the seminiferous epithelium (Saïd et al., 1993). These latter cases suggest that a premeiotic developmental process is also perturbed by structural heterozygosity. Recently, incompatibilities in chromosomal hybrids were detected at the whole developmental level. In Tunisia, chromosomal hybrids in wild mice exhibited higher levels of asymmetry than parental races (Chatti et al., 1999b). Fluctuating asymmetry is an indicator of developmental stability (DS), which is defined as a set of mechanisms selected to ensure phenotypic constancy of replicated parts of an organism (e.g. left and right sides of bilateral organisms), in spite of small and random developmental irregularities (Debat & David, 2001). The higher fluctuating asymmetry of chromosomal hybrids in Tunisia was interpreted as a consequence of developmental incompatibilities between the two races, 2n = 22 (RB22) and 2n = 40 (ST40) (Chatti et al., 1999b). It indicated that perturbations in these hybrids affected more than meiosis and reproduction. However, Chatti et al. (1999b) stressed that, in Tunisia at least, the developmental incompatibilities could not be attributed with certainty to chromosomal differentiation alone because ST40 and RB22 populations presented a slight genic differentiation that may explain the hybrid dysgenesis observed for development. Allozyme data showed that the genetic distance between the Tunisian races (RB22 and ST40) was greater than that between neighbouring RB22 and ST40 chromosomal races in Europe (Saïd et al., 1999). Mitochondrial DNA (mtDNA) confirmed this observation, with haplotype frequencies being different between the two
Tunisian races (Saïd et al., 1999). Hence, two mechanisms were proposed to account for these incompatibilities. The first was related to genic divergence between parental races. The second hypothesis considered that structurally differentiated chromosomes are located in different functional domains in the nucleus, impairing the expression of genes involved in fertility components (Capanna & Redi, 1994), but also DS (Saïd et al., 1999). In the Madeira archipelago, a new chromosomal hybrid zone was discovered. On the main island, Madeira, several Rb races are present in allopatry (Britton-Davidian et al., 2000). The most eastern race (2n = 22) has nine fixed Rb fusions. This race hybridizes with individuals from the standard race (2 n = 40) in the city of Funchal, which is the main harbour of the island. Hence, Madeira provides a situation very similar to that of Tunisia concerning the chromosomal divergence. The present study aimed to test the role of chromosomal differentiation in generating developmental incompatibilities in chromosomal hybrids, using two approaches. First, we performed the clines of frequency in chromosomal number in both hybrid zones. The difference in slopes of the clines was used as an estimate of the difference in selection against hybrids in the two geographical systems. Second, we compared the asymmetry levels between Madeiran and Tunisian mice. If developmental incompatibilities are due to chromosomal differentiation per se, a similar level of asymmetry is expected in the hybrids between RB22 and ST40, independently of their geographical origin. Conversely, if the level of DS in hybrids, relatively to that of parental groups, differs in the two systems (Madeira and Tunisia), it would suggest that another mechanism is involved in the emergence of the incompatibilities present in Tunisia. The levels of DS in the Madeiran chromosomal groups were appraised using the fluctuating asymmetry of tooth measurements. The traits measured were the same as in the Tunisia study and the sample sizes were similar, allowing us to compare the two chromosomal systems.
MATERIAL AND METHODS SAMPLES Wild mice were trapped in the Madeira archipelago during several field trips (1998–99). Individuals belonging to the Rb race (2 n = 22) and hybrids originated from 30 localities on the eastern part of the main island (Madeira). For the asymmetry study, due to the scarcity of individuals belonging to the standard race (2n = 40) on Madeira (two individuals), sampling was extended to mice originating from the island of Porto Santo, which is 50 km distant from Madeira. According to molecular data (Gündüz et al., 2001),
© 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 88, 33–43
DEVELOPMENTAL STABILITY IN MOUSE HYBRIDS there is no evidence that animals from Porto Santo are those which hybridize with the mice of the Funchal Rb race. However, all haplotypes from both these islands fell within the same monophyletic clade (Gündüz et al., 2001). Hence, mice from Porto Santo island were used as a reference for the nearest standard mouse population. A total of 145 individuals was included in the analysis; 24 individuals belonged to the standard race (2n = 40), 48 to the Rb one (2n = 22), and 73 to the hybrids. In further analyses, these samples are encoded ST40, RB22, and HYB, respectively. For the clinal analysis, data of diploid numbers and distances were those already used by Gazave et al. (2003) for Madeira and by Chatti et al. (1999a) for Tunisia.
KARYOTYPES Chromosomes were prepared from yeast-stimulated bone marrow cells, using the air-drying technique (Lee & Elder, 1980). For each individual, the identification of chromosomes according to the nomenclature of Cowell (1984) was performed using the G-banding method (Seabright, 1971). Metaphase chromosomes were analysed using a Zeiss Axiophot microscope and karyotyped with the Genevision software (Applied Imaging).
CLINE
OF FREQUENCY
Clinal variation in diploid number was investigated following the procedure commonly used in this species (Chatti et al., 1999a; Gündüz et al., 2001; Gazave et al., 2003). We assumed no difference in the migration rate in the two hybrid zones. Hence, the width of the clines, defined as the inverse of their maximum slope, can be compared and used as an estimate of the difference in selection against hybrids in both geographical systems. The analysis was computed using C-fit software (devised by T. Lenormand), which estimates the maximum likelihood fit for each hybrid zone by a Metropolis algorithm, adapted from N. H. Barton (Szymura & Barton, 1986). A likelihood ratio test was performed to determine the significance of the differences in cline width and slope.
TOOTH
CHARACTERS
Mandibles were manually cleaned. For each individual, the maximum length and width of the three lower molars were measured, which are classically used in rodent studies on fluctuating asymmetry. These six traits, labelled LM1, LM2, LM3, WM1, WM2, and WM3, were measured on each side (left and right mandible), respectively, labelled by the indices ‘L’ and ‘R’. Each tooth was measured twice independently, by the same operator (E. G.).
PRELIMINARY
35
ANALYSES
Due to the handling procedure, one tooth was missing in some individuals, leading to an unequal sample size among characters in a given group. Measurement error and directional asymmetry(DA) estimation A two-way mixed model analysis of variance (ANOVA) was performed to estimate measurement error and DA, for each character in each chromosomal group, according to the procedure proposed by Palmer (1994). This analysis involved the individual and side (left or right) effects and their interaction as independent variables, explaining the raw left and right values of the two sessions of measurement. Due to the low measurement error value (see below), the mean for each individual of the values of each session of measurement was used in all the following analyses. Distribution normality Normality (Shapiro–Wilks’ test), skewness and kurtosis of the (R–L) distributions were appraised for the 18 distributions corresponding to the six measured characters in the three groups considered. These preliminary analyses were computed with STATISTICA software (release 4.3). In order to limit type I errors, the Bonferroni technique (Rice, 1989) was used considering sets of k = 6 trait-related tests.
FLUCTUATING
ASYMMETRY ESTIMATES
The analysis performed here combined the approaches recommended by Graham et al. (1998) and Leung, Forbes & Houle (2000), as applied in Nunes et al. (2001). The variable ‘chromosomal group’, which involved three modalities (ST40, RB22, and HYB) was encoded CG. Six regressions of R = ƒ(L + CG + L*CG) were performed, in order to appraise the uniformity of DA across the three groups (R and L indicating the right and left of a given trait). Although DA was present in a few cases, it was shown not to differ among groups for a given trait (see Results section); thus, the data of the three groups were pooled for each of the six traits. Principal Component Analyses (PCA) were performed on the L and R dataset for each trait. The scores obtained on the first and second principal component (PC1 and PC2) represent, respectively, the size and the asymmetry of each individual. By summing the absolute values of PC2 scores across the six traits, we obtained a composite index of fluctuating asymmetry (FA) analogous to CFA2 proposed by Leung et al. (2000). Summing raw values of PC1 provided a composite indicator of size, encoded SIZE in the following treatments. Because the six characters were pooled for each individual, one missing datum for one character
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resulted in the removal of the whole individual from the analysis. Thus, the PCA was performed on 125 individuals only. All these analyses were computed with R software, version 1.3.0 (Gentleman & Ihaka, 1996).
RELATIONSHIP BETWEEN CFA2 AND OTHER VARIABLES For the whole sample To verify that SIZE had no influence on CFA2, four regressions of SIZE on CFA2 were performed, one per chromosomal group, and one on the overall sample (Chatti et al., 1999b). We tested the effect of altitude (ALT), site (ST), and sex (SX), which were the main identifiable sources of variation among individuals. ST was tested alone, because individuals per site were too few to allow testing a model with interactions. The effects of ALT and SX were explored using a two-way mixed model ANOVA. We tested the difference in FA level among chromosomal groups (RB22, HYB, ST40) with a one-way ANOVA model. A trend in the increase of the level of fluctuating asymmetry with increasing values of chromosomal heterozygosity was reported in previous studies (Chatti et al., 1999b). Hence, the explanative variable HTZ (number of heterozygous fusions) was considered as a quantitative variable, and tested in a linear regression model. Within the hybrid group We explored the effect of HTZ with a linear regression, for the reason given immediately above. The effect of each fusion was also tested performing one-way ANOVAs.
RELATIONSHIP BETWEEN SIZE AND OTHER VARIABLES
ference in slopes is highly significant (slope = 1.912 in Tunisia, slope = 1.334 in Madeira; χ2 = 28.3, d.f. = 1, P < 10−5), suggesting a higher selection on hybrids in Tunisia (Fig. 1). The width in Tunisia estimated in the current analysis (0.5233 km) is slightly higher that reported by Chatti et al. (1999a) (0.4200 km). This is due to differences in the method of calculation between the two studies. The estimate in our analysis is conservative compared to that of Chatti et al. (1999a). All the (R–L) variables were normally distributed, except for LM1 in the RB22 group and LM2 in the HYB group (Table 1). These two distributions were both skewed and leptokurtic, precluding the occurrence of antisymmetry. Leptokurtic distributions may reflect a mixture of several heterogeneous FA distributions. Because departures from normality did not concern one trait across groups, nor all traits for one group in particular, the entire sample was used in the FA analysis. The significance of the ‘side’ effect in the two-way mixed ANOVA revealed the presence of DA for WM1 in the three groups, as well as for WM2 and WM3 in ST40 (Table 2). Because unequal DA among groups could lead to invalid FA differences (Graham et al., 1998), the uniformity of the DA in the three groups was tested for each character. No difference in regression coefficients and intercepts was found among the three groups (Table 3). Hence, for each trait, all the individuals were pooled and PCAs were performed on these datasets, in order to obtain the CFA2 described in Leung et al. (2000). No relationship was found between size (SIZE) and asymmetry (CFA2), either in the overall sample (F1,123 = 0.33, P = 0.57) or within each group (HYB: F1,65 = 0.97, P = 0.33; ST40: F1,15 = 0.48, P = 0.50;
The effects of ALT and SX were tested using a two-way mixed model ANOVA. To investigate a possible effect of Bergmann’s rule, a regression of SIZE on ALT was performed across groups (i.e. on the overall sample). Our second aim was to examine the effect of CG on size. The factor CG was found strongly correlated with ALT. An analysis of covariance involving group effect and altitude as covariants was not performed because of serious discrepancies among the slopes (size vs. altitude) among the three chromosomal groups. Therefore, an ANOVA on the residuals of the regression of SIZE on ALT across groups was undertaken to evaluate the effect of size among groups once the effect of altitude was removed. A Tukey HSD test was performed to detect differences among the three groups.
RESULTS The clines of frequency of metacentric chromosomes between Madeira and Tunisia were compared. The dif-
Figure 1. Changes in frequency of Rb fusions as a function of distance (km). The dashed line and solid line are, respectively, the fitted function for Tunisia and Madeira. 0 km represents Funchal harbour (Madeira) and the periphery of the city Monastir (Tunisia).
© 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 88, 33–43
DEVELOPMENTAL STABILITY IN MOUSE HYBRIDS
37
Table 1. Preliminary analyses: skewness, kurtosis and distribution normality (R–L) distributions Group
N
Trait
Mean (× 1000)
Skewness ± SE†
Kurtosis ± SE†
Normality‡ (Shapiro-Wilks’ W)
23 23 17 24 23 17
LM1 LM2 LM3 WM1 WM2 WM3
−1.02 3.78 4.56 −14.04 −3.30 3.94
0.47 ± 0.48 NS −0.68 ± 0.48 NS −0.32 ± 0.55 NS 0.19 ± 0.47 NS 0.46 ± 0.48 NS −0.34 ± 0.55 NS
1.56 ± 0.93 NS 0.64 ± 0.93 NS −1.24 ± 1.06 NS 0.46 ± 0.92 NS 0.41 ± 0.93 NS 0.88 ± 1.06 NS
0.950 NS 0.967 NS 0.935 NS 0.955 NS 0.963 NS 0.963 NS
72 72 68 72 72 69
LM1 LM2 LM3 WM1 WM2 WM3
−0.36 −0.17 2.48 −5.92 −1.81 −1.51
−0.31 ± 0.28 NS −1.32 ± 0.28*** −0.41 ± 0.29 NS −0.65 ± 0.28* 0.12 ± 0.28 NS −0.90 ± 0.29***
0.74 ± 0.56 NS 4.23 ± 0.56*** 0.92 ± 0.57 NS 1.89 ± 0.56** 0.14 ± 0.56 NS 2.60 ± 0.57***
0.988 NS 0.925 ** 0.979 NS 0.975 NS 0.988 NS 0.962 NS
47 46 44 46 47 42
LM1 LM2 LM3 WM1 WM2 WM3
−1.44 5.88 6.16 −6.45 −4.53 6.11
2.54 ± 0.35*** −0.21 ± 0.35 NS 0.22 ± 0.36 NS 0.46 ± 0.35 NS 0.39 ± 0.35 NS −0.26 ± 0.37 NS
10.80 ± 0.68*** 0.06 ± 0.69 NS −0.15 ± 0.70 NS −0.60 ± 0.69 NS 0.05 ± 0.68 NS −0.38 ± 0.72 NS
0.758*** 0.983 NS 0.980 NS 0.951 NS 0.976 NS 0.979 NS
ST40
HYB
RB22
†Mean trait and SE. ‡NS, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.00 All P-values are adjusted using the sequential Bonferroni procedure.
RB22: F1,39 = 1.35, P = 0.25). The factors ALT and ST had no effect on CFA2, nor did the chromosomal group (CG) affect CFA2 (Table 4). The factor sex (SX) is close to significance. Absence of a sex effect on FA is generally the rule in mice (J.-C. Auffray, pers. comm.). The number of heterozygous fusions (HTZ) had no effect on the global asymmetry indices CFA2 (Table 5), either when the three groups were pooled, or within the hybrid group only (Fig. 2). Furthermore, none of the nine fusions (individually tested) had an effect on CFA2 (all 0.0596 < F1,65 < 3.88, 0.808 > P > 0.053). Hence, among the factors tested that potentially may influence asymmetry of the individuals, none was shown to have an effect on the composite indices of asymmetry. By contrast to asymmetry, size appeared to be dependent on both altitude and chromosomal groups. The positive relationship between SIZE and ALT was highly significant in the pooled sample (Table 6). The biogeographical distribution of chromosomal number is not independent of altitude, with RB22 being found at higher altitudes than ST40, and HYB being located in between (Fig. 3). To appraise the effect of CG on SIZE, it was necessary to take into account the strong correlation between CG and ALT. The ANOVA performed on the residuals of the linear regression of
SIZE on ALT was also highly significant, showing that size differed more among groups than expected from an altitude effect alone (Table 4). The Tukey HSD test showed that the three groups were different (Table 6), with RB22 being larger than ST40 whereas the hybrids were intermediate.
DISCUSSION CLINES
OF FREQUENCY
The clines of frequency of the Rb fusions suggested a stronger selection against hybrids in Tunisia than in Madeira. This is concordant with the results obtained in the asymmetry study. Both approaches suggest that the hybrids from Tunisia are more affected than those from Madeira by chromosomal differentiation whereas, in both cases, the karyotypic divergence between parental races consists of the same number of fusions (nine).
EFFECT
OF ALTITUDE
The island of Madeira is extremely mountainous, and sampling sites were distributed over a range of more than 800 m in altitude. Chromosomal number varied with altitude, with RB22 being captured at higher
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E. GAZAVE ET AL.
Table 2. Preliminary analyses: measurement error and directional asymmetry estimates Side (d.f. = 1)
Individual Trait RB22 LM1 LM2 LM3 WM1 WM2 WM3 HYB LM1 LM2 LM3 WM1 WM2 WM3 ST40 LM1 LM2 LM3 WM1 WM2 WM3
d.f. MS ( × 1000) F †
Interaction
MS ( × 10 000) F †
46 45 43 45 46 41
12.96 5.07 7.78 7.35 7.04 4.36
114.88*** 44.00*** 20.29*** 49.02*** 60.27*** 49.20***
71 71 67 71 71 68
8.63 20.07 7.90 6.08 5.30 4.72
22 22 16 23 22 16
10.37 5.42 6.78 1.81 1.59 1.56
0.97 15.91 16.69 19.11 9.65 15.66
Error
d.f. MS ( × 10 000) F †
d.f. MS ( × 10 000)
0.10 NS 3.419 NS 1.22 NS 6.86* 5.24* 5.91*
46 45 43 45 46 41
9.89 4.65 13.72 2.79 1.84 2.65
8.76*** 4.04*** 3.58*** 1.86*** 1.58*** 2.99***
94 92 88 92 94 84
1.13 1.18 3.83 1.50 1.17 0.89
46.12*** 0.02 240.52*** 0.09 18.62*** 4.18 68.98*** 25.26 65.43*** 2.35 25.45*** 1.57
0.00 NS 0.02 NS 0.46 NS 9.93** 1.02 NS 0.30 NS
71 71 67 71 71 68
6.31 4.11 9.06 2.54 2.30 5.25
3.37*** 4.92*** 2.14*** 2.89*** 2.84*** 2.83***
144 144 136 144 144 138
1.87 0.83 4.24 0.88 0.81 1.85
168.64*** 0.24 35.18*** 3.29 23.40*** 3.53 15.40*** 47.32 31.50*** 2.51 7.41*** 2.64
0.09 NS 0.86 NS 0.89 NS 15.24*** 0.80 NS 0.66 NS
22 22 16 23 22 16
2.79 3.83 3.98 3.11 3.13 4.03
4.53*** 2.48*** 1.37 NS 2.64*** 6.21*** 1.92 NS
46 46 34 48 46 34
0.62 1.54 2.90 1.18 0.50 2.10
†NS, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001 All P-values are adjusted using the sequential Bonferroni procedure.
Table 3. Uniformity of directional asymmetry testing
Intercept ST40 Intercept HYB Intercept RB22 Slope ST40 Slope HYB Slope RB22
Estimate ± SE LM1
Estimate ± SE LM2
Estimate ± SE LM3
Estimate ± SE WM1
Estimate ± SE WM2
Estimate ± SE WM3
0.28 ± 0.13 0.08 ± 0.14 NS 0.36 ± 0.15 NS 0.81 ± 0.09 0.95 ± 0.09 NS 0.77 ± 0.10 NS
−0.01 ± 0.13 0.12 ± 0.14 NS 0.19 ± 0.15 NS 1.01 ± 0.14 0.87 ± 0.15 NS 0.83 ± 0.16 NS
−0.07 ± 0.13 0.16 ± 0.14 NS 0.13 ± 0.15 NS 1.12 ± 0.20 0.76 ± 0.22 NS 0.82 ± 0.23 NS
0.33 ± 0.12 0.10 ± 0.12 NS 0.18 ± 0.12 NS 0.62 ± 0.13 0.89 ± 0.14 NS 0.81 ± 0.14 NS
0.34 ± 0.12 0.09 ± 0.13 NS 0.10 ± 0.13 NS 0.62 ± 0.14 0.90 ± 0.14 NS 0.90 ± 0.14 NS
0.26 ± 0.14 −0.02 ± 0.15 NS 0.04 ± 0.16 NS 0.60 ± 0.23 1.02 ± 0.24 NS 0.95 ± 0.25 NS
The table shows the estimates, standard error and significance of the difference in slope and intercept between HYB and RB22 vs. that of ST40 (NS: P > 0.05).
altitudes than ST40 whereas hybrids were located at intermediate levels. As predicted by Bergmann’s rule, an increase in size with altitude was observed. Consequently, size was also related to the chromosomal group with respect to altitude (on average, RB22 are the largest, ST40 the smallest, and hybrids intermediate). Interestingly, once the global effect of altitude was removed, the CG factor remained significant. This indicates that RB22 were larger and ST40 smaller than expected by an altitude effect alone. This is
mainly due to RB22 individuals that were larger even at low and intermediate altitudes. Our analysis shows no influence of ALT on CFA2, the composite indices of asymmetry, suggesting that altitude was not a source of DS impairment. Thus, in Madeiran mice, altitude does not appear to be particularly stressful. Bergmann’s rule, which is interpreted as a physiological adaptation to altitude (Hoffman & Parsons, 1991), would thus explain why the animals found at higher altitudes did not exhibit an increased
© 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 88, 33–43
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DEVELOPMENTAL STABILITY IN MOUSE HYBRIDS Table 4. Results of the fluctuating asymmetry analysis: analysis of variance models
Dependent variable: CFA2 Effects and interactions Site Error Sex Altitude Sex × Altitude Error CG Error Residual SIZE Error Dependent variable: SIZE Sex Altitude Sex × Altitude Error
d.f.
MS
F
29 95 1 1 1 121 2 122 2 122
2.91 2.43 9.07 0.96 0.72 2.52 0.80 2.58 263.41 17.33
1 1 1 121
148.26 500.90 19.22 21.23
P
1.20
0.25
3.60 0.38 0.28
0.06 0.54 0.59
0.31
0.73
15.20
< 10−3
6.98 23.59 0.91
0.009 < 10−3 0.34
Table 5. Results of the fluctuating asymmetry analysis: linear regression models Estimates ± SE
Independent variable Dependent variable: CFA2 HTZ (hybrids only) HTZ (all individuals) Dependent variable: SIZE ALT (all individuals)
Intercept ± SE Slope ± SE Intercept ± SE Slope ± SE
5.06 ± 0.36 −0.12 ± 0.11 4.78 ± 0.18 −0.04 ± 0.07
Intercept ± SE Slope ± SE
FA. Although expected, an unvarying FA despite a size increase with altitude has not been systematically observed in rodents. In the mole-rat, Spalax erhenbergi, FA increased with altitude despite Bergmann’s rule (Auffray et al., 1999). These authors suggested that genetic or environmental stresses covarying with altitude were present and that the populations were not well adapted. Such stresses are probably not present in Madeira.
−2.72 ± −4.11 0.0098 ± 0.0019
P
< 10−3 0.30 < 10−3 0.57 < 10−3 < 10−3
mice, hybrids between RB22 and ST40 presented a significantly higher level of developmental instability than parental groups. For a similar chromosomal differentiation between parental groups, different levels of asymmetry in Tunisian and Madeiran hybrids were observed, showing that the existence of developmental perturbations is not the rule in such types of hybrids.
THE
ROLE OF CHROMOSOMAL HETEROZYGOSITY IN DEVELOPMENTAL INCOMPATIBILITIES
EFFECT
OF CHROMOSOMAL DIFFERENTIATION
Chromosomal differentiation (CG factor) had no effect on the level of asymmetry between ST40 and RB22 in Madeira. Furthermore, the hybrids displayed a level of FA identical to those of the parental groups. This is interpreted as an absence of genomic incompatibilities between parental groups in Madeira. Conversely, Chatti et al. (1999b) demonstrated that, in Tunisian
In testing the effect of the number of heterozygous fusions on FA, we investigated whether the accumulation of chromosomal heterozygosity might affect DS. This is because structural heterozygosity is considered to be responsible for perturbations observed in hybrids between chromosomally differentiated groups. Chatti et al. (1999b) reported a significant positive relationship between asymmetry and the number of heterozy-
© 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 88, 33–43
E. GAZAVE ET AL.
0 5
SIZE
5
10
40
ST40 HYB
10
RB22
0
200
400
600
800
ALT
Figure 2. Effect of the number of heterozygous (HTZ) fusions on the FA indices (CFA2), on the hybrid sample.
Table 6. Results of the fluctuating asymmetry analysis: Tukey HSD test Groups
Means
Comparisons
P
ST40 HYB RB22
−3.74 −0.61 2.54
ST40 − HYB HYB − RB22 RB22 − ST40
0.016 < 10−3 < 10−3
gous fusions for some traits, but not for others. When the P-values provided by Chatti et al. (1999b) are combined over all traits following the methods described by Manly (1985), this relationship becomes highly significant (χ210 = 40.81, P < 0.0001). This result was consistent with observations concerning fertility components. However, in Madeiran hybrids, a clear absence of correlation between FA and HTZ was observed. This result is interesting for two reasons. First, although many independent observations on the effects of structural heterozygosity on fertility exist, little is known about its effects on development and nonreproductive functions. Second, for the same level of structural heterozygosity, we obtain different responses in terms of DS, depending on the geographical system studied.
EFFECT
OF GENIC DIVERGENCE IN
TUNISIA
The absence of developmental instability in the Madeiran mice suggests that the incompatibilities observed in the Tunisian hybrids have a genic rather
Figure 3. Effect of altitude on the composite estimate of size.
than a chromosomal origin. However, two points do not support this conclusion. First, hybrids between Mus musculus musculus and M. m. domesticus display heterosis both in size and DS (Alibert et al., 1994, 1997). Because the genetic differentiation between these two subspecies of the house mouse (Dnei = 0.34, Bonhomme et al., 1984) is much greater than that between the RB22 and ST40 races in Tunisia (D = 0.07, Saïd et al., 1999), it is difficult to explain the incompatibilities in the Tunisian mice by genetic divergence alone. Second, contrary to the other locations investigated in Tunisia, recent allozyme data on the mice from Kairouan (Tunisia) have shown that, in this city, the two parental karyomorphs are not genetically differentiated in this city (Ould Brahim et al., 2005). However, the width of the hybrid zone, used as an indicator of incompatibility between RB22 and ST40, is as narrow in Kairouan as in Monastir (Chatti et al., 1999a). This strongly suggests that the incompatibilities in the Tunisian hybrids are not explained by the genetic divergence that was accumulated between parental populations. The possibility remains that a small number of genes affecting development has evolved in antagonistic directions in RB22 and ST40 in Tunisia. If these genes are restricted to a small portion of the genome, their divergence may not be revealed by the allozyme study. This shows that overall estimates of genetic divergence, as measured by allozymes or molecular data, do not constitute an accurate predictor of the level of incompatibilities affecting morphological development in hybrid mice.
© 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 88, 33–43
DEVELOPMENTAL STABILITY IN MOUSE HYBRIDS
THE
ROLE OF FUSION EFFECT
The contrasting results between Tunisia and Madeira may result from the two systems compared in the present study being equivalent in their karyomorphic divergence but not identical, because eight of the nine Rb fusions are not common to the two races. Strong variation in aneuploidy rates were reported (Gropp & Winking, 1981; Redi & Capanna, 1988) for the same number of heterozygous fusions, depending on the genetic background, age, or sex of the individual carrying the fusions, or even the nature of the fusion. Thus, Capanna & Redi (1994) proposed a nonmeiotic process for the disturbances in Robertsonian heterozygotes. These authors suggested that a fusion could induce a repositioning of chromosomes within the nucleus. This could result in altered gene expression in premeiotic cells, and thus could have different effects on reproductive functions and fertility, depending on the acrocentrics involved in the fusion. This may explain part of the variation recorded in aneuploidy rates for the same number of heterozygous fusions. Although never demonstrated, the fusion effect proposed by Capanna & Redi (1994) is consistent with recent cytogenetic studies. The organization of chromosomes in the nucleus is nonrandom (Nagele et al., 1995; Bridger & Bickmore, 1998; Parada & Misteli, 2002), and the nuclear position of a gene can influence its activity (Brown et al., 1997; Andrulis et al., 1998). Thus, it could be postulated that the fusion of two acrocentric chromosomes may affect the expression of the genes located on these chromosomes due to the shift in chromosome position. In this case, changes would become effective at the first stages of embryogenesis, and it would be expected that chromosomal differentiation not only affects reproductive-related functions in hybrids, but also the whole development. This may account for the pattern observed in Tunisia. When Chatti et al. (1999b) tested the effect of each fusion on asymmetry in Tunisia, they did not find an effect for any single one. Similar tests performed on Madeiran mice showed no effect of a particular fusion on asymmetry, although significant effects, if present, are unlikely to be detected given the small sample size per fusion. However, the effect of one particular fusion is sometimes observed. For example, Chatti et al. (1999a) noticed that reduction in testis weight in Tunisian hybrids was significantly associated with the presence of heterozygous Rb(4.6). Recently, Le Roy et al. (2001) mapped a QTL on chromosome 4, contributing to 17.5% of the total variance of testicular weight in the mouse. The expression of this locus on chromosome 4 may be altered when it is associated with chromosome 6, such as in Rb(4.6) in Tunisia. The effect of individual Rb fusions on DS is poorly documented. The spontaneous emergence of one
41
Rb(4.19) in laboratory-bred progeny of wild mice allowed Auffray et al. (2001) to compare asymmetry levels of Rb(4.19) carriers to that of Rb(4.12) carriers, and of these two groups with all-acrocentric mice. The authors found no influence of these fusions on DS, whatever their state (homozygous or heterozygous). However, the presence of a single heterozygous Rb has also been shown to have little or no effects on aneuploidy and fertility (Wallace et al., 1992), suggesting that its effect on FA may similarly be small and undetectable. Furthermore, because the fusions focused on by Auffray et al. (2001) are not present in Tunisia or Madeira, it may be assumed that only some acrocentric combinations lead to morphological incompatibilities, among which neither the Rb(4.19), nor the Rb(4.12) are included. In conclusion, the presennt study shows that structural heterozygosity per se does not necessarily impair DS. Besides the divergence of a specific gene or gene system affecting development in chromosomal hybrids, the data are also compatible with newly emerging explanations proposing that incompatibilities may originate from particular chromosomes, the fusion of which has altered the expression of some genes. Special care must be taken in future studies to appraise the effects of individual fusions and the compartments of the organism that are or not affected by chromosomal hybridization.
ACKNOWLEDGEMENTS We are grateful to R. Capela, M. Biscoito, A. I. Galvão, G. Ganem, C. Marques, and J. B. Searle for field assistance and karyotyping; to J. Prudêncio for mandibles preparation; and to Wendy Bickmore for reading the manuscript. The present report comprises publication ISEM 2005-019.
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