Morphological Integration of the Mandible in Yellow-Necked Field Mice: the Effects of B Chromosomes

By Jojic, Vida Blagojevic, Jelena; Ivanovic, Ana; Bugarski-Stanojevic, Vanja; Vujosevic, Mladen

As a complex skeletal organ consisting of 2 functional and developmental units (ascending ramus and alveolar region), the mandible represents a well-established model in morphological integration studies. The concept of morphological integration assumes that developmentally or functionally related traits are more correlated than others and hence evolve together. We compared the level and pattern of mandibular morphological integration between groups of adult yellow-necked field mice (Apodemus flavicollis), with and without B chromosomes (Bs) in a population from Mt. Avala, Serbia. Bs are dispensable supernumerary chromosomes characterized by irregular and non-Mendelian modes of inheritance. The level of morphological integration was higher in animals with Bs. One of the 2 regions of the mandible tested (alveolar region) was significantly more affected by the presence of Bs than the other, with an increase in intensity of integration of 41.61% versus 15.86%. The hypothesis of morphological integration, which postulates disunion of the mandible into 2 distinct functional and developmental modules, was confirmed in animals with Bs. Bs probably have a function because they affect mandible phenotype (although the mechanism is unknown), increase variability within populations, and could lead to selective advantage. Key words: Apodemus flavicollis, B chromosomes, mandible, morphological integration

Notable numbers of plant, animal, and fungi species are distinguished by the presence of supernumerary chromosomes, known as B chromosomes (Bs-Jones 1995; Jones and Rees 1982). Widespread occurrence of Bs is accompanied by a variety of characteristics that are never shared among all Bs. Dispensability is accepted as the only defining feature of all Bs, a characteristic that allows them to evolve more or less independently from the rest of the genome (Camacho et al. 2000). Other important features are non-Mendelian inheritance, absence of pairing or recombination with A chromosomes, heterochromatic nature, and genetic silence. However, the amount of data that question the last 2 attributes is increasing (Graphodatsky et al. 2005; Green 1988; Miao et al. 1991a, 1991b; Plowman and Bougourd 1994; Ruiz-Rejon et al. 1980; Tanic et al. 2005).

Beukeboom (1994) estimated that Bs are present in 15% of living species. However, Bs have been scored in only 1.2% (55 species) of mammals, most of which are rodents (Vujosevic and Blagojevic 2004). Within the genus Apodemus, Bs are found in 6 of 21 known species. Bs have been reported in almost all populations of yellow-necked field mice, Apodemus flavicollis (Melchior, 1834), studied in former Yugoslavia (Vujosevic et al. 1991; Vujosevic and Zivkovic 1987) and also over a wide area throughout the range of this species (Kartavtseva 2002; Wojcik et al. 2004). The standard karyotype of A. flavicollis is composed of 48 acrocentric chromosomes; its Bs also are acrocentric, euchromatic, and of the same size as the 5 smallest autosomes. Based on homology in the distribution of G and C bands between B and A chromosomes, it has been hypothesized that Bs originated from the standard chromosome set (Vujosevic and Zivkovic 1987). However, Bs fail to pair with A chromosomes and during meiosis they appear as univalents or bivalents (Vujosevic et al. 1989).

The effects of Bs are rarely manifested in the phenotype. In the plant Haplopappus gracilis (Jackson and Newmark 1960), Bs change the color of the achenes, whereas maize plants with Bs develop striped leaves (Staub 1987). Data on the effects of mammalian Bs are particularly scanty (Volobujev 1980; Vujosevic and Blagojevic 2004). In fact, there is no simple method for their detection. Possible effects of Bs could be important at the population level, and would have to be scored quantitatively. Shellhammer (1969) and Suva and Yonenaga-Yassuda (2004) failed to establish a connection between the presence of Bs and morphological traits. However, Shellhammer (1969) suggested that Bs produce physiological or behavioral effects, which was confirmed in silver foxes (Vulpes vulpes-Belayev et al. 1974a, 1974b; Volobujev and Radjabli 1974; Volobujev et al. 1976). Moreover, investigations of populations of yellow-necked field mice carried out in the last 10 years do not support the idea of genetic inertness of Bs. Rather, it has been shown that Bs have a role in regulating population dynamics in conditions of stress produced by overcrowding (Blagojevic and Vujosevic 1995). Furthermore, it was found that the presence of Bs affects different phenotypic characters (Blagojevic and Vujosevic 2000, 2004; Blagojevic et al. 2005).

In our study, we investigated phenotypic correlations among quantitative traits. When quantitative genetic data are not easily obtainable, which is the case in studies of natural populations, phenotypic variation and covariation are often used as an approximation of quantitative genetic variation and covariation (Ackermann and Cheverud 2000). Cheverud (1988, 1995, 1996a) found that phenotypic and genetic correlation matrices are very similar. Under the assumption that Bs, as additional genetic material, may act upon genetic and phenotypic correlations, we used morphological integration of the mandible to study their potential phenotypic effects.

Morphological integration refers to the relationships and connections among morphological traits within any complex morphological structure (Chernoff and Magwene 1999; Marroig and Cheverud 2001; Olson and Miller 1958). The rodent mandible, as a complex skeletal organ, represents a particularly advantageous system for studying morphological integration (Atchley and Hall 1991; Ehrich et al. 2003; Klingenberg et al. 2001, 2003, 2004). It is composed of multiple parts, with different embryonic origins and timing of differentiation, assembled into 2 functional modules: the alveolar region, which supports teeth roots, and the ascending ramus, which articulates with the rest of the skull and participates in the attachments of muscles (Atchley 1993; Atchley and Hall 1991). However, some findings supported the hypothesis that 2 separate modules can be recognized within the mandible (Atchley et al. 1985; Cheverud et al. 1997; Leamy 1993; Mezey et al. 2000), whereas others did not (Klingenberg and Leamy 2001; Klingenberg et al. 2001).

The purpose of our study was to identify the potential effects of Bs upon morphological integration of the mandible within a population of A. flavicollis. Three questions were addressed: Is there any difference in the level of morphological integration between animals with and without Bs? How well do groups of animals with and without Bs fit the model of a partitioned mandible? Does the pattern of morphological integration differ between animals with and without Bs?

MATERIALS AND METHODS

Specimens and measurements.-Yellow-necked field mice (A. flavicollis) were collected on Mount Avala (44[degrees]41′N, 20[degrees]31′E), Serbia, during 1998, 2000, and 2002. Animals were captured and treated following procedures approved by the American Society of Mammalogists (Animal Care and Use Committee 1998). Mice were trapped in custom-made Long-worth-like traps. Traps baited with sardines and wheat were set before dusk and checked early the next morning. All specimens were euthanized with ether. Chromosomes were prepared directly from bone marrow using the standard procedure (Hsu and Patton 1969). The presence of Bs was determined by scoring 30 metaphase figures. Animals with standard karyotype (without Bs) were marked as BO group, and those with Bs as B+ group. Sex ratios for both groups were close to 1. The frequency of B+ animals was 0.44 in 1998, 0.50 in 2000, and 0.22 in 2002. In this study only adults (n = 112) with complete eruption of the 3rd molar were included. The age of the animals was estimated from the weight of the dry eye lens, which is considered the best indicator of age in many rodents (Nabaglo and Pachinger 1979).

Mandibles were cleaned using dermestid beetles. Right mandibles were used in subsequent analyses. Images of the mandibles in lateral view, together with the scale in millimeters (640 x 480 pixels resolution), were obtained with a Nikon COOLPIX4500 digital camera (Refot B, Belgrade, Serbia). Ten 2-dimensional landmarks were digitized around the outline of the mandible and 13 interlandmark distances were calculated (TpsDig, version 1.40., F. J. Rohlf, http:/ /life.bio.sunysb.edu/ morph/). Landmarks were classified by type according to Bookstein (1991). Two landmarks were type I (juxtapositions of tissues), 5 were type II (maxima of curvature and valleys of invaginations), and 3 were type III (extremal points). The measurements were divided into 2 subsets (Table 1; Fig. 1) based on the developmental and functional composition of the mammalian mandible (Atchley 1993; Atchley and Hall 1991; Klingenberg et al. 2003).

Precision.-To estimate measurement error, a subsample of 22 mandibles (~20% of the total) was digitized on 3 separate occasions and model II analysis of variance (ANOVA) with repeated measures was performed. Variance due to measurement error estimated as a percentage of the total variation was evaluated for 2 traits determined with different types of landmarks. For superior molar alveolus length (m9) determined with type I landmarks, it was 1.57%; for inferior condylar length (m^sup 2^) determined with type II and type III landmarks, it was 1.58%. Statistical analyses.-A Kolmogorov- Smirnov test showed that the analyzed datasets satisfied assumptions of normality. Mean values and standard deviations for mandibular traits are given in Table 2. The preliminary analysis of morphometric variation within the total sample was done by factorial ANOVA, with the effects of sex, year of collection (time), and presence or absence of Bs as the fixed factors including their interactions. Subsequently, Bonferroni adjustment was made. The sex (and interactions) effects were not significant for any of the traits and therefore both sexes were pooled throughout all subsequent analyses. The significant time (F = 6.24, d.f. = 2,100, P = 0.04), B genotype (F = 10.20, d.f. = 1, 100, P = 0.03), and their interaction (F = 9.78, d.f. = 2, 100, P = 0.001) effects were found only for superior molar alveolus length (m9). Also, 1-way ANOVA on the weight of dry lenses indicated no difference in age between the studied groups (F = 0.03, d.f. = 1, 110, P = 0.87).

Principal component analysis of the correlation matrices of mandibular traits was used to assess the level of morphological integration, as well as the contribution of size to total mandibular integration. The degree of morphological integration was estimated by the index of integration (I-Cheverud et al. 1983) using eigenvalues derived from the correlation matrices. Higher values of I reflect tighter correlations among traits. However, the index of integration could be affected and biased by the sample size (Cheverud et al. 1989) and higher value of I might be the result of lower sample size. In our case, the sample size of B+ was lower than that of BO group (ratio between B+ and BO animals was almost 1:3). To verify obtained values of I in B+ animals and to overcome unbalanced sample sizes, we performed the following procedure. From the sample of individuals without Bs (n = 83), we randomly sampled (without replacement) 1,000 subsamples of the same sample size as the sample of B carriers (n = 29). We calculated indices of integration for all randomly derived subsamples. Then, the original I-values for the B carriers were compared with the estimated distribution of I. If the calculated value of I for the original sample of B carriers fell in the upper 5% tail of this distribution, we considered the influence of Bs on the level of morphological integration to be significant. Conducting the described procedure, we estimated the effects of Bs on the level of morphological integration free of effects of sample size.

According to the hypothesis, the mandible consists of 2 separate modules, an ascending ramus and an alveolar region. Following the approach by Cheverud (1995, 1996a) and the classification of traits in Table 1, 2 theoretical matrices (13 x 13) for the ascending ramus and alveolar region were constructed. If 2 traits belonged to the specified mandibular module, a value of 1 was entered; otherwise, a value of O was entered. With the aim to test the hypothesis of total morphological integration, a single theoretical connectivity matrix was constructed in the following manner: the value of 1 is entered if 2 traits share the same functional and developmental trait set, otherwise the value of O is entered. These 3 theoretically derived matrices were compared to the observed correlation matrices to obtain the matrix correlation (R) as a measure of the structural similarity between pairwise matrix correlations.

The statistical significance of the observed matrix correlation for all comparisons was assessed using the quadratic assignment procedure (Mantel’s test), under the null hypothesis of no correlation between compared matrices (Mantel 1967). This procedure includes 1,000 random permutations of 1 matrix followed by correlation of each randomized matrix with the reference matrix to generate a distribution of matrix correlations (Cheverud et al. 1989). If the observed matrix correlation exceeds 95% of the random correlations, the structures of 2 matrices are considered to be significantly similar (Marroig and Cheverud 2001).

Additionally, the overall mean of the observed Pearson’s correlation coefficients for functionally and developmentally related and unrelated traits in each of the empirically derived matrices were calculated (Marroig et al. 2004).

We also compared the matrix correlation pattern between BO and B+ animals by calculating elementwise matrix correlation between the observed correlation matrices (Ackermann and Cheverud 2000; Marroig and Cheverud 2001). Observed matrix correlations are always estimated with error (Cheverud et al. 1989), and matrix repeatability has been suggested as a technique for estimating the impact of such error (Cheverud 1996a; Marroig and Cheverud 2001). This technique is based on comparison of observed matrix correlations (R^sub obs^) to maximum correlations (R^sub max^) to obtain the adjusted correlations (R^sub adj^). For any 2 matrices 1 and 2, R^sub max^ is defined as a function of their repeatability (t), where R^sub max^ = (t^sub 1^t^sub 2^)^sup 0.5^. In this study, matrix repeatabilities were obtained using the method of autocorrelation (Marroig and Cheverud 2001; Young 2004). Specimens were resampled with replacement from the original data set for each group, and 1,000 resampled data sets were obtained. Correlation matrices were calculated from resampled data sets and compared to the original data set using a matrix correlation. Repeatabilities (t^sub 1^ for BO group; t^sub 2^ for B+ group) were evaluated as the mean values of the obtained R (PopTools, version 2.6.2., distributed by G. M. Hood, http://www.cse.csiro.au/poptools).

RESULTS

Chromosome analyses.-Chromosome analyses revealed that in the total sample of 112 specimens, 83 animals were without Bs (45 males and 38 females), whereas 29 mice had Bs (14 males and 15 females). Among the carriers of Bs, 58.6% had 1 B, 34.5% had 2 Bs, and 6.9% had 3 Bs. The average frequency of B+ animals was 0.26.

Level of morphological integration.-The level of morphological integration was higher in the group of B+ animals than in the BO group for both mandibular units and for the whole mandible as well (Table 3). Comparisons with the derived distributions of I (obtained for 1,000 randomly generated subsamples) revealed that the presence of Bs significantly affected the integration of the alveolar region only. The obtained I-value for the alveolar region in the group of B+ animals exceeded 97.5% of the values of I calculated for the BO animals. Precisely, the values of I for 999 randomly generated subsamples (n = 29) of BO animals were lower than the I-value obtained in the sample of B+ animals (n = 29). Thus, the higher level of morphological integration for the alveolar region could not be the result of the smaller sample size, but was related to the presence of Bs. The values of I obtained for the ascending ramus, and for the whole mandible, fell into 89.3% and 82% of the derived distributions, respectively, and therefore the increases in the level of morphological integration were not statistically significant. The percentage differences in the degree of morphological integration between these 2 groups were 15.86% for the ascending ramus, 41.61% for the alveolar region, and 14.94% for the whole mandible.

After performing principal component analysis, the principal components were extracted from the correlation matrices. The 1st principal component (PCl; component of size) captured 49.7% and 57.6% of the total variation of mandibular traits in the BO and B+ specimens, respectively.

Testing the hypothesis of morphological integration.-The matrix correlations between the observed correlation matrices and theoretically derived matrices, as well as the average correlations among functionally and developmentally linked traits, are presented in Table 4. The observed correlations were significantly correlated with the theoretically postulated pattern of total morphological integration in B+ animals. Additionally, within the overall mandible, the average correlations of functionally and developmentally related traits were 22.0% higher in B+ animals than the average correlations of unrelated traits. The hypothesis of overall morphological integration was not confirmed in BO animals.

Within the alveolar region the average correlations of related traits versus those of unrelated traits were 16.4% (P = 0.096) higher in B+ animals, whereas in the BO group the average correlations of unrelated traits exceeded the average correlations among related traits. Correlations among functionally and developmentally related traits within the ascending ramus were higher than among unrelated traits in both groups.

The degree of similarity in pattern of correlation.-Similarity of the matrix correlation pattern between BO and B+ animals was highly statistically significant (R^sub obs^ = 0.481, P = 0.001). Correlation matrix repeatabilities, t^sub 1^ for BO and t^sub 2^ for B+, were 0.922 and 0.834, respectively (R^sub max^ = 0.877). The adjusted matrix correlation (R^sub adj^) was 0.548.

DISCUSSION

The effects of Bs are rarely manifested in phenotypes either qualitatively or quantitatively. A scarcity of reported cases is especially evident for mammals. Our previous results (Blagojevic and Vujosevic 2000, 2004) indicate that impacts of Bs inA.flavicollis should be sought at the level of populations. An earlier study of western harvest mice (Reithrodontomys megalotis), as well as a recent study of the Brazilian rodent Akodon montensis, failed to establish a correlation between the presence of Bs and variance of external and cranial traits (Shellhammer 1969; Silva and Yonenaga- Yassuda 2004). However, application of a more sophisticated approach based on phenotypic correlations among mandibular traits has enabled better evaluation of the effects of Bs. Our results show that the level of morphological integration in the mandible is increased in the presence of Bs. However, the alveolar region is significantly more affected by the presence of Bs than is the ascending ramus. Blagojevic (1997) also found that, in this species, the level of correlation among cranial traits is higher in B+ animals. According to the hypothesis of morphological integration (Chernoff and Magwene 1999; Cheverud 1996b; Olson and Miller 1958; Riedl 1978) and quantitative genetic theory (Cheverud 1984; Lande 1979, 1980), traits that function and develop together will tend to be inherited together. Coinheritance of functionally and developmentally related traits arises from pleiotropy and linkage disequilibrium (Cheverud 1996b; Marroig and Cheverud 2001). The higher indices of integration that were obtained in animals possessing Bs could be explained by changes in pleiotropic effects or linkage disequilibrium, or both, produced either by the presence of Bs or their potential genetic activity. In support of this hypothesis could be the finding of Tanic et al. (2005) that Bs in A. flavicollis alter the expression of 3 genes. In many cases, the presence of Bs is associated with changes in chiasma frequency. Camacho (2005) proposed that in cases where chiasma frequency is decreased in the presence of Bs this could favor the maintenance of beneficial Bs. The reduction of chiasma frequency could increase linkage disequilibrium and thus contribute to the higher correlations among traits.

Although examination of our data indicates a general stability of correlation pattern within A. flavicollis, the hypothesis of total morphological integration (2-modular organization of the mandible) was confirmed in B+ animals only. The correspondence between observed and hypothetical correlation patterns in B+ animals is largely due to higher correlations for alveolar-region traits. Thus, once again it appears that the presence of Bs contributes preferentially to tighter connections among alveolar-region traits rather than among ascending-ramus traits. To highlight genetic mechanisms behind the phenotypic integration of complex morphological structures, a number of recent studies focused on analysis of quantitative trait loci of the mouse mandible (Murren and Kover 2004). Each of the 19 autosomes in mice carries significant quantitative trait loci for aspects of mandibular morphology (Cheverud et al. 1997; Enrich et al. 2003). It was found that the effects of some quantitative trait loci extend over the entire mandible, whereas other quantitative trait loci affect localized regions (Cheverud 2000; Cheverud et al. 1997; Ehrich et al. 2003). The already complex genetic architecture of the mandible is additionally complicated by the presence of Bs.

Overall, it seems that Bs affect the phenotype of the mandible by contributing to tighter relationships among mandibular features that share the same development and function, although the mechanism remains unknown. Morphological integration may facilitate adaptive evolution (Cheverud 1995; Marroig et al. 2004; Murren 2002; Oison and Miller 1958; Wagner 1996). The mandible is a complex morphological structure, with functions closely related to the fitness of an individual. Therefore, animals with more integrated mandibular traits (in our case B carriers) could be favored by natural selection. Vujosevic and Blagojevic (2000) showed that the frequency of B carriers is correlated with certain climatological conditions and increases with altitude. Boyeskorov et al. (1994) also found that the frequency of animals possessing Bs is higher in peripheral populations of the same species. This indicates that the presence of Bs in A. flavicollis has beneficial effects in certain environmental conditions and that the benefit could come from their contribution to higher correlations among different cranial and mandibular features.

Furthermore, if we accept that the ascending ramus and alveolar region are partially autonomous and that the modularity is a matter of degree (Klingenberg et al. 2003, 2004), then we can predict that Bs tend to separate the modules to a greater extent. The observed trend of B carriers to have more independent mandibular modules compared to noncarriers could mean that 2 correlation patterns are present among mandibular traits. Vujosevic and Blagojevic (2004) proposed that Bs contribute to the genetic variability of species possessing them. Increased genetic variability could be a possible reason for their long-term presence in populations, especially in variable environmental conditions and in peripheral populations that are subject to frequent variation in population density. Because genetic and phenotypic covariance structure may be adaptive within a certain environment (Berg 1960; Cheverud 1984; Wagner 1988) and can constrain evolutionary change in response to a new environment (Arnold 1992; Maynard Smith et al. 1985), the observed divergence in correlation patterns of A. flavicollis could provide diverse responses to local fluctuating selection pressures.

ACKNOWLEDGMENTS

This work was supported by the Ministry of Science and Environmental Protection of the Republic of Serbia (grant 14301 IG).

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Submitted 19 January 2006. Accepted 2 October 2006.

Associate Editor was Craig L. Frank.

VIDA JONC,* JELENA BLAGOJEVIC, ANA IVANOVIC, VANJA BUGARSKI- STANOJEVIC, AND MLADEN VUJOSEVIC

Department of Genetic Research, Institute for Biological Research “Sinisa Stankovic,”

Bulevar despota Stefana 142, 11060 Belgrade, Serbia (VJ, JB, VB- S, MV)

Institute of Zoology, Faculty of Biology, University of Belgrade, Studentski trg 16,

11000 Belgrade, Serbia (AI)

* Correspondent: vjojic@ibiss.bg.ac.yu

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