Biological Journal of the Linnean Society, 2009, 97, 555–570. With 6 figures
Morphological variation in house mice from the
Robertsonian polymorphism area of Barcelona
bij_1237
555..570
MARIA ASSUMPCIÓ SANS-FUENTES1*, JACINT VENTURA2,
MARÍA JOSÉ LÓPEZ-FUSTER1 and MARCO CORTI3†
1
Departament de Biologia Animal, Facultat de Biologia, Universitat de Barcelona, Avda. Diagonal
645, 08028 Barcelona, Spain
2
Departament de Biologia Animal, de Biologia Vegetal i d’Ecologia, Facultat de Biociènces,
Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain
3
Dipartimento di Biologia Animale e dell’Uomo, Università di Roma ‘La Sapienza’, via A. Borelli 50,
00161 Roma, Italy
Received 19 September 2008; accepted for publication 11 December 2008
Morphometric variation in the Robertsonian polymorphism zone of Barcelona of Mus musculus domesticus was
studied by geometric morphometrics. This system is characterized by populations of reduced diploid number
(2n = 27–39) surrounded by standard populations (2n = 40). We investigated the morphological variation in mice
from this area, as well as the effect of geographical distance and karyotype on this variation. We also investigated
the degree of co-variation between the two functional units of the mandible to explore the origin of this system
(primary intergradation or secondary contact). The size and shape of the cranium, mandible and scapula were
analysed for 226 specimens grouped by population, chromosome number and structural heterozygosity. Size was
estimated as the centroid size, and shape was estimated after Procrustes superimposition. No significant
differences in size between populations or chromosomal groups were detected. Diploid number, structural heterozygosity and local geographical isolation contributed to the differentiation in shape. Morphological differentiation between standard mice and Robertsonian specimens was observed, suggesting genetic isolation between these
groups. Co-variation between the ascending ramus and alveolar region of the mandible was quantified by the trace
correlation between landmark subsets of these modules. The trace values showed an ascending trend, correlated
with the distance from the centre of the polymorphism area, a pattern consistent with a primary intergradation
scenario. © 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 97, 555–570.
ADDITIONAL KEYWORDS: co-variation – geometric morphometrics – morphological variation – Mus
domesticus – primary intergradation – Robertsonian fusion.
INTRODUCTION
The house mouse, Mus musculus domesticus (also
referred to in the literature as M. m. domesticus), has
a standard karyotype of 40 chromosomes (19 autosomal pairs and a sexual pair). Robertsonian (Rb)
*Corresponding author. Current address: Department of
Ecology and Evolutionary Biology, University of Arizona,
Tucson, AZ 85721, USA. E-mail: [email protected]
†Marco Corti died before the final version of this article was
completed.
translocations, i.e. centric fusion between two nonhomologous acrocentric chromosomes to form a metacentric, leading to a reduction in the diploid number
in the carriers, are frequent in this species (Capanna,
1982; Piálek, Hauffe & Searle, 2005). Rb translocations can be present in structural heterozygosity,
when the homologous chromosomes of the Rb fusion
are the corresponding acrocentrics, or in structural
homozygosity, when the homologue is the same Rb
translocation. A group of contiguous populations
sharing the same set of Rb chromosomes, all in a
homozygous state, form a chromosome race (Hausser
© 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 97, 555–570
555
556
M. A. SANS-FUENTES ET AL.
et al., 1994). In Eurasia and North Africa, 97 metacentric populations have been described (see Piálek
et al., 2005). Hybrid zones between metacentric and
standard races, or between different metacentric
races, are frequent and well known (Spirito et al.,
1980; Corti, Ciabatti & Capanna, 1990; Saïd &
Britton-Davidian, 1991; Searle, 1991; Castiglia &
Capanna, 1999). These areas are characterized by
intermediate diploid number between the two parental types and structural heterozygous individuals. Rb
fusions are believed to be involved in speciation,
because the accumulation of different fusions in two
races can lead to hybrid hypofertility or sterility as a
result of malsegregation during meiosis, producing a
reduction in gene flow between races (Garagna et al.,
1989, 1990; Castiglia & Capanna, 2000). In addition,
there is evidence of recombination suppression
around the centromeres of Rbs, in either heterozygous
or homozygous state, thus leading to a reduction in
gene flow in the genes of the pericentromeric region
(Yamamoto & Miklos, 1978; Davisson & Akeson, 1993;
Choo, 1998; Castiglia, Annesi & Capanna, 2002;
Dumas & Britton-Davidian, 2002; Franchini et al.,
2006).
Among all of the Rb populations described, the Rb
polymorphism zone of Barcelona is localized in the
northeast of the Iberian Peninsula, and is surrounded
by standard populations (Gündüz et al., 2001; SansFuentes et al., 2007). This area covers over 5000 km2
and the diploid number ranges between 27 and 39,
showing the lowest 2n in the populations located at
the centre of the distribution (Fig. 1). A number of
combinations of seven Rb fusions account for these
karyotypes, in either the heterozygous or homozygous
state [Rb(3.8), Rb(4.14), Rb(5.15), Rb(6.10), Rb(7.17),
Rb(9.11) and Rb(12.13)]. In this area, staggered clines
for Rb chromosomes have been described (Gündüz
et al., 2001; M. A. Sans-Fuentes, unpubl. data) and, to
date, no parental chromosomal Rb race has been
found that is consistent with the race definition by
Hausser et al. (1994). Therefore, this area is considered to be an Rb polymorphism zone rather than a
typical hybrid zone (Sans-Fuentes et al., 2007), representing a particular Rb scenario that might have
originated by primary intergradation, although secondary contact cannot be excluded a priori. Thus, it
may represent a case showing a raciation process,
eventually leading to the formation of an Rb race
without geographical isolation. It also represents an
interesting case study to highlight how phenotypic
differentiation occurs during raciation.
Phenotypic variation has a genetic basis, and Rb
translocations can favour changes in allele frequency
in genes regulating the developmental process of mor-
Figure 1. The house mouse Robertsonian (Rb) polymorphism area of Barcelona. Diploid number ranges per sampled
population are shown in parentheses. Locality names are shown in Table 1. Asterisk indicates the estimated centre of the
Rb polymorphism area (M. A. Sans-Fuentes, unpubl. data).
© 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 97, 555–570
MORPHOLOGICAL VARIABILITY IN ROBERTSONIAN MICE
phological traits, either by reproduction unfitness of
hybrids or recombination suppression in Rb translocations (Lane & Eicher, 1985; Eppig & Eicher, 1988;
Bidau et al., 2001; Castiglia & Capanna, 2002; Dumas
& Britton-Davidian, 2002). House mice constitute an
appropriate model to study morphological variation,
as the genetics of this species is well known. A body of
quantitative trait loci for the skull (Leamy et al.,
1999) and mandible (Klingenberg et al., 2001) has
already been mapped onto the house mouse chromosomes, part being located near the centromere. It can
be expected that genes placed in these quantitative
trait loci suffer a change in allele frequency or linkage
disruption, or the effect of both. Therefore, these
alleles can determine a new common expression
pattern in Rb mice, leading to a different morphological phenotype. We already know from non-metric
traits of the skull that, in the Rb polymorphism zone
of Barcelona, there is morphological differentiation
related to karyotypic diversity, especially between
standard and Rb mice (Muñoz-Muñoz et al., 2003). In
the present study, we seek to go further in the knowledge of how the shape and size of different osseous
structures can be related to geography and karyotype
characteristics. We also wish to determine whether
the patterns of morphological variation are similar in
cranial and postcranial osseous structures that differ
widely in both origin and function.
In addition, the analysis of both shape differentiation and co-variation of osseous structures may
provide information useful for disentangling the
557
origins of this Rb polymorphism area. With this goal
in mind, the mandible and scapula can provide more
reliable information than the cranium, as they have
mainly masticatory and locomotor function, respectively; meanwhile, the cranium is a more complex
region of the skeleton, containing different organs
(brain, sense organs, feeding apparatus). Thus, it is
expected that the evolution of these two structures
will be more indicative of the natural history of this
area. In the present study, we attempt to understand
the origins of the Rb polymorphism zone of Barcelona,
focusing on the study of the mandible, as information
regarding its development is readily available.
Two main functional units compose the final structure of the mandible: the alveolar region, i.e. the
anterior part bearing the teeth, and the ascending
ramus, which articulates with the cranium (Klingenberg, Mebus & Auffray 2003; Fig. 2C). Because the
shape of the mandible depends on the coordinate
development of different bone elements and soft
tissues (Richtsmeier et al., 2002), co-variation between these two units is expected. Although there
is no conclusive information regarding the molecular
cascades controlling bone formation, it appears that a
precise interaction between genes is necessary for the
correct development of the mandible (Fukuhara et al.,
2004). Thus, hybridization between two different
genomes that have evolved independently might
decrease developmental efficacy through the disruption of genomic co-adaptation, i.e. the overall genetic
balance resulting from the selective processes over
Figure 2. Landmarks used in the analyses for each bone structure: A, dorsal view of cranium; B, ventral view of cranium;
C, lingual view of mandible (the two main functional units are shown); D, dorsal aspect of scapula.
© 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 97, 555–570
558
M. A. SANS-FUENTES ET AL.
the evolutionary histories of populations (Alibert &
Auffray, 2003). Therefore, the study of the patterns of
co-variation throughout the polymorphism region
may provide valuable information about its origin. We
make the hypothesis that, if this Rb polymorphism
zone is the result of secondary contact, we will find
co-variation reduction between the two units of the
mandible in mice from localities situated at an intermediate distance between the centre of the polymorphism area and standard localities. In these localities,
in which mice have an intermediate 2n between 26
(hypothetical race) and 40, i.e. 2n = 33–34, we expect
that several alleles from both parental races will meet
in high frequency, resulting in the disruption of
genetic co-adaptation. Thus, animals from localities
closer to parental races would have higher levels of
co-variation, as they have experienced more generations of recombination and selection than the intermediate populations, which buffer the disruption of
genetic co-adaptation. By contrast, if the polymorphism area is the result of a primary intergradation,
we expect a decay of co-variation closer to the centre
of the Rb polymorphism area, where fusions are
arising. Moreover, we expect a progressive increase in
co-variation correlated with the geographical distance
from the centre. We assume that the Rb fusions that
are widely distributed and are present in the populations of the periphery are the oldest. Thus, the
peripheral populations have undergone more generations of recombination and selection, allowing for the
buffering of the disruption of genetic co-adaptation
that could have appeared after hybridization with
standard populations.
With all this in mind, the goals of this study are to
investigate: (1) the degree of morphological variation
in the cranium, mandible and scapula in mice from
the Rb polymorphism zone of Barcelona; (2) the effect
of geographical distance, diploid number and the
structural composition of the karyotype on this variation; and (3) the degree of co-variation between the
two functional units of the mandible, in an attempt to
explore the origin of this area.
MATERIAL AND METHODS
SOURCE OF SPECIMENS
Data were collected from mice live-trapped at 16 sites
(farms) in the Rb polymorphism zone of Barcelona
between 1996 and 1999 (Fig. 1, Table 1). Each specimen was characterized by its own karyotype
(see Gündüz et al., 2001; Muñoz-Muñoz et al., 2006;
Sans-Fuentes et al., 2007), and their skeletons were
cleaned by exposure to desmestid larvae (Coleoptera:
Dermestidae). Dorsal and ventral views of the
cranium, the lingual view of the mandible and the
dorsal aspect of the scapula were studied.
The specimens were divided into three age classes
according to their reproductive state (by gonadal
study) and body size (head and body length, HBL):
Table 1. Characteristics of sampling localities
Locality
Latitude/longitude
Distance to the
centre (km)*
2n mean†
H‡
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
41°17′N,
41°14′N,
41°22′N,
41°23′N,
41°23′N,
41°25′N,
41°13′N,
41°18′N,
41°21′N,
41°21′N,
41°11′N,
41°32′N,
41°34′N,
41°44′N,
41°27′N,
41°29′N,
5.98
10.14
10.38
11.18
11.52
15.75
20.19
22.39
33.12
38.53
39.15
44.17
44.23
57.84
70.29
72.46
30.217
31.840
30.881
32.125
30.155
34.714
36.000
30.200
38.640
39.645
40.000
38.333
38.556
39.500
40.000
40.000
1.85
1.32
1.79
1.46
1.22
1.57
1.30
1.33
1.17
0.29
0.00
1.14
0.56
0.17
0.00
0.00
Garraf (GA)
Vilanova i la Geltrú (VG)
La Granada (LG)
Lavern (LV)
St. Pau d’Ordal (SP)
St. Sadurní d’Anoia (SS)
Calafell (CF)
Gavà (GV)
Les Pobles (LP)
Bellaterra (BE)
La Riera (LR)
Sta. Coloma de Queralt (SC)
Sabadell (SA)
Calaf (CA)
Fulleda (FU)
L’Espluga Calba (EC)
1°50′E
1°43′E
1°43′E
1°46′E
1°48′E
1°47′E
1°34′E
2°00′E
1°24′E
2°06′E
1°22′E
1°23′E
2°06′E
1°31′E
1°01′E
1°00′E
*Distance to the centre of the Robertsonian polymorphism area (see Fig. 1; M. A. Sans-Fuentes, unpubl. data).
†Mean diploid number (2n).
‡Heterozygosity calculated according to Gündüz et al. (2001).
© 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 97, 555–570
MORPHOLOGICAL VARIABILITY IN ROBERTSONIAN MICE
young, subadult and adult. Young correspond to age
class I (HBL ⱕ 73 mm, including only males without
spermatozoids in seminiferous tubules and females
without maculae cianosae or embryos). Subadults
correspond to age class II (73 < HBL < 86 mm, including only males with spermatozoids in seminiferous
tubules and all females regardless of the absence of
maculae cianosae or embryos). Adults correspond to
age class III (HBL ⱖ 86 mm, including only males
with spermatozoids in seminiferous tubules and all
females regardless of the absence of maculae cianosae
or embryos). Because of the small sample size of age
class I (N = 23), only animals of age classes II and III
were analysed (Table 2).
The effect of geographical variation was studied
by grouping specimens according to locality, as
represented in Table 2. The effect of structural
homozygosity–heterozygosity (SHH) was analysed
according to the presence of homozygous and/or heterozygous Rb translocations by pooling specimens as
follows: structural Rb homozygotes (HH), animals
with a diploid number between 28 and 38 and with all
their Rb fusions in the homozygous state; heterozygotes I (HTI), animals with one heterozygous fusion;
heterozygotes II (HTII), specimens with two heterozygous fusions; heterozygotes III (HTIII), mice with
three or more heterozygous fusions; specimens with
a 2n = 40 karyotype but from localities in which Rb
mice also occur (40H); and standard homozygotes
(40ST), animals from localities in which Rb mice do
not occur (Table 2). The HTI, HTII and HTIII heterozygote groups were formed irrespective of their 2n
value and the possible occurrence of other fusions in
the homozygous state. Although the 40H and 40ST
groups have the same 2n, they are separated because
40H mice may have originated from structural heterozygotes and not necessarily from 2n = 40 parents.
To study the effect of diploid number (2n analysis),
the animals were assigned to six groups by establishing chromosomal ranges as follows: 28–30, 31–33,
34–37, 38–39, 40H and 40ST. The groups of animals
with 40 chromosomes are the same as those of the
SHH classification (Table 2).
DATA
ACQUISITION
Digital images from the dorsal and ventral views of
the cranium, the lingual view of the mandible and the
dorsal aspect of the scapula were taken with a Pixera
Professional camera (Pixera Corporation) and a
135 mm Nikon lens, by placing the object at a distance of 53 cm. Twenty-two landmarks were recorded
from the dorsal view of the cranium, twenty-seven
from the ventral view, sixteen from the left mandible
and ten from the right scapula using TpsDig (Rohlf,
1996; Fig. 2).
DATA
559
ANALYSIS
The left and right sides of the cranium were averaged
to avoid effects of lateral asymmetry. This was performed by rotating configurations to have the tip of
the rostrum set at the origin, and the posterior edge
of the foramen magnum lying along the x axis, and
then averaging the corresponding landmarks across
the midline. These half configurations were used for
all subsequent analyses.
The form was decomposed into its shape and size
components, which were examined separately. The
size of each osseous structure was estimated by the
centroid size (CS) using TpsSmall (Bookstein, 1991;
Rohlf, 1998).
Sexual dimorphism and age effects on CS were
studied by two-way analysis of variance (ANOVA)
using only 40ST mice to avoid the possible effect
caused by Rb fusions. Normality and homogeneity of
variances were tested by the Kolmogorov–Smirnov and
Levene tests, respectively. Differences in CS between
localities or chromosomal groups for each osseous
structure were investigated by one-way ANOVAs.
To study shape changes, specimens were scaled to
unitary CS and aligned with respect to the consensus
configuration (the average configuration of landmarks) using generalized Procrustes analysis (Rohlf
& Slice, 1990). The closeness of the approximation of
the shape space to tangent space was estimated using
TpsSmall (Rohlf, 1998).
Sex and age effects on shape were analysed for a set
of relative warps (RWs; Rohlf, 1993) only in 40ST
animals by means of a two-way multivariate analysis
of variance (MANOVA). RWs were used instead of the
augmented weight matrix (W⬘) to reduce the number of
variables in relation to the sample size, because of the
small number of standard mice (N ª 30; Table 2). RWs
were obtained by TpsRelw (Rohlf, 2002).
Shape differences between localities or chromosomal groups in each osseous structure were tested by
one-way MANOVAs on W⬘, which accounts for total
shape. Trends in shape changes were investigated by
canonical variate analysis (CVA) and visualized by
regressing W⬘ onto canonical vectors (CVs) using
TpsRegr (Rohlf, 2000). Multiple comparisons between
groups were performed by Hotelling T2 test on unbiased Mahalanobis distances, corrected by sample size
(Marcus, 1993). Bonferroni correction was applied
on significance levels of Mahalanobis distances
(Rice, 1989; Marcus, 1993). The phenetic relationships between groups were shown by means of
minimum-length spanning trees (MSTs; Gower &
Ross, 1969) superimposed on CVA plots.
In MANOVAs, a significant P value for the locality
factor can be a result of geographical effects, for
example isolation by distance, rather than different
© 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 97, 555–570
(17, 18)
(22, 26)
(5, 10)
(9, 6)
(7, 3)
(6, 3)
0)
0)
6)
1)
1)
(3,
(4,
(4,
(3,
(0,
(6, 8)
(7, 13)
(5, 8)
(3, 5)
(5, 3)
(13, 9)
(3, 4)
(16, 12)
(3, 8)
(5, 4)
(5, 3)
(13, 9)
(7, 9)
(1, 3)
(4, 2)
2)
2)
2)
1)
7)
4)
(2,
(3,
(2,
(2,
(8,
(2,
37
49
14
14
9
10
17
44
32
21
9
10
10
0
7
4
3
15
6
11
31
8
3
6
0
21
2
6
(18, 19)
(22, 27)
(5, 9)
(9, 5)
(6, 3)
(7, 3)
(10, 7)
(22, 22)
(11, 21)
(11, 10)
(6, 3)
(7, 3)
(11, 10)
(2, 0)
(4, 2)
(2, 5)
(2, 2)
(3, 0)
(4, 11)
(3, 3)
(1, 10)
(17, 14)
(5, 3)
(2, 1)
(4, 2)
(7, 3)
16
21
13
9
7
21
7
28
13
11
7
21
3
5
4
3
15
8
0
3
4
11
3
1
0
15
5
7
0)
0)
7)
1)
1)
2)
2)
2)
1)
7)
5)
(7, 9)
(14, 7)
(5, 8)
(3, 6)
(4, 3)
(12, 9)
(3, 4)
(15, 13)
(4, 9)
(7, 4)
(4, 3)
(12, 9)
(6, 9)
(2, 3)
(5, 2)
(3,
(4,
(4,
(2,
(0,
(1,
(3,
(2,
(2,
(8,
(3,
39
50
16
13
9
10
15
48
33
22
9
10
11
0
10
4
4
17
6
11
31
8
2
4
0
20
2
7
(18, 21)
(23, 27)
(6, 10)
(7, 6)
(6, 3)
(7, 3)
(8, 7)
(24, 24)
(11, 22)
(11, 11)
(6, 3)
(7, 3)
(10, 10)
(2, 0)
(5, 2)
(3, 7)
(2, 2)
(3, 1)
(5, 12)
(3, 3)
(1, 10)
(17, 14)
(5, 3)
(2, 0)
(2, 2)
(7, 4)
Cl. II
17
19
12
8
9
24
7
26
14
9
9
24
4
6
4
4
15
8
0
3
4
9
5
1
0
15
3
8
0)
0)
5)
1)
1)
2)
2)
2)
1)
7)
5)
(8, 9)
(13, 6)
(6, 6)
(3, 5)
(6, 3)
(15, 9)
(3, 4)
(15, 11)
(6, 8)
(6, 3)
(6, 3)
(15, 9)
(7, 8)
(1, 2)
(6, 2)
(3,
(4,
(4,
(4,
(0,
(2,
(4,
(2,
(3,
(8,
(3,
Cl. III
Lingual view of mandible
30
42
14
9
7
8
9
40
28
18
7
8
9
0
10
2
3
15
0
11
22
6
3
0
2
22
1
4
7)
1)
1)
10)
(15, 15)
(17, 25)
(5, 9)
(5, 4)
(5, 2)
(5, 3)
(4, 5)
(22, 18)
(7, 21)
(9, 9)
(5, 2)
(5, 3)
(0, 2)
(11, 11)
(1, 0)
(3, 1)
(1, 10)
(12, 10)
(5, 1)
(2, 1)
(3,
(1,
(2,
(5,
(6, 3)
Cl. II
11
19
11
10
8
20
5
25
12
9
8
20
4
5
4
3
13
6
0
2
3
9
4
0
3
14
4
5
0)
0)
6)
3)
2)
2)
2)
1)
6)
3)
(7, 4)
(14, 5)
(5, 6)
(5, 5)
(5, 3)
(10, 10)
(3, 2)
(16, 9)
(6, 6)
(6, 3)
(5, 3)
(10, 10)
(3, 0)
(10, 4)
(2, 2)
(3, 2)
(2,
(3,
(3,
(1,
(2,
(3,
(2,
(2,
(7,
(3,
Cl. III
Dorsal view of scapula
The number of animals for each group is shown. The number of females (left) and the number of males (right) are indicated in parentheses. Cl. II, age class
II; Cl. III, age class III. For the codification of chromosomal groups, see Material and methods.
35
48
15
15
10
9
14
20
13
8
8
22
4
5
4
3
15
6
0
3
4
10
4
1
0
16
4
6
Diploid number
28–30
31–33
34–37
38–39
40H
40ST
(11, 10)
(2, 0)
(4, 2)
(2, 6)
(2, 2)
(3, 0)
(4, 10)
(3, 3)
(1, 9)
(16, 14)
(5, 3)
(1, 1)
(4, 2)
(8, 4)
7
28
11
9
8
22
12
0
8
4
3
14
6
10
30
8
2
6
0
21
2
6
Structural homozygosity–heterozygosity
HH
16 (8, 8)
HTI
44 (23, 21)
HTII
32 (11, 21)
HTIII
21 (11, 10)
40H
10 (7, 3)
40ST
9 (6, 3)
Locality
Bellaterra
Calaf
Calafell
L’Espluga Calba
Fulleda
Garraf
Gavà
Lavern
La Granada
Les Pobles
La Riera
Sabadell
Santa Coloma de Queralt
Sant Pau d’Ordal
Sant Sadurní d’Anoia
Vilanova i la Geltrú
Cl. III
Cl. II
Cl. II
Cl. III
Ventral view of skull
Dorsal view of skull
Osseous structure
Table 2. Sample size per established mice group and osseous structure
560
M. A. SANS-FUENTES ET AL.
© 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 97, 555–570
MORPHOLOGICAL VARIABILITY IN ROBERTSONIAN MICE
chromosomal compositions in each locality. Thus,
to determine whether the differences between populations were a result of isolation by distance, Mantel
tests (10 000 permutations) were performed between
Mahalanobis distance matrices for each osseous
structure and geographical distance matrices among
localities.
To explore the possible origin of this Rb polymorphism area (primary intergradation or secondary
contact), we quantified the co-variation between the
alveolar region and ascending ramus of the mandible
as the trace correlation (rT) between the coordinates
of these two subsets of landmarks, following the
approach of Klingenberg et al. (2003). For this
purpose, we used IMP software (H. David Sheets,
http://www3.canisius.edu/~sheets/moremorph.html).
Unlike the procedure employed by Klingenberg et al.
(2003), who used the landmark data themselves as
the variables when calculating the trace correlation,
IMP uses the partial warp scores of the specimens as
the variables (for a discussion, see IMP help). The
significance of the observed trace correlation rT was
assessed by permutation tests (1000 permutations;
Good, 1994). We calculated rT throughout the localities of the Rb polymorphism area. As the number of
mice available from some localities is low, we grouped
the Rb localities by the distance from the centre of the
polymorphism area. All the standard localities were
pooled together. Thus, four groups were made: group
1, with localities located 5–15 km from the centre
(GA, VG, LG, LV, SP); group 2, localities located
15–23 km from the centre (SS, CF, GV); group 3,
localities located 33–58 km from the centre (LP, BE,
SC, SA, CA); and group 4, localities composed only of
40ST specimens (LR, FU, EC) (Table 1). It is worth
mentioning that the mean diploid number increases
from group 1 to group 4.
RESULTS
SIZE
ANALYSIS
Significant age class differences in CS were found for
dorsal and ventral views of the cranium (F = 9.47,
P < 0.01; F = 17.35, P < 0.001; respectively), mandible (F = 13.37, P < 0.001) and scapula (F = 20.21,
P < 0.001). In all osseous structures, the CS values
were higher in age class III. No significant effect of
sex and its interaction with age was found. Therefore,
subsequent analyses of CS were performed only in
adults (age class III) and by grouping sexes. ANOVAs
showed no significant differences for all osseous structures in locality, SHH or diploid number.
SHAPE
ANALYSIS
The approximation of the shape space by the tangent
space was almost perfect for the cranium (dorsal and
561
ventral views), mandible and scapula (r = 0.999 in all
osseous structures). Two-way MANOVAs on the first
six RWs (accounting for 74.6% of the variance for the
dorsal view of the cranium, 78.8% for the ventral view
of the cranium, 74.95% for the mandible and 89.63%
for the scapula) did not reveal any evidence of sexual
dimorphism or age class effect. Therefore, sexes and
age classes were pooled together in subsequent analyses. MANOVAs performed on W⬘ were always significant for the locality factor, and either SHH or diploid
number (Table 3). A further investigation through
Hotelling T2 test on Mahalanobis distances between
localities revealed significant differences at P < 0.001
in all 105 pairwise comparisons for the ventral view
of the cranium after Bonferroni correction. For the
dorsal view of the cranium and for the mandible,
almost all the pairwise comparisons were significant
at P < 0.001 (only eight and three pairwise comparisons were not significant for these structures, respectively). In the scapula, only 42 of the 91 pairwise
comparisons were significant (P < 0.001). Nevertheless, the Mantel test between geographical and
Mahalanobis distances did not show any significant
correlation for any osseous structure (dorsal view of
the cranium: Z = 0.2775, P = 0.3780; ventral view of
the cranium: Z = 0.2449, P = 0.4050; mandible: Z =
0.8494; P = 0.2010; scapula: Z = -0.9854; P = 0.8490).
Therefore, the morphological differentiation found
between localities was not related to geographical
isolation by distance.
With regard to morphological differentiation
between chromosomal groups, Hotelling T2 test on
Mahalanobis distances showed significant differences
at P < 0.001 in all pairwise comparisons for the
cranium and mandible, in both the SHH and 2n
analyses. Concerning the scapula, in SHH analysis,
significant differences were found between all pairwise
comparisons at P < 0.001, except between HTIII and
the other groups, and the comparisons HH/HTI and
HH/HTII, which showed P < 0.01. In addition, for the
scapula, all comparisons between groups in the 2n
analysis showed significant differences at the 0.001
level, except for the comparison between 38–39 and
40H (P < 0.05). Although, in both SHH and 2n analyses, all the Mahalanobis distances between groups
were statistically significant, the pattern of divergence
was not coincident for all osseous structures. On the
one hand, the magnitude of Mahalanobis distances
differed between the structures analysed, showing
higher values of divergence in most of the pairwise
comparisons for the ventral view of the cranium and
the mandible. On the other hand, morphological differentiation between chromosomal groups was not
homogeneous for all structures. For example, although
for the ventral view of the cranium and mandible,
40ST mice showed the highest divergences, for the
© 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 97, 555–570
1.542*
1.750*
1.982*
1.808*
1.683*
2.620*
2.730*
2.444*
SHH analysis
Dorsal view of cranium
Ventral view of cranium
Mandible
Scapula
2n analysis
Dorsal view of cranium
Ventral view of cranium
Mandible
Scapula
(min.
(min.
(min.
(min.
34.78%;
43.74%;
31.82%;
18.52%;
max.
max.
max.
max.
63.89%)
70.97%).
73.53%)
70.77%)
(min. 50.00%; max. 85.71%)
(min. 42.86%; max. 100)
(min. 57.14%; max. 100%)
(min. 20%; max. 80.56%)
46.76 % (min. 29.03%; max. 63.24%).
62.20% (min. 40.74%, max. 77.42%)
60.47% (min. 44.44%; max. 73.53%)
54.20% (min. 36.84%; max. 68.29%)
44.12%
54.61%
50.92%
43.04%
70.58%
85.87%
83.75%
61.90%
3
4
4
3
2
2
2
1
8
11
8
6
(77.32%)
(91.76%)
(93.79%)
(83.57%)
(60.83%)
(65.52%)
(69.67%)
(50.65%)
(89.81%)
(97.15%)
(90.91%)
(85.76%)
Number of
significant CVs
36.24%
37.09%
49.17%
50.10%
31.59%
45.21%
53.20%
50.65%
21.64%
21.89%
21.90%
24.60%
CV1
20.70%
21.56%
20.23%
18.48%
29.24%
20.31%
16.46%
17.39%
18.43%
18.06%
20.66%
CV2
20.38%
19.69%
12.91%
14.99%
15.30%
13.44%
14.08%
14.55%
CV3
13.41%
11.47%
10.82%
11.38%
10.61%
10.91%
CV4
*P < 0.001.
The posterior probability membership based on generalized distances, the number of significant canonical vectors (CVs; the percentage of variance explained by
all the significant CVs is given in parentheses) and the percentage of total among-group relative to within-group variance explained for each of the first four CVs
when significant are shown.
3.021*
4.160*
3.361*
2.807*
Mean posterior
probability membership
Wilk’s lambda
MANOVA
Population analysis
Dorsal view of cranium
Ventral view of cranium
Mandible
Scapula
CVA
MANOVA analysis
Table 3. Multivariate analysis of variance (MANOVA) and canonical variate (CVA) for each structure and established mice group
562
M. A. SANS-FUENTES ET AL.
© 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 97, 555–570
MORPHOLOGICAL VARIABILITY IN ROBERTSONIAN MICE
dorsal view of the cranium and scapula, 40H mice
presented the highest divergence values, in both SHH
and 2n analyses (data not shown).
The CVA for overall shape differences allowed the
discrimination between localities and chromosomal
groups. The posterior probability membership showed
higher mean values in the locality analysis. The percentage of variance explained by the total number of
significant CVs was higher for both the site and 2n
analyses than for SHH analysis (Table 3).
For locality analysis, the different CVs did not seem
to segregate sites following any pattern related to the
geographical distribution (data not shown), which is
consistent with the results of the Mantel test. For the
SHH analysis, CV1 accounted for the differences
between the 40-chromosome groups (40ST and 40H)
and those with Rb fusions in both the dorsal view of
the cranium and the scapula (Fig. 3). Although CV2
was not statistically significant in the scapula, in the
dorsal view of the cranium it separated 40H and
HTIII groups with respect to the other groups. An
MST superimposed on the CVA scatter plot showed
that, for the scapula, both 40ST and 40H had HTIII
as the nearest group, but 40ST had a larger distance
from HTIII than 40H. In the dorsal view of the
cranium, the nearest group to 40ST was 40H. The
deformation grids showed that the main morphologi-
563
cal differences between 40-chromosome specimens
and Rb specimens with respect to the scapula concerned the acromion. In the dorsal view of the
cranium, the differences between these two main
groups were located in the neurocranium, specifically
the interparietal and orbitary regions and the posterior width of the posterior origin of the zygomatic
arch (Fig. 4). The ventral view of the cranium and the
mandible showed a similar pattern for CVA (Fig. 3).
In both structures, CV1 discriminated 40ST from the
others. CV2 did not show a clear pattern of discrimination in both cases. The MST showed that 40ST was
the most differentiated group for both structures. The
main difference between the MST of the ventral view
of the cranium and mandible was the relative position
of 40H. Although, in both structures, the nearest
neighbour to 40H is HTI, for the mandible, 40H had
an intermediate position between 40ST and HTI and,
for the ventral view of the cranium, 40H was only
directly related to HTI. The relationships between
HTI, HTII, HTIII and HH were similar in both structures. The deformation grids showed that the main
morphological differences between 40ST and the
other chromosomal groups concerned the anterior and
posterior widths of the zygomatic arch, the incisive
foramen length, and the coronoid and condylar processes of the mandible (Fig. 4).
Figure 3. Canonical variate analysis (CVA) of W⬘ for dorsal and ventral views of cranium, lingual side of mandible and
dorsal aspect of scapula between structural homozygosity–heterozygosity (SHH) groups. Graphs show the mean canonical
punctuation per group for the first two canonical vectors (CV1 vs. CV2). Minimum-length spanning trees (MSTs) are
represented by broken lines. Distances between neighbour groups are indicated.
© 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 97, 555–570
564
M. A. SANS-FUENTES ET AL.
Figure 4. Deformation grids belonging to negative and positive extremes of the first canonical vector (CV1) for each bone
structure for structural homozygosity–heterozygosity (SHH) analysis (see Fig. 3). Shape changes have been augmented
three times to emphasize differences.
In the 2n analysis, CV1 showed a trend in shape
change associated with a reduction in diploid
number (from negative to positive scores in the
cranium and scapula, and from positive to negative
in the mandible; Fig. 5). Ordination onto the remaining CVs did not exhibit any pattern. MST in
the scapula analysis highlighted two main groups:
one composed of 38–39, 40H and 40ST, and the
other composed of 34–37, 31–33 and 28–30. In the
dorsal view of the cranium, 40H was the most differentiated, with 40ST its closest group. 40ST was
joined to the group composed of 28–30, 31–33, 34–37
and 38–39. MST on the ventral view of the cranium
showed the distance of 40ST from the remaining
groups, and showed 40H as nearest to 38–39. For
the mandible, 40ST was the most differentiated
group, and the closest group to 40H was 38–39. The
group 38–39 was linked to the set 28–30, 31–33 and
34–37. The deformation grids showed that the shape
changes had a similar pattern to the SHH analysis
(Fig. 6).
QUANTIFICATION
OF CO-VARIATION IN THE MANDIBLE
The degree of co-variation between the landmark
subsets of the alveolar region and ascending ramus
was low in the populations that were close to the centre
of the Rb polymorphism area and increased according
to the distance to the centre: group 1, rT = 0.144,
P < 0.001; group 2, rT = 0.254, P < 0.001; group 3,
rT = 0.276, P < 0.001; group 4, rT = 0.341, P < 0.001.
DISCUSSION
AGE
AND SEXUAL DIMORPHISM IN SIZE AND SHAPE
In the study area, males and females did not differ
significantly with regard to the cranium, mandible
and scapula. These results are concordant with those
reported in other Rb hybrid zones, obtained by either
geometric morphometrics on both the cranium and
mandible (Auffray, Alibert & Latieule, 1996; Corti &
Rohlf, 2001), or traditional morphometrics on the
mandible (Hauffe et al., 2002). Significant differences
© 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 97, 555–570
MORPHOLOGICAL VARIABILITY IN ROBERTSONIAN MICE
565
Figure 5. Canonical variate analysis (CVA) of W⬘ for dorsal and ventral views of cranium, lingual side of mandible and
dorsal aspect of scapula between 2n groups. Graphs show the mean canonical punctuation per group for the first two
canonical vectors (CV1 vs. CV2). Minimum-length spanning trees (MSTs) are represented by broken lines. Distances
between neighbour groups are indicated.
between subadults and adults were focused on the CS
of all osseous structures analysed and not on their
shape. This shows that specimens soon reached their
adult shape, in accordance with Davis (1983), who
indicated that little shape variation occurs after 2
months from birth.
SIZE
AND SHAPE ANALYSIS BETWEEN
CHROMOSOMAL GROUPS
Contrary to other studies in Rb areas, no size differences in any osseous structure related to geography or
karyotype were found. In a previous study on the
skull of mice Rb populations from the Rhaetic Alps,
differences in CS were detected between chromosomal
races of adult mice (Corti & Rohlf, 2001), showing
that standard mice have larger skulls than animals
with Rb fusions (2n = 22 and 2n = 26). This discordance between results could be due to differences in
the metacentric composition between the two studied
Rb areas.
Shape differentiation was found for localities and
chromosomal groups. It was difficult to unravel the
geography, SHH and diploid number effects, because of
the intrinsic structure of the Rb polymorphism area of
Barcelona. Morphological differentiation among localities was not caused by geographical isolation by dis-
tance, that is, animals that were from distant localities
were not more differentiated than animals that were
from closer localities. Therefore, the morphological
differentiation found between sites could be the result
of karyotype differentiation, local geographical isolation or a combination of both factors. As all mice
analysed were from commensal populations and
shared the same habitat, the differences found across
this area cannot be attributed to adaptation to different environments. Moreover, all the localities are
under Mediterranean climate conditions.
The significant discrimination obtained by CVAs in
SHH and 2n analyses, and the significant Mahalanobis distances between chromosomal groups, suggest
that both diploid number and structural heterozygosity contribute to the morphometric differentiation. In
all structures analysed, 2n analysis showed better
discrimination than SHH analysis. Therefore, it can
be assumed that the reduction in 2n is a more important factor for shape variation than SSH. In the
locality analysis, 2n and heterozygosity were implicit
factors. This explains the highest a posteriori mean
probability membership obtained for this analysis.
Nevertheless, we cannot rule out an effect of local
geographical isolation.
CVA combined with MST, for SSH and 2n analysis,
showed a clear morphological differentiation of all-
© 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 97, 555–570
566
M. A. SANS-FUENTES ET AL.
Figure 6. Deformation grids belonging to negative and positive extremes of the first canonical vector (CV1) for each bone
structure for 2n analysis (see Fig. 5). Shape changes have been augmented three times to emphasize differences.
acrocentric mice with regard to Rb specimens in most
of the osseous structures. For the ventral view of the
cranium and mandible, the most differentiated group
was 40ST. These results are concordant with those on
the variability of the epigenetic characters of the skull
in the same study area, revealing a clear separation
between standard and Rb mice (Muñoz-Muñoz et al.,
2003). All this indicates a reduction in gene flow
between standard and Rb mice, including 40H
animals. Reduced fertility, low fitness of structurally
heterozygous mice in Rb hybrid zones, and a reduction in recombination derived from the presence of
Rb translocations have been considered as important
barriers to gene flow between populations differing in
karyotype (Davisson & Akeson, 1993; Searle, 1993;
Hauffe & Searle, 1998). Moreover, fertility studies
performed on Rb males of the study area showed a
reduction in the spermatid/spermatocyte rate (M. A.
Sans-Fuentes, unpubl. data). Therefore, these factors
could play an important role in the morphological
differentiation of Rb mice.
With regard to 2n analysis, the general pattern
showed that animals with low 2n (28–37) were
similar in shape. With the exception of the dorsal
view of the cranium, the closest group to 38–39 was
40H. Moreover, Mahalanobis distances between 40H
and 40ST were higher than between 40H and 38–39
(data not shown). This suggests that 40H mice
originate from mating between heterozygous mice
with high 2n, that is with 2n = 38–39, rather than
from 40ST, and supports the distinction made here
between 40ST and 40H. The general pattern for SHH
analysis showed that HH, HTI, HTII and HTIII had
similar shapes. Nevertheless, relationships of 40H
mice with the other groups varied according to the
structure analysed.
The patterns obtained by CVA and MST were not
completely coincident for all of the analysed structures. This can be attributed to an alteration of
the morphological integration in some chromosomal
groups. It is well known that the cranium, mandible
and scapula are highly modular and highly integrated
structures (Klingenberg, Leamy & Cheverud, 2004;
Young, 2004; Hallgrímsson et al., 2007). Although
these modules are independent as a result of their
distinct embryonic origins, different processes of development and diverse functional roles, they interact
considerably during development, generating a highly
© 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 97, 555–570
MORPHOLOGICAL VARIABILITY IN ROBERTSONIAN MICE
integrated whole. Thus, alterations in the patterns of
integration can imply that different modules evolve in
diverse directions. The skull, for example, comprises
three partially independent units that behave as
modules – the basicranium, the neurocranium and the
face – and that may interact unequally via epigenetic
interactions (for a review, see Hallgrímsson et al.,
2007). The different CV ordinations found for the
dorsal and ventral views of the cranium correspond to
differences in the shape of the rostrum. The deformation grids of the dorsal view of the cranium show that
the rostral region is not very different between Rb and
standard mice. Nevertheless, in the ventral view of the
cranium, greater changes in the rostrum can be
observed between groups, specifically in the incisive
foramen length, suggesting that the co-variation
between both views of the cranium is low for the rostral
region. This agrees with other studies which found
that this is the most variable region of the skull, and
has fewer and weaker partial correlations with aspects
of the neurocranium and basicranium (Howells, 1973;
Hallgrímsson et al., 2007). Different patterns of variation can also be observed for the posterior part of the
cranium. The shape of the neurocranium showed
differences between Rb and standard mice, but the
basicranium practically did not differ between these
groups. The variation in shape observed in the mandible resembled the variation found in the ventral view
of the cranium. The mandible is part of the face, but
any change in the shape of this structure is constrained by its feeding function, which is shared by the
facial part of the ventral view of the cranium. Although
the origin and function are completely different
(Goodrich, 1986), changes in shape followed the same
general pattern as observed in the other structures
analysed, with the 2n = 40 animals being the most
differentiated.
In spite of differences detected in the CV ordination
patterns between chromosomal groups among structures, the general trend was a noticeable separation
of 40-chromosome mice with respect to Rb groups.
This suggests that the morphological differentiation
between groups is mainly driven by genetic variability underlined by the presence of Rb fusions. Although
this result is similar to that found in other M. m.
domesticus Rb systems (Thorpe, Corti & Capanna,
1982; Corti & Thorpe, 1989; Nance et al., 1990;
Auffray et al., 1996; Fel-Clair et al., 1996; Saïd et al.,
1999; Corti & Rohlf, 2001; Hauffe et al., 2002), it is
difficult to identify a common evolutionary pattern for
all Rb hybrid zones of M. m. domesticus. This is
because other variables, such as metacentric composition, local geographical isolation, and historical, ecological, and anthropogenetic factors, also play an
important role in the morphological differentiation
between Rb populations.
CO-VARIATION
567
ANALYSIS
Endler (1977) argued that both secondary contact
and primary intergradation produce non-differentiated patterns, unless a hybrid zone can be observed
in the first 100 generations since the secondary
contact was produced. Nevertheless, the characteristics of the Rb polymorphism zone of Barcelona
support the hypothesis that it is the product of a
primary intergradation. Thus, this area is geographically widespread (5000 km2), staggered clines for the
Rb chromosome have been described, supporting the
hypothesis of a sequential formation of Rb translocations (Gündüz et al., 2001) and, until now, no chromosomal race (as defined by Hausser et al., 1994) has
been found. Moreover, the results obtained in the
present study regarding co-variation between different integrative units of the mandible reinforce this
hypothesis. The co-variation between mandible functional units can be estimated by analysing either the
co-variation of fluctuating asymmetry or the variation of co-variation among individuals. The former
provides information about co-variation caused by
direct developmental interaction, such as partitioning
of a precursor tissue or inductive signalling from one
tissue to an adjacent tissue. The variation among
individuals refers to co-variation from both direct
developmental interaction and parallel variation in
separate developmental pathways as, for example,
from allelic variation in a gene that has a function in
both pathways (for details, see Klingenberg et al.,
2003). In the present study, our interest is in the
second type of co-variation, as we suggest that epistatic interactions between genes that participate in
the development of two modules can be disrupted as
a result of the presence of Rb fusions altering the
co-variation between the two modules.
The values of trace correlation between the
ascending ramus and alveolar region of the mandible, calculated for the population groups, show an
ascending trend correlated with the distance from
the centre of the polymorphism area of Barcelona.
The trace value for chromosomal group 4, which
includes only standard populations, is slightly lower
than the value obtained by Klingenberg et al. (2003)
for individual variation. This value decreases progressively in the populations that are closer to the
centre, especially in group 1, comprising individuals
in the localities closest to the centre. This pattern is
expected under the hypothesis of primary intergradation. It is probable that mice from populations
closer to the centre carry younger Rb fusions which
are less spread geographically. By contrast, mice
from populations further from the centre carry older
fusions, which have undergone more generations of
recombination and selection, allowing buffering of
© 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 97, 555–570
568
M. A. SANS-FUENTES ET AL.
the disruption of genetic co-adaptation that could
have appeared after hybridization with standard
populations.
Alternatively, the low trace value in group 1 may be
the effect of a larger number of Rb chromosomes in
the animals in this group. During the past decade,
accumulating evidence has supported the view that
nuclear architecture provides another level of epigenetic gene regulation (for example, Cremer & Cremer,
2001). It is expected that both a larger number of Rb
metacentrics and a higher heterozygosity can distort
the nuclear architecture and regulation gene pathways during development. Group 1 had the largest
number of Rb metacentrics and the highest mean
heterozygosity (mean 2n = 30–32; mean H = 1.528).
However, groups 2 and 3 did not differ substantially
in the trace values, but differed in the mean 2n
and mean heterozygosity (group 2: mean 2n = 30–36,
mean H = 1.4; group 3: mean 2n = 38–39, mean
H = 0.666). These results suggest that the number of
Rb chromosomes is not the main cause of the mandible co-variation detected.
In addition, the hypothesis of primary intergradation was supported by the results obtained from the
2n analysis for the mandible and scapula. These two
structures showed a trend of morphological differentiation correlated with the increase in 2n, which is in
accordance with gradual gene flow from the centre
of the Rb polymorphism area to the periphery, as
expected in a primary intergradation scenario.
Nevertheless, additional morphological studies on
these structures and others based on neutral genetic
markers are required to finally answer the question
about the origin of the Rb polymorphism zone of
Barcelona.
ACKNOWLEDGEMENTS
Special thanks go to Dr Paolo Colangelo for his suggestions on the manuscript and Hoshin Gupta for
the English correction. This study was supported by
a grant from the Spanish Ministerio de Ciencia y
Tecnología (BMC2000-0541), and by an FPI grant to
MASF from the Spanish Ministerio de Ciencia y
Tecnología.
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