RESEARCH ARTICLE
A comparative study of the recombination pattern
in three species of Platyrrhini monkeys (primates)
Raquel Garcia-Cruz & Sarai Pacheco & Miguel Angel Brieño & Eliana R. Steinberg &
Marta D. Mudry & Aurora Ruiz-Herrera & Montserrat Garcia-Caldés
Received: 1 March 2011 /Revised: 30 May 2011 /Accepted: 23 June 2011 /Published online: 7 July 2011
# Springer-Verlag 2011
Abstract Homologous chromosomes exchange genetic
information through recombination during meiotic synapsis,
a process that increases genetic diversity and is
fundamental to sexual reproduction. Meiotic studies in
mammalian species are scarce and mainly focused on
human and mouse. Here, the meiotic recombination events
were determined in three species of Platyrrhini monkeys
(Cebus libidinosus, Cebus nigritus and Alouatta caraya) by
analysing the distribution of MLH1 foci at the stage of
pachytene. Moreover, the combination of immunofluorescence
and fluorescent in situ hybridisation has enabled us to
construct recombination maps of primate chromosomes
that are homologous to human chromosomes 13 and 21.
Our results show that (a) the overall number of MLH1 foci
varies among all three species, (b) the presence of
heterochromatin blocks does not have a major influence
on the distribution of MLH1 foci and (c) the distribution of
crossovers in the homologous chromosomes to human
chromosomes 13 and 21 are conserved between species of
the same genus (C. libidinosus and C. nigritus) but are
significantly different between Cebus and Alouatta. This
heterogeneity in recombination behaviour among Ceboidea
species may reflect differences in genetic diversity and
genome composition.
Introduction
Meiosis is a specialised cell division by which haploid
gametes (containing a single copy of each chromosome) are
generated. In most eukaryotes, homologous chromosomes
exchange genetic information through recombination during
meiosis, a process that increases genetic diversity by
breaking haplotypes. Recombination provides physical
connections between homologues during the first meiotic
division, contributing to correct chromosome segregation.
Although recombination can occur at the somatic level
(such as recombination produced in the immune system),
only those recombination events occurring in the germ line
will have effects in genome diversity and composition.
Homologous recombination is triggered by programmed
double-strand breaks early in meiosis (Bishop and
Zickler 2004). Studies in the budding yeast have revealed
that the broken ends are processed, and approximately half
of them result in double Holliday junctions and crossovers
Communicated by: S. Keeney
A. Ruiz-Herrera and M. Garcia-Caldés contributed equally to this work.
R. Garcia-Cruz :S. Pacheco :M. A. Brieño :A. Ruiz-Herrera :
M. Garcia-Caldés
Departament de Biologia Cellular, Fisiologia i Immunologia,
Universitat Autònoma de Barcelona,
UAB Campus, Bellaterra,
08193 Barcelona, Spain
A. Ruiz-Herrera :M. Garcia-Caldés
Institut de Biotecnologia i Biomedicina (IBB),
Universitat Autònoma de Barcelona,
UAB Campus, Bellaterra,
08193 Barcelona, Spain
E. R. Steinberg :M. D. Mudry
Grupo de Investigación en Biología Evolutiva (GIBE), CONICET,
Departamento de Ecología, Genética y Evolución, FCEyN,
Universidad de Buenos Aires,
Buenos Aires, Argentina
A. Ruiz-Herrera (*) :M. Garcia-Caldés (*)
Departament de Biologia Cellular, Fisiologia i Immunologia,
Universitat Autònoma de Barcelona,
UAB Campus,
08193 Cerdanyola del Vallès, Spain
e-mail: aurora.ruizherrera@uab.cat
e-mail: Montserrat.Garcia.Caldes@uab.cat
Chromosoma (2011) 120:521–530
DOI 10.1007/s00412-011-0329-6
(COs) (Petes 2001). In this organism, COs exhibit three
main features: (a) they are not randomly distributed but
rather concentrated in hotspots, (b) there is always at least
one CO per pair of homologous chromosomes (so-called
obligatory chiasmata) to ensure the proper disjunction of
homologous chromosomes and (c) they present “interference”
(COs tend to follow an evenly spaced distribution)
(Petes 2001). Studies in mammals have shown, on the
other hand, that the total number and distribution of COs
in a specific chromosome depend on several factors, such
as chromosomal size, gender and age of the individual
under study (Lynn et al. 2004; Paigen and Petkov 2010).
Larger chromosomes tend to accumulate larger numbers of
COs, and each chromosome arm generally presents at least
one CO (Sun et al. 2004). Early studies (Dutrillaux 1986)
demonstrated a strong correlation between the number of
chiasmata and the haploid number of chromosome arms.
Following this view, Pardo-Manuel de Villena and Sapienza
(2001) suggested that the recombination rate was proportional
to the number of chromosome arms, after analysing a
wide range of mammalian species. Such assumptions are of
relevance given that perturbations in the recombination
pattern have been shown to be linked to non-disjunction
problems in humans (Lamb et al. 2005).
Recombination maps elaborated using LD analyses or/
and sperm typing are now available for a variety of species
(Frazer et al. 2007; Megens et al. 2009; Qanbari et al. 2010;
Rogers et al. 2006; Wu et al. 2010). While LD analyses
represent indirect evidence of the recombination process,
sperm typing directly detects recombinant DNA molecules.
LD-based maps estimate the location of recombination
events in the progeny (see Lynn et al. 2004 for a review)
and represent an integration of population-level processes
over several generations (Clark et al. 2010). However, if our
intention is to analyse the recombination process as it
occurs, alternative methods to genetic linkage analysis
should be used. This is the case of the in situ
immunolocalisation of recombination proteins on spermatocyte
cells (Lynn et al. 2002), which allows for the
recognition of physical COs during the first meiotic
prophase in each cell individually by analysing the
distribution of MLH1 foci, a marker of CO events (Baker
et al. 1996). Recombination maps using this last direct
approach have been made in humans, both female (Robles
et al. 2009; Tease et al. 2002; Lenzi et al. 2005) and male
(Barlow and Hulten 1998; Codina-Pascual et al. 2006;
Ferguson et al. 2009; Hassold et al. 2004; Lynn et al. 2002;
Sun et al. 2004), mouse (Anderson et al. 1999; Froenicke
et al. 2002) and, more recently, in birds (Pigozzi 2007;
2008; Pigozzi and Solari 2005), marsupials (Franco et al.
2007), dogs (Basheva et al. 2008), the American mink
(Borodin et al. 2009) and the common shrew (Borodin et
al. 2008a,b) as well as several rodent species (Dumont and
Payseur 2011). However, meiotic studies in non-human
primates are scarce and are based on classical techniques,
such as Giemsa and silver staining (Armada et al. 1987;
Lima and Seuanez 1991; Mudry et al. 1998, 2001; Rahn et
al. 1996; Seuánez et al. 1983; Steinberg et al. 2007, 2008).
Only until very recently has in situ immunolocalisation of
recombination proteins been applied in non-human primates
(Garcia-Cruz et al. 2009; Hassold et al. 2009).
Therefore, experimental descriptions of recombination
maps in other mammalian species are needed if the
ultimate goal is to understand the mechanisms underlying
chromosomal evolution and speciation.
In an attempt to shed light on the dynamics of
mammalian meiosis and its implication in chromosome
evolution, we have analysed the recombination features
during meiosis in three Ceboidea species: Cebus libidinosus
[formerly C. paraguayanus (Groves 2001)], Cebus
nigritus and Alouatta caraya (Platyrrhini, Primates).
Immunoflorescence against different meiotic proteins
involved in the synaptonemal complex (SC) formation
and recombination were performed on testicular material
of these species. Moreover, the application of fluorescence
in situ hybridisation with the 13 and 21 human wholechromosome
painting probes in spermatocytes of all three
species allowed for the construction of the recombination
map of these specific chromosomes from an evolutionary
perspective, since their chromosomal homologies are
known (de Oliveira et al. 2002; Garcia et al. 2000, 2002;
Stanyon et al. 2011). Using this approach, we were able to
investigate whether the cytological distribution of COs in
homologous chromosomes is conserved among these
species and whether the presence of extracentromeric/
interstitial heterochromatin regions influences the location
of COs.
Materials and methods
Sample processing
One male individual from each species was included in this
study, all of them born in the wild within the southern-most
natural geographic distribution. The adult specimen of A.
caraya (ACA) was 15 years old and was kept in captivity at
the Centro de Reeducación del Mono Aullador Negro
(Córdoba, Argentina), where it has sired nine offspring. The
typical lifespan of Alouatta has been recorded to be
approximately 20 years (Chebez and Ruiz Blanco 2008).
Both C. libidinosus (CLI) and C. nigritus (CNI) adult
specimens were kept in captivity at the Córdoba Zoo
(Córdoba, Argentina). The CLI specimen was 10 years old
and has sired one offspring, whereas the CNI specimen was
7 years old and with unrecorded offspring. The lifespan of
522 Chromosoma (2011) 120:521–530
Cebus can range from 15 to 25 years, depending on the
species (Canevari and Fernández Balboa 2003; Fedigan and
Jack 2004).
Testicular biopsies were obtained from each male according
to the protocols of the “Guide for Care and Use of Experimental
Animals”, as promulgated by the Canadian Council of Animal
Care and the Principles for the Ethical Treatment of non-human
Primates of the American Society of Primatologists (ASP).
Biopsies were taken by the veterinarian Miguel Carelo
(Córdoba Zoo) after administration of a dose of anaesthetics
(Ketamine, Holliday Scott; Argentina; 10 mg kg−1
day−1
) and
transferred to GIBE (FCEyN, UBA; Buenos Aires), where
they were kept at −80°C until use. Testicular tissues were
processed as previously described (Garcia-Cruz et al. 2009) in
order to obtain spermatocyte spreads.
Immunostaining
Immunostaining of meiocytes was performed following
Garcia-Cruz et al. (2009). Different sets of antibodies were
used: rabbit poyclonal serum against central element
protein SYCP3 (a kind gift of C. Heyting), mouse
monoclonal antibody against MLH1 (BD Pharmingen)
and human calcinosis, Raynaud’s phenomenon, oesophageal
dysfunction, sclerodactyly and telangiectasia (CREST)
serum (a kind gift of M. Fritzler). Fluorochrome-conjugated
secondary antibodies were used for detection (all from
Jackson ImmunoResearch Laboratories). Primary and secondary
antibodies were diluted in PBTG (0.2% bovine
serum albumin, 0.05% Tween, 0.2% gelatin in phosphatebuffered
saline). Primary antibodies were incubated overnight
at 4°C or at room temperature in a humid chamber.
Secondary antibodies were incubated for 1 h at 37°C in a
humid chamber. DNA was counterstained with antifade
solution (Vector Laboratories) containing 0.1 μg/ml 4′,6′diamidino-2-phenylindole
(Sigma).
Fluorescence “in situ” hybridisation
Whole-chromosome painting probes specific to human
(HSA) chromosomes 13 and 21 (Metasystems) were hybridised
on previously immunostained preparations to identify
SCs of homologous chromosomes in ACA (chromosomes 20
and 21, respectively) and CLI/CNI (chromosomes 17 and 11,
respectively). A first denaturation of the slides was performed
in 70% formamide in a 2× saline sodium citrate (SSC)
solution for 5 min at 70°C. The slides were washed in water,
and treatment with NaSCN 1 M for 3 h at 65°C was applied. A
second denaturation step was then performed as stated above.
Dehydration of the slides was carried out before applying the
denatured probe. Hybridisation was performed at 37°C in a
humid chamber for 72 h. Post-hybridisation washes were
performed in 4× SSC.
Data processing
Preparations were visualised using a Nikon eclipse 90i
epifluorescence microscope equipped with the appropriate
filters and a charged coupled device camera. Images were
captured and produced by Isis software (Metasystems).
The Micromeasure 3.3 computer application (Reeves
2001) was used for the analysis of MLH1 foci positions
and for the measurement of the length of the heterochromatin
blocks in CLI and CNI chromosomes. For each SC
analysed, the position of each MLH1 focus was recorded as
a relative position (percentage of total length) from the
centromere, identified by the CREST signal in each
preparation. To convert the MLH1 foci to genetic distances,
the number of MLH1 detected per SC was multiplied by 50
map units (1 crossover=50 cM). Additionally, the Kolmogorov–Smirnov
test (KS test) was used to determine whether
the distribution of MLH1 foci differs significantly among
species by constructing plots of cumulative frequency.
Results and discussion
In this report, the recombination maps of three primate
species (C. libidinosus, C. nigritus and A. caraya) are
described by analysing the number and distribution of
MLH1 foci in the SCs of pachytene spermatocytes. The
Cebus genus belongs to the Cebidae, and it has been
regarded as retaining the putative ancestral karyotype of all
Platyrrhini species (Clemente et al. 1990; Couturier and
Dutrillaux 1981; Garcia et al. 2002; Neusser et al. 2001).
Within this genus, C. libidinosus (CLI) and C. nigritus
(CNI) share virtually the same karyotype (2n=54, aFN=
72), with differences mainly due to variation in size of noncentromeric
heterochromatic regions (Mudry 1990; Nieves
et al. 2010; Ponsà et al. 1995), specifically regarding
chromosomes 17 and 11. A. caraya, on the other hand, is a
member of the Atelidae family, and it is currently
considered that its karyotype (2n=52, aFN=66) has
suffered multiple reorganisations since its divergence from
the Platyrrhini common ancestor (de Oliveira et al. 2002;
Stanyon et al. 2011).
In a first experiment, the number of MLH1 foci was
scored in a total of 366 cells, 116 in CNI, 85 in CLI and
165 in ACA (Fig. 1). During meiosis, the X and Y
chromosomes in Cebus spermatocytes form a sex body
with a human-like morphology (Seuánez et al. 1983). A.
caraya, on the other hand, presents a complex sexchromosome
system, which forms a convoluted sex body
involving four chromosomes (Mudry et al. 1998, 2001;
Rahn et al. 1996), which folded back onto itself, not
allowing for a correct visualisation of the MLH1 foci
(Fig. 2a). Thus, given the complexity of the morphology
Chromosoma (2011) 120:521–530 523
adopted by the sexual complement in A. caraya spermatocytes,
the SCs involved in the sexual complement were not
included in the present analysis, and MLH1 counts
correspond to autosomal SCs in all three species. The
mean MLH1 foci number per autosomal set was found to
be significantly different among species (χ2
=12.92, p=
0.002): 41.3±4.8 (SD) for CLI, 39.2±3.3 (SD) for CNI and
40.6±4.3 (SD) for ACA, with a range of 31–52, 31–46 and
Fig. 1 Quantification of the
numbers of MLH1 foci detected
per cell in all three Platyrrhini
species analysed. The horizontal
bars indicate the means. CLI C.
libidinosus, CNI C. nigritus,
ACA A. caraya
Fig. 2 Immunolocalisation of
meiotic recombination events.
a, b Sequential image of a
spermatocyte from ACA depicting
a triple immunostaining with
SYCP3 (red), MLH1 (green)
and CREST (blue) and b fluorescent
in situ hybridisation
(FISH) with the paintings of
human chromosomes 13 (green)
and 21 (red). Note how the
sexual complement in Alouatta
caraya spermatocytes is folded
back onto itself, not allowing for
a correct visualisation of the
MLH1 foci. c, d Selected chromosomes
that are homologous
to c HSA13 and d HSA21 in
ACA, CLI and CNI spermatocytes,
as identified by sequential
immunostaining (SYCP3 in red,
MLH1 in green and CREST in
blue) and FISH. Human chromosome
13 is homologous to
ACA20, CLI17 and CNI17,
whereas human chromosome 21
is homologous to ACA21,
CLI11 and CNI11. Note the
identification of the heterochromatin
blocks (Het) in CLI and
CNI chromosomes by the
absence of a hybridisation signal
with the commercial painting
probes
524 Chromosoma (2011) 120:521–530
31–50 MLH1 foci per cell, respectively (Fig. 1). It is well
known that recombination rates present inter- and intraspecific
variation, although the factor(s) affecting these
differences remain elusive (Paigen and Petkov 2010).
MLH1 recombination studies similar to the present work
performed in humans, macaques and rodents are an
example of such diversity (Hassold et al. 2009; Lynn et
al. 2002; Sun et al. 2004; Dumont and Payseur 2011). The
present data correspond to one representative specimen of
each species, and some caution has to be taken when
generalising to the whole species. Nevertheless, and
assuming that one MLH1 focus can be translated into 50
centimorgans (cM) in genetic length, these recombination
events would correspond to autosomal genetic lengths of
2,065 cM for CLI, 1,960 cM for CNI and 2,030 cM for
ACA. The values of genetic lengths in the Cebidae and
Atelidae species analysed in the present study and in a
previous study of our group (Garcia-Cruz et al. 2009) are
considerably lower than the 2,500 cM (corresponding to 50
MLH1 foci/cell) estimated for autosomal chromosomes in
human males by cytological studies (Sun et al. 2004). A
similar reduction in the recombination rate, with respect to
human, has been found in the rhesus macaque (1,950 cM)
(Hassold et al. 2009).
In order to explain the differences found among species,
the effect of chromosomal number variation on recombination
was explored, given that a strong correlation between the
number of COs and the haploid number of chromosome arms
has been reported in the literature (Dutrillaux 1986; PardoManuel
de Villena and Sapienza 2001). Under this assumption,
we would expect Cebus species (aFN=72, 16 acrocentric,
eight submetacentric and two metacentric autosomal
chromosome pairs) to have a higher number of COs than A.
caraya (aFN=66, 15 acrocentric, seven submetacentric and
two metacentric autosomal chromosome pairs). Our results
of MLH1 foci scoring indicate that the minimum requirement
of one CO per chromosome is accomplished in all
three species, but the recombination rate does not seem
related to the number of chromosome arms. Therefore,
although our study is based on one specimen as a
representative of each species, there must be additional
elements, such as the karyotype evolution of each species
(the final outcome of diverse fusion/fission events and their
own genomic makeup), and not only its fundamental
chromosome number, that influence the recombination rate.
Once the cytological distribution of COs was established
for each primate species, the distribution of MLH1 foci on
specific chromosomes was studied by combining immunostaining
and fluorescence in situ hybridisation (FISH)
with whole-chromosome paints (Fig. 2a, b). Our efforts
were concentrated on those Platyrrhini chromosomes
homologous to human chromosomes 13 and 21, both
chromosomes frequently involved in human disorders
(mainly aneuploidies) and whose recombination and pairing
behaviour are well known in male humans (Codina-Pascual
et al. 2006; Ferguson et al. 2009). These chromosomes are
evolutionarily conserved as a whole entity among the three
Platyrrhini species, so their analysis can be used to assess
the influence of different genetic backgrounds on meiotic
recombination and vice versa. In addition, these chromosomes
present differences in size of non-centromeric
heterochromatic blocks between C. libidinosus and C.
nigritus homologous pairs, but not in A. caraya, where
these heterochromatin blocks are absent (Fig. 2c, d). By this
way, the comparison of the recombination patterns among
homologous chromosomes in all three species was also
used to determine the possible effect of heterochromatin on
meiotic recombination. Human chromosome 13 (HSA13) is
homologous to an entire acrocentric chromosome in all
three Platyrrhini species, corresponding to CNI17, CLI17
and ACA20 (de Oliveira et al. 2002; Garcia et al. 2000).
Fig. 3 Comparison of the percentage of cells with a different number
of MLH1 foci in conserved chromosomes. Data from the homologous
human counterparts have been extracted from Codina-Pascual et al.
(2006). HSA Homo sapiens, CLI C. libidinosus, CNI C. nigritus,
ACA A. caraya
Chromosoma (2011) 120:521–530 525
Additionally, the homologous chromosomes in both Cebus
species present differences in the size of a terminal
heterochromatin block, which can be confirmed by the
absence of hybridisation signal in Cebus preparations
(Fig. 2c). Likewise, the chromosomes homologous to
HSA21, corresponding to CNI11, CLI11 and ACA21, are
acrocentric in all three species, but associated with a
fragment of HSA3 (de Oliveira et al. 2002; Garcia et al.
2000), a conservation of chromosomal synteny (3/21)
considered to be ancestral for all eutherian mammals
(Robinson and Ruiz-Herrera 2008). C. libidinosus presents
a distal or terminal heterochromatin block in chromosome
11, which takes up to 75% of the total chromosome length,
while this heterochromatic region is absent in C. nigritus
and A. caraya (Fig. 2d).
A total of 403 cells was analysed, 99 in CNI, 107 in CLI
and 197 in ACA, with sequential immunofluorescence and
FISH. The analysis of MLH1 foci for these specific
chromosomes in all three species revealed that chromosomes
homologous to HSA13 usually contain two MLH1
foci (mean number of MLH1 foci per SC is 1.85 for CLI,
1.70 for CNI and 1.71 for ACA), while those chromosomes
homologous to HSA21 present one MLH1 focus (mean
number of MLH1 foci per SC is 1.12 for CLI, 1.02 for CNI
and 1.04 for ACA) (Fig. 3). Chromosomes with nonexchanges
were either not detected or were very rare.
Interestingly, MLH1 frequencies in human chromosomes
13 and 21 (Codina-Pascual et al. 2006; Sun et al. 2004) are
similar to those reported here for their CLI, CNI and ACA
counterparts (Fig. 3). Thus, the overall reduction in the
genetic length found in the spermatocytes of these Platyrrhini
species, with respect to human spermatocytes, could
not be attributed to a general reduction of the recombination
rate in all chromosomes.
After the total number of MLH1 foci per chromosome
was determined for each chromosome homologous to
HSA13 and HSA21, their relative positions along the SC
were analysed (Fig. 4, left panel). Using the centromere as a
reference, the positions of each MLH1 focus were
calculated along the SC, from the centromere to the
Fig. 4 Chromosomal distributions of recombination events. Left
panel Histogrammes showing the distribution of MLH1 foci in
chromosomes, which are homologous to HSA13 and HSA21 in all
three Platyrrhini species (ACA, CLI and CNI). The X-axis represents
the length of the synaptonemal complex (SC) from the centromere to
the telomere, while the Y-axis shows the percentage of total MLH1
foci. Each chromosome is divided into 5% intervals of SC length.
Note that SC length refers to the SC length of the euchromatic region.
Arrows in the X-axis show the position of heterochromatin blocks
(Het) in chromosomes homologous to HSA13 (in CLI and CNI) and
HSA21 (in CLI). Right panel Male recombination maps for human
chromosomes 13 and 21 based on SNP data (data extracted from
Kong et al. 2010). The X-axis represents the length of each human
chromosome expressed in base pairs (bp), whereas the Y-axis indicates
the standard recombination rate
526 Chromosoma (2011) 120:521–530
telomere. In those chromosomes with non-centromeric
heterochromatin blocks (CLI11, CLI17 and CNI17),
MLH1 foci were always found to be located within the
euchromatic regions. Thus, for comparison, the MLH1
position was expressed as the relative position of each focus
in the euchromatin of the chromosome. For each chromosome,
the length of the SC has been divided into 5%
intervals. The general pattern of MLH1 distribution in these
chromosomes varies among species depending on the
chromosome studied (Fig. 4). The recombination pattern
in primate chromosomes homologous to HSA13 shows the
effect of interference, which leads to the highest rates of
recombination in two separate regions, the first one
proximal to the centromere, with the other one being more
terminal (Fig. 4). It is important to note, however, that in
the case of both Cebus species, a small percentage of COs
is also localised in the central region of the SC. This
distribution shows similar features with the male recombination
map of human chromosome 13 inferred from
genome-wide single-nucleotide polymorphism (SNP) data
at a resolution of 10 kilobases (kb) (Kong et al. 2010) (right
panel in Fig. 4). Although SNP data and MLH1 foci
represent different levels of resolution and units of
measurement (axis length for the cytological measurements
vs. DNA length for the genetic analysis), similar patterns
can be appreciated. In the case of chromosomes homologous
to HSA21, COs are more evenly distributed in all
instances, although differences among species were found.
In both Cebus species, COs are distributed along the whole
length of the SCs, whereas in ACA, the MLH1 foci
concentrate in the terminal region of the chromosome.
These differences among species are more evident when
plotting the data into cumulative frequency graphs (Fig. 5).
This type of representation allows for the statistical
estimation of similitudes between MLH1 distributions
among homologous chromosomes of the three species.
The analysis revealed that the distribution of MLH1 in
chromosomes homologous to HSA13 and HSA21 was
similar between both Cebus, regardless of whether chromosomes
contain a heterochromatin block in both species
(p=0.727 for chromosomes homologous to HSA13,
Kolmogrov–Smirnov) or not (p=0.953 for chromosomes
homologous to HSA21, Kolmogrov–Smirnov). However,
the distribution of MLH1 in these chromosomes differs
between both Cebus species and ACA (p<0.01 for both
chromosomes, Kolmogrov–Smirnov). In chromosomes
homologous to HSA13, which normally contain two
MLH1 foci in all three species, the first focus tends to
appear closer to the centromere while the second focus
tends to appear slightly closer to the telomere in ACA,
when compared to Cebus. Nevertheless, differences in
MLH1 distribution between the species are more evident
in chromosomes homologous to HSA21. In that chromosome,
which normally contains one MLH1 focus,
exchanges are displaced towards distal positions in ACA,
when compared to the homologous chromosome in Cebus.
In any case, our results indicate that recombination is
suppressed at the centromeric region in all species (Fig. 5),
mirroring results previously obtained in human (Ferguson
et al. 2009) and mouse (Froenicke et al. 2002).
From our data, it is also inferred that the presence or
absence of the interstitial or terminal heterochromatin
blocks does not have a major influence on the distribution
of MLH1 foci in euchromatic region of homologous
chromosomes in either Cebus species. Heterochromatin
shows a particular behaviour during meiotic pairing and
synapsis. In humans, Codina-Pascual et al. (2006) noted
that non-centromeric heterochromatin regions are the last
regions to synapse and, when unsynapsed, gaps and splits
are observed as in the SCs, showing differences in timing
along synapsis, depending on the chromosomes analysed.
Mirroring these results, a previous study from our research
group in another specimen of C. libidinosus (=C. paraFig.
5 Cumulative frequency plots in conserved chromosomes in the
Platyrrhini species. Note that SC length refers to the SC length of the
euchromatic region. CLI C. libidinosus, CNI C. nigritus, ACA A.
caraya. The inset in each plot represents the G-banding pattern for
each of the chromosomes analysed. Arrows in the X-axis show the
position of heterochromatin blocks (Het) in chromosomes homologous
to HSA13 (in CLI and CNI) and HSA21 (in CLI)
Chromosoma (2011) 120:521–530 527
guayanus) (Garcia-Cruz et al. 2009) showed how this
pattern of delayed pairing and synapsis is also conserved in
non-human primates. Despite this particular behaviour,
heterochromatic regions do not interfere with the general
progress of pairing and synapsis (Garcia-Cruz et al. 2009)
nor the recombination process itself (present data).
The relative distribution of MLH1 foci in homologous
chromosomes does not show significant differences in the
same genus, regardless of the presence or absence of
heterochromatin blocks, while it differs in homologous
chromosomes from different families. These differences
among species may be caused by the intrinsic genetic
characteristics of each chromosome as a result of their
different evolutionary histories since their common divergence.
Atelidae and Cebidae Platyrrhini families are
thought to have diverged approximately 20 million years
ago (Opazo et al. 2006), enough time to accumulate genetic
divergences and chromosomal reorganisations. The conserved
karyotype in both Cebus species may explain the
similarities in their CO distribution when analysing specific
chromosomes. On the other hand, it is currently considered
that the karyotype of A. caraya has suffered multiple
chromosomal rearrangements since its divergence from the
Platyrrhini common ancestor, and its genomic landscape in
terms of chromosomal homologies is different from its
Cebidae relatives. Indeed, the fact that recombination
hotspots are not conserved among primate species has been
described in previous studies based on linkage disequilibrium
analysis in human and chimpanzees (Paigen and
Petkov 2010; Ptak et al. 2005). Although both species share
99% of genetic similarities, their karyotypes differ in
chromosome number due to several inter- and intrachromosomal
reorganisations (Clemente et al. 1990; Feuk
et al. 2005; Yunis and Prakash 1982). In light of our data,
the recombination rate as well as the number of COs per
individual chromosome might be influenced by the chromosomal
reorganisations that are shaping the genomic
evolutionary landscape of each species. Further analysis
including other primate species would be of great interest to
deepen our knowledge of the influence of meiotic recombination
in chromosomal evolution and vice versa.
Acknowledgments We are grateful to Dr. C. Heyting and Dr. M.
Fritzler for their generous gifts of antibodies. We thank L.Uroz, G.
Elkorchi and J. del Rey for technical support. Ignasi Roig is
acknowledged for comments on an earlier version of this manuscript
and Mariela Nieves for her suggestions on the last draft. We are also
thankful to the editor and two anonymous reviewers whose comments
and suggestions improved the manuscript. All research reported in this
manuscript has met the appropriate national and institutional guidelines
for the legal acquisition and use of laboratory animals and
authorised study of wild animals. The authors also adhered to the
Guide for Care and Use of Experimental Animals, as promulgated by
the Canadian Council of Animal Care, and to the American Society of
Primatologists (ASP) Principles for the Ethical Treatment of Nonhuman
Primates. The work, which took place in Argentina, was done
in accordance with the laws of Argentina. We have received funds
from PCI2005-A7-O428, MDM PIP 5012, UBACyTX107, PRP2006-
02, BFU2006-12951, 2009SGR1107 and CGL2010-20170. The
English of this manuscript has been proofread by a native, Englishspeaking
University Instructor of English.
References
Anderson LK, Reeves A, Webb LM, Ashley T (1999) Distribution of
crossing over on mouse synaptonemal complexes using immunofluorescent
localization of MLH1 protein. Genetics 151:1569–
1579
Armada JL, Barroso C, Lima M, Muniz J, Seuanez HN (1987)
Chromosome studies in Alouatta belzebul. Am J Primatol
13:283–296
Baker SM, Plug AW, Prolla TA et al (1996) Involvement of mouse
Mlh1 in DNA mismatch repair and meiotic crossing over. Nat
Genet 13:336–342
Barlow AL, Hulten MA (1998) Crossing over analysis at pachytene in
man. Eur J Hum Genet 6:350–358
Basheva EA, Bidau CJ, Borodin PM (2008) General pattern of meiotic
recombination in male dogs estimated by MLH1 and RAD51
immunolocalization. Chromosome Res 16:709–719
Bishop DK, Zickler D (2004) Early decision; meiotic crossover
interference prior to stable strand exchange and synapsis. Cell
117:9–15
Borodin PM, Karamysheva TV, Belonogova NM, Torgasheva AA,
Rubtsov NB, Searle JB (2008a) Recombination map of the
common shrew, Sorex araneus (Eulipotyphla, Mammalia).
Genetics 178:621–632
Borodin PM, Karamysheva TV, Rubtsov NB (2008b) Immunofluorescent
analysis of meiotic recombination and interference in the
domestic cat. Tsitologiia 50:62–66
Borodin PM, Basheva EA, Zhelezova AI (2009) Immunocytological
analysis of meiotic recombination in the American mink
(Mustela vison). Anim Genet 40:235–238
Canevari M, Fernández Balboa C (2003) 100 mamíferos argentinos.
Ed Albatros, Buenos Aires
Chebez J, Ruiz Blanco M (2008) Carayá-pitá. In: Chebez JC. Los que
se van. Fauna argentina amenazada, vol. 3. Albatros, Buenos
Aires, pp 64–69
Clark AG, Wang X, Matise T (2010) Contrasting methods of
quantifying fine structure of human recombination. Annu Rev
Genomics Hum Genet 11:45–64
Clemente IC, Ponsa M, Garcia M, Egozcue J (1990) Evolution of the
Simiiformes and the phylogeny of human chromosomes. Hum
Genet 84:493–506
Codina-Pascual M, Campillo M, Kraus J, Speicher MR, Egozcue J,
Navarro J, Benet J (2006) Crossover frequency and synaptonemal
complex length: their variability and effects on human male
meiosis. Mol Hum Reprod 12:123–133
Couturier J, Dutrillaux B (1981) Conservation of replication chronology
of homologous chromosome bands between four species of
the genus Cebus and man. Cytogenet Cell Genet 29:233–240
de Oliveira EH, Neusser M, Figueiredo WB et al (2002) The
phylogeny of howler monkeys (Alouatta, Platyrrhini): reconstruction
by multicolor cross-species chromosome painting.
Chromosome Res 10:669–683
Dumont BL, Payseur BA (2011) Evolution of the genomic recombination
rate in murid rodents. Genetics 187:643–657
Dutrillaux B (1986) Role of chromosomes in evolution: a new
interpretation. Ann Genet 29:69–75
528 Chromosoma (2011) 120:521–530
Fedigan L, Jack K (2004) The demographic and reproductive context
of male replacements in Cebus capucinus. Behaviour 141:755–
775
Ferguson KA, Leung S, Jiang D, Ma S (2009) Distribution of MLH1
foci and inter-focal distances in spermatocytes of infertile men.
Hum Reprod 24:1313–1321
Feuk L, MacDonald JR, Tang T et al (2005) Discovery of human
inversion polymorphisms by comparative analysis of human
and chimpanzee DNA sequence assemblies. PLoS Genet 1:
e56
Franco MJ, Sciurano RB, Solari AJ (2007) Protein immunolocalization
supports the presence of identical mechanisms of XY body
formation in eutherians and marsupials. Chromosome Res
15:815–824
Frazer KA, Ballinger DG, Cox DR et al (2007) A second generation
human haplotype map of over 3.1 million SNPs. Nature
449:851–861
Froenicke L, Anderson LK, Wienberg J, Ashley T (2002) Male mouse
recombination maps for each autosome identified by chromosome
painting. Am J Hum Genet 71:1353–1368
Garcia F, Nogues C, Ponsa M, Ruiz-Herrera A, Egozcue J, Garcia
Caldes M (2000) Chromosomal homologies between humans and
Cebus apella (Primates) revealed by ZOO-FISH. Mamm Genome
11:399–401
Garcia F, Ruiz-Herrera A, Egozcue J, Ponsa M, Garcia M (2002)
Chromosomal homologies between Cebus and Ateles (primates)
based on ZOO-FISH and G-banding comparisons. Am J Primatol
57:177–188
Garcia-Cruz R, Robles P, Steinberg ER, Camats N, Brieno MA,
Garcia-Caldes M, Mudry MD (2009) Pairing and recombination
features during meiosis in Cebus paraguayanus (Primates:
Platyrrhini). BMC Genet 10:25
Groves C (2001) Primate taxonomy. Smithsonian Institution Press,
Washington, p 350
Hassold T, Judis L, Chan ER, Schwartz S, Seftel A, Lynn A (2004)
Cytological studies of meiotic recombination in human males.
Cytogenet Genome Res 107:249–255
Hassold T, Hansen T, Hunt P, VandeVoort C (2009) Cytological studies
of recombination in rhesus males. Cytogenet Genome Res
124:132–138
Kong A, Thorleifsson G, Gudbjartsson DF et al (2010) Fine-scale
recombination rate differences between sexes, populations and
individuals. Nature 467:1099–1103
Lamb NE, Sherman SL, Hassold TJ (2005) Effect of meiotic
recombination on the production of aneuploid gametes in
humans. Cytogenet Genome Res 111:250–255
Lenzi ML, Smith J, Snowden T, Kim M, Fishel R, Poulos BK, Cohen
PE (2005) Extreme heterogeneity in the molecular events leading
to the establishment of chiasmata during meiosis i in human
oocytes. Am J Hum Genet 76:112–127
Lima MM, Seuanez HN (1991) Chromosome studies in the red howler
monkey, Alouatta seniculus stramineus (Platyrrhini, Primates):
description of an X1X2Y1Y2/X1X1X2X2 sex-chromosome
system and karyological comparisons with other subspecies.
Cytogenet Cell Genet 57:151–156
Lynn A, Koehler KE, Judis L et al (2002) Covariation of synaptonemal
complex length and mammalian meiotic exchange rates.
Science 296:2222–2225
Lynn A, Ashley T, Hassold T (2004) Variation in human meiotic
recombination. Annu Rev Genomics Hum Genet 5:317–349
Megens HJ, Crooijmans RP, Bastiaansen JW et al (2009) Comparison
of linkage disequilibrium and haplotype diversity on macro- and
microchromosomes in chicken. BMC Genet 10:86
Mudry MD (1990) Cytogenetic variability within and across populations
of Cebus apella in Argentina. Folia Primatol (Basel)
54:206–216
Mudry MD, Rahn M, Gorostiaga M, Hick A, Merani MS, Solari AJ
(1998) Revised karyotype of Alouatta caraya (Primates: Platyrrhini)
based on synaptonemal complex and banding analyses.
Hereditas 128:9–16
Mudry MD, Rahn IM, Solari AJ (2001) Meiosis and chromosome
painting of sex chromosome systems in Ceboidea. Am J Primatol
54:65–78
Neusser M, Stanyon R, Bigoni F, Wienberg J, Muller S (2001)
Molecular cytotaxonomy of New World monkeys (Platyrrhini)—
comparative analysis of five species by multi-color chromosome
painting gives evidence for a classification of Callimico goeldii
within the family of Callithrichidae. Cytogenet Cell Genet
94:206–215
Nieves M, Muhlmann M, Mudry MD (2010) Cebus paraguayanus
and Cebus nigritus (Primates, Platyrrhini): a comparative
genomic hybridization analysis. Cytogenet Genome Res
128:214–220
Opazo JC, Wildman DE, Prychitko T, Johnson RM, Goodman M
(2006) Phylogenetic relationships and divergence times among
New World monkeys (Platyrrhini, Primates). Mol Phylogenet
Evol 40:274–280
Paigen K, Petkov P (2010) Mammalian recombination hot spots:
properties, control and evolution. Nat Rev Genet 11:221–233
Pardo-Manuel de Villena F, Sapienza C (2001) Recombination is
proportional to the number of chromosome arms in mammals.
Mamm Genome 12:318–322
Petes TD (2001) Meiotic recombination hot spots and cold spots. Nat
Rev Genet 2:360–369
Pigozzi MI (2007) Localization of single-copy sequences on
chicken synaptonemal complex spreads using fluorescence in
situ hybridization (FISH). Cytogenet Genome Res 119:105–
112
Pigozzi MI (2008) Relationship between physical and genetic
distances along the zebra finch Z chromosome. Chromosome
Res 16:839–849
Pigozzi MI, Solari AJ (2005) The germ-line-restricted chromosome in
the zebra finch: recombination in females and elimination in
males. Chromosoma 114:403–409
Ponsà M, Garcia M, Borrell A et al (1995) Heterochromatin and
cytogenetic polymorphisms in Cebus apella (Cebidae, Platyrrhini).
Am J Primatol 37:325–331
Ptak SE, Hinds DA, Koehler K et al (2005) Fine-scale recombination
patterns differ between chimpanzees and humans. Nat Genet
37:429–434
Qanbari S, Pimentel EC, Tetens J, Thaller G, Lichtner P, Sharifi AR,
Simianer H (2010) The pattern of linkage disequilibrium in
German Holstein cattle. Anim Genet 41:346–356
Rahn MI, Mudry M, Merani MS, Solari AJ (1996) Meiotic behavior
of the X1X2Y1Y2 quadrivalent of the primate Alouatta caraya.
Chromosome Res 4:350–356
Reeves A (2001) MicroMeasure: a new computer program for the
collection and analysis of cytogenetic data. Genome 44:439–
443
Robinson TJ, Ruiz-Herrera A (2008) Defining the ancestral eutherian
karyotype: a cladistic interpretation of chromosome painting and
genome sequence assembly data. Chromosome Res 16:1133–
1141
Robles P, Roig I, Garcia R et al (2009) Analysis of recombination
along chromosome 21 during human female pachytene stage.
Reprod Biomed Online 18:784–794
Rogers J, Garcia R, Shelledy Wet al (2006) An initial genetic linkage
map of the rhesus macaque (Macaca mulatta) genome using
human microsatellite loci. Genomics 87:30–38
Seuánez HN, Armada JL, Barroso C, Rezende C, da Silva VF (1983)
The meiotic chromosomes of Cebus apella (Cebidae, Platyrrhini).
Cytogenet Cell Genet 36:517–524
Chromosoma (2011) 120:521–530 529
Stanyon R, Garofalo F, Steinberg ER, Capozzi O, Di Marco S, Nieves M,
Archidiacono N, Mudry MD (2011) Chromosome painting in two
genera of South American monkeys: species identification, conservation
and management. Cytogenet Genome Res 134:40–50
Steinberg ER, Nieves M, Mudry MD (2007) Meiotic characterization and sex
determination system of neotropical primates: Bolivian squirrel monkey
Saimiri boliviensis (primates: Cebidae). Am J Primatol 69:1236–1241
Steinberg ER, Cortes-Ortiz L, Nieves M et al (2008) The karyotype of
Alouatta pigra (Primates: Platyrrhini): mitotic and meiotic
analyses. Cytogenet Genome Res 122:103–109
Sun F, Oliver-Bonet M, Liehr T et al (2004) Human male
recombination maps for individual chromosomes. Am J Hum
Genet 74:521–531
Tease C, Hartshorne GM, Hulten MA (2002) Patterns of meiotic
recombination in human fetal oocytes. Am J Hum Genet
70:1469–1479
Wu ZK, Getun IV, Bois PR (2010) Anatomy of mouse recombination
hot spots. Nucleic Acids Res 38:2346–2354
Yunis J, Prakash O (1982) The origin of man: a chromosomal pictorial
legacy. Science 215:1525–1530
530 Chromosoma (2011) 120:521–530