Blackwell Science, LtdOxford, UKBIJBiological Journal of the Linnean Society0024-4066The Linnean Society of London, 2003?

2003
80?
107124
Original Article
HETEROCHROMATIN and EVOLUTION IN PRIMATES
F. GARCA
ET AL.

Biological Journal of the Linnean Society, 2003, 80, 107124. With 3 figures

Qualitative analysis of constitutive heterochromatin and

primate evolution
F. GARCA1, M. GARCIA1, L. MORA1, L. ALARCN2, J. EGOZCUE1 and M. PONS1*
1

Departament de Biologia Cel. lular, Fisiologia i Immunologia and Institut de Biotecnologia i

Biomedicina, Unitat de Biologia Cel.lular. Facultat de Cincies., Universitat Autnoma de Barcelona,
08193- Bellaterra (Barcelona), Spain
2
Departament de Gentica i Microbiologia, Universitat Autnoma de Barcelona, 08193 Bellaterra
(Barcelona), Spain
Received 5 July 2002; accepted for publication 11 February 2003

The results of qualitative heterochromatin analysis in 16 species of primates: Homo sapiens, Pan troglodytes and
Gorilla gorilla (F. Hominidae), Hylobates syndactilus (F. Hylobatidae), Macaca fascicularis, M. tibetana, Mandrillus
sphinx, M. leucophaeus, Cercopithecus aethiops, C. sabaeus and C. albogularis (F. Cercopithecidae), Cebus apella,
Ateles belzebuth hybridus, Aotus azarae, Saimiri sciureus and Lagothrix lagothricha (F. Cebidae) are presented in
this work. We characterized heterochromatin using: (a) in situ digestion with restriction enzymes AluI, HaeIII, RsaI
and Sau3A, and (b) chromosome staining with DA/DAPI on unbanded chromosomes, on C-banded chromosomes and
on sequentially G-C-banded chromosomes. The aim of this work was to relate the qualitative characteristics of constitutive heterochromatin observed with the cytogenetic evolutive processes in the primate group. Results obtained
show that (1) in the family Cercopithecidae, Papionini species do not present chromosomal rearrangements when
their karyotypes are compared and the heterochromatin characteristics are uniform, while Cercopithecini species
show a high number of chromosomal reorganizations, but they have the same heterochromatic characteristics; (2) the
Platyrrhini species analysed show variability in their karyological and heterochromatic characteristics; (3) the Hominoidea present two different situations: Pan, Gorilla and Homo with few chromosomal reorganizations among their
karyotypes but with a high variability in their heterochromatin characteristics, and Hylobates with low heterochromatin variability and a highly derived karyotype. Speciation processes related to chromosome changes and heterochromatin variations in different groups of primates are discussed. 2003 The Linnean Society of London,
Biological Journal of the Linnean Society, 2003, 80, 107124.

ADDITIONAL KEYWORDS: chromosomes cytogenetics DA/ DAPI heterochromatin restriction enzymes.

INTRODUCTION
Comparative cytogenetic studies on primates based on
chromosome banding (Dutrillaux, 1979; Pons et al.,
1986; Clemente et al., 1990; Medeiros et al., 1997) or
fluorescent in situ hybridization (FISH) using whole
human chromosome probes (ZOO-FISH) (Wienberg &
Stanyon, 1997; Garca et al., 2000; Ruiz-Herrera et al.,
2002) have shown that euchromatic regions seem to be
conserved during evolution without any significant
loss or gain of material, although they have undergone
many chromosome reorganizations.

*Corresponding author. E-mail: montse.ponsa@uab.es

Concerning primate heterochromatic chromosomal

regions, it is well known that they show frequent variations in localization and quantity (Marks, 1985;
Pons et al., 1995). However, there are a limited number of publications analysing qualitative characteristics of primate heterochromatin at the cytogenetic
level (Pieczarka et al., 1996, 2000; Toder, Xia &
Bausch, 1998; Garca et al., 1999). While C-banding
has been used extensively to identify the size (quantitative analysis) and position of the heterochromatic
regions, fluorochrome staining and in situ digestion
with restriction enzymes (REs) are useful to detect
qualitative heterochromatin heterogeneity. Fluorochrome staining could provide information about
DNA sequence distribution throughout chromosomes

2003 The Linnean Society of London, Biological Journal of the Linnean Society, 2003, 80, 107124

107

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F. GARCA ET AL.

(Schmid et al., 1986; Luke & Verma, 1995). Type II

REs digest fixed chromosomes and induce specific
banding patterns which seem to be the result of the
differential distribution of target sequences throughout the chromosomes, the accessibility to the target
region and the size of the fragments generated related
to the possibility of being removed (Mezzanotte et al.,
1983; Babu & Verma, 1990; Stuppia et al., 1991; Pieczarka et al., 1996, 2000; Garca et al., 1999). Regions
which remain undigested after RE digestion appear
darkly stained, while regions lightly stained or not
stained are regions enriched in the target sequences
accessible to the restriction enzymes from which DNA
has been removed.
The aim of this work was to analyse qualitative variations of constitutive heterochromatin in different primate species, and to relate the results obtained with
the cytogenetic evolutive processes in this group.

MATERIAL AND METHODS

The species, taxonomic group, sex, sample origin, and
number of specimens analysed in this study are summarized in Table 1.

Chromosome preparations were obtained from

standard lymphocyte cultures (RPMI 1640 medium,
72 h incubation, 0.2 mg mL-1 Colcemid for 20 min)
and from standard fibroblast cultures (EMEM
enriched with 20% FBS, 0.12 mg mL-1 Colcemid for
3 h). To identify each chromosome pair, metaphases
were stained using a sequential G-C banding technique adapted from Seabright (1971) and Sumner
(1972).

CONSTITUTIVE

HETEROCHROMATIN

CHARACTERIZATION

In situ digestion with restriction enzymes

The REs used were AluI, HaeIII, RsaI and Sau3A
(Boehringer Mannheim). Each RE was dissolved in
the buffer indicated by the manufacturer to obtain a
concentration of 0.5 U mL-1, and 100 mL of this solution was placed on each slide. Digestion was carried
out at 37C in a moist chamber for 1214 h. After incubation, coverslips were removed in distilled water. The
slides were air-dried and stained with Leishman for
3 min. Control slides followed the same treatment, but
only with buffer.

Table 1. Primate species analysed

Catarrhini

Family

Species

Code

2n

Specimens
analysed

Hominidae

Homo sapiens
Pan troglodytes
Gorilla gorilla
Hylobates syndactylus
(Papionini)
Macaca fascicularis
Macaca tibetana
Mandrillus leucophaeus
Mandrillus sphinx
(Cercopithecini)
Cercopithecus aethiops
Cercopithecus sabaeus
Cercopithecus albogularis
Cebus apella
Ateles belzebuth hybridus
Aotus azarae
Lagothrix lagothricha
Saimiri sciureus

HSA
PTR
GGO
HSY

46
48
48
50

4
, 1 
2
, 1 
1
, 1 
1

MFA
MTI
MLE
MSP

42
42
42
42

1
, 1 
1

1

1

CAE
CSA
CAL
CAP
ABH
AAZ
LLA
SSC

60
60
74
54
34
50
62
44

1
1

1

7
, 4 
4
, 2 
1

1

1

Hylobatidae
Cercopithecidae

Cercopithecidae

Platyrrhini

Cebidae

Origin of the specimens:

PTR, GGO, HSY, MTI, MLE, MSP y ABH: Parc Zoolgic de Barcelona (Spain).
MFA: CIDA,S.A. (Barcelona, Spain).
CAE y CSA: Zoolgico de la Casa de Campo (Madrid, Spain).
CAP: Zoolgico de la Casa de Campo (Madrid, Spain), Marineland Catalunya (Palafolls, Barcelona, Spain) and Aqualen
(Albinyana, Tarragona, Spain).
CAL y AAZ: private collections (Madrid, Spain).
LLA (AG05356) and SSC (AG05311): Coriell Institute for Medical Research.
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HETEROCHROMATIN AND EVOLUTION IN PRIMATES

Distamycin A/DAPI staining technique
Distamycin A/DAPI was performed on unbanded chromosomes (DA/DAPI) (Schweizer, Ambros & Andrle,
1978), on C-banded chromosomes (C + DA/DAPI)
(Bella & Gosalvez, 1991), and on sequentially G-C
banded chromosomes (G-C + DA/DAPI) (Garca et al.,
1999). In DA/DAPI stained chromosomes which had
been previously G-C banded, control of bands was
made by staining half of the slide with Wright
(G-bands) or Giemsa (C-bands). The other half of the
slide was stained with DA/DAPI.
The same banding techniques (in situ digestion with
REs or DA/DAPI staining) were performed on human
chromosomes which were used as controls.

CLADISTIC

ANALYSIS

Cladistic analysis was performed using the PAUP*

4.0b8 programme (Swofford, 2001). The discrete characters used were: (a) the presence of each type (defined
in Table 2) of constitutive heterochromatin, (b) its
chromosomal localization: centromeric, pericentromeric, interstitial or terminal (Table 2), and (c) the
number of localizations where each heterochromatin
type is observed. Characters are defined in Appendix
1. Discrete characters which were absent in all species
studied were not included in the series. These characters for all the constitutive heterochromatin types are
defined in Table 2, and the analysis was based on a
presence-absence character matrix (Appendix 2).
Those taxa having a particular character were
assigned a 1, and those lacking it a 0.
Parsimony analysis was conducted using the
branch-and-bound algorithm with the following
options: MULTREES, furthest addition, MAXTREES
= 100. Statistical support for nodes of the trees was
assessed with the bootstrap method (retaining groups
with a frequency >50% from trees compiled from 1000
bootstrap replications; Felsenstein, 1985). Bootstrap
replicates were also obtained with the branch-andbound method (PAUP*, Version 4.0b8).

109

heterogeneity (more than one type) of the heterochromatin and its chromosome localization are shown.
The qualitative characteristics of centromeric heterochromatin have been compared in homologous
chromosomes from Homo sapiens, Pan troglodytes,
Gorilla gorilla (F. Hominidae) and Cebus apella
(F. Cebidae) based on chromosomal homologies among
HSA, PTR and GGO determined by G-banding comparison (Yunish & Prakash, 1982) and confirmed by
ZOO-FISH (Wienberg et al., 1990; Jauch et al., 1992),
and chromosomal homologies between HSA and CAP
by G-banding comparison and ZOO-FISH (Clemente
et al., 1987 and Garca et al., 1999, 2000). Results are
shown in Table 4, where it can be seen that, in general, homologous chromosomes from different species
do not conserve the same type of heterochromatin.

CLADISTIC

ANALYSIS

Appendix 2 shows the data matrix that results from

the analysis of the presence or absence of the characters (Table 2 and Appendix 1).
The phylogenetic analysis yields 12 equally mostparsimonious trees 216 steps long, with a consistency
index of 0.778. Figure 3 shows the strict consensus of
these trees; only the nodes with a frequency >50%
have been retained. According to this topology, the
Cercopithecidae species from the Papionini tribe
(MFA, MTI, MLE, MSP) are grouped in a cluster (bootstrap support, 72%). Cercopithecidae species from the
Cercopithecini tribe (CAE, CSA and CAL) are grouped
in a cluster (bootstrap support, 83%). The Platyrrhini
CAP and ABH are grouped in a cluster (bootstrap support, 63%). Papionini and Cercopithecini species, CAP,
ABH, AAZ and SSC (Platyrrhini species), and HSY
(Hylobatidae species) are grouped in a cluster, in this
case with minor support (bootstrap support, 56%). In
this analysis, the relationship among HSA, PTR, GGO
and LLA, and with the rest of the species analysed,
could not be determined.

DISCUSSION
RESULTS
QUALITATIVE

ANALYSIS OF CONSTITUTIVE

HETEROCHROMATIN

Different types of heterochromatin (Table 2) were

defined according to the results obtained after RE
(AluI, HaeIII, RsaI and Sau3A) (Fig. 1) and DA/DAPI
(DA/DAPI, C + DA/DAPI and G-C + DA/DAPI) (Fig. 2)
treatments. In Table 2, species, heterochromatin types
and localization in the chromosomes (centromeric,
pericentromeric, interstitial or terminal as well as
arm affected) are summarized.
In Table 3, the results obtained in relation to the
species analysed, the homogeneity (only one type) or

EVOLUTION

OF CONSTITUTIVE HETEROCHROMATIN

RELATED TO CHROMOSOMAL LOCALIZATION

The qualitative characteristics of heterochromatin are

not related to their chromosomal localization. Some
species show the same characteristics (homogeneous)
in the heterochromatin localized in interstitial or terminal (ITH), centromeric (CH) or pericentromeric
(PCH) regions, while in others species it is heterogeneous (Table 3).
Interstitial and terminal heterochromatin is, in general, homogeneous, different from CH and speciesspecific. Our data and data from Pieczarka et al.
(1996) and Garca et al. (1999) suggest that the origin

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F. GARCA ET AL.

Table 2. Types and patterns of constitutive heterochromatin detected in the primate species analysed, and their localization in the karyotype
Type

Pattern

Species

Localization

++++ +++

PTR

Cen (2p, 7, 1317, 21, 22)

p (13, 14, 21, 22)

GGO

Cen (5, 14, 16)

Pericen q (14, 16)
Ter p (9, 15, 18)

HSA

Cen (9, 16)

AAZ

Pericen p (4, X)

LLA

Cen (13)

PTR

Cen (9)

HSA

Cen (1)
Pericen q (1, 16)

GGO

Ter p (2p, 13)

HSA

Cen (15)
Pericen (9)

AAZ

Pericen p (10, 12, 13, 14, 16, 17, 19, 21, 22, 23, 25, 26)
Pericen q (3)

LLA

Pericen (2)

II

III

+++ +++

+++ +++

IV

+++ +++

PTR

Cen (19, 20)

+++ +++

AAZ

Cen
Pericen p (9)

SSC

Cen (5)

VI

++ +++

AAZ

Pericen p (15, 18, 20)

VII

++++ ++

PTR

Cen (18)

GGO

Cen (2p, 4, 9, 13, 15, 21, 22)

Ter q (17, 21, 22)

CAP

Cen (9, 10)

AAZ

Pericen q (2)

PTR

Cen (11)

GGO

Ter p (2q)

CAE

Cen
Pericen

CSA

Cen
Pericen
Ter

CAL

Cen

CAP

Cen (1126)

LLA

Cen (1621, 24)

SSC

Cen (16)

VIII

+++ ++

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HETEROCHROMATIN AND EVOLUTION IN PRIMATES

111

Table 2. Continued
Type

Pattern

Species

Localization

IX

++ ++

AAZ

Int

+++ ++

PTR

Cen (10, 12)

GGO

Cen (1, 3, 6, all from group [8, 1012, X] except one pair, 1820)

HSA

Cen (7, 19)

CAP

Cen (8, X)

LLA

Cen (1, 5, 6, 7, 9, 10, 14, 15, 22, 2530, X)

GGO

Ter p (6)

CAP

Cen (1, 3, 4, 6)

ABH

Cen (1, 2, 48, 1016, X)

GGO

Cen (one pair from group [8, 1012, X], 17)

HSA

Cen (3, 4, 5, 10)

HSY

Cen (one pair from group [20, 2224])

LLA

Int q (16, 17, 18, 24)

PTR

Cen (1, 4, 6, 8)

GGO

Cen (7)

HSA

Cen (12)

XI

XII

XIII

XIV

++ ++

++ ++

+ + ++

++ ++

XV

+ ++

HSY

Cen (one pair from group [410], 13, two pairs from group
[1519])

XVI

+ ++

PTR

Cen (3, X)

HSY

Ter
Int (12)

HSA

Cen (11, X)

MFA

Cen

MTI

Cen

MLE

Cen

MSP

Cen

LLA

Cen (2, 8)

HSY

Cen (21, three pairs from group [20, 2224])

CAP

Cen (2, 5, 7)

ABH

Cen (3, 9)

PTR

Cen (2q, 5)

HSY

Cen (13, six pairs from group [410], 11, 12, 14, three pairs from
group [1519], X)

HSA

Cen (8)

LLA

Int q (10)

XVII

XVIII

+ ++

++

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F. GARCA ET AL.

Table 2. Continued
Type

Pattern

Species

Localization

XIX

+++ +

PTR

Ter p (2p, 2q, 8, 11, 12, 16, 19, 20)

Ter q (1922)
Int q (13)

XX

++ +

PTR

Ter p (15)

XXI

++

HSA

Cen (2,6)

XXII

+ ++

HSA

Cen (13, 14, 17, 18, 2022)

XXIII

++

CAP

Int
Ter

XXIV

++ +

PTR

Ter p (10, 17)

Ter q (16, 17)
Int q (7)

XXV

++ ++

ABH

Int p
Int q
Ter p

XXVI

+ ++

GGO

Ter q (2p)

XXVII

++

GGO

Ter p (3)
Ter q (3)

XXVIII

+ ++

GGO

Ter p (5)
Ter q (9, 1316)

XXIX

++ ++

GGO

Ter q (5)

XXX

+++ ++

GGO

Ter p (14)

XXXI

++ ++

GGO

Ter p (16, 17, 20)

Ter q (20)

XXXII

+++ ++

GGO

Ter p (19)
Ter q (18, 19)

XXXIII

+++ ++

LLA

Ter p (1, 11)

XXXIV

+ +++

LLA

Cen (3, 4)

XXXV

++ +++

LLA

Cen (11, 12, 23)

XXXVI

+++

LLA

Int q (2123)

XXXVII

++ +

SSC

Cen (1, 1012)

XXXVIII

+++ +

SSC

Ter p (5, 914)

Ter q (5)
Int p (6, 14)
Int q (6)

XXXIX

++

SSC

Cen (6, 9, 13)

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HETEROCHROMATIN AND EVOLUTION IN PRIMATES

113

Table 2. Continued
Type

Pattern

Species

Localization

XL

SSC

Cen (7)

XLI

+++ ++

SSC

Ter p (7)

XLII

+++

SSC

Cen (15, 17, 18)

XLIII

+++ +

SSC

Cen (19, 20, 21)

XLIV

+ +

SSC

Cen (X)

Pattern: symbols +, or represent the result from each treatment used to analyse constitutive heterochromatin. From left
to right: In situ digestion with AluI, HaeIII, RsaI and Sau3A (+ indicates digestion, indicates no digestion, and indicates
partial digestion), and fluorescent staining with DA/DAPI on unbanded, C-banded and G-C banded chromosomes
(+ indicates bright staining, indicates no bright staining).
Species codes are given in Table 1.
Cen = centromeric heterochromatin, Pericen = pericentromeric heterochromatin, Ter = terminal heterochromatin at p or
q arms, Int = interstitial heterochromatin, p = heterochromatic p arm.
In the species where the number of the chromosome pair is specified, the karyotypes have been arranged as follows: HSA:
standard arrangement; PTR and GGO: according homologies with human chromosomes; HSY: according van Tuinen &
Ledbetter (1983); CAP: according Matayoshi et al. (1986); ABH: according Medeiros et al. (1997); AAZ: following Ma (1981);
LLA: according Clemente et al. (1987); SSC: following Ma & Jones (1975).

of ITH is recent, a fact which could explain the homogeneity observed in nearly all the species, and that it
may have occurred after speciation or was perhaps, in
some cases, related to the process of speciation. The
heterogeneity observed in the interstitial or terminal
heterochromatin of PTR, GGO, LLA and SSC species
could be due to changes which occurred after their
speciation.
Different possibilities can be postulated to explain
the origin of PCH: (1) PCH comes from CH by amplification in chromosomes with identical CH and PCH
(PTR, GGO, CAE and CSA species, Tables 2 and 3), or
by mutation and amplification in chromosomes with a
different type of CH and PCH (AAZ, LLA and HSA
species, Tables 2 and 3). Alternatively, (2) a different
(non-centromeric) origin can be postulated for chromosomes with different types of CH and PCH in the same
chromosome (AAZ, LLA and HSA species, Tables 2
and 3).
In some species, several chromosomes show PCH or
ITH with the same qualitative characteristics (the
same type of heterochromatin). That is the case of
PCH in Aotus azarae and ITH in Cebus apella, Ateles
belzebuth hybridus and Aotus azarae (Table 2), where
PCH and ITH could have an internal or external origin, but its dispersion in the karyotype seems to be the
consequence of amplification and jumping events.
Except in the Cercopithecidae and Aotus azarae
(Platyrrhini), the primate species analysed show high

variability in CH characteristics (Table 3), but in

these species we observed that acrocentric chromosomes and chromosomes with a heterochromatic p
arm are homogeneous in the CH characteristics, while
non-acrocentric chromosomes present variability in
CH (except in LLA and SSC species). Some authors
have suggested that acrocentric chromosomes can
associate and interchange repetitive DNA (Schweizer
& Loidl, 1987). Interchange of repetitive DNA could
explain the high degree of uniformity observed in the
heterochromatin of acrocentric chromosomes and in
chromosomes with a heterochromatic p arm. This kind
of association has been observed in Cebus apella
metaphases (this work), in other primate species
(Henderson, Warburton & Atwood, 1976) and in the
pig (Jantsch et al., 1990). Heterochromatin associations affecting the p arms of chromosome pairs 13, 14,
21 and 22 have also been observed in PTR.

EVOLUTION

OF CONSTITUTIVE HETEROCHROMATIN

RELATED TO SPECIES EVOLUTION

In the Platyrrhini monkeys, the high interspecific and

even intraspecific chromosome variability (Pons
et al., 1995) and the high variability in the qualitative
characteristics of constitutive heterochromatin
(mainly in the CH), allow postulation that probably in
this group both changes in euchromatin and in heterochromatin are involved in the speciation process.

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F. GARCA ET AL.

A
1

10

11

12

13

14

15

16

19

20

21

22

23

24

25

26

27

28

29

17

18

XY

10

11

12

13

14

15

16

19

20

21

22

23

24

25

26

27

28

29

17

18

XY

Alu I

Hae III

Rsa I

Sau3A

Figure 1. G-C sequential banding (A) and in situ digestion with restriction enzymes (B) in Cercopithecus aethiops.

2003 The Linnean Society of London, Biological Journal of the Linnean Society, 2003, 80, 107124

HETEROCHROMATIN AND EVOLUTION IN PRIMATES

115

X X

10

11

12

13

14

15

X X

10

11

12

13

16

14

15
*

*
*

16

*
*

D1

D2
*

* *

*
*
*

*
*

*
*

D3

Figure 2. G-C sequential banding (A) and chromosome staining with DA/DAPI on unbanded (D1), on C-banded (D2) and
on sequentially G-C-banded (D3) chromosomes (B) in Ateles belzebuth hybridus.

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F. GARCA ET AL.

Table 3. Presence of one or more types of heterochromatin (homogeneous or heterogeneous) in the primate species
analysed, according to their chromosome localization

Species

Centromeric
heterochromatin

Pericentromeric
heterochromatin

Interstitial
heterochromatin

Terminal heterochromatin

Homogen

Homogen

Homogen

Homogen

HSA

I, II, III, X,
XIII, XIV,
XVI, XVIII
XXI, XXII
I, II, IV,
VII, VIII,
X, XIV,
XVI, XVIII
I, VII, X,
XIII, XIV

PTR

GGO

HSY
MFA
MTI
MLE
MSP
CAE
CSA
CAL
CAP
ABH
AAZ
LLA

SSC

Heterogen

Heterogen

p arm
I

XIX, XXIV

XIX, XX,
XIV

I, III, VII,
VIII, XII,
XXVI-XXXII
One pair
XVI

XVI

VIII
VIII

VIII

VII, VIII, XI,

XII, XVII
XII, XVII

XXIII

I, III, V,
VI, VII
I, VIII, XI,
XVI, XXXIV,
XXXV
V, VIII,
XXXVII, IXL,
XL, XLII,
XLIII, XLIV

Heterogen

II, III

XIII, XV,
XVII, XVIII
XVI
XVI
XVI
XVI
VIII
VIII
VIII

Heterogen

One pair
XXIII
XXV

XXV
IX

One pair
III

XIII, XVIII,
XXXVI
XXXVIII

XXXIII

XXXVIII, XLI

Roman numerals indicate types of heterochromatin (from Table 2).

All species from the Papionini tribe (2n = 42) have

almost identical karyotypes (Dutrillaux et al., 1979;
Clemente et al., 1990), indicating that chromosomal
reorganizations practically have not occurred in this
group. In our study, Macaca and Mandrillus species
share the same type of constitutive heterochromatin
(Type XVI, Tables 2 and 3), indicating that in this
group neither the euchromatin nor probably the heterochromatin are involved in speciation.
Chromosomal evolution in the Cercopithecini
species (2n = 4872) has occurred mainly by fusionfission rearrangements (Pons et al., 1986). The
Cercopithecini species studied in this work (CAE, CSA

and CAL) share the same type of heterochromatin

(Type VIII, Tables 2 and 3), indicating that in this
group speciation is related to changes in euchromatin
but not in the qualitative characteristics of heterochromatin. Cercopithecus aethiops and C. sabaeus,
both species from the aethiops group, have the same
karyotype (Sineo, Stanyon & Chiarelli, 1986); the only
difference is the presence of terminal heterochromatin
in four chromosome pairs in C. sabaeus. Differences in
heterochromatin localization have been related to taxonomic differentiation in other primates, for example,
in Cebus apella paraguayanus and Cebus apella nigritus (Matayoshi et al., 1987; Mudry et al., 1991; Pons

2003 The Linnean Society of London, Biological Journal of the Linnean Society, 2003, 80, 107124

HETEROCHROMATIN AND EVOLUTION IN PRIMATES

117

Table 4. Types of centromeric heterochromatin in the homologous chromosomes

of HSA, PTR, GGO and CAP
Homologous chromosome/centromeric heterochromatin type
HSA

PTR

GGO

CAP

1/II

1/XIV

1/X

2/XXI
3/XIII

2p/I
2q/XVIII
3/XVI

2p/VII
2q/
3/X

14/VIII
22/VIII
5/XVII

4/XIII
5/XIII
6/XXI
7/X
8/XVIII

4/XIV
5/XVIII
6/XIV
7/I
8/XIV

4/VII
5/I
6/X
7/XIV
8/X or XIII

9/I
10/XIII

9/II
10/X

9/VII
10/X or XIII

11/XVI
12/XIV
13/XXII
14/XXII
15/III

11/VIII
12/X
13/I
14/I
15/I

11/X or XIII
12/X or XIII
13/VII
14/I
15/VII

16/I

16/I

16/I

17/XXII
18/XXII
19/X
20/XXII
21/XXII
22/XXII
X/XVI

17/I
18/VII
19/IV
20/IV
21/I
22/I
X/XVI

17/XIII
18/X
19/X
20/X
21/VII
22/VII
X/X or XIII

18/VIII
11/VIII
2/XVII
1/XII
3/XII
15/VIII
7/XVII
8/XI
19/VIII
4/XII
26/VIII
16/VIII
12/VIII
17/VIII
6/XII
24/VIII
6/XII
4/XII
5/XVII
21/VIII
7/XVII
9/VII
10/VII
11/VIII
25/VIII
X/XI

Species codes are given in Table 1.

Roman numerals indicate types of heterochromatin (from Table 2).
The number of each chromosome pair was established as follows: HSA: standard,
PTR and GGO: according to their homologies with human chromosomes, and CAP:
following Matayoshi et al. (1986). HSA, PTR and GGO homologies have been
defined by Yunish & Prakash (1982). HSA and CAP homologies have been defined
by Garca et al. (2000).
In the specimens of Gorilla gorilla analysed, chromosome 2q did not have detectable centromeric heterochromatin.

et al., 1995). In these cases a new localization of heterochromatin can be related to the speciation process,
even if qualitative characteristics have not changed.
The topology obtained with PAUP shows that the
Cercopithecidae are grouped in two clusters, one with
the species of the Papionini tribe (72% bootstrap support) and the other with those of the Cercopithecini
tribe (83%) (Fig. 3). These relationships are consistent

with the conventional taxonomic classification of

primates.
In the Hominidae, only a small number of inversions are needed to make their karyotypes homologous
(Yunish & Prakash, 1982); however, heterochromatin
is highly variable, indicating that changes in both
euchromatin and heterochromatin can be involved in
speciation. Hylobates present a highly derived karyo-

2003 The Linnean Society of London, Biological Journal of the Linnean Society, 2003, 80, 107124

118

F. GARCA ET AL.
MFA
72

MTI
MLE
MSP
CAE

83
56

CSA
CAL

63

CAP
ABH
HSY
AAZ
SSC
HSA
PTR
GGO
LLA

Figure 3. Strict consensus of the 12 equal mostparsimonious trees. Percentage bootstrap values are
given on the nodes. MFA = Macaca fascicularis, MTI =
Macaca tibetana, MLE = Mandrillus leucophaeus, MSP =
Mandrillus sphinx, CAE = Cercopithecus aethiops, CSA =
Cercopithecus sabaeus, CAL = Cercopithecus albogularis,
CAP = Cebus apella, ABH = Ateles belzebuth hybridus,
HSY = Hylobates syndactilus, AAZ = Aotus azarae, SSC
= Saimiri sciureus, HSA = Homo sapiens, PTR = Pan
troglodytes, GGO = Gorilla gorilla, LLA = Lagothrix
lagothricha.

type (Jauch et al., 1992) while heterochromatin characteristics are less variable than in the Hominidae
species. In Hylobates, changes in euchromatin seem to
be more important in the speciation process than are
heterochromatin variations.
Homologous chromosomes in different species generally do not share the same heterochromatin characteristics. This is the case when HSA, PTR, GGO and
CAP karyotypes are compared (Table 4). This is in
agreement with other data published showing different heterochromatic characteristics in homologous
chromosomes of the great apes (Archidiacono et al.,
1995; Toder et al., 1998). The same occurs when comparing Macaca and Mandrillus species (Papionini)
with Cercopithecus species (Cercopithecini): homologous chromosomes do not share the same heterochromatin characteristics. Changes can be related to the
separation of both tribes.

CONCLUSIONS
In summary, we can conclude the following.
1 Interstitial and terminal heterochromatin (ITH)
shows less qualitative variability than does centro-

meric heterochromatin (CH). This fact can be related

to a more recent evolutive origin for ITH than for CH.
2 With regard to cytogenetic characteristics analysed
(qualitative analysis of constitutive heterochromatin
and chromosomal homologies) different combinations
were observed. (a) In the Cercopithecidae family
there are species without chromosomal reorganizations and with the same qualitative characteristics of
heterochromatin (Papionini tribe), and there are species with a high number of chromosomal reorganizations when their karyotypes are compared but with
the same characteristics of heterochromatin (Cercopithecini tribe). (b) The Platyrrhini species analysed
show different karyotypes with a high number of
rearrangements and with qualitative characteristics
of constitutive heterochromatin that are highly variable. (c) The Hominoidea show two patterns: species
with a low number of chromosomal reorganizations
when their karyotypes are compared and with a
considerable variability in the characteristics of
heterochromatin (Hominidae: PTR, GGO and HSA),
and HSY, which shows a highly derived karyotype
and the lowest variability in the constitutive
heterochromatin.
3 Concerning the qualitative characteristics of the
constitutive heterochromatin, PAUP shows a monophyletic origin for the Papionini and Cercopithecini
species, and a polyphyletic origin for the Platyrrhini
and Hominoidea species.
4 Our results suggest the possibility that changes in
heterochromatin characteristics have contributed to
the speciation process.

ACKNOWLEDGEMENTS
The authors are grateful to the staff of the Zoo de la
Casa de Campo (Madrid), Parc Zoolgic de Barcelona,
Marineland Catalunya, S.A., CIDA, S.A., Aqualen,
and Veterinary clinic of L. Monsalve for providing primate blood samples. Financial support was received
from DGES (PB96/1170) and DGES (BXX 2000
0151), and from the Universitat Autnoma de
Barcelona (grant to L. Mora). We would also like to
thank Mr Chuck Simmons, an English instructor of
this university, for reviewing the English of this
manuscript.

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APPENDIX 1
Characters (C) used for the construction of the data matrix.
C1
C2
C3
C4
C5
C6
C7
C8
C9
C10
C11
C12
C13
C14
C15
C16
C17
C18
C19
C20
C21
C22
C23
C24
C25
C26
C27
C28
C29
C30
C31
C32
C33
C34
C35

Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence

of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of

type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type

I heterochromatin
I heterochromatin; centromeric localization
I heterochromatin; pericentromeric localization
I heterochromatin; terminal localization
I heterochromatin; p arm localization
I heterochromatin; 12 localizations
I heterochromatin; 315 localizations
II heterochromatin
II heterochromatin; centromeric localization
II heterochromatin; pericentromeric localization
II heterochromatin; 12 localizations
II heterochromatin; 315 localizations
III heterochromatin
III heterochromatin; centromeric localization
III heterochromatin; pericentromeric localization
III heterochromatin; terminal localization
III heterochromatin; 12 localizations
III heterochromatin; 315 localizations
IV heterochromatin
IV heterochromatin; centromeric localization
IV heterochromatin; 12 localizations
V heterochromatin
V heterochromatin; centromeric localization
V heterochromatin; pericentromeric localization
V heterochromatin; 12 localizations
V heterochromatin; >15 localizations
VI heterochromatin
VI heterochromatin; pericentromeric localization
VI heterochromatin; 315 localizations
VII heterochromatin
VII heterochromatin; centromeric localization
VII heterochromatin; pericentromeric localization
VII heterochromatin; terminal localization
VII heterochromatin; 12 localizations
VII heterochromatin; 315 localizations

2003 The Linnean Society of London, Biological Journal of the Linnean Society, 2003, 80, 107124

HETEROCHROMATIN AND EVOLUTION IN PRIMATES

Appendix 1 Continued
C36
C37
C38
C39
C40
C41
C42
C43
C44
C45
C46
C47
C48
C49
C50
C51
C52
C53
C54
C55
C56
C57
C58
C59
C60
C61
C62
C63
C64
C65
C66
C67
C68
C69
C70
C71
C72
C73
C74
C75
C76
C77
C78
C79
C80
C81
C82
C83
C84
C85
C86
C87
C88
C89
C90
C91

Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence

of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of

type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type

VIII heterochromatin
VIII heterochromatin; centromeric localization
VIII heterochromatin; pericentromeric localization
VIII heterochromatin; terminal localization
VIII heterochromatin; 12 localizations
VIII heterochromatin; 315 localizations
VIII heterochromatin; >15 localizations
IX heterochromatin
IX heterochromatin; interstitial localization
IX heterochromatin; 12 localizations
X heterochromatin
X heterochromatin; centromeric localization
X heterochromatin; 12 localizations
X heterochromatin; 315 localizations
XI heterochromatin
XI heterochromatin; centromeric localization
XI heterochromatin; 12 localizations
XI heterochromatin; >15 localizations
XII heterochromatin
XII heterochromatin; centromeric localization
XII heterochromatin; terminal localization
XII heterochromatin; 12 localizations
XII heterochromatin; 315 localizations
XIII heterochromatin
XIII heterochromatin; centromeric localization
XIII heterochromatin; interstitial localization
XIII heterochromatin; 12 localizations
XIII heterochromatin; 315 localizations
XIV heterochromatin
XIV heterochromatin; centromeric localization
XIV heterochromatin; 12 localizations
XIV heterochromatin; 315 localizations
XV heterochromatin
XV heterochromatin; centromeric localization
XV heterochromatin; 315 localizations
XVI heterochromatin
XVI heterochromatin; centromeric localization
XVI heterochromatin; interstitial localization
XVI heterochromatin; terminal localization
XVI heterochromatin; 12 localizations
XVI heterochromatin; >15 localizations
XVII heterochromatin
XVII heterochromatin; centromeric localization
XVII heterochromatin; 12 localizations
XVII heterochromatin; 315 localizations
XVIII heterochromatin
XVIII heterochromatin; centromeric localization
XVIII heterochromatin; interstitial localization
XVIII heterochromatin; 12 localizations
XVIII heterochromatin; >15 localizations
XIX heterochromatin
XIX heterochromatin; interstitial localization
XIX heterochromatin; terminal localization
XIX heterochromatin; 315 localizations
XX heterochromatin
XX heterochromatin; terminal localization

2003 The Linnean Society of London, Biological Journal of the Linnean Society, 2003, 80, 107124

121

122

F. GARCA ET AL.

Appendix 1 Continued
C92
C93
C94
C95
C96
C97
C98
C99
C100
C101
C102
C103
C104
C105
C106
C107
C108
C109
C110
C111
C112
C113
C114
C115
C116
C117
C118
C119
C120
C121
C122
C123
C124
C125
C126
C127
C128
C129
C130
C131
C132
C133
C134
C135
C136
C137
C138
C139
C140
C141
C142
C143
C144
C145
C146
C147

Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence

of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of

type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type

XX heterochromatin; 12 localizations
XXI heterochromatin
XXI heterochromatin; centromeric localization
XXI heterochromatin; 12 localizations
XXII heterochromatin
XXII heterochromatin; centromeric localization
XXII heterochromatin; 315 localizations
XXIII heterochromatin
XXIII heterochromatin; interstitial localization
XXIII heterochromatin; terminal localization
XXIII heterochromatin; 315 localizations
XXIV heterochromatin
XXIV heterochromatin; interstitial localization
XXIV heterochromatin; terminal localization
XXIV heterochromatin; 315 localizations
XXV heterochromatin
XXV heterochromatin; interstitial localization
XXV heterochromatin; terminal localization
XXV heterochromatin; 315 localizations
XXVI heterochromatin
XXVI heterochromatin; terminal localization
XXVI heterochromatin; 12 localizations
XXVII heterochromatin
XXVII heterochromatin; terminal localization
XXVII heterochromatin; 12 localizations
XXVIII heterochromatin
XXVIII heterochromatin; terminal localization
XXVIII heterochromatin; 315 localizations
XXIX heterochromatin
XXIX heterochromatin; terminal localization
XXIX heterochromatin; 12 localizations
XXX heterochromatin
XXX heterochromatin; terminal localization
XXX heterochromatin; 12 localizations
XXXI heterochromatin
XXXI heterochromatin; terminal localization
XXXI heterochromatin; 315 localizations
XXXII heterochromatin
XXXII heterochromatin; terminal localization
XXXII heterochromatin; 315 localizations
XXXIII heterochromatin
XXXIII heterochromatin; terminal localization
XXXIII heterochromatin; 12 localizations
XXXIV heterochromatin
XXXIV heterochromatin; centromeric localization
XXXIV heterochromatin; 12 localizations
XXXV heterochromatin
XXXV heterochromatin; centromeric localization
XXXV heterochromatin; 315 localizations
XXXVI heterochromatin
XXXVI heterochromatin; interstitial localization
XXXVI heterochromatin; 315 localizations
XXXVII heterochromatin
XXXVII heterochromatin; centromeric localization
XXXVII heterochromatin; 315 localizations
XXXVIII heterochromatin

2003 The Linnean Society of London, Biological Journal of the Linnean Society, 2003, 80, 107124

HETEROCHROMATIN AND EVOLUTION IN PRIMATES

Appendix 1 Continued
C148
C149
C150
C151
C152
C153
C154
C155
C156
C157
C158
C159
C160
C161
C162
C163
C164
C165
C166
C167
C168

Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence

of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of

type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type
type

XXXVIII heterochromatin; terminal localization

XXXVIII heterochromatin; interstitial localization
XXXVIII heterochromatin; 315 localizations
XXXIX heterochromatin
XXXIX heterochromatin; centromeric localization
XXXIX heterochromatin; 315 localizations
XL heterochromatin
XL heterochromatin; centromeric localization
XL heterochromatin; 12 localizations
XLI heterochromatin
XLI heterochromatin; terminal localization
XLI heterochromatin; 12 localizations
XLII heterochromatin
XLII heterochromatin; centromeric localization
XLII heterochromatin; 3158 localizations
XLIII heterochromatin
XLIII heterochromatin; centromeric localization
XLIII heterochromatin; 315 localizations
XLIV heterochromatin
XLIV heterochromatin; centromeric localization
XLIV heterochromatin; 12 localizations

2003 The Linnean Society of London, Biological Journal of the Linnean Society, 2003, 80, 107124

123

124

F. GARCA ET AL.

APPENDIX 2
Data matrix of heterochromatin characters in 16 primate species.

HSA
PTR
GGO
HSY
MFA
MTI
MLE
MSP
CAE
CSA
CAL
CAP
ABH
AAZ
LLA
SSC

HSA
PTR
GGO
HSY
MFA
MTI
MLE
MSP
CAE
CSA
CAL
CAP
ABH
AAZ
LLA
SSC

11111111112222222222333333333344444444445555555555666666666677777777778888888888899999
12345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345
11000101110111101000000000000000000000000000011100000000001100111100001100100000110100000000111
11001011101000000011100000000110010110010000011100000000000000011010001100100000110101111111000
11110010000010011000000000000110101100110000011010000101101101011100000000000000000000000000000
00000000000000000000000000000000000000000000000000000000001101000001111011011101110010000000000
00000000000000000000000000000000000000000000000000000000000000000000001100010000000000000000000
00000000000000000000000000000000000000000000000000000000000000000000001100010000000000000000000
00000000000000000000000000000000000000000000000000000000000000000001100010000000000000000000000
00000000000000000000000000000000000000000000000000000000000000000000001100010000000000000000000
00000000000000000000000000000000000111000100000000000000000000000000000000000000000000000000000
00000000000000000000000000000000000111100100000000000000000000000000000000000000000000000000000
00000000000000000000000000000000000111000100000000000000000000000000000000000000000000000000000
00000000000000000000000000000110010110000100000001110110010000000000000000001101000000000000000
00000000000000000000000000000000000000000000000000001100100000000000000000011100000000000000000
10100100000010100100011101111101010000000011100000000000000000000000000000000000000000000000000
11000100000010101000000000000000000110001000000001101000001010100000001100100000101100000000000
00000000000010100000011010000000000110010000000000000000000000000000000000000000000000000000000
111111111111111111111111111111111111111111111111111111111111111111111
9999000000000011111111112222222223333333333344444444445555555555666666666
6789012345678901234567890123456789012345678901234567890123456789012345678
1110000000000000000000000000000000000000000000000000000000000000000000000
0000000111100000000000000000000000000000000000000000000000000000000000000
0000000000000001111111111111111111110000000000000000000000000000000000000
0000000000000000000000000000000000000000000000000000000000000000000000000
0000000000000000000000000000000000000000000000000000000000000000000000000
0000000000000000000000000000000000000000000000000000000000000000000000000
0000000000000000000000000000000000000000000000000000000000000000000000000
0000000000000000000000000000000000000000000000000000000000000000000000000
0000000000000000000000000000000000000000000000000000000000000000000000000
0000000000000000000000000000000000000000000000000000000000000000000000000
0000000000000000000000000000000000000000000000000000000000000000000000000
0001111000000000000000000000000000000000000000000000000000000000000000000
0000000000011110000000000000000000000000000000000000000000000000000000000
0000000000000000000000000000000000000000000000000000000000000000000000000
0000000000000000000000000000000000001111111111110000000000000000000000000
0000000000000000000000000000000000000000000000001111111111111111111111111

Presence of the character = 1, absence of the character = 0, number of characters = 168.

2003 The Linnean Society of London, Biological Journal of the Linnean Society, 2003, 80, 107124