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Cytogenetic comparison and phylogeny of three species of hylobatidae.

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Cytogenetic Comparison and Phylogeny of Three Species of
Department of Anthropology, University of Wisconsin, Madison, Wisconsin,
53706 (I? VT), Department of Cell Biology, University of Texas System
Cancer Center, M.D. Anderson Hospital and Tumor Institute (F? V T ) and
Department of Medicine, Baylor College of Medicine, Houston, Texas, 77030
(D.H. LJ
Hylobatidae, Primate cytogenetics
Representatives of three subgenera of Hylobatidae, Hylobates
(Symphalangus) syndactylus, H. (Nomascus) concolor and H. (Hylobates) agilis
were compared karyotypically by G-banding, C-banding, and silver staining.
A greater degree of similarity (30-55%) was found among these groups than
previous reports suggest; however, these figures are still considerably lower
than chromosome similarities characteristic of all other catarrhine groups.
Inversion, translocation, fission, and fusion have all played a role in restructuring hylobatid chromosomes since a common hominoid ancestor. H. syndactylus and H. concolor show the greatest G-band correspondence, and in addition
share a n unusual C-band distribution and a n extremely rare nucleolar organizing region placement (on the Y chromosome). The latter two are probably
shared derived traits, suggesting that these two species shared a common
ancestor not shared by other hylobatids. These data sugest a branching order
for these three hylobatid groups different from those derived by other morphological and biochemical methods.
The gibbons and siamang (family Hylobatidae) constitute a sister taxon to the great
apes (Pongidae) and humans (Hominidae) in
the primate superfamily Hominoidea. While
few fossil specimens have been accepted a s
ancestors of Hylobatidae, many other aspects
of gibbon biology are well studied with regard to their taxonomic position. Amino acid
sequencing, immunodiffusion, and DNA hybridization (Darga et al., 1973; Benveniste
and Todaro, 1976) all corroborate morphological and phenotypic analyses of hylobatids
(Groves, 1972)and demonstrate their natural
grouping with other Hominoidea.
By contrast, karyotypic studies of the Hylobatidae have been noteworthy in revealing
karyotypic heterogeneity within this group
and surprisingly little karyotypic similarity
to other anthropoid apes and humans (Hamerton et al., 1963; Chiarelli, 1963; Dutrillaux
et al., 1975a; Warburton et al., 1975; Tantravahi et al., 1975; Myers and Shafer, 1979;
Couturier et al., 1982). This is unexpected
since the great apes (and monkeys as remotely related to humans as Macaca, Papio,
0 1983 ALAN R. LISS. INC.
and Cercopithecus) share extensive chromosome and gene map similarity with each
other and with humans (Stock and Hsu, 1973;
Finaz et al., 1977; Dutrillaux et al., 1978a,b).
An analysis of conventionally stained chromosome preparations by Chiarelli (1972) indicated that the hylobatids had differentiated
into a t least three karyotypic groups: H y b
bates syndactylus (2n = 501, H. concolor
(2n= 521, and most other Hylobates (lar,
agilis, and klossi) (2n = 44). Hylobates pileatus Warkvong, 1973)and H. muelleri (Wolkin
and Myers, 1980) also have 2n = 44. These
44 chromosome species (with the exception of
H. klossi) and H. moloch constitute the socalled “lar group.” The identification of a
fourth group occupied by H. hoolock (2n =
38) has recently been made (Prouty et al.,
The conventionally stained karyotypes of
the 44-chromosome species appear to be very
similar or identical (Chiarelli, 1972). This
Received December 1,1982; accepted April 4, 1983.
identity has been confirmed by G-banding
(Tantravahi et al., 1975) between H. lar and
an animal termed H. moloch, the latter probably a misidentified H. muelleri (Alan Mootnick, personal communication). However,
changes in diploid number have been accompanied by a surprising degree of structural
rearrangement, as shown in comparisons between banded preparations of H. pileatus and
H. concolor (Dutrillaux et al., 1975a) where
only seven autosomal homologies were observed. Only one chromosome was thought
to be shared by H. muelleri and H. syndactylus (Myers and Shafer, 19791, a n unusual
finding in species this closely related. The
structural differences between these three
groups were confirmed by Couturier et al.
(1982),where only four to six autosomes were
similar between any two groups.
While both H. syndactylus and H. concolor
have appeared karyotypically dissimilar to
the 2n = 44 group, the similarity in diploid
number of the former two suggests that they
might share considerable chromosome homology with each other. Ledbetter (1981)
noted that H. syndactylus possesses a nucleolar organizing region (NOR) on the Y chromosome, a rare finding in mammals and the
first reported case in a primate. DeBoer and
Van Oostrum-Van der Horst (1975) suggested
that the Y chromosome of H. concolor participated with other small acrocentrics in “satellite associations,” but the presence of an
NOR was not confirmed by more precise
methods, e.g., silver staining or in situ hybridization.
In this study we employ G-banding, Cbanding, and silver staining in a systematic
comparison of the karyotypes of three major
groups of Hylobatidae-H. syndactylus, H.
concolor, and H. agilis-in order to reevaluate the degree of karyotypic divergence
within this family. In addition, the mechanisms of chromosomal evolution in Hylobatidae are investigated, and several criteria
are considered by which the phylogenetic relationships among the three groups can be
Cytogenetic studies were performed on one
male and one female from each of the three
hylobatid species. Lymphocyte cultures from
a male and female H. syndactylus, a male H.
concolor, and a female H. agilis were grown
in RPMI 1640 20% fetal calf serum (FCS)
and harvested after 72 hours by standard
procedures. Fibroblast cultures from the
same male H. syndactylus, a female H. concolor, and a male H. agilis were initiated in
15%FCS followed by convenHam’s F-10
tional harvest. All animal specimens were
obtained from the Houston Zoo, with the exception of the male H. agilis, which was obtained from the Vilas Park Zoo, Madison,
G-banding (Seabright, 19711, C-banding
(Arrighi and Hsu, 1971) and silver staining
(Bloom and Goodpasture, 1976) were performed by standard procedures. The size order and percentage total chromosomal length
(%TCL) of G-banded chromosomes were determined from caliper measurements taken
of five well-spread metaphases of moderate
contraction. The following abbreviations
were used in accordance with the Paris Conference (19711, Supplement (19751, when referring to chromosomes of particular species:
HSY = Hylobates syndactylus, HCO = Hylohates concolor, HAG = Hylohates agilis.
The three banding methods employed were
treated as independent cytogenetic traits in
the assessment of phylogenetic relationships
among the Hylobatidae. The functions of
chromosomal rearrangements, as they are
reflected in alteration of G-banding patterns,
are not known, but they may serve to reinforce reproductive barriers between isolated,
diverging populations. In any case, G-banding permits the tracing of chromosomally homologous regions that have undergone
rearrangement during evolution; thus it can
be used to compare the distributions of particular rearrangements in different taxa. Gbanding can be used to determine overall
similarity of karyotypes of different taxa, but
in addition, and more importantly, it is used
in the present study to determine both ancestral and derived chromosome banding patterns. The advantage of this cladistic
approach to primate phylogenetic reconstruction from comparative banding has recently
been pointed out by Marks (1982).
C-banding stains what is generally regarded to be genetically inert chromosomal
material (heterochromatin) (John and Miklos, 19791, and as such minor quantitative
variation in C-band blocks is usually of little
phylogenetic significance within and between taxa. For this reason such variation is
usually disregarded in cytogenetic comparisons in evolutionary studies (Stock and Hsu,
1973). However, major qualitative differences in the chromosomal distribution or lo-
cation of C-band material has been used to
distinguish mammalian species independently of other banding methods (Stock,
1981); when ancestral and derived distributions of heterochromatin can be determined,
then common possession of unusual patterns
may be indicative of close phylogenetic relationship.
Similarly, quantitative variation in the intensity of silver staining of nucleolar organizing regions (NORs), where ribosomal RNA
genes reside, is a common finding, while major differences in the location of these regions
often exist between primate species (Tantravahi et al., 1976). Unusual, shared derived
patterns of NOR distribution may indicate
descent from a common ancestor with a derived distribution.
The significance of comparative chromosome banding analysis to the study of phylogeny was evaluated using the ability of banding to indicate ancestral (plesiomorphic) and
derived (apomorphic) chromosomal configurations. Briefly, chromosomal character
states were considered to be symplesiomorphic (shared, ancestral) if they occurred
generally in both Hylobatidae and other Anthropoidea. Synapomorphies (shared derived
traits), on which the grouping of species is
based, were defined a s those traits that had
a restricted distribution in two or more species, and that, empirically, were found too
rarely in other anthropoid primates to be the
result of convergent evolution. It is assumed
throughout this analysis that Hylobatidae
shared with Pongidae and Hominidae a n
ancestor not also shared by any Cercopithecoidea. Although this can be demonstrated
karyotypically, it is our purpose only to make
those out-group comparisons that are relevant in determining phylogeny within the
Hylobates (Symphalangus) syndactylus
(2n = 50)
The G-banding pattern of H. syndactylus
(Fig. l a ) appears to be identical to that reported by Myers and Shafer (1979). One or
both terminal regions of all pairs, including
the X and Y, are darkly stained after Gbanding, and several pairs are obviously dimorphic for the size of this region (e.g., 5 and
17). A comparison to the C-bands (Fig. l b )
shows that these terminal regions are in fact
heterochromatic (Myers and Shafer, 19791, a
distribution like that in some chromosomes
of the chimpanzee and gorilla (Miller et al.,
1974; Miller, 1977). By contrast, centromeric
or juxtacentromeric C-bands are faint or
nearly absent in many chromosomes of H.
syndactylus, unlike the situation in humans,
great apes, and Old World monkeys. The long
arm of 12 bears a n interstitial block of heterochromatin (shown by arrows in Fig. lb).
Interstitial heterochromatin is infrequently
found in catarrhine primates but is reported
in the gorilla and chimpanzee (Miller et al.,
1974; Miller, 1977; Seuanez, 1979).
Silver staining (Fig. lc, arrows) demonstrates the unusual distribution of NORs in
H. syndactylus on a n autosomal pair of acrocentrics (Klinger, 1963; Henderson et al.,
1976) and on the Y chromosome in the male
as reported by Ledbetter (1981)for this same
animal. In the male these three chromosomes participate in NOR association, while
in the female only the two autosomal acrocentrics bear NORs and participate in associations.
Hylobates (Nomascus) concolor (2n = 52)
The G-banding pattern of H. concolor is
shown in Figure 2a. Comparison to the Q- or
R-banded preparations of Dutrillaux et al.
(1975a) is difficult, but there appears to be
good correspondence between the results of
these two studies. The C-banding (Fig. 2b)
shows that, like H. syndactylus, H. concolor
possesses terminal heterochromatin on most
pairs and small quantities of centromeric
heterochromatin. Three autosomal pairs have
interstitial C-bands (Fig. 2b, arrows).
Figure 2c shows the presence of five NORs
(shown by arrows for the autosomes) in a
male H. concolor. The smallest element of
the karytoype, the Y, is silver positive and
participates in NOR associations with two
other acrocentric pairs, 24 and 25. This is
only the second report of a n NOR-bearing Y
chromosome in a primate. The G-banding
patterns of 24 and 25 closely resemble those
of human 22 and 21, respectively, and the
fact that they are silver positive (like human
21 and 22) seems to confirm their homology
with these human chromosomes, as proposed
by Dutrillaux et al. (1975a).
Hylobates (Hylobates) agilis (2n = 44)
The G-banding patterns of a female H.
agilis (Fig. 3a) are virtually identical to the
published karyotypes of H. lar and H. muelleri (Warburton et al., 1975; Tantravahi et
al., 1975; Myers and Shafer, 19791, confirm-
Fig. 1. The banded chromosomes of male H. syndactylus. a. G-banding demonstrates dark terminal staining
and heteromorphism for terminal heterochromatin in
pairs 5 and 17 (long arms). b. C-banding showing terrni-
nal heterochromatin and an interstitial region on pair
12 (arrows). c. Silver staining showing nucleolar organizing regions (NORs) on the Y chromosome and pair 21
Fig. 2. The banded chromosomes of H. concolor a. Gbanding of a male reveals dark terminal (heterochromatic) staining of most chromosome arms, with heteromorphism for the terminal region of pair 14. b. C-banding
in a female showing interstitial bands on three pairs
(arrows). Y chromosome from the male is shown in inset.
c. Silver staining in the male showing an NOR on the Y
chromosome and two autosomal pairs (arrows).
Fig. 3. The banded chromosomes of H. ugilis. a. Gbanding of the female. Pairs 8 and 15 from the male are
represented (inset) to demonstrate heteromorphism, by
inversion or complex rearrangement, for two different
pairs in this animal. b. C-banding in the male showing
only centromeric locations, and the Y chromosome being
barely visible due to lack of heterochromatin. c. Silver
staining showing NORs on pair 13 (arrows) and absence
of NOR on the Y chromosome.
ing the close relationship within this group.
With two exceptions, the G-banding patterns
of our female are identical to those of our
male. As Figure 3a (inset, from a single cell)
shows, homologs of pairs 8 and 15 in the male
are consistently found to match poorly, and
it is evident that at least two independent
rearrangements are represented in this animal. It is interesting that one of the rearranged elements (8)is apparently homologous
to a chromosome involved in pericentric inversion in H. muelleri (Tantravahi et al.,
1975); the rearrangements appear identical
and could constitute a polymorphism.
C-bands in H. agilis, unlike H. syndactylus
and H. concolor, are present only a t the centromeres of many chromosomes, a distribution like that found in H. lar and H. muelleri
(Tantravahi et al., 1975; Myers and Shafer,
1979; Fig. 3b). The secondary constriction of
the marker chromosome (pair 13) often appears heterochromatic in the female, as noted
by Tantravahi et al. (1975) in H. muelleri,
while in our male it was most often achromatic.
The NOR region was found a t the predicted
location, i.e., at the secondary constriction of
pair 13 (Fig. 3c, arrows), known to carry
rRNA genes in H. lar (Warburton et al., 1975)
and H. muelleri (Tantravahi et al., 1976).The
Y chromosome in the male was not silver
positive nor did it form associations with
members of pair 13. In both the male and
female frequent association was noted between the 13s.
A composite illustration of all proposed Gband homologies is shown in Figure 4, while
one example in which the ancestral form can
be determined and the subsequent rearrangement traced is illustrated in Figure 5.
As a group, the three species show significant banding pattern conservation for only
six autosomal pairs (Fig. 4a). Two of these
(HSY 1/HCO 4/HAG 6 and HSY 14MCO 16/
HAG 16) have been retained intact, while
1;HSY 11MCO 2MAG 5; and HSY 21/HCO
22/HAG 3 have diverged by chromatin loss
or addition. It is of interest that the X and Y
have undergone rearrangement in the Hylobatidae. While H. syndactylus and H. agilis
possess the typical catarrhine primate X
chromosome, H. concolor is unique in possessing a n X with a pericentric inversion,
which has placed the proximal region of the
short arm onto the long arm. As noted earlier
the Y chromosomes of both H. syndactylus
and H. concolor contain heterochromatin in
addition to a n NOR on the short arm, and
their morphology, a s shown in Figure 4a,
suggests a pericentric inversion difference
between them. The comparatively small size
of the H. agilis Y chromosome is due to the
absence of both heterochromatin and NOR
In addition to chromosomes shared by all
three species, there are additional similarities revealed when comparisons between two
species are made. Eight chromosomes of H.
syndactylus and H. concolor show some homology (Fig. 4b), three of these without modification (HSY 12/HCO 12; HSY 13MCO 14;
and HSY 15/HCO 17). Two others (HSY 3/
HCO 11and HSY 4/HCO 7) show identity for
their short arms only. HSY 18 is identical t o
HCO 8 except for the lack, in HSY 18, of the
terminal region of the long arm of HCO 8.
HSY 5 and HCO 6 share distinctive, lightly
stained regions and differ by a pericentric
inversion and the absence, in HCO 6, of the
terminal region of the short arm of HSY 5.
The banding pattern of HSY 22 is consistent
with its being the fusion product of HCO 23
and HCO 24, with concomitant loss of the
NOR region of HCO 24.
H. concolor and H. agilis share three autosoma1 pairs not found in H. syndactylus (Fig.
4c). H. syndactylus and H. agilis share only
three autosomal pairs not also found in H.
concolor (Fig. 4d). A discrepancy in terminal
staining is seen between H. syndactylus and
H. agilis, and it is important to note that the
proposed homologies of these two species are
more clearly evident when the terminal dark
(heterochromatic) bands of H. syndact-ylus are
disregarded in the comparison.
Finally, autosomes of the three species that
have no match in the group, even ignoring
heterochromatin, are presented (Fig. 4e, H.
syndactylus; 4f, H. concolor; 4g, H. agilis). At
least two facts are evident from these figures:
(1)Chromosomes with no match are distributed throughout the size range, and (2) a
relatively large proportion (%TCL) of the
genome of H. agilis remains unmatched.
In order to further quantify and evaluate
these pairwise comparisons of G-bands, we
have calculated the percentage of total chromosomal length (%TCL) represented in the
similar chromosomes of these three species.
From caliper measurements of five G-banded
cells from each animal, we estimate that H.
Fig. 4. Haploid composite showing proposed G-banded
pairings between species (a-d) and remaining unpaired
chromosomes (e-g). a. Chromosomes common to all three
species are ordered H. syndactylus (HSY; left), H concc
lor (HCO; middle), and H. agilis (HAG; right). b. Similarities between HSY (left) and HCO (right). Note fusion of
HCO 23 and 24 to form HSY 22. c. Three chromosomes
are unique to HAG (left) and HCO (right). d. Excellent
matching is evident between three chromosomes of HSY
(left) and HAG (right) when terminal heterochromatin
in HSY 19,23,and 24 is disregarded. Chromosomes from
H. syndactylus (el, H. concolor (0, and H. agilis (g) that
have no interspecific match in the Hylobatidae. (See
Discussion for explanation of differences in banding patterdsize of proposed pairings.)
syndactylus and H. concolor show conservation of G-banded chromosomes for 55-60% of
their TCL. For H. syndactylus and H. agilis
this figure drops to about 30-35%, while H.
concolor and H. agilis show obvious banding
pattern conservation for about 45% of the
total length of G-banded chromosomes. While
we have not presented the individual %TCL
figures for each chromosome, we have found
excellent agreement between the proposed
homologies based on G-band patterns and
the comparison of TCL for all such homologous chromosomes.
ships among hylobatids and between them
and other catarrhine primates have heretofore permitted only tentative statements. The
present study is the first to our knowledge to
present a comparison of G-banded karyotypes of three major hylobatid karyotypic
groups. This study reveals a degree of intergroup conservation not previously detected,
while at the same time confirming previous
reports of relatively extensive karyotypic restructuring in the Hylobatidae. In addition,
several interesting cytogenetic features
unique among catarrhine primates have been
found in this group.
While the karyotypic relationships among
the hominoids (excluding hylobatids) and the
Old World monkeys have been well characterized, karyotypic data regarding relation-
Taxonomy of hylobatidae
Chiarelli (1963, 1968) allied the Hylobatidae with the Colobinae, a leaf-eating subfamily of Cercopithecoid monkeys, on the basis
of resemblance of unbanded karyotypes. This
view has been criticized by many authors
(Groves, 1972; Andrews and Groves, 1976),
and has been dismissed on cytogenetic
grounds by comparison of R-banded preparations between Colobus uellerosus, C. palliatus, and hylobatids (Dutrillaux et al., 1981).
In a review of the biology and taxonomy of
the Hylobatidae, Groves (1972)confirms their
hominoid status and argues for subgeneric
distinction between the three recognized ma-
Fig. 5. Proposed scheme of derivation of HCO 1 and
HSY 9 from the ancestral element HAG 1. a. One arm of
HAG 1 (left) shows identity to a hominoid chromosome
(orangutan equivalent of human chromosome 7, shown
right in the brackets) that is ancestral to extant pongids
and man, thereby establishing this as the ancestral configuration in these hylobatids. b. HCO 1 (right) can be
derived from HAG 1 (left) by a single deletion (translocation ?) from this ancestral arm, with the break point
indicated by a dash. c. HSY 9 (right) shares this break
with HCO 1, and in addition is differentiated from the
latter by a deletion (indicated by dash) of the other arm,
which occurred after these two lineages diverged.
Fig. 6. Four alternative phylogenetic schemes for
three major hylobatid groups (A, H. ugilis and other “lar
group” gibbons; C, H. concolor; S, H. synductylus). a.
Three-way split, suggested by Chivers (1977) and Lucotte e t al. (1982). b. Common branch shared by “lar
group” and H. concolor, suggested by Creel and Preuschoft (1976) and Bruce and Ayala (1979). c. Common
branch shared by “lar group” and H. syndactylus, suggested by Groves (1972) and Haimoff et al. (1982). Two
jor groups (the designation adopted here). In
attempting to discern ancestral relationships
from morphological traits he tentatively ordered the divergence of hylobatids as follows:
H. concolor was the first to split from the
ancestral hylobatid stock, after which H. syndactylus and the 44-chromosomegibbons split
in the remaining branch. In this scheme,
then, H. syndactylus and H. agilis are somewhat more closely related to each other than
either is to H. concolor; that is, they share a
more recent common ancestor (Fig. 6c). The
same result has recently been obtained by
Haimoff et al. (1982)using character compatibility analysis of 55 morphological and behavioral traits. However, the results of Creel
and Preuschoft (1976) based on cranial morphometric traits seem to suggest that H. syndactylus was the first to split from the
ancestral stock, after which H. concolor and
the 44-chromosome species shared a common
ancestor for a short time (Fig. 6b).
Two biochemical studies that have encompassed all three groups likewise differ in
their conclusions. The results of electrophoretic comparison of 23 blood proteins (Bruce
and Ayala, 1979) suggest that H. lar shows
the greatest genetic identity to H. concolor
(Fig. 6b). However, in a comparison of 18
blood proteins Lucotte et al. (1982) found no
especially close relationship between any two
of the three species, suggesting all three were
equidistant from each other (Fig. 6a). Thus
no consensus on the exact branching order of
phenotypic traits, length of 0s penis (op) and scrota1 sac
morphology (ss) support this scheme in character compatibility analysis (Haimoff et al., 1982). d. Common
branch shared by H. concolor and H. syndacty1u.s (this
study). Qualitative traits of terminal heterochromatin
(h),Y chromosome NOR (n), and translocation involving
ancestral hylobatid chromosome HAG 1 (t) support this
branching order.
Hylobatidae has been achieved from previous studies of biochemical and morphological traits. However, as pointed out by
Haimoff et al. (1982) these various phylogenetic schemes have in common an early separation of both H. syndactylus and H.
concolor from a n ancestor of the 44-chromosome “lar group” (including H. agilis).
Karyology and cladistics
The cytogenetic data support this early
separation but suggest a branching of H. syndactylus and H. concolor which is different in
detail from those of previous studies. The
results of G-banding, C-banding, and silver
staining all suggest that H. concolor and H.
syndactylus share several derived cytogenetic traits not present in H. agilis or in
other 44-chromosome gibbons. To our knowledge this is the first suggestion of close phylogenetic relationship between H. concolor
and H. syndactylus (Fig. 6d).
The greatest correspondence of G-bands between any two species is that between H.
syndactylus and H. concolor (55-60% TCL).
While this is quantitatively greater than the
relationship of H. agilis to either H. syndactylus (35% TCL) or H. concolor (45% TCL), it
is difficult to assess the importance of these
figures. Of course, a more cladistic method of
assessing phylogenetic relationship would
require interpretation of the nature of the
rearrangements by which the karyotypes differ. Unfortunately chromosomal restructuring in the Hylobatidae has been so extensive
as to make it impossible t o recognize neartotal homology with confidence or to trace all
the changes from one species’ karyotype to
the next.
However, at least one G-banded chromosome, traceable in all three groups, is informative regarding the cladistic relationships
among the Hylobatidae. As seen in Figure
4a, HSY 9 and HCO 1 are similar in sharing
the same deletion from the long arm of HAG
1.The long arm of HAG 1 is probably ancestral for all hominoids as shown by R-banding
(Dutrillaux et al., 1975a) and confirmed by
us with G-banding as shown in Figure 5a.
Therefore, the simplest scheme for the derivation of these three chromosomes is that
HAG 1 is a conserved ancestral hylobatid
chromosome, and that in a lineage common
to H. syndactylus and H. concolor a deletion
of a portion of long arm (probably by translocation) occurred (Fig. 5). After separation
of H. concolor from H. syndactylus, this deriv-
ative chromosome underwent a n additional
translocation involving the other arm, resulting in the morphology seen in HSY 9.
It is abundantly clear from many studies
that heterochromatin is predominantly distributed a t or near the centromeres of chromosomes of most primates. Variation in this
pattern as seen in C-banding of platyrrhine
and catarrhine primates (Ma, 1981; Ponsa et
al., 1981; Seuanez, 1979) is usually attributable to small but detectable quantitative differences in the amounts of this heterochromatin. The pattern of primarily centromeric
C-band locations was probably ancestral in
Catarrhini, and has been retained in H. agilis
and other 44-chromosome gibbons.
The C-band distributions in H. syndactylus
and H. concolor, on the other hand, differ
from this pattern in a major qualitative way.
In these species C-bands are not only reduced
in most centromeric regions, but more significantly, large blocks of terminal heterochromatin are present in both arms of all but a
few chromosomes. The probable mechanism
for deriving this pattern is not known, but
the most parsimonious conclusion is that the
C-bands in H. syndactylus and H. concolor
were altered in a systematic fashion in a
common ancestor not shared by other hylobatids.
Terminal C-bands have also been found on
many chromosomes of the chimp and gorilla,
but there are differences apparent between
C-bands of pongids and hylobatids. In these
pongids the presence of terminal heterochromatin has not been associated with reduction
of centromeric heterochromatin as in hylobatids. In addition, the terminal C-bands of
Pan and Gorilla are not nearly so prominent
in G-banded preparations as they are in H.
syndactylus and H. concolor (Seuanez, 1979;
our own observations). These differences, and
the absence of terminal heterochromatin in
Pongo and Homo, suggest that it was an independent evolutionary occurrence in pongids and hylobatids, and do not weaken the
hypothesis that centromeric heterochromatin was the ancestral distribution in Hominoidea.
The exclusive presence in H. syndactylus
and H. concolor of a n NOR-bearing Y chromosome is a shared trait unknown in other
primates. It is found very rarely in other
mammals, where in several instances closely
related species have conserved a commonly
derived NOR-bearing Y chromosome (Pathak et al., 1982). There is no detectable sil-
ver staining of the Y chromosome in the male
H. agilis reported here, nor is there evidence
in other published reports for a n NOR-bearing Y chromosome in any 2n = 44 Hylobates.
Here too, the most parsimonious explanation
for this distribution is that the Y chromosome NOR arose in a common ancestor of H.
syndactylus and H. concolor after its separation from the branch leading to 2n = 44
Hylo bates,
It is pertinent to consider the disagreement
between this and other phylogenetic studies
of Hylobatidae. Of the contrasting techniques employed, it appears that neither the
electrophoretic procedures (Bruce and Ayala,
1979; Lucotte et al., 1982) nor the craniometric comparison (Creel and Preuschoft, 1976)
have determined rigorously the ancestral and
derived states of the traits employed. Character compatibility analysis (Haimoff et al.,
1982) is perhaps the most rigorous attempt
to deduce hylobatid phylogeny from determination of ancestral and derived morphological and behavioural traits. Although that
analysis suggested early divergence of H.
concolor from a common ancestor of H. symphalangus and H. agilis, we would like to
point out that the length of the apparent
common trunk shared by the latter two is
very short; only two character state transitions define this common trunk, whereas a t
least 20 character state transitions define
the common trunk leading to H. agilis and
other 44 chromosome gibbons. We emphasize
this to indicate that the results of their study
and ours are actually fairly close in that both
recognize a n early divergence of both H. syndactylus and H. concolor from 44-chromosome gibbons.
Although we cannot critically evaluate
character compatibility analysis, consideration of our study and of other modern karyotypic studies employing multiple banding
methods permits a reevaluation of the four
karyotypic traits among the 55 traits employed by Haimoff et al. (1982). Strictly
speaking, it is not accurate to designate as
primitive and derived the diploid numbers
and autosomal arm numbers (their first two
traits) found in hylobatids. Apparent tendencies in the same direction by two species
may or may not be commonly derived, and
this can only be determined by banding analysis. In fact, the assumptions by Haimoff et
al. (1982) concerning primitive diploid number and arm number in hylobatids are now
known to be in error in light of modern com-
parative banding analysis and have apparently led to spurious agreement between
their karyotypic traits and their phylogeny.
Their third trait, satellites (now known to be
NORs), has been shown in the present study
to be complex, and previously undetected
NORs lead to a conclusion different from that
of Haimoff et al. (1982). The presence or absence of acrocentrics (trait 4) is not independent of diploid number in these species, and
for the same reasons should be excluded.
We would like to consider the possibility
that reanalysis of the data of Haimoff et al.
(1982) with the inclusion of modern karyotypic data might bring the results of our
two studies in closer agreement. Since only
29 of 55 characters employed by that study
fully agree with their phylogeny, and the
alternative phylogenies generated by the
other 26 characters are not shown, we cannot
estimate the likelihood that a phylogeny
similar to ours would emerge from their data
upon reanalysis. Since according to Haimoff
et al. (1982) their analysis is easily repeated
with new or modified data, it seems worthwhile to pursue this promising method. Similarly, additional karyotypic data should be
sought as well from previously incompletely
described hylobatids in order to expand the
basis of karyotypic comparison.
Mechanisms of rearrangement
While karyotypic evolution in the Hylobatidae has been too extensive to allow identification of all chromosomal rearrangements
in this group, several mechanisms of rearrangement have been identified. Pericentric
inversion, the most frequent type of alteration encountered in hominoid evolution (DUtrillaux, 1975b; Yunis and Prakash, 19821, is
found in several hylobatid elements. The
most obvious of these involves the X chromosome of H. concolor (Dutrillaux, 1975a Fig.
4a), making it the the only exception to the
typical catarrhine configuration for this
Among the autosomes pericentric inversions account for a t least two differences
among the hylobatids (HSY 5 B C O 6; HSY
21/HCO 22). In addition, paracentric inversion may explain the dissimilarity in the long
arms of HSY 3/HCO 11and HSY 4HCO 7.
The metacentric chromosome HSY 22 is
apparently a tandem fusion of HC023 and
HC024, the latter of which is not strictly
acrocentric but possesses a euchromatic short
arm. A true Robertsonian centric fusion has
apparently occurred forming HAG 3. AS Figure 4a shows, one arm of this chromosome is
a precise match to HSY 21, which as noted
earlier is acrocentric and bears a n NOR. The
other arm of HAG 3 is similar to, and may
be homologous to, human (and pongid) 13
(Tantravahi et al., 19751, which is also acrocentric and bears NORs. It is reasonable to
suggest that these two acrocentrics were
present in ancestral hylobatids, and that centric fusion occurred in the lineage leading to
H. agilis and other 2n = 44 species. The
tendency of acrocentric NOR-bearing chromosomes to undergo fusion is well documented in human populations.
The existence of homology between chromosomes of disparate size (HSY 5/HCO 6;
HSY 7/HCO 15/HAG 15; HSY 11/HCO 2/
HAG 5; HSY 9/HCO l/HAG 1)suggests noncentric fission or fusion, or translocation.
HSY 7 differs from its smaller counterparts
(HCO 15/HAG 15) by a small chromatin addition, suggesting donation of a piece resulting from translocation. Conversely, HSY 11
differs from its larger counterparts (HCO 2/
HAG 5) by a small terminal deletion, with
the smaller piece presumably incorporated
elsewhere in the genome. It is unlikely that
fission and centromeric activation alone can
account for this change, since the smaller
product is not apparently present as a discrete element. It should be noted here that
the direction of change (i.e., deletion or addition) can be inferred from the fact that H.
concolor and H. agilis, which are in different
clades within the Hylobatidae, share identity
for these elements, which must therefore be
the ancestral forms for all three species.
These instances of partial homology are
the strongest evidence so far that translocation has been a factor in the differentiation
of karyotypes in the Hylobatidae, as suggested by Dutrillaux et al. (1975a) and Couturier et al. (1982). Many other karyotypic
differences that are not yet traceable are
probably also due to translocation events,
since the other classical modes of rearrangement such as inversion or fusiordfission do
not readily explain these differences.
This is a surprising observation, since only
one instance of fixation of a reciprocal translocation has been noted in other hominoids,
between chromosomes 5 and 17 in the gorilla
(Yunis and Prakash, 1982).It is puzzling why
karyotypic evolution in Hylobatidae should
be characterized by a class of rearrangement
so rarely found in other hominoids. That such
translocation may be a frequent recurring
event in hylobatids is evidenced by the finding of a H. concolor leucogenys x H. c. gabriellae subspecies hybrid that is heterozygous for a reciprocal translocation (Couturier et al., 1982).
Finally, it can be stated confidently that
fission played a role in the evolution of diploid numbers in H. syndactylus and H. concolor. It is fairly clear, based on cercopithecoidhominoid comparison (Dutrillaux,
1978a,b), that the last common ancestor of
all living Catarrhini possesed 42-48 chromosomes. To generate diploid numbers of 50
and 52 would have required at least one or
two fission events. The failure to ascertain
fission products with certainty in these species may indicate rearrangement subsequent
to such fission.
Intraspecific variation
The finding of a H. agilis with two apparent inversion heteromorphisms is noteworthy, given the small number of animals
studied. One of the inversions in the present
study is identical to that reported by Tantravahi et al. (1975) in a H. muelleri, the only
other such report in 2n = 44 hylobatids. A
review of the banded karyotypes of 2n = 44
gibbons in previous published reports suggests maintenance of two morphs of chromosome 8 in living populations. Three Hylobates
lar (Tantravahi et al., 1975; Benirschke and
Hsu, 1977; Warburton et al., 1975), three H.
pileatus (one by Dutrillaux et al., 1975a; two
by our own unpublished observations), and
one termed “gibbon” (Pearson, 1973) are apparently homomorphic for the submetacentric form, while one H. agilis (present study)
is homomorphic for the metacentric form. A
H. agilis (present study) and a H. muelleri
(Tantravahi et al., 1975) are heteromorphic
for this pair-i.e., both forms are present. A
H. muelleri abbotti, male parent to a siamang-gibbon hybrid (Myers and Shafer,
1979), was evaluated from G-banded photographs (kindly provided by David Shafer),
and appears also to be heteromorphic for pair
8. Thus, individuals assigned to two H y l e
bates species (H. muelleri and H. agilis) probably possess both forms of chromosome 8.
Several explanations may be offered for
this finding. Since these two groups are
placed in H. lar a s subspecies by Groves
(1972), the inversion may reflect a widespread polymorphism maintained in the various populations of H. lar. Alternatively, the
two forms of 8 may actually distinguish different subspecies of H. lar, with heteromorphism occurring mainly by hybridization
in captivity. This is now known to be the
situation between Sumatran and Bornean
subspecies of orangutans, which have been
geographically separated for at least 8,000
years and which differ by a n inversion (Seuanez, 1979). However, if H. muelleri and H.
agilis are distinct species as Haimoff et al.
(1982)recognize, then the maintenance of the
same polymorphism in both is probably evidence of the relative recency of their separation. Many more gibbons of verifiable provenance will have to be studied in order to
determine the relationship of chromosome
variation to population structure.
The degree of detectable karyotypic similarity between three major groups of Hylobatidae has been greatly increased with the
aid of multiple chromosome banding methods. Considerable disparity still exists, however, between the banded karyotypes of these
three groups, and there is now stronger evidence that translocation has been a major
factor in karyotypic evolution in this family.
The existence of two heteromorphisms in a
single individual in this study is in accord
with other studies showing structural heteromorphisms, suggesting that extant populations of hylobatids are karyotypically variable. Karyotypic differences between H. syndactylus, H. concolor, and H. agilis (and other
“lar group” species) are consistent with previous assignments of these three to distinct
subgenera. However, all three banding methods employed reveal shared derived traits
that suggest the existence of a common
ancestor to H. syndactylus and H. concolor
not shared by H. agilis and other 44-chromosome species, a phylogenetic scheme which
differs from other recent schemes based on
morphological or biochemical comparisons.
The authors would like to thank the Houston Zoological Gardens and the Henry Vilas
Zoo, Madison, for their cooperation in obtaining tissue specimens used in this study. We
would like to thank Dr. T.C. Hsu for much
help and encouragement throughout the duration of this study. We thank Dr. Leonard
Prouty and Dr. Elliott Haimoff for providing
data prior to publication. The invaluable assistance of Chris Fuscoe in the preparation
of this manuscript is gratefully acknowledged. This work was supported in part by
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