Aust. J. Zool., 1983, 31, 533-40
Some Karyotypes of Australian Agamids
(Reptilia : Lacertilia)
Geofley J. Witten
Department of Anatomy, University of Sydney, N.S.W. 2006.
Abstract
Karyotypes of 22 species of Australian agamids are described. Most of these exhibit a
macrochromosome complement of six pairs. Lophognathus differs in having 10 pairs of
macrochromosomes. Most Australian agamids possess 20 microchromosomes but Physignathus and
Gonocephalus retain 24. This difference confirms the division of the Australian Agamidae into a large
endemic radiation and a small group more closely related to Asian genera.
Introduction
The phylogenetic relationships within the Australian Agamidae are not well known.
The study of karyotypic variation within some groups has been useful in elucidating
relationships not readily apparent by the study of morphology (e.g. King 1977). Only two
descriptions of Australian agamid karyotypes have been published (Witten 1978; King
1981). An investigation of the karyotypes of Australian agamids was undertaken to
determine whether phylogenetic relationships could be demonstrated by karyotypic
patterns.
Materials and Methods
Most karyotypes were prepared by an in vivo technique as follows:
Blood was removed from the animal by means of a capillary tube inserted into the posterior angle
of the eyelids, and pushed posteriorly into an orbital blood sinus. This was assumed to promote an
immune response involving cell division in the spleen. An intraperitoneal injection of 0 1%colchicine
was administered, at a dosage rate of 0.05 ml per gram body weight. The animal was killed 5 h after
the colchicine injection. The spleen, and in males a testis, was removed, chopped finely and placed
in 0.9% sodium citrate solution for 10 min. Air-dried smears were prepared by the techniques of
Baker et al. (1971), being fixed in 3 : 1 methanol :acetic acid. The smears were stained in a 4% aqueous
solution of Gurr's improved Giemsa stain for 10- 15 min, and mounted in canada balsam after being
dried for at least 1 h on a hotplate at 40-45OC.
Some early results were obtained by a method similar to that of Baker et al. (1971). Bone marrow
from the femur was flushed out with 0 9% sodium citrate solution, after the animal had been bled
and treated with phytohaemagglutinin for 2 days. Results from this method were poor, with the
exception of those for Physignathus lesueurii, and the method described above was subsequently
developed.
At least 10 divisions were examined from each preparation. The clearer of these preparations were
photographed under oil immersion. The photomicrographs were printed and the lengths of
chromosomes measured with dial calipers used in a stepwise fashion. The total length of each
chromosome arm was recorded, and the percentage of total macrochromosome length and centromeric
indices were calculated for each division. Where possible somatic preparations were used for analysis,
0004-959X/83/040533$02.00
but many second meiotic divisions were also used. Some meiosis I cells were used to obtain estimates
of relative chromosome lengths, although the centromeres were not observable on such preparations.
(c)
(4
Fig. 1. Chromosome figures: ( a ) somatic complement from bone marrow of Physignathus lesueurii;
(b) first meiotic division from testis of Pogona barbata; (c) second meiotic division from testis of
Amphibolurus muricatus; (d) second meiotic division from testis of Lophognathus gilberti. There are
24 microchromosomes in (a), but only 10 in (b) and (c). The two largest microchromosomes in (c)
appear to be metacentric. The break in size between macro- and microchromosomes is less distinct
in (d) than in the other types. Scale line, 10 pm.
Results
The nomenclature adopted for this paper is that of Storr (1982), except that the genera
Lophognathus and Amphibolurus are retained [for justification see Witten (1982a)I.
Karyotypes of 90 individuals of 22 species were obtained. Three distinct karyotypes were
encountered.
Physignathus lesueurii and Gonocephalus spinipes possess 12 metacentric macrochromosomes and 24 microchromosomes (2n = 36; 12M, 24m), with a distinct break in
size between the larger and smaller chromosomes (Fig. la). The second largest pair of
macrochromosomes is submetacentric.
Karyotypes of Australian Lizards
The great majority of endemic Australian species possess a karyotype similar to that
of Physignathus, but with four fewer microchromosomes (2n = 32; 12M, 20m; Figs lb,
lc). This karyotype is present in species of Amphibolurus, Diporiphora, Chlamydosaurus,
Ctenophorus, Pogona and Tympanocryptis (Table 1).
Table 1. Karyotypes of Australian agamids
Species
Total
Number of animals
Somatic
Meiotic
cells
cells
2n
Description
Gonocephalus spinipes
Physignathus lesueurii
Tympanocryptis tetraporophora
T. diemensis
Ctenophorus decresii
C. pictus
C. fordi
C. femoralis
C. isolepis
C. cristatus
C. clayi
C. nuchalis
Pogona barbata
P. vitticeps
Amphibolurus muricatus
A, nobbi
Chlamydosaurus kingii
Diporiphora australis
D. bennettii
D,bilineata
D. magna
Lophognathus g, gilberti
L. g. centralis
*One L. g. gilberti was studied. The few cells on the preparation showed a number of telocentric chromosomes,
which were seen in the above species only in this specimen and in L. g. centralis. An accurate count was not possible.
Lophognathus gilberti centralis possesses a karyotype with a diploid number of 2n =
40. There is a less distinct break in size between macrochromosomes and microchromosomes (Fig. Id), but there appear to be 20 macrochromosomes and 20 microchromosomes. Most macrochromosomes are telocentric or subtelocentric, with only three
pairs metacentric. Some of the larger microchromosomes also appear to be metacentric.
Analysis of chromosome lengths did not reveal consistent differences among those taxa
with 12 macrochromosomes (Table 2). In all such species analysed the second largest pair
of chromosomes is submetacentric(Table 2). All other macrochromosomes are metacentric.
At least the larger microchromosomes appear to be metacentric in those species with only
20 microchromosomes (Fig. 1).
The Lophognathus karyotype was more difficult to analyse. The larger number of
chromosomes meant that fewer cells were suitable for measurement, as chromosomes were
more often superimposed on one another. The largest two chromosomes represent a
proportion of the total macrochromosome length similar to pairs I11 and IV in the other
agamid species analysed (Table 3).
Discussion
Data have been published for 36 agamid species in 12 genera (Table 4). Several agamid
genera possess the karyotype suggested by Gorman (1973) to be primitive for lizards, with
AData from Krishna Rao and Aswathanarayana (1979) included for comparison.
Physignathus lesueurii
Tympanocryptis diemensis
Amphibolurus muricatus
A. nobbi
Ctenophorus decresii
C. pictus
C. fordi
Pogona barbata
P. vitticeps
Psammophilus dorsalis+
Calotes versicolofi
Species
Physignathus lesueurii
Tympanocryptis diemensis
Amphibolurus muricatus
A. nobbi
Ctenophorus decresii
C. pictus
C. fordi
Pogona barbata
P. vitticeps
Psammophilus dorsalis
Calotes versicolor
Species
Table 2. Relative macrochromosome lengths, and centromeric indices of agamids
L, length as percentage of total macrochromosome length, N, number of divisions analysed. Ic, centromeric index
Karyotypes of Australian Lizards
12 metacentric macrochromosomes and 24 microchromosomes. In fact, this karyotype is
probably better considered as primitive only for the suborder Iguania (King 1981).
The form of the macrochromosomes appears to be remarkably similar in all those
agamids retaining six pairs. The second largest pair is submetacentric and the other five
are metacentric. A comparison between the analyses of Krishna Rao and Aswathanarayana
(1979) for two Indian agamids and the Australian species with 12 macrochromosomes
(Table 2) reveals few differences. The centromeric indices for the Australian species are
lower for the second pair, but the absence of statistical data for the Indian species means
that no meaningful comparison is possible. The general description of the form of the
macrochromosomes is apparently applicable to most other agamids with six pairs of
macrochromosomes. Figures in Moody and Hutterer (1978; Lyriocephalus), Sokolovsky
(1975; Agama) and Ha11 (1970; Leiolepis) all show the second largest macrochromosome
as submetacentric. The same basic structure occurs also in at least some iguanids (Gorman
1973).
Table 3. Lophognathus gilberti centralis chromosome data
Percentage is that of total macrochromosome length. Ic, centromeric index
Chromosome
number
Relative length
SD
Percentage
Centromeric index
Ic
SD
I
I1
111
IV
v
VI
VII
VIII
IX
X
Most agamids with more than 12 macrochromosomes have largely telocentric or
submetacentric chromosomes. Most of these karyotypes are at least theoretically derivable
from the primitive arrangement by centric fission, the primitive complement of 24
macrochromosome arms being retained in most species. For example, Japalura (Makino
and Momma 1949) and most Phrynocephalus (Sokolovsky 1974) have 24 telocentric
macrochromosomes. Changes in the number of macrochromosome arms have more often
been reductions. Gorman and Shochat (1972) reported reductions in some species of
Agama: 22 in A. agama, A. sanguinolenta and A. sinaita; 18 in A. atricollis [(A.
sanguinolenta from Sokolovsky (1975)l. Phrynocephalus contains the other two species
which depart from the normal number of macrochromosomal arms: P. helioscopus with
a reduced number of 22 and P, mystaceus with an increased number of 26 (Sokolovsky
1974). Lophognathus gilberti is thus the only other species with an increased number (26)
of macrochromosomal arms. Although P. mystaceus and L. gilberti share the same number
of arms, the karyotypes are quite different (P. mystaceus: 2V+221+24m; L. gilberti:
6V+ l4I+2Om).
The Lophognathus karyotype could have been derived from the primitive karyotype
with centric fissions in four of the six pairs of chromosomes. A pencentric inversion could
subsequently have caused the ninth pair to become metacentric. However, the increase
in the number of macrochromosomal arms suggests that other mechanisms may have been
involved. Lophognathus exhibits several morphological features which are almost certainly
derived with respect to other Australian agamids (Witten 1982a). Its karyotype is therefore
unlikely to be primitive.
Many agamid species previously studied have 24 microchromosomes. A significant
minority have a reduced microchromosome complement of 22 (Table 4). Only
Table 4. Published karyotype data on the family Agamidae
Description: V, metacentric or submetacentric macrochromosomes; I, telocentric or subtelocentric macrochromosomes; m, microchromosomes
Species
Agama agama
A. atricollis
A. caucasica
A.
A.
A.
A.
A.
erythrogastra
hymalayana
lehmanni
planiceps
pa Nida
A. ruderata
A. sanguinolenta
A. savignii
A. sinaita
A. tuberculata
Amphibolurus nobbi
Calotes versicolor
C, jerdoni
Ctenophorus caudicinctus
Japalura swinhonis
J. polygonata
Leiolepis belliana
Lyriocephalus scutatus
Phrynocephalus guttatus
P. helioscopus
P. interscapularis
P. mystaceus
P. raddei
P. reticularis
P. rossikowi
P. versicolor
Psammophilus dorsalis
Ptyctolaemus gularis
Sitana ponticeriana
Uromastyx aegyptius
U, hardwickei
U. ornatus
2n
Description
Source
Gorman and Shochat 1972
Gorman and Shochat 1972
Arronet-Kulikova 1965A
Sokolovsky 1975
Sokolovsky 1975
Sokolovsky 1975
Sokolovsky 1975
Gorman and Shochat 1972
Gorman and Shochat 1972; Gorman
1973
Bhatnagar and Yoniss 1977
Sokolovsky 1975
Gorman and Shochat 1972; Gorman
1973
Gorman and Shochat 1972; Gorman
1973
Matthey 1931A
Gorman and Shochat 1972
Dutt 1969A
Witten 1978
Makino and Asana 1948; Knshna Rao
and Aswathanarayana 1979; S h m a
and Nakhasi 1980
Sharma and Nakhasi 1980
King 1981
Nakamura 1935A
Makino and Momma 1949
Hall 1970
Moody and Hutterer 1978
Sokolovsky 1974
Arronet-Kulikova 1965A
Sokolovsky 1974
Sokolovsky 1974
Sokolovsky 1974
Sokolovsky 1974
Sokolovsky 1974
Sokolovsky 1974
Sokolovsky 1974
Krishna Rao and Aswathanarayana
1979
Sharma and Nakhasi 1980
Makino agd Asana 1948
Gorman and Shochat 1972
Matthey 1931A; Sharma and Nakhasi
1980
Makino and Asana 1948
Gorman and Shochat 1972
-
ARde Gorman 1973.
Lyriocephalus and Psammophilus had previously been reported to possess fewer than 22
microchromosomes. In the case of Lyriocephalus, Moody and Hutterer (1978) suggested
that several microchromosomes may have fused to form the larger, apparently metacentric
Karyotypes of Australian Lizards
539
microchromosomes of that genus. This explanation is apparently not applicable to
Psammophilus, where all microchromosomes are telocentric (Krishna Rao and
Aswathanarayana 1979). These latter authors suggested that the extra two or four
microchromosomes may have fused with the macrochromosomes, although they presented
no data to substantiate this claim.
Of the Australian species karyotyped, Physignathus and Gonocephalus possess a 'full'
complement of 24 apparently telocentric microchromosomes. The other species karyotyped,
including Lophognathus, possess 20 microchromosomes, the largest pair of which are
apparently metacentric. It therefore appears likely that the reduced complement of these
species is due to the fusion of four pairs of telocentric microchromosomes to form two
pairs of slightly larger metacentric microchromosomes (Fig. lc).
Centric fission appears to be a common phenomenon in agamid karyotypes. There are
at least three different karyotypes reported in the literature which may represent the results
of centric fission (Table 4). The different degrees of fission within Phrynocephalus
(Sokolovsky 1974) and Agama (Gorman and Shochat 1972) probably represent separate
evolutionary events, demonstrating a predisposition in at least these genera towards centric
fission. The centric fissions presumed to have occurred in the evolution of the Lophognathus
karyotype are therefore not unusual for the family. However, the reduced number of
microchromosomes in the Lophognathus karyotype indicates a close relationship with most
other Australian agamids.
The reduced number of microchromosomes in all Australian agamids except
Physignathus and Gonocephalus suggests that these species represent a single radiation.
The only other species with a karyotype of 2n = 32, Psammophilus dorsalis (Krishna Rao
and Aswathanarayana 1979), differs from the Australian radiation in the possession of
telocentric microchromosomes. The other karyotype which resembles that of the Australian
radiation, Lyriocephalus (Moody and Hutterer 1978), does not correspond exactly with
the most common Australian configuration. There is a strong possibility that the number
of microchromosomes was reduced in a similar way in the two groups, but the reduction
has been greater in Lyriocephalus. The differing karyotypes increase the probability that
they represent completely separate but parallel evolutionary events. There is little other
information which suggests that Lyriocephalus and the Australian radiation are related.
The Australian agamid fauna appears to be made up of two groups. Most agamids
belong to a large radiation, which appears to have evolved entirely within the Australian
region (Witten 1982a, 1982b). This group is characterized by a karyotype possessing 20
microchromosomes, including two pairs which are metacentric. The other group has
apparently amved in Australia relatively recently, and is represented by relatively few
species. The latter group is characterized by the possession of the presumed primitive
iguanian karyotype.
Acknowledgments
I thank Jim Bull for introducing me to the basic techniques of karyology. M. J. Blunt
and H. G. Cogger provided assistance and guidance throughout this study. Drafts of this
paper were read and criticised by M. Arnold and L. A. Moffat. B. Bowdern and M. A.
Witten assisted in the collection of specimens. Financial and technical assistance were
provided by the Department of Anatomy, University of Sydney.
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Manuscript received 9 August 1982; accepted 24 December 1982