Nucleolar organiser region (NOR) location in karyotypes of Australian ground frogs
(Family Myobatrachidae)
M. J. Mahony & E. S. Robinson
School of Biological Sciences, Macquarie University, North Ryde, New South Wales 2113, Australia
Abstract
Nucleolar organiser regions (NORs) were examined in over 90~ of the species of Australian ground frogs
(familiy Myobatrachidae), including representatives from all twenty currently recognised genera and the three
subfamilies. Throughout the family, location of the NOR within the karyotype showed considerable variation
yet karyotype morphology showed uniformity. The precise mechanism(s) whereby variation in NOR location
evolved while karyotype morphology was unchanged remains uncertain. Comparison of the two major subfamilies showed that the Limnodynastinae had a greater diversity of NOR location than the Myobatrachinae.
The limnodynastine genus, Heleioporus, was the only one to show multiple NOR sites in several species.
NOR location was particularly stable within most polytypic genera. Differences in NOR location within the
remaining polytypic genera (Heleioporus, Limnodynastes, Neobatrachus, Philoria'and Ranidella) pointed to
taxonomic discriminations that were generally consistent with recent proposals based on other criteria.
Introduction
Ribosomal DNA genes, usually present as tandem repeats, code for the rRNA of interphase
nuleoli and the region of a chromosome containing
these genes is termed the nucleolar organiser region
(NOR). NORs can be easily detected in metaphase
chromosomes by a quick and simple treatment with
silver nitrate (Goodpasture & Bloom, 1976) which
appears to stain nucleolar phospho-proteins B23
and C23 associated with rDNA transcription
(Schwarzacher & Wachtler, 1983, Busch et al.,
1982). A few examples of silver staining of chromosomal regions other than NORs have been
reported (Varley & Morgan, 1978; Medina et al.,
1983; H a a f et al., 1984) but in the vast majority of
cases it is specific for those rDNA genes that were
actively transcribing during the previous interphase. A comparison of silver stained mitotic
metaphase spreads with those conventionally
stained with for example, aceto-orcein or Giemsa
Genetica 68, 119-127 (1986).
~ Dr W. Junk Publishers, Dordrecht. Printed in The Netherlands.
shows that NORs are usually located within regions
long referred to as secondary constrictions. Silver
stained NORs are however more readily identified
tllan are secondary constrictions and not all secondary constrictions silver stain (see later). It is the
specificity of silver staining for a complex gene locus that makes the NOR potentially useful as a
chromosome marker for cytogenetic and cytotaxonomic studies.
In studies which have investigated the location of
the NOR in anurans one feature has consistently
emerged; in closely related species (species complexes or species groups), the NOR is almost always
localised in the same region of the same chromosome pair. Schmid (1983) has argued that, 'exceptions to this rule gave indications of chromosomal
rearrangements having occurred in the NORcarrying chromosome segments in the evolution of
the Anura'. Schmid (1978 a & b, 1982) examined
species from a wide variety of genera and families,
while King (1982) concentrated on the members of
120
a single but large and diverse genus. To date no
study has examined NOR location in an extensive
array of genera from one family. The Australian
myobatrachid (ground) frogs are a suitable group
for such a comparative study of NOR location.
These frogs are generally recognised as an old
southern fauna which has evolved in isolation on
the Australian continent since the breakup of
Gondwanaland. Furthermore, this family has in recent times been the subject of comparative studies
in morphology, biochemistry and behavior, which
have resulted in various taxonomic revisions.
Karyotypic analysis has provided little assistance in
taxonomic resolution because of the well known
stability in diploid number, relative chromosome
lengths and morphology. However, Morescalchi
and Ingram (1978) used the limited karyotypic data
to point to some generic affinities. For a summary
of the limited previous work on the chromosomes
of myobatrachid frogs see Mahony and Robinson
(1980).
The present study was undertaken (a) to establish
the extent of variation in NOR location in a large
number of genera and species of Myobatrachidae,
and (b) to determine whether NOR location has
any taxonomic and phylogenetic value, particularly
at the generic level, in a family where the current
state of nomenclature and phylogenetic relationships are distinctly unstable (Tyler, 1979).
Materials and methods
lus, Lechriodus Loveridge, 1935; L. fletcheri, L.
aganoposis, L. melanopyga, Limnodynastes Fitzinger, 1843. (peroni group)L, convexiusculus, L.
fletcheri, L. peroni, L. tasmaniensis, (dorsalis
group) L. dorsalis, L. dumerillii, L. interioris, L.
terraereginae, (ornatus group)L, ornatus, L. spenceri," L. salmini. Megistolotis Tyler, Martin & Davies, 1979; M. lignarius. Mixophyes Gunther, 1864;
M. balbus, M. fasciolatus, M. iteratus, M. schevilli.
Neobatrachus Peters, 1863; N. aquilonius, N. centralis, N. pelobatoides, N. pictus, N. sudelli, N. sutor, N. wilsmorei. Notaden Gunter, 1873: N. benettii, N. melanoscaphus, N. nichollsi. Philoria
Spencer, 1901; P. frosti, P. kundagungan, P.
Ioveridgei, P. spagnicolus.
Subfamily Myobatrachinae
Arenophryne Tyler, 1976; A. rotunda. Assa Tyler,
1972; A. darlingtoni. Crinia Tschudi, 1938; C. georgiana, Geocrinia Blake, 1973; G. leai, G. laevis, G.
lutea, G. rosea, G. victoriana. Metacrinia Parker,
1940; M. nichollsi. Myobatrachus Schlegel, 1850;
M. gould#. Paracrinia Heyer and Liem, 1976; P.
haswelli. Pseudophryne Fitzinger, 1843; P. australis, P. bibroni, P. coriacea, P. corroboree, P. dendyi,
P. guentheri, P. major, P. occidentalis, P. semimarmorata. Ranidella Girard, 1853; R. bilingua, R.
glauerti, R. insignifera, R. parinsignifera, R.
pseudinsignifera, R. remota, R. riparia, R. signifera, R. tasmaniensis. Taudactylus Straughan &
Lee, 1966; T. acutirostris, 77. eungellensis, 77. liemi,
T. rheophilus. Uperoleia Gray, 1841; U. crassa, U.
inundata, U. laevigata, U. lithomoda, U. rugosa.
Animals
Frogs were collected between 1979 and 1984 from
localities across the Australian continent with most
sites in the south-eastern and south-western
regions. Identifications were confirmed where
necessary from Cogger (1983) and the taxonomy
adopted here is the same as that of Cogger et aL,
(1983). Identified specimens have been deposited in
the Australian Museum, Sydney, New South Wales.
The chromosomes of the following species were
examined.
Subfamily Limnodynastinae
Adelotus Ogilby, 1907; A. brevis, Heleioporus
Gray, 1941; H. albopunctatus, H. australiacus, H.
barycragus, H. eyrei, H. inornatus, H. psammophi-
Subfamily Rheobatrachinae
Rheobatrachus Liem, 1973; R. vitellinus
Techniques
Live sl~ecimens were transported to the laboratory and processed as soon as possible after capture.
Mitotic spreads were obtained from duodenal
epithelium using a technique described by Mahony
and Robinson (1980). At least five karyotypes were
prepared and measured for each species usually
from several specimens. Relative lengths of chromosomes were calculated as a percentage of total
haploid length. For silver staining of mitotic
spreads the method of Bloom and Goodpasture
(1976) was used.
121
(Relative Length range 16% to 80/0) while the rem a i n d e r are s m a l l e r (R.L. range 7 % to 4 % ) . M o s t
c h r o m o s o m e s are m e t a c e n t r i c b u t s u b m e t a c e n t r i c s
a n d s u b a c r o c e n t r i c s are quite c o m m o n a n d
a c r o c e n t r i c s o c c u r in a few species. M i c r o c h r o m o s o m e s a n d h e t e r o m o r p h i c sex c h r o m o s o m e s were
not observed. A t y p i c a l k a r y o t y p e ( 2 n = 2 4 ) is
s h o w n in Fig. 1.
In Table 1 the site o f the N O R is represented di-
Results
L Limnodynastinae
C h r o m o s o m e n u m b e r is not u n i f o r m t h r o u g h o u t
this subfamily. M o s t o f the 40 species e x a m i n e d
have a d i p l o i d n u m b e r o f 2n = 24 b u t a few have 22.
K a r y o t y p e s o f the m a j o r i t y o f genera show two distinct size classes: c h r o m o s o m e s 1 to 6 are large
a
II
;; ;;
xx
6
!
b
1
4
6
7
12
C
I
X|
7
XX
*8
**
*~*
**
12
r
"1
Fig. 1. Typical karyotypes of the three subfamilies: (a) Limnodynastinae (Adelotus brevis);-(b) Myobatrachinae (Pseudophryne corroboree);-(c) Rheobatrachinae (Rheobatrachus vitellinus). The NOR bearing pair is shown to the right in each case. Arrowheads indicate the major secondary constrictions and arrows the silver stained NORs. Scale bar represents 10 u.m.
122
Table 1. Variation in NOR location in the subfamily Limnodynastinae (excluding the genus Heleioporus).
Genus
A delotus
Number of species
Haploid chromosome number and NOR location
Total
1
1
Examined
2
3
4
5
6
7
8
9
10 11 12
1
II
o
Lechriodus
4
3
Limnodynastes peroni group
5
4
dorsalis group
5
5
ornatus group
2
2
(-)
1
(-)
(-)
LI
(L. salmint)
Megistolotis
1
1
Mixophyes
4
4
Neobatrachus
7
6
iN. centralis)
1
Notaden
3
3
Philoria
4
3
II
(P. frostO
1
agrammatically for each genus (except Heleioporus
which is shown in Table 2). Location of the N O R
is quite variable, with 13 different sites and 8 chromosomes involved (Table 1). No particular pair or
chromosomal site appears to be favoured.
The two monotypic genera (Adelotus, and
Megistolotis) have distinctive NORs as do the polytypic genera Lechriodus, Notaden and Mixophyes.
NOR location is variable within four of the polytypic genera: in both Neobatrachus and Philoria a
single species is different; in Limnodynastes recognisable species groups are involved; and in
Heleioporus variation includes multiple N O R sites
in addition to differences in N O R location.
1 2 3 4
5
1
5
2
6
7
7
8
9
10 11 12
9
10 11 12
In Table 2 the site of the NORs is represented diagrammatically for each species of Heleioporus.
Four species, H. albopunctatus, H. eyrei, H. inoratus and)H, psammophilus, have NORs in the same
position on chromosomes 1, 2, 3 and 11. H. albopunctatus and H. eyrei have an additional NOR
on c h r o m o s o m e 4. H. australiacus has only one
N O R site, but it is notable that this site is the same
as that observed on c h r o m o s o m e 11 in the four species above. The remaining species, H. barycragus,
has only one NOR, which is not located in any of
the sites identified in the other species.
123
Table 2. NOR location and c h r o m o s o m e morphology within the genus Heleioporus.
Species
C h r o m o s o m e morphology and N O R location
1
2
3
4
5
6
7
8
9
10
11
12
H~H~HHHHHH~
H. albopunctatus
H. eyrei
H. inornatus
H~NHHHH~~
HHNHHHHH~,~
HNHHHHH~~
H. psammophilus
H. australiacus
H. barycragus
a
1
2
3
7
6
11
12
r
!
r
I
b
-11-1t-Ii
!
I|
7
2
iI
3
II
it
II
6
s~ .,Jr ~.
11
12
Fig. 2. Karyotypes of Heleioporus psarnmophilus stained with (a) aceto orcein, (b) silver. Arrowheads indicate the major secondary
constrictions and arrows the silver stained NORs. Scale bar represents 10 p.m.
124
IL Myobatrachinae
All 39 species examined had a diploid number of
2n = 24 and little variation in karyotype m o r p h o l o gy was observed throughout the subfamily. Almost
all species possessed a karyotype similar to that
shown (Fig. 2), with two size classes: a group of
larger chromosomes - Nos. 1-6 (Relative Lengths
range 15~ to 9~ and a group of smaller chromosomes - Nos. 7-12 (R.L. range 7~ to 4~
The
only clear departure from this standard karyotype
was that in a few genera c h r o m o s o m e 12 was distinctly smaller than the rest.
In Table 3 the site of the N O R is represented di-
agrammatically for each genus. Location of the
N O R is more stable than that observed in the Limnodynastinae with 6 of the 12 genera having the
N O R on the short arm of c h r o m o s o m e 4, but the
variation is still considerable with the N O R located
on 5 chromosomes in the remaining genera.
Five genera are polytypic; Geocrinia, Pseudophryne, Taudactylus, Uperoleia and Ranidella
and with the exception of Ranidella, N O R location
is stable within each genus. In Ranidella, two species, namely R. remota and R. tasmaniensis have
N O R locations different from one another and
from other members of the genus.
Table 3. Variation in NOR location in the subfamily Myobatrachinae.
Genus
Number of species
Haploid chromosome number and NOR location
Total
1 2 3 4 5 6 7 8 9 1 0 1 1 1 2
Examined
A renophryne
I
1
A ssa
1
1
Crinia
1
1
Geocrinia
5
5
Metacrinia
1
1
Myobatrachus
1
1
Paracrinia
1
1
Pseudophryne
10
10
Ranidella
13
7
(R. rernota)
1
(R. tasrnaniensis)
1
Taudactylus
Uperoleia
5
4
15
6
H
H
H
H
M
2 3 4 5
2
45
67
89
101112
1112
125
III. Rheobatrachinae
This subfamily includes only the genus Rheobatrachus which until recently consisted only of the
type R. silus. The karyomorphology of R. silus was
presented by Morescalchi and Ingrain (1974). A
new species, Rheobatrachus vitellinus, found and
identified by the senior author has been described
and the chromosomes of one specimen have been
examined (Mahony et al., 1984).
Both Rheobatrachus species have a diploid number of 2n=24, with the chromosomes in two size
classes: a group of larger chromosomes - Nos. 1 to
6 (Relative Length range 15% to 9%) and a group
of smaller chromosomes - Nos. 7 to 12 (R.L. range
6% to 3%). Variation in chromosome morphology
occurs between R. silus and the newly described
species; chromosome 6 is acrocentric, and 10 metacentric in R. silus (Morescalchi & Ingram, 1974),
whereas in the newly described species they are submetacentric and acrocentric respectively. The NOR
is located on the short arm of pair 6 in the new species, but its position in R. silus is unknown.
Discussion
Viewing the family as a whole, the major conclusion to be drawn from this comprehensive survey of
myobatrachid karyotypes is that diversity in NOR
location is accompanied by uniformity in karyotype morphology. No particular chromosome, or
chromosome arm appears to show a preference for
NOR location, indeed the NOR is found on almost
all members of the haploid complement but relative chromosome sizes and centromere positions
were remarkably constant. To account for this
NOR variation throughout the family against a
background of karyotypic homogeneity, the 'conventional' types of restructuring such as translocations and inversions are unappealing mechanisms,
since they would be expected to lead to a spectrum
of changes in karyotype morphology. Other
mechanisms, for example multiple cryptic structural rearrangements or minute insertions (King,
1980), reintegration of amplified rDNA genes during oogenesis (Nardi et al., 1977; Schmid, 1978) or
activation of latent nucleolar sites (King, 1980) may
well be involved in myobatrachids but in the ab-
sence of adequate G-banding data the actual source
of NOR transposition or activation remains uncertain. Unfortunately these animals have proved to be
extremely resistant to adequate G-banding.
Early in this study it became clear that NOR location was uniform within many lower-order taxa.
Schmid (1978 a, b) and King (1980) had already observed in other anuran families that NOR position
was stable amongst closely related species or within
species complexes. As our work on myobatrachids
expanded, we found that NOR uniformity extended
to the generic level. Indeed, so consistent was NOR
location within almost all polytypic genera that we
examined the status of exceptional species in detail
to see if NOR position could be a useful taxonomic
indicator. We consider that the NOR evidence supports some generic groupings not widely accepted
in the current literature.
Perhaps the clearest example of NOR uniformity
reflecting generic limits is shown in the genus Limnodynastes, the type genus of the subfamily Limnodynastinae. This genus is currently divided into
three species groups, namely the 'peronf, 'dorsalis'
and 'ornatus' groups although Tyler et al., 1979
suggested that the groups 'possibly merit elevation
to generic status' based on internal morphology.
The NOR data support this elevation in that the
members of each group share an NOR location distinct from the other two groups. The degree of
difference in NOR position between the three
groups is at least as great as differences between
many genera in the family and in the case of the
'dorsalis' and 'ornatus' group, generic status is further strengthened by a difference in chromosome
number. Names are available for the three groups
(proposed earlier on morphological grounds) and
the following suggestions are made: Limnodynastes Fitzinger, (sensu stricto) 1841 refers to
the 'peronf group; Platyplectron Peters, 1863 is the
first available name to refer to the 'dorsalis' group;
and Platyplectrum Gunther, 1863 refers to the 'ornatus' group. One species currently regarded as a
member of the 'peronl~ group (Limnodynastes
salmint) has a sufficiently distinct NOR location to
warrant inclusion in a new genus.
Several other generic revisions in the subfamily
Limnodynastinae are favoured on the basis of
NOR location. A division of Philoria agrees with
the recognition of a separate genus Kyrannus
Moore, 1958 for the three species that have their
126
NOR on chromosome 1, but the validity o f the genus name is under question (Cogger, 1983). A division of Neobatrachus would resuIt in generic separation of N. centralis and in the genus Heleioporus,
one species, H. barycragus shows a distinctive site
and may also warrant separate generic status.
In the subfamily Myobatrachinae NOR location
is particularly stable within genera with the exception of two species of Ranidella (R. remota and R.
tasmaniensis). The genus Raniclella has been the
centre of a number of morphological studies and
the generic affiliations of several species remain uncertain (see Thompson, 1981). In fact, even the use
of the name Ranidella (rather than Crinia) has recently been challenged (Heyer et al., 1983). On the
basis of NOR location, R. remota and R. tasmaniensis should be removed from the genus
Ranidella.
Proposals regarding the phylogenetic relationships o f myobatrachid genera based on NOR location alone cannot be made without a knowledge of
the chromosomal mechanisms involved and their
temporal sequence. However, some general statements can be made about likely ancestral and derived states, particularly when taken together with
some recent microcomplement fixation (MCF) data
of Daugherty and Maxson (1983). For example, the
genera in the subfamily Myobatrachinae are more
conservative in NOR location than the Limnodynastinae. Six myobatrachine genera have the
NOR located on the same arm of the same chromosome and in four of these genera (Arenophryne,
Metacrinia, Myobatrachus and Pseudophryne) it
appears to be in exactly the same position. Immunological studies (Daugherty & Maxson, 1983)
indicate that these genera are closely affiliated and
that the primary generic diversification within the
Myobatrachinae may have occurred as long ago as
the Cretaceous, thus predating the diversification
of the extant mammalian orders.
In sharp contrast, the subfamily Limnodynastinae is notably diversified in NOR location and includes the only example of a genus with multiple
NOR sites (Heleioporus). Multiple sites are not uncommon in mammals (Goodpasture & Bloom,
1975) but in over 90O7o of frogs examined, the NOR
is found to occur at only one locus (Schmid, 1982a
& b). The origin and functional significance of additional NOR sites is still a matter of conjecture. In
this case, as in others (Hsu et al., 1975), it would
appear that they represent a derived condition. The
evidence in Heleioporus indicates that their origin
in four o f the species, H. albopunctatus, 14. eyrei,
H. inornatus and H. psammophilus, occurred in
their ancestor and has been maintained subsequent
to their speciation. Taken together with examples of
reduced diploid chromosome number and o f polyploidy, the Limnodynastinae, while still showing
conservatism in chromosome morphology, are
much less conservative karyotipically than the Myobatracbinae.
Acknowledgements
We gratefully acknowledge the help provided by
numerous people with the collection of specimens,
especially M. Davies, S. Donnellan, H. Ehrmann,
D. Roberts and M. Tyler. Several useful taxonomic
suggestions were made by H. Cogger.
We also wish to thank E G. Johnston for reading
the manuscript, R. Oldfield for advice with photography and M. Minard for secretarial help.
Support for this study was provided by the Australian Biological Resources Study, Australian
Museum Postgraduate Research Awards and the
Peter Rankin Trust Fund.
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Received 10.1.1985.
Accepted 13.6.1985.