Chromosome morphology in Gilbert’s Potoroo, Potorous gilbertii (Marsupialia: Potoroidae)

Abstract

Chromosome morphology was examined for male and female Gilbert’s potoroo, Potorous gilbertii, to infer taxonomic and evolutionary relationships among the extant taxa within the genus Potorous. P. gilbertii has the same number of chromosomes as P. tridactylus, 2n = 12,13. Giemsa-banding patterns were very similar in P. gilbertii and P. tridactylus; however, differences were noted between the sex chromosomes. Given that the relationships among extant Potorous are unresolved, we mapped karyotypes onto two alternative phylogenies to suggest methods of karyotype evolution within this group. Karyotypes and molecular-based information from the now ‘presumed extinct’ P. platyops or sequencing of multiple gene regions for phylogenetic analysis within the Potoroidae would provide valuable information for resolving the issue of rooting, and hence drawing conclusions on the evolution of karyotypes within this group.

Full text

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Chromosome morphology in Gilbert’s potoroo,
Potorous gilbertii (Marsupialia : Potoroidae)
E. A. SinclairA, A. R. MurchB, M. Di RenzoB, and M. PalermoB
ADepartment of Zoology, University of Western Australia, Nedlands, WA 6907, Australia.
Present address: Department of Zoology, Brigham Young University, Provo, UT 84602, USA.
BDepartment of Cytogenetics, King Edward Memorial Hospital, Subiaco, WA 6008, Australia.
Abstract
Chromosome morphology was examined for male and female Gilbert’s potoroo, Potorous gilbertii, to infer
taxonomic and evolutionary relationships among the extant taxa within the genus Potorous. P. gilbertii has
the same number of chromosomes as P. tridactylus, 2n = 12,13. Giemsa-banding patterns were very similar
in P. gilbertii and P. tridactylus; however, differences were noted between the sex chromosomes. Given
that the relationships among extant Potorous are unresolved, we mapped karyotypes onto two alternative
phylogenies to suggest methods of karyotype evolution within this group. Karyotypes and molecular-based
information from the now ‘presumed extinct’ P. platyops or sequencing of multiple gene regions for
phylogenetic analysis within the Potoroidae would provide valuable information for resolving the issue of
rooting, and hence drawing conclusions on the evolution of karyotypes within this group.
Introduction
Gilbert’s potoroo, Potorous gilbertii, was originally recognised by John Gilbert as a full
species, Hypsiprymnus gilbertii (1840). The description was, however, based on a single
animal. Its taxonomic status was later re-interpreted, and it was synonymised with the long-
nosed potoroo, P. tridactylus, on the basis of morphology (Ride 1970; Johnston and Sharman
1976). The difficulties in determining the correct taxonomic status of Gilbert’s potoroo have
principally arisen through a lack of specimens: less than ten specimens were known to have been
collected (Clem Fisher, personal communication). Some subfossil material has also been
collected (Baynes et al. 1975), but the last known living specimens were collected last century.
Gilbert’s potoroo was rediscovered in 1994 at Two Peoples Bay in Western Australia
(Sinclair et al. 1996). A subsequent study of the genetic relationship between this animal and its
extant relatives in eastern Australia, using allozyme electrophoresis and DNA sequencing, was
carried out by Sinclair and Westerman (1997). Results showed that Gilbert’s potoroo was as
different from the two eastern Australian species (P. tridactylus and the long-footed potoroo, P.
longipes) as these two were from each other. It was concluded that Gilbert’s potoroo should
again be recognised as a full species, but its relationships to other taxa were not fully resolved.
Chromosome morphology of Australian macropods has been extensively studied, and
provides a window into both evolution between the major groups (Rofe 1978; Sharman 1989),
as well as relationships among closely related species (e.g. within the genus Petrogale: Eldridge
et al. 1991a, 1991b). In both American (north and south) and Australian species, the most
common karyotype number is 2n = 14 (Rofe and Hayman 1985). The relative uniformity
(highly conserved G-banding patterns) and wide distribution of this karyotype across
superfamilies and families suggest that it may be the ancestral karyotype for marsupials
(Hayman and Martin 1974; Rofe and Hayman 1985). Several marsupial species have a high
chromosome number, including the rufous bettong, Aepyprymnus rufescens (2n = 32: Hayman
and Martin 1969), the banded hare-wallaby, Lagostrophus fasciatus (2n = 24: Sharman 1961),
and P. longipes (2n = 24: Seebeck and Johnston 1980). It may be inferred that A. rufescens and
L. fasciatus have derived karyotypes (see Hayman and Martin 1974), while Johnston et al.
(1984) concluded that the ancestral Potorous karyotype was similar to 2n = 24 of P. longipes.
Australian Journal of Zoology, 2000, 48, 281–287
10.1071/ZO99049 0004-959X/00/030281
© CSIRO 2000

The higher chromosome numbers observed in the above species would have to result from
fission events. Imai and Crozier (1980) have suggested that fission events played a major role in
mammalian chromosome evolution.
Within the potoroos, P. tridactylus (2n = 12F,13M: Sharman et al. 1950; Sharman and
Barber 1952) and P. longipes are both clearly distinguishable on the basis of karyotypes, while
the two subspecies of P. tridactylus can not be chromosomally differentiated on the basis of
their karyotypes (Sharman and Barber 1952; Shaw and Krooth 1964). Johnston et al. (1984)
artificially constructed the P. tridactylus karyotype from that of P. longipes and suggested that
the ancestral karyotype was probably similar to that of P. longipes. Therefore, fusions, primarily
centric, had played an important role in the evolution of the Potorous karyotype.
Most marsupials have a typical XY sex-determining mechanism (Sharman and Barber 1952),
in which males are the heterogametic sex. However, several exceptions have been reported: P.
tridactylus, Wallabia bicolor, and Macrotis lagotis all have a second Y chromosome while
Lagorchestes conspicillatus has multiple X chromosomes (Martin and Hayman 1966). In this
study, we obtain karyotypes for male and female P. gilbertii and make comparisons with the two
extant congenerics.We statistically test two alternative hypotheses for relationships within
Potorous. Karyotypes will be mapped onto the phylogenetic trees to make inferences about
their evolutionary history.
Materials and Methods
Fresh peripheral blood was collected from one female and one male P. gilbertii trapped at Two Peoples
Bay, Western Australia (Fig. 1) and from two captive P. tridactylus from the Caversham Wildlife Park,
Western Australia.
282 E. A. Sinclair et al.
Fig. 1. The distribution of Potorous species in Australia (from Strahan 1995). Karyotypes of
the taxa are included (see text for references).

Standard thymidine synchronised lymphocyte cultures were set up and prepared for cytogenetic analysis
using the technique of Wheater and Roberts (1987) with minor modifications. The technique gave an
acceptable yield of good-quality metaphases. The trypsin banding technique of Seabright (1971) was used
for G-banding and the barium hydroxide method of Sumner (1972) with minor modifications for C-banding.
Given that the relationships among these taxa are not fully resolved, we have tested alternative rooting
hypotheses under conditions of maximum likelihood, using PAUP* 4.0b3 (Swofford 1999). The computer
program Modeltest ver. 2.1 (Posada and Crandall 1998) was used to estimate the model of evolution that
best explains the variation in cytochrome bsequences from Sinclair and Westerman (1997). Trees with two
alternative rootings tested the hypotheses that P. gilbertii and P. longipes were sister taxa (consistent with
the cytochrome btree), and that P. gilbertii and P. tridactylus were sister taxa (one possible tree from the
allozyme data). A Kishino–Hasegawa test (Kishino and Hasegawa 1989) was performed to test whether
there was a significant difference between the topology of these two trees. This test compares the log-
likelihood scores for trees with different topologies.
Results
Metaphase spreads for males and females were examined for both species (Fig. 2). P.
gilbertii has a karyotype of 2n = 12 for females and 2n = 13 for males, where the male has two
Y chromosomes, the same arrangement as previously reported by Sharman et al. (1950) for both
subspecies of P. tridactylus. G-banding patterns were similar for P. tridactylus and P. gilbertii.
No consistent differences in the banding patterns of the autosomes or the X chromosomes could
be detected. However, the Y1chromosome in P. gilbertii appears to be about twice the size of
that in P. tridactylus and also appears to have two extra dark G-bands. The Y2chromosomes of
both species appear identical and their banding patterns appear very similar to the pattern seen
on the long arm of the X chromosome.
C-banding was unsuccessful in the P. tridactylus samples, but the C-banding patterns obtained
for P. gilbertii (Fig. 3) were compared with those published for P. tridactylus by Johnston et al.
(1984). Heterochromatin was restricted to small amounts around the centromeres in the autosomes
in both species. There were also similar blocks of heterochromatin in the long arm of the X
283Chromosome morphology in Gilbert’s potoroo
Fig. 2. Comparison of G-banded karyotypes for Potorous gilbertii and Potorous tridactylus for (a) male
and (b) female.

284 E. A. Sinclair et al.
Fig. 3. C-banded karyotypes of Potorous gilbertii: (a) male and (b) female.
Fig. 4. Phylogenetic trees for the two hypotheses on rooting (a) P. gilbertii and P. longipes as sister taxa,
and (b) P. gilbertii and P. tridactylus as sister taxa, with karyotypes mapped onto them.

chromosome. However, P. gilbertii lacked the large block of heterochromatin that makes up most
of the short arm of the X in P. tridactylus. Almost all of Y1is heterochromatin in P. tridactylus,
but P. gilbertii has a euchromatic region distal to the heterochromatin on the long arm.
Alternative hypotheses for tree rooting are presented in Fig. 4. A Tamura and Nei (1993)
plus gamma model was selected as the most appropriate model of evolution by Modeltest
(gamma shape distribution = 0.293). The log-likelihood score for the tree that supported P.
gilbertii and P. longipes as sister taxa was 1060.58, and that where P. gilbertii and P. tridactylus
were sister taxa was 1066.48. The Kishino–Hasegawa test was not significant, P = 0.34,
indicating that there was no significant difference in the topology of these two trees. Therefore,
any discussion on the evolution of chromosome karyotypes within potoroids remains speculative
until the issue of rooting this tree can be resolved.
Discussion
The karyotype of P. gilbertii is essentially the same as for P. tridactylus (2n = 12, 13). The
sex-determining mechanism is also XX : XY1Y2. These karyotypes are clearly distinguishable
from the P. longipes form, which Johnston et al. (1984) hypothesised was ancestral. The 2n =
12, 13 karyotype is widespread, being present in Potorous from eastern and western Australia
and on Tasmania and islands in Bass Strait (see Fig. 1).
We acknowledge that there are problems associated with phylogenies derived from single
mitochondrial trees (see Avise 1994; Krajewski and Woods 1995). However, in the absence of
further data at present, we make the following suggestions. The phylogeny of Sinclair and
Westerman (1997), in which mtDNA sequence data support the idea that P. gilbertii and P. longipes
are sister taxa, indicates that 2n = 24 is the derived karyotype. (The allozyme tree was unrooted so
there are two equally parsimonious trees, depending on the rooting.) This refutes the conclusion of
Johnston et al. (1984) that the ancestral Potorous karyotype was similar to that of P. longipes.
Increases in chromosome number, as a result of chromosomal fissions have been postulated in other
mammalian groups including canids (Todd 1970), primates (Matthay 1973), and kangaroo rats
(Patton and Rogers 1993). Although the mechanisms for an increase in chromosome number have
not yet been demonstrated in marsupials (Sharman 1973), this may be an example.
If, however, P. gilbertii and P. tridactylus are sister taxa, which is supported by the
karyotype data, then 2n = 24 is ancestral, and consistent with Johnston et al. (1984). The 2n =
12, 13 derived karyotype could then be hypothesised to have occurred either once (in eastern
Australia and then dispersed westward) or twice. Maynes (1989) suggests that zones of contact
between the south-west and south-east have resulted in many of the western species, including
Setonix brachyurus, Macropus irma, and Potorous tridactylus (now gilbertii). Given that the
autosomes are very similar in both species, it seems unlikely that the exact same chromosomes
would fuse twice independently. For example, in Macropus species that have a common
karyotype of 2n = 16, G-banding patterns indicate that this karyotype has been derived from no
fewer than eight different fusion events in the autosomes (see Rofe 1978).
Divergence estimates, from cytochrome bsequence data (Sinclair and Westerman 1997), are
much older than the last known ‘contacts’ of the Pleistocene, suggesting that these species (or
their immediate ancestors) have been isolated for a much longer period. During the Pleistocene,
a land bridge was present south of what is now the Nullarbor Plain. A continuous distribution of
potoroos may have been present across this region, consistent with a single fusion event. At the
end of this period, potoroos with the 2n = 12, 13 karyotype could remain in the east and west,
but become isolated. However, there is no evidence from fossil or subfossil material to indicate
that either P. tridactylus or P. gilbertii occurred in central southern Australia. Extant Potorous
spp. first appear in the fossil record during the Pleistocene (Flannery 1984). The broad-faced
potoroo (P. platyops) is the only Potorous species identified from Pleistocene and Holocene
cave deposits in the Nullarbor Plain, which has been dated at between 38 000 and 7500 years
ago (Lundelius and Turnbull 1989). P. platyops (and one other unidentified potoroid) is also
reported from surface deposits between Esperance and the Eyre Peninsula, dated at between 390
285Chromosome morphology in Gilbert’s potoroo

± 210 years ago (Baynes 1987) and 180 ± 76 years ago (Merrilees 1970). So the fossil record,
as it stands presently, provides little support for either explanation.
It seems that two approaches could help resolve this issue of how to root the phylogenetic tree,
and hence draw conclusions on the evolution of karyotypes within this group. Karyotyping and
molecular-based information from the now ‘presumed extinct’ P. platyops would be very
valuable, since it is the only potoroo species that is known to have had a distribution across the
arid parts of southern Australia. It is extremely unlikely, however, that suitable material will ever
become available to karyotype this species, although it may be possible to obtain DNA sequences
from museum material. A more feasible option would be to collect DNA sequence data from
multiple gene regions (nuclear and mitochondrial) and from more taxa to obtain a more robust
phylogeny of the Potoroidae. Known karyotypes (including information on chromosome
painting, such as that provided by Rens et al. 1999) can then be mapped onto this phylogeny.
Acknowledgments
Thank you to Jeff Middleton, Jackie Courtenay and Leigh Whisson for their help in the field;
the Potoroo Recovery Team; David Thorn at the Caversham Wildlife Park for P. tridactylus
blood samples; Jack Sites, Keith Crandall, Duke Rogers, and two reviewers for their
constructive comments on this manuscript. This work was supported by the University of
Western Australia, ALCOA of Australia, and an Australian Post-graduate Research Award to
EAS.
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Manuscript received 16 August 1999; accepted 19 May 2000
287Chromosome morphology in Gilbert’s potoroo
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