Conservation Genetics 4: 655–657, 2003. © 2003 Kluwer Academic Publishers. Printed in the Netherlands.
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A rapid PCR-RFLP diagnostic test for distinguishing sympatric bandicoot species (Marsupialia: Peramelidae) in southeastern Australia Kyall R. Zenger1,2∗ , Mark D.B. Eldridge1 & Peter G. Johnston1 1 Department
of Biological Sciences, Macquarie University, 2109 NSW, Australia; 2 Current address: Reprogen – Faculty of Veterinary Science, The University of Sydney, 2570 NSW, Australia (∗ Author for correspondence, E-mail:
[email protected])
Key words: bandicoots, PCR-RFLP, Peramelidae, marsupial, species-detection The southern brown bandicoot (Isoodon obesulus) has a fragmented coastal distribution in Australia. It is found on Cape York Peninsula in north Queensland (QLD), in southeastern New South Wales (NSW), Victoria (VIC), Tasmania, southeastern South Australia (SA) and in southwest Western Australia. Although I. obesulus was originally widespread along the southeast coast of NSW (Strahan 1995), the species has declined since the European settlement of Australia and is now restricted to two small populations, located on the northern outskirts of Sydney and the far southeast corner of the state. As a result, I. obesulus is listed as an endangered species (Schedule 1) of the Threatened Species Act 1995 (NSW). Morphologically, I. obesulus and the northern brown bandicoot (I. macrourus) are similar and this has contributed to confusion about their distribution in the Sydney region and has also led to misclassifications being made. Atkins (1998) reviewed available distribution data and concluded that I. macrourus occurred to the north and I. obesulus to the south of the Hawkesbury River just north of Sydney. Although unconfirmed reports persist of I. obesulus north of the Hawkesbury, the recent southerly range extension of 350 km for I. o. peninsulae on Cape York peninsula (Pope et al. 2001) makes it imperative that identity of bandicoots distributed along the eastern coast of Australia be reassessed. Since I. macrourus and I. obesulus are sympatric at Lamb Range in northern Queensland, it is possible that there are small disjunct populations of I. obesulus north of the Hawkesbury River which have previously escaped detection. In addition, the long nosed bandicoot (Perameles nasuta) is sympatric with both Isoodon species throughout
much of eastern Australia. As a result, the identity of specimens is often confused and the precise geographic boundaries of these species distributions remain unknown. Rapid molecular diagnostic tests provide valuable information for conservation management and have been used successfully in many species (i.e., Greig et al. 2002). The development of a simple reliable molecular technique for unequivocally identifying each of the three extant species of bandicoots found in NSW would resolve the problem of correctly identifying specimens and provide valuable information regarding their current distribution. In this technical note, we seek to produce a rapid and reliable genetic test to identify each of the three potentially sympatric bandicoot species found in the Sydney region. The mitochondrial cytochrome b gene was chosen because it has been recently shown to be very useful in differentiating these species (Westerman and Krajewski 2000; Pope et al. 2001). Ear biopsies were obtained from I. obesulus (n = 6), I. macrourus (n = 5) and P. nasuta (n = 2) individuals sampled across their range (see Figure 1). DNA was extracted from the tissue via the ‘salting out’ method of Sunnucks and Hales (1996). Mitochondrial DNA was amplified by PCR using primers to amplify the cytochrome b gene region (L14724 and H15149; Irwin et al. 1991). PCR was performed in 40 µL reactions containing; ∼ 400 ng of genomic DNA, 2.5 mM MgCl2 , 10 mM Tris-HCL (pH 8.3), 50 mM KCL, 0.1% Triton X100, 0.1% Tween 20 and NP40, 200 µM each of dATP, dCTP dGTP and dTTP, 0.5 µM of each primer, and 2.0 units of Taq polymerase (Promega). PCR amplifications were carried out using an MJ Research PTC100 thermocycler, with an initial 94 ◦ C denatur-
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Figure 1. Variable sites of partial 447 bp segment of mtDNA cytochrome b region. Variable nucleotide position 1 is relative to position 14155 within complete I. macrourus mtDNA sequence (GenBank accession no. AF358864). Dots represent identical bases to the first sequence. Im prefix within animal ID refers to I. macrourus, while Io and Pn are I. obesulus and P. nasuta respectively. Individuals 3–8, 12 and 13 were obtained from the Sydney region, while individuals 1 and 2 were obtained from south coast QLD and animals 9–11 originated from south coast SA. Shaded or filled areas indicate polymorphic sites used to distinguish species via restriction enzymes. Grey shaded indicates MseI, while black areas are SspI.
ation for 3 minutes, 35 cycles of 94 ◦ C denaturation for 30 seconds, 60 ◦ C annealing temperature for 45 seconds and an extension step of 72 ◦ C for 1 minute. On completion of the last cycle a final extension of 72 ◦ C for 3 minutes was carried out with the PCR held at 15 ◦ C until removed. Products were then purified (Geneworks – Ultraclean gel purification kit) and sequenced using BigDye termination (Perkin-Elmer Applied Biosystems) and resolved on an ABI 377 sequencer. Amplification and sequencing of the mtDNA cytochrome b gene from the three bandicoot species revealed 68 variable sites between all individuals (Figure 1). Of the 447 bp fragments sequenced, I. obesulus differed from I. macrourus by 7.31% and P. nasuta by 10.89%, while I. macrourus differed from P. nasuta by 11.41%. Of all the restriction enzymes capable of unequivocally distinguishing the three species apart, two alternatives – MseI (5’T TAA-3’) and SspI (5’-AATATT-3’) were chosen based on cost, availability and enzyme properties. Figure 1 indicates the respective polymorphic sites used to differentiate the species, while Figure 2 indicates the band sizes and patterns produced following digestion and electrophoresis on a 4% agarose gel. Verification and robustness of this technique for differentiating the three bandicoot species was accomplished by screening known individuals from broad geographical regions. These areas included, the
Figure 2. Restriction enzyme digestion pattern following electrophoresis on a 4% agarose gel. For each species, a male and female individual is included respectively. Sizes of relative fragments are compared against 100 bp marker. (A) Digestion with MseI produced 2 bands for I. macrourus (111 & 375 bp), 1 band for I. obesulus (486 bp) and 3 bands for P. nasuta (79, 87 and 320 bp). (B) Digestion with SspI produced 3 bands for I. macrourus (76, 95 and 315 bp), 4 bands for I. obesulus (76, 95, 127 and 188 bp) and 2 bands for P. nasuta (171 and 315 bp).
657 Sydney region (I. macrourus n = 29, I. obesulus n = 32 and P. nasuta n = 28), southern VIC (I. obesulus n = 17), southeastern SA (I. obesulus n = 11), southeastern QLD (I. macrourus n = 3) and far northern QLD (I. macrourus n = 10 and I. o. peninsulae n = 3). All animals were able to be unequivocally distinguished from each other based on the diagnostic tests described. The only discrepancy was the far northern QLD population of I. macrourus located at Lambs Range that could not be distinguished from I. obesulus using MseI. Sequencing confirmed this was attributed to a unique mutation (T→C position 337, Figure 1) within the I. macrourus restriction site inhibiting digestion. However, correct identification was obtained when using the other restriction enzyme, SspI.
Acknowledgements We thank the NSW National Parks and Wildlife Service, National Parks and Wildlife Service SA, Forestry SA and Fisheries and Wildlife Victoria for their assistance and co-operation. We are also grateful to Khia Atkins, David Paull, Barry Grigg, Mark Adams, Andrew Claridge, Andy Murray, Natasha
Funke, Ross Wellington and Lisa Pope for providing samples or assisting us with sample collection. This research was supported by a grant from NSW National Parks and Wildlife and a Macquarie University Research grant to PGJ.
References Atkins KL (1998) Habitat Preference and Distribution of Prameles Nasuta and Isoodon Obesulus in Bushland Remnants in Sydney. MSc thesis, Macquarie University, Sydney Australia. Greig C, Robertson JM, Banks MA (2002) Rapid PCR-based species tests for threatened sympatric salmonids. Conserv. Genet., 3, 83–86. Irwin DM, Kocher TD, Wilson AC (1991) Evolution of the cytochrome b gene of mammals. J. Mol. Evol., 32, 128–144. Pope L, Storch D, Adams M, Moritz C, Gordon G (2001) A phylogeny for the genus Isoodon and a range extension for I-obesulus peninsulae based on mtDNA control region and morphology. Aust. J. Zool., 49, 411–434. Strahan R (ed.) (1995) The mammals of Australia. Reed Books, Sydney. Sunnucks P, Hales DF (1996) Numerous transposed sequences of mitochondrial cytochrome oxidase I–II in aphids of the genus Sitobion (Hemiptera: Aphididae). Mol. Biol. Evol., 13, 510–524. Westerman M, Krajewski C (2000) Molecular relationships of the Australian bandicoot genera Isoodon and Perameles (Marsupialia: Peramelina). Aust. Mammal., 22, 1–8.
