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Supplementary Material
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Avoidance of nuclear copies of mtDNA
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Several approaches were taken to guard against using nuclear copies of mtDNA regions
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in this analysis. Firstly we aligned our partial ND2 seqeunce with known complete
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Macropus robustus (common wallaroo) ND2 sequence from GenBank (Accession
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number NC_001794.1) to check for indels or premature top codons which would
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indicate a pseudogene. Our second approach was to analyse two independent
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mitochondrial regions (ND2 and CR) to assess concordance of these markers, given the
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unlikely chance that both amplified regions (i.e. different primer combinations) would
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amplify nuclear pseudogene within one individual. Given that relative divergences and
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relationships were concordant between these markers we were satisfied that it was
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unlikely any nuclear pseudogenes were present in our dataset. Finally, we performed a
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comparative serial dilution on the template DNA of a subset of individuals to test for the
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presence of numts (nuclear pseudogenes of mtDNA). After each dilution we attempted
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to amplify the mtDNA CR and a known amplifiable nDNA region, the X linked
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glucose-6-phosphate dehydrogenase (G6PD; Loebel and Johnston 1997). Once the
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nDNA fragment failed to amplify, the mtDNA fragment amplified from the same
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template dilution was sequenced to compare with the original sequence. Results show
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that all putative mtDNA sequences obtained were from the mitochondrial genome.
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Identity of ABTC101599
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The identity of the P. burbidgei sample ABTC101599 was puzzling given its high
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divergence from all other P. burbidgei, P. brachyotis and P. concinna sampled. We are
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confident that this sequence is not a numt. In addition to the approaches taken above, we
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were unable to successfully amplify any nuclear DNA loci from this specimen due to
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relatively poor template quality. The voucher specimen associated with this specimen
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(WAM 54836) is a small rock-wallaby of the P. burbidgei/P. concinna type but appears
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to be immature and so cannot be definitively identified to species at this stage.
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There are several possible explanations for this result. Firstly, populations of P.
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burbidgei may be more highly structured for mtDNA than previously thought or this
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sequence may represent a retained ancestral state within P. burbidgei. Secondly, this
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individual may have been misidentified in the field and actually represents a specimen
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of P. concinna monastria (Kimberley subspecies), which also occurs in the Prince
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Regent River region of the Kimberley and is morphologically very similar to P.
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burbidgei (Kitchener and Sanson 1978). This would explain its significant divergence
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from P. burbidgei and the sister relationship of this lineage to P. c. canescens (Arnhem
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Land subspecies). Thirdly, this specimen may represent a currently unknown species in
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this paraphyletic species complex. Further sampling of P.burbidgei throughout its range
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and also P. concinna especially from the Kimberley is now required to resolve this
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anomaly and clarify the relationships and taxonomic classification of these small rock-
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wallabies.
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References
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Loebel, DAF, Johnston, PG (1997) Analysis of the intron-exon structure of the G6PD
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gene of the wallaroo (Macropus robustus) by polymerase chain reaction. Mammalian
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Genome, 8, 146-147.
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Kitchener, DJ, Sanson, G (1978) Petrogale burbidgei (Marsupialia, Macropodidae) a
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new rock wallaby from Kimberley, Western Australia. Records of the Western
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Australian Museum, 6, 269-285.
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