http://informahealthcare.com/mdn ISSN: 1940-1736 (print), 1940-1744 (electronic) Mitochondrial DNA, Early Online: 1–2 ! 2014 Informa UK Ltd. DOI: 10.3109/19401736.2014.947583 MITOGENOME ANNOUNCEMENT The complete mitochondrial genome of the eastern grey kangaroo (Macropus giganteus) William G. Dodt 1 , Bennet J. McComish 2 , Maria A. Nilsson 3 , Gillian C. Gibb 4 , David Penny 5 , and Matthew J. Phillips 1 1 School of Earth, Environmental and Biological Sciences, Queensland University of Technology, Brisbane, Australia, 2 School of Physical Sciences, University of Tasmania, Hobart, Australia, 3 Biodiversity and Climate Research Center, BiK-F, Senckenberg Museum, Frankfurt am Main, Germany, 4 Institute of Agriculture and Environment, Massey University, Palmerston North, New Zealand, and 5 Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand Abstract We present the complete mitochondrial genome (accession number: LK995454) of an iconic Australian species, the eastern grey kangaroo (Macropus giganteus). The mitogenomic organization is consistent with other marsupials, encoding 13 protein-coding genes, 22 tRNA genes, 2 ribosomal RNA genes, an origin of light strand replication and a control region or D- loop. No repetitive sequences were detected in the control region. The M. giganteus mitogenome exemplifies a combination of tRNA gene order and structural peculiarities that appear to be unique to marsupials. We present a maximum likelihood phylogeny based on complete mitochondrial protein and RNA coding sequences that confirms the phylogenetic position of the grey kangaroo among macropodids. Keywords Grey kangaroo, Macropus, mitochondrion, mt genome, RNA-editing History Received 17 June 2014 Revised 17 July 2014 Accepted 19 July 2014 Published online 8 August 2014 The eastern grey kangaroo (Macropus giganteus) is a grazing marsupial found in grassland and open forests in southern and eastern Australia. This highly gregarious species is the second largest extant marsupial (second to the red kangaroo) and is the member of the kangaroo and wallaby family (Macropodidae) that is most frequently encountered by humans. Genomic DNA was isolated from muscle tissue supplied by EcoMeats in Canberra. The mitogenome was amplified in six overlapping fragments (primer sequences and reaction conditions are available upon request). Gel extracted PCR products were quantified and combined with a mixture of total genomic DNAs from divergent (non-mammalian) taxa. 100 bp paired-end reads were generated from an Illumina HiSeq run following standard protocols. De-novo assembly of mitochondrial (mt) genomes from the mixed samples followed a modified version of the pipeline described in McComish et al. (2010). M. giganteus mt reads were present in high coverage (averaging 1000 Â across the mt genome), and assembled to give a single contig. Geneious 6.1.7 (Biomatters Ltd, Auckland, New Zealand) was used to align the assembled contig against a M. robustus reference mt genome and to perform preliminary annotation. The final mt assembly was verified by mapping the full set of sequence reads back to the assembled contig using BWA (Li & Durbin, 2009). Phylogenetic reconstruction was performed with RAxML 7.0.4 (Stamatakis, 2014), using the GTR + I + G model across four partitions – the three codon positions and the RNA coding sequences. M. giganteus was placed with the wallaroo (M. robustus) to the exclusion of Lagorchestes, with 100% bootstrap support (Figure 1). This confirms relationships from studies using several mitochondrial and nuclear genes (Meredith et al., 2008; Phillips et al., 2013) and demonstrates the statistical power of complete mitogenomes for resolving macropodid relationships. We confirm that the arrangement of the tRNA genes, tryptophan (W), alanine (A), asparagine (N), cysteine (C) and tyrosine (Y), and the origin of L-strand replication (OriL) is in the marsupial specific ACW-OriL-NY configuration (Erickson et al., 2011), distinct from the WANCY arrangement observed in placental and monotreme mammals (Kirsch & Poole, 1972). An unusual characteristic of marsupials is that the anti- codon of the Aspartate (Asp) tRNA (5 0 -GCC-3 0 ) differs from all other vertebrates (5 0 -GTC-3 0 ; Casavant et al., 2000). The Asp tRNA undergoes RNA-editing in marsupial mt genomes, such that 50% of transcripts have the correct anticodon (Mo ¨rl et al., 1995). This feature appears to be unique to marsupials. A peculiarity of australidelphian marsupial mt genomes is that tRNA, Lysine (Lys), does not have the appropriate anticodon, indicating that this tRNA may be a pseudogene within this clade. Functional lysine may be imported from the nucleus into the mitochondrion (Do ¨rner et al., 2001). Further, we show that the control region is devoid of repetitive sequences, which are often found in the mt genomes of other mammals. To date, no kangaroo mt genomes have been shown to contain repetitive sequences. Correspondence: William G. Dodt, School of Earth, Environmental and Biological Sciences, Queensland University of Technology, 2 George Street, Brisbane 4001, Australia. Tel: +4917 629493158. E-mail: william.dodt@senckenberg.de Mitochondrial DNA Downloaded from informahealthcare.com by QUT Queensland University of Tech on 08/18/14 For personal use only.