DNA extraction and mtDNA amplification

Genomic DNA was extracted using Qiagen DNeasy Blood and Tissue kit (Qiagen GmbH, Hilden, Germany) following standard protocols or according to a high salt method [50]. An ~850 base pair (bp) fragment of Domain 1 of the mtDNA CR (from the tRNA proline to the end of the central conserved region) was amplified using the marsupial-specific primers L15999M and H16498M [51]. PCRs were carried out in 25 μl reactions using 100–500 ng of genomic DNA, 1 x Reaction Buffer (Bioline My taq Red Reagent Buffer; Bioline, Australia), 2 pmol primers and Bioline My Taq Red DNA polymerase (0.5 unit). Negative controls were included in each PCR. Thermocycling was performed on an Eppendorf Mastercycler EpS (Eppendorf, Hamburg, Germany) under the following conditions; initial denaturation (94°C for 2 min); 38–45 cycles of denaturation (94°C for 20 s); annealing (60°C for 40 s) and extension (72°C for 50 s) followed by a final extension (5 min at 72°C). PCR products were cleaned using ExoSap-IT^© (USB Corporation, Cleveland, Ohio, USA). Sequencing was resolved on an AB 3730xl Sequencer at AGRF Sydney.

Additionally, sequence data for the mtDNA CR was obtained from five museum specimens (GenBank {"type":"entrez-nucleotide-range","attrs":{"text":"KJ530551 to KJ530556","start_term":"KJ530551","end_term":"KJ530556","start_term_id":"634743615","end_term_id":"634743620"}}KJ530551 to KJ530556; [38,52]). These individuals dated from 1870 to 1938 and the dried museum skin specimens were provided by the Bohusläns Museum, Gӧteborg Museum, Museum of Comparative Zoology at Harvard University, Queensland Museum and Stockholm Museum. All work was carried out in a laboratory dedicated to ancient DNA experiments (in the Leibniz Institute for Zoo and Wildlife Research) to avoid the risk of contamination with modern DNA. The DNA extraction, library and bait preparation and hybridisation procedures, and bioinformatics analyses for these samples are described in Tsangaras et al. [38] and Tsangaras et al. [52]. Hybridization capture PCR product baits were generated from modern koala genomic DNA (SN265; {"type":"entrez-nucleotide","attrs":{"text":"KJ530552.1","term_id":"634743616","term_text":"KJ530552.1"}}KJ530552.1) with CR primers (PCI-CR-NF:5′-CATCAACACCCAAAGCTGAT-3′ and PCI-CR-NR: 5′-TTCTAGGTACGTCCGCAATCT-3′). Subsequent library and bait preparation, and hybridisation capture are described in [52], with sequencing on an Illumina MiSeq platform at University of Copenhagen National High-throughput DNA Sequencing Center. Bioinformatics procedures are described in [52] with consensus sequences for all samples generated using Geneious v 6.1.8 [53].