The mitochondrial genome sequence of the Tasmanian tiger (Thylacinus cynocephalus)

Abstract

We report the first two complete mitochondrial genome sequences of the thylacine (Thylacinus cynocephalus), or so-called Tasmanian tiger, extinct since 1936. The thylacine's phylogenetic position within australidelphian marsupials has long been debated, and here we provide strong support for the thylacine's basal position in Dasyuromorphia, aided by mitochondrial genome sequence that we generated from the extant numbat (Myrmecobius fasciatus). Surprisingly, both of our thylacine sequences differ by 11%-15% from putative thylacine mitochondrial genes in GenBank, with one of our samples originating from a direct offspring of the previously sequenced individual. Our data sample each mitochondrial nucleotide an average of 50 times, thereby providing the first high-fidelity reference sequence for thylacine population genetics. Our two sequences differ in only five nucleotides out of 15,452, hinting at a very low genetic diversity shortly before extinction. Despite the samples' heavy contamination with bacterial and human DNA and their temperate storage history, we estimate that as much as one-third of the total DNA in each sample is from the thylacine. The microbial content of the two thylacine samples was subjected to metagenomic analysis, and showed striking differences between a wild-captured individual and a born-in-captivity one. This study therefore adds to the growing evidence that extensive sequencing of museum collections is both feasible and desirable, and can yield complete genomes.

Figure 1.

Dorsal and ventral images of the skin of USNM 125345, young adult male, which died in 1905 at the National Zoo; thylacine specimen 1 sequenced in our study (courtesy National Museum of Natural History Photographic Services).

Figure 2.

Thylacine specimen 2 sequenced in our study, from the mammal collection of the Swedish Museum of Natural History (Stockholm), NRM 566599, which died in 1893 at the London Zoo. (Photo: Staffan Waerndt, ©Swedish Museum of Natural History.)

Figure 3.

(A) A maximum likelihood phylogeny of marsupials based on the reduced-bias mitochondrial DNA matrix that places Thylacinidae inside Dasyuromorphia. Didelphimorphia was used as the outgroup, based on results of published nuclear and mitochondrial studies (Amrine-Madsen et al. 2003; Nilsson et al. 2004; Phillips et al. 2006). Bootstrap support values followed by Bayesian posterior probabilities (BPP) are shown above respective nodes. The first bootstrap value is derived from the reduced-bias matrix used to generate the tree, and the second bootstrap value is based on the unmodified sequence of the coding mitochondrial genes. The first and second BPP values correspond to the RY-coded and non-RY-coded (mixed model) analyses. Nodes with 100% bootstrap values and 1.0 posterior probability are marked with asterisks. Branch lengths were estimated under the general-time-reversible + gamma + invariants model of sequence evolution. (ns) Not significant, with bootstrap support below 50. (B,C) Maximum likelihood phylogeny derived from (B) 12S rRNA and (C) cytb sequences. Note the large divergence between the sequences from this study (labeled ThylacinusNew) and those previously published (ThylacinusKre) (Krajewski et al. 1997, 2000).

Figure 4.

Comparative metagenomics study of the two biomes detected in the hair of thylacine specimens 1 and 2.