Late Pleistocene Australian Marsupial DNA Clarifies the Affinities of Extinct Megafaunal Kangaroos and Wallabies

Abstract

Understanding the evolution of Australia's extinct marsupial megafauna has been hindered by a relatively incomplete fossil record and convergent or highly specialized morphology, which confound phylogenetic analyses. Further, the harsh Australian climate and early date of most megafaunal extinctions (39-52 ka) means that the vast majority of fossil remains are unsuitable for ancient DNA analyses. Here, we apply cross-species DNA capture to fossils from relatively high latitude, high altitude caves in Tasmania. Using low-stringency hybridization and high-throughput sequencing, we were able to retrieve mitochondrial sequences from two extinct megafaunal macropodid species. The two specimens, Simosthenurus occidentalis (giant short-faced kangaroo) and Protemnodon anak (giant wallaby), have been radiocarbon dated to 46-50 and 40-45 ka, respectively. This is significantly older than any Australian fossil that has previously yielded DNA sequence information. Processing the raw sequence data from these samples posed a bioinformatic challenge due to the poor preservation of DNA. We explored several approaches in order to maximize the signal-to-noise ratio in retained sequencing reads. Our findings demonstrate the critical importance of adopting stringent processing criteria when distant outgroups are used as references for mapping highly fragmented DNA. Based on the most stringent nucleotide data sets (879 bp for S. occidentalis and 2,383 bp for P. anak), total-evidence phylogenetic analyses confirm that macropodids consist of three primary lineages: Sthenurines such as Simosthenurus (extinct short-faced kangaroos), the macropodines (all other wallabies and kangaroos), and the enigmatic living banded hare-wallaby Lagostrophus fasciatus (Lagostrophinae). Protemnodon emerges as a close relative of Macropus (large living kangaroos), a position not supported by recent morphological phylogenetic analyses.

Identifying remains of extinct kangaroos in Late Pleistocene deposits using collagen fingerprinting

“…Most of these fragments are within the size range expected for M. rufogriseus and V. ursinus, but two bones were a closer match for Macropus giganteus in size (Garvey, 2006). Biomolecular methods hold great potential for identifying nondescript bone fragments to species, as has been demonstrated recently in Australia using ancient DNA (aDNA)-based methods (Murray et al, 2013;Grealy et al, 2015;Llamas et al, 2015). Nevertheless, archaeological and palaeontological sites with good aDNA preservation are rare in non-frozen landscapes (Poinar and Stankiewicz, 1999).…”

An assessment of ancient DNA preservation in Holocene–Pleistocene fossil bone excavated from the world heritage Naracoorte Caves, South Australia

“…We have also shown that the DNA fragmentation at RCEC is not entirely dependent on age, meaning that the DNA loss in megafaunal bones from the older layers may have been slower than the younger layers. Improvements in DNA technology such as target enrichment through hybridization capture and higher sequencing coverage may improve the chances of retrieving low‐copy number DNA (Llamas et al ., ), particularly mtDNA. Post‐excavation conditions will also influence the retrieval of aDNA.…”

“…At these sites, aDNA preservation is clearly exceptional; in comparison, much shorter fragments were retrieved from much younger samples at RCEC. More recently, 37–121 unique mtDNA sequences averaging 37–40 bp were recovered from 40 000–50 000 cal a BP megafaunal bone from high altitude caves in Mt Cripps, Tasmania (Llamas et al ., ); however, no DNA was able to be amplified through PCR, and libraries were enriched for endogenous DNA. While there were too few endogenous sequences to estimate k for Layer 6, we still managed to retrieve mtDNA and nuDNA from bones over 18 000 years old without enrichment.…”

“…Such models can be useful to predict the probability of recovering informative DNA from megafaunal species (e.g. sthenurine kangaroos) in the 40 000–50 000 cal a BP period over which they became extinct (Llamas et al ., ). Assuming that the decay rate of DNA in older layers is comparable to those in Layers 1 or 3, modelling (Fig.…”

“…Furthermore, the use of aDNA has had implications for the conservation and management of endangered animal populations (Shepherd and Lambert, 2008). With advances in DNA sequencing techniques, aDNA is being extracted from increasingly older fossil material in increasingly poor preservation conditions (Orlando et al, 2011;Murray et al, 2012), including the relatively warm, moist microclimates of many Australian cave systems (Murray et al, 2013;Haouchar et al, 2014;Llamas et al, 2014;Grealy et al, 2015).…”