Climatic and evolutionary processes are inextricably linked to conservation. Avoiding extinction in rapidly changing environments often depends upon a species’ capacity to adapt in the face of extreme selective pressures. Here, we employed exon capture and high-throughput next-generation sequencing to investigate the mechanisms underlying population structure and adaptive genetic variation in the koala (Phascolarctos cinereus), an iconic Australian marsupial that represents a unique conservation challenge because it is not uniformly threatened across its range. An examination of 250 specimens representing 91 wild source locations revealed that five major genetic clusters currently exist on a continental scale. The initial divergence of these clusters appears to have been concordant with the Mid-Brunhes Transition (∼ 430–300 kya), a major climatic reorganization that increased the amplitude of Pleistocene glacial-interglacial cycles. While signatures of polygenic selection and environmental adaptation were detected, strong evidence for repeated, climate-associated range contractions and demographic bottleneck events suggests that geographically isolated refugia may have played a more significant role in the survival of the koala through the Pleistocene glaciation than in situ adaptation. Consequently, the conservation of genome-wide genetic variation must be aligned with the protection of core koala habitat to increase the resilience of threatened populations to accelerating anthropogenic threats. Finally, we propose that the five major genetic clusters identified in this study should be accounted for in future koala conservation efforts (e.g. guiding translocations), as existing management divisions in the states of Queensland and New South Wales do not reflect historic or contemporary population structure.

Genomic DNA was extracted from koala tissue samples (n=226) using either the Bioline Isolate II Genomic DNA Kit (Bioline, Eveleigh, Australia) or according to a high salt method (Sunnucks & Hales, 1996). Dry specimens (skins or residual tissue on skeletal elements) from natural history collections (n=33) were processed using the QIAGEN DNeasy Blood & Tissue Kit (Qiagen, Hildon, Germany) according to the protocol developed by Fulton, Wagner and Shapiro, (2012). A custom in-solution exon capture procedure was designed according to Bragg et al., (2017). Genomic libraries (700-1400 ng total) were prepared for each koala sample following the methods devised by Meyer and Kircher, (2010), with modifications from Bi et al., (2013). Individually indexed libraries were pooled in equimolar ratios and hybridized against the SeqCap EZ probes (Roche NimbleGen, Pleasanton, USA) to enrich for target exons, as described by Potter et al., (2018). Sequencing was performed as 100-bp paired-end reads on the HiSeq 2500, NextSeq 500 or NovaSeq 6000 platforms (Illumina, San Diego, CA). The resulting short-read sequence data were deposited in the NCBI SRA database (SRA accession: PRJNA816339) and processed using a variety of software packages and scripts (included with the Dryad package).