Future-proofing the koala: synergizing genomic and environmental data for effective species management

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.

Methods
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).

气候与演化过程与生物保护密不可分。在快速变化的环境中避免灭绝，往往取决于物种应对极端选择压力的适应能力。本研究采用外显子捕获（exon capture）技术与高通量下一代测序（high-throughput next-generation sequencing），探究澳大利亚标志性有袋类动物考拉（树袋熊，Phascolarctos cinereus）的种群结构与适应性遗传变异机制——该物种因其分布区内受威胁程度不均一，成为独特的保护学研究对象。
对覆盖91个野生采样点的250份标本进行分析后发现，当前大陆尺度上存在5个主要遗传聚类群。这些聚类群的初始分化时间似乎与中布容尼斯转型期（Mid-Brunhes Transition，约43万~30万年前）相一致，该重大气候重组事件增大了更新世冰期-间冰期循环的振幅。研究虽检测到多基因选择（polygenic selection）与环境适应的信号，但反复出现的、与气候相关的分布区收缩及种群瓶颈（demographic bottleneck）事件的有力证据表明，地理隔离的避难所（refugia）对于考拉在更新世冰期的存续，可能比原地适应发挥了更关键的作用。因此，全基因组遗传变异的保护必须与核心考拉栖息地保护相结合，以提升受威胁种群应对日益加剧的人为威胁的恢复力。最后，本研究提出，未来的考拉保护工作（例如指导物种易位（translocation）计划）应纳入本次研究鉴定出的5个主要遗传聚类群，因为昆士兰州与新南威尔士州现有的管理分区并未反映其历史或当代种群结构。
方法
本研究从226份考拉组织样本中提取基因组DNA，所用试剂为Bioline Isolate II基因组DNA提取试剂盒（Bioline，澳大利亚伊夫利），或采用高盐法（Sunnucks & Hales, 1996）。针对自然历史馆藏的33份干燥标本（皮肤或骨骼残留组织），则参照Fulton、Wagner与Shapiro（2012）开发的实验流程，使用QIAGEN DNeasy血液与组织试剂盒（Qiagen，德国希尔德）进行处理。本研究参照Bragg等人（2017）的方案设计了定制化溶液内外显子捕获流程。参照Meyer与Kircher（2010）的方法并结合Bi等人（2013）的改进方案，为每份考拉样本制备基因组文库（genomic library，总质量700~1400 ng）。将带有独特索引标签的文库以等摩尔浓度混合，参照Potter等人（2018）的描述，与SeqCap EZ探针（Roche NimbleGen，美国普莱森顿）进行杂交以富集目标外显子序列。测序采用100 bp双端读长，在HiSeq 2500、NextSeq 500或NovaSeq 6000平台（Illumina，美国加利福尼亚州圣地亚哥）上完成。所得短读长测序数据已提交至NCBI SRA数据库（SRA登录号：PRJNA816339），并通过多种软件包与脚本进行处理（相关文件已随Dryad数据包一同公开）。