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Reversing the decline of threatened koala (Phascolarctos cinereus) populations in New South Wales: Using genomics to enhance conservation outcomes

Cite this dataset

Lott, Matthew et al. (2024). Reversing the decline of threatened koala (Phascolarctos cinereus) populations in New South Wales: Using genomics to enhance conservation outcomes [Dataset]. Dryad. https://doi.org/10.5061/dryad.ht76hdrph

Abstract

Genetic management is a critical component of threatened species conservation. Understanding spatial patterns of genetic diversity is essential for evaluating the resilience of fragmented populations to accelerating anthropogenic threats. Nowhere is this more relevant than on the Australian continent, which is experiencing an ongoing loss of biodiversity that exceeds any other developed nation. Using a proprietary genome complexity reduction-based method (DArTSeq), we generated a data set of 3,239 high quality Single Nucleotide Polymorphisms (SNPs) to investigate spatial patterns and indices of genetic diversity in the koala (Phascolarctos cinereus), a highly specialised folivorous marsupial that is experiencing rapid and widespread population declines across much of its former range. Our findings demonstrate that current management divisions across the state of New South Wales (NSW) do not fully represent the distribution of genetic diversity among extant koala populations, and that care must be taken to ensure that translocation paradigms based on these frameworks do not inadvertently restrict gene flow between populations and regions that were historically interconnected. We also recommend that koala populations should be prioritised for conservation action based on the scale and severity of the threatening processes that they are currently faced with, rather than placing too much emphasis on their perceived value (e.g., as reservoirs of potentially adaptive alleles), as our data indicate that existing genetic variation in koalas is primarily partitioned amongst individual animals. As such, the extirpation of koalas from any part of their range represents a potentially critical reduction of genetic diversity for this iconic Australian species.

README

1) DArTSeq Fastq files

  • Demultiplexed DArTSeq fastq sequencing files for 314 koala specimens and 60 technical replicates.

2) Sequence processing & SNP calling

  • This folder contains the scripts and files required to process the fastq sequencing files and identify SNPs. Stacks v2.64 was used to process the demultiplexed and adapter trimmed short‐read sequence data supplied by DArT. The reads were truncated to 69bp in length and low-quality data (based on the PHRED scores provided in the FASTQ files) were identified and discarded using the process_radtags program. Burrows-Wheeler aligner (BWA) v0.7.15 was then used to align the read data from the previous step to the koala reference genome (GCA_002099425.1_phaCin_unsw_v4.1). The alignments were subsequently converted to BAM format using SAMtools v1.6. Finally, the reference-aligned data were used to assemble the sequences into loci and identify SNPs using the ref_map.pl pipeline in Stacks. A single SNP was extracted from each locus for downstream processing and analysis using the populations program, also implemented in Stacks.

3) R software scripts for analysing the SNP data

  • The R software scripts for identifiyng candidate outlier SNPs that might represent loci under selection (PCAdapt), assessing population structure (DAPC), detecting departure from Hardy–Weinberg equilibrium (HWE), identifying "genetically diagnostic units" in koalas by fixed difference analysis, calculating pairwise FST indices, testing for hierarchical partitioning of genetic variation (AMOVA), examining the relationship between geographical and genetic distances (Mantel test), calculating individual-based measures of genetic variation (homozygosity by locus), and investigating the relationship between genetic diversity in koalas and several key aspects of their environment using a multilevel mixed-effects linear model have all been collated here.

Methods

Genomic DNA was extracted from 314 koala samples using either the Bioline Isolate II Genomic DNA Kit (Bioline, Eveleigh, Australia) following the manufacturer’s protocols, or a standard high-salt precipitation procedure (Sunnucks and Hales, 1996). Genotyping was performed using the Diversity Arrays Technology platform (DArTseq™). DNA was processed as per Kilian et al. (2012), using paired adaptors which corresponded to two different restriction enzyme overhangs: PstI and SphI. The PstI-compatible adapter included an Illumina flow cell attachment sequence, a sequencing primer binding site, and a varying length barcode region. The reverse adapter contained a SphI-compatible overhang sequence and a flow cell attachment region. A digestion–ligation reaction was performed at 37 °C for 2 h with ~100–200 ng of gDNA per sample. The DNA fragments that were successfully cut by both PstI and SphI were then amplified by 30 cycles of polymerase chain reaction (PCR), and the PCR products were sequenced as 77‐bp or 138-bp single‐end reads on the HiSeq 2500 and Novaseq 6000 platforms, respectively (Illumina, San Diego, USA). The resulting short-read sequence data were processed using a variety of software packages and scripts which have been included with the Dryad package.

Funding

Government of New South Wales, Award: Grant Agreement No. KR_2019_01, New South Wales Koala Strategy

Australian Museum Foundation