DOI: 10.5061/dryad.h18931zqx
title: Immediate and long-term genetic consequences of linear transport infrastructure: Can fauna crossing mitigate its cost?

description: The genetic consequences of the subdivision of populations are regarded as
 significant to long-term evolution, and research has shown that the scale
 and speed at which this is now occurring is critically reducing the
 adaptive potential of most species which inhabit human-impacted
 landscapes. Here, we provide a rare, and to our knowledge, the first
 analysis of this process while it is happening and demonstrate a method of
 evaluating the effect of mitigation measures such as fauna crossings. We
 did this by using an extensive genetic dataset collected from a koala
 population which was intensely monitored during the construction of linear
 transport infrastructure which resulted in the subdivision of their
 population. First, we found that both allelic richness and effective
 population size decreased through the process of population subdivision.
 Second, we predicted the extent to which genetic drift could impact
 genetic diversity over time and showed that after only 10 generations the
 resulting two subdivided populations could experience between 12–69% loss
 in genetic diversity. Lastly, using forward simulations we estimated that
 a minimum of 8 koalas would need to disperse from each side of the
 subdivision per generation to maintain genetic connectivity close to zero
 but that 16 koalas would ensure that both genetic connectivity and
 diversity remained unchanged. These results have important consequences
 for the genetic management of species in human-impacted landscapes by
 showing which genetic metrics are best to identify immediate loss in
 genetic diversity and how to evaluate the effectiveness of any mitigation
 measures.
description: <em><strong>The study koala
 population.</strong></em> We were able to
 ask these questions because of the rareness of the dataset which was
 obtained during and after construction of a linear transport
 infrastructure (e.i. rail line) which resulted in the subdivision of a
 large population of koala. The dataset was collected as part of an
 extensive Koala Management Program (Beyer et al. 2018), in which all
 free-ranging koalas (<em>Phascolarctos</em>
 <em>cinereus</em>; n = 503 koalas) were captured and healthy
 animals released and monitored between 2013 and 2017 for the purpose of a
 rail infrastructure project located in the eastern Moreton Bay Region
 (-27.234°; 153.036°, Queensland, Australia). These koalas formed part of a
 single breeding population (Schultz et al. 2022) inhabiting a mixture of
 urban and peri-urban koala habitat remnants along 12.6 kms of the linear
 transport infrastructure project footprint (Hanger et al. 2017).
 Koala-proof fencing was installed alongside the rail line corridor to
 prevent koala death from crossing attempts, and underpasses were built at
 strategic locations along the rail line (Hanger et al. 2017). Because all
 koalas were VHF and/or GPS tracked during all phases of construction (pre,
 during and post), it afforded us detailed knowledge of how and why the
 impacted koala population’s size varied throughout the rail line
 construction phases: (1) all deaths and births and causes of death (Hanger
 et al. 2017; Beyer et al. 2018), (2) which koalas were translocated as a
 result of the rail line infrastructure project, (3) their locations pre
 and post-construction (whether koalas occupied habitat on one side or the
 other of the rail line), and (4) rail crossing events by koalas after
 establishment of the rail barrier using both dedicated fauna crossings and
 hydrology culverts (Dexter et al. 2017). In total, 291 koalas out of the
 461 processed (57.8% population size decline) died or were euthanised
 during the four-year monitoring program: 182 from predation, 84 from
 disease, 14 from trauma, and 11 unknown causes of death (Beyer et al.
 2018). Twenty-eight were translocated as a result of their core home range
 directly overlapping with the rail line and/or because current land-use
 and imminent future land-use precluded their long-term viability if left
 <em>in situ</em>. Full protocols are
 available in the technical report by Hanger et al. (2017), and scientific
 permits and ethics approvals for catching, handling, veterinary
 examination and treatment, and monitoring of koalas as follows: scientific
 research permits issued by Queensland Department of Environment and
 Heritage Protection WISP-11525212, WISP-16125415, WISP-13661313,
 WITK-14173714, WISP-17273716; animal ethics approvals from Queensland
 Department of Agriculture and Fisheries CA-2012/03/597, CA-2013/09/719,
 CA-2014/06/777, CA-2015/03/852 and CA-2016/03/950.
 <em><strong>Koala subsampling, genotyping and final
 genetic dataset.</strong></em> Single
 nucleotide polymorphism (SNP) genotyping data were generated from blood or
 tissue samples, collected during routine veterinary examinations from 452
 of the 503 monitored individual koalas for which blood or tissue samples
 remained. Blood samples were stored at -20 °C, and tissue samples were
 stored in 70% ethanol. DNA was extracted using the DNeasy Blood and Tissue
 Kit (QIAGEN), following the manufacturer’s protocol, and DNA extracts were
 stored at -80° C. SNP genotyping was conducted as in Kjeldsen et al.
 (Kjeldsen et al. 2019) by Diversity Arrays Technology, Canberra, using
 their proprietary DArTseq<sup>TM</sup> technology.
 DArTseq<sup>TM</sup> utilises a combination of next-generation
 sequencing platforms and DArT complexity-reduction methods (Kilian et al.
 2012; Courtois et al. 2013; Cruz et al. 2013; Raman et al. 2014). The
 protocol is also optimised for organism and application by selecting the
 most appropriate complexity reduction method. This is assessed based on
 minimising skewed size ranges, non-ideal numbers of fragments, and
 percentages of repetitive elements. Samples were then processed as per
 Kilian et al. (2012). SNP genotyping produced a total of 8649 SNPs. We
 then filtered those SNPs for minor allele frequency ≥ 0.05, 90% individual
 call-rate, ≥ 99% technical replication average which resulted in 3655 SNPs
 from 367 koalas in total. We further removed 42 koalas which, while
 included in the Koala Management Programme, where found in areas not
 contiguous with the main population, and subsequently used as
 translocation sites for koalas whose home ranges overlapped the rail
 infrastructure footprint. Finally, to increase the accuracy of our
 simulations (see below), we removed individual koalas with any missing
 data in their genotypes, resulting in 270 individuals.
 To isolate the effect of population subdivision caused by the
 construction of the linear infrastructure from mortality events caused by
 predation, disease, trauma from roads and fighting and unknown causes, we
 organised the koala genotypes into three genetic datasets. As our point of
 reference, we created Dataset 1 (n = 270) which contains all the genotypes
 from the monitored koalas collected during the 4 years. To account for
 death by predation, disease, trauma and unknown events, we created Dataset
 2 (n = 114) which contains all genotyped koalas from dataset 1 minus
 koalas which died of from predation, disease, trauma and unknown causes.
 Comparing changes in the genetic composition of dataset 1 and 2 allows us
 to understand the impact of population decline due to mortality events
 caused by predation, disease, trauma and unknown causes. To then estimate
 the immediate and longer-term effect of the linear infrastructure on our
 studied koala population, we created Dataset 3 which contains all
 genotyped koalas from dataset 2 minus those that were translocated because
 their core home range sat in the linear transport infrastructure footprint
 and any new koalas born after the linear transport infrastructure was
 built. Koalas in dataset 3 were then divided into two to represent their
 locations above (n = 27) and below (n =75) the linear transport
 infrastructure. Comparing changes in the genetic composition of dataset 2
 and 3 (above and below) allowed us to isolate the immediate and predict
 the longer-term genetic consequences of the linear transport
 infrastructure project (i.e. population size reduction by translocation
 and population subdivision). This left 102 koalas that were previously
 genetically connected, then became subdivided on either side of the
 rail-line.