Novel mitochondrial haplotype of spotted-tailed quoll ( Dasyurus maculatus ) present on Kangaroo Island (South Australia) prior to extirpation

Abstract

Spotted-tailed quolls (Dasyurus maculatus) – cat-sized, carnivorous marsupials – occupied Kangaroo Island (KI), South Australia, for over 50,000 years but became locally extinct following European settlement of the island in 1836. As the largest mammalian predator on KI when the Europeans colonised it, spotted-tailed quolls would have played a significant role in maintaining healthy ecosystem function. The reintroduction of spotted-tailed quolls to KI could redress some of these ecological benefits and would establish a refuge population of the species, which is considered endangered by the Australian government. However, before a reintroduction could be considered, the genetic relationship between KI’s spotted-tailed quolls and the currently recognised extant subspecies needs to be established. While subspecies are difficult to differentiate by skeletal morphology, they are genetically distinct. Here, we extracted ancient DNA from five left dentaries excavated from Kelly Hill Cave (KI) that were morphologically identified as D. maculatus. Following genetic confirmation of these identifications, we sequenced a 450-bp region of the mitochondrial D-loop to determine the subspecific genetic affiliation(s) of KI’s D. maculatus, and therefore the subspecies that may be the most appropriate candidate for reintroduction. We find that all five specimens are most closely related to the Tasmanian subspecies, but form a distinct monophyletic clade that may represent a new subspecies. Further research (including genotyping spotted-tailed quoll specimens from mainland South Australia and Western Victoria) is required before decisions are made regarding the sourcing of individuals for reintroduction to KI.

Keywords

conservation extinction extirpation fossil mitochondria next-generation sequencing reintroduction sub-fossil tiger quoll

Introduction

Spotted-tailed quolls (or tiger quoll, Dasyurus maculatus) are mainland Australia’s largest extant carnivorous marsupial. Like many of Australia’s marsupials, spotted-tailed quoll populations significantly declined following European colonisation and today they are considered endangered under the Australian Environment Protection and Biodiversity Conservation Act (Australian Federal Government, 1999). Prior to genetic analysis, two subspecies of spotted-tailed quoll were recognised: D. m. maculatus, occupying Tasmania and much of the east coast of mainland Australia, and the critically endangered D. m. gracilis, occupying a small, isolated region in northern Queensland.

However, following the first intra-specific genetic analysis of spotted-tailed quolls from across their range using the mitochondrial control region and microsatellites, Firestone et al. (1999) found that the Tasmanian populations comprised a monophyletic clade, genetically distinct from all mainland populations, while D. m. gracilis fell within the mainland D. m. maculatus clade. These results suggest that the two subspecies of spotted-tailed quoll be reclassified as Tasmanian or mainland subspecies (Figure 1). This finding is critical for informing conservation decisions, such as which populations would benefit from admixture with others through translocation (Firestone et al., 1999, 2000).

Figure 1.

The location of the Kelly Hill Cave, Kangaroo Island, South Australia. The distribution of extant D. maculatus subspecies (according to data available from the Atlas of Living Australia) is shown in black. The historic distribution of D. maculatus according to Firestone et al. (1999) is shown in grey. Locations sampled by Firestone et al. (1999) are shown in yellow alongside the identified D-loop haplotype.

The mainland range of spotted-tailed quolls is estimated to have been reduced by up to 90% since European settlement (Jones et al., 2001), and although they are only found on the east coast and Tasmania today, the Holocene distribution of spotted-tailed quolls encompassed a large area of South Australia (Jones et al., 2001), including the Murray River (Fusco et al., 2017), south-east (Reed and Bourne, 2009) and mid-north of South Australia (Peacock and Abbott, unpublished accounts), and Kangaroo Island (KI) (Adams et al., 2016; McDowell, 2013; Peacock et al., 2018; Walshe, 2014). Spotted-tailed quolls were extirpated from the 440,500 ha KI sometime after 1836 (Peacock et al., 2018; Walshe, 2014), most likely because of human persecution (Peacock and Abbott, 2014; Peacock et al., 2018). The extinction of apex predators has been shown to contribute to ecosystem dysfunction (Estes et al., 2011; How et al., 2009), for example, by increasing the intensity of herbivory (Terborgh et al., 2001) or by causing ‘outbreaks’ of mesopredators (Johnson et al., 2007; Ripple et al., 2014; Ritchie and Johnson, 2009). The reintroduction of D. maculatus to KI could have several potential benefits in this regard: for instance, one of the drivers of decline in the KI narrow-leaved mallee (Eucalyptus cneorifolia) woodland – listed as a ‘critically endangered ecological community’ (Commonwealth of Australia, 2014) – is over-grazing of vegetation by herbivores, which the presence of apex predators such as the spotted-tailed quoll may help to suppress. D. maculatus also depredates common brush-tail possums (Trichosurus vulpecula; Belcher, 1995), which are a pest on KI because they eat the eggs and chicks of endangered native birds and compete for nesting hollows (Garnett et al., 1999). Suppressing hyperabundant possums may boost the numbers of birds, which in turn could have a positive effect on re-wilding the landscape on KI by dispersing seed, assisting the regeneration of native vegetation that has been cleared in the recent past. Thus, the reintroduction of spotted-tailed quolls to KI could not only establish an additional population that may help mitigate the threat of extinction elsewhere, but also help restructure the island’s ecosystems (Wood et al., 2017).

Reintroductions by definition seek to return missing or deficient local species, often taking into consideration the genetics of subspecies or subpopulations (Spencer et al., 2017; Woolley et al., 2015). However, the subspecies of D. maculatus that existed on KI in the past is unknown: KI D. maculatus specimens may have belonged to (1) the mainland subspecies, (2) the Tasmanian subspecies, (3) both the mainland and Tasmanian subspecies (coexisting) or (4) a new, genetically distinct subspecies. This question must be addressed to guide any successful re-establishment of a population of D. maculatus on KI. In this endeavour, ancient DNA (aDNA) information retrieved from fossils is necessary, particularly when taxa are morphologically cryptic. For example, aDNA has been used to identify the subspecies of rock wallaby (Petrogale lateralis) that inhabited Depuch Island (Western Australia) prior to extirpation by foxes (Haouchar et al., 2013). Because aDNA is degraded, the authors targeted mitochondrial loci as there are many more copies of the mitogenome than the nuclear genome. Using a similar approach, we extracted aDNA from D. maculatus bone specimens excavated from Kelly Hill Cave, KI (Figure 2), sequenced regions of the mitochondrial D-loop and compared the genetic information with published data from extant mainland and Tasmanian D. maculatus in order to ascertain the subspecies status of this population.

Figure 2.

Dasyurus maculatus Holocene-aged sub-fossil dentaries excavated from Kelly Hill Cave, Kangaroo Island (South Australia) that were sampled for aDNA: specimen (a) MB3660, (b) MB3662, (c) MB3664, (d) MB3661 and (e) MB3663.

Materials and methods

Samples

Five left dentaries (Figure 2) morphologically identified as D. maculatus by Matthew McDowell were excavated in 2013 from the Kelly Hill Cave complex, KI, South Australia (35°59′S, 136°55′E; Figure 1). All samples originated from sediments overlying a radiocarbon age of 1100–950 cal BP (McDowell et al., 2013). Samples were wet sieved on-site with no personal protective equipment used during sample collection.

A modern fur sample of D. maculatus was obtained for use as a positive control upon which to optimise primers. Fur was donated by Softfoot Marsupial Sanctuary, South Australia, and was collected by a veterinarian while the animal was under sedation for unrelated purposes. The animal originated from a population in New South Wales.

Sample preparation

Bone samples were prepared for DNA extraction in a designated ultraclean facility at Curtin University, Western Australia. Teeth were carefully removed from each dentary, then surface-decontaminated using 10% household bleach followed by 70% ethanol. One or two whole teeth were ground into fine powder using a mortar and pestle. Between 100 and 200 mg of tooth powder was placed into a 2.0 mL Safelock Eppendorf tube and stored at −20°C until extraction. Procedures for contamination avoidance as stipulated by Shapiro and Hofreiter (2012), Cooper and Poinar (2000) and Willerslev and Cooper (2005) were abided.

DNA extraction

Ancient samples

Tooth powder was digested for 24 h at 55°C in 1 mL of buffer consisting of 0.25 mg/mL Proteinase K in 0.5 M EDTA. After digestion, each sample was centrifuged for 5 min at maximum speed in a benchtop centrifuge to pellet cell debris. The supernatant was transferred to 13 mL of a buffer containing final concentrations of: 5 M Guanidine Hydrochloride, 40% isopropanol, 0.05% Tween-20, and 90 mM sodium acetate pH 5.2 in ultrapure water. DNA was then extracted from the samples according to Dabney et al. (2013) with minor changes described by Grealy et al. (2017).

Modern sample

In a physically separate laboratory, the modern fur sample was rinsed in ultrapure water prior to digestion in 1.6 mL of buffer containing final concentrations of: 10 mM Tris-HCl, 10 mM NaCl, 5 mM CaCl, 2.5 mM EDTA, 10% Proteinase K, 40 mM DTT and 2% SDS in ultrapure water. The sample was incubated with rotation at 55°C for 24 h. After digestion, the sample was centrifuged for 5 min at maximum speed in a benchtop centrifuge to pellet cell debris. The supernatant was transferred to a Vivaspin 500 column (MWCO 30,000 kDa) and centrifuged at 13,000 r/min until concentrated to 50 μL. This concentrate was combined with 250 μL of PB buffer (QIAGEN) and passed through a QIAquick spin column (QIAGEN) by centrifugation at 13,000 r/min for 1 min. The membrane was washed by passing 750 μL of AW1 buffer through the column as above, followed by 750 μL of AW2 buffer. After a dry-spin at 13,000 r/min for 1 min, DNA was eluted in 50 μL of EB buffer (QIAGEN). The concentration of the DNA was quantified using a QIAxpert spectrophotometer.

PCR amplification

In order to confirm the identity of the samples and gauge the fragment length of amplifiable DNA, a 100-bp barcoding region of the mitochondrial 12 S rRNA gene was amplified using a vertebrate-specific primer set (12SV5: forward 5′-TAGAACAGGCTCCTCTAG-3′, reverse 5′-TTAGATACCCCACTATGC-3′; Riaz et al., 2011). qPCR reactions of 25 μL were prepared in duplicate containing final concentrations of: 1 M Betaine, 0.4 mg/mL BSA, 1× PCR buffer, 2 mM MgCl₂, 0.4 μM of each primer, 0.25 mM dNTPs, 2 U AmpliTaq Gold DNA polymerase, 0.6 μL of 5× SYBR Green in DMSO and 2 μL of DNA in water. Thermal cycling conditions included denaturation at 95°C for 5 min, followed by 50 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s, followed by a final extension step at 72°C for 10 min.

A 451-bp region of the mitochondrial D-loop (described by Firestone et al., 1999) was amplified in six short, overlapping fragments from all samples (Figure S1, available online). Primers were designed to target products less than 170 bp in length (Table S1, available online). Primers were each ordered with unique 8 bp, 5’ multiplex identifiers. PCR conditions were optimised for each primer set using the modern D. maculatus DNA sample as a positive control. For all primer sets, amplification was carried out in duplicate 25-μL qPCR reactions containing final concentrations of: 1 M Betaine, 0.4 mg/mL BSA, 1× PCR buffer, 2 mM MgCl₂, 0.4 μM of each primer, 0.25 mM dNTPs, 2 U AmpliTaq Gold DNA polymerase, 0.6 μL of 5× SYBR Green in DMSO and 2 μL of DNA in water. Thermal cycling conditions included denaturation at 95°C for 5 min, followed by 50 cycles of 95°C for 30 s, 54°C for 30 s and 72°C for 30 s, followed by a final extension step at 72°C for 10 min. Extraction controls and DNA-free reactions were used as negative controls to test for laboratory and cross-contamination.

Library preparation and sequencing

Post PCR, reaction duplicates were combined and purified using the QIAquick PCR purification kit (QIAGEN) according to the manufacturer’s instructions, eluting in 30 μL EB buffer. Purified reactions were pooled in approximately equimolar concentrations after quantification on a QIAxpert spectrophotometer according to the manufacturer’s instructions. Fragments greater than 170 bp were size-selected using the PippinPrep (Sage Science) e-gel system according to the manufacturer’s instructions, in order to remove non-specific products and primer dimer. Products were again purified using the QIAquick PCR purification kit and quantified, as above. Illumina sequencing adapters were then ligated to the ends of the library following the NEBNext ligation protocol (New England Biolabs) with minor changes (SI I). In order to remove adapter dimer and non-ligated product, fragments above 280 bp in length were selected using the PippinPrep and purified as above. The concentration of the final library was quantified using a Qubit fluorometer (Thermo Fisher), according to the manufacturer’s instructions. The library was diluted to 2 nM in EB buffer and was paired-end sequenced on Illumina’s MiSeq using a 300-cycle v2 nanosequencing kit following the manufacturer’s instructions.

Bioinformatics and data analysis

Paired-end reads were trimmed of primer sequences in Geneious v10.0.5 (Kearse et al., 2012) and merged using Usearch v9 (Edgar, 2010). Sequence data was filtered using a previously published pipeline (Grealy et al., 2016) that included quality filtering, chimera filtering, abundance filtering and dereplication. For the 12SV5 primer set, sequences were aligned against NCBI’s reference genetic database GenBank as of November 2017 (Benson et al., 2006) using the blastn v2.7.1 + algorithm (Altschul et al., 1990) displaying the top 25 hits and evoking the options –perc_identity 90 and –qcov_hsp_perc 100. Taxa were assigned to the identity of the reference match with the highest bit score.

For each D-loop region, unique sequences were aligned and the consensus (bases that agree among 75% of sequences; typically identical to the most abundant sequence) was extracted. Each region of each sample was aligned with remaining regions from that sample to create a 472-bp contiguous sequence. D-loop sequences were then aligned in Geneious using the default parameters alongside previously published D. maculatus control region sequences (Firestone et al., 1999; GenBank accession # AF082751-70), and an outgroup D. viverrinus sequence (AF082774). Phylogenetic trees were generated using neighbour-joining, UPGMA, maximum evolution, and maximum likelihood methods coupled with various genetic distance models (Tamura-Nei, Jukes-Cantor, HKY) using 5000 bootstrap replicates to test the statistical significance of the evolutionary relationships inferred. Phylogenetic analyses were carried out using Geneious Tree Builder as well as MEGA v6 (Tamura et al., 2011). A minimum spanning network of haplotypes was drawn using PopART v4.8.5 (French et al., 2014).

Results

Confirmation of specimen identity through DNA barcoding

A 100-bp barcoding region of the mitochondrial 12 S rRNA gene was amplified from all five D. maculatus bone specimens, as well as a modern fur sample used as a positive control for downstream assays. On average, over 100,000 raw reads were obtained per sample, which collapsed to an average of 40 unique filtered reads per sample.

Sequences best matching to published Dasyurus maculatus 12 S rDNA were retrieved from all five ancient samples and the modern sample, confirming the morphological identification of the bone specimens as D. maculatus, and demonstrating that fragments at least 120 bp in length were preserved. However, sample MB3664 yielded relatively few D. maculatus reads (i.e. one unique read of 10 copies matched D. maculatus), indicating that the endogenous DNA was present in very low copy numbers or was swamped by the presence of highly abundant contaminants (human and rat; Table S2, available online). This interpretation is supported by the fact that subsequent assays targeting D. maculatus-specific DNA amplified successfully.

Some contaminating taxa (including other marsupials and rodents; Table S2, available online) were also amplified from several specimens; this is not unexpected for ancient samples and probably arose from the excavation process or depositional context (where diagenetic processes can mobilise and co-mingle DNA from the remains of other species buried in cave sediments), rather than the laboratory environment, as extraction controls and DNA-free PCR controls failed to amplify or amplified human DNA only (Table S2, available online). Because the primers designed to amplify the D. maculatus D-loop are species-specific, contaminants were not amplified in subsequent PCR assays. Low abundance hits that could only be assigned to higher taxonomic levels, such as Dasyurus and Dasyuridae, probably represent background levels of error arising from post-mortem damage (Gilbert et al., 2003), polymerase error (D’Abbadie et al., 2007) or sequencing error (Fox et al., 2014).

Reconstruction of the mitochondrial D-loop from D. maculatus bones excavated from Kelly Hill Cave, KI

All primer sets designed to amplify short regions of the mitochondrial D-loop amplified sequences of expected size in the modern D. maculatus positive control sample, as determined via agarose gel electrophoresis. In contrast, no primer sets amplified using control DNA from other species. We retrieved a total of nearly 700,000 merged reads with, on average, three unique sequences each post quality control (Table S3, available online). Regions that failed to amplify (Regions 2 and 3 for MB3663 and Region 2 for MB3664) were treated as missing data in the alignment. For region 1, MB3662 and the positive control produced a single unique fragment of expected size (118 bp); however, all other samples produced a highly abundant sequence sporting an 11-bp deletion between 64A and 76A, with some (MB3660 and MB3664) yielding both sequence variants; separate phylogenies were reconstructed with and without this deletion masked (see section ‘Phylogenetic placement of KI D. maculatus’). No-template controls failed to amplify for all but two primer sets; however, several orders of magnitude fewer reads were obtained from these negative controls compared with the samples (Table S3, available online) indicating the presence of low-level cross-contamination. Nevertheless, to be conservative, phylogenetic trees were also reconstructed excluding these regions (see section ‘Phylogenetic placement of KI D. maculatus’).

The six amplified regions were concatenated to produce a 472-bp contiguous sequence of the mitochondrial control region from the D. maculatus bone specimens, as well as the modern positive control.

Phylogenetic placement of KI D. maculatus

The total alignment of all D. maculatus sequences (including previously published sequences and outgroup taxon) was 522 bp in length (Figure S2, available online). Any sites where data were not available for more than two sequences were masked, as was the deletion observed in Region 1, producing a final alignment with 445 informative sites (Figure S3, available online).

Phylogenies inferred from all methods and genetic distance models (Table S4, available online) produced the same consensus tree showing that the KI D. maculatus bone specimens form a reciprocally monophyletic clade (100% bootstrap support) sister to the Tasmanian subspecies with high statistical support (89%; Figure 3). The modern D. maculatus positive control was placed within the mainland clade alongside specimens from NSW with high support, as expected (Figure 3). Phylogenetic trees with almost identical topologies were also reconstructed regardless of whether the deletion observed in Region 1 was masked or not (Figure S4, available online); because of this, and for the sake of conservancy, the region beginning with 61 C (an SNP associated with the deletion) and ending with 75 C was masked to produce the final tree (Figure 3). Furthermore, disregarding the data entirely from both Regions 2 and 5 (where low-level cross-contamination was detected in the negative control) produced the same topology shown in Figure 3 (with 366 informative sites), albeit with slightly lower bootstrap support at the last common ancestor between the Tasmanian and KI clades (87% vs 89%; Figure S5, available online).

Figure 3.

Phylogenetic tree generated using 451 bp of the Dasyurus maculatus control region. The tree was generated in Geneious v10. Evolutionary history was inferred using UPGMA tree with evolutionary distances computed with the HKY model. A total of 5000 bootstrap replicates were performed with bootstrap support displayed at each node as a percentage. The tree is drawn to scale, with branch lengths in the units of the number of base substitutions per site. Accession numbers of extant D. maculatus were taken from Firestone et al. (1999). Sequences generated in this study from the positive control (hair silhouette) and ancient specimens from Kangaroo Island are incorporated (dentary silhouettes).

Within D. maculatus, for the 471-bp region examined by Firestone et al. (1999), 34 variable sites were described. Not considering the deletion in Region 1, we identified two new variable sites in the bone specimens from KI (118G and a deletion at 132 bp), as well as an additional KI-specific SNP (361 C; Figure 4; Figure S6, available online). All variable sites were fixed among the five KI D. maculatus specimens. As such, a new haplotype has been identified in the KI specimens, here called Hap KI (Figure 4). The positive control sample was identical to AF082754, AF082758 and AF082760, and therefore was assigned to Hap C from New South Wales, as expected (Firestone et al., 1999).

Figure 4.

An alignment of the new D. maculatus haplotype found within a 471-bp region within the mitochondrial D-loop found from KI specimens (Hap KI). Numbers across the top indicate the site position (bp) in an alignment excluding the outgroup D. viverrinus (cf. Figure S6, available online). The consensus is the most common base among all D. maculatus samples. The number in brackets next to the name describes the number of specimens assigning to that haplotype. KI-specific SNPs are highlighted in grey. Asterisks denote SNPs common only to KI and Tasmanian specimens. Crosses denote SNPs common only to KI and mainland specimens.

A minimum spanning network of haplotypes shows that there is a minimum of six nucleotide substitutions (i.e. 98.6% identity) between the KI haplotype and the Tasmanian haplotypes (Figure 5, E and F), while there is a minimum of 10 substitutions between the KI haplotype and mainland haplotypes (i.e. a maximum of 97.2% identity; Figure 5). Comparatively, haplotypes within each subspecies differ from one another by between one and five nucleotide substitutions. The KI haplotype shares four SNPs that were hitherto unique to the Tasmanian subspecies (29T, 35G, 122C, 352A), but also share three SNPs that were hitherto unique to the mainland subspecies (56T, 268C, 361C).

Figure 5.

Minimum spanning network generated in PopART depicting relationships between D. maculatus haplotypes, including 12 previously published haplotypes (Firestone et al., 1999) and 1 new haplotype derived here from ancient specimens on Kangaroo Island. The geographic location of haplotypes is indicated by the grey rectangles. Crosshatches represent nucleotide substitutions between haplotypes, as does the (approximate) length of the connections.

Discussion

This study reports novel genetic information retrieved from D. maculatus bone specimens excavated from Kelly Hill Cave, KI, where the species persisted until some years after European colonisation. Our aim was to determine the past subspecific affiliation of D. maculatus from KI, and in light of these fossil data, re-evaluate Evolutionary Significant Units (ESUs) within the species with a view to inform future conservation efforts.

Five independent bone specimens from KI gave genetic sequences that differed not only from the positive control sample but also from all modern quolls, indicating that these sequences are authentically ancient and not artefacts from DNA damage or cross-contamination from modern quolls. Because the KI specimens form a monophyletic grouping with high statistical support, and because the genetic distance within the KI clade is 6–10 times lower than the genetic distance between the KI clade and either extant subspecies, the KI specimens may represent a unique subspecies. This genetic distance becomes even higher if we consider the sequence variant containing a SNP at 61 C and associated deletion in Region 1 to be a true haplotype (as opposed to an amplification artefact, such as a nuclear copy of a mitochondrial gene or ‘NuMt’, or as a result of mitochondrial heteroplasmy). This sequence variant may be a true haplotype as it was amplified in four of the five specimens, and where both variants were amplified, the one with the deletion was represented by more than 10 times the number of copies than the variant without the deletion (Table S3, available online); in these samples (MB3660 and MB3664), the variant without the deletion could have resulted from low-level cross-contamination from MB3662 (where only the variant without the deletion was amplified). The use of an independent primer set to amplify the region containing the deletion in Region 1 will help determine whether this is a true mitochondrial mutation, which would be evolutionarily significant and indicate that at least two mitochondrial haplotypes were present on KI in the past. Alternatively, sequencing the whole mitochondrial genome of D. maculatus could show whether the control region has undergone a duplication, potentially clarifying the origin of the deletion in Region 1. Additional specimens throughout time and across KI from sites such as Mt Taylor, Bales Bay and Seton Rock Shelter (where Dasyurus sp. fossils have been found; Hope et al., 1977; McDowell et al., 2015; Walshe, 2014) will also need to be genotyped to better gauge the level of past genetic diversity within the species on KI, which could show undiscovered variation that may nest the KI haplotypes within one of the extant clades. Although it is unlikely that the five specimens sampled here are related to one another given that they span a 150-year time period, additional sampling would also be required to rule out this possibility. It also remains possible that the low mitochondrial diversity observed here is because of cross-contamination from one specimen to the rest, or from the depositional environment; however, given the high number of reads amplified from all specimens across most of the regions (particularly relative to the negative controls), the lack of amplification in the extraction controls, and the fact that teeth surfaces were bleached prior to powdering, this possibility seems unlikely.

Whether the KI spotted-tailed quolls represent a new subspecies or not, our research suggests that at least some D. maculatus individuals from KI were more closely related to Tasmanian individuals than their extant mainland counterparts, despite being geographically closer to the latter. While it is possible that KI and Tasmanian populations were once connected by dispersal across the exposed continental shelf during times when the sea level was low (prior to 9,000–13,000 years ago; Belperio and Flint, 1999; Davies, 1974), it is also possible that there was never direct gene flow between these populations: both may be descended from a common mainland ancestor (or ancestors) that is now extinct, or has (or have) not yet been genotyped. The phylogenetic relationships we have observed may be a natural consequence of sampling fragmented populations that were formerly part of a geographically widespread, genetically connected distribution, as was shown to be the case for brush-tailed bettongs (Bettongia penicillata; Haouchar et al., 2016). Indeed, there are no published genotypes of D. maculatus from South Australia or western Victoria although the South Australian museum alone holds at least ten 19th−20th century specimens from South Australia (www.ozcam.org.au). Thus, fossil and museum specimens, as well as extant individuals, from sites on mainland South Australia (e.g. within the Fleurieu Peninsula (Fusco et al., 2016) and Naracoorte Cave World Heritage area (Macken and Reed, 2013)), as well as western Victoria (such as Fern Cave, Swain’s Cave, McEachern’s Cave, and Glenelg; Jones et al., 2001) and the continental islands of the Bass Strait (King, Flinders, Deal, and Cape Barren Islands; Hope, 1974; Peacock et al., 2018), need to be genotyped in the future.

Furthermore, while there is concordance between the phylogenetic trees inferred from the mitochondrial control region (genotyped here) and microsatellite loci (Firestone et al., 1999), phylogenies inferred from the maternally inherited mitochondria really only reveal the evolutionary history of matriarchal lineages. Consequently, population structuring appears exaggerated in animals that display ‘strong male-biased dispersal’ (Pacioni et al., 2015). D. maculatus is one such species where females hold home ranges but males are transient and can traverse up to 5 km in 2 days (Glen and Dickman, 2005) and range across 359–2561 ha (Claridge et al., 2005). The genotyping of nuclear loci can provide greater resolution of intra-specific genetic diversity and better reflect whole species evolution, particularly where population structure is influenced by differences in male and female behaviour. Sequencing the complete genome of D. maculatus should therefore be considered a research priority as it would not only help in the identification of such phylogenetically informative nuclear loci, but would also help in dating when populations diverged from one another – both of which are crucial for identifying the factors driving evolution within D. maculatus. However, retrieving SNP and microsatellite data from fossil specimens poses considerable technical challenges, and will probably require a shotgun approach coupled with enrichment via hybridisation capture in the future; a reference genome for D. maculatus would assist significantly in this endeavour.

While the list of threatened mainland marsupials that have been successfully introduced to islands (Cardoso et al., 2009; Thomas, 2014; Woinarski et al., 2014) continues to grow, the risks, benefits and feasibility of reintroducing spotted-tailed quolls need to be considered further (Eldridge, 2010; Louys et al., 2014). To do this, a comprehensive picture of the genetic diversity that existed in D. maculatus across time and space is needed. Such knowledge would not only assist in establishing a successful, sustainable insurance population of spotted-tailed quolls on KI, but could also have implications for the conservation of today’s extant mainland and Tasmanian populations (Weeks et al., 2016), which are currently being managed separately.

Conclusion

Australian islands have acted as a haven for many species that are in danger of extinction on the mainland and play an important part in modern conservation. Ancient DNA extracted from fossil specimens of KI D. maculatus was used to elucidate their evolutionary relationship with extant subspecies, with the view to inform decisions regarding re-establishing a population on KI, if such a reintroduction were proposed. D. maculatus bone specimens from KI were found to be close relatives of the Tasmanian subspecies, but were a genetically distinct group in their own right that with further research may support its designation as a novel subspecies. Elucidating the processes that have resulted in this phylogeographic pattern requires further DNA analysis of recent and historical specimens from mainland South Australia and Western Victoria. While we argue that reintroduction of D. maculatus to KI is a feasible venture (sensu Peacock et al., 2018), we recommend a thorough evaluation of the existing mitochondrial and nuclear genetic variation within mainland and Tasmanian populations, as well as an assessment of their interbreeding success and suitability to KI’s environment. Reintroducing D. maculatus to KI may improve conservation of the species and could restore ecological connections lost when this species was extirpated from KI after European settlement; however, further research is required before deciding if and how a translocation should happen.

Supplemental Material

Grealy_et_al_The_Holocene_Supplementary_Information_Revision – Supplemental material for Novel mitochondrial haplotype of spotted-tailed quoll (Dasyurus maculatus) present on Kangaroo Island (South Australia) prior to extirpation

Supplemental material, Grealy_et_al_The_Holocene_Supplementary_Information_Revision for Novel mitochondrial haplotype of spotted-tailed quoll (Dasyurus maculatus) present on Kangaroo Island (South Australia) prior to extirpation by Alicia Grealy, Matthew McDowell, Clancy Retallick, Michael Bunce and David Peacock in The Holocene

Footnotes

Acknowledgements

The authors thank Frederik Seersholm for a critical reading of the manuscript.

Author contributions

The study was conceived by DP and MM. Bone was excavated and identified by MM. Modern quoll fur was provided by CR. AG designed and conducted the genetic experiments. AG analysed the data. AG wrote the manuscript with contributions from all the authors.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received the following financial support for the research, authorship and/or publication of this article: The study was supported financially by Softfoot Sanctuary and an Australian Research Council grant (DP160104473) awarded to MB.

ORCID iD

Alicia Grealy

Data accessibility statement

The DNA sequences have been deposited on GenBank (accession numbers: TBA). Data generated can be accessed through DataDryad at doi: TBA. Correspondence and requests for material should be addressed to AG (alicia.grealy@uqconnect.edu.au).

Supplemental material

Supplemental material for this article is available online.

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