Post on 19-Jan-2023
RESEARCH ARTICLE
Phylogeographic evidence links the threatened ‘Grampians’Mountain Dragon (Rankinia diemensis Grampians)with Tasmanian populations: conservation implications in south-eastern Australia
Julienne Ng • Nick Clemann • Stephanie N. J. Chapple •
Jane Melville
Received: 26 May 2013 / Accepted: 8 October 2013 / Published online: 7 November 2013
� Springer Science+Business Media Dordrecht 2013
Abstract The importance of protecting genetic diversity
within a species is increasingly being recognised by con-
servation management authorities. However, discrepancies
in conservation policy between authorities, such as state
versus national bodies, can have significant implications for
species management when they cross state boundaries. We
conducted a phylogeographic study of the south-eastern
Australian lizard Rankinia diemensis to identify evolu-
tionary significant units (ESUs), including the endangered
population from the Grampians National Park in western
Victoria. Phylogenetic analyses of two gene regions
(mtDNA: ND2; nuclear: RAG1) revealed high levels of
genetic divergence between populations, indicating isola-
tion over long evolutionary time frames. Based on criteria
of genetic divergence and isolation, R. diemensis contains at
least two ESUs that require specific management. We found
that R. diemensis from the Grampians are closely related to
Tasmanian populations, but that the divergence between
these regions is great enough (3.7 % mtDNA) that they
should be considered separate ESUs. However, we believe
the close evolutionary ties between these two regions needs
to be taken into account; yet under current practises, con-
servation management of subspecific ESUs relies on state-
level efforts. We argue that another population that occurs
on the Victorian coast also qualifies as an ESU and requires
targeted conservation action. Rankinia diemensis provides a
case-in-point of the discrepancy between the state-level
approach of maintaining genetic variation within a species
and the more conservative Commonwealth focus on con-
serving biodiversity at the species level.
Keywords Agamidae � Bass Strait � Conservation
management � Evolutionary significant units (ESUs) �Genetic variation � Threatened species protection
Introduction
Conservation management authorities are increasingly
recognising the importance of protecting genetic diversity
within a species. By using molecular studies to identify
populations that represent independent evolutionary lin-
eages, or evolutionary significant units (ESUs), manage-
ment of these populations can be prioritised to maintain the
genetic diversity and evolutionary potential of the species
(Whelan et al. 2009). For example, Australia’s Biodiversity
Conservation Strategy 2010–2030 (Natural Resource
Management Ministerial Council 2010) recognises the
long-term benefits of biodiversity conservation and the
maintenance of evolutionary potential. Australian wildlife
can be listed as threatened at a state or national level, or
both. National listing occurs under the Federal Environ-
ment and Biodiversity Conservation Act 1999 and various
States list species as threatened under their own legislation.
Electronic supplementary material The online version of thisarticle (doi:10.1007/s10592-013-0544-1) contains supplementarymaterial, which is available to authorized users.
J. Ng � S. N. J. Chapple � J. Melville (&)
Department of Sciences, Museum Victoria, GPO Box 666,
Melbourne, VIC 3000, Australia
e-mail: jmelv@museum.vic.gov.au
Present Address:
J. Ng
Department of Biology, University of Rochester,
Rochester, NY 14627-0211, USA
N. Clemann
Department of Sustainability and Environment, Arthur Rylah
Institute for Environmental Research, Heidelberg, VIC 3084,
Australia
123
Conserv Genet (2014) 15:363–373
DOI 10.1007/s10592-013-0544-1
Relevant to this study, Tasmania lists threatened species
under its Threatened Species Protection Act 1995, and
Victoria does likewise under the Flora and Fauna Guar-
antee Act 1988; both have a legislated commitment to
retain evolutionary potential and genetic diversity. Con-
servation management of taxa listed as threatened at a state
level typically only occurs within that state and not within
the remainder of their range. This can have implications for
the management of species where ESUs cross state borders,
as co-operative interstate management plans can be diffi-
cult to establish and coordinate (Whelan et al. 2009).
Such management issues are particularly challenging in
south-eastern Australia where the distribution of some
species spans Bass Strait. These species occur on the island
state of Tasmania, the southern Australian mainland and,
frequently, on islands within Bass Strait. For example,
Orange-bellied Parrots (Neophema chrysogaster) breed in
south-western Tasmania and overwinter in the south-east of
mainland Australia (Drechsler 1998). And with a similar
life-history, the Swift Parrot (Lathamus discolor) breeds in
the forests of eastern Tasmania and over-winters in
southern regions of mainland Australia (Mac Nally and
Horrocks 2000). Research on both species has highlighted
the importance of conservation management in both the
breeding and over-wintering parts of their range. Man-
agement of these species is facilitated by being listed as
threatened at a national level, allowing conservation strat-
egies to be developed across States (Orange-bellied Parrot
National Recovery Team 2006; Saunders and Tzaros
2011).
Tasmania is of particular importance in the conserva-
tion of Australian species because of its long history of
isolation from mainland Australia (*14,000 years),
resulting in high levels of endemicity and providing a
refuge from many invasive species since European set-
tlement. Species whose distributions span Bass Strait are
of particular interest in terms of whether the Tasmanian
populations constitute ESUs that are distinct from main-
land populations. For example, the Striped Marsh Frog
(Limnodynastes peronii) is listed as endangered in Tas-
mania but is common and widespread along the eastern
seaboard of mainland Australia. Molecular work showed
that the Tasmanian populations of L. peronii form a clade
with populations in western Victoria and South Australia
(Schauble and Moritz 2001), but that there is a significant
genetic divergence between the western Victorian/South
Australian populations and the more eastern mainland
populations. Thus, it is appropriate to ask whether the
Tasmanian and western Victorian/South Australian pop-
ulations should be considered a single ESU? Or should
the endangered Tasmanian populations be managed sep-
arately? Other small terrestrial vertebrate species, such as
the Tussock Skink (Pseudemoia pagenstecheri) and
Glossy Grass Skink (Pseudemoia rawlinsoni) that are
listed as threatened in Victoria and Tasmania (but not
listed nationally) also provide challenges about whether
(and how) threatened species should be managed across
jurisdictions, especially when separated by obvious bar-
riers such as Bass Strait.
The Mountain Dragon (Rankinia diemensis) occurs in
south-eastern Australia, from Tasmania and the Bass
Strait Islands to southern Victoria, then along the Great
Dividing Range to near Tamworth in New South Wales
(Wilson and Swan 2010; Fig. 1). The isolated and dis-
junct nature of some Victorian populations of R. diem-
ensis suggests that this species might consist of multiple
ESUs (Clemann 2003). Currently the ‘Grampians’ form,
an isolated population known only from the Grampians
National Park (NP) in western Victoria, is listed as crit-
ically endangered in Victoria (Department of Sustain-
ability and Environment 2013). Rankinia diemensis is not
listed as threatened nationally or in any Australian State
other than Victoria. Another isolated population at An-
glesea in southern Victoria is also of conservation interest
because it is located on the fringe of a popular coastal
town where it may be threatened by residential develop-
ment, human disturbances, altered fire regimes and pre-
dation by domestic dogs and cats (Clemann 2003). The
Anglesea population is unique amongst Victorian popu-
lations as it inhabits coastal heathland instead of upland
Fig. 1 Distribution of the samples sequenced in the current study for
Rankinia diemensis. Symbols represent different mtDNA clades
identified in Fig. 2: Clade 1—New South Wales (NSW) (triangle);
Clade 2—Victoria (Anglesea: black circle; central VIC: gray circle;
Big River SF: white circle); and Clade 3—Grampians NP (asterisks)
and Tasmania (Tas) (Flinders Is.: white square; mainland: black
square). Shading represents the current distribution of R. diemensis
(Wilson and Swan 2010)
364 Conserv Genet (2014) 15:363–373
123
heaths and sclerophyll forest (Wilson and Swan 2010). In
addition, there are multiple isolated populations that occur
in Bass Strait, including on the Furneaux Group of
islands, Flinders Island and other nearby islands. Thus,
the isolated nature of Tasmania, the Bass Strait islands,
Anglesea and the Grampians populations suggests poten-
tial for significant genetic and adaptive divergence from
conspecific populations and, consequently, that some R.
diemensis populations may qualify as ESUs.
We conducted a phylogeographic study of R. diemensis
to identify ESUs in this species. Specifically, we compare
populations across the range of this species, including
samples from Tasmania, Flinders Island in Bass Strait,
Anglesea and the Grampians NP. We undertook extensive
phylogenetic analyses using mitochondrial and nuclear
markers to assess evolutionary relationships and levels of
genetic divergence between R. diemensis populations. Our
study resolves longstanding debate over the presence of
ESUs (as defined by Moritz 1994) in this species, where we
identify independent historic lineages that form mono-
phyletic groups when analysed with mtDNA markers and
display divergence at nuclear loci. We also provide
Fig. 2 Maximum likelihood
phylogenetic tree for Rankinia
diemensis based on *1,400 bp
mitochondrial DNA (ND2).
Samples sequenced in the
current study are designated by
tissue or museum registration
numbers and outgroups have
been previously published (see
Table S1 for details). ML
bootstraps ([70 %) are
provided above the branches
and posterior probabilities
([90 %) below the branches.
Scale bars represent
substitutions per site. Symbols
on the clades are those from
Fig. 1
Conserv Genet (2014) 15:363–373 365
123
definitive information to guide the conservation manage-
ment of distinct ESUs across State boundaries.
Materials and methods
Study species and tissue samples
Rankinia diemensis belongs to the family Agamidae and is
the southernmost agamid in the world. Until recently, it had
been placed within the genus Tympanocryptis. However,
molecular analyses indicated that the species was a distinct
lineage (Melville et al. 2001; Schulte et al. 2003) and it was
placed in the monotypic genus Rankinia (Melville et al.
2008).
Rankinia diemensis is distinctive amongst Australian
agamids as it favours cool highland habitats of south-
eastern Australia and occurs in some areas that receive
annual snowfall (Clemann 2003). On mainland Australia,
the species’ distribution ranges from the uplands of New
South Wales, along the eastern highlands to north-east of
Melbourne (Fig. 1). There are also isolated populations to
the north-west of Melbourne at Wombat State Forest (SF),
to the south-west at Anglesea, and in western Victoria in
the Grampians NP. Further south, the species occurs on
islands in the Bass Strait and in the north and east regions
of mainland Tasmania (Welling 1999; Clemann 2003).
Field-collected tissue samples (tail tips) and preserved
tissue samples (liver) from museum collections were
included in our study to provide coverage across the dis-
tribution of R. diemensis (Fig. 1; Table S1). A total of 36
genetic samples of R. diemensis were included in this study
from New South Wales; central and eastern Victoria; An-
glesea in southern Victoria; the Grampians NP in western
Victoria; Flinders Island; and Tasmania. Our analyses also
included two previously published outgroups: Amphibolu-
rus muricatus (GenBank#s—ND2: AF128468; RAG1:
HQ662426) and Pogona barbata (GenBank#—ND2:
AF128474).
Laboratory protocols and alignment of DNA sequences
Genomic DNA was extracted from tail tips or liver samples
using a DNAeasy tissue extraction kit (Qiagen) as per
manufacturer’s instructions or using a using Proteinase K
digestion and chloroform–isoamyl alcohol extraction. For
all specimens, a fragment (*1,200 bp) of the mtDNA
genome was targeted that includes the entire protein-cod-
ing gene ND2 (NADH dehydrogenase subunit two) and
flanking genes encoding tRNATrp, tRNAAla, tRNAAsn,
tRNACys, tRNATyr. For a subset of specimens, we
sequenced a *1,200 bp of recombination activating gene-
1 (RAG1) exon in the N-terminal domain (see Melville and
Hale 2009). Oligonucleotide primer pairs used in PCR
amplification and sequencing of mitochondrial and nuclear
genes are detailed in Melville et al. (2011). Amplifications
were performed in 25 ll volumes in the presence of
1.5 mM MgCl2, 0.2 mM dNTPs, 0.2 lM of forward and
reverse primer, 19 Qiagen PCR buffer and 1 Unit of
HotStarTaq DNA polymerase (Qiagen). Thermal cycling
conditions consisted of an initial denaturing and enzyme
activation step at 95 �C for 15 min followed by 40 cycles
of denaturing at 95 �C for 20 s, annealing at 48 �C (RAG1)
or 55 �C (ND2) for 20 s, and extension at 72 �C for 90 s,
using a Corbett thermocycler. Negative controls were run
for all amplifications. PCR amplifications were visualised
on a 1.2 % agarose mini-gel and amplified products were
purified using GFX spin columns or using SureClean Plus
(BIOLINE). Purified product was sent to Macrogen
(Korea) for sequencing.
Sequence chromatograms were edited using Geneious
6.1.2 (Biomatters Ltd.) to produce a single continuous
sequence for each specimen. Mitochondrial DNA sequences
were aligned using tRNA secondary structure models (Ma-
cey et al. 1997) and protein-coding regions were translated
to amino acids to check alignment and for stop codons.
Phylogenetic analyses
Phylogenetic analysis of all samples for the mtDNA gene
region incorporating ND2 was undertaken using maximum
likelihood in PhyML (Guindon and Gascuel 2003) imple-
mented in Geneious 6.1.2 (Biomatters Ltd.) and Bayesian
analyses in MrBayes 3.2 (Ronquist et al. 2012). Amphib-
olurus muricatus and Pogona barbata served as outgroups
as they are from the closest related genus to R. diemensis
(Melville et al. 2001). Maximum likelihood phylogenetic
trees were estimated using a BEST topology search.
Analyses were performed using a general time-reversible
model of sequence evolution (GTR; Tavare 1986). This
model was chosen based on preliminary analyses using
jModeltest 2.1.3 (Darriba et al. 2012) that chose the
TIM2 ? C as the optimal model with the Akaike Infor-
mation Criterion. However, current implementation of
PhyML (Guindon and Gascuel 2003) in Geneious 6.1.2
(Biomatters Ltd.) does not allow for specification of the
TIM2 substitution model. A gamma distribution was fixed
using the jModeltest estimates and all other model
parameter values were estimated from the data. Bootstrap
resampling (Felsenstein 1985) was applied to assess sup-
port for individual nodes in each above-mentioned analysis
using 100 bootstrap replicates in PhyML with the same
settings as above. We considered a bootstrap value of
C95 % as strongly supported (Felsenstein and Kishino
1993),\95 to C70 % as moderately supported, and\70 %
as weakly supported.
366 Conserv Genet (2014) 15:363–373
123
Bayesian analyses were performed using the evolution-
ary model selected by jModelTest with parameters esti-
mated from data during the analysis. Four Markov chains
were used in each of two simultaneous runs starting from
different random trees. Analyses were run for 10 million
generations for each dataset. Standard deviation of split
frequencies was used as a convergence diagnostic to con-
firm suitability of run length. For all analyses, it was
confirmed that potential scale reduction factor values were
close to 1.0, indicating that an adequate sample of the
posterior probability distribution had been achieved (Ron-
quist et al. 2012). In addition, the output was examined
using Tracer v1.3 (Drummond and Rambaut 2003) to
check that stationarity had been reached.
We used a Bayesian framework for species tree estima-
tion, incorporating both gene regions (ND2 and RAG1), to
determine whether the predicted ESUs constitute evolu-
tionary lineages across the two gene regions. To do this
analysis we used a reduced ND2 dataset, matching sequence
data for individuals in the RAG1 dataset, resulting in two
datasets of 25 individuals. We used *BEAST, enabled in
BEAST v1.7.5, to co-estimate the two gene trees embedded
in a shared species tree (see Heled and Drummond 2010).
Unlinked substitutions models were employed across the
loci and GTR ? C (ND2) and GTR ? C ? I (RAG1)
models of sequence evolution were implemented. These
models were chosen based on preliminary analyses using
jModelTest. A Yule process species tree prior was specified
and the gene tree priors were automatically specified by the
multispecies coalescent. The analysis was run for 50 million
generations. The output was examined using Tracer v1.3
(Drummond and Rambaut 2003) to check that stationarity
had been reached. An estimate of the species tree was
obtained using TreeAnnotater.
Results
Mitochondrial DNA
To investigate phylogeographic relationships within R.
diemensis, 35 new sequences and 3 published sequences
(Melville et al. 2001) were analysed for the ND2 protein
coding gene. The alignment comprised 1,367 characters:
427 characters were variable and 286 characters were par-
simony informative. A TIM2 ? C model was selected as the
best fitting model for likelihood analysis using AIC criteria.
Model parameters were: gamma = 0.2160; substitution
rates = 0.26481, 3.85451, 0.26481, 0.10000, 2.94408; and,
nucleotide frequencies 0.3508, 0.3077, 0.1034.
The mtDNA Bayesian (mean ln-likelihood -6,192.54) and
ML (ln-likelihood -6,155.21153) analyses recovered three
well supported monophyletic lineages (Fig. 2), consisting of
samples from: (1) NSW; (2) central and eastern Victoria; and
(3) western Victoria, Flinders Island in Bass Strait, and Tas-
mania. A sample from Big River, Victoria, forms the basal
lineage of clade (2), but this is not well supported with boot-
strap resampling or Bayesian inference. Clade (3) is resolved
as the sister lineage to the remainder of R. diemensis, but this
relationship is not well supported. Within two of the clades,
there are well supported subclades. The NSW clade consists of
two well supported subclades, one from Goonoo, NSW, and
the other consisting of two samples (Lawson and Sydney,
NSW). Similarly, clade (3) contains two well supported
subclades, one from western Victoria (Grampians NP) and the
second consisting of samples from Flinders Island and
Tasmania.
Genetic divergences between all the clades and subc-
lades is significant (Table 1), ranging between 7.1 and
9.1 %, with the exception of the divergence between the
western Victoria (Grampians NP) and Flinders Island/
Tasmania subclades that have a divergence level of 3.7 %.
The single sample from Big River (NMVD 71904), central
Victoria, is highly divergent from all clades (Table 1), with
divergences ranging between 8.2 and 9.1 %, including
from the relatively nearby King Spur (MNVD74165)
sample, which is *110 km away, and is 8.2 % divergent.
Estimate of species tree
A reduced ND2 dataset, matching sequence data for indi-
viduals in the RAG1 dataset, was used, resulting in two
datasets of 25 individuals. A new jModelTest was conducted
on this reduced mtDNA dataset to estimate the optimal
model of evolution. A TrN ? C model was selected as the
best fitting model for likelihood analysis using AIC criteria.
Model parameters were: gamma = 0.1520; substitution
rates = 0.1000, 0.245, 0.1000, 0.1000, 0.1694; and, nucle-
otide frequencies = 0.3545, 0.3016, 0.1012.
Twenty-five new sequences and one previously pub-
lished sequence were analysed for the RAG1 nuclear gene.
The alignment comprised 1,227 characters: 59 characters
were variable and 36 characters were parsimony informa-
tive. A TPM3uf ? I ? C model was selected as the best
fitting model for likelihood analysis using AIC criteria.
Model parameters were: gamma = 0.7300; proportion of
invariant sites = 0.8310; substitution rates = 0.34379,
3.94226, 0.10000, 0.34379, 3.94226; nucleotide frequen-
cies = 0.3246, 0.2218, 0.2181.
The posterior parameter value estimates from the
*BEAST species tree analysis were characterised by high
([200) effective sample sizes and convergence of the indi-
vidual runs was confirmed from assessments using Tracer.
The maximum clade credibility trees from the posterior sets
of species trees inferred from both genes were similar
(Fig. 3). The topology of the mtDNA tree was the same as
Conserv Genet (2014) 15:363–373 367
123
that for the full dataset. In the topology of the RAG1 tree,
however, three samples (JN4 Anglesea, NMVD71902
Wombat State Forest (SF) and NMVD71908 Wombat SF)
form a poorly supported clade, which does not occur in the
mtDNA tree. In addition, the remainder of the central and
eastern Victorian samples were not resolved as monophy-
letic in the RAG1 tree. However, no branches received
strong branch support (i.e.,[95 %). In the RAG1 tree only
two clades received strong support: the NSW samples
(97.9 %) and a clade containing Tasmanian, Flinders Island
and Grampians NP samples (99.8 %). These well supported
clades also occur in the mtDNA tree.
Most nodes in the species tree inferred using both ND2
and RAG1 gene regions were well supported (Bayesian
posterior probability [95 %), with the exception of the
clade containing central and eastern Victorian populations
including the Big River sample (72.2 %). However, the
clade containing the central and eastern Victorian popu-
lations, excluding the Big River sample, received strong
support (99 %). Within regions, sister relationships were
not highly supported for Tasmania and Flinders Island
(92.7 %) and Kinglake and Wombat SF (38.7 %). The
clade containing Tasmanian, Flinders Island and Grampi-
ans NP samples was highly supported (99.6 %).
Discussion
Phylogeographic structure and historical biogeography
The mtDNA phylogenetic analyses revealed high levels of
genetic differentiation between populations of R. diemensis,
indicating isolation over long evolutionary time frames.
Genetic divergences between major geographic regions
(i.e., Tasmania, central/eastern Victoria and NSW) averaged
8–9 %. Based on fossil-calibrated molecular clock estima-
tions in related Australian agamid groups (e.g., Amphibo-
lurus—Melville et al. 2011), divergences between these R.
diemensis populations would have occurred during the late
Miocene or the Pliocene. We found a matching level of
divergence within Victoria between the Grampians NP and
populations in the rest of Victoria (range 7.6–8.2 %).
However, divergences between the Grampians NP popula-
tion and its sister lineage (Tasmanian and Flinders Island
populations) was only about 3.7 %. This suggests that the
common ancestor of the Grampians NP and Tasmanian
populations diverged from the central and eastern Victorian
populations during the late Miocene to Pliocene, while the
divergence between Grampians and Tasmanian population
is of Pleistocene origin.
Genetic divergences across Bass Strait during the Pleis-
tocene would have been mediated by climatic changes from
glacial to inter-glacial cycling. The cool to cold climates of Ta
ble
1U
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8.3
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.9±
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nd
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and
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(8.6
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1(8
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368 Conserv Genet (2014) 15:363–373
123
Tasmania extend back into the Pliocene, escalating with the
establishment of the Pleistocene glacial oscillations. Tas-
mania was the only part of Australia that experienced sig-
nificant glaciation during the Plio-Pleistocene. Tasmania
was isolated from mainland Australia from the time of the
penultimate deglaciation at about 135–43 kya (Lambeck and
Chappell 2001). The first sustained land connection on the
eastern side occurred at about 43 kya. At the last glacial
maximum (*21 kya), most of the Bassian Plains were
exposed, sea levels were *120 m lower than present and
land area increased by 25 % (Williams 2000). At 17.5 kya
the relative sea level had risen sufficiently for it to enter the
Bass Basin from the west and form an estuarine environment,
cutting a western dispersal route between Tasmania and
Fig. 3 Gene and species tree
phylogenies based on the
25-individual datasets inferred
using *Beast: a mtDNA (ND2);
b nuclear (RAG1); and c species
tree. Clade posterior
probabilities are indicated on
branches. Scale bars represent
substitutions per site. Symbols
on the clades are those from
Fig. 1
Conserv Genet (2014) 15:363–373 369
123
Victoria. Then, at 14 kya, the sea level rise reached the
barrier in the east and Tasmania became isolated from the
Australian mainland (Lambeck and Chappell 2001). These
changes in sea level during the Pleistocene shaped the bio-
geographic history of small vertebrate species, such as rep-
tiles and amphibians (Rawlinson 1967, 1974; Littlejohn and
Martin 1974).
There is a growing body of phylogeographic research
examining the historical biogeography of reptile and frog
species in south-eastern Australia. However, only a handful
of studies have investigated phylogeographic patterns
across Bass Strait (Table 2). A study of the Common
Froglet (Crinia signifera) found that Tasmanian popula-
tions comprised a monophyletic lineage, sister to southern
Victorian and South Australian populations (Symula et al.
2008). These authors concluded that although Tasmania
was connected to Victoria as recently as 14 kya, the Tas-
manian radiation of C. signifera was approximately
6 million years old. Thus, the divergence of Tasmanian
populations could not be attributed to the inundation of the
Bassian Plains alone. In contrast, Chapple et al. (2011)
found that Tasmanian populations of the Delicate Skink
(Lampropholis delicata) shared haplotypes with eastern
Victorian populations, indicating a connection between
these two regions until relatively recently (*12–15 kya;
Table 2). Although L. delicata colonised Tasmania during
the late Pleistocene, Chapple et al. (2011) were unable to
eliminate the possibility that L. delicata became estab-
lished in Tasmania through human-assisted colonisation.
Most of these studies (Table 2) have found a genetic link
between south-eastern Victorian and Tasmanian popula-
tions; only a study of two frogs species (Limnodynastes
peronii and L. tasmaniensis) found that Tasmanian popu-
lations were most closely related to western Victoria pop-
ulations (Schauble and Moritz 2001). The genetic pattern
occurring in L. peronii and L. tasmaniensis is very similar
to what we found in R. diemensis, with an older divergence
between eastern and western Victorian populations than
between Tasmanian and western Victorian populations.
Volcanic activity in western Victoria during the late
Pliocene and continuing until the Holocene could have led
to divergences between the Grampians NP and central/
eastern Victorian populations of R. diemensis. This volca-
nic activity created the young basalt units known as the
Newer Volcanic Province, which extends from central
Victoria, including Melbourne, through the south-west of
Victoria to South Australia (Rosengren 1999). Extensive
grasslands formed across these lava beds (Jones 1999),
which could have led to the contraction of R. diemensis
into remnant forests and heathlands, and subsequent
divergence from eastern populations.
This volcanic activity may also have led to the vicari-
ance of the Anglesea population from populations further Ta
ble
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atio
n
370 Conserv Genet (2014) 15:363–373
123
north. The population at Anglesea forms a shallow but well
supported clade in the mtDNA, suggesting a relatively
recent isolation event. The geographically closest popula-
tion to Anglesea, Wombat SF, exhibits a 0.5 % genetic
divergence in mtDNA from Anglesea. Based on fossil-
calibrated molecular clock estimations in related Australian
agamid groups (e.g., Amphibolurus—Melville et al. 2011),
the isolation of the Anglesea population probably occurred
in the late Pleistocene or more recently during the Holo-
cene. A barrier to dispersal may have been created by
volcanic activity that occurred in the Newer Volcanic
Province. This basalt plain extends approximately
15,000 km2 across Victoria and South Australia, and 400
eruption points have been identified (Joyce 1988). Most of
the volcanoes north of Anglesea are believed to have
erupted in the Holocene or late Pleistocene (Joyce 1988)
and are likely to have caused the isolation of the Anglesea
population.
We found significant genetic structure across the
remainder of the distribution of R. diemensis in eastern
Victorian and into NSW. In particular, one specimen col-
lected from Big River SF in eastern Victoria was highly
divergent from all other samples ([7.8 % mtDNA;
Table 1), including a sample from nearby King Spur
(*110 km distance). Deep genetic breaks, estimated to
have occurred in the late Miocene–Pliocene have been
found across a number of taxa in eastern Victoria,
including White’s skink (L. whitii, Chapple et al. 2005), the
common froglet (C. signifera, Symula et al. 2008), the
spotted grass frog (L. tasmaniensis, Schauble and Moritz
2001) and the garden skink (L. guichenoti, Chapple et al.
2011). It has been suggested that these genetic divergences
may be due to repeated marine incursions of the Gippsland
Basin (Chapple et al. 2011). The sample of R. diemensis
from Big River SF sits within the geographic distribution
of the main eastern Victorian clade (Fig. 1), thus, it is
difficult to make any conclusions about this one highly
divergent sample. It is possible that there are multiple
sympatric ‘‘taxa’’ in eastern Victoria or that there is just a
high level of haplotypic diversity in this region, although, it
must be noted that the Big River SF specimen was sup-
ported as a divergent lineage in the species tree analysis.
More sampling in this region is required to determine the
phylogeographic and biogeographic history of R. diemensis
in eastern Australia.
Similarly, there were deep genetic divergences between
Victorian and NSW samples ([8.2 % mtDNA uncorrected
sequence divergence), probably dating to the late Miocene-
Pliocene. Multiple phylogeographic studies have also found
deep genetic breaks in southern NSW (e.g., Chapple et al.
2005; Symula et al. 2008). However, the sampling in our
study is not detailed enough in NSW and north-eastern
Victoria to determine the location of the genetic break
between the Victorian and NSW clades of R. diemensis. Our
study highlights the need for further phylogeographic sam-
pling in the northern regions of R. diemensis’ distribution.
ESUs and management implications
The importance of protecting populations to maintain
intraspecific diversity raises the question of whether iso-
lated R. diemensis populations are sufficiently distinct to
merit particular conservation attention. The ESU concept
was developed to answer such questions and provide a way
to prioritise populations of conservation importance (Ryder
1986). Moritz’s (1994) ESU definition aims to identify and
protect evolutionary heritage. Moritz (1994) advocates that
those populations that have been physically isolated over a
long period can produce unique genotypes, or combina-
tions of genotypes, which cannot be replaced. To recognise
these populations, the level of genetic divergence between
the isolated population and conspecific populations can be
analysed with mtDNA and nuclear markers. Populations
that form a monophyletic group when analysed with
mtDNA markers, and display divergence at nuclear loci,
are considered to represent independent historic lineages,
and thus are important to conserve. Based on these criteria
of genetic divergence and isolation, R. diemensis probably
consists of at least two ESUs that require specific man-
agement (i.e., Grampians NP and Anglesea).
Grampians National Park
This sole mainland occurrence of this R. diemensis lineage
is isolated in the Grampians NP surrounded by agricultural
landscapes. The Grampians NP populations are subject to a
several threats (exemplified by several large, intense fires
in recent years and concentrated efforts to control exotic
predators in the Grampians NP). Although listed in the
highest threat category in Victoria (Department of Sus-
tainability and Environment 2013), the populations in the
Grampians NP have no national listing, which lessens the
likelihood of this ESU being prioritised for conservation
actions. We believe it is important to further investigate
genetic diversity, including population genetic studies,
within the Grampians NP and Tasmanian populations of R.
diemensis, and between these populations and eastern
Victorian populations, to determine the comparative levels
of diversity. In addition, further field surveys are needed to
understand the distribution of R. diemensis within the
Grampians NP and similar habitats in western Victoria.
It is unknown whether the western Victorian population is
restricted to the Grampians NP, or if it extends to nearby
peaks such as Mts Cole and Langi Ghiran. Also, little is
known about the extent of occurrence and specific habitat
preferences of this species in the Grampians NP. Whilst our
Conserv Genet (2014) 15:363–373 371
123
samples were collected in upland areas, R. diemensis were
caught in pitfall traps in Victoria Valley in the Grampians
NP several decades ago (G. Gillespie pers. comm.). We
also recommend maintaining or intensifying control efforts
for exotic predators in this area.
Anglesea
As well as the samples used in this study, Museum Victoria
holds specimens of R. diemensis from localities on the
outskirts of Anglesea. Atypically for this species in Vic-
toria, our samples and these specimens were collected in
coastal heathlands. Examining the Anglesea population
solely in genetic terms suggests a borderline ESU status.
The Anglesea population, despite being reproductively
isolated and forming a well-supported monophyly in
mtDNA, exhibits a relatively low level of divergence from
both the Kinglake and Wombat SF populations. This sug-
gests that its isolation was a recent occurrence, likely to be
fostered by volcanic activity in the Holocene or late
Pleistocene period. However, Moritz (1994) emphasised
that it is not solely the extent of sequence divergence that
qualifies a population as an ESU but also the pattern of the
sequence divergence that identifies distinct lineages. Based
on the substitution rates of mtDNA, monophyletic groups
identified with mitochondrial markers have an inference
between thousands to millions of years (Crandall et al.
2000), and thus the monophyletic pattern of sequence
divergence will reflect a historical lineage. The phyloge-
netic trees represent the Anglesea population as a mono-
phyletic group with the mtDNA gene ND2 but this
monophyly was not supported with the nuclear gene RAG1.
However, the Anglesea population displays genetic diver-
gence and is a naturally isolated population that occupies a
different ecological niche to their geographically closest
conspecific populations. We recommend that the distinc-
tiveness of the Anglesea population ecologically, geo-
graphically and genetically means that this population
should be considered an ESU under threat from coastal
development, inappropriate fire regimes and exotic preda-
tors. Further surveys to refine understanding of these
population’s distribution and habitat preferences will
facilitate effective management of these populations.
Conclusions
The Grampians NP population of R. diemensis clearly fits
Moritz’s (1994) definition of an ESU, with geographic
isolation and deep genetic divergences reflected in both
mitochondrial and nuclear DNA. The mtDNA divergence
between Tasmania and the Grampians NP is great enough
(3.7 %) that these two regions might be considered
separate ESUs, however, there are obviously close evolu-
tionary ties between these two regions. National threatened
species listing rarely accommodates between-population
intraspecific variation. Consequently, under current prac-
tises, conservation management of subspecific ESUs relies
on state-level efforts, and it is challenging to generate the
resources to manage across state boundaries. We believe
that the Grampians NP/Tasmanian evolutionary relatedness
in R. diemensis provide a case-in-point of the discrepancy
between the state-level approach of maintaining variation
within a species, and the more conservative Common-
wealth focus on conserving biodiversity at only species or
subspecies levels. Finally, we believe that there needs to be
further research on north-eastern Victorian and NSW
populations of R. diemensis to establish how many ESUs
occur in this region and where the genetic breaks occur, as
well as investigating the geographic extent of the western
Victorian and Anglesea populations.
Acknowledgments We thank R. Rose, R. Swain, D. Goodall, G.
Heard, G. Peterson, B. Malone, M. Scroggie and J. Poon for assis-
tance in the field, R. Ayres for commenting on the manuscript, and J.
Austin for advice on molecular work. We also thank J. Van-Buskirk
for contributions to the project, P. Robertson for expert advice on the
lizard distributions, and J. Stuart-Smith, R. Sadlier (Australian
Museum) and G. Gillespie for providing tissue samples. Funding was
provided by the Australian Research Council to JM and the Peter
Rankin Trust Fund for Herpetology to JN.
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