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

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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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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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