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Phylogeography of the World's Tallest Angiosperm, Eucalyptus Regnans

Phylogeography of the World's Tallest Angiosperm, Eucalyptus Regnans

Journal of Biogeography (J. Biogeogr.) (2010) 37, 179–192

ORIGINAL Phylogeography of the world’s tallest ARTICLE angiosperm, regnans: evidence for multiple isolated Quaternary refugia Paul G. Nevill1,2,3*, Gerd Bossinger1 and Peter K. Ades4

1School of and Ecosystem Science, ABSTRACT University of and Cooperative Aim There is a need for more phylogeography studies, Research Centre for Forestry, Creswick, , 2Botanic Gardens and Parks particularly in Australia, where, unlike much of and North America, ice Authority, Kings Park and Botanic Gardens, sheet cover was not extensive during the Last Glacial Maximum (LGM). This West Perth, Australia, 3School of Biology, study examines the phylogeography of the south-east Australian montane The University of , Nedlands, . The work aimed to identify any major evolutionary Australia and 4School of Forest and Ecosystem divergences or disjunctions across the species’ range and to examine genetic Science, University of Melbourne and signatures of past range contraction and expansion events. Cooperative Research Centre for Forestry, Location South-eastern mainland Australia and the large island of . Parkville, Australia Methods We determined the chloroplast DNA haplotypes of 410 E. regnans individuals (41 locations) based on five chloroplast microsatellites. Genetic structure was examined using analysis of molecular variance (AMOVA), and a statistical parsimony tree was constructed showing the number of nucleotide differences between haplotypes. Geographic structure in population genetic diversity was examined with the calculation of diversity parameters for the mainland and Tasmania, and for 10 regions. Regional analysis was conducted to test hypotheses that some areas within the species’ current distribution were refugia during the LGM and that other areas have been recolonized by E. regnans since the LGM. Results Among the 410 E. regnans individuals analysed, 31 haplotypes were identified. The statistical parsimony tree shows that haplotypes divided into two distinct groups corresponding to mainland Australia and Tasmania. The distribution of haplotypes across the range of E. regnans shows strong geographic patterns, with many populations and even certain regions in which a particular haplotype is fixed. Many locations had unique haplotypes, particularly those in East in south-eastern mainland Australia, north-eastern Tasmania and south-eastern Tasmania. Higher haplotype diversity was found in putative refugia, and lower haplotype diversity in areas likely to have been recolonized since the LGM. Main conclusions The data are consistent with the long-term persistence of E. regnans in many regions and the recent recolonization of other regions, such as the Central Highlands of south-eastern mainland Australia. This suggests that, in spite of the narrow ecological tolerances of the species and the harsh environmental conditions during the LGM, E. regnans was able to persist locally or contracted to many near-coastal refugia, maintaining a diverse genetic

*Correspondence: Paul G. Nevill, Botanic structure. Gardens and Parks Authority, Kings Park and Keywords Botanic Garden, West Perth, WA 6005, Australia. Chloroplast microsatellite, climate change, Eucalyptus, phylogeography, Pleis- E-mail: [email protected] tocene refugia, south-eastern Australia, Tasmania.

ª 2009 Blackwell Publishing Ltd www.blackwellpublishing.com/jbi 179 doi:10.1111/j.1365-2699.2009.02193.x P. G. Nevill et al.

INTRODUCTION

Climatic oscillations during the Quaternary have had a strong influence on species distributions and evolution (Hewitt, 1996). In south-eastern Australia, the Pleistocene climate fluctuated between cool-dry (glacial) and warm-wet (intergla- cial) weather conditions (Bowler, 1982; Frakes et al., 1987; Markgraf et al., 1995), with the Last Glacial Maximum (LGM) (maximum extent of ice sheets during the last glaciation) occurring between 23 and 16 ka (Barrows et al., 2004). Temperatures during glacial maxima were up to 6–6.5C cooler than those experienced today (Colhoun et al., 1996), and rainfall, particularly in summer, was reduced (Hubbard, 1995). In contrast to the case in central and northern Europe and much of North America, glaciation in south-eastern Australia was not extensive, being confined to small areas in the Snowy Mountains region of continental Australia (Barrows et al., 2001) and to the Central Plateau of Tasmania (Colhoun et al., 1996; Duller & Augustinus, 1997), and, as a result, there was not the removal of all vegetation from large areas. In south-eastern Australia, the combination of aridity and lower temperatures had a much more important effect on species distributions than did glaciation (Kershaw & Nanson, 1993). Tree species are thought to have responded to changing climate by contracting to suitable refugia during the LGM and Figure 1 Current natural range of Eucalyptus regnans (indicated then re-colonizing the landscape when conditions became by grey shading) in south-eastern Australia and Tasmania and more favourable. Lowland species are thought to have the location of the 10 geographic regions used in the analyses contracted to coastal refugia or onto the exposed continental (Otway Ranges, Central Highlands, south Central Highlands, shelf, whereas montane species made elevational migrations or South Gippsland, East Gippsland, northern Tasmania, north- survived in suitable microsites (McKinnon et al., 2004). These eastern Tasmania, eastern Tasmania, south-eastern Tasmania and range contractions and recolonization events should have left central Tasmania) (map source Boland et al., 2006). genetic signatures in the genomes of these species. For example, the low genetic diversity found in tree species in areas of northern Europe recolonized since the LGM is thought Tasmania is similar and shows clear links. Natural vegeta- to have arisen from a series of bottlenecks associated with tion in low- to moderate-rainfall areas is typically founder events (Hewitt, 1996), which results in only a subset of forest or (10–30 m tall). In higher-rainfall areas and the diversity present in refugia being found in recolonized protected microsites in low- to moderate-rainfall areas, tall areas (Petit et al., 2003). It is generally thought that refugia are eucalypt forest (30–80 m), and, to a very limited extent, cool- indicated by the present-day derived populations containing temperate forest can be found. Alpine vegetation exists in high levels of diversity or low levels of diversity but unique south-eastern mainland Australia above 1800 m a.s.l., whereas divergent haplotypes. By studying these genetic signatures it in Tasmania it is above 1100 m a.s.l., owing to its more will be possible to gain a better understanding of how eucalypt southerly position and thus cooler climate. species survived and then flourished following such severe During the LGM, Tasmania and south-eastern mainland climatic changes. Australia were colder and generally more arid than at present. The area in this study is situated in south-eastern mainland On the mainland, and records suggest that Australia and on the large island of Tasmania (320 km · much of the Victorian Central Highlands were treeless and 320 km) between the latitudes 37 and 4338¢ S (Fig. 1). dominated by alpine vegetation. are thought to have Tasmania and the mainland are separated by the , persisted at low elevations in the south of the region, near sea which is currently 250 km wide but generally less than 100 m level (McKenzie, 1997, 2002). In the Otway Ranges region, deep. Since the early , Tasmania and the mainland fossil pollen records indicate that eucalypts persisted along have been repeatedly separated and linked by changes in sea with rain forest taxa (McKenzie & Kershaw, 1997, 2000, 2004). level (Baillie, 1989). A land-bridge joining South Gippsland, Pleistocene vegetation history in East Gippsland is less well Flinders Island and north-east Tasmania in the east and the known; however, a recently published study from the west of Otway Ranges, King Island and north-west Tasmania in the that region indicates that, as in the south Central Highlands, west existed as recently as 7 ka (Jackson, 1999). Consequently, eucalypts survived but were restricted to small areas of suitable the vegetation of south-eastern mainland Australia and habitat and were not widespread (Kershaw et al., 2007).

180 Journal of Biogeography 37, 179–192 ª 2009 Blackwell Publishing Ltd Phylogeography of Eucalyptus regnans

Forests in south-eastern mainland Australia are thought to In this study we examine in detail the phylogeography of the have expanded from southern, near-sea-level refugia, with the montane Australian tree species Eucalyptus regnans F. Muell. major phase of expansion between 7 and 5 ka (McKenzie & Eucalyptus regnans is endemic to mountainous regions of Kershaw, 2000). In Tasmania, glaciation was extensive on the south-eastern Australia (Fig. 1) and is discontinuously dis- central plateau and the was close to current sea level tributed in south-eastern, mainland Australia at elevations (Kirkpatrick & Fowler, 1998). Pollen records indicate that the between 200 and 1100 m a.s.l. on and south of the Great midlands were treeless, and, like the Central Highlands on the Dividing Range in the east and in the Otway Ranges in the mainland, dominated by alpine vegetation (Macphail & west. In Tasmania the species is found in the north, north-east, Jackson, 1978; Sigleo & Colhoun, 1981). Modelling of the south and south-east between sea level and 700 m. Eucalyptus distribution of vegetation types during the LGM indicates that regnans is out-crossing and pollinated by non-specific , wet eucalypt could have survived in Tasmania in the birds and small (Ashton, 1975b; Griffin, 1980), with south-east, on the west coast and in small refugia in the north- seed dispersal by gravity limited to around two tree heights east (Kirkpatrick & Fowler, 1998). (Cremer, 1966). The species is second only to Californian Chloroplast DNA (cpDNA) has been used to examine redwoods in height and is the world’s tallest angiosperm, genetic variation in selected species in the eucalypt subgenera growing to between 90 and 100 m (Ashton, 1958). In less Symphyomyrtus and Eucalyptus, with studies focusing on favourable areas it is often confined to small, disjunct south-western Australia (Byrne & Macdonald, 2000; Byrne & populations in gullies and sheltered slopes. Unlike many other Hines, 2004; Wheeler & Byrne, 2006) and Tasmania (Steane eucalypt species it is highly sensitive to fire and regenerates et al., 1998; Jackson et al., 1999; McKinnon et al., 1999, 2001a; almost exclusively by seed. High-intensity fires will kill all , Freeman et al., 2001; Rathbone et al., 2007). A review of resulting in an even-aged stand, whereas fires of lower intensity Tasmanian eucalypt phylogeography has been published by will produce stands of multiple age classes, with individuals McKinnon et al. (2004). The phylogeography of only one living for up to 400 years (McCarthy & Lindenmayer, 1998; widely distributed, south-eastern Australian species, the Boland et al., 2006). predominantly lowland , has been studied In our study we used cpDNA microsatellites (cpSSR) in detail (Freeman et al., 2001). That study on E. globulus and developed for eucalypt species (Steane et al., 2005) to examine some less intensive studies of other south-eastern Australian chloroplast haplotype diversity and structure for 41 popula- species (Steane et al., 1998; Jackson et al., 1999; McKinnon tions of E. regnans. Specifically, this work aimed to: (1) et al., 1999, 2001a; Rathbone et al., 2007) found: (1) chloro- identify any major evolutionary divergences or disjunctions plast DNA is variable within species and geographically across the species’ range; and (2) examine signatures of refugia structured; (2) the distribution and diversity of haplotypes is and expansion to infer historical migration and colonization associated with locations of likely glacial refugia in the events for montane species in south-eastern Australia. The Otway Ranges, and on the south and east coasts of Tasma- results will be interpreted in the light of the climatic and nia; (3) there are lower levels of diversity or fixation of geological history of south-eastern Australia. chloroplast types in hypothesized recolonized areas, including central Tasmania; and (4) chloroplasts are shared between MATERIALS AND METHODS species, and geographical location is more important than species in determining the clade to which the chloroplast is Sampling strategy assigned. Less is known about the effect of past climate changes on Forty populations and ten individuals per population (n = 410) widespread south-eastern Australian montane species, includ- representing the entire geographic distribution of E. regnans ing Eucalyptus regnans and E. delegatensis. In contrast to were sampled in south-eastern, mainland Australia and E. globulus, these species are now extensively distributed in Tasmania. The sampling strategy included many small, outlier areas thought to have been treeless during the LGM (namely populations found across the species’ discontinuous distribu- the Central Highlands and central Tasmania). They are also tion. The locations of sampled populations are listed in Table 1 present in areas in which few or no fossil pollen data are and shown in Fig. 2. These locations coincide as closely as available (East Gippsland and South Gippsland in south- possible with those of the collections conducted by CSIRO in the eastern, mainland Australia, and northern, north-eastern, east mid-1970s, which were subsequently used in provenance trials and south-eastern Tasmania). These montane species have a (Griffin & Johnson, 1979; Raymond et al., 1997). higher cold tolerance than E. globulus, and it is possible that In order to avoid sampling trees that had been regenerated they were able to persist in some regions by means of after harvesting using seed possibly transferred from other elevational migration or contraction to suitable micro-sites places, sample trees were restricted to those estimated to be at that may not be evident in fossil pollen assemblages. If least 40 years of age, as seed transfer was not extensively carried montane eucalypt species were able to persist in suitable out before the 1960s. Where possible, selected trees were at micro-sites or multiple near-coastal refugia, then this history is least 100 m apart so as to avoid sampling closely related likely to be discernable in the geographic pattern of chloroplast individuals. However, at a few sites this was not possible owing genetic diversity. to the limited number of suitable remaining trees.

Journal of Biogeography 37, 179–192 181 ª 2009 Blackwell Publishing Ltd P. G. Nevill et al.

Table 1 Geographic location, population code and region of the 41 Eucalyptus regnans populations used in this study. Number of haplotypes (n/hap) is the number of distinct haplotypes found in individuals sampled at (1) that location and (2) that region. Number of unique haplotypes (n/unique) is the number of haplotypes found only in (1) that population and (2) that region. ‘Haplotypes’ indicates the distinct haplotypes found in a population.

Population Latitude S Longitude E n/hap n/unique Haplotypes

1 Powelltown )37.78 145.82 2 1 H4 H9 2 Kallista )37.92 145.38 2 0 H2 H3 3 Mt Erica )37.92 146.37 1 0 H4 South Central Highlands (R) 4 1 4 Toolangi )37.52 145.52 1 0 H4 5 Mt Disappointment )37.42 145.20 1 0 H4 6 Rubicon )37.33 145.93 1 0 H4 7 Barkly River )37.45 146.48 1 0 H4 8 Mt Useful )37.68 146.53 1 0 H4 Central Highlands (C) 1 0 9 Valencia Creek )37.55 147.00 2 0 H4 H8 10 Dargo )37.46 147.25 2 0 H4 H8 11 Quarry Creek )37.43 147.65 4 4 H10 H11 H12 H13 12 Buchan )37.23 148.05 2 2 H14 H15 13 Yalmy River )37.35 148.45 2 2 H16 H17 14 Bendoc )37.30 148.97 1 1 H18 East Gippsland (R) 11 10 15 Wilsons Promontory )39.10 146.37 1 1 H7 16 Mirboo East )38.52 146.38 4 1 H2 H3 H4 H5 17 Traralgon Creek )38.43 146.52 1 1 H6 South Gippsland (R) 6 3 18 Carisbrook )38.60 143.75 2 0 H1 H2 19 Upper Ford River )38.65 143.48 2 0 H1 H2 20 Parker Spur )38.75 143.52 2 1 H1 H31 Otway Ranges (R) 2 2 Mainland Total 19 21 Smithton )41.02 145.37 1 0 H20 22 Ferndene )41.15 146.02 2 0 H19 H20 23 Beaconsfield )41.30 146.75 1 0 H20 Northern Tasmania (C) 2 0 24 Lisle )41.25 147.37 2 1 H19 H23 25 Ben Nevis )41.42 147.55 3 2 H20 H21 H22 26 Dan’s Valley )41.38 147.88 1 0 H19 27 Gould’s Country )41.22 148.15 2 0 H19 H20 North-eastern Tasmania (R) 5 3 28 Levendale )42.52 147.53 3 1 H24 H25 H26 29 Nugent )42.70 147.82 1 0 H26 30 Royal George )41.93 147.93 1 0 H24 31 Swan Port )42.40 147.85 1 0 H24 Eastern Tasmania (R) 3 2 32 Lorina )41.58 146.08 1 0 H20 33 Florentine Valley )42.48 146.45 1 0 H20 34 Styx )42.82 146.60 2 0 H20 H26 35 Stoneyhurst Creek )42.67 147.07 1 0 H20 36 Moogara )42.78 146.90 2 1 H20 H27 Central Tasmania (C) 3 1 37 Strathblane )43.37 146.97 2 0 H20 H28 38 Kaoota )43.02 147.15 1 0 H29 39 Fern Tree )42.92 147.25 2 0 H20 H28 40 Bruny Island )43.40 147.27 2 0 H20 H29 41 Murdunna )42.97 147.95 2 1 H26 H30 South-eastern Tasmania (R) 5 3 Tasmania Total 12

The 41 populations used in analyses are allocated to one of the following 10 regions based on their geographical location: Otway Ranges, Central Highlands, south Central Highlands, South Gippsland, East Gippsland, northern Tasmania, north-eastern Tasmania, eastern Tasmania, south-eastern Tasmania, central Tasmania. (R) following the name of a region indicates a hypothesized refugium. (C) indicates a hypothesized recolonized region.

182 Journal of Biogeography 37, 179–192 ª 2009 Blackwell Publishing Ltd Phylogeography of Eucalyptus regnans

Figure 2 Geographic distribution of chloroplast DNA haplotypes found in Eucalyptus regnans. Population numbers are the same as those used in Table 1. The size of sections of the pie charts corresponds to the number of individuals with that haplotype. The colour coding of haplotypes is the same as in Fig. 3.

Journal of Biogeography 37, 179–192 183 ª 2009 Blackwell Publishing Ltd P. G. Nevill et al.

Figure 3 Statistical parsimony tree of haplo- types of Eucalytpus regnans based on chloro- plast microsatellites. Circle size indicates relative haplotype frequency. Branch length indicates the number of nucleotides between haplotypes. Haplotypes present in fewer than five individuals are represented with a circle of the same size. Latent nodes (unobserved haplotypes), indicated by small, unlabelled red circles, are included to complete the tree.

PCRs were performed in 12.5-ll reaction volumes contain- DNA extraction ing 1.25 lLof10· PCR buffer (Invitrogen, Carlsbad, CA,

Total genomic DNA was extracted from cambial scrapings USA), 3 mm of MgCl2 (Invitrogen), 0.24 mm of dNTP mix following the protocol described by Tibbits et al. (2006). (Invitrogen), a primer concentration of 0.2 lm for each of the Modifications were made to the protocol, including: (1) forward and reverse primers, 40 ng of template DNA, 0.5 lgof increasing the buffer-to-sample ratio (600 lL buffer/200 lL bovine serum albumin and 1 unit of Taq DNA polymerase sample) to reduce the co-purification of phenolic compounds (Invitrogen). PCR was carried out in a Mastercycler 5330 and carbohydrates, thus improving DNA quality; and (2) (Eppendorf) thermocycler using the following reaction condi- extending the time spent at the initial -bath step to 12 h tions: initial denaturation for 5 min at 94C, followed by 39 to improve cell lysis and DNA yield. cycles of 94C for 30 s, an annealing temperature of 55C for 30 s, 72C for 1 min, and a final extension of 72C for 5 min. The amplified products were combined and separated on an Chloroplast microsatellite analysis ABI 3730 DNA analyzer (Perkin Elmer Applied Biosystems), Chloroplast microsatellites were amplified with primers and allele sizes were determined using Genemapper version designed for Eucalyptus species (Steane et al., 2005). Chloroplast 4.0 (Perkin Elmer Applied Biosystems). DNA is maternally inherited in most angiosperms, including eucalypts (Byrne et al., 1993; McKinnon et al., 2001b). A total Genetic data analysis of 10 primer pairs (EMCRC59cp, EMCRC60cp, EMCRC62cp, EMCRC65cp, EMCRC67cp, EMCRC74cp, EMCRC84cp, A haplotype is defined as a distinct combination of the alleles EMCRC85cp, EMCRC86cp and EMCRC90cp) were initially at a set of loci. Haplotype composition, number of haplotypes screened against 20 individuals of E. regnans from across the and number of unique haplotypes were calculated for each species’ range for their ability to amplify a product. Primers were population. A unique haplotype was defined as one found in also screened against individuals from two other species in the only that population. Alleles at microsatellite loci can be subgenus Eucalyptus (E. obliqua and E. delegatensis) to assess analysed as unordered, where comparisons do not account for their suitability for a subsequent study on chloroplast sharing in variation in allele size, or ordered, where the assumed number eucalypts. Polymerase chain reaction (PCR) amplification was of mutational steps between alleles provides additional infor- assessed on 1.4% agarose gel. From the initial 10 primers, 5 mation. Mean within-population genetic diversity, species primer pairs (EMCRC60cp, EMCRC67cp, EMCRC74cp, total genetic diversity, and population differentiation were

EMCRC86cp and EMCRC90cp) that produced polymorphic, calculated treating alleles as unordered (hS, hT and GST) and strong and reproducible amplification profiles in all three ordered (vS, vT, and RST), following the method of Pons & Petit species were selected and screened against all E. regnans samples. (1996) using permut (available at http://www.pierroton. The forward primer of each primer pair was labelled with inra.fr//labo/Software/PermutCpSSR/). The differenti-

6-FAM or HEX fluorescent (Perkin Elmer Applied ation statistics GST and RST were compared to test for Biosystems, Foster City, CA, USA). phylogeographic structure. A higher RST than GST means that

184 Journal of Biogeography 37, 179–192 ª 2009 Blackwell Publishing Ltd Phylogeography of Eucalyptus regnans more closely related haplotypes occur in the same population, were selected based on the absence of stutter peaks, which can indicating phylogeographic structure (Pons & Petit, 1996). To make scoring of microsatellites difficult. Wherever allele scores visualize the number of base-pair differences between haplo- were ambiguous, samples were reamplified. If allele scoring types, a haplotype tree was constructed in Network 4.2.0.1 remained ambiguous after reamplification, the samples were (available at http://www.fluxus-engineering.com/sharepub. excluded from the study. The combination of 28 different alleles htm#a10) using the median-joining network algorithm. The produced 31 unique haplotypes, with 19 haplotypes found in method reconstructs all maximum parsimony trees from a south-eastern mainland Australia, 12 in Tasmania and none given dataset. The distance between each pair of haplotypes common between the two. The haplotype frequency varied was the sum of nucleotide differences between them over the greatly, with haplotype H4 the most frequent in mainland five chloroplast microsatellite loci. samples (45% of 200 mainland individuals) and haplotype H20 Geographic structure in population genetic diversity was characterizing Tasmanian samples (44% of 210 Tasmanian examined through the calculation of diversity parameters for individuals) (see Appendix S1 in Supporting Information). south-eastern mainland Australia and Tasmania, and for The haplotype tree shows that haplotypes are divided into the 10 regions shown in Fig. 1. Populations were assigned two distinct groups corresponding to the mainland and a priori to one of each of the regions based on their geographic Tasmania, with the exception of haplotypes H1 and H31, location (Table 1). Regional analysis was conducted to test found in the Otway Ranges, which are located in the hypotheses that areas within the species’ current distribution Tasmanian group (Fig. 3). The haplotype structure is simpler were refugia during the last LGM and that other areas have in Tasmania than on the mainland, with most haplotypes been recolonized by E. regnans since the LGM. These regional separated from one another by only one to two base pairs. On classifications were postulated based on previous studies the mainland, many of the haplotypes are connected to at least examining fossil pollen and charcoal records (Macphail & two others and are separated by at least two base pairs. There is Jackson, 1978; Sigleo & Colhoun, 1981; Kershaw et al., 1991, evidence of homoplasy in alleles. For example, size variant 146 2007; McKenzie, 1997, 2002; McKenzie & Kershaw, 1997, 2000, (locus EMCRC86cp) found at both Kaoota (38) and Bruny 2004; Fletcher & Thomas, 2007), on a number of studies on Island (40) in southern Tasmania is also found at Bendoc (14) chloroplast variation in eucalypts (Steane et al., 1998; Jackson in far East Gippsland. However, homoplasy in haplotypes does et al., 1999; McKinnon et al., 1999, 2001a, 2004; Freeman not appear to have occurred. et al., 2001) and on Quaternary climate modelling (Kirkpatrick & Fowler, 1998). The regions were classified as colonized (C) Haplotype diversity and geographic distribution or refugia (R) and are as follows: for south-eastern, mainland Australia, East Gippsland (R), Otway Ranges (R), South The distribution of haplotypes across the range of E. regnans Gippsland (R), south Central Highlands (R) and Central shows strong geographic structure (Fig. 2). Populations con- Highlands (C); and for Tasmania, north (C), north-east (R), tained from one to four haplotypes, with Quarry Creek (11) in east (R) south-east (R) and central (C). Differences in the East Gippsland and Mirboo East (16) in South Gippsland each mean within-region diversity values were tested by comparing having four haplotypes (Fig. 2 and Table 1). At many hypothesized refugial regions with regions thought to have locations, and even in certain regions, only a single haplotype been recolonized using t-tests. was found in all sampled individuals. In particular, samples To examine whether haplotype distribution was geograph- from the Central Highlands of the mainland were fixed for ically structured, a haplotype frequency map was constructed. haplotype H4. Likewise, the central Tasmanian region was Analysis of molecular variance (AMOVA) was performed to fixed for haplotype H20, with the exception of Moogara (36) examine the distribution of genetic variation within and and Styx (34), which each contain an additional haplotype. among populations and between south-eastern mainland Many populations had unique haplotypes, particularly so in Australia and Tasmania. Analyses were performed using the East Gippsland, South Gippsland, north-eastern Tasmania and program GenAlEx 6 (Peakall & Smouse, 2006), and signifi- south-eastern Tasmania (Fig. 2 and Table 1). cance tests were conducted using 9999 permutations. Overall, there was low within-population diversity

(hS = 0.18, vS = 0.07), but high total diversity (hT = 0.89, v = 0.89). Populations were highly differentiated, with RESULTS T GST = 0.79 and RST = 0.93 (Table 2). The mainland had similar levels of within-population (h = 0.20, v = 0.14) and Haplotype variation and relationships among S S total (h = 0.82, v = 0.82) diversity to Tasmania (h = 0.17, haplotypes T T S vS = 0.16 and hT = 0.72, vT = 0.72) (Table 2), and the differ- The five microsatellite loci assayed in 410 E. regnans individuals ences were not statistically significant (P-values all > 0.4605). produced a total of 28 different alleles, with the loci At a regional level on the mainland, the south Central

EMCRC60cp, EMCRC67cp, EMCRC74cp, EMCRC86cp and Highlands (hT = 0.73, vT = 0.75), East Gippsland (hT = 0.96, EMCRC90cp producing seven, seven, three, seven and four vT = 1), the Otway Ranges (hT = 0.62, vT = 0.62) and South alleles, respectively. Even though many alleles were only 1 bp Gippsland (hT = 1.00, vT = 1.00) were far more diverse than apart, the assigning of allele sizes did not cause problems. Loci the Central Highlands (hS =0, vS = 0 and hT =0, vT =0)

Journal of Biogeography 37, 179–192 185 ª 2009 Blackwell Publishing Ltd P. G. Nevill et al.

Table 2 Chloroplast microsatellite marker haplotype diversity (within-population diversity hS, vS and total diversity hT, vT) and differ- entiation (GST, RST) for 41 Eucalyptus regnans populations in south-eastern Australia.

Region n R/C hS hT GST vS vT RST

East Gippsland 60 R 0.38 (0.11) 0.96 (0.04) 0.60 (0.12) 0.01 (0.01) 1 (0.01) 0.99 (0.01) Central Highlands 50 C 0.00 0.00 0.00 0.00 0.00 0.00 South Central Highlands 30 R 0.26 (0.16) 0.73 (0.19) 0.64 (0.14) 0.19 (0.13) 0.75 (0.11) 0.75 (0.15) Otway Ranges 30 R 0.30 (0.16) 0.62 (0.06) 0.49 (0.37) 0.30 (0.16) 0.62 (0.06) 0.49 (0.37) South Gippsland 30 R 0.24 (0.24) 1.00 (0.14) 0.76 (0.24) 0.08 (0.08) 1.00 (0.35) 0.92 (0.05) Total Mainland 200 0.20 (0.06) 0.82 (0.08) 0.76 (0.07) 0.14 (0.10) 0.82 (0.25) 0.83 (0.11) North-eastern Tasmania 40 R 0.26 (0.10) 0.63 (0.09) 0.59 (0.29) 0.44 (0.20) 0.58 (0.16) 0.23 (0.26) South-eastern Tasmania 50 R 0.29 (0.09) 0.87 (0.09) 0.67 (0.11) 0.42 (0.36) 0.84 (0.49) 0.50 (NC) Northern Tasmania 30 C 0.07 (0.07) 0.60 (0.24) 0.89 (NC) 0.07 (0.07) 0.60 (0.24) 0.89 (NC) Eastern Tasmania 40 R 0.53 (0.09) 0.62 (0.08) 0.14 (0.14) 0.53 (0.13) 0.62 (0.11) 0.14 (0.19) Central Tasmania 50 C 0.04 (0.04) 0.04 (0.04) 0.00 (NC) 0.04 (0.04) 0.04 (0.04) 0.00 (NC) Total Tasmania 210 0.17 (0.04) 0.72 (0.08) 0.76 (0.06) 0.16 (0.09) 0.72 (0.15) 0.78 (0.08) Total for all populations 410 0.18 (0.04) 0.89 (0.03) 0.79 (0.04) 0.07 (0.04) 0.89 (0.08) 0.93 (0.04) n, number of individuals; R/C, region classification as refugia (R) or recolonized (C). Standard errors over populations are given in parentheses. NC, not calculable.

Table 3 Analysis of molecular variance (AMOVA) of chloroplast otypes in the Central Highlands of south-eastern mainland microsatellite data for 41 populations of Eucalyptus regnans in Australia and near fixation in the central regions of Tasmania south-eastern Australia. suggest that these areas were recolonized after a warmer and wetter climate developed following the LGM (Hubbard, 1995). Sum of Percentage Source d.f. squares of variation P This finding is consistent with previous studies of fossil pollen, charcoal records and climate modelling, which have indicated Among regions 1 305.921 67 0.0100 that these two regions were likely to have been treeless during Among populations 39 215.759 25 0.0100 the LGM (Macphail & Jackson, 1978; Sigleo & Colhoun, 1981; within regions McKenzie, 1997, 2002; Kirkpatrick & Fowler, 1998). The trend Within populations 369 64.500 8 0.0100 of low relative haplotype diversity in hypothesized colonized areas has been seen in many tree species (reviewed in Petit (Table 2) (P-values all < 0.0001). A similar trend of high- and et al., 2005) and was found also in E. globulus (Freeman et al., low-diversity regions was found in Tasmania, with the north 2001). Low diversity is thought to arise from successive

(hT = 0.60, vT = 0.60), north-east (hT = 0.63, vT = 0.58), east founder events, which are likely to be an important part of the

(hT = 0.62, vT = 0.62) and south-east (hT = 0.87, vT = 0.84) colonization process. being more diverse than the central region of the island In south-eastern mainland Australia, eucalypt forests are

(hT = 0.04, vT = 0.04) (Table 2) (P-values all < 0.0046). Over- known to have persisted in the Otway Ranges and south all, diversity was lower in regions postulated as recolonized Central Highlands during the LGM. Palynological studies do than in regions postulated as refugia, with the exception of not provide information on which eucalypt species survived, northern Tasmania. however, owing to the homogenous appearance of eucalypt

In a test for phylogeographic structure, RST (0.93) was pollen. In this study, the finding of high haplotype diversity significantly greater than GST (0.79) (P < 0.05), indicating that and many unique haplotypes in populations in South Gipps- more closely related haplotypes are more likely to be present in land and East Gippsland indicates that these regions were likely the same population. An AMOVA showed strong differenti- to have been refugia or near refugia on the coastal plain. It is ation between the mainland and Tasmania and between possible that populations with high diversity represent suture populations within the mainland and Tasmania (Table 3). zones, where distinct maternal lineages converge (Remington, 1968; Hewitt, 1996). Little is known about vegetation in South Gippsland during DISCUSSION the LGM, with palynological studies in the region confined to Wilsons Promontory (Hope, 1974; Ladd, 1979), the most Refugia and recolonization southerly point on the Australian mainland. The deposits The cpSSR data strongly suggest refugia and areas of recolon- examined in these studies extend back to only 13 ka and ization. In some regions all populations were fixed for a indicate that eucalypt forest was present at that time and has particular haplotype, whereas in other regions adjacent pop- persisted in the area until the present day. The high differen- ulations were highly differentiated (Fig. 2). Fixation of hapl- tiation between populations found in this study suggests that

186 Journal of Biogeography 37, 179–192 ª 2009 Blackwell Publishing Ltd Phylogeography of Eucalyptus regnans there were multiple refugia or in situ survival of E. regnans in was able to penetrate East Gippsland as far east as Dargo (10) this region. The history of the coastal haplotype H2 at Kallista and Valencia Creek (9), where it encountered individuals (2) in the south Central Highlands, Mirboo East (16) in South carrying the H8 haplotype. Gippsland, and Carisbrook (18) and Upper Ford River (19) in In Tasmania, the high haplotype diversity and the number the Otway Ranges is difficult to discern. Haplotype H2 could of unique haplotypes found in the north-east, east and south- be an ancestral haplotype present in these locations prior to the east of the island are consistent with these areas being refugia LGM. This conclusion is supported by the haplotype’s internal for E. regnans during the LGM. Climate modelling (Kirkpa- position in the haplotype tree. There is also the possibility that trick & Fowler, 1998) and previous molecular studies (Steane there has been flow between these regions. et al., 1998; Jackson et al., 1999; McKinnon et al., 1999, 2001a, The history of eucalypt forest in the East Gippsland region is 2004; Freeman et al., 2001) supported the existence of glacial also poorly understood. There has been only one palynological refugia for eucalypts in eastern and south-eastern Tasmania. study, in the west of the region (Kershaw et al., 2007), and very There have been no fossil pollen studies in north-east limited cpDNA work, which is confined to only one individual Tasmania and only very limited phylogeographic studies of each of E. globulus (Freeman et al., 2001) and E. elata,a other species. Tentative support for refugia in the region comes species within subgenus Eucalyptus (McKinnon et al., 1999). from a study by Kirkpatrick & Fowler (1998), who used The East Gippsland E. regnans populations are found at the palynological and ecological information to test alternative highest elevations of any across the species’ range, and are climatic models and to predict the distribution of various small and isolated, each being separated by at least 30 km from vegetation types. their nearest neighbours, and by 100 km from the nearest large In this study we sampled four populations in the north-east occurrence of the species. Therefore, it was expected that they region of Tasmania and a total of 40 individuals. A north- would be differentiated genetically from each other and from eastern haplotype H19 is widely distributed in the region and is populations in the other regions. Furthermore, if the popu- found in three of the four populations (Dan’s Valley, Lisle and lations were small for long periods there may be low within- Gould’s Country). It is found either in all sampled individuals population diversity. Prior to this study it was not known if the from a population or in a mixture with the widespread H20 species had survived in situ during previous climate oscillations haplotype and H21, H22 and H23, which were found in only or had recolonized the region, possibly during the same period one, two and one individuals, respectively. This north-eastern as the recolonization of the Central Highlands. If the species haplotype is separated by only 1 bp from the widespread H20 had recolonized East Gippsland during the same event as the haplotype found in the north, centre and south of the island. Central Highlands colonization it would raise interesting There is the possibility that this haplotype is recent, but a questions about the ability of E. regnans and eucalypts in mutation that arose in one individual is unlikely to have spread general to respond to climate change and, in particular, about through an already occupied landscape. It is more likely that the rate at which they could migrate across the landscape. If the E. regnans survived in the north-east at low numbers and that region had been recolonized following the LGM we would there was post-glacial expansion from one or more refugia. expect to find low haplotype diversity and low population The only occurrence of haplotype H19 outside the north- differentiation. eastern region is in the near-coastal Ferndene (22) population We found that each of the four most easterly populations in almost 100 km to the west. The northern Tasmanian popu- the region have unique haplotypes, which suggests that there is lations of Ferndene (22), Smithton (21) and Beaconsfield (23) no seed-mediated gene flow between the populations, and that do not appear to have been refugia for the species, as all time and isolation have allowed the accumulation of different individuals have either the widespread H20 haplotype, or, at mutations in each. During the LGM, either in situ survival of Ferndene (22), haplotype H19, and there are no unique the species or an elevational migration to distinct coastal haplotypes in the region. It is possible that individuals with the refugia for each of the populations is possible. Our results are H19 haplotype recolonized parts of northern Tasmania from consistent with the findings of a palynological study at the north-east at the end of the LGM, during conditions Caledonia Fen in the far west of the region (Kershaw et al., warmer and wetter than those of the present day, and that the 2007). Kershaw et al. (2007) found a continuous presence of species contracted to a few near-coastal locations with the eucalypts throughout the pollen sequence, which covers the onset of drier conditions c. 5 ka. The widespread occurrence of last interglacial/glacial cycle (140,000 years). They proposed H20 in Tasmania and its central position in the network that eucalypts were able to persist in small sheltered patches suggests that this is the most likely ancestral lineage. It is close to the site. possible that this haplotype was present in the north-east and The haplotype H4, which is fixed in the Central Highlands, south-east before the LGM, and that the north and centre of is found as far east as Dargo (10) and Valencia Creek (9) but at Tasmania have been recolonized from these regions. a higher frequency than H8, which is unique to these two Given that eucalypts demonstrate widespread sharing of populations. It is possible that East Gippsland was the source chloroplast lineages between species (Steane et al., 1998; of the Central Highlands haplotype. It is also possible that the Jackson et al., 1999; McKinnon et al., 1999, 2001a), it is haplotype invaded the Central Highlands from the south, as it possible that some of the haplotypes found in this study, is found in the Powelltown and Mirboo East populations, and particularly highly divergent ‘tip’ haplotypes (e.g. H11 at

Journal of Biogeography 37, 179–192 187 ª 2009 Blackwell Publishing Ltd P. G. Nevill et al.

Quarry Creek), have been acquired from other species. areas in central and northern Tasmania, and particularly the , which hybridizes readily with E. regnans, Central Highlands of south-eastern mainland Australia, there is a likely donor of cpDNA and could be the source of some of is high population differentiation and the local restriction of the haplotype diversity found in this study. A comparative haplotypes (31 haplotypes found in 41 populations and 410 phylogeography study, examining a number of co-occurring individuals). This suggests that, in spite of the narrow eucalypt species, is underway. ecological niche of the species and the harsh environmental conditions during the LGM, E. regnans was able to persist locally or contracted to many near-coastal refugia, maintaining Cryptic refugia and the effect of climate and a diverse genetic structure. This is in strong contrast to the low recolonization processes on patterns of genetic haplotype diversity and fixation of haplotypes over large areas diversity of Europe found in studies of species including common ash, There is now considerable evidence for the survival of plant excelsior (12 haplotypes found in 201 populations and and animal species during the LGM at locations with climatic 1280 individuals across Europe using four chloroplast micro- parameters near or beyond their present ecological tolerances. satellites; Heuertz et al., 2006), five species of white , Numerous studies indicate that thermophilius plant and Quercus spp., in France and the Iberian peninsula (11 animal species were able to persist in northern Europe and at haplotypes found in 92 populations and 367 individuals using high latitudes in North America by contraction to favourable six chloroplast microsatellites; Grivet et al., 2006) and silver microsites in areas of suitable topography (reviewed by , Betula pendula (13 haplotypes found in 47 populations Stewart & Lister, 2001). Studies in Australia are more limited, and 431 individuals across Europe using RFLPs; Palme´ et al., but at Tallaganda in south-eastern , 2003). The differences between the genetic structure of phylogeographic studies on log-dwelling invertebrates that E. regnans and that of these European species can be accounted require cool and moist conditions suggest the long-term for by several factors. South-eastern Australia, in contrast to persistence of wet eucalypt forest (Garrick et al., 2004; much of Europe, did not experience the wholesale removal of Sunnucks et al., 2006). A similar scenario for E. regnans in all vegetation from large areas as a result of widespread a number of regions is suggested by the results of our study. glaciation or periglacial conditions. Furthermore, the scale of For example, the high haplotype diversity and unique recolonization events is much smaller than that of those in haplotypes found only in the north-east of Tasmania suggest Europe. The largest distance between likely refugia and the the long-term survival of the species in the region. The most isolated recolonized area is c. 100 km, with most north-east of Tasmania is thought to have been drier during E. regnans populations within 60 km of the coast. Therefore, much of the Quaternary period than in the present day, with rather than the waves of recolonization or long-distance longitudinal dunes in the region interpreted as indicating dispersal experienced in Europe, E. regnans, with the exception semi-arid conditions during the LGM (Bowden, 1983). Most of the Central Highlands region of mainland Australia, does of the area is likely to have been a particularly difficult place not appear to have migrated great distances. This contrast for the drought- and fire-intolerant E. regnans to survive, between a south-eastern Australian tree species and forest tree because unlike most eucalypts it has poor osmotic control species of Europe emphasizes the importance of climate and (Ashton, 1975a) and is currently confined to high-rainfall recolonization processes in determining a species’ genetic areas. The species may have been able to contract to deep diversity. valleys or suitable microsites where escape from the low temperatures and aridity of the LGM was possible. A similar Link between mainland Australia and Tasmania scenario is likely in the South Gippsland region of main- land Australia. There are widespread linear dune systems The question of the direction and location of the most recent north of Wilsons Promontory and in south-east Gippsland, gene flow between Tasmania and the mainland is of great suggesting arid conditions in the past and probably during interest, and this study on a species found on both sides of the LGM (Hill & Bowler, 1995). In addition, the low Bass Strait provides an excellent opportunity for addressing elevation of the compared to the Central it. Prior to relatively recent molecular studies, the eastern link Highlands would have reduced the effect of orographic between Tasmania and continental Australia via what is now rainfall (Worth et al., 2009). It is possible, but unlikely, that Flinders Island was seen as the most likely route for gene flow the unique haplotypes found in this region were present (Marginson & Ladiges, 1982) as it was likely to have been elsewhere, perhaps in areas considered more suitable for the above sea level longer than any potential link on the western survival of the species during the LGM, such as the south side of Bass Strait (Freeman et al., 2001). In spite of this, a Central Highlands of mainland Australia, and have since study by McKinnon et al. (1999) on chloroplast sharing disappeared or are at low frequencies and were not detected between several species in subgenus Eucalyptus tentatively in this study. suggested (the study was limited to only 23 samples) a It is interesting to compare the genetic structure observed stronger connection across the western side of the land- for E. regnans with that of forest tree species in Europe bridge, with samples from the western half of (Stewart & Lister, 2001). With the exception of the recolonized belonging to a Tasmanian clade, and those from the eastern

188 Journal of Biogeography 37, 179–192 ª 2009 Blackwell Publishing Ltd Phylogeography of Eucalyptus regnans half forming a clade restricted to mainland Australia. A more whether locations such as Mirboo East (16) are refugia or extensive study conducted by Freeman et al. (2001) on whether their high diversity has arisen from admixture of E. globulus found sharing of exactly the same haplotype maternal lineages. between the Otway Ranges and the central-west coast of This study is one step in the development of a gene pool Tasmania but no sharing of haplotypes between South management strategy for the species, which will be further Gippsland and north-east Tasmania. This sharing of improved with the addition of differentially inherited markers haplotypes between mainland and Tasmanian populations such as nuclear microsatellites and information associated with indicates that seed-mediated gene flow between populations . The issue of seed transfer over large geographical of E. globulus has occurred more recently via this western distance is of particular concern for this species, as E. regnans link. Freeman et al. (2001) suggested that the last, large-scale can be a poor and inconsistent producer of seed (Ashton, migration of E. globulus occurred from the Otway Ranges, 1975b). and seed development in the species is a long across the western side of the Bassian Plain, south along the and complicated process taking four to five years (Ashton, west coast of Tasmania, and then northwards again along the 1975b). This means that careful planning is required prior to east coast. However, Freeman et al. (2001) do not speculate forestry operations to ensure that suitable seed is available. on the timing of this migration and it is possible that it Clear felling is the preferred method of harvesting in tall pre-dates the LGM. eucalypt forest. Following the cutting and removal of logs, a Unlike E. globulus, which has no extant populations along regeneration burn of the remaining vegetation and residue the north coast of Tasmania, E. regnans has scattered stands from harvesting is conducted to create an ash bed that provides throughout the north-west, north and north-east. In this study nutrients for seedling growth. Seed is then dispersed by hand there was no sharing of identical haplotypes between the or helicopter (Flint & Fagg, 2007). Our results are compatible mainland and Tasmania, but the statistical parsimony tree with current strategies for that suggest that seed places haplotypes H1 and H31, found only in the Otway should be obtained from the stand that has been harvested or Ranges (Figs 2 and 3), with the major Tasmanian group. This from the immediate area. This would preserve existing patterns finding suggests a closer genetic link between north-west of intra-specific chloroplast types and diversity, which are Tasmania and the mainland than between north-east Tasmania particularly apparent in some regions. This ‘local is best’ and the mainland. The peripheral position of haplotypes H1 strategy is also important in the light of possible local and H31, which appear to be derived from the Tasmanian environmental adaptation, which would not be detectable haplotype H20, suggests that the direction of the seed- with the of genetic marker used in this study. However, mediated gene flow was from north-west Tasmania to the global climate change and the possibility that local genotypes western Otway Ranges. will no longer be ‘best’ (Aitken et al., 2008) may warrant a re- The sharing of more recently diverged but not identical examination of this policy. haplotypes could mean that gene flow between E. regnans in the Otway Ranges and north-west Tasmania occurred prior ACKNOWLEDGEMENTS to the last major migration of E. globulus between the mainland and Tasmania. Another possibility is that the faster We thank Tim Davis and Mark Neyland at Forestry Tasmania, mutation rate of microsatellite regions compared with other Peter Fagg at the Department of Sustainability and the regions of the chloroplast genome (Provan et al., 1999), Environment Victoria, Michael Ryan at Vicforests, as well as including the JLA region used in other studies on eucalypts, Mark Watkins, Tom Wright, Tom Nevill, Callum Nevill and is causing a loss of evolutionary information. Miranda Nevill for assistance in sample collection. This work was supported by the Cooperative Research Centre for Forestry and an Australian Postgraduate Award. Implications for conservation

These results have implications for the conservation and REFERENCES management of E. regnans genetic resources. Phylogeography can assist in the development of management and conserva- Aitken, S., Yeaman, S., Holliday, J., Wang, T. & Curtis- tion strategies as it examines the links between historical McLane, S. (2008) Adaptation, migration or extirpation: processes, geography and the distribution of genetic diversity climate change outcomes for tree populations. Evolutionary (Byrne, 2007). The genetic distinctiveness and diversity Applications, 1, 96–111. within certain regions such as East Gippsland and north- Ashton, D. (1958) The ecology of Eucalyptus regnans F. Muell.: east Tasmania suggest that they should be given high priority the species and its frost resistance. Australian Journal of in the formulation of gene pool management strategies. A , 6, 154–176. nuclear microsatellite study on the species would test the Ashton, D. (1975a) The seasonal growth of Eucalyptus regnans genetic isolation in terms of pollen-mediated gene flow of F. Muell. Australian Journal of Botany, 23, 239–252. some locations. Information on levels of nuclear diversity Ashton, D. (1975b) Studies of the flowering behaviour in would also assist in the identification of populations that Eucalyptus regnans F. Muell. Australian Journal of Botany, have been through genetic bottlenecks and potentially clarify 23, 399–411.

Journal of Biogeography 37, 179–192 189 ª 2009 Blackwell Publishing Ltd P. G. Nevill et al.

Baillie, P. (1989) Jurassic–Cainozoic. Geology and mineral Garrick, R., Sands, C., Rowell, D., Tait, N. & Greenslade, P. resources of Tasmania (ed. by C. Burrett and E. Mar- (2004) Phylogeography recapitulates topography: very fine- tin), pp. 339–409. Geological Society of Australia, scale local endemism of a saproxylic ‘giant’ springtail at Melbourne. Tallaganda in the Great Dividing Range of south-east Aus- Barrows, T., Stone, J., Fifield, L. & Cresswell, R. (2001) Late tralia. Molecular Ecology, 13, 3329–3344. Pleistocene glaciation of the Kosciuszko Massif, Snowy Griffin, A.R. (1980) Floral phenology of a stand of Mountain Mountains, Australia. Quaternary Research, 55, 179–189. Ash (Eucalyptus regnans F. Muell.) in Gippsland, Victoria. Barrows, T., Stone, J. & Fifield, L. (2004) Exposure ages for Australian Journal of Botany, 28, 393–404. Pleistocene periglacial deposits in Australia. Quaternary Griffin, A.R. & Johnson, K.W. (1979) Co-operative provenance Science Reviews, 23, 697–708. studies of Eucalyptus regnans F. Muell. Establishment Boland, D., Brooker, M., Chippendale, G., Hall, N., Hyland, B., Report. CSIRO division of Forest Research, Gippsland Johnston, R., Kleinig, D., McDonald, M. & Turner, J. (2006) Regional Station, Traralgon, Australia. Forest trees of Australia, 5th edn. CSIRO Publishing, Grivet, D., Deguilloux, M., Petit, R. & Sork, V. (2006) Con- Melbourne. trasting patterns of historical colonization in white Bowden, A. (1983) Relict terrestrial dunes: legacies of a former (Quercus spp.) in and Europe. Molecular Ecology, climate in coastal northeastern Tasmania. Geomorphology, 15, 4085–4093. 45, 153–174. Heuertz, M., Carnevale, S., Fineschi, S., Sebastiani, F., Haus- Bowler, J. (1982) Aridity in the late Tertiary and Quaternary of man, J.F., Paule, L. & Vendramin, G.G. (2006) Chloroplast Australia. Evolution of the flora and fauna of arid Australia DNA phylogeography of European ashes, Fraxinus sp. (ed. by W. Barker and P. Greenslade), pp. 35–45. Peacock (Oleaceae): roles of hybridization and life history traits. Publications, Adelaide. Molecular Ecology, 15, 2131–2140. Byrne, M. (2007) Phylogeography provides an evolutionary Hewitt, G. (1996) Some genetic consequences of ice ages, and context for the conservation of a diverse and ancient flora. their role in divergence and speciation. Biological Journal of Australian Journal of Botany, 55, 316–325. the Linnean Society, 58, 247–276. Byrne, M. & Hines, B. (2004) Phylogeographical analysis of Hill, S. & Bowler, J. (1995) Linear dunes at Wilsons Prom- cpDNA variation in (). ontory and South-East Gippsland, Victoria: relict landforms Australian Journal of Botany, 52, 459–470. from periods of past aridity. Proceedings of the Royal Society Byrne, M. & Macdonald, B. (2000) Phylogeography and con- of Victoria, 107, 73–81. servation of three oil taxa, Eucalyptus kochii ssp. Hope, G. (1974) The vegetation history from 6000 B.P. to kochii, ssp. plenissima and E. horistes. Australian Journal of present of Wilsons Promontory, Victoria, Australia. New Botany, 48, 305–312. Phytologist, 73, 1035–1053. Byrne, M., Moran, G. & Tibbits, W. (1993) Restriction map Hubbard, N.N. (1995) In search of regional palaeoclimates – and maternal inheritance of chloroplast DNA in Eucalyptus Australia, 18,000 yr BP. Palaeogeography, Palaeoclimatology, nitens. Journal of Heredity, 84, 218–220. Palaeoecology, 116, 167–188. Colhoun, E.A., Hannan, D. & Kiernan, K. (1996) Late Wis- Jackson, W.D. (1999) Paleohistory of vegetation change: the consin glaciation of Tasmania. and Proceedings of the last 2 million years. Vegetation of Tasmania (ed. by J.B. Reid, Royal Society of Tasmania, 130, 33–45. R.S. Hill, M.J. Brown and M.J. Hovenden), pp. 64–88. Flora Cremer, K.W. (1966) Dissemination of seed from Eucalyptus of Australia Supplementary Series no. 8, Australian Biolog- regnans. Australian Forestry, 30, 33–37. ical Resource Study, . Duller, G. & Augustinus, P. (1997) Luminescence studies of Jackson, H.D., Steane, D.A., Potts, B.M. & Vaillancourt, R.E. dunes from north-eastern Tasmania. Quaternary Science (1999) Chloroplast DNA evidence for reticulate evolu- Reviews, 16, 357–365. tion in Eucalyptus (Myrtaceae). Molecular Ecology, 8, 739– Fletcher, M. & Thomas, I. (2007) Holocene vegetation and 751. climate change from near Lake Pedder, south-west Tasma- Kershaw, A.P. & Nanson, G.C. (1993) The last full glacial cycle nia, Australia. Journal of Biogeography, 34, 665–677. in the Australian region. Global and Planetary Change, 7, Flint, A. & Fagg, P. (2007) Mountain Ash in Victoria’s State 1–9. Forests, Silvicultural Reference Manual No 1. Department of Kershaw, A.P., D’Costa, D.M., McEwen-Mason, J. & Wagstaff, Sustainability and the Environment, Melbourne. B.E. (1991) Palynological evidence for Quaternary vegeta- Frakes, L., McGowran, B. & Bowler, J. (1987) Evolution of tion and environments of mainland southeastern Australia. Australian environments. (ed. by G. Dyne Quaternary Science Reviews, 10, 391–404. and D. Walton), pp. 1–16. Australian Government Pub- Kershaw, A.P., McKenzie, G.M., Porch, N., Roberts, R.G., lishing Service, Canberra. Brown, J., Heijnis, H., Orr, M.L., Jacobsen, G. & Newall, Freeman, J.S., Jackson, H.D., Steane, D.A., McKinnon, G.E., P.R. (2007) A high-resolution record of vegetation and cli- Dutkowski, G.W., Potts, B. & Vaillancourt, R. (2001) mate through the last glacial cycle from Caledonia Fen, Chloroplast DNA phylogeography of Eucalyptus globulus. southeastern highlands of Australia. Journal of Quaternary Australian Journal of Botany, 49, 585–596. Science, 22, 481–500.

190 Journal of Biogeography 37, 179–192 ª 2009 Blackwell Publishing Ltd Phylogeography of Eucalyptus regnans

Kirkpatrick, J.B. & Fowler, M. (1998) Locating likely glacial Palme´, A., Su, Q., Rautenberg, A., Manni, F. & Lascoux, M. forest refugia in Tasmania using palynological and ecologi- (2003) Postglacial recolonization and cpDNA variation of cal information to test alternative climatic models. Biological silver birch, Betula pendula. Molecular Ecology, 12, 201–212. Conservation, 85, 171–182. Peakall, R. & Smouse, P.E. (2006) GENALEX 6: genetic analysis in Ladd, P. (1979) A Holocene vegetation record from the eastern Excel. Population genetic software for teaching and research. side of Wilsons Promontory, Victoria. New Phytologist, 82, Molecular Ecology Notes, 6, 288–295. 265–276. Petit, R.J., Aguinagalde, I., de Beaulieu, J.-L., Bittkau, C., Macphail, M. & Jackson, W. (1978) The Late Pleistocene and Brewer, S., Cheddadi, R., Ennos, R., Fineschi, S., Grivet, D., Holocene history of the midlands of Tasmania, Australia. Lascoux, M., Mohanty, A., Muller-Starck, G., Demesure- Proceedings of the Royal Society of Victoria, 90, 287–300. Musch, B., Palme, A., Martin, J.P., Rendell, S. & Vendramin, Marginson, J. & Ladiges, P. (1982) Morphological and geo- G.G. (2003) Glacial refugia: hotspots but not melting pots of graphical disjunctions in forms of Eucalyptus nitida Hook. f. genetic diversity. Science, 300, 1563–1565. (Myrtaceae): with special reference to the evolutionary sig- Petit, R., Duminil, J., Fineschi, S., Hampe, A., Salvini, D. & nificance of Bass Strait, southeastern Australia. Proceedings Vendramin, G. (2005) Comparative organisation of chlo- of the Royal Society of Victoria, 94, 155–167. roplast, mitochondrial and nuclear diversity in plant pop- Markgraf, V., McGlone, M. & Hope, G. (1995) ulations. Molecular Ecology, 14, 689–701. paleoenvironmental and paleoclimatic change in southern Pons, O. & Petit, R. (1996) Measuring and testing genetic temperate ecosystems – a southern perspective. Trends in differentiation with ordered versus unordered alleles. Ecology and Evolution, 10, 143–147. Genetics, 144, 1237–1245. McCarthy, M.A. & Lindenmayer, D. (1998) Multi-aged Provan, J., Soranzo, N., Wilson, N.J., Goldstein, D.B. & Powell, Mountain Ash forest, wildlife conservation and timber W. (1999) A low mutation rate for chloroplast microsatel- harvesting. Forest Ecology and Management, 104, 43–56. lites. Genetics, 153, 943–947. McKenzie, G.M. (1997) The late Quaternary vegetation history Rathbone, D., McKinnon, G., Potts, B., Steane, D. & Vaillan- of the south-central highlands of Victoria, Australia. 1. Sites court, R. (2007) Microsatellite and cpDNA variation in is- above 900 m. Australian Journal of Ecology, 22, 19–36. land and mainland populations of a regionally rare eucalypt, McKenzie, G.M. (2002) The late Quaternary vegetation history Eucalyptus perriniana (Myrtaceae). Australian Journal of of the south-central highlands of Victoria, Australia. II. Sites Botany, 55, 513–520. below 900 m. Austral Ecology, 27, 32–54. Raymond, C.A., Volker, P.W. & Williams, E.R. (1997) Prov- McKenzie, G.M. & Kershaw, A.P. (1997) A vegetation history enance variation, genotype by environment interactions and and quantitative estimate of Holocene climate from Chapple age–age correlations for Eucalyptus regnans on nine sites in Vale, in the Otway region of Victoria, Australia. Australian south eastern Australia. Forest Genetics, 4, 235–251. Journal of Botany, 45, 565–581. Remington, C.L. (1968) Suture-zones of hybrid interaction McKenzie, G.M. & Kershaw, A.P. (2000) The last glacial cycle between recently joined biotas. Evolutionary biology (ed. by from Wyelangta, the Otway region of Victoria, Australia. T. Dobzhansky, M. Hecht and W. Steere), pp. 321–428. Palaeogeography, Palaeoclimatology, Palaeoecology, 155, 177– Plenum, New York. 193. Sigleo, W. & Colhoun, E. (1981) A short pollen diagram from McKenzie, G.M. & Kershaw, A.P. (2004) Holocene pollen Crown Lagoon in the Midlands of Tasmania. Papers and record from cool , Aire crossing, the Proceedings of the Royal Society of Tasmania, 115, 181–188. Otway region of Victoria, Australia. Review of Palaeobotany Steane, D.A., Byrne, M., Vaillancourt, R.E. & Potts, B.M. and Palynology, 132, 281–290. (1998) Chloroplast DNA polymorphism signals complex McKinnon, G.E., Steane, D.A., Potts, B.M. & Vaillancourt, R.E. interspecific interactions in Eucalyptus (Myrtaceae). (1999) Incongruence between chloroplast and species phy- Australian Systematic Botany, 11, 25–40. logenies in Eucalyptus subgenus Monocalyptus (Myrtaceae). Steane, D.A., Jones, R.C. & Vaillancourt, R.E. (2005) A set of American Journal of Botany, 86, 1038–1046. chloroplast microsatellite primers for Eucalyptus (Myrta- McKinnon, G.E., Vaillancourt, R.E., Jackson, H.D. & Potts, ceae). Molecular Ecology Notes, 5, 538–541. B.M. (2001a) Chloroplast sharing in the Tasmanian euca- Stewart, J. & Lister, A. (2001) Cryptic northern refugia and the lypts. Evolution, 55, 703–711. origins of the modern biota. Trends in Ecology and Evolution, McKinnon, G.E., Vaillancourt, R.E., Tilyard, P.A. & Potts, 11, 608–613. B.M. (2001b) Maternal inheritance of the chloroplast Sunnucks, P., Blacket, M.J., Taylor, J.M., Sands, C.J., Ciavaglia, genome in Eucalyptus globulus and interspecific hybrids. S.A., Garrick, R.C., Tait, N.N., Rowell, D.M. & Pavlova, A. Genome, 44, 831–835. (2006) A tale of two flatties: different responses of two McKinnon, G.E., Jordan, G.J., Vaillancourt, R.E., Steane, D.A. terrestrial flatworms to past environmental climatic fluctu- & Potts, B.M. (2004) Glacial refugia and reticulate evolu- ations at Tallaganda in montane southeastern Australia. tion: the case of the Tasmanian eucalypts. Philosophical Molecular Ecology, 15, 4513–4531. Transactions of the Royal Society B: Biological Sciences, 359, Tibbits, J., McManus, L., Spokevicius, A. & Bossinger, G. 275–284. (2006) A rapid method for tissue collection and high

Journal of Biogeography 37, 179–192 191 ª 2009 Blackwell Publishing Ltd P. G. Nevill et al.

throughput genomic DNA isolation from mature trees. BIOSKETCHES Plant Molecular Biology Reporter, 24, 1–11. Wheeler, M.A. & Byrne, M. (2006) Congruence between Paul Nevill is a PhD student with an interest in phylogeog- phylogeographic patterns in cpDNA variation in Eucalyptus raphy, conservation genetics and the management of biodi- marginata (Myrtaceae) and geomorphology of the Darling versity. Plateau, south-west of Western Australia. Australian Journal of Botany, 54, 17–26. Gerd Bossinger is Associate Professor and Group Leader for Willis, J. (1967) Typification of eight Victorian species names Forest Molecular Biology and Genetics at the University of in Eucalyptus. Muelleria, 1, 167. Melbourne Department of Forest and Ecosystem Science. His Worth, J.R., Jordan, G.J., McKinnon, G.E. & Vaillancourt, R.E. research group investigates the molecular control of meristem (2009) The major Australian cool temperate rainforest tree differentiation and pattern formation, concentrating on the cunninghamii withstood Pleistocene glacial vascular cambium and formation. More recently, the aridity within multiple regions: evidence from the chloro- relevance of gene structure and function for patterning genetic plast. New Phytologist, 182, 519–532. diversity has moved into focus.

Peter Ades is a senior lecturer in forest genetics at the SUPPORTING INFORMATION University of Melbourne Department of Forest and Ecosystem Science. His research interests include modelling gene flow Additional Supporting Information may be found in the and estimating risks to remnant eucalypt stands arising from online version of this article: introgression from , developing methodologies for Appendix S1 Characteristics of haplotypes detected with five identifying patterns of adaptive variation in tree species, and chloroplast microsatellites in Eucalyptus regnans. conservation genetics of forest species.

Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting materials supplied Editor: Pauline Ladiges by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

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