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Australian Journal of Botany, 2006, 54, 17–26

Congruence between phylogeographic patterns in cpDNA variation in Eucalyptus marginata (Myrtaceae) and geomorphology of the Darling Plateau, south-west of Western Australia M. A. WheelerA and M. ByrneB,C A School

of Biology and Biotechnology, Murdoch University, South Street Murdoch, WA 6150, Australia. Division, Department of Conservation and Land Management, Locked Bag 104, Bentley Delivery Centre, WA 6983, Australia. C Corresponding author. Present Address: PO Box 65, Rosebank, NSW 2480, Australia. Email: [email protected]

B Science

Abstract. Phylogeographic patterns in the cp genome of Eucalyptus marginata Don ex Sm., a species common in the mesic region of south-western Australia, were investigated by using RFLP analysis. The chloroplast diversity was structured into two geographically distinct lineages and nested clade analysis inferred historical fragmentation as the major influence on the phylogeographic pattern. The lineages were separated along the geomorphological boundary of the Darling Scarp, which separates the Coastal Plain from the Darling Plateau. The divergence between the lineages is consistent with uplifting of the Darling Plateau in the late Neogene. Further geographic structuring in haplotype distributions was evident in the forest lineage on the Darling Plateau, where one sublineage was present in the central forest region and another was restricted to the south-eastern region. The level of divergence between these sublineages was similar to that between divergent lineages that have been identified in comparative phylogeographic studies of cpDNA variation in three species widespread throughout south-western Australia. In these species, divergence was attributed to the influence of significant changes in climatic oscillations across the semi-arid region during the mid-Pleistocene. The divergence identified in this study indicates that the influence of climatic change was widespread throughout south-western Australia, including the mesic, higher-rainfall region.

Introduction The analysis of chloroplast DNA often shows intra-specific variation in spite of its slow mutation rate (Soltis et al. 1991; Ennos et al. 1999) and, because of the predominantly maternal inheritance and lack of recombination within the chloroplast genome, this variation is ordered in genealogical lineages (Schaal et al. 1998). The influence of historical processes on the evolution of populations and species at lower taxonomic levels can be deduced from phylogeographic analyses of genealogical lineages (Avise 2000), particularly in association with expectations derived from coalescent theory (Templeton et al. 1995). Comparative studies over a range of taxa from several families can elucidate shared patterns indicative of common response to factors influencing evolutionary processes within regions. Such comparative studies have identified glacial refugia in the Pacific Northwest of America (Soltis et al. 1997), and postglacial recolonisation routes throughout Europe (Ferris et al. 1993, 1998; Demesure et al. 1996; Dumolin-Lapègue et al. 1997; King and Ferris 1998). Phylogeographic studies at the species © CSIRO 2006

level have been carried out in the following three species from three different families in south-western Australia: Santalum spicatum (R.Br.) A.DC. (Byrne et al. 2003), Eucalyptus loxophleba Benth. (Byrne and Hines 2004) and Acacia acuminata Benth. (Byrne et al. 2002). Comparison of their phylogeographic patterns has identified common historical fragmentation events in the semi-arid region between the dry arid zone (less than 300 mm rainfall) to the north and east and the higher-rainfall (greater than 600 mm rainfall) mesic area to the south-west, despite no obvious geographic barrier. The shared patterns indicate a common response to historical fragmentation in these widespread species, despite more recent secondary contact between lineages. It has been hypothesised that this fragmentation was induced by climatic fluctuations during the mid-Pleistocene, corresponding with a change to more extreme climatic oscillations and the shift from short to longer cycles of greater amplitude (Bowler 1982). Significant climatic change would lead to fragmentation of species distribution and subsequent divergence of lineages 10.1071/BT05086

0067-1924/06/010017

18

Australian Journal of Botany

that is still detectable, even though later expansion may result in secondary contact. These effects are consistent with other evidence of major transformation in vegetation and climate between the late Pliocene and the mid–late Pleistocene (Macphail et al. 1993; Dodson and Kershaw 1994; Jordan 1997), and fossil evidence that shows significant change in vegetation associated with glacial and inter-glacial periods during the mid–late Pleistocene (Dodson 1994). Support for historical fragmentation within the flora of south-western Australia is also found in the many species with divergent genetic lineages and the high level of genetic diversity within species, both in the nuclear and chloroplast genomes (Byrne et al. 1999, 2001; Coates 2000). Climatic instability in south-western Australia would be expected to be most extreme in the transitional semi-arid zone between the mesic region in the far south-west and the arid region to the north-east. This would suggest there may be little genetic structuring in species whose distributions are wholly within the arid or mesic regions as the intensity of the climatic changes would be less than in the semi-arid zone. However, the influence of climatic perturbations on change in vegetation appears to have been widespread across southern Australia; hence, some influence of climatic oscillations may also be detectable in the mesic and arid regions. Analysis of cpDNA variation in species with distributions restricted to the mesic or arid regions could provide information on the extent of climatic fluctuations across south-western Australia. Eucalyptus marginata is one such example as it is a dominant species of the forests in the mesic, higher-rainfall region in the south-west of Western Australia. It has a relatively continuous distribution and often grows in almost mono-specific stands on the laterite soils of the Darling Plateau. The species probably had a larger distribution during wetter periods since there are outlier populations to the east of the main distribution, which would be included in the main distribution if mean annual rainfall were increased by only 75 mm (Churchill 1968). Analysis of cpDNA variation in eucalypts can be confounded by hybridisation. Although there is evidence of recent hybridisation between many eucalypt species, there is some debate over the evolutionary significance of hybridisation in the genus, and it is difficult to distinguish between genetic patterns resulting from hybridisation and lineage sorting of ancestral polymorphism since the pattern of incongruence is similar (Wendel and Doyle 1998). The widespread species, E. loxophleba Benth., showed congruent phylogeographic patterns with other species in Western Australia, which indicates that the pattern of cpDNA variation detected was due to phylogeographic factors rather than species specific factors such as hybridisation (Byrne and Hines 2004). Hybridisation is not likely to be a significant issue in this study because E. marginata is not known to hybridise extensively

M. A. Wheeler and M. Byrne

with other species, although three hybrid combinations are recognised by the Western Australian Herbarium (E. buprestium F.Muell. × E. marginata, E. marginata × E. megacarpa F.Muell., E. marginata × E. pachyloma Benth., see Florabase, http://florabase.calm.wa.gov.au). These hybrids have very restricted distributions and occur at the margins of the range of E. marginata in the Stirling Range and at Cape Naturaliste. A study of genetic diversity in the nuclear genome showed genetic differentiation between E. marginata and its sister species E. staeri (Maiden) Kessell & C.A.Gardner (Wheeler et al. 2003) even though they have sympatric distributions. This study assessed the pattern of cpDNA variation throughout the range of E. marginata to investigate whether it is consistent with the hypothesis of little structure in cpDNA variation in species occupying the mesic climatic region of south-west of Western Australia. Materials and methods Plant material The informal classification of Ladiges et al. (1987) placed E. marginata and its sister species E. staeri in subgenus Monocalyptus, section Diversifolia, series Marginatinae. Three subspecies of E. marginata are recognised on morphological grounds (Brooker and Hopper 1993); subspecies marginata is a tall forest tree with dark green leaves that occurs in the southern and part of the western sections of the total distribution area for the species, subspecies thalassica has a weeping habit with blue-green leaves and occurs in the north-eastern part of the distribution area, and subspecies elegantella is a small tree with small, narrow, olive-green leaves, that occurs in a narrow strip of the eastern part of the Swan Coastal Plain. Analysis of diversity in the nuclear genome showed high genetic identity at the species level, with no genetic differentiation between the three subspecies (Wheeler et al. 2003). For this study, samples of E. marginata were collected from adult trees from 15 populations that were selected from throughout the range encompassing all three subspecies, and including two outlying populations from the eastern edge of the range (Fig. 1). In addition, samples were collected from one population of the sister species E. staeri, and two populations of E. todtiana F.Muell. for use as an outgroup (Table 1). In their informal classification Ladiges et al. (1987) placed E. todtiana in Superseries Todtianicae, related to Superseries Marginaticae (containing series Marginatinae and one other eastern Australian species) in an unresolved polytomy. DNA was extracted from the leaves of five individuals for each population, following the methods outlined in Byrne et al. (1998). For each sample, 2 µg of DNA was digested with six restriction enzymes (BclI, BglII, EcoRI, XbaI, EcoRV and HindIII), subjected to electrophoresis, Southern blotted and hybridised with heterologous probes covering the single copy regions of the chloroplast genome. The probes used were P1, P3, P4, P6, P8 and P10 from Petunia (Sytsma and Gottlieb 1986), and pTBa1 from tobacco (Sugiura et al. 1986). Restriction digestion and hybridisation were as described in Byrne and Moran (1994) and probe inserts were amplified and then labelled with 32 P by using the random priming method. Data analysis For each probe–enzyme combination polymorphism in fragments was identified as length or restriction-site mutations. Fragment patterns for

Phylogeography in Eucalyptus marginata


+18(S)

Swan Coastal Plain Darling Scarp Map Area 3(K)

1(N)

+

17(S)

4(J)

PERTH 2(N) )

15(H) 8(C)

Bunbury

9(A) 14(I) )

)

13(E,H)

12(C,G) 16(D)

Albany

Fig. 1. Collection locations and distribution of clades identified by cpDNA analysis for Eucalyptus marginata. Population symbols: F represents E. marginata populations in the main forest clade (Clade I), 䊏 represents E. marginata populations in the coastal clade (Clade II), 䉬 represents population of E. staeri, + represents populations of E. todtiana. Population numbers as in Table 1. Letters in parentheses following the population collection number are the haplotypes present in the population. Populations in Subclades Ia and Ib are encompassed by lines.

Table 1. Location of populations of Eucalyptus marginata, E. staeri and E. todtiana sampled for analysis of chloroplast diversity Population

Taxon

Latitude (S)

Longitude (E)

1. Yanchep 2. Beeliar 3. Julimar Forest 4. Mundaring Forest 5. Welshpool Road 6. Jarradale 7. North Dandalup 8. Dwellingup 9. Collie 10. Nannup 11. Bridgetown 12. Mount Frankland 13. Perup Nature Reserve 14. Katanning 15. Jilakin Rock 16. Mount Frankland 17. Wanneroo 18. Moora

ssp. marginata ssp. marginata ssp. thalassica ssp. thalassica ssp. elegantella ssp. thalassica ssp. elegantella ssp. marginata ssp. marginata ssp. marginata ssp. marginata ssp. marginata ssp. marginata

31◦ 30 01 32◦ 09 41 31◦ 27 35 31◦ 59 06 32◦ 00 15 32◦ 20 13 32◦ 30 13 32◦ 42 24 33◦ 14 52 33◦ 47 05 34◦ 01 45 34◦ 49 09 34◦ 09 00

115◦ 41 22 115◦ 49 48 116◦ 13 43 116◦ 10 09 116◦ 00 51 116◦ 04 39 115◦ 58 46 116◦ 06 33 116◦ 08 58 115◦ 25 44 115◦ 59 01 116◦ 46 38 116◦ 34 37

ssp. marginata ssp. marginata E. staeri E. todtiana E. todtiana

33◦ 40 46 32◦ 39 53 34◦ 47 12 31◦ 39 28 29◦ 46 03

117◦ 34 23 118◦ 19 34 116◦ 43 10 115◦ 44 16 115◦ 51 54

19

consecutive cp probes were compared to ensure that each polymorphism was correctly interpreted and counted only once. Where a length mutation was detected by more than one restriction enzyme, it was counted as only one polymorphism. Haplotypes were identified by presence or absence of polymorphisms. Nucleotide diversity was calculated by using restriction-site mutations with HAPLO (Lynch and Crease 1990), and partitioned within and between populations. Haplotype diversity was calculated by Nei’s gene diversity measures (Nei 1978) for haplotypes in the total sample and in each population. A parsimony analysis of haplotype relationships characterised by presence or absence of each polymorphism was undertaken by using PAUP (Swofford 1991). Bootstrap analysis used 100 replications and heuristic search, with TBR branch swapping and MULPARS on. To test for association between phylogenetic position of haplotypes and their geographic distribution, a nested clade analysis (Templeton et al. 1995; Templeton 1998) was carried out. The nested cladogram was drawn from the PAUP cladogram based on criteria described in Templeton et al. (1992) where haplotypes are clustered together into clades working from the tips of the cladogram inward, and this is repeated at higher nesting levels until the total cladogram level is reached. Geographic association of haplotypes was determined by permutation analyses based on 1000 re-samples, using the program GeoDis (Posada et al. 2000) and interpretation of the nested clade analysis followed the inference key of Posada and Templeton (2004).

Results Polymorphism in cpDNA Analysis of restriction fragments in the cp genome of E. marginata revealed polymorphism with all enzymes used. In total, 11 site mutations, 30 length mutations and one pattern that could only be interpreted as a missing band were detected (Table 2). These polymorphisms were distributed over 17 haplotypes. One haplotype was detected in the E. staeri population, and this was characterised by 11 polymorphisms, of which two were unique length mutations and nine were mutations shared with E. marginata. The samples of E. todtiana represented one haplotype, which was differentiated from the E. marginata haplotypes by a length mutation. Within E. marginata, the most common haplotype, A, was present in three populations and represented 18% of the samples (Table 2). There were four haplotypes that were present in two populations, C (10% of samples), H (11% of samples), N (14% of samples) and O (7% of samples). All other haplotypes were present in only one population, with frequencies ranging from 1 to 7%. In general, haplotypes were fixed in populations but in five populations two haplotypes were sampled and three haplotypes were present in one population. Haplotype relationships A phylogenetic parsimony analysis of haplotype relationships gave one tree of length 44, with a consistency index of 0.977 as none of the polymorphisms showed any homoplasy (Fig. 2). The phylogeny showed that the haplotypes present in

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Table 2. CpDNA haplotypes of Eucalyptus marginata, E. staeri and E. todtiana Haplotype

Mutations

Populations (no. of individuals)

A B C D E F G H I J K L M N O P Q R S

22,23,24,42,43,10,12,40,20,41,35,44 22,23,24,42,43,10,12,40,20,41,35, 44,30 22,23,24,42,43,10,12,40,20,41 22,23,24,42,43,10,12,40,20,41,25,32 22,23,24,42,43,10,12,40,1,34,5,7,35,9,15 22,23,24,42,43,10,12,40,1,34,5,7,35,30 22,23,24,42,43,10,12,40,1,34,14,6 22,23,24,42,43,10,12,40,1,34,14,21 22,23,24,42,43,10,12, 40,1,34,17 22,23,24,42,43,19 22,23,24,42,43,2,3,4 22,23,24,42,43,26,33,36,39 22,23,24,42,43,27 22,28,29,38 22,28,29,38,13,8,11 22,28,29,38,13,8,11,16 22,28,29,38,13,8,11,18 22,28,29,38,13

Jarrahdale (3), Bridgetown (5), Collie (5) Jarrahdale (1) Dwellingup (5), Mt Frankland (2) E. staeri (5) Perup (1) Nannup (1) Mt Frankland (2) Jilakin Rock (4), Perup (4) Katanning (5) Mundaring Forest (5) Julimar Forest (5) Nannup (3) Nannup (1) Yanchep (5), Beeliar (5) Nth Dandalup (2), Welshpool Rd (3) Welshpool Rd (1) Welshpool Rd (1) Nth Dandalup (3) E. todtiana (8)

31

Ia

80

C Dwellingup (8), Mount Frankland (12)

20,41

D Eucalyptus staeri (16)

25,32 92 10,12

92

9,15

5,7, 37 30

Ib

78 1,34

64

6

14 21

23,24,, 3 42,43

F Nannup (10) G Mount Frankland (12) H Jilakin Rock (15), Perup (13)

J Mundaring (4))

19

K Julimar Forest (3)

2,3,4

ssp. thalassica

L Nannup (10)

26,33,36,39

M Nannup (10)

27

100

E Perup (13)

I Katanning (14)

17

95

I

Bridgetown (11), Collie (9), Jarrahdale (6) ssp. thalassica B Jarrahdale (6) A

89 35,44

N Yanchep (1), Beeliar (2)

22

O

96

II 28,29, 38

62 13

78 8,11

16 18

North Dandalup (7) Welshpool Rd (5)

P Welshpool Rd (5)

ssp. elegentella

Q Welshpool Rd (5) R North Dandalup (7) S Eucalyptus todtiana (17,18)

Fig. 2. Phylogenetic parsimony tree of haplotype relationships in Eucalyptus marginata, with E. todtiana used as outgroup. Haplotypes designated by letters A–S are according to Table 2. Numbers above lines in italics represent bootstrap values greater than 50%, numbers below lines represent mutations according to Table 2 (site mutations in bold). Numbers in parentheses indicate population numbers according to Fig. 1.

E. marginata and E. staeri were clearly separate from that in the outgroup E. todtiana. Within E. marginata the haplotypes were separated into two main clades (Fig. 2) that represent separation of the Swan Coastal Plain populations from the

Darling Plateau forest populations. Within the forest clade (Clade I) there was structuring with two associated subclades (Subclades Ia and Ib), and a polytomy of four additional branches. One subclade (Subclade Ia) contained haplotypes present in populations from the central (Jarrahdale, Collie and Dwellingup) and southern (Bridgetown, and Mount Frankland) forests, including ssp. marginata, ssp. thalassica, and E. staeri. The other (Subclade Ib) contained haplotypes present in populations from the southern (Perup, Nannup and Mount Frankland) forest and the two outlier populations (Katanning and Jilakin Rock), and included only ssp. marginata. The most southerly sampled population at Mt Frankland contained haplotypes from both the subclades. The remaining haplotypes in the unresolved polytomy were present in the most south-western population at Nannup and in the two most northern populations of ssp. thalassica. The second main clade (Clade II) contained haplotypes present in all the sampled populations on the Swan Coastal Plain, and all except Yanchep and Beeliar were classified as being ssp. elegantella by Brooker and Hopper (1993). Within this clade (Clade II), haplotypes in the ssp. marginata populations of Yanchep and Beeliar (the northern section of the Swan Coastal Plain) were differentiated from those in the ssp. elegantella populations at Welshpool and North Dandalup. The haplotypes present in the outlier populations at Jilikan Rock and Katanning showed no differentiation from the other haplotypes in E. marginata (Fig. 2). The haplotype present in the E. staeri population at Mt. Frankland was a unique haplotype that was not present in any sampled individuals of E. marginata, including those collected from the same area, although the haplotype was related to



those present in E. marginata as it occurred in the same clade (Fig. 2).

of contiguous range expansion was made for Clade 3-2 because of the significantly small dispersal value for the interior Clade 2-4 and the large displacement value for the tip Clade 2-5, which represents Haplotype L present in the most south-western population at Nannup. Inference of past fragmentation was made for Clade 3-1 because of the mostly non-overlapping distribution of Clade 2-1 with Clades 2-2 and 2-3. Inference of past fragmentation was also made for the total cladogram, owing to the small dispersion value of the tip Clade 4-2 and its non-overlapping distribution with Clade 4-1. The inference of past fragmentation at this level is also supported by the large number of mutations that separate the clades. The geographic distribution of the clades influenced by past fragmentation is shown in Fig. 1.

Geographic distribution of haplotypes To test the strength of the geographic pattern, a nested clade analysis was carried out. The cladogram of nested clade structure is shown in Fig. 3. Significant association between haplotype distribution and geographic structure was identified at the total cladogram level and at lower nesting levels for a single one-step clade, three two-step clades and both three-step clades. The dispersion (Dc) and displacement (Dn) values, and their probability of being significantly large or small, for the clades showing significant geographic structure are given in Table 3. Interpretation of historical process influencing the geographical structure of haplotypes at each clade level was made on the basis of the significance of the dispersion and displacement values, using the inference key of Posada and Templeton (2004) (Table 3). Clade 4-1 in the nested clade analysis is equivalent to Clade I in the parsimony analysis and Clade 4-2 is equivalent to Clade II. At lower nesting levels in the cladogram, association between haplotypes or clades and their geographic distribution was generally influenced by restricted gene flow or was inconclusive. At higher nesting levels an inference

3-1

E

1-3

21

Nucleotide diversity Nucleotide diversity, the average number of nucleotide differences per site between two sequences (Nei 1978), can be determined for restriction sites but not for length mutations. The average substitution/nucleotide site for pairs of random haplotypes, averaged over all pairs of individuals in E. marginata, was 0.201%. The majority of this diversity was distributed between populations, with only 0.0128% within populations. The proportion of nucleotide diversity between

4-1

2-2

0

0

4-2

F

3-2 0

1-5

2-4 0

G

0

0

H

0

1- 4

0

1- 6

I

0

3-3

K 1-8

0

0

1-9 J

0

0

2-3 M 0 0

2-6

2-7 Q

0

0

0

0

0

N 1-12

R

0 1-11

O

P 1-10

C 0 0 1-2 0 A L 1-7

2-1

B 1-1

2-5

Fig. 3. Haplotype network showing nested clades for Eucalyptus marginata. Haplotype designations are those from Table 2. Interior haplotypes not detected in the samples are represented by 0. Each line connecting haplotypes represents a single polymorphism. One-step clades are indicated by thin-lined boxes, two- and three-step clades by heavier-lined boxes.

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Table 3. Phylogeographical inferences inferred from nested clade analysis of Eucalyptus marginata Dc, Clade distance (dispersion); Dn, nested clade distance (displacement); L, probability of larger than expected value; S, probability of smaller than expected value. Significant probabilities in bold. Numbers in key steps refer to options in inference key of Posada and Templeton (2004) Nested clade

Clade/haplotype

Clade 1-4 Clade 2-1

Clade 2-3

Clade 2-4

Clade 3-1

Clade 3-2

Clade 4-1

Total cladogram

Dc

Probability

Dn

Probability

Hap G (tip) Hap H (tip) Clade 1-1 (tip) Clade 1-2 (interior) Interior v. tip Clade 1-4 (tip) Clade 1-6 (interior) Interior v. tip Clade 1-8 (tip) Clade 1-9 (interior) Interior v. tip Clade 2-1 (tip) Clade 2-2 (tip) Clade 2-3 (interior) Interior v. tip Clade 2-4 (interior) Clade 2-5 (tip) Interior v. tip Clade 3-1 (tip) Clade 3-2 (interior) Interior v. tip

0.0000 114.2860 65.7093 107.8590 42.1497 123.9578 0.0000 −123.9578 0.0000 58.7768 58.776 76.9570 56.6449 90.5520 15.3613 47.1423 0.0000 47.1423 109.1700 98.2708 −10.8992

S0.3020 S0.1680 S0.1420 L0.0270 L0.0580 L0.0000 S0.0000 S0.0000 S0.0020 S0.4650 L0.0020 S0.0000 S0.3970 S0.0680 L0.2050 S0.0140 S0.0630 L0.3470 S0.0070 S0.0760 S0.3130

141.2541 119.1533 64.2389 102.3932 38.1542 124.4491 12.5888 −111.8603 50.8852 44.0232 −6.8620 100.7311 98.3600 121.9879 21.4630 78.3064 171.4734 −93.1670 111.6750 150.8375 39.1625

L0.0280 S0.0280 S0.0950 L0.0790 L0.0790 L0.0000 S0.0000 S0.0000 L0.4650 S0.4650 S0.4650 S0.0850 S0.4170 L0.0610 L0.0670 S0.0140 L0.0140 S0.0140 S0.0110 L0.0010 L0.0040

Clade 4-1 (interior) Clade 4-2 (tip) Interior v. tip

121.6436 35.5962 86.0475

L0.1790 S0.0000 L0.0000

123.2646 101.8188 21.4458

L0.0810 S0.0720 L0.0730

the populations (NST ) was 94.4%. Diversity between the two main lineages, I and II, was 0.285%. There was more variation within Clade I (0.114%) than within Clade II (zero because there were no site mutations within haplotypes in Clade II). Diversity between the two forest subclades (Subclades Ia and Ib) was 0.135%, with greater variation within Subclade Ia (0.042%) than within Subclade Ib (0.01%). Discussion The level of chloroplast variation identified within E. marginata was higher than has been observed in other eucalypt studies that have been assayed by RFLPs and a similar sampling strategy. This study detected 41 mutations distributed over 17 haplotypes, compared with E. nitens (H.Deane & Maiden) Maiden, which had 25 mutations in 13 haplotypes (Byrne et al. 1993), E. kochii Maiden & Blakely with 31 mutations in 18 haplotypes (Byrne and MacDonald 2000) and E. loxophleba with 22 mutations in 15 haplotypes (Byrne and Hines 2004). The level of nucleotide diversity in E. marginata (0.201%) was also higher than has been observed in other woody species in Western Australia, such as E. loxophleba (0.088%, Byrne and Hines 2004), E. kochii (0.081%, Byrne and MacDonald 2000), Acacia acuminata (0.079%, Byrne et al. 2002) and Santalum spicatum (0.084%, Byrne et al. 2003).

Key steps and inference 1, 2, 11, 17 no Inconclusive 1, 2, 3, 4 no Restricted gene flow 1, 2, 11, 17 no Inconclusive 1, 2, 3, 4, no Restricted gene flow 1, 2, 3, 4, 9 no Allopatric fragmentation 1, 2, 11, 12 no Contiguous range expansion 1, 2, 3, 4 no Restricted gene flow 1, 19 Fragmentation

The high level of chloroplast DNA variation within E. marginata showed significant geographic structure with partitioning into two main lineages that separated according to the geomorphological boundary represented by the Darling Scarp (Fig. 1). One lineage was restricted to the sandy Swan Coastal Plain (Clade II/4-2) and the other was present throughout the lateritic Darling Plateau (Clade I/4-1), including the outlier populations. At this deepest level in the phylogeny, phylogeographic analysis led to an inference of past fragmentation as having a significant effect on the geographical distribution of the clades. The two clades have non-overlapping distributions, are separated by a large number of polymorphisms and the nucleotide diversity was high. Nucleotide divergence can provide an estimate of time since separation by using a molecular clock, which is a measure of the rate of evolution along the branches of a molecular phylogeny. There are limitations with the use of a molecular clock since it assumes that rates of evolution are constant between evolutionary lineages, and it is difficult to calibrate without reliable independent evidence from biogeographical or fossil data. A rate of evolution for the whole cp genome is difficult to estimate but there is some evidence to suggest it is slightly slower than the rate of synonymous substitutions for cp genes, which range from 0.08 to 0.16%, probably owing to lower substitution rates in non-coding regions (Zurawski


et al. 1984; Zurawski and Clegg 1987; Doebley et al. 1990). An estimate of 0.1% nucleotide divergence representing one million years separation was made for non-coding regions of the rbcl gene (Zurawski et al. 1984) and this may be the best estimate for restriction-site data from across the genome as used in this study. With this estimate as a broad indicator, recognising the assumptions and limitations of a molecular clock, the divergence between the two main lineages in E. marginata indicates separation ∼2.85 million years ago and corresponds with geological features of the Darling Scarp in the late Pliocene. The Darling Scarp occurs along the Darling Faultline and separates the Darling Plateau from the Coastal Plain. The faultline was active in the Cretaceous but separation of the plain and the plateau occurred with uplifting of the Darling Plateau, beginning in the mid-Neogene. During the late Neogene and early Pleistocene the Darling Scarp formed coastal cliffs (Playford et al. 1976) and there was gradual uplift of the Perth Basin, and widespread, shallow transgressions or marine flooding, finishing in the early Pleistocene with regression of the coastline (Quilty 1974; Kendrick et al. 1991). The time of separation of the two lineages in the late Neogene corresponds with the geological activity that led to the separation of the Swan Coastal Plain and the Darling Plateau. The correlation of divergence between lineages with a geomorphological event enables calibration of the molecular clock estimate in this landscape that has not been possible in previous studies. Genetic divergence times can be greater than actual separation times since the presence of ancestral diversity will lead to greater time to coalescence than to separation (Avise 2000). The divergence time estimated here and the estimates for the time of uplifting of the Darling Plateau are very similar, and indicate that use of evolutionary rates from cp genomes of other plants are applicable in eucalypts in this landscape. Use of divergence rates based on cp gene sequence data from other plants also provided reasonable estimates in Helianthus (Rieseberg et al. 1991) where they gave similar divergence times to those estimated from isozyme data. At shallower levels within the phylogeny there was also significant geographical association of haplotypes in the forest clade (Clade I/4-1), with one sublineage present throughout the central forest and another occurring in the south-east. The nested clade analysis inferred past fragmentation as the significant influence on the distributions of these sublineages. They have mostly separate distributions, although they overlap in the southern part of their distributions, which could have occurred through later expansion of the previously fragmented lineages. The two sublineages appear to have evolved from an internal clade (Clade 1-9) containing haplotypes (Haplotypes J and M) that were detected at low frequency and in geographically separated populations. The patchy distribution of two related haplotypes in widely separated populations in the north


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and south-west of the range is unusual. Even though the haplotypes in the clade are not widespread or frequent, characteristics that are generally associated with ancestral haplotypes (Schaal et al. 1998), they have an internal position in the phylogeny suggesting ancestry, and their presence in both the northern and southern areas is consistent with their retention as ancestral haplotypes in these regions and extinction in other populations where they have evolved into other lineages. The presence of other derived haplotypes from other clades in the same populations also supports this interpretation. Patchy distribution of haplotypes in peripheral populations has been identified in other studies (Masta et al. 2003; Byrne and Hines 2004) and complicated the interpretation of phylogeographic patterns. The nested clade analysis inferred contiguous range expansion for the three-step nesting level for this clade, owing to the small dispersal and displacement values for the clade as an internal clade, but this is more likely to be a result of haplotype extinction rather than range expansion. Lineage sorting through localised haplotype extinction would be likely to have a strong influence on haplotype distribution in south-western Australia where climatic influences during the Pleistocene would have caused repeated contraction and expansion of both population and species range, leading to complex patterns at lower levels in phylogenies. The nucleotide divergence between the forest sublineages suggests separation 1.35 million years ago, which is similar to the divergence times between lineages identified in the previous studies of widespread species occupying the semi-arid zone (Byrne et al. 2002, 2003; Byrne and Hines 2004). This suggests that the divergence of the forest lineages occurred in response to historical factors at a similar time to the divergence of lineages in other species, indicating that climatic instability may have had a significant impact in the mesic area as well as in the semi-arid zone. The coastal lineage (Clade II/4-2) showed little variation in comparison to the forest clade, with only five of the total of 18 haplotypes. There was a geographic pattern with the ssp. marginata populations in the northern part of the Coastal Plain differentiated from the ssp. elegantella populations at the edge of the Scarp. However, the nested clade analysis did not identify this as a significant geographic association of haplotypes, possibly because of the limited geographic spread of the clade. Compared with the ssp. marginata populations in the northern part of the Coastal Plain that showed no haplotype variation, the ssp. elegantella populations showed considerable variation with four haplotypes present in the 10 sampled trees. In comparison to the populations of ssp. elegantella that formed a separate clade within the coastal lineage, the populations of ssp. thalassica were not genetically differentiated from other populations within the forest

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lineage. Although the clustering of populations of spp. elegantella indicate common ancestry in this subspecies, the haplotypes of both ssp. thalassica and ssp. elegantella were nested within those of ssp. marginata, and the cp phylogeny does not support differentiation of the subspecies. This is consistent with the results of the nuclear RFLP study (Wheeler et al. 2003) that also showed little differentiation between the subspecies, and indicates that the morphological differences are not indicative of differentiation across the genome. The outlying populations of Jilikan Rock and Katanning showed no differentiation from populations within the main distribution of E. marginata, suggesting that the isolation of these populations has been recent. This is also consistent with the lack of differentiation of these populations in the nuclear study (Wheeler et al. 2003). The genetic similarity of the outlier populations to those in the main range supports the hypothesis of recent contraction of the E. marginata (jarrah forest) range (Churchill 1968), which may be caused by recent climatic change resulting in increased aridity in the Holocene. The level of diversity and differentiation identified in the cp genome is greater than that detected in a study of the nuclear genome (Wheeler et al. 2003). The patterns in the two studies reflect differing time scales. The cpDNA study reflects influences over longer historical time frames (potentially millions of years) owing to the low rate of evolution, low effective population size, uniparental inheritance and low dispersal though seed (Schaal et al. 1998). Patterns of diversity in the nuclear genome reflect influences over more recent time frames owing to faster rate of evolution, higher effective population size, recombination and gene flow through pollen dispersal. Hence, the nuclear study did not detect the differentiation of lineages on the Coastal Plain and in the forest because of the impacts of these factors in more recent historical time following expansion of the species after fragmentation events in earlier times. The sample of E. staeri collected from Mt Frankland did not share any haplotypes with its close neighbour, E. marginata, at Mt Frankland, even though the samples were collected only 7 km apart. This suggests that there has been no recent hybridisation between the two species in this area. However, affinity between E. staeri and E. marginata is evident since the haplotype of E. staeri is nested within the E. marginata forest lineage. This affinity could be a result of common ancestry or could reflect historical hybridisation, and it is difficult to separate these explanations as they result in similar genetic patterns (Wendel and Doyle 1998). The same samples of E. staeri were used in a study of nuclear variation and the two species showed clear differentiation in the nuclear genome (Wheeler et al. 2003), suggesting that common ancestry is a more likely explanation for the affinities of haplotypes detected in the cp genome. Common ancestry is also a likely explanation as the two species are


sister species in the morphological cladogram of Ladiges et al. (1987). The differentiation between E. todtiana and E. marginata detected in this study also indicates that hybridisation and introgression are not significant factors in the evolution of these western monocalypt species, since one of the collections of E. todtiana was made at a site where it co-occurred with E. marginata. Conclusion The patterns seen here in the chloroplast DNA of E. marginata reflect the evolutionary history of the region. The separation of the Coastal Plain from the Darling Plateau by the uplifting along the Darling Scarp has led to divergence of two lineages in E. marginata. There was also structuring in cpDNA patterns within the lineage distributed throughout the forest region, indicating that the influence of climatic changes in the mid-Pleistocene was widespread in south-western Australia and not confined to the semi-arid region. It would be expected that other widespread species that exist in the mesic south-west of Western Australia would show similar patterns of separation between the coast and the main forest areas, owing to the major geomorphological boundary of the Darling Scarp, and it would be interesting to determine whether such species also show influence of fragmentation through the forest region, owing to historical climatic influences. There are implications for selection of E. marginata germplasm for seed orchards arising from this work since the isolation of populations on the Coastal Plain would be likely to result in adaptive differences between the populations on the Coastal Plain and those in the forest. Indeed, it has been observed that germplasm collected from the Swan Coastal Plain has poor growth in comparison to other provenances when planted in trial sites on the Darling Plateau (Alcoa World Alumina Australia, unpubl. data). Acknowledgments We thank Bronwyn Macdonald for technical assistance and M. French for assistance with field identification. M. A. W. was in receipt of an APA(I) scholarship while undertaking this work. References Avise JC (2000) ‘Phylogeography. The history and formation of species.’ (Harvard University Press: Cambridge, MA) Bowler JM (1982) Aridity in the late Tertiary and Quaternary of Australia. In ‘Evolution of the flora and fauna of arid Australia’. (Eds WR Barker, PJM Greenslade) pp. 35–45. (Peacock Publications: Frewville, SA) Brooker MIH, Hopper SD (1993) New series, subseries, species and subspecies of Eucalyptus (Myrtaceae) from Western Australia and from South Australia. Nuytsia 9, 1–68. Byrne M, Hines B (2004) Phylogeographic analysis of cpDNA variation in Eucalyptus loxophleba (Myrtaceae). Australian Journal of Botany 52, 459–470. doi: 10.1071/BT03117


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Manuscript received 13 May 2005, accepted 8 September 2005

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