CARACTERÍSTICAS AMBIENTALES

8.1. Introduction

The south-west of Western Australia is one of the most botanically diverse Mediterranean regions in the world, with roughly 5700 species, of which 52.5% are endemic (Beard, 2000). The nutrient-poor soils and the long period of geomorphologic stability are thought to be the main reasons for the high rate of endemism (Beard, 2000). Sclerophyllous plants were common in the Eocene period, much earlier than in the eastern part of Australia, because of the increasing aridity in the region (Hill et al., 1999; Hopper et al., 1996). Separation of the Australian and Antarctic plates proceeded in the Eocene (Playford et al., 1976). This was followed by epeirogenic uplift of the Yilgarn Block and at least some parts of the Perth Basin during the Tertiary which has possibly continued until the present (Kendrick et al., 1991; Murray-Wallace, 2002; Playford et al., 1976). In turn, the Darling Plateau (as part of the Yilgarn Block) was uplifted approximately 300m along with extensive formation of lateritic soils (Hopper, 1979; Playford et al., 1976). The area was tectonically stable compared to other parts of Australia and the world during the Pleistocene (Murray-Wallace, 2002). The Darling Scarp is situated on one of the major crustal fault lines of the earth, the Darling Fault line, extending north-south for over 1000km with a spread of up to 15,000 metres (Playford et al., 1976). The Pleistocene period saw the region undergo several climatic changes with the more rapid northward movement of the Australian continent, but without the volcanic

activity or periods of glaciation that occurred in the eastern part of the continent (Hope, 1994; Hopper, 1979). From 20,000 to 10,000 years BP the area experienced the onset of more arid conditions, and until 14,000 BP intense widespread aridity affected much of Western Australia (Wyrwoll, 1979). From 10,000 years BP until the present, there have been several climatic changes, with wetter periods being experienced when sea levels and sea surface temperatures rose during minor interglacials, and drier conditions during glaciation, although no actual glaciation occurred in south-west Western Australia (Wyrwoll, 1979). Coastal landforms were in a state of flux during this time as a result of fluctuating sea levels (Hopper, 1979).

Eucalyptus marginata inhabits the more mesic areas of south-western Australia and has had greater stability in its environment than have the more semi-arid species of the region.

The Eucalyptus marginata forest has a less diverse flora than other parts of the south west of Western Australia, such as the semi-arid lands or sandy heathlands, possibly because it occupies much of the more mesic areas. It often grows in almost monospecific stands with a low floristic diversity understorey on the laterite soils of the Darling Plateau. The species probably had a much larger distribution during wetter periods, and if the rainfall increased by 75mm per annum, all the present outliers would be included within the main distribution (Churchill, 1968). Pollen records show that it underwent a substantial increase in distribution from 500 BC to 1400 AD (Churchill, 1968). There are three subspecies of E. marginata recognised on morphological grounds (Brooker and Hopper, 1993); subspecies marginata is a tall forest tree with dark green leaves, subspecies thalassica has a weeping habit with

blue-green leaves, and subspecies elegantella is a small tree with small, narrow, olive-green leaves. Distributions of the subspecies are shown in Figure 7.1 (Chapter 7).

The geological history of the area would be expected to have some impact on the evolution of the regional flora. Fragmentation has occurred in south western Australian species, particularly in the semi-arid areas of the region, and particularly species with restricted distributions, due to the fluctuating climate and gradual tectonic movement. Widespread species have also undergone fragmented periods during times of increased aridity, or uplifting of the Darling Plateau. This differs from the kind of catastrophic events seen in eastern Australia caused by glaciation and volcanic activity that caused rapid geographic barriers separating some species, and bringing others into close proximity to each other. Studies in eastern Australian eucalypts have found that many species can share haplotypes, with a geographic pattern evident (Freeman et al., 2001; Jackson et al., 1999; McKinnon et al., 1999;

2001; Steane et al., 1998; Stokoe pers. comm.). This has been attributed to hybridisation and introgression, similar to the processes seen in the European oaks (Ferris et al., 1993), and the Tellima grandiflora complex in California (Soltis et al., 1992). Isolated remnants that were confined to glacial refugia, in California, during glacial periods are thought to have hybridised. Tellima grandiflora showed a pattern of very little nuclear differentiation but well defined chloroplast differentiation, and it is possible that this occurred when one of the clades hybridised with a species of Mitella, which is a separate genus but part of the same Heuchera group of genera.

Eastern Australia has also seen periods of glaciation that similarly could have caused isolation of sympatric eucalypt populations of different species in glacial refugia.

The analysis of chloroplast DNA can be used to assess the influence of historical processes, since the rate of evolution in the chloroplast genome is approximately one fifth of that of the nuclear genome (Ennos et al., 1999, Soltis et al., 1992). Studies of plant phylogenies, including eucalypts, have been successfully undertaken using RFLP chloroplast markers (Butcher et al., 2002; Byrne, 1999; Byrne et al, 1994;

Byrne et al., 1995; Byrne et al., 1998; Hines and Byrne, 2001; Jackson et al, 1999;

McKinnon et al., 1999; Reiseberg, 1998). Cp DNA studies can provide insight into complex evolutionary histories that are present in the south-west Australian flora, and are able to distinguish patterns that exist between widely divergent taxa, implying that the patterns are from common geomorphologic or climatic events (Byrne et al., 2002; Byrne et al., 2003; Byrne and Hines, 2003). These patterns are indicative of historical fragmentation that was caused by increasing, but fluctuating aridity in the region, and have also identified relictual taxa in species with restricted or disjunct distribution (Byrne et al., 1999; Byrne et al., 2000; Elliot and Byrne, 2003a).

South-western Australia has not been affected by large scale glaciation or volcanic activity, and climatic changes have been limited to changes in sea levels, temperatures and rainfall, causing expansion and contractions of distributions.

Studies of other widely distributed species in this region, such as Acacia acuminata and Santalum spicatum have identified phylogeographic patterns (Byrne et al., 2002;

Byrne et al., 2003), with the separation of lineages corresponding with historical fragmentation. These two species have similar distributions but are from different families, so it is likely that the phylogeographic patterns are caused by historical events, rather than genetic processes limited to the taxa concerned. Some south-western eucalypts also show similar phylogeographic patterns. CpDNA studies on south western Australian eucalypts, such as Eucalyptus loxophleba and E.

angustissima have provided more evidence that the fragmented species have similar evolutionary patterns to species that have similar distributions, but are not taxonomically closely related (Byrne and Hines 2003; Elliot and Byrne, 2003a). This suggests that incomplete lineage sorting (Wendel and Doyle, 1998) is a more likely process to have taken place in this area than hybridisation, which is a species specific process.

Chloroplast DNA studies can show patterns identifying influences of historical processes. It is therefore valid to use cpDNA studies for phylogeographic assessments in a widespread species such as Eucalyptus marginata since widespread species can also have undergone historical fragmentation in the semi arid areas of the south western Australian region. Climatic fluctuations in the mesic areas, such as the distribution area for E. marginata were not as significant as in the arid zone. Little differentiation was identified in the nuclear genome of E. marginata (Chapter 7) indicating that there may have been good gene flow during the past few thousand years, and that the species may be approaching a gene-flow/drift equilibrium (Hutchinson and Templeton, 1999). Investigation of the chloroplast DNA was undertaken to determine whether E. marginata has been influenced by historical

processes due to fluctuations during the Pleistocene, in common with many other species in the semi-arid areas of the south western region of Australia, that show a higher level of structuring in the nuclear genome.

8.2. Materials and Methods

8.2.1. Plant material

Collections were made as for Chapter 7 and in addition, two populations of E.

todtiana were used as an outgroup (Figure 8.1). The DNA was extracted from five individuals for each population as outlined in Byrne et al. (1998), as for Chapter 7.

The genomic DNA samples were digested with six enzymes, Bcl I, Bgl II, EcoR I, Xba I, EcoR V, and Hind III, Southern blotted, and hybridised with six heterologous petunia probes, which cover the single copy regions of the chloroplast genome. The probes used were P1, P3, P4, P6, P8, and P10 (Sytsma and Gottlieb, 1986), and one tobacco probe, pTBa1 (Suguira et al., 1986). Restriction digestion and hybridisation were as described in Byrne and Moran (1994), and probe inserts were amplified then labelled with ³²P using the random priming method, as for Chapter 7.

Perth

Bunbury

Albany Darling Scarp Swan Coastal Plain

Figure 8.1. Map showing collection locations and distribution of clades identified by cpDNA variation for Eucalyptus marginata. Represents E.

marginata trees in the main forest clade, with the subclades grouped, represents E. marginata trees in the coastal clade, represents the site for E. staeri,

and represents sites for populations of E. todtiana. E. marginata sites are as for Figure 7.1.

8.2.2. Data analysis

Mutations were identified into length or restriction site mutations, and from this haplotypes were identified. Fragment patterns for consecutive cp probes were compared to ensure that each mutation 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 mutation. Nucleotide diversity was calculated for restriction site mutations using HAPLO (Lynch and Crease, 1990), and partitioned within and between populations. Haplotype diversity was calculated with Nei’s gene diversity measures (Nei, 1978) for haplotypes in the total sample and in each population.

A parsimony analysis characterised by presence or absence of each mutation was undertaken with the haplotypes using PAUP (Swofford, 1991). Bootstrap analysis used 100 replications and heuristic search, with TBR branch swapping and MULPARS on. Eucalyptus todtiana, which belongs to the same subgenus as E.

marginata, was defined as the outgroup.

8.3. Results

8.3.1. Polymorphism in cpDNA

A total of 11 site mutations, 30 length mutations and one missing band (Table 8.1) were detected, distributed over 17 haplotypes (Table 8.2) for Eucalyptus marginata, with 11 mutations (an extra two length mutations as well as those shared with E.

marginata) and one haplotype detected for E. staeri. Two individuals were removed from the analysis, one from Mount Frankland, and the other from Jilakin Rock, because they were not E. marginata as thought when collected.

Table 8.1. Mutations observed in the cpDNA study of Eucalyptus marginata.

Abbreviations for populations are as follows: Mt. Fr – Mount Frankland, Kat – Katanning, Nan – Nannup, Jil. R – , Jilakin Rock, Perup – Perup, Ju. F – Julimar Forest, Nth. Dand – North Dandalup, Welsh Rd – Welshpool Road, Jar – Jarrahdale, Dw – Dwellingup, Br – Bridgetown, Col – Collie.

No. Probe/enzyme Mutation type

Fragment sizes (kb)

Populations and individual numbers

1 P10/EcoR I length 1 v. 1.1 Mt. Fr 1-2, Kat 1-5, Nan 1, Jil. R 1-5,