All Content © CRC for Forestry 2007

Stringy bark diversity study winds up: New data on genetic diversity in Eucalyptus obliqua on the island of Tasmania

Justin A Bloomfield1, Paul Nevill2,3, René E Vaillancourt1, Dorothy A Steane1,4, Brad M Potts1

1School of Plant Science and Cooperative Research Centre for Forestry, University of Tasmania, Private Bag 55, Hobart, Tasmania 7001, Australia
2School of Forest and Ecosystem Science and Cooperative Research Centre for Forestry, University of Melbourne, Parkville, Victoria 3010, Australia 
3Current address: Botanic Gardens and Parks Authority, Kings Park and Botanic Gardens and School of Plant Biology, The University of Western Australia, Western Australia, Australia
4Author for correspondence: Dorothy.Steane@utas.edu.au


Fig1_m

Figure 1. This statistical parsimony tree shows relationships between chloroplast haplotypes found in E. obliqua in Tasmania. The size of a circle represents the relative frequency of a haplotype; each haplotype is represented by a unique colour; haplotype numbers correspond to haplotypes identified by Nevill et al. (unpubl.; Haplotypes 19 - 42) and new haplotypes determined in this study (Haplotypes 45 - 55); each line joining two haplotypes indicates a putative cpDNA mutation event (i.e., the insertion or deletion of a single basepair at a microsatellite locus).

Eucalyptus obliqua dominates much of the wet sclerophyll forest managed for forestry purposes by Forestry Tasmania (Neyland et al. 2009). Clearfelling followed by high intensity burning and aerial sowing has been the dominant silvicultural system used for E. obliqua forests in Tasmania since the 1960s (Hickey and Wilkinson 1999).  However, aggregated retention systems, where patches of intact forest are retained within a harvested coupe, have been adopted recently as standard practice for
Fig2_m

Figure 2. Nineteen chloroplast haplotypes were found in 65 Tasmanian E. obliqua populations. Pie charts indicate the haplotype composition of each sampled population; regions demarcated by green lines are Forestry Tasmania's seed zones. Within the black line there is no putative ancestral haplotype (Haplotype 20) and the region is dominated by Haplotype 24 (red); the broken line indicates that the suggested boundary defining this region is bisecting seed zones; the round dotted line in the south demarcates Forestier Peninsula which is dominated by Haplotype 30.

harvesting (retaining contiguous and/or free standing patches) in the majority of old growth wet Eucalyptus forests on State Forest land  (Forestry Tasmania 2009; Neyland et al. 2009). Regardless of silvicultural system, coupes are artificially re-sown, usually using seed collected on site before harvesting. However, in cases where this is not possible, Forestry Tasmania has developed a seed zone system based on geography and environmental attributes (e.g. altitude, rainfall and geology) to guide off-site seed transfer (Forestry Tasmania 2010). When seed is not available from within the same seed zone, the emphasis is on transferring seed from another seed zone that has similar environmental attributes, to obtain seed as closely genetically adapted to the target environment as possible (Forestry Tasmania 2010).

We have been studying the patterns of molecular genetic variation in E. obliqua to determine whether there are further considerations which need to be taken into account to better conserve local gene pools and patterns of diversity that exist in native forest tree gene pools across Tasmania. Reciprocal field trials with E. obliqua have identified local differentiation in adaptive traits occurring at a fine scale (e.g. within coupe or seed zone) (Wilkinson 2008; Strich 2006). However, despite the phenotypic diversity observed in E. obliqua (Nicolle 2006), our recent molecular study based on putatively neutral nuclear microsatellites (which reflect combined seed and pollen dispersal) showed very little broad-scale differentiation across Tasmania (Bloomfield et al. 2011b). This result suggests that the seed zone system is conservative for E. obliqua and environmental matching is likely to be the key consideration for seed transfer.  However, there was a limited degree of spatial structure evident in the chloroplast DNA (cpDNA), which may reflect factors such as historic migration routes or refugia.  This genetic structuring can be overlaid on the seed zone system to better maintain the natural pattern of genetic variation in E. obliqua.  CpDNA often shows more spatial structuring than nuclear DNA because it is haploid (does not undergo recombination), maternally inherited and dispersed only by seed. We increased the number of populations in our cpDNA survey so that we could refine the delineation of the spatial structure observed in the cpDNA in E. obliqua, so this new layer of information could be integrated into the Forestry Tasmania seed transfer guidelines.

Fig3_m

Figure 3. Contour map of chloroplast DNA haplotype richness in E. obliqua across Tasmania.  Haplotype richness was calculated using FSTAT (Goudet 2001). Individual values indicate haplotype richness per population (rarefied to a sample size of four); a value of 1.0 indicates that a population is fixed for a single haplotype.  Red colouring on the map indicates regions of relatively high haplotype richness (i.e., more haplotypes per region), while blue indicates regions of lower haplotype richness.

An additional 34 populations of E. obliqua were sampled from throughout Tasmania and their cpDNA microsatellite data combined with that of Bloomfield et al. (2011b).  In total, 5 – 10 individuals from 65 populations have now been analysed using cpDNA microsatellites, producing 19 unique cpDNA haplotypes in Tasmanian E. obliqua (Table 1). The statistical parsimony tree (Figure 1) displays the relationships among the 19 Tasmanian cpDNA haplotypes identified. While nuclear DNA microsatellites showed little spatial structure, presumably because pollen flow is extensive in this species (Bloomfield et al. 2011b), some structure was evident in the cpDNA (Figure 2).  We found an area in central eastern Tasmania, also encompassing the Tasman Peninsula, which lacks the putative ancestral haplotype (blue haplotype, haplotype 20; see outlined area in Figure 2). The suggested boundary drawn in Figure 2 to enclose this area includes many of the eastern Tasmanian seed zones but may bisect up to three seed zones (broken line in Figure 2). Furthermore, the Forestier Peninsula (see round-dotted line in Figure 2) has a high frequency of a well-derived haplotype (Haplotype 30) which is not found anywhere else and is fixed in two populations. Other similarly derived haplotypes (Haplotypes 52 and 53) are only found in single populations and are not fixed in their respective populations.

The areas of greatest haplotype richness for Tasmanian E. obliqua are in the south-east, north and north-east, where (except for the Launceston area) glacial refugia have been postulated (Figure 3).  Furthermore, an area characterised by distinct haplotypes was found in the central-east. While trials have not revealed any adverse fitness effects of transferring seed across seed zones of similar environment (e.g. Forestier Peninsula and Lune River - Wilkinson 2008; Strich 2006), the natural patterns of genetic variation in E. obliqua would be better preserved with an additional guideline to prioritise seed zone transfers between similar environments within the same major chloroplast DNA regions as indicated in Figure 2 (as defined by primarily haplotypes 24 and secondarily haplotype 30).


Acknowledgements
We wish to thank David McElwee for assistance with field sampling, Sasha Wise for assistance with laboratory work and Mark Neyland and Lachie Clark for helpful discussion.

References
Bloomfield J, Nevill P, Vaillancourt RE, Steane DA, O’Reilly-Wapstra J, Potts BM (2011a) Genetic diversity in Eucalyptus obliqua on the island of Tasmania.  Poster presented at Annual Conference of Ecological Society of Australia, Hobart, Tasmania, 21-25 November 2011.

Bloomfield JA, Nevill P, Potts BM, Vaillancourt RE, Steane DA (2011b) Molecular genetic variation in a widespread forest tree species Eucalyptus obliqua (Myrtaceae) on the island of Tasmania. Australian Journal of Botany 59: 226-237.

Forestry Tasmania (2009) A New Silviculture for Tasmania’s Public Forests: a review of the 610 variable retention program. Forestry Tasmania, Hobart

Forestry Tasmania (2010) Eucalypt seed and sowing. Native Forest Silviculture Technical Bulletin No. 1, Forestry Tasmania, Hobart.

Goudet J (2001) FSTAT, a program to estimate and test gene diversities and fixation indices.  Version 2.9.3 . Available at http://www2.unil.ch/popgen/softwares/fstat.htm (last accessed 3 March 2011).

Hickey JE, Wilkinson GR (1999) The development and current implementation of silvicultural practices in native forests in Tasmania. Australian Forestry 62: 245–254.

Neyland M, Hickey J, Beadle C, Bauhus J, Davidson N, Edwards L (2009) An examination of stocking and early growth in the Warra silvicultural systems trial confirms the importance of a burnt seedbed for vigorous regeneration in Eucalyptus obliqua forest. Forest Ecology and Management 258: 481-494.

Nicolle D (2006) A classification and census of regenerative strategies in the eucalypts (Angophora, Corymbia and Eucalyptus – Myrtaceae), with special reference to the obligate seeders. Australian Journal of Botany 54: 391-407.

Strich P (2006) Local adaptive differentiation within Eucalyptus obliqua. Honours Thesis, University of Melbourne.

Wilkinson GR (2008) Population differentiation within Eucalyptus obliqua: implications for regeneration success and genetic conservation in production forests. Australian Forestry 71: 4-15.


Biobuzz issue fifteen, December 2011