Eucalypts for Ornamental Horticulture: Selection, Interspeciftc Hybridisation and Postharvest Testing
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Eucalypts for Ornamental Horticulture: Selection, InterspecifTc Hybridisation and Postharvest Testing
Kate Louise Delaporte
Playford Memorial Trust Scholar in Ornamental Horticulture
submitted in fulfilment of the requirement for the degree of Doctor of Philosophy
Department of Horticulture, Viticulture and Oenology w ai'le ^î:T:::';ï:TJ rn s'Íi'[u'Íe
September 2000 Frontspiece Arrangemeft of Eucalypfirs buds, flowers and leaves (8. kruseana, E.Iesouefü, E. tetragona and E. youngiana) collected from the Laidlaw Plantation, October, 1999. Table of Contents
Table of Contents
Abstract I Declaration and authorisation of access to copying üi Acknowledgements iv Dedication v List of Tables vi List of Figures vüi List of Plates xüi Glossary of Terms xiv
Chapter One Introduction 1 l.l. Introduction 1 1.2. Native plants and ornamental horticulture 1.2.1. Introduction 2 1.2.2. Australian natives for cut flowers and foliage 2 1.2.3. Development of ornamental Eucalyptus species 2 1.2.4. Native ornamental breeding and selection 3 1.3. Aims 5
Chapter Two Literature Review 6 2.1. The genus Eucalyptus 6 2.2. Taxonomy 2.2.1. Introduction 7 2.2.2. Taxonomy and Eucalyptus 8 2.3. Interspecifichybridisation 2.3.1. Introduction 9 2.3.2. Interspecific hybridisation tn Eucalyptus 10 2.4. Molecular Biology 2.4.1. Introduction t2 2.4.2. RAPD-PCR and Eucalyptus r3 2.5. Postharvestphysiology 2.5.1. Introduction t4 2.5.2. Postharvest treatment of Eucalyptus r6
Chapter Three Plant Material 18 3.1. Introduction 18 3.2. Species used in the study and their natural distribution 18 3.3. Selection criteria for species chosen 34 3.4. Location of material 3.4.1. The Waite Arboretum 47 3.4.2. The Monarto Woodland 48 3.4.3. The Laidlaw Plantation 49 Trble of Confenf-s
Chapter Four Selection within species for cut bud and flower production 52 4.I. Abstract 52 4.2. Introduction 53 4.3. Materials and methods 4.3.1. Plant material 55 4.3.2. Planting design 55 4.3.3. Pre-planting treatment 55 4.3.4. Irrigation 56 4.3.5. Fertiliser 56 4.3.6. Weed control 56 4.3.7 . Plant measurements 56 4.3.8. Weather data 56 4.4. Results 4.4.L. Between species comparison 4.4.1.1. Growth 59 4.4.1.2. Flowering 60 4.4.1.3. Pest susceptibility 60 4.4.2. Selection of superior individuals within species 60 4.4.3. Summary of each species 64 4.4.4. Further testing 67 4.5. Discussion 74 4.6. Conclusion 75
Chapter Five Eucalyptus .Urrbrae Gem': Parentage determination through RAPD analysis 76 5.1. Abstract 76 5.2. Introduction 76 5.3. Materials and methods 5.3.1. Plant material 78 5.3.2. DNA extraction 8l 5.3.3. RAPD analysis 83 5.3.4. Hierarchical distance analysis 84 5.3.5. Non-hierarchical distance analysis 85 5.4. Results 5.4.1. RAPD analysis 87 5.4.2. Hierarchical distance analysis 88 5.4.3. Non-hierarchical distance analysis 9t 5.5. Discussion 94 5.6. Conclusion 96
Chapter Six Eucalyptzs 6Urrbrae Gem': Parentage determination through morphological analysis 97 6.1. Abstract 97 6.2. Introduction 98 6.3. Materials and methods 6.3.1. Plant material 98 6.3.2. Adult morphology t02 Tshle of Confenfs
6.3.3. Controlled pollination t02 6.3.4. Seedling morphology 103 6.3.5. Data analysis to4 6.4. Results 6.4.1. Adult morphology analysis 108 6.4.2. Controlled pollination seed data tL6 6.4.3. Seedling morphology 116 6.5. Discussion t25 6.6. Conclusion 13r
Chapter Seven InterspecifTc hybridisation: E. macrocørpa, E. pyriformis and E. youngianø 132 7.1. Abstract 132 7.2. Introduction r33 7.3. Materials and methods 7.3.1. Plant material 137 7.3.2. Controlled pollination 137 7.3.3. Seed data analysis 138 7 .3.4. Seedling morphology r39 7.3.5. Data analysis 140 7.4. Results 7.4.1. Controlled pollination seed data 140 7.4.I.1. Between species comparison 140 7 .4.1.2. Within species comparison 143 7 .4.2. Seedling morphology analysis 7 .4.2.1. Summary of all crosses 148 7.4.2.2. Summary of each cross 158 7.5. Discussion 183 7.6. Conclusion 188
Chapter Eight Interspecific hybridisation: subgenus Symphy omyrtus, sections
B is e ctaria and Adnatarin 189 8.1. Abstract 189 8.2. Introduction 190 8.3. Materials and methods 8.3.1. Plant material 192 8.3.2. Controlled pollination 192 8.3.3. Seed data analysis 192 8.3.4. Seedling morphology 194 8.3.5. Data analysis 194 8.4. Results 8.4.1. Seed data analysis 195 8.4.2. Seedling morphology analysis 8.4.2.1. E. gillii as the female parent 198 8.4.2.2. E. socialis as the female parent 207 8.4.2.3. Summary of crosses 2t5 8.5. Discussion 217 8.6. Conclusion 2t9 Table of Contents
Sect. Bis¿ctaria and Adnataria E. gillii dendrograms 220 Sect. Bis¿ctaria and AdnatariaE. gillü MDS ordinations 228 Sect. Bisectaria and AdnatariaE. socialis dendrograms 235 Sect. Bisectaria and AdnatariaE. socialis MDS ordinations 240
Chapter Nine Postharvest yase life of four flowering Eucalyptus species 248 9.1. Abstract 248 9.2. Introduction 249 9.3. Materials and methods 9.3.1. Plant material 253 9.3.2. Harvest 253 9.3.3. Vase life of flowers and leaves 254 9.3.4. Experimental treatments 9.3.4.1. Continual sucrose 254 9.3.4.2. Sucrose pulsing and cold dry storage 254 9.3.5. Statistical analysis 255 9.4. Results 9.4.1. Continual sucrose 256 9.4.2. Sucrose pulsing and cold dry storage 263 9.4.3. Observations of other species 267 9.5. Discussion 267 9.6. Conclusion 2t2
Chapter Ten General Discussion 274
Bibliography 280
Appendix 301 1.1 Anova tables for section 4.4. (Table 4.L.) 30r r.2. Anova tables for section 7.3. (TablesT .4 to 7 .7) 302 1.3. Anova tables for section 9.4. (tables 9.1. to 9.4) 2.r. Sect. Bisectaria and AdnatariaE. giUii PCC graphs 309 2.2 Sect. Blsectaria and AdnatariaE. socialis PCC graphs 322 3.1. Presentations 337 3.2. Posters 338 3.3. Papers 338 Abstract Abstract
Of nine ornamental Eucalyptøs species, E. lesouefü was the fastest growing, while E
yalatensís and E. youngiana were the slowest. Seventeen plants from three species showed
superior characters, specifically bud size, shape and colour, precocity and floriferousness.
The parentage of the ornamental hybrid Eucalyptus 'Urrbrae Gem' was investigated using
RAPD-PCR, adult morphology and seedling morphology. The results were analysed using
hierarchical (UPGMA) and non-hierarchical distance methods (multidimensional ordination
and minimum spanning tree analysis). The female parent was known to be E erythronema
var. erythronema, but previous opinion placed the male parent as either E. stricklandii or E.
gomphocephala. All results indicated that E. stricklandü is the male parent of E. 'Urrbrae
Gem'.
Crosses between Eucalyptus macrocarpa, E. pyriformis and E. youngiana were conducted,
with all combinations producing fertile seed. The 166 seedlings, when measured for a range
of leaf and stem characters at three different nodes, showed strong evidence of intermediacy
between parents, with l.2Vo grouped with the male parenf, 94.67o clustered between the parent
species, and the remaining 4.2Vo with the female parent.
Eucalyptus gillii and E. socialis were used as female parents in a controlled pollination
program using pollen collected from sixteen species. The 425 seedlings produced were
measured at three months for fifteen seedling characters. The UPGMA dendrograms showed
225 seedlings clustered with neither the male nor female parent seedlings, suggesting intermediacy. The remaining 200 seedlings clustered with the female parent. The multivariate analysis supported the UPGMA results, with the ordination point clusters
I Ahsfrsef remaining consistent with the dendrogram groupings.
Continuous application of 0.5 to 5.0Vo sucrose in the vase solution reduced vase life of E.
tetragona flowers by three days, while vase life of E. youngiana was not affected. Pulsing with 0.5 to LOTo sucrose, in conjunction with cold dry storage at 3oC for one to two weeks, had no effect on vase life of E. tetragonø flowers. Preliminary trials with E forrestiana
subsp. forrestiana and E stoatei indicate that pulsing with sucrose at 2.0 to 5.0Vo may be beneficial to vase life. Sucrose did not increase flower opening after harvest of any species tested, nor did citric acid affect vase life. Significant differences in vase life were found between plants within a species.
It Declaration
Declaration and authorization of access to copying
This work contains no material which has been accepted for the awa¡d of any other degree or diploma in any university or other tertiary institution and, to the best of my knowledge and beliel contains no material previously published or written by another person, except where due reference has been made in the text.
I give consent to this copy of my thesis, when deposited in the University Library, being available for loan and photocopying.
Signed: Date: flru0
tu A cknowledsements
Acknowledgements
This work was conducted with support from the Playford Memorial Trust Scholarship in
Horticulture, with additional support from the Australian Landscape Trust and the Bookmark
Biosphere Reserve Trust. I would like to thank my supervisors, Professor Margaret Sedgley,
Drs Graham Collins, John Conran and Andreas Klieber, for their wisdom and advice; Jennifer
Cashmore AO and Don Laidlaw AO and members of the Playford Memorial Trust Board for their enthusiasm; Denis Tricks and Natalie Peate for their support and encouragement; the staff of the Waite Campus I & CC for photography and printing; Dr Jennifer Gardner for information and access to the Waite Arboretum; Jeff Fairlamb and State FloraÆorestrySA for information on, and access to, the Monarto Woodland; and the staff of the Department of
Horticulture, Viticulture and Oenology including Orchard staff. On a more personal note, I would like to thank Michelle Wirthensohn, Alison Kellow and Cassie Collins and others in the Department for their friendship and encouragement; Peta Adams and Richard Glatz for their help with pollinations; Kirsty Neaylon for her assistance in seedling morphology measurements and analysis; and Val Morris, Louise Gooding, Noel Lothian and Alan Gray for their unique contributions to this work. A very special thanks to my mother, Joan, for her unwavering faith in me, my family and friends for helping me with priorities and keeping me sane and last, but not least, Dave Sinclair, for absolutely everything.
tv Dedication Dedication
To my father, who would be so proud,
To Mr Stuckey, for his inspiration, and
To Sir Thomas Playford, for his example.
Y List of Tables
List of Tables
Table 3.1. Relatednes s of Eucalyplzs species investigated. t9 Table 3.2. Leaf characters of all species investigated. 20 Table 3.3. Inflorescence characters of all species investigated. 22 Table 3.4. Capsule characters of all species investigated. 24 Table 3.5. Cultivation characters of species investigated. 26 Table 3.6. Use in project and distribution information for all species investigated. 28 Table 4.1. Summary of data for nine species in the Laidlaw Plantation. 59 Table 4.2. Season of appearance of macroscopic buds for nine species in the Laidlaw Plantation. 63 Table 4.3 Allocation of individuals within species to four primary selection categories. 64 Table 4.4. Eucalyptus species with ornamental merit suitable for further testing. 68 Table 5.1. Eucalyptus species located near the female parent of E. 'Urrbrae Gem', Waite Arboretum, 1955. Applies to Figure 5.1. 79 Table 5.2. Individuals used in the E 'Urrbrae Gem' analysis. 8l Table 5.3. Species used in the RAPD analysis. 88 Table 6.1. Characters used in phenetic analysis (seedling and adult) with codes. 106 TabLe 6.2 Adult morphological characters of E. 'Urrbrae Gem' and putative relatives (from Table 5.1), with correlation coefficient (r2), Kruskal-Wallis f/ statistic' and probability (P). 113 Table 6.3 Seed data from E. erythronema var. erythronema x E. stricklandii and E. erythronema vaÍ. erythronema x E. gomphocephala controlled pollinations and open pollinations of putative parent species. 117 Table 6.4. Seedling morphological characters of E. 'Urrbrae Gem' and putative relativeõ (from Tabie 6.1) with correlation coefftcient 1r2¡, Kruskal- Wallis Il statistic and probability (P). r20 Table 6.5. Number of seedlings of E. 'Urrbrae Gem' and putative relatives in each dendrogram cluster (Figure 6.4). t2r Table 7.1. Plants used in controlled pollination matrix from subgenus
Sy mp hy omyrT¿¿s, sect. B i s e c t ari a, ser . M ac r o c arp ae . 138 Table7.2 Characters and codes used in phenetic analysis (seedling), measured at nodes 3, 5 and 7, three months after germination. r39 Table 7.3 Summary of flower, capsule, seed and seedling data for interspecific, intraspecific and open pollination of E. macrocarpa, E. pyriformis and E. youngiana. t4t TabLe7.4. Effect of male parent on controlled pollination of E macrocarp& 144 Table 7.5. Effect of male parent on controlled pollination of E. pyriformís. 145 Table 7.6. Effect of male parent on pollination of E youngiana. 146 Table7.7. Comparison of female parent on controlled pollination of E. macrocarpa, E. pyriþrmis and E. youngíana. t47 Table 7.8 Seedling morphological characters of seedlings from E. macrocarpa and putative parents. 149 Table 7.9. Seedling morphological characters of seedlings from E pyriformis and putative parents. 151
vt List of Tables
Table 7.10. Seedling morphological characters of seedlings from E. youngiana and putative parents. 153 Table 7.11. Summary of information ftom UPGMA dendrograms, MDS ordination and MST distance values for all nine crosses. 155 Table7.l2. Summary of variant (Y) and invariant (-) characters and significance (*) from correlation coefficient 12. 157 Table 8.1. Eucalyptus species used in controlled pollinations. 193 Table 8.2. Characters and codes used in phenetic analysis (seedling), measured at node 3,7 and 10, three months after germination. r95 Table 8.3. Summary of flower, capsule, seed and seedling data for interspecific, intraspecific and open pollination with E. gillii as the female parent. Data for one tree only, other two did not set seed. 196 Table 8.4. Summary of flower, capsule, seed and seedling data for interspecific, intraspecific and open pollination with E. socialis as the female patent. Data for one tree only, other two did not set seed. r97 Table 8.5 Summary of data for E gillii: Gower UPGMA dendrogran¡ MDS plot and MST analysis. 201 Table 8.6. Summary of measured variant (Y) and inva¡iant (-) seedling characters (Table 8.2.) and significance (*) from correlation coefficient 1r2¡ for E. giUü. 202 Table 8.7. Seedüng morphological characters of seedlings from crosses between E. giUii and other species. 203 Table 8.8 Summary data for E. socialis: UPGMA dendrogran¡ MDS plot and MST analysis. 209 Table 8.9. Summary of measured variant (Y) and invariant (-) seedling characters (Table 8.2) and significance (*) from correlation coeffrcient 1r2¡ for E. socialis. 210 Table 8.10. Seedling morphological characters of seedlings from E socialis and putative male parent species. 2ll Table 8.11. Number of hybrids produced in relation to phylogenetic placement by Pryor and Johnson (1971). 216 Table 9.l. Effect of continual sucrose on vase life of flowers and leaves, flowers opening after harvest and time to negative weight change for Eucalyptus tetragona and E youngiana stems. 257 Table9.2. Effect of plant on vase life of flowers and leaves, flowers opening after harvest and time to negative weight change in continual sucrose trials for E. tetragona and E. youngianø stems. 258 Table 9.3. Effect of sucrose pulsing and cold storage on vase life of flowers and leaves, flowers opening after harvest and time to negative weight change for Eucalyptus tetragor?d stems. 264 Table 9.4. Effect of plant on vase life of flowers and leaves, flowers opening after harvest and time to negative weight change in sucrose pulsing trial for Eucalyptus tetragona stems. 264 Appendix 1.1 Anova tables for Chapter 4.4.1. (Table 4.1.). 301 Appendix 1.2 Anova tables for Chapter 7 .3.3 (Tables 7 .4,7 .5,7 .6 and 7 .7). 302 Appendix 1.3 Anova tables for Chapter 9 (Tables 9.1,9.2,9.3 and9.4). 304
vll List of Fieures
List of Figures
Figure 1.1. The ornamental eucalypt breeding flow chart. 4 Figure 3.1. Eucalyptus species location maps. 3t Figure 3.2. Flowering times of all species studied. 33 Figure 3.3. The Monarto Woodland, Callington, South Australia. 50 Figure 4.1. The Laidlaw Plantation. 57 Figure 4.2. Monthly averages of minimum and maximum air temperatures at the Waite Agricultural Research Institute weather station - March '96 to March '99. 58 Figure 4.3. Monthly averages of rainfall and evaporation at the Waite Agricultural Research Institute weather station - March '96 to March '99. 58 Figure 4.4. Monthly averages of hours of sunshine and solar radiation at the Waite Agricultural Research Institute weather station - March '96 to March '99. 58 Figure 4.5. Growth rate (height) per month for nine species. 6L Figure 4.6. Growth rate (trunk diameter) per month for nine species. 6L Figure 4.7. Progressive tree heights for nine species. 62 Figure 4.8. Progressive trunk diameters for nine species. 62 Figure 5.1. The Waite Arboretum (1955): section of arboretum showing layout of plantings near the female parent of E. 'Urrbrae Gem'. 80 Figure 5.2. Dendrogram of RAPD data showing genetic similarity. 92 Figure 5.3. MDS ordination with MST linkages of RAPD data. 93 Figure 6.1. UPGMA dendrogram of 20 adult individuals measured. 110 Figure 6.2. Semi-strong hybrid multidimensional scaling of 2l individuals into their five taxonomic groups based on 54 adult characters. l1l Figure 6.3. Principal canonical correlation plot for the SSH ordination based on the adult characters measured. I12 Figure 6.4 UPGMA dendrogram representing 486 seedlings. l2l Figure 6.5 SSH multidimensional scaling of 20 groups means of 486 seedlings using alljuvenile characters. 123 Figure 6.6. Principal canonical correlation plot for the semi-strong hybrid ordination based on the seedling characters measured. 124 Figure 7.1. Map of natural distribution of E macrocarpa, E. pyriformis and E.youngiana. 136 Figure7.2. Mean number of seeds per flower pollinated and mean seed weight (mg) for each cross. 142 Figure 7.3. Mean seed weight (mg) and percent germination of seed for each cross. I42 Figure7.4. Dendrogram of E macrocarpa x E. pyriformis seedlings and parent seedlings. 162 Figure 7.5. Multidimensional ordination of E. macrocarpa x E. pyrifonzis seedlings and parents. 162 Figure 7.6. Principal component correlation graphs of E. macrocarpa x E. pyriformis seedlings and parents. 163 Figure 7.7. Dendrogram of E macrocarpa x E. youngiana seedlings and parent seedlings. 164 Figure 7.8. Multidimensional ordination of E. macrocarpa x E. youngiana seedlings and parent seedlings. 164 Figure 7.9. Principal component correlation graphs of E. macrocarpa x E. youngiana seedlings and parents. 165 Figure 7.10. Dendrogram of E. macrocarpa x E. macrocarpø seedlings and parent seedlings. 165
Yltt List of Fisures
Figure 7.11. Multidimensional ordination of E. macrocarpa x E. macrocarpa seedlings and parent seedlings. 167 Figtre7.l2. Principal component correlation graphs of E. macrocarpa x E. macrocarpa seedlings and parents. 168 Figure 7.13. Dendrogram of E pyriformis x E. macrocarpa seedlings and parent seedlings. 169 Figure 7.14. Multidimensional ordination of E. pyriformis x E. macrocarpa seedlings and parent seedlings. 169 Figure 7.15. Principal component correlation graphs of E. pyriformis x E. macrocarpa seedlings and parents. 170 Figure 7.16. Dendrogram of E pyriformis x E. youngiana seedhngs and parent seedlings. l7l Figure 7.17. Multidimensional ordination of E. pyriformis x E. youngiana seedlings and parent seedlings. l7l Figure 7.18. Principal component correlation graphs of E. pyríformis x E. youngiana seedlings and parents. 172 Figure 7.19. Dendrogram of E pyriformis x E. pyriforrnis seedlings and parent seedlings. 174 Figure 7.20. Multidimensional ordination of E. pyriformis x E. pyriformis seedlings and parent seedlings. 174 Figure7.2l. Principal component correlation graphs of E. pyriformis x E. pyriformis seedlings and parents. I75 Figure 7.22. Dendrogram of E. youngiana x E. macrocarpa seedlings and parent seedlings. 176 Figure7.23. Multidimensional ordination of E. youngiana x E. macroca4pa seedlings and parent seedlings. 176 Figure7.24. Principal component correlation graphs of E. youngianax E. macrocarpa seedlings and parents. 177 Figure7.25. Dendrogram of E youngiana x E. pyriformis seedlings and parent seedlings. 178 Figure7.26. Multidimensional ordination of E. youngianax E. pyríformrs seedlings and parent seedlings. 178 Figure 7 .27 . Principal component correlation graphs of E. youngiana x E. pyriformis seedlings and parents. 179 Figure 7.28. Dendrogram ofE youngianax E. youngiana seedhngs and parent seedlings. 181 Figure7.29. Multidimensional ordination of E. youngianax E. youngianø seedlings and parent seedïngs. 181 Figure 7.30. Principal component correlation graphs of E. youngiana x E. youngiana seedlings and parents. 182 Figure 8.1. Dendrogram of E. gillii x E. caesia seedlings and parent seedlings. 220 Figure 8.2. Dendrogram of E. gillii x E. eremophila seedlings and parents. 220 Figure 8.3. Dendrogram of E. gillii x E. sideroxylon seedlings and parents. 220 Figure 8.4. Dendrogram of E. gillii x E. websteriareø seedlings and parents. 221 Figure 8.5. Dendrogram of E. gillii x E. youngianø seedlings and parents. 221 Figure 8.6. Dendrogram of E. gillii x E. orbifolia seedlings and parents. 221 Figure 8.7. Dendrogram of E. gillii x E. transcontinentalis seedlings and parents . 222 Figure 8.8. Dendrogram of E. gillii x E. oldfieldü seedlings and parents. 223 Figure 8.9. Dendrogram of E. gillii x E. gardneri seedlings and parents. 224 Figure 9.10 Dendrogram of E. gillii x E. kruseana seedlings and parents. 225 Figure 8.1I Dendrogram of E. gillií x E. polyanthemos seedhngs and parents. 225
lx f icf nf Í'imrrac
Figure 8.12. Dendrogram of E. gillii x E. socialis seedlings and parents. 226 Figure 8.13. Dendrogram of E. gillíi x E. giUii seedlings and parents. 227 Figure 8.14. Multidimensional ordination of E. gillii x E. caesia seedlings and parents. 228 Figure 8.15. Multidimensional ordination of E. gillii x E. eremophila seedltngs and parents. 228 Figure 8.16. Multidimensional ordination of E. gillii x E. siderorylon seedlings and parents. 229 Figure 8.17. Multidimensional ordination of E. gillii x E. web ste riana seedlings and parents. 229 Figure 8.18. Multidimensional ordination of E. gillii x E. youngiana seedltngs and parents. 230 Figure 8.19. Multidimensional ordination of E. gíIlii x E. gardnen seedlings and parents. 230 Figure 8.20. Multidimensional ordination of E. gillii x E. kruseana seedlings and parents. 231 Figure 8.21. Multidimensional ordination of E. gillii x E. polyanthemos seedlings andparents. 231 Figure 8.22. Multidimensional ordination of E. gillii x E. socialis seedlings and parents. 232 Figure 8.23. Multidimensional ordination of E. gillii x E. oldfieldii seedlings and parents. 232 Figure 8.24. Multidimensional ordination of E. gillii x E. orbifolia seedlings and parents. 233 Figure 8.25. Multidimensional ordination of E. gillii x E. transcontinentalis seedlings and parents. 233 Figure 8.26. Multidimensional ordination of E. gillii x E. gillii seedlings and parents. 234 Figure 8.27. Dendrogram of E. socíalís x E. caesia seedlings and parents. 235 Figure 8.28. Dendrogram of E. socialís x E. eremophila seedhngs and parents. 235 Figure 8.29. Dendrogram of E. socialis x E. macrocarpa seedlings and parents. 235 Figure 8.30. Dendrogram of E. socialis x E. oldfieldii seedlings and parents. 236 Figure 8.31. Dendrogram of E. socialis x E. orbifolía seedlings and parents. 236 Figure 8.32. Dendrogram of E. socialis x E. siderorylon seedlings and parents. 236 Figure 8.33. Dendrogram of E. socialis x E. polyanthemos seedlings and parents 237 Figure 8.34. Dendrogram of E. socialis x E. websteriana seedlings and parents. 237 Figure 8.35. Dendrogram of E. socialis x E. youngiana seedhngs and parents. 237 Figure 8.36. Dendrogram of E. socialis x E. gardnen seedlings and parents. 238 Figure 8.37. Dendrogram of E. socialis x E. gillii seedlings and parents. 238 Figure 8.38. Dendrogram of E. socialis x E. kruseanø seedlings and parents. 238 Figure 8.39. Dendrogram of E. socialis x E. steedmanii seedlings and parents. 239 Figure 8.40. Dendrogram of E. socialis x E. transcontinentalis seedlings and parents. 239 Figure 8.41. Dendrogram of E. socialis x E. socialis seedlings and parents. 239 Figure 8.42. Multidimensional ordination of E. socialis x E. caesia seedlings and parents. 240 Figure 8.43. Multidimensional ordination of E. socialis x E. eremophila seedlings and parents. 240 Figure 8.44. Multidimensional ordination of E. socialis x E. macrocarpa seedlings and parents. 241 Figure 8.45. Multidimensional ordination of E. socialis x E. oldfieldii seedlings and parents. 241 Figure 8.46. Multidimensional ordination of E. socialis x E. orbifolia seedlings and parents. 242
x List of tr''isures
Figure 8.47. Multidimensional ordination of E. socialis x E. polyanthemos seedlings and parents. 242 Figure 8.48. Multidimensional ordination of E. socialis x E. siderorylon seedlings and parents. 243 Figure 8.49. Multidimensional ordination of E. socialis x E. websteriana seedlings and parents. 243 Figure 8.50. Multidimensional ordination of E. socialis x E. youngiana seedlings and parents. 2M Figure 8.51. Multidimensional ordination of E. socialis x E. gardneri seedlings and parents. 244 Figure 8.52. Multidimensional ordination of E. socialis x E. gillü seedlings and parents. 245 Figure 8.53. Multidimensional ordination of E. socialis x E. kruseana seedlings and parents. 245 Figure 8.54. Multidimensional ordination of E. socialis x E. steedmanü seedlings and parents. 246 Figure 8.55. Multidimensional ordination of E. socialis x E. transcontinentalis seedlings and parents. 246 Figure 8.56. Multidimensional ordination of E. socialis x E. socialis seedlings and parents. 247 Figure 9.1. Vase life (days) of E. tetragona with sucrose. 259 Figure 9.2. Percent flowers opening of E. tetragona with sucrose. 259 Figure 9.3. Time to negative weight change of E. tetragona with sucrose. 259 Figure 9.4. Vase life (days) of E. tetragonaplants with sucrose. 260 Figure 9.5. Percent flowers opening of E. tetragona plants with sucrose. 260 Figure 9.6. Time to negative weight change of E. tetragona plants with sucrose. 260 Figure 9.7. Vase life (days) of E. youngiana with sucrose. 26t Figure 9.8. Percent flowers opening of E. youngiana with sucrose. 261 Figure 9.9. Time to negative weight change of E. youngiana with sucrose. 26t Figure 9.10 Vase life (days) of E. youngianaplants with sucrose. 262 Figure 9.11 Percent flowers opening of E. youngiana plants with sucrose. 262 Figure 9.12 Time to negative weight change of E. youngiana plants with sucrose. 262 Figure 9.13 Vase life (days) of E. tetragona with sucrose and cold storage. 265 Figure 9.14 Percent flower opening of E. tetragona with sucrose and cold storage. 265 Figure 9.15 Time to negative weight change of E. tetragona with sucrose and cold storage. 265 Figure 9.16. Vase life (days) of E. tetragona plants with sucrose and cold storage. 266 Figure 9.17. Percent flower opening of E. tetragona plants with sucrose and cold storage. 266 Figure 9.18. Time to negative weight change of E. tetragona plants with sucrose and cold storage. 266 Appendix 2.1. Figure 8.57. Principal canonical correlation graphs of E. gillii x E. caesía seedlings and parents. 309 Figure 8.58. Principal canonical correlation graphs of E. giUii x E. eremopåila seedlings and parents. 310 Figure 8.59. Principal canonical correlation graphs of E. gillii x E. gardneru seedlings and parents. 311 Figure 8.60. Principal canonical correlation graphs of E. gillii x E. gillii seedlings and parents. 312 Figure 8.61. Principal canonical correlation graphs of E. gillii x E. kruseana seedlings
xl List of Fieures
and parents. 313 Figure 8.62. Principal canonical correlation graphs of E. gillii x E. oldfieldii seedlings and parents 314 Figure 8.63. Principal canonical correlation graphs of E. gillii x E. orbiþlia seedlings and parents 315 Figure 8.64. Principal canonical correlation graphs of E. gillii x E. polyanthemos seedlings and parents. 316 Figure 8.65. Principal canonical correlation graphs of E. giUii x E. siderorylon seedlings and parents. 317 Figure 8.66, Principal canonical correlation graphs of E. gillii x E. socíalls seedlings and parents. 318 Figure 8.67. Principal canonical correlation graphs of E. gillii x E. transcontinentalis seedlings and parents. 319 Figure 8.68. Principal canonical correlation graphs of E. gillii x E. websteriana seedlings and parents. 32O Figure 8.69. Principal canonical correlation graphs of E. gillii x E. youngiana seedlings and parents. 321 Appendix 2.2. Figure 8.70. Principal canonical correlation graphs of E. socialis x E. caesia seedlings and parents. 322 Figure 8.71. Principal canonical correlation graphs of E. socialis x E. eremophila seedlings and parents. 323 Figure 8.72. Principal canonical correlation graphs of E. socialis x E. gardneri seedlings and parents. 324 Figure 8.73. Principal canonical correlation graphs of E. socialis x E. gil/ii seedlings and parents. 325 Figure 8.74. Principal canonical correlation graphs of E. socialis x E. kruseana seedlings and parents. 326 Figure 8.75. Principal canonical correlation graphs of E. socialis x E. macrocarpa seedlings and parents. 327 Figure 8.76. Principal canonical correlation graphs of E. socialis x E. oldfieldii seedlings and parents. 328 Figure 8.77. Principal canonical correlation graphs of E. socialis x E. orbifolia seedlings and parents. 329 Figure 8.78. Principal canonical correlation graphs of E. socialis x E. polyanthemos seedlings and parents. 330 Figure 8.79. Principal canonical correlation graphs of E. socialis x E. sideroryIon seedlings and parents. 331 Figure 8.80. Principal canonical correlation graphs of E. socialis x E. socialis seedlings and parents. 332 Figure 8.81. Principal canonical correlation graphs of E. socialis x E. steedmanii seedlings and parents. 333 Figure 8.82. Principal canonical correlation graphs of E. socialis x E. transcontinentalis seedlings and parents. 334 Figure 8.83. Principal canonical correlation graphs of E. socialis x E. websteriana seedlings and parents. 335 Figure 8.84. Principal canonical correlation graphs of E. socialis x E. youngiana seedlings and parents. 336
xu List of Pletes
List of Plates
Plate 3.1. A: E. caesia subsp. caesia; B: E. eremophila; C: E. erythrocorys; D: E.fonestiana subsp.þrrestiana. 4l Plate3.2. A: E. erythronema var. erythronema; B: E. erythronema, var. marginata; C: E. gardneri; D: E. giUii. 42 Plate 3.3. A: E. gomphocephala; B: E. kruseana; C: E. leptophylla; D: E.lesouefü. 43 Plate3.4. A: E. macrocarpa; B: E. oldfieldii; C: E. orbiþIia; D: E. polyanthemos. 4 Plate 3.5. A: E. pyriformis;B; 8. sideroryIon;C: E. steedmanii;D: E. stoatei. 45 Plate 3.6. A: E. stricklandii;B: E. tetragona;C: E. yalatensis;D: E.youngiana. 46 Plate 3.7. The Laidlaw Plantation: Eucalyptus cut flower and bud section, October, 1998, corresponds to Figure 4.1. Scale 1:400. 5l Plate 4.1. A: E. anceps (3.3);B: E. erythrocorys(5.1); C: E.leptophylla (l.a); D: E. stoatei(Plant2.2). 69 Plate 4.2. A; E. forrestiana subsp. dolichorþncha (2.2);B: E. fonestiana subsp. dolichorhyncha (2.2); C: E. forre stiana subsp. dolicho rhyncha (3.L); D: E.forrestiana subsp. dolichorhyncha(3.1). 70 Plate 4.3. A: E lesouefii (1.4 );B: E.Iesouefii (3.3 ); C: E. lesouefii Ø.Ð; D: E.lesouefii (5.1). 7l Plate 4.4. A: E. tetragona (1.2); B: E. tetragona (2.1)C: E. tetragona (3.1): D: E. tetragona buds (Plant 5.4). 72 Plate 4.5. A: E. yalatensis (a.1); B: E. yalatensis (a.Ð; C: E. youngiana (5.2). 73 Plate 5.1. Agarose gels of RAPD primers A: OPD-I1 and B: OPD-14. 89 Plate 5.2. Agarose gels of RAPD primers A: OPD-10 and B: OPD-19. 90 Plate 6.1. Flowers of E. 'Urrbrae Gem' (photo courtesy of Dr. J. Gardner). 101 Plate 6.2. Seedling leaves of A: a) open pollinated E. erythronema vaÍ. erythronema, b) E. erythronema var. erythronema x E. stricklandii and c) open pollinated E. stricklandii, and B: a) open pollinated E. erythronema vat. erythronema, b) E. erythronemavar. erythronemaxE. gomphocephala and c) open pollinated E. gomphocephala. ll9 Plate7.l. A'. E. macrocarpa flower; B: E. pyriformis flower;C: E. youngiana flower. Bar = lcm. 135 Plate7.2. Hybrid seedlings six weeks after germination, E. macrocarpa x E. pyriformis and E macrocarpa x E. youngiana. L66 Plate 7.3. Hybrid seedlings six weeks after germination, E. pyriformis x E. macrocarpa and E. pyriformis x E. youngiana. 173 Plate7.4. Hybrid seedlings six weeks after germination, E. youngiana x E. macrocarpa and E. youngiana x E. pyriformis. 180 Plate 7.5. A: Putative hybrid between E. pyriformis and E. youngiana and B: Putative hybrid between E. youngiana and E. macrocarpa, both from the Waite Arboretum. 187 Plate 9.1. Species used in postharvest trials. A: E. tetragona; B: E. youngíana; C: E.forrestiana subsp.forrestiana;D: E. stoatei . 252
xul Glossarv of Terms
Glossary of Terms
acuminate: tapering gradually/suddenly to a protracted point, sides straight or convex, angle of convergence >90" acute: with sides equally curved convexly to the base, whole included in 90 degree angle; sharp, terminating at once in a point not abruptly but not tapering; with 2 almost straight lines converging at an angle of <90o
Adnataria: one of the 8 informal sections of subgenera Symphyomyrtus adult: one of the four recognised leaf phases of Eucalyptus plants, when the plant is physiologically mature and reproductive alternate: leaves arranged in a spiral (1 at each node); placed singly and alternately one above
the other on some cornmon body, i.e. leaves on stem
anther: part of the stamen that bears pollen
apex: tip or point of leaf, opposite of base
apiculate: terminating abruptly in a little point
Arboretum: collection of cultivated trees
ascending: having a direction upward with an oblique base
attenuate: with convex curved sides narrowed gradually and concavely to the base
angular : having projected longitudinal angles
apiculate: ending abruptly in a short point axillary: arising from the axil of ordinary foliage leaves
basal rim: rim at base of operculum
beaked: pointed
binary: character coded by two states, usually present (1) or absent (0)
Bisectaria: one of the 8 informal sections of subgeneru Symphyomyrtus
bloom: white covering of wax; a flower or inflorescence
þugl: immature flower, consisting of hypanthium and operculum, prior to operculum lift and anthesis
campanulate: bell shaped, inflated and gradually enlarged into a limb, base not being conical calyx: the outermost of the floral envelopes
capsule: a dry, many seeded fruit, or 'gum nut', which opens at maturity at the valves channelled: regularly ridged citric acid: a six carbon tricarboxylic acid, from citrus plants (lemons)
xrv Glossarv of Terms clavate: club shaped clonal propagation: the production of new plants by tissue culture clone: a population of genetically identical cells or individuals concave: rounded inward concolorous: same colour throughout conical: like a true cone conspicuous: obvious, highly visible continuous: character measured in specific units e.g. length mm controlled pollination: placing the pollen on the stigma manually flower: single structure, consisting of operculum, hypanthium, stamens, pistil and pedicel fruiÍ: alternative name for capsule fusiÍorm: spindle shaped i.e. na¡rower at both ends than at center, tapering to each end genotype; the genetic constitution of an organism as opposed to its physical appearance. genus; the smallest natural group containing distinct species glandular: covered with hairs bearing glands at the tips glaucous; with blue-ish green sheen or bloom globular'. spherical
Gower Metric: a range standardised Manhattan distance measure used to generated a matrix showing dissimilarity between individuals or operational taxonomic units (OTUs) see Appendix I habit: form or manner of growth hemispherical: half a sphere, smoothly rounded hierarchical classification: a system into which individuals are grouped into an ascending series of successively larger and broader categories, based on the affrnities of the component units hypanthium: flat or cup shaped receptacle, contains the ovary inconspicuous: present but not readily visible inflexed: suddenly bent inwards inflorescence: ¿urangement of flowers on the floral axis, group of flowers infrageneric: within a genus inserted: inside the mouth of the capsule internode: the portion of the stem between the level of insertion of 2 successive leaves or leaf pairs (or branches of an inflorescence) inter: between
xv Glossarv of Terms intermedíate: one of the four recognised leaf phases of Eucalypføs plants, when the plant is between the juvenile leaves and the adult leaves, rarely seen, in some species may be physiologically mature and reproductive intra: within invariant: not variable or different juvenilg one of the four recognised leaf phases of Eucalyptus plants, when the plant is between the seedling leaves and the intermediate leaves, in some species may be physiologically mature and reproductive
Iamina: the blade of a leaf, thickness often measured lanceolate: tL=4-6Wl narrowly elliptic, tapering equally to each end leaf margin: edge of leaf, may be smooth or jagged IeaÍpetiole angle: angle between the leaf petiole and the midrib level: withrespect to valves - neither exserted nor inserted, same height as the disc Iinear: t3l6l-:Wl n¿urow, short, with the 2 opposite margins parallel lobed: party divided with a determinate number of segments
mallee: a eucalypt with several stems arising from a lignotuber manipulated hybridisation: controlled pollination using pollen from a different plant or
species midrib (midvein): the central, and usually most prominent, vein of a leaf or leaf-like organ
minimum spanning tree: a set of lines, representing the pair-wise associations from a group of
objects, which interconnect all the objects. mucronatq abruptly terminated with a hard short point; (mucronulate): diminutive multidimensional scaling: ordination on more than one axis, usually three multistate; character measured in more than two states, coded by numbers multivariate anabtsis: analysis using a number of variates (characters) M)¡rtaceae: family to which Eucalyptus genus belongs
nectary: nectar secreting organ
node: a point of the stem from which one or more leaves arise numerical taxonomy: a method of classification based on the numerical analysis of the
variation of a large number of characters in a group of organisms ob-'. prefrx meaning inverted oblong: tI-2-3Wl elliptical, obtuse at each end, sides almost parallel oblique: where the degree of inequality in the 2 sides is slight obscure: not prominent
xvt fll¡rsssrv of Terms obtuse: terminating gradually in a blunt end, rounded enough to include a 90o angle obovate: [I-II/ZW] the reverse of ovate, broadest above the middle, narrow below oil glands: a gland that secretes essential oil, usually on leaf surface operculum: the budcap, or lid, formed by the fusion of the petals; an outer operculum may be formed by the calyx, or both calyx and corolla may be united opposite: leaves arranged in 2 opposite rows (2 at each node) orbicular: tI=Wl perfectly circular ordination: technique used to relate individuals or groups, with individuals ordered in the way that best reflects their similarities and differences OTU: operational taxonomic unit, in this study an individual plant ovary: ovule bearing unit of the pistil; simple if formed from a single carpel, compound if formed from more than one ovate: U.F<2\VI oblong or elliptical, egg shaped; broadest at the lower end ovoid; ovate, egg shaped PA[N: computer program used for multivariate ordination pÊdieel: attaches flower to umbel pgdunele: attaches flower or umbel to branch pgrydule!Å: downward hanging, weeping pøal: individual organs that form the corolla perta\sIe: having petioles peliple: the stalk portion of a leaf phenaqry: the expression of characters of an organism determined by an interaction between the environment and the genotype, and between dominance and epistatic relationships within the genotype pistil: collective term for ovary, style and stigma pith glands: oil glands within the branchlets, twigs or stems postharvest: occurring after harvest precocious: forward in development preharve st: occurring before harvest
UalntLent: highlY visible plovg4anca: origin, source, place where found or produced, as a cultivar or selection of a taxon prugtesp: with deposits of powder or flakes of wax, often like flour and easily detached (farinose, mealy, scurfy)
xvu Glossary of Terms
Wramidal: like a cone with angular sides quadrangular: four sided figure ramet: any individual belonging to a known clone, preferred for studies of phenotypic plasticity in relation to environmental factors RAPD: random amplified polymorphic DNA, method for generating DNA fingerprints receptacle: the more or less enlarged or elongated end of the stem or floral axis on which some or all of the flower parts are borne
recurv ed; curved backwards
reflexed: bent abruptly backwards at more than 90o
retuse: terminating in a round end ribbed: having ribs rhomboid: tl=lvl oval, a little angular in middle rostrate: terminating gradually in a hard long straight point seedling: young plant, from germination to 12 months; one of the four recognised leaf phases of Eucalypløs plants sepals: the individual organs that form the corolla senescence: the period between maturity and death of a plant or plant part series; informal level of classification, below section, above species sessile: flat, adjacent to surface, no attaching stalk (petiole or peduncle or pedicel) shining: glossy surface, highly reflective, not dull or glaucous SMC: simple matching coefficient, smooth: smooth, not wrinkled, ridged or warty spathulate; [L=4-6W] oblong with lower end much attenuated species: the basic unit of classification; a population or system of populations that normally interbreed
square: four equal sides stamen: collective term for anther and filament stigma: the part of the pistil that receives the pollen; variable in shape and form according to
the mode of pollination, with as many stigmatic surfaces provided as carpels unless fusion of
the surfaces has taken place
stressi 7o degree of distortion of OTUs ordinates during multidimensional scaling, should be
<20Vo style; narrow constricted "neck" between the stigma and ovary, sometimes absent when the stigma is sessile; often with internal cavities (stylar canals) to facilitate penetration of pollen
xvut Glossarv of Terms tubes s¡¿å: below subspecies: informal level below species to.x,on; general term applied to any taxonomical element, population or group irrespective of its classification level (taxa) terete: slender cylindrical, but not so slender as filiform (threadlike) truncate: as ifcut straight across turbinate: inversely conical with contraction towards the point umbel: an inflorescence composed of a cluster of flowers whose pedicels arise from the same point umbonate: round with projecting point in the centre uncinate: hooked, curved back suddenly at the point undulate: having an uneven alternating convex and concave margin
UPGMA: unweighted pair group method with arithmetic average urceolate: same as campanulate but more contracted at orifice with an erect limb valves: openings that dehisce to allow seed to be disseminated vase life: period of time (days) a stem (including flowers and leaves) remains marketable after harvest variate: character variant: not all the same variet))i a "botanical variety' is a category below that of species, a 'cultivated variety' is a cultivar vegetative propagation: the production of new plants without using seeds; cuttings or grafting venation: collective vein pattern on leaves ventral: upper surface wart.y'. small lumps on surface, not smooth weeping: hanging vertically, pendulous, not erect whorled; radial affangement around a stem with 2 pairs of leaves at each node winged; having wings on surface
Adapted from Brooker and Kleinig (1990), Tootill (1984), Soule and Sherman (1978)
xrx Chapter One (_l Introduction !lBR h'
1..1. Introduction
Ornamentals are an expanding sector of the Australian horticulture industry. Within the ornamental sector is the area of floriculture, where plants are grown specifically to produce cut stems with flowers,'buds and foliage. The Eucalyptus genus has received recent attention for use in floriculture; however, previous studies have focused on foliage rather than bud and flower production. The potential to expand exists; but before such potential can be realized, research is needed into relevant aspects, including selection of superior genotypes from suitable species, clonal propagation and post harvest physiology.
Selecting the most suitable specimen is the first step in plant improvement, and thus studies of growth habit are important prior to selection. Eucalyptus specimens suitable for floriculture are initially selected for their morphological appearance, such as the shape and colour of leaves and flowers. Attention should then be given to the postharvest physiology of the specimens, as this will influence their vase life. Aspects that require investigation relate to the rates of wilting, senescence and abscission of leaves, buds and flowers. The postharvest physiology of some Eucalyptus foliage species has been investigated, but the flowers and buds of a wider range of species require research.
The Eucalypføs forestry industry has investigated interspecific hybridisation and clonal propagation for improvement in timber production. The methodology involved could be applied to ornamental production, enabling the development and propagation of new hybrids.
Molecular biology techniques can also be used to generate DNA profiles of plants, and to identify parentage or clarify hybrid status.
I fnfrrvlrrefion 1 -2- 1.2. Native plants and ornamental horticulture
I.2.I. Introduction
The continent of Australia has been isolated for over 50 million years, and this has enabled the evolution of a diverse range of flora suited to a wide range of geographical and climatic conditions. This provides opportunities for development of new and unusual species for amenity horticulture.
1.2.2. Australian natives for cut flowers and foliage
Although sales of Australian native cut flowers worldwide are valued at $400m (wholesale), only around llVo is produced in Australia. In 1997-1998, total flower exports from Australia were valued at$.27.4m, with 93Vo native flowers and exotic proteaceae (FECA, 1999). The top native flowers and exotic proteaceae exported, by volume and value terms in 1997-1998, are waxflower, kangaroo paw, Thryptomene, Stirlingia, Protea, Banksia, Leucodendron,
Koala fern and Scholtzia, with unspecific eucalypt product tenth and general foliage rated fifteenth.
L2.3. Development of ornamental Eøc¿l.vprus species
There are no superior varieties of eucalypt for cut flower production available in Australia
(Sedgley, 1998), resulting in lack of uniformity and yield of product. A large number of
Eucalyptus species show potential, however, development requires selection at both species and genotype level, for a number of criteria. Eucalypt selection programs are subject to long generation times and propagation difficulties and these must be overcome if ornamental eucalypts are to find their place on world markets.
1.2.4. Native ornamental breeding and selection
Breeding and selection occur consecutively, following a series of logical steps (Figure 1.1)
) Intrrxluction 1.2-
(Sedgley, 1996). Selection initially occurs at the species level, where the gene pool from wild and cultivated populations is searched for species fitting the desired floral criteria. Selection proceeds to the genotype level, where plants of the same species are compared and assessed at primary and secondary levels. For this study, it was assumed that the phenotype expressed for any plant was fully indicative of the genotype of that plant in all criteria. Primary
selection focuses on flowering, plant form and growth rates. Genotypes exhibiting superior characters at this level are moved on to secondary selection, where the response of the plant to cultivation, postharvest testing and clonal propagation is determined. Selected genotypes can be included in inter and intra specific breeding programs, for the development of hybrids
combining superior characters from different plants. Once the selected genotype meets all
criteria at all levels, it is registered as a cultivar and can be multiplied for commercial release.
3 Introduction 1.2.
Selection at Species Selection for Floral Criteria '' Species level Gene Pool - Wild and Cultivated'populations
.- Selection at Genotype Level Hybrid Production through C ontrolled Pollination
Superior Development of Propagation M ethods Genotype
Postharvest Testing
PBR Comparative Trials Registration
Multiplication and Release to Industry
Figure l.l. Ornamental eucalypt breeding flow chart.
4 Infrrvluefion 1^3- 1.3. Aims
The project aims to improve species from the Eucalyptus genus for ornamental horticulture by addressing three aspects of plant improvement:
1. Species and genotype selection: to grow a range of species suitable for the cut flower and bud industry under similar conditions, and to observe and record their growth and development over a three year period, with selection of genotypes for further development.
2. Interspecific hybridisation and hybrid identification: to investigate interspecific hybridisation, through controlled pollination, as a method to produce hybrids with increased ornamental merit, and to clarify hybrid status using DNA fingerprinting, and adult and seedling morphology comparisons.
3. Post harvest physiology and treatments: to perform postharvest trials on cut stems of
Eucalyptus, with flowers and buds, to establish the optimal postharvest holding conditions for increased vase life and flower opening.
5 f ilo¡qftrro f,laviou 1 1
Chapter Two Literature Review
2.1. The genus Eucalyptus
The genus Eucalyptus consists of over 700 species (Brooker and Klienig, 1999), almost all endemic to Australia. They are grown all over the world as ornamentals and as a valuable source of timber, fuel and shade (Brooker and Kleinig, 1990). V/ithin Australia, eucalypts are widespread in all areas except the desert zone; their distribution depends mainly on climatic environment, with other factors, such as soil type, determining species placement and mix
(Williams and Brooker, 1997).
Eucalyptus species are evergreen woody perennials, ranging from very large trees (e.g.
Eucalyptus regnans up to 100 m) to small mallee types (e.g. E. tetraptera vp to 3 m). Most can be described as forest (30 - 50 m) or woodland (10 - 25 m) species. Many are heteroblastic, that is the juvenile foliage is very different from that of the adult. Adult leaves have a distinctive appearance; they are petiolate, falcate-lanceolate, and mainly isobilateral.
Juvenile foliage refers to that which occurs on seedlings up to I m tall (Pryor, 1976) and is particularly useful in species identification (Williams and Brooker,1997). Juvenile characters are strongly inherited and can be identified a few months after germination. Within some species (e.g.E. cinerea), adult type leaves are not produced on some individuals, despite them being ontogenetically mature. Juvenile leaves are often round and glaucous, making them particularly attractive for floriculture.
Individual species are identified according to flower morphology and overall appearance.
Stamens and anthers are important features, as are cotyledon shape, operculum shape, and ovule number and position. Overall appearance is determined by noting the main characters of
6 f iforofrrro Doviou î ? 1 plant growth habit, bark, leaves, inflorescences, buds, fruit and seeds (Chippendale, 1988).
Pryor and Johnson (1971) developed a classification system for the eucalypts, which they used to identify the following subgroups: subgenus, section, series, subseries, species and subspecies. There are currently seven informal subgenera (Blakella, Corymbia, Eudesmia,
Guabaea, Idiogenes, Monocalyptus and Symphyomyrtus) one of which, Corymbia, has recently been elevated to generic level (Hill and Johnson, 1995). Symphyomyrtus is by far the largest subgenus, containing eleven sections, and is the most important commercially.
Revisions to the classification continue, but Pryor and Johnson's (1971) phylogeny will be followed in this research.
2.2. Taxonomy
2.2.1. Introduction
The term taxonomy, the study of the principles and practices of classification, is often used synonymously with systematics, which is the scientific study and description of the variation in living organisms and the relationships that exist between them. The field of taxonomy includes various sections and methods. Numerical taxonomy is a classification method based on the numerical analysis of a large number of characters in a group of organisms. To be highly predictive, a classification must be based on a large number of characters, each of equal weight (Toothill, 1984). A data matrix is compiled from measurements and observations of a range of characters for the individuals or operational taxonomic units
(OTUs) used in the study. The data matrix can be subjected to a number of mathematical analyses, using different coefficients, to provide a measure of the similarity or dissimilarity between all OTUs, with the end product one or more dendrograms. Some analysis programs enable the generation of ordination plots for clearer resolution of the relationships between
7 Literature Rewew 2-2.2-
OTUs, as well as providing information regarding the significance of particular characters
2.2.2. Taxonomy and Eucalyptus
Eucalypts were first described by Charles L'Héritier in 1788, with the first classification of the whole genus based largely on staminal characters (Bentham, 1867). Later revisions were based on studies of other macroscopic characters, particularly cotyledons (Maiden, 1903-33), buds, fruits and anthers (Blake, 1953; Blakely, 1955). Other taxonomic investigations revealed a wider range of potentially diagnostic characters, including anatomical structures such as the seed coat (Gauba and Pryor, 1958, 1959, 196l), leaf waxes (Hallam and
Chambers, 1970), trichomes (Ladiges, 1984), oil glands and ducts (Carr and Can, 1969) and inflorescences or parts thereof (Can and Carr, 1959, 1962; Boland and Sedgley, 1986). An informal classification suggesting an infrageneric hierarchy appeared in the early 70s (Pryor and Johnson, l97l) and has been widely adopted. The most recent classification of the genus was published by Chippendale (1988) as part of the Flora of Australiø series, and presents a formal classification at the series level. Revisions to particular sections and series have been published since, including revisions of ser. Dumosae (Brooker, l97l), subgenus
Symphyomyrtus sect. Bisectaria (Hill and Johnson, 1992), subgenus Eudesmia (Hill and
Johnson, 1998), and particularly controversial, the revision of the bloodwoods and the separation of subgenus Corymbia as the genus Corymbia (Hill and Johnson, 1995). Other clarifications of relationships between species and series through studies of the morphology of both seedling and adult plants have been documented (Chappill and Ladiges, 1996; Ladiges et al., 1984; Ladiges et aI., 1987). Morphological characters are used to study geographical variation within species/complexes (Boland, 1978; Jordan et aI., 1993: Potts, 1985) for intergrade classification (Doran and Burgess, 1993) and hybrid identification (Hopper et aL,
1978).
8 T iforof rr¡o flor¡iaw I 1 1
The advent of techniques enabling the study of plants at a molecular level has resulted in an influx of data regarding taxonomic relationships within the Eucalypf¡ls genus. Genetic diversity within species has been investigated using techniques such as allozymes (Moran and
Hopper, 1983; Sampson et al., 1989, 1990; Kennington and James, 1997a, b, 1998), while linkage maps (Byrne et al., 1995; Marques et al., 1998) are used for studying genome structure and QTL studies. The combination of morphological and molecular techniques to determine relationships between individuals and species will enable further clarification of the hierarchy of Eucalyptus.
This investigation will use numerical taxonomy to determine similarities and dissimilarities between individuals and species. The resulting association matrices will enable the generation of a phenetic dendrogram and a multidimensional ordination clarifying the relationships of the individuals investigated.
2.3. Interspecifïc hybridisation
2.3.1 Introduction
Hybridisation, whether natural or artificial, is an important component in the development of superior lines of many horticultural plants. When two different species or strains are crossed, the resultant offspring may display high levels of variability, and may exhibit increased growth, improved vigour and adaptability, and higher resistance to diseases and pests than either or both of the parents. The offspring can be selected for further development based on superior characteristics, such as growth rate and habit, early flowering and unusual flowers or buds.
9 Literature Review 2.3.2.
2.3.2. Interspecific Hybridisation in Eøc¿l.vprøs
Interspecific hybridisation can occur naturally in Eucalypføs, often where two populations merge at the perimeters of their respective ecosystems. Natural hybrids have been studied and identified between Eucalyptus preissiana and E. bruprestium (Hopper et aI., 1978), E.obliqua and E. pulchella (Potts and Reid, 1983), E. regnans and E. macrorhynclra (Ashton and
Sandiford, 1988), and between E. tetraptera and E. stoatei (Bennett, 1995). Most of these hybrids display morphological characters intermediate between the parent species. Natural hybridisation is rather rare across the genus, with only l57o of possible combinations expected on geographic/taxonomic grounds being recorded, and 37Vo of these are known from only a single herbarium record (Griffin et a1.,1988).
There are several breeding barriers to natural hybridisation in eucalypts: geographical isolation, ecological isolation and reproductive isolation. Geographical isolation is a major deterrent to natural hybridisation (Griffin et aI., 1988), the most striking example being the lack of natural hybridisation between species from the east and west of Australia, due to the presence of the Nullarbor Plain. The expanse of the plain means that pollen from either side cannot cross by natural means, and so natural cross pollination cannot occur. Ecological isolation is also important, as different habitat conditions (e.g. soil type) mean that some species will rarely or never grow in proximity to each other. Reproductive isolation occurs as different flowering periods, or by more complex post mating barriers and systematic affinity.
Post mating barriers within Eucalyptus are sometimes unilateral and relate to floral morphology. Large flowered species have longer styles than small flowered species; the barrier to hybridisation can occur when the larger flowered species is used as the female. The pollen tubes of the small flowered, short styled species are unable to penetrate the ovary of the large, long styled flower. This has been suggested as the simplest explanation for the
10 f .ifernf¡rre Review 2-3-2^ unilateral failure of hybridisation (Gore et al., 1990). Boland and Sedgley (1986) determined that the anatomy of the stigma has implications with regard to the breeding systems of eucalypts. This may also affect the success of interspecific hybridisation.
Within Eucalyptus, the subgeneric groups are probably reproductively isolated. The frequency of natural hybridisation generally reflects the hierarchy of taxonomic affinities; although important exceptions have been noted in the subgenera Monocalyptus and Corymbía
(Griffin et al., 1988). There is considerable variation in rates of inter- and intra- sectional hybridisation within the Symphyomyrtus subgenus (Griffin et aI., 1988). The relationship between species, according to the particular systematic groups to which they belong, is an index of likely capacity to interbreed. With this in mind, manipulated interspecific hybridisation has been attempted, with most examples coming from the Symphyomyrtus subgenus (Griffin et aI., 1988). For example, Beardsell et al. (1979) performed crosses between Eucalyptus caesia, E. sideroxylon and E. Ieucorylon to produce a small attractive tree suitable for street planting. E. caesia was found to be suitable as the male parent and E. sideroxylon and E. leucoxylon as females. Other work by Tibbits (1988) found that E. nitens
(maternal parent) was capable of producing viable crosses with other closely related species from within the same series. The conclusion was reached that it is possible to artihcially combine different species, or separate populations of the same species, that may be incapable of cross breeding naturally because of geographicaVecological isolation or differences in flowering times (Tibbits, 1988). However, there may be a higher frequency of viability problems with both seed and offspring with increasing taxonomic distance.
The offspring of interspecific hybridisation will most often exhibit morphological characters intermediate between the parents. Flower size and colour, leaf shape and growth habit are the most common morphological characters assessed to indicate hybrid status. Assessment can
1,L f ,iferafrrre Review 2-4-1- also be carried out at an anatomical level, and these data examined statistically; and it may be supported by other characters such as the nature of the chemical constituents of the plant (e.g
Ashton and Sandiford, 1988).
This project will study interspecific hybridisation within the Symphyomyrtus subgenus, with the dual aim of understanding limits to compatibility and of producing new and unique hybrids with increased ornamental merit.
2.4. Molecular Biology
2.4.1. Introduction
The accurate, fast, reproducible and cost effective identification of plant populations and varieties is essential in agriculture as well as pure plant research (Morell et al., 1995).
Traditional methods of varietal identification involve assessing a range of morphological characters but these may have limitations, such as subjectivity in character analysis and delays in the expression of certain diagnostic characters (e.g. flower shape or fruit colour) particularly in long lived species. The environment in which the plant grows may also influence the morphological characters of the plant.
Varietal identification can use morphological characters in conjunction with molecular biology techniques, which have been reported in phylogenetic studies and varietal identification. Techniques include isozyme analysis (Eldridge, 1976; House and Bell, 1994), restriction fragment length polymorphisms (RFLP) (Steane et al., I99l; Byrne et aI., 1994;
Sale, 1995), simple sequence repeat (SSR) - anchored polymerase chain reaction amplification (Zietkiewicz et a\.,1994), microsatellite polymerase chain reaction (Wu et aI.,
1994; Weising er al., 1995), DNA amplified fingerprinting (Luro et al., 1995); amplified
t2 Literature Revtew 2.4.2. fragment length polymorphisms (AFLP) (Vos et al., 1995), arbitrarily primed polymerase chain reaction (AP-PCR) (Welsh and McClelland, 1990), and random amplified polymorphic
DNA polymerase chain reaction (RAPD-PCR) (Williams et a1.,1990).
The introduction of RAPD-PCR and AP-PCR has enabled a wider range of species to be studied genetically than previously, as no prior knowledge of the genome is needed (Morell et aL, 1995; Vos el aI., 1995). Both techniques utilize short primers of known sequence, arbitrarily chosen, to amplify specific regions of DNA. With appropriate primers, polymorphic DNA profiles can be developed within and between species. DNA fragments are separated on agarose gels and visualized under uv light by ethidium bromide staining to reveal distinct profiles (Rafalski and Tingey,1993).
Since its inception, RAPD-PCR has been widely used for hybrid identification: in plants such as rice (Wang et al., 1994); Theobroma cacao (N'Goran et aI., 1994); Annona sp. (Ronning and Schnell, 1995); grapevine (Moreno et aI., 1995); Pinus sylvestris (Lu et al., L995);
Rabbiteye blueberry (Aruna et aL,1995); and Rhododendron spp. (Iqbal et aI., 1995). Doubts have arisen concerning the accuracy and reproducibility of the RAPD-PCR technique (Heun and Helentjaris, 1993; Levi et aL, 1993: Kng, 1994); however problems can be limited by carefu I protocol establishment.
2.4.2. RAPD-PCR and Eøcal.vprzs
Several studies have been conducted involving RAPD-PCR and Eucalyptus, looking into aspects including genetic linkage maps (Grattapaglia and Sederoff, 1994; Grattapaglia et aI.,
1995), genotype discrimination and verification (Keil and Griffin, 1994), genetic variation
(Nesbitt et a1.,1995) and hybrid verification (Sale, 1995). The DNA extraction procedure and the PCR protocol have varied slightly between studies, although the method described by
r3 f ,iteratrrre Review 2-5.1-
Doyle and Doyle (1990) remains the central link in terms of DNA extraction. PCR protocols generally follow that described by Williams et aI. (1990) with some variations.
RAPD-PCR will enable identification of interspecific hybrids of Eucalypføs when used in conjunction with morphological characters. Seedling morphological characters are currently used as the initial step in hybrid identification. As the plant matures, adult morphological characters are used for further analysis. Identifying varieties and hybrids using RAPD-PCR will enable the separation of hybrid genotypes from self or parental genotypes in the offspring of breeding programs at an early stage. This will reduce the time and resources currently required to develop a new variety, as non-desirable genotypes can be culled quickly. New varieties qualify for Plant Breeders Rights registration; and molecular methods such as
RAPD-PCR have the potential to supplement morphological examination and clarify hybrid status while the plant is still immature. RAPD-PCR may also provide a specific marker that identifies a variety (Sedgley, 1995), providing an easy method of varietal identification.
This project will use RAPD-PCR to develop DNA profiles of all parent plants used in the controlled interspecific hybridisation project, and all offspring produced. The profiles will be used in conjunction with morphological examination as a means to clarify hybrid status.
2.5. Postharvestphysiology
2.5.L. Introduction
General post harvest physiology and its principles have been discussed at length for most horticultural produce, including the main lines of cut flowers. Halevy and Mayak (1979,
1981) reviewed aspects of cut flower physiology, determining the main principles of
senescence in cut flowers. Flowers differ from most horticultural produce as they are often
t4 T.ifernfr¡re Review 2-5^1 - picked at an immature stage and are extremely perishable. Flower and leaf tissues are highly susceptible to water loss and maintaining appropriate water relations is crucial to the maintenance of tissue quality and longevity (Halevy and Mayak, 1981). Other important components to be considered in the preservation of blooms are: the maturity of the bloom, nutrient supply, temperature, presence of ethylene gas, growth processes, physical injury and
disease attack (Halevy and Mayak, 19J9, 1981; Goszczynska and Rudnicki, 1988; Reid,
1991; Joyce, 1994; V/illiamson and Milburn, 1995). The maturity of the bloom can have an
effect on the type and amount of nutrient required. Blooms picked at the bud stage require
more carbohydrates, in order to continue their development, than those picked at maturity
(Halevy and Mayak, L979;1981; Han, 1992; Doi and Reid, 1995). Blooms and foliage to be
stored for long periods often require carbohydrates before and after, but rarely during, storage.
Certain species are susceptible to the presence of endogenous (from the flower itself and
exogenous ethylene (from other ethylene producing products such as climacteric fruits or car
exhaust fumes) as this increases the rate of senescence. Such blooms should be treated with
an inhibitor of ethylene formation, such as STS (Halevy and Mayak, l98l; Tingley and
Prince, 1990; van Doorn and Woltering, l99l) and kept well ventilated, away from ethylene
producing products. Cool temperatures reduce metabolic processes and slow senescence, and
must be maintained at all times (Goszczynska and Rudnicki, 1988). Most flowers can be kept
at 0 to 2oC; others, such as tropical flowers, should be kept between 7 and l2"C to avoid
chilling injury. Long term cold storage can be achieved, either wet or dry, if blooms are
properly pre-treated before storage with ca¡bohydrates and biocides (Jones, l99l; Rudnicki ¿r
aI., l99l Jones and Hill, 1993). Blooms should be handled carefully to prevent physical
injury; this may increase ethylene production or provide avenues for entry of disease
organisms.
15 Literature Review 2.5.2. Once harvested, blooms and foliage are often transported long distances to market.
Maintenance of cool temperatures and a supply of a vase solution (containing water, a biocide and carbohydrates) will enable the product to arrive at its final destination in a marketable condition with suitable vase life.
2.5.2. Post harvest treatment of Eøcøl.vptøs
Recent studies on the post harvest treatment of Australian native plants have focused on flowering species, such as Banksia spp. (Barth, 1992: Delaporte, 1995), Telopea speciosissimø (Faragher, 1986a; 1986b; Jones & Faragher, 1991), Anigozanthøs spp. cultivars
(Teagle et a\.,1991), Thryptomene sp. (Jones and Faragher, 1991), Verticordia sp. (Jones and
Faragher, 1991) and Geraldton Wax cultivars (Jones and Faragher, l99l). Foliage species of
Eucalyptus and Acacia are now receiving more research attention in terms of post harvest treatment, especially long term cold storage and carbohydrate supply requirements.
Few Eucalyprøs species have been fully investigated and most work has addressed foliage vase life. Trials into the cold storage and post harvest solution requirements of E crenulata and E gunnii foliage have been conducted (Jones et al.,1993; Jones et al., 1994); as well as trials to assess the effect of dry storage and floral preservatives on the vase life of E. gunnii,
E. delegatensis, E. rubida, E 'Silver Drop', E. viminalis and E. globulus foliage (Forrest,
1991). Wirthensohn et aI. (1996) treated a range of foliage species with sucrose and dry storage. Current evidence suggests that species react differently to treatments, although most can be dry or wet stored for 4 weeks at 1 - 5oC without damage (Forrest, 1991; Jones et al.,
1993; Jones et al., 1994; Wirthensohn et al., 1996). Pulsing with inhibitors of ethylene formation has little effect, and the addition of biocide to the solution may enhance the vase life of some species but not others (Jones et al., 1993: Jones et al., 1994). Further trials with a wider range of species are required to facilitate the development of a standard protocol for
t6 f ,ilerature Review 2-5-2- post harvest care of foliage, buds and flowers.
Eucalyptus species, suitable for foliage production, generally have numerous, small, roundish leaves and a 'blue' colouring which can be caused by the presence of leaf waxes. If leaf waxes are present, they may be damaged during post harvest handling (Jones & Sedgley,
1993) reducing the quality of the stem. Some species of Eucalyptus have attractive buds, flowers and fruit, however, their suitability for floriculture has not been investigated. The development of handling protocols and appropriate post harvest solutions for flowers and buds is required if the potential of this aspect of the Eucalyptus genus is to be reahzed.
There is an urgent need for further information into post harvest treatments of Eucalyptus species for foliage, flowers, bud and fruit production. This project will study the effect of carbohydrates, such as sucrose, and dry cold storage on the vase life of flowering Eucalyptus stems.
l7 Dlonf llrfofo¡iol 1 1
Chapter Three Plant Material
3.1. Introduction
Of the 500 or so species of Eucalyptus, many exhibit varying levels of horticultural potential.
This may take the form of showy or large buds, flowers or inflorescences, attractive leaves or capsules, or a neat compact tree form possibly with interesting bark or trunk shape. The major focus of this study is the cut bud and flower industry, although foliage and amenity potential is also acknowledged. Limited selection of species for floriculture has been undertaken, although several eucalypt species are already produced commercially for their foliage and many are grown as garden specimens. Selection at species level is required for floriculture, leading to selection of superior genotypes from suitable species.
3.2. Species used in the study and their natural distributions
For this study, thirty species, as named by Chippendale (1988), \ryere assessed for their
horticultural potential. Species for the hybridisation programs were selected on the basis of
their floricultural characteristics as well as their taxonomic relatedness, according to the
classification of Pryor and Johnson (1971) (Table 3.1). The morphological characteristics of
all species studied are presented in Tables 3.2. (leaf), 3.3. (inflorescence) and 3.4. (capsule).
General cultivation notes are presented in Table 3.5, and the use of each species in the project,
their common name and natural distribution are listed in Table 3,6. The natural distributions
of each species are illustrated with maps in Figure 3.1. Flowering times are illustrated in
Figure 3.2. General notes on desirable horticultural characters are discussed. The sources of
the plant material are described in section3.4.
t8 Plant Material 3.2.
Table 3.1. Relatedness of Eucalypløs species investigated (Pryor & Johnson l97l)
Subgenus Section Series Species Subspecies Eudesmia Quadraria Tetragonae tetragona erythrocorys
Symphyomyrtus Bisectaria Cornutae gomphocephala Occidentales steedmanii eremophila Erythronemae erythronema erythronema marginatQ Reduncae gardneri Grossae stricklandii Kruseanae kruseana Oleosae transcontinentalis socialis siUii Macrocarpae orbiþIia websteriqna caesía caesta macrocarpa oldfieldii pyriþrmis youngiana Foecundae Ieptophylla
Dumaria Dumosae anceps lesouefii Incrassatae stoatei forrestiana forrestiana dolichorrhyncha
Adnataria Pruinosae prutnosa Polyanthemae polyanthemos Melliodorae sideroxvlon sideroxvlon E. yalatensis ser. Subulatae (Chippendale, 1988) was identif,red after the classification by Pryor and Johnson (1971), associated species include E. gillii, E. socialis and E. transcontinentalis.
19 Table 3.2.l-naf characters of all from 1973;1988 Brooker and I Adult xW Petiole xW anceps lanceolate, thick, acuminate; shining, green;5-12 x l-2.5 elliptic to ovate; light green terete or quadrangular; 10-19 caesia subsp. caesia lanceolate, sometimes falcate, acute or acuminate; grey-green; 7-12 x 1.2-2.5 alternate; petiolate; orbicular or cordate; shining, green glaucous; 1040 eremophíla narrowly lanceolate to elliptical, acuminate or uncinate; green, glandular; narrowly lanceolate slightly flattened; 6-8 x l-1.7 5-r0
erythrocorys opposite; narrow lanceolate, often falcate, acuminate or uncinate; bright ovate; green; stellate hairy quadrangular; 10-30 green; 12-25 x 14
erythronema var. nanowly lanceolate, uncinate; shining, green, glandular; lanceolate slightly flattened; 6-9 erythronema 6-8 x 0.8-1.5
erythronema var. narrowly lanceolate, uncinate; shining, green, glandular; lanceolate slightly flattened; 6-9 marginata 6-8 x 0.8-1.5 forresliana subsp. lanceolate, apiculate; shining, deep green, glandular; ovate; green flattened; 10-20 forrestiana 6-9 x 1.2-2 forrestiana subsp. lanceolate, apiculate; shining, deep green, glandular; ovate; green flattened; 10-20 dolichorhyncha 6-9 x 1.2-2
gardneri lanceolate, uncinate; blue-green; 7-9 x0.8-2 not seen terete; l0-15 gillíi higher mature branches; lanceolate, acute; glaucous;6-8 x 1.2-2 opposite; sessile; cordate or broadly ovate; glaucous sessile gomphocephala lanceolate, acuminate; 9-L6 x 1.6-2.5 alternate; petiolate; ovate, often cordate; discolorous flattened or channelled; 15- kruseana opposite; orbicular, cordate; glaucous; 1.5-2x 1.5-2 opposite; orbicular, persistent sessile Ieptophylla narrowly lanceolate to lanceolate, moderately thick; shining, green; 4-8 x 0.4- opposite; lanceolate; green terete; 5-10 Iesouefii lanceolate, sometimes falcate, acuminate, thick; dull, green, glandular; 10-12 lanceolate; glaucous terete; 15-20 x 0.5-2.5
nurcrocarpa opposite; broadly ovate to elliptic-ovate, apiculate; glaucous; opposite; broadly elliptic to suborbicular; grey-green and sessile 8-12 x 5-8 glaucous
oldfieldii f anceolate to broadly lanceolate, acuminate; dull, grey-green; 7-10 x 1.2-2.5 alternate; petiolate; lanceolate-ovate; pale-green terete; 15-20 orbiþlia alternate, sometimes opposite; suborbicular, retuse or emarginate; grey-green; opposite; petiolate; suborbicula¡; grey-green terete; 10-20; pruinose 2.5-3.8 x 2.54.3 Continued Table3.2. Continued Species Adult(LxWcm) Juvenile Petiole(LxWmm) polyanthemos ovate to broadly lanceolate, apiculate; grey or glaucous; alternate; petiolate; orbicular, emarginate; dull, grey- terete or flattened; l5-25 5.5-9 x 1.5-3.5 green, concolorous
Prurnosa broadly ovate to elliptic-ovate, cordate, apiculate; grey-green to pruinose; opposite; ovate or broadly lanceolate; glaucous, absent 5-13 x2.5-9 concolorous pyriþrmis lanceolate to ovate-lanceolate, acuminate; light green; alternate; petiolate; ovate to lanceolate, flattened: 12-25 6-8 x 1.3-2.5 sometimes orbicular; green
siderorylon lanceolate to narrowly lanceolate, acuminate or uncinate; green, grey-green or alternate; petiolate; lanceolate to linear; grey or grey-green terete; 10-20 subsp. siderorylon blue-green; 7-14 x 1.2-1.8
socialis lanceolate, uncinate; dull, grey-green;6-9 x 1.2-2 alternate; sessile to shortly petiolate; elliptic to ovate; dull, terete; l0-20 green
steedmanii narrowly elliptic or lanceolate; 4.5-6.5 x 0.8-1.3 alternate; petiolate; lanceolate or ovate; green flattened;2-6 sloatei oblong or elliptic to ovate or broadly lanceolate, apiculate; shining, dark alternate; petiolate; ovate; green quadrangular; l3-16 green; 6-8 x 2-3
stricklandii lanceolate; thick; shining, green; l0-13 x 2-3 alternate; petiolate; elliptic to ovate terete; 20-35 tetragona opposite; broadly elliptic, apiculate, thick; glaucous or green; 7-15 x2.5-7 similar to adult edges continuous into stems; to-20
transcontínentalis lanceolate to narrowly lanceolate, acuminate; blue-grey or grey-green, dull, alternate; petiolate or opposite; sessile; decurrent, ovate; ierete; 15-25 glandular; 7-15 x l-2.2 blue-green
websteriana alternate, sometimes opposite; obovate, retuse; dull, grey-green; 24 x 1.5-2.5 opposite to alternate; petiolate; orbicular; subglaucous terete; 6-15 yalatensis lanceolate, uncinate; grey-green, dull, glandular;6-lI x l-2 subopposite or alternate; shortly petiolate, ovate or broadly terete or slightly flattened; lanceolate; green 8-r2
younS,.ana lanceolate, to falcate, acuminate; pale green; l0-15 x 2-3.5 not seen terete; 20-30 Table 3.3. Inflorescence characters of all species investigated (adapted from Chippendale, 1973; 1988; Brooker and Kleinig, 1990) Species # buds/ Bud shape; pedicel Operculum Hypanthium Peduncle (L mm) umbel (L mm) (L x \il mm) (LxWmm) anceps 7 cylindrical; to 2mm or obscure conical, fai ntly striate; cylindrical or hemispherical, faintly slightly flattened; 5 - l2 34x4-5 striate, 4-5 x 4-5
caesia subsp. caesia 3 clavate; glaucous; l0-30; conical;8-10 x l0-12 obconical or campanulate;8-12 x lO-12 terete; 20-30 eremophila '1 horn-shaped; 4-10 horn-shaped; 10-25 x 3-5 hemispherical; 5-8 x 4-6 slightly flattened; 15 - 25 erythrocorys 3 broadly turbinate nrgose, depressed hemispherical, quadrangular, ribbed; 15 x 15; green quadrangular; l0-30 cross shaped; l0 x l0; red
erythronema var. 7 conical or + fusiform; often conical; l0-15 x 7-10 obconical; 6-7 x 8-9 terete or angular, recurved; 15-20 erythronema quadrangular, smooth; l0-l 8
erythronema vat. 3 conical or t fusiform; often conical; expanded basal rim; obconical;6-7 x8-9 terete or angular, recurved; l5-20 marginata quadrangular, faintly ribbed; l0-l 8 l0-15 x 7-10 forrestiana subsp. l, rarely 3 pyriform, quadrangular, 4-winged; pyramidal to hemispherical or obpyramidal or obovate, quadrangular, 4- flattened at top, recurved; 30-50 forrestiana absent flattened;5-10 x 12-15 winged; 3040 x 12-15 forrestiana subsp. l, rarely 3 pyriform, quadrangular, 4-winged; narrowly rostrate; obpyramidal or obovate, quadrangular, 4- flattened at top, recurved: dolichorhyncha absent 12-22 x7-14 winged; 13-15 x7-17 30-50
gardneri 't-tt narrowly horn-shaped; 2-3 incinate; l0-15 x 3-4 obconical; 4-5 x34 flattened; l0-15 gíllii 7-ll glaucous; 14 rostrate; 5-8 x 4-5 cylindrical; 34 x 4-5 terete; 6-15 gomphocephala 7 clavate; absent or to 4 hemispherical; obconical or campanulate, often faintly fTattened; 13-27 8-10 x 9-13 ribbed; 13-22x13-17
kruseana 7 ovoid to conical; glaucous; 3-6 conical; 4-6 x 4-5 obconical; 34 x 4-5 terete or flattened; 3-10 leptophylla 7-t3 fusiform; terete;24 conical;34x34 obconical; 34 x34 quadrangular; 6-8 lesouefii 7 broadly fusiform; 2-6; glaucous conical, ribbed; l0-15 x l0-12 obconical, ribbed; 6-7 - 6-9 slightly flattened or angular mocrocarpa I ovoid; pedicel absent conical-hemi spherical ; hemispherical or obconical; terete, thick; glaucous; 25-35 x2540 15-25 x2540
oldfieldii J globular to ovoid; pedicel absent hemispherical, shortly rostrate; hemispherical; 5-8 x 9-12 terete, thick; 6-12 6-lO x9-12
Continued Table 3.3. Continued Species # budV Bud shape; pedicel Operculum Hypanthium Peduncle (L mm) umbel (L mm) (LxWmm) (L x \ü mm)
orbiþlia 7 globular to ovoid, sometimes shortly conical or hemispherical-conical; hemispherical; 3-5 x 6-8 terete; 10-25 umbonate;4-7 7-13 x 6-8 polyanthemos 7 paniculate; clavate to fusiform, conical; l-2 xto2 obovoid to obconical; 3 x 3 terete; 5-10 glaucous; 2-5
Prutnosa 7 fusiform to pyriform, pruinose; 5-10 conical, sometimes slightly obconical; 4-6 x3-6 sli ghtly fl atten ed to tercte; 12-25 rostrate; 4-6 x 3-6 pyriþrmis J pyriform; thick 8-35 hemispherical, umbonate, ridged; obconical or obpyramidal, ridged; 20-30 x terete, thick, pendulous; 20-65 20-3O x25-38 25-38
siderorylon subsp. 7 ovoid, rostrate; 2-15 conical to rostrate; 3-5 x 4-5 ovoid to hemispherical; 4-6x4-5 7-20 siderorylon
socialis 7 -13 fusiform; 2-8 conical, rostrate; 5-8 x 4-5 hemispherical1' 4-5 x 4-5 slightly fl attened; 8-20 steedmonii 3-7 ovoid or pyramidal, 4-winged; pyramidal; 10-15 x 5-8 turbinate: l0-18 x 8-12 down curved, flattened; 15-30 stoatei I pyriform or turbinate; 2-10 conical, smooth or shallowly pyriform, ribbed (6-10 ribs): flattened, dilated, recurved; 20- ribbed; 5-10 x 8-19 2O-30 x l5-2O 30 stricklandii 7 cylindrical; absent conical; glaucous; 10-15 x 7-10 campanulate; glaucous; 8-10 x 7-10 glaucous, thick, flattened; l0-20 tetragona J clavate, quadrangular, glaucous; hemispherical, striate, cross; 4-angled, toothed at corners; flattened or angular; angular 4-10 34 x6 5-6 x 5-6 5-15
transcontinentalis 7 glaucous;4-7 hemispherical at base, t cylindrical or suburceolate; terete or angled; narrowly rostrate; 8-13 x 5-6 4-5 x 5-6 7-r3 websteriana 7 globular or broadly ovoid; glaucous; hemispherical-conical ; shallow hemispherical; terete; 10-15 angular, 6-8 34 x 4-6 2-3 x3-5
yalatensis 7-tt fusiform;2-3 conical or rostrate; 4-6 x34 obconical or hemispherical; 2-3 x 34 terete or quadrangular; 4-8 younqrana J subglobular; pedicels absent or thick; hemispherical or sometimes hemispherical to turbinate, strongly terete, thick, recurved; to8 conical, strongly ribbed; 20-25 x ribbed; 20-25 x25-25 lo-20 20-35 Table 3.4. characters of all from 1973 1988 Brooker and T Species Fruit(LxWmm) Valves Disc Seed anceps cylindrical, 5-10 x 5-7 level: 3 or 4 descending D shaped; red brown caesia subsp. caesia urceolate; glaucous;20-30 x 18-25 included; 5-6 descending irregular crescent; grey-black eremophila subpyriform or cylindrical; 8-10 x 7-9 exserted; 34 obscure crescent; brown erythronema var. obconical or turbinate, striate; just exserted; 4-5 broad, level or obliquely crescent; brown erythronema 7-12 x lO-15 descending erythronema var. obconical or turbinate, striate; winged byexpanded just exserted; 4-5 broad, level or obliquely crescent; brown marginata horizontal rjm:7-12 x l0-15 descending erythrocorys broadly campanulate, 4-tooth ed: 25 40 x 30-50 enclosed;4 broad, prominently domed pyramid; da¡k brown fotestiana subsp. pyriform, quadrangular, 4-winged; ;3or4 orbicula¡, pyramid, winged forrestiana 30-50 x 15-35 orbicular, pyramid, winged forrestiana subsp. pyriform, quadrangular, 4-winged; ;3or4 dolichorhyncha 30-50 x 15-35 gardneri pyrimidal or cylindrical; 7-8 x 5ó t level; 34 descending round, oval; light brown gillii globular; glaucous; 5-7 x5-7 exsefed;34 descending gomphocephala campanulate or cylindrical, often faintly ribbed; 7-9 x level or slightly exserted; 4 broad, level, convex or ascending round - oval; black 7-8 kruseana cylindrical to obconical; 6-7 x6-7 included or exserted; 34 narrow; descending crescent; brown Ieptophylla hemispherical or subglobular; 3-6 x 3-6 exserted; 3-4 descending elliptic; brown lesouefii hemispherical, ribbed; glaucous; 8-10 x 8-10 exserted; 3 or 4 D shaped, teardrop; dark red brown nøcrocarPa shallowly hemispherical or tubinate; exserted;4-7 ascending orbicula¡ or irregular pyramidal, 30-50 x 50-90 ribbed on ventral surface, narrowly winged; brown oldfieldii subglobular, obconical or hemispherical; lO-12 x L2- exserted;4 ascending irregular crescent or orbicular; l8; usually sessile brown orbiþIia hemispherical or campanulate; pruinose; 6-ll x 12- exserted; 34 ascending irregular crescent; l8 grey-brown polyanthemos obconical to pyriformis, often glaucous;4-7 x3-6 broad pruinosa cylindrical to almost obconical; glaucous when just exserted; 3-5 narrow, descending 7-10 x 6-8 Continued Table 3.4. Continued Species Fruit(LxWmm) Valves Disc Seed pyriþrmis broadly turbinate, strongly ribbed; level or exserted;4-6 steeply ascending irregular pyramidal, narrowly 25-38 x 30-65 winged, ridged on ventral surface; da¡k brown síderoxylon ovoid, subglobular or urceolate; included; 5 subsp. siderorylon 5-ll x 6-10 socialis globular; 5-8 x 5-8 exserted; 34 descending compressed ovoid; grey - dark brown steedmanii turbinate,4-winged; l0-18 x l0-15 exserted; 4 nanow, descending crescent; brown sloatei pyriform or turbinate, strongly ribbed; 25-35 x25-30 :3 pyramid, winged; black stricklandii campanulate, faintly several ribbed; exserted; 3-4 broad, descending crescent; red brown l3-15 x l0-13 tetragona subglobular; glaucous; l2-18 x I l-18 included;4 broad, descending obliquely pyramid; dark brown transcontinentalis globular; 5-8 x 5-8 exserted; 3-4 broad, descended D shaped, elliptic; dark brown websleriana shallow hemispherical ; glaucous ; exserted; 4 ascending, convex irregular crescent: 5-8 x 8-12 red brown yalatensis hemispherical or obconical; 4-5 x 5-6 exserted; 34 broad, descended younStana turbinate or hemispherical, strongly ribbed; 25-38 x exserted;4-6 concave, ascending irregular crescent, narrowly 35-70 winged, ridged on ventral surface: brown-dark brown Table 3.5. Cultivation characters qf species investigated (adapted from Chippendale, 1973; 1988; Brooker and Kleinig, 1990). Species Habit Rainfall Months of flowering; flower colour Soil type Tolerance lmml anceps Mallee to 6m 300-500 24:cream sandy loam - clayey flats drought, frost, coastal, wind
caesia subsp. caesia Mallee to lOm 2s0-500 6-9; pink shallow sandyJoam drought, frost, clay
eremophila Tree to 4.5m 200-500 6-10; yellow, pink drought, frost, coastal, wind
erylhrocorys Tree to 8m 470 3-5; yellow sandy drought, frost, coastal
e ryt hronema vat. ery t hronema Mallee or tree to 6m 300-380 9-l 1; red sandy drought, frost, wind
e rythronemn var. mar ginata Mallee of tree to 6m 300-380 l0-1; red, yellow sandy drought, frost, wind for rest iana subsp. for re s t i ana Tree to 5m 330400 l2-3; yellow drought, frost, wind, saline fo rre s t iana stbsp.dol ichorhyncha Tree to 5m 330400 l2-3; yellow drought, frost, wind, saline
gardnerí Tree to 9m 330-500 5-6; yellow-green sandyJoam, gravelly drought, frost, wind, timber
gillii Mallee or tree to 7.5m 330400 5-10; creamy drought, frost, wind
gomphocephala Tree to 40m 750-1000 2-3; creamy limestone, sandy drought, coastal, saline
kruseana Mallee to 2.5m 200-250 4-7; yellow-green drought, frost
leptophylla Mallee to 5m, tree to 8m 200 3-8; creamy drought, frost, saline
lesouefii Tree to l8m 230-300 l0-12; cream drought, frost, saline
macrocarpa Mallee to 5m 380-500 9-12: red drought, frost
oldfieldii Mallee or tree to 6m 7-9; cream sandy drought, frost, wind
orbþlia Mallee or tree to 6m 230-250 5-8; cream sandy drought, frost, shallow soil
Continued Table 3.5. Continued
Species Habit Rainfall Months of flowering; flower colour Soil type Tolerance polyanthemos Tree to 25m 9-12; white prutnosa Tree to lOm 4-6; cream yellow pyriþrmis Mallee to 4.5m 280400 7-10; red, yellow, peach drought, frost s ideroxylon subsp. s iderorylon Tree to 35m variable 5-10; white, pink, red or pale yellow socialis Mallee to 9m or tree to l2m 7-10; creamy white drought, frost, wind steedmanii Tree to l2m 300 l2-l; yellow drought, frost, coastal, wind stoatei Tree to 6m 4lt l0-3; yellow drought, frost, coastal, wind stricklandii Tree to llm 250-280 I l-l; yellow-green sandy, clay drought, frost, saline, tetraSona mallee to 3m 330-690 ll-l; white sandy drought, frost, coastal, wind
1 ranscontinentalis Tree to 25m 250-380 8-l l; cream drought, frost websteriana Mallee to 6m 250 4-9: cream sandy drought, frost, shallow soil yalatensis Mallee or tree to 6m 300 l2-2; creamy coastal younSwno Mallee or tree to I lm 200-230 8-l; red, yellow, peach sand, sandyJoam drought, frost Table 3.6. Use in and distribution information for all from 1973 198 Brooker and I Species Authority Common name Natural distribution Use in Project Location t2345ABC * :È anceps (R.Br. ex Kangaroo Island W.4., S.4., Williams, northeast to Bendering, southeast to Stirling Maiden) Blakely Mallee Ranges, east to coastal and subcoastal S.4., east to Murray Bridge 32"-37"5, 1160-142"8 * caesia subsp. caesia Benth. Gungumr W.4., central, northern and eastern wheatbelt 31'-33oS, l16'-ll9"E * eremophila (Diels) Maiden W.4., widespread in wheatbelt and goldfields of southwest to east of Zanthus to south coastal plains 29"-34"5, ll5"-124"8 * erythrocorys F. Muell. Illyarrie W.A.; restricted distribution in western coastal areas from Cockleshell Gully in south to northeast ofDongarra 29"-31"S, l10o-ll7oE * erythronema var. Ttrcz. Red flowered W.4., east towards Southern Cross, southern wheatbelt erythronemo Mallee 3l'-3305, l16'-ll9'E * * erythronemo var. Benth. White Mallee W.4., northern distribution compared to above, northern wheatbelt marginata 300-320s, l16"-ll9"E * * forrestiana subsp. Diels Fuchsia Gum W.4., scattered distribution in southern subcoastal areas from northeast forrestiana of Ravensthorpe, eastw¿rÍds to Mt Beaumont 32o-34os, l2l"-123"8 * ,< forrestiana subsp. Brooker Fuchsia Gum W.4., scattered distribution south of Salmon Gums, occurs separate to dolichorhyncha above 32"-33oS, l2l"-122"E + + * gardneri Maiden Blue Mallet W.4., scattered distribution, south of Williams to Ravensthorpe Range, also disjunct occurrence near Cadoux 32"-34"5, I l6o-120'E * giUii Maiden Curly Mallee N.S.W., S.4., restricted and disjunct distribution in Northern Flinders * * Range and Barrier Range north ofBroken Hill 3lo-33os, 137"-139"8 gomphocephala A. DC. Tuart W.4., Jurien in nofh to Ludlow in south, coastal dunes and sub coastal + * plains 32'-37'5, 116"-142"8 * kruseana F. Muell. Bookleaf W.4., restricted distribution east and southeast from Kalgoorlie, from Mallee Cardunia Rock north of Ka¡onie to Binyarinlnna Rock and east of Higginsville, north and northeast ofNorseman 3l'-33"S, 122"-124"8 + leptophylla syn. foecunda N.S.W., S.4., Vic., W.4., dry country of southern Australia from east Schauer Goldhleds to central N.S.W. and Vic., also Mt Lofty foothills, S.A. 27"-37"5, 123"-147"8
Continued Table 3.6. Continued Species Authority Common name Natural distribution Use in Project Location l2345ABC * lesouefii Maiden Goldfields W.4., widespread in the Central and Southern Goldfields, from about Blackbutt [æonora and l¿ke Minigwal in the north, towards Cundeelee in the east, south to the Norseman area and east to the Fraser Range 28'-33'5, l2lo-124"8 * {. nncrocarpa Hook Mottlecah W.4., scattered but widespread occurrence in western part of wheatbelt, * from Piawaning in the north to Wagin Kulin in the south, with an outlier northwest ofBadgingarra on the northern sandplain 280-34'5, ll5'-ll7'E oldfieldii F. Muell. Oldfield's W.4., widespread distribution from the sandplains north of Kalba¡ri * * Mallee National Park and northeast of Wannoo, southeastwards to the Northern Wheatbelt and eastwards to the Great Victoria Desert 26"-32"5, ll2"-127"8 orbiþlia F. Muell. Round leaved N.T., S.4., W.4., scattered and widespread distribution in Northern .f Mallee Goldfields, extending eastwards to northwest S.A. and Central Australia 22" -26" S, 126o -134"8 and 28"-32oS, I I 8o- I 20'E polyanthemos Schauer Red Box N.S.W., Vic., central and Southern tablelands, from Grampian Ranges * *. east to Great Dividing Range and intervening plains 320-38'S, 143"-148'E pruinosa Schauer Silver Box N.T., W.A., Qld., 14"-22"5, 125"-148'E :ß pyriformis Turcz. Dowerin Rose, W.4., scattered distribution in the northern wheatbelt from Morawa in * Pear-fruited the north to Dowerin in the south, more rarely north of Geraldton Mallee 27"-3205, I 150-l l70E * * siderorylon Cunn. Ex. Woolls Red konbark, N.S.W., Qld., Vic., widespread on western slopes and plains of N.S.W., subsp.sideroxlyon Mugga extending sporadically into S.E. Qld, and west of Sydney towards Blue Mountains and down to Wodonga 29"-37"5, l44o-l50oE socialis F. Muell. Ex. Red Mallee N.S.W., Vic., S.4., W.4., widespread distribution * * Miq. 20"-37"5, ll5"-147'E steedmanii C. Gardner Steedman's W.4., pure stands, 80 km east of Hyden 32'-33"5, 119'-120'E * * Gum stoatei C. Gardner Scarlet Pear W.4., restricted distribution east and northeast of Ravensthorpe, to south * * * * Gum of I¿ke 33" 120"-l2l"E Continued Table 3.6. Continued Species Authority Common name Natural distribution Use in Project Location 1234548C stricklandii Maiden Strickland's W.4., scattered distribution south and east of Coolgardie and Kalgoorlie + * * Gum towards Norseman, and east towards Zanthus, and one disjunct occurrence between Menzies and Diemals 30"-33oS, l2l"-l24oB tetragona (R. Br) F. Muell. Tallerack W.A.; sandplains of southwest; disjunct occunence in north to Eneabba 't * * and south from Pingarin to Esperance 32o-35os, 116"-123"8 websteriana Maiden Webster's W.4., Goldfields 29o-33"S, l2l"-123"8 * * Mallee yalatensis Boomsma S.4., scattered and widespread distribution in western S.A. from Elliston 4( and Hinck's Conservation Pa¡k on southern Eyre Peninsula, westwards around southern part 3lo-34os, 125"-136oB younStana F. Muell. L-arge-fruited S.4., W.4., scattered and widespread distribution from north of * * * * :ß Mallee Kalgoorlie eastwards, north of the Nullarbor Plain and throughout the Great Victoria Desert l2l"-136"8
I = Growth trials (Chapter 4) A = Waite Arboretum, Urrbrae, S.A. 2 = E. 'Urrbrae Gem' analysis (Chapter 5 & 6) B = Laidlaw Plantation, Urrbrae, S.A. 3 = Bisectaria and AdnatanZ (Chapter 8) C = Monarto Woodland, Monarto, S.A. 4 = Macrocarpae (Chapter 7) 5 = Postharvest trials (Chapter 9) Plant Materiàl3.2.
Figure 3.1. Eucalyprøs species location maps.
L E. anceps 2. E. caesia subsp. caesia 3. E. eremophila
V f'r H
4. E. erythrocorys 5. E. erythronemavar 6. E. erythronemavaÍ. erythronema marginata
V V V
7 . E. forrestiana stbsp. 8. E. forrestiana subsp. 9. E. gardneri þruestiana dolichorhyncha
V V fr
10. E. giUii lL. E. gomphocephala 12. E. kruseanø
v trr V
13. E.leptophylla 14. E.lesouefii 15. E. macrocarpa
È-l \-/ v
31 Plant MateriÐ13.2.
16. E. oldfieldii 17. E. orbiþlia 18. E. polyanthemos
V
19. E. pruinosa 20. E. pyriformis 21. E. sideroxylon
ç--r V
22. E. socialis 23. E. steedmanii 24. E. stoatei
V V
25. E. stricklandii 26. E. tetragona 27 . E. transcontinentalis
,V È-T V
28. E. websteriana 29. E. yalatensis 30. E. youngiana
V V V
From: Chippendale, (1988): Flora of Australia Volume 19, Myrtaceae, Eucalyptus, Ang ophora. Australian Government Printing Service, Canberra ( 1 988).
32 Figure 3.2. Flowering times of species used
Feb øcePs caesia stbsp. cresia erewphila erythrocorys erythroretm vú.erythrm vú. etytltonema erythronema v æ. mqginalz vú narginalq tbsp Jone s tiøø sfup. fone s ti ana lomstiæø stbsp dolichonþncha Jo n e stima stbsp. d o li c ho nhync ha garùteri Ctilü gomphocephala bnxeru lepøpþlla Iesouefi mærocarpa ruErocarPa oldfieldü orbifoÍia poþmthem Prwnosa pyrilomis si de rory lon stbsp - sí de rory lon sfiry.siderorylm socialis sleedwii sloatei sf¡icklødii letragona trwontìrønlalis websleriM yalatensis
Jul Aug sep Oct Nov
mtuml flowering
(data on flowering times adâpted from Gardnc, 1990; Brmker urd Kleinig, 1990; Chippørdale, 1988) Plant Material 3.3.
3.3. Selection criteria for species chosen
E. anceps
Desirable characters: floriferous; small, coloured buds; mallee habit.
Project use: selection for superior forms for floriculture (Chapter 4).
Horticultural use: garden plant.
E. caesia subsp. caesia
Desirable characters: small, weeping habit; dark red stems; powdery new growth, buds and fruit; soft pink flowers; small leaves; attractive bark.
Project use: pollen donor for hybridisation to more vigorous species to introduce desirable characters (Chapter 8).
Horticultural use: garden plant, floriculture Plate 3.1
E. eremophila
Desirable characters: clusters of buds with long opercula, opening to pink or yellow flowers with sparse, wide spreading anthers.
Project use: pollen donor for hybridisation to introduce desirable characters (Chapter 8).
Horticultural use: garden tree if size can be reduced; moderately showy. Plate 3.1
E. erythrocorys
Desirable characters: terminal flowers with bright red cross-shaped operculum; vivid yellow stamens and bright green buds; variation in tree habit and size, as well as flower size flowering period.
Project use: selection for superior cut flower and garden forms (Chapter 4).
Horticultural use: possible pollen donor for hybridisation with E. tetragona (the only other closely related species with horticultural potential), garden plant, floriculture. Plate 3.1
34 Plant Materiat 3.3.
E. erythronema vaÍ. erythronema
Desirable characters: many dark red buds opening to yellow to dark red flowers; attractive habit and trunk; reported to be a highly promiscuous parent.
Prdect use: female parent of E. 'Urrbrae Gem', used in molecular and morphological analysis
(Chapters 5 and 6).
Horticultural use: pollen donor or female parent in floricultural crossing programs, or development as a garden tree. Plate3.2
E. erythronema var. marginata
Desirable characters: as above
Project use: sister taxa to female parent of E. 'Urrbrae Gem', used in molecular and
morphological analysis (Chapters 5 and 6).
Use: as above Plate 3.2
E. forrestiarza subsp. forrestiana
Desirable characters: pendulous bright red buds with showy hypanthium; short operculum;
yellow stamens; small habit; glossy leaves; some variation.
Project use: selection for superior cut flower and garden forms, postharvest testing (Chapters
4 and 9).
Horticultural use: garden or pot plant, or floriculture. Plate 3.1
E. forrestiarea subsp. dolicho rhyncha
Desirable characters: as above, but with longer operculum.
Project use: as above
Horticultural use: as above
35 Plant Material3.3.
E. gardneri
Desirable characters: masses of green buds and yellow flowers; grey-blue foliage.
Project use: pollen donor for vigour, floriferousness and foliage (Chapter 8).
Horticultural use: garden plant. Plate 3.2
E. siUii
Desirable characters: small roundish grey foliage; numerous small white flowers; good coppicing ability.
Project use: pollen donor to impart vigour, floriferousness and foliage (Chapter 8).
Horticultural use: foliage plant. Plate3.2
E. gomphocephala
Desirable characters: attractive flowers and juvenile foliage.
Project use: putative male parent of E. 'Urrbrae Gem' (Chapters 5 and 6).
Horticultural use: large garden tree. Plate 3.3
E, kruseana
Desirable characters: small habit; green flowers; small round grey-blue-purple leaves; variable form.
Project use: pollen donor to impart foliage and habit characters (Chapter 8).
Horticultural use: foliage production, floriculture. Plate 3.3
E.leptophylla
Desirable characters: compact habit; bright yellow or red buds.
Project use: selection for superior cut flower and garden forms (Chapter 4).
Horticultural use: floriculture or garden plant. Plate 3.3
36 Plant Mater¡al3.3.
E.lesouefií
Desirable characters: attractive buds of various colours and levels of glaucousness; floriferous; precocious; variable tree habit.
Project use: selection for superior cut flower and garden forms (Chapter 4).
Horticultural use: breeding with closely related smaller species, floriculture. Plate 3.3
E. macrocarpa
Desirable characters: large, bright red flowers; glaucous leaves, buds, stems and fruit.
Project use: female parent and pollen donor in breeding programmes (Chapters 7 and 8).
Horticultural use: breeding with closely related smaller species; garden plant or floriculture.
Plate3.4
E. oldfieldii
Desirable characters: small mallee;, medium round yellow flowers, green buds.
Project use: pollen donor to impart tree habit and flower size (Chapter 8).
Horticultural use: garden plant or floriculture. Plate3.4
E. orbifolia
Desirable characters: medium-sized, round, glaucous buds with creamy stamens; heart shaped leaves; long flowering season; attractive bark; variation within species.
Project use: pollen donor or female parent to impart above characters (Chapter 8).
Horticultural use: floriculture or garden plant. Plate 3.4
E. polyanthemos
Desirable characters: interesting grey-green foliage; numerous terminal small white buds; attractive round juvenile foliage.
37 Plant Material 3.3.
Project use: pollen donor to investigate limits to breeding capability, impart terminal flower position (Chapter 8).
Horticultural use: large garden tree. Plate3.4
E. pruinosa
Desirable characters: large terminal clusters of yellow flowers; good coppicing ability.
Project use: pollen donor to impart terminal flower position (Chapter 8).
Horticultural use: foliage production, floriculture.
E. pyriformis
Desirable characters: considerable variation within species for flower shape, size and colour, as well as tree habit.
Project use: breeding with closely related species to impart vigour and floral characters
(Chapter 7).
Horticultural use: breeding, floriculture or garden plant. Plate 3.5
E. sideroxylon subsp. sideroxylon
Desirable characters: attractive pendulous flowers and silvery-grey juvenile foliage.
Project use: pollen donor for flower colour, juvenile foliage (Chapter 8).
Horticultural use: large garden tree. Plate 3.5
E. socialis
Desirable characters: considerable variation within species for leaf size and shape, as well as tree habit and level of glaucousness.
Project use: pollen donor or female parent (Chapter 8).
Horticultural use: foliage production, Sarden plant.
38 Plnnf Mnferiql 3-î-
E. steedmanii
Desirable characters: attractive yellow buds and flowers; small tree habit
Project use: pollen donor (Chapter 8).
Horticultural use: floriculture or garden plant Plate 3.5
E. stoatei
Desirable characters: pendulous bright red buds with showy hypanthium; blunt operculum; yellow stamens; small habiti glossy leaves; some variation.
Project use: selection for floriculture or as a garden plant, postharvest testing (Chapters 4 and e).
Horticultural use: garden or pot plant or floriculture. Plate 3.5
E. stricklandii
Desirable characters: masses of yellow buds on glaucous new growth forming many months prior to flowering.
Project use: putative male parent of E. 'Urrbrae Gem" (Chapters 5 and 6).
Horticultural use: garden plant or floriculture. Plate 3.6
E. tetragona
Desirable characters: glaucous stetrts, buds, flower and fruit; Christmas flowering, small tree habit;, within species variation.
Project use: selection for superior forms for floriculture and garden plants, postharvest testing
(Chapters 4 and 9).
Horticultural use: breeding with closely related species, such as E. erythrocorys, garden plant or for floriculture. Plate 3.6
39 Plent Meferial 3-3-
E. transcontinentalis
Desirable characters: attractive small yellow buds and small round juvenile foliage.
Project use: pollen donor to impart above characters (Chapter 8).
Horticultural use: floriculture.
E. websteriana
Desirable characters: small grey-green round to heart-shaped leaves; small glaucous buds opening to cream flowers; interesting bark; small, neat habit; variation within species; very similar to E. orbifolía.
Project use: pollen donor or female parent for above characters (Chapter 8).
Horticultural use: floriculture or garden plant.
E. yalatensis
Desirable characters: compact habit; blue foliage with contrasting yellow or red stems; many yellow or red buds opening to cream flowers.
Project use: selection for garden plant or for floriculture (Chapter 4).
Horticultural use: garden plant or floriculture. Plate 3.6
E. youngiana
Desirable characters: large flowers with variability in flower and bud size, shape and colour.
Project use: breeding with closely related species for above characters; selection for garden plants and floriculture, postharvest testing (Chapters 4,7,8 and 9).
Horticultural use: garden plant or floriculture. Plate 3. 6
40 Plant Material 3.3.
Plate 3.L A: E. caesir¿ subsp. caesia;B: E. eremophila; C: E. erythro corys ; D : E. forrestiana subsp. /orr es tiana 4l Plant Material 3.3
l¡ Plate 3.2. A: E. erytltronelne var, erythronelno; B: E. erythronema vat. tnarginata,, C: E. gardneri;D: E. gillii
42 Flant [\44úenial 3.3,
:its
oo
*;'.
ü üf
t
{
Plate 3.3. A,: E. gomphocephctla,'B: E. lcruseancL; C: E. leptopltylLa; l): E' lesouefii
43 Plant Material 3.3.
Plate 3.4. A: E. macrocarpa;B: E. old/ieldii; C: E. orbifolia;D: E. polyanthemos
44 Plant Material3.3.
Plate 3.5. A: E. pyriþrmis;B: E. sideroxylon; C: E. steedmanii;D: E. stoatei
45 Plant Material 3.3
ic D
* I ( t I \ ) ! ^.q' I ,' /l I \ \ \ \ t, t a ;l ¡t .x J;
Plate 3.6. A: E. striclclanclii;B: E. tetragona; C: E. yalatensis; D: E. youngiana
46 Plant Material 3.4.1.
3.4. Location of material
3.4.I. The Waite Arboretum
The rWaite Arboretum (a reference collection of trees and shrubs cultivated for scientific, horticultural and scientific purposes) was established in 1924, along with the Waite
Agricultural Research Institute, on land bequeathed to the University of Adelaide by Peter
Waite (Gardner, 1990). The Arboretum consists of 27 ha of public land, located in the foothills of Adelaide (34o58'S 138'38'E) 100-110m above sea level. The average annual rainfall is 625 mm, falling mostly in winter, and the area has a mild climate with little or no frosts and warm dry summers. The soil type varies across the area, consisting mainly of red brown earths, with 25 cm or more of a hne sandy loam topsoil and prismatic structured clay subsoil (Litchfield, 1952). The soil is generally free from gravel or stone, with a pH in the surface horizon of 5-8. A small area consists of mosaics of red brown earths and lime enriched black earths, with a pH of 6-9.
Genera from similar micro climes are planted throughout the grounds, with the Eucalyptus
and Corymbia genera represented by over 800 specimens from 370 species. The various
species from these genera are native to all states, and cover a range of Series not usually
occurring in close proximity in their endemic locations. The mild climate also encourages
long flowering seasons. The potential exists for interspecific hybridisation to occur naturally,
as reproductive barriers like temporal and spatial isolation are minimised. The Waite grounds
have extensive plantings around the perimeter of Australian genera, including Eucalyptus,
Acacia, Banksia, Melaleuca, Dryandra, Hakea, Chamelaucium and Casuarina, the majority
of which were planted around 1990.
47 Plant Material3,4.2.
3.4.2. The Monarto Woodland 'Woodland The Monarto (Figure 3.3), planted between 1974 and 1979, is located on the eastern margin of the South Mt. Lofty Ranges, South Australia (35"58'5 139'25'E) (see
Chittleborough et al,. 1976). The majority of the Woodland is situated along the Bremer escarpment (planted in 1974) and eastwards to Murray Bridge along the South Eastern
Freeway (planted 1975-77). The climate in the region is described as being of a
Mediterranean type (Bureau of Meteorology, 1975) with hot dry summers and cool winters with most of the rainfall occurring during the cooler periods. Rainfall across the area varies, from <300 mm in the north east, to 420 mm along the Bremer esca4)ment, but can be irregular. Prevailing winds vary with season: May-October winds are strong and from the northwest to southwest, November-April winds are lighter and from the southeast or northeast. Intense rainfall can occur during the spring months.
The soils of this area are of reddish brown loamy sand or sandy loam, shallow and very low in organic matter. At around 15 cm there is a sharp break to a red-brown clay layer, 10-20cm thick. Below this is a yellow-brown, highly calcareous layer , grading to bedrock between 50-
100 cm deep. Slight variations are present in this general soil type, frequently with shallower layers of topsoil and clay, resulting in soil with low water holding capacity. The lower lying areas are of differing soil types, one being a fine sandy loam or loam, greyish-brown in colour, shallow, with a highly calcareous subsoil of pale brown clay loam and a schist bedrock. The other soil type exists on the gently sloping plains near Monarto South, consisting of clayey sands or sandy clays to 10 cm and dark reddish brown in colour that may crack in summer. The sub layer is a dark reddish brown medium clay, with carbonate visible form 30 cm grading to a heavy sandy clay at around 100cm. This soil has good water holding capacity.
48 Plant Materia.I3.4.2.
During 1974, 1975 and 1977, rows of varying numbers of plants of a single species were established and monitored to enable the assessment of different species under the local conditions. Genera planted werc Acacia, Agonis, Atriplex, Brachychiton, Callistemon,
Callitris, Casuarina, Eremophila, Eucalyptus, Hakea, Leptospermum, Melaleuca, Myoporum,
Pinus, and Tamarix. No records of provenance of seed were available.
Specimens from the Monarto \ù/oodland came from Plots 3, 7, 8 and 10 (Figure 3.3). Prior to planting, the ground was ripped to a depth of 50 cm. Each planting site was 'bowled' and mulch, consisting of a square of black plastic sheeting, put over the bowl. The tree was planted in the centre of the 'bowl' and watered in. Watering continued during years I and 2, and some inter-row ploughing was carried out, however further maintenance was restricted due to labour costs. Currently, the area is minimally managed, with fire prevention maintenance occurring during spring.
3.4.3. The Laidlaw Plantation
In L997, the section of land containing all Eucalypts and acacias planted by students of the
Department of Horticulture, Viticulture and Oenology as part of the native plant breeding
program was dedicated in the name of Donald Hope Laidlaw AO, in recognition of his work
for the Playford Memorial Trust Scholarship in Horticulture. The Laidlaw Plantation (Plate
3.7) is the site for the eucalypt bud and flower species trials. The soil type and climate are the
same as the Waite Arboretum.
49 Plant Materiat 3.4.
Figure 3.3. The Monarto'Woodland, Callington, South Australia. Areas planted highlighted, plant material from plots 3, 7, 8 and 10 (blue). õ 0 gN.D a Ð o
a o @ +z
t\\il\
?
o N
N -¿ \
50 Ptrant Material 3.4.
Plate 3.7. The Laidlaw Plantation: Eucalyptus cut flower and bud section, October, 1998, corresponds to Figure 4.1. Scale 1:400
51 Selecfinn
Selection at Species Selection for Floral Criteria S pecies level Gene Pool - V/ild and Cultivated populations
Criteria Selection at Genotype Selection for Floral Genotype Level
Hybrid Production through C ontrolled Pollination
H yb rid/Parent Identific ation/C onfirm ation
S uperior Development of Propagation Methods Genotype
Postharvest Testing
PBR Registration
Multiplication and Release to Indu stry
Ornamental eucalypt breeding cycle flow chart
Areas covered by this chapter are highlighted. Selection 4.1.
Chapter Four Selection within species for cut bud and flower production
4.1. Abstract
Nine Eøcalyptus species, recommended by the Australian cut flower industry as species requiring further development, were grown at the Waite Campus in a randomised complete block design with regular irrigation and fertiliser application. Measurements of tree height and trunk diameters, as well as observations of general growth, health and presence of buds and flowers \/ere made every three months. Analysis of the data showed E. lesouefü to be the fastest growing species in both height and trunk diameter, while E. yalatensis was the slowest in height and E. youngiana the slowest in trunk diameter. The remaining species were variable. Insect and disease attack were observed on E. anceps, E. youngiana, E. stoatei and
E. forrestiana, with potentially serious consequences. All species had produced buds by the end of the trial, a little over four years after germination, with the majority producing buds within three years. Seventeen plants from three species showed superior characters across all primary selection criteria, specifically bud size, shape and colour, precocity and floriferousness, and merit further testing for secondary criteria with the aim of selection of named cultivars. These included individuals of the following species: plants numbered 2.2,
2.3,3.1 and 5.2fromE.foruestiana subsp. dolichorhyncha; plants numbered 1.3,I.4,2.3,2.4,
3.I,3.3,4.1,4.4 and 5.1 from E. lesouefii; and plants numbered 1.2,2.1, 3.1 and 5.4 from E. tetragona.
52 Selpefinn 4-2-
4.2. Introduction
Horticultural plant breeding relies on the selection of superior individuals or populations, based on gross morphological or agronomic characters, from either wild or cultivated populations (Woodson, 1991; von Hentig, 1995). Individuals exhibiting extremes of the desired characters are noted, with seed or vegetative material collected for further study. The individual may be grown under a range of conditions to test its response to environmental factors. Selection at species level requires a range of genotypes to be grown and assessed for the desired characters, with genotypes with superior attributes selected for further testing.
Development of eucalypts for forestry (Cossalter, 1996; Davidson, 1996: Matheson et al.,
1996) or ornamental horticulture uses a combination of selection from established plantations and from natural populations. The stock can be grown in seed orchards for further seed production, or vegetative propagation can be trialed. Plantations provide an opportunity for controlled selection: seed from selected parents are grown under specific, often optimal, conditions. This enables those individuals most suited to the conditions, or exhibiting other desirable characters (depending on end use), to be measured, and their characters quantified and compared.
Selection at species level of eucalypts for cut flower and bud production, requires that species meet floral criteria, specifically relating to bud and flower characters. Bud criteria range from
5 mm to 100 mm in diameter, generally with an inverse correlation between bud size and number per stem. They should be brightly coloured (red, orange, burgundy, green) with shaped, textured or ridged operculum and/or hypanthium. Open flower criteria include bright colours, or contrasting with bud, stem or leaf colour. The fruit are also of interest, as are the leaves and stems.
53 Selecfion 4-2-
Selection at genotype level takes selection further, dividing the criteria into primary and secondary levels. Primary level selection criteria are separated into three main categories: flowering, where the focus is on floriferousness, precocity, buds (shape, colour and position on the stem) and flowers (colour); plant form, covering general plant habit (mallee or tree), stems (number, colour, strength and orientation), leaves (size, colour and shape) and growth rate; and plant health, encompassing the plant's response to attack by common insect pests and diseases. Selection at the secondary level focuses on the response of the plant to cultivation (pruning, harvesting, including time to regrowth of commercial length stems with buds or flowers, watering and fertiliser), postharvest testing (sucrose levels and cold storage) and propagation (cuttings, grafting and tissue culture).
Genotypes that exhibit exceptional characters for some criteria, but poor characters for others, could be incorporated in inter and intraspecific breeding programs. The resulting hybrid offspring may combine the exceptional characters of both parents, producing a plant superior to both. Other genotypes may be more suited to production as garden specimens or pot plants.
The aim of this study was to grow a range of species recornmended as suitable for the cut flower and bud industry under similar conditions, and to observe and record their growth and development over a three year period. From these data, selection of species and primary selections of genotypes could be made. Those individuals exhibiting exceptional characters in the areas of flowering, plant form and plant health could go on to selection at the secondary level.
54 Selecfion 4-3-^5-
4.3. Materials and methods
4.3.I Plant Material
Nine species of eucalypts recommended by the Australian native cut flower industry were planted in the Laidlaw Plantation in October 1996. The species planted were E anceps, E. erythrocorys, E. forrestiana subsp. dolichorhyncha, E. leptophylla, E. Iesouefii, E. stoatei, E. tetragona, E. yalatensis, and E. youngiana. A tenth species, E. uncinata. was included, however as the trial progressed the plants of this species bore little resemblance to true to type seedlings, and so it was removed from the analysis. Plants were grown from seed collected
'Western by Nindethana Seed Company, Australia, from unspecified provenances. As the provenance of seed was unknown, it could not be determined whether the trees would be wholly representative of the variation within each species. Despite this, the level of variation expressed within each species in the trial was sufficient that it may applied to the species as a whole.
4.3.2. Planting design
Twenty plants from each species were planted in a randomised complete block design, consisting of five replications of four plants from each species (Figure 4.1). Rows were spaced 3.5 m apart, with trees planted 2.5 m apart, with two buffer rows to the north, east and west and three buffer rows to the south.
4.3.3. Pre-planting treatment
The soil type is uniform across the plot consisting of red brown earth, with 25 cm or more of a
fine sandy loam topsoil and prismatic structured clay subsoil generally free from gravel or
stone, with a pH in the surface horizon of 5-8. As the plot had previously been used for
pasture and cropping, the area was deep ripped to a depth of 0.5 m along the rows, the whole
area was rotary hoed to a depth of 0.3m to break up the soil.
55 Selection 4.3.5.
4.3.4. Irrigation
Irrigation was supplied by 2Llh pressure compensating drippers (Netafim), once monthly for
12 hours for the first summer after planting, every six weeks the second summer after planting, and every eight weeks the third suÍìmer after planting.
4.3.5. Fertiliser
The trees were mulched around the base with oat straw for the first 12 months. Blood and
Bone (Pivot) was applied at the rate of 100 gm per tree every six months
4.3.6. Weed control
A pre-emergent herbicide, Surflan (Agro Sciences), was applied post planting, with applications of Roundup (Monsanto) along the rows each winter to control spring weed growth. The plantation was mown when required.
4.3.7. Plant measurements
At three monthly intervals each tree was measured for height and trunk diameter (10 cm above ground), and lignotuber diameter (if present). Records of the presence of new growth, the appearance of flower buds and flowers, insect pests and diseases were noted at this time.
Data from each individual within a species was compared to identify superior genotypes, while averages from each species were analysed using Analysis of Variance (Genstat 5
Release 4.2. (PctWindows NT, 1997, Lawes Agricultural Trust, Rothamsted Experimental
Station) with L.S.D. used where appropriate.
4.3.8. Weather data
Rainfall and evaporation, hours of sunshine and solar radiation, and maximum and minimum temperatures were recorded daily at the Waite Agricultural Research Institute Weather
56 Selectinn 4-3-^5- station. The monthly averages of these data over 36 months, from March 7996 to March
1999, are displayed in Figures 4.2. to 4.4.
Figure 4.1. The Laidlaw Plantation: layout of eucalypt cut bud and flower section as a randomised complete block design of five sections each containing four plants of each species, with two or three buffer rows. Rows and irrigation run East - West. Figure corresponds to Plate 3.7.
N A B C D E F G H I J K L M N 1 1 2 ) 7 7 3 3 665 5 4 4 6 6 2 2 2 7 7 3 3 665 5 4 4 6 6 3 7 7 5 ) 10 8 964 I 3 7 2 2 4 7 7 5 2 10 8 964 I 3 7 2 2 5 3 3 5 2 10 8 964 I 3 7 3 3 6 3 3 5 2 10 8 964 1 J 7 3 3 7 3 3 4 l0 I 5 726 3 9 8 4 4 I 3 3 4 l0 I 5 726 J 9 8 4 4 9 4 4 4 l0 I 5 726 3 9 8 5 5 10 4 4 4 10 I 5 726 3 9 8 5 5 11 2 2 5 4 3 7 r098 1 2 6 7 7 t2 2 2 5 4 3 7 1098 1 2 6 7 7 13 3 3 5 4 3 7 1098 1 2 6 2 2 l4 3 3 5 4 3 7 1098 I 2 6 2 2 15 2 2 6 3 I 5 9810 4 7 2 5 5 T6 2 2 6 3 1 5 9810 4 7 ) 5 5 L7 2 2 6 3 1 5 9810 4 7 ) 3 3 18 2 2 6 3 I 5 9810 4 7 2 3 3 t9 5 5 9 4 2 I 3107 8 6 5 4 4 20 5 5 9 4 2 I 3107 8 6 5 4 4 2t 7 7 9 4 2 I 3107 8 6 5 7 7 22 7 7 9 4 2 1 3107 8 6 5 7 7 23 J^ 3 3 3 2 2 332 2 J 3 I 1 24 3 3 J 3 2 2 332 2 3 5 I I 25 4 4 4 4 4 4 444 4 4 4 4 4
Key: number indicates species I = E. anceps 6 = E. stoatei 2 = E. erythrocorys 7= E. tetragona 3 = E. forrestiana subsp. dolichorþncha 8 = E uncinataa 4 = E. leptophylla 9 = E. yalatensis 5= E. lesouefii I0 = E. youngiana u All E. uncinata plants were removed from the analysis as not true to type.
s7 35
30 *minyr I
25 *minyr2 o20 3 o minyr g bÒ 3ls {-maxyr I
l0 -_l-maxyr 2
5 maxyr 3
0 Mar Apr May Jun Jul Aug Sep Oct Nov Deo Jan Fcb
Figure 4.2. Monthly averages of minimum and maximum air tempteratures at the Waite Agricultural Research Institute weather station March'96 to March'99
140 I rain yr 'l 120 1
6 I rainyr 100 7 G' 5 g minyr Eao J 4 o -#evap .E 60 & 1 t: -1 d o *evap 40 )
20 1 *erap J
0 0 Mar Apr May Jun Jul Aug Sep Oot Nov Deo Jan Feb
Figure 4.3. Monthly averages of rainfall and evaporation at the Waite Agricultural Research Institute weather station March'96 to March'99
30 12
t< 10 sola¡ rad
E. yrl bb 20 I o o Ê Isolarrad Þ Ë yi2 Ê o É 6 solar rad o 815d yr3 o ë10 4 Ê hor¡rs yr1 ) 5 hours yr2 0 0 --Fhours Mar Apt May Jun Jul Aug Sep Oot Nov Deo Jan Feb yr3
Filg. 4.4 . Monthly averages of hours of sunshine and solar radiation at the Waite Agricultural Research Institute weather station March '96 to March '99 Selection 4.4.1.
4.4. Results
4.4.1. Between species comparison
4.4.1.1. Growth: The averages of the three monthly tree height and trunk diameter for each species are summarised in Table 4.1. E. lesouefil grew at the fastest rate for both tree height (79.3 mm/month) (Figure 4.5) and trunk diameter (2.I mm/month) (Figure 4.6), and achieved the greatest height (mean 3125 mm) (Figure 4.7) and trunk diameter (66.9 mm)
(Figure 4.8) in the 30 month trial. E erythrocorys recorded the second best results for growth in all four categories. At the other end of the spectrum, E. yalatensis produced the lowest rate of vertical growth (39.3 mm/month) and the shortest height (1617 mm), while E. youngiana showed the least trunk thickening (46.0 mm at 1.3 mm/month). E. tetragona produced a thick trunk of intermediate height, and all other species were consistent in their ordering, with little difference between species.
Table 4.1. Summary of data for nine species in the Laidlaw Plantation.
Tree height' Trunk diameter* Species Final range rate Final range rate E. anceps I 805d 650-26s0 42.1' 50.0* 26.8-62.3 1.5"o
E. erythrocorys 2777b 1900-3650 74.7u 64.3" 46.5-74.2 2.0u E. foruestiana 2054d 1200-3300 443.d 55.5b' 36.8-69.7 1.7b"
E.leptophylla l6t7' 750-2250 40.2d 5 1.g'd 32.0-69.8 1.5"d E.lesouefti 3125^ 1800-4000 79.3^ 66.9^ 52.3-82.s 2.tu
E, stoatei 20g5"d r400-2650 53.5b" 52.g'd 3s.4-68.7 1.6"d E. tefiagona 1853d 800-2700 46.P 623ù 46.0-99.8 l.gub
E. yalatensis 1659" 1300-2200 39.3d 49.2"d 38.9-69.0 1.5"d
E. youngiana 2363" 1500-2800 6l.gb 46.0d 33.5-71.3 1.3d F pr: <0.001 <0.001 <0.001 <0.001 S.E.M. 103, I 3.4 2.6 0.1 L.S.D (57o) 288.0 9.4 7.2 o.2 *Data are averages 20 plants for each species, planted in 5 blocks (as replications)
Different superscripts indicate significant differences in a column at P<0.05.
Anova table Appendix 1.1
59 Selection 4.4.2.
4.4.1.2. Flowering: Time to first flowering varied both within and between species
(Table 4.2). Almost half the E. lesouefii specimens had produced macroscopic buds by the first spring after planting, less than two years after germination, along with three E. fotestiana trees. The majority of plants within each species had produced their first buds by the second spring after planting, or less than three years after germination. Only E. erythrocorys took longer, flowering in the third summer after planting (four years after germination).
4.4.1.3. Pest susceptibiliry: The presence of pest and disease problems on each tree were recorded at each measurement period. Insect pests were sawflies (Perga spp.), found in clumps on E. Iesouefii in late spring, although they did not appear to be feeding on this species, and leaf miners (Perthida spp.), feeding on the young new growth of E tetragona.
The most coflìmon pests, and possibly most damaging from a production perspective, were the constant presence of ants and scale (Eriococcus confusus), and the associated sooty mould. E. anceps were highly affected at planting, becoming less susceptible as they grew.
Specimens of E. forrestiana, E. stoatei and E. youngiana became affected during the summer
months, with some plants highly susceptible, while others were not affected.
4.4.2. Selection of superior individuals within species
Over the 30 months of the trial, different levels of variation within species became evident.
Some variation is valuable at the selection stage, as genotypes with more desired characters
can be identified. Individuals within species were allocated to four categories, based on the
primary selection criteria (Table 4.3). Individuals in category I merit further testing for
secondary selection criteria. Individuals in category II and III could be incorporated into
breeding programs; offspring produced would require re- assessment for primary criteria.
Individuals in category IV show no ornamental merit in this trial and should be disregarded.
60 tal lrJ oa om/month 0a mm/month t Þaèuo{æ€ oI p o o ôooooooÔo È p È 9 !r, o o E. øtceps o E. øtceps o á ã ä Þ ês (D (D E. erlhrocorys E. erythrocorys ) o ,r' ùa E(! € I (D E.fonestiøta E. ã Ð forrestiøta o o
ö' I o' E. leptophylla E. leptopþlla ì o a (Ð E Ø (D C) (! lesouefii c) E. øo E. lesouefi (D ?
E. stoalei E. sloalei
E. tetragona E. tetragono
E. yalalensis E. yalalensis
E.youngiøa E-yormgiøa 3500
3000 +8. (mcpes 1I-8. erythrocorys
2500 #8.þrresliana +8. leptophylla +8. lesoueJìi 2000 *E.stoatei eç +8. tebagona Þ o 1500 *E,yalotensls lE.youngiana
1000
500
0 \o \Ô c\ È- r t- a oo æ Ø o\ ô q a o\ ô\ o\ a t èD É I Þo îÐ Iô èú) o o o -g É Ét it i¡ É 'É È H E 5 Þ. Þ. rt) Þ Þ ah E â Ø H = È .h U) at)
Figure 4.7. Progressive tree heights for nine $pecies
70
60 #8. øtceps
+8. erythrocorys s0 *8.þrrestiana +8. leptoplrylla Ë40 g +8. Iesouefi o I .*.8, stoalei E30 +8. telragona *E.yalatensis 20 *E.youngiana
l0
0 \o \o r F- r t'.- 00 oo û ú Or ô ô ô o\ o\ a o\ o o bo t á É o è0 Io o o o PP ù .Ë E 'É É E ÊI 'tr tr ø b. E I É Á .t) E Ø ) F û ) E at) Þ v) .t)
Figure 4.8, Progressive trunk diameters for nine species Table 4.2. Season of appearance of macroscopic buds for nine species in the Laidlaw Plantation.
Species Winter Spring Summer Autumn Winter Spring Summer Autumn Winter Spring Summer totalb 96 96 97 97 97 97 98 98 98 98 99 E. anceps I 3 2 2 2 2 12t20 E. erythrocorys 5 5t20 E. forrestiana subsp 3^ I I 5 3 I 16t20 dolichorþncha E.Ieptophylla 4 13 1 I 20t20 E.lesouefii 9 3 I 2 2 19t20 E. stoafei 2 2 2 6t20 E. tetragona t7 17t20 E. yalatensis 7 I I 2 2 2 18t20 E. youngiana 8 3 3 2 16t20 u Number of plants showing new buds in that season. o Total number of plants for that species showing buds in duration of trial. Table 4.3. Allocation of individuals within species to four primary selection categories. Plant E. anceps E. E. forrestiana E. leptophylla E. lesouefii E. stoatei E. tetragona E. yalatensis E. youngiana number erythrocorys subsp.
1.1 IV III IV III III u II m ilI 1.2 IV III IV il II IV I III il 1.3 IV m II m I II II III il 1.4 IV il III il I III II m III 2.t IV il il m II II I il il 2.2 IV ru I III III II m III II 2.3 IV il I m I III III ru III 2.4 IV ru il m I IV m il IV 3.1 IV ru I III I IV I u n 3.2 IV II IV UI II II m ilI m 3.3 IV il IV il I II il ilI II 3.4 IV II II III II III IV II IV 4.r IV m m m I IV il u m 4.2 IV II IV u ry ry m m IV 4.3 IV m II ilI IV m II m IV 4.4 IV m IV III I il ilI II ry 5.1 IV II IV m I IV u n IV 5.2 IV III I il II il ru III m 5.3 IV II II III II ilI il il m 5.4 IV III IV m IV IV I ilI m [= Genotlpes/plants excel in all primary selection criteria, straight on to secondary selection ||= Genotypes/plants superior in most primary selection criteria, require intraspecific hybridisation to refine genotypes III= Genotypes/plants more suited to amenity selection or interspecific hybridisation IV= Genotypes/plant not suitable for commercial development Selection 4-4-3.
Most within species variation was seen in bud and flower colour, as well as time of flowering and positioning of flowers. For example, E. youngiana plants showed either red or yellow flowers, with hypanthium size ranging from 40 to 100 mm in diameter. The buds of E. lesouefii varied slightly in size and greatly in bud colour - from glossy orange to highly glaucous purple. Out of the twenty E. Iesouefíi plants measured, 12 different genotypes were observed, while very little variation was app¿uent between E. stoatei plants. This highlights the degree of variation within some species and the need for selection at genotype level prior to commercial release.
The 17 plants/genotypes placed in category I may now undergo selection at the secondary level. The plants should undergo rigorous postharvest testing, to assess vase life under a range of different conditions. They should be assessed for suitability to clonal propagation, to enable reproduction of the plant without loss of desirable characters. Finally the plants should be tested for their response to production techniques, such as pruning and harvesting.
4.4.3. Summary of each species
E. anceps Plants growing for this species varied in height and growth rate, but
maintained a mallee appearance. The presence of flower buds was first recorded in spring
'97, with the buds taking at least twelve months to mature and flower. Flower buds were first
produced in leaf axils at the growing tip, but as the branch overgrew, the mature buds were
located within the canopy. After the trial, E. anceps was found not suitable for further testing,
with all plants in category IV. Plate 4.1.
E. erythrocorys Plants for this species grew quickly with single trunks. Appearance of
flower buds was first recorded in summer '99, with buds taking less than three months to
reach maturity and flower. Flower buds were axillary or terminal, but found at the top of the
õ5 Seleclinn 4-4-3- tree. E. erythrocorys shows promise for further development, however the long period to flowering and the large size of the plants are undesirable characters. Five plants were placed in category II, requiring within species breeding and further assessment, with the remaining fifteen plants in category III. Plate 4.1.
E.forrestiarza subsp. dolichorhyncha All plants from this species grew slowly, with uniform shape and single trunks. Flower buds fnst appeared in spring '96, maturing within twelve months. Flower buds were initially terminal, as the branch overgrew, more buds developed in each leaf axil, giving each branch many buds at different stages of maturity.
The buds changed colour from green to bright red prior to flowering. Four plants from this species were suitable for further testing, as they showed superior primary characters. A further five plants were placed in category II and two in category III. The remaining plants were considered unsuitable for as they suffered badly from infestations of scale and sooty mould. Plate 4.2.
E.leptophylla All plants from this species grew slowly in height, but spread out to form low, wide rounded plants. Buds first appeared in summer'97, and took up to twelve months to reach maturity. Buds formed terminally, but as the branch overgrew mature buds were located within the canopy. Plants form this species were considered highly ornamental, but of little use for cut flower and bud production, due to short stems and over growing. All plants were in category III. Plate 4.1.
E.lesouefit Wide variation was observed within this species for tree height and habit, as well as flower bud shape, colour and position. The first appearance of buds was recorded in spring '96, with buds taking up to twelve months to mature and flower. Buds were initially positioned at the end of each branch, but the branches overgrew, leaving the
66 Selecfinn 4-4-3- mature buds within the canopy. Eight plants were considered worthy of further testing
(category I), six in category II and two in category III, with the remaining three plants not suitable. Plate 4.3.
E. stoatei Plants grew moderately fast with uniform shape. Buds first appeared winter
'97, taking over twelve months to mature. Buds were initially positioned at the end of each branch, but the branches overgrew, leaving the mature buds within the canopy. Seven plants from this species were placed in category II, requiring within species breeding and further assessment, the main detraction being the non-terminal placement of buds, however this may change as the plants age. Plate 4.1.
E. tetragona Plants from this species grew slowly, with uniform shape but varying height.
The plants quickly became top-heavy and leaned over. Buds first appeared in spring '97, maturing and flowering within three months. Buds were terminal. Four plants showed exceptional characters during this study, and were placed in category I. A further seven plants have potential, but may be more suited to within species breeding. Plate 4.4.
E. yalatensis All plants grew into low, rounded mallees. Buds fkst appeared in spring '97, maturing within twelve months. Buds were initially positioned at the end of each branch, but the branches overgrew, leaving the mature buds within the canopy. All plants were considered highly ornamental, with four plants requiring within species breeding and re-
assessment prior to further testing for cut bud or flower production, The remaining plants
were placed in category III. Plate 4.5.
E. youngiana Plants grew quickly, with a single trunks or gtowing from a lignotuber
with three or four trunks. The first buds appeared in spring '97, taking up to twelve months to
67 Selecfinn 4-4-4- mature, with additional buds maturing to flower in less than six months. Buds varied in position, commonly low on the branch. Five plants were placed in category II, with potential for breeding and selection for cut buds and flowers. Nine plants would be suitable for ornamental production, with the remaining six plants suffering severely from scale. Plate 4.5.
4.4.4. Further testing
Other species with ornamental potential, not included in this study, but which merit further testing are listed in Table 4.4.
Table 4.3. Eucalyptus species with ornamental merit suitable for further testing. E. orbiþIia E. macrocarpa E. corrugata E. bunacoppinensis E. ewartina E. websteriana E. rhodantha E. synandra E. orymitra E. pachyphylla E. preissiana E. pyiformis E. campaspe E. cocinna E. lane-poolei E, kruseana E. erythroncma E. tetraptera E. megacarpa E. drummondii E. gillii E. stricklandii E. pruirwsa E. eudesmoides E. kingsmillü E. crucis E. caesia E. rameliana E. nutans E. coronata E. clelandii E. oldfieldii E.leptopoda E sessi/is
68 Selection 4.4
I
4t ç 4
,s t r¡ f-. I ç 1.t t t .t u 'tÌ. al . 4ó { l-l -t, l^ "t ¿' h, 'ì .rl / å ,l t Þ a T,
t t.l {
¡
J Plate 4.1. A: E. anceps (3.3);B: E. erythrocorys (5.1); C: E.leptophylla (Plant 1,4); D: E. stoatei (Plant2.2)
69 Selection 4.4
Plate 4.2. A: E.forrestiana subsp. dolichorhyncha (2.2);B: E. fotestiana subsp. dolichorhyncha (2.2); C: E.forrestiana subsp. dolichorhyncha (3.1); D: E.forrestiarea subsp. dolichorhyncha (3.1).
70 Selection 4,4
{
\
I
Plate 4.3. A: E. lesouefii (1 4 ), B: E. lesouefii (3.3 ) C: E. lesouefii @.Q;D: E, Iesouefii (5.1),
7l Selection 4.4
C
a ¡\ tt, b ú Ji Á'7 g I ì I f t I j :{ 4 ç¿
t
Plate 4.4. A.: E. tetragona (1.2); B: E. tetragona (2.I) C: E. tetragona (3.1); D: E. tetragona buds (Plant 5.4)
72 Selection 4.4
Plate 4.5. A: E, yalatensis (a.1); B: E. yalatensis (4.4) C: E. youngiana (5.2).
73 Selection 4.5.
4.5. Discussion
The nine species trialled in this study were recommended by the Australian cut flower industry as species with merit for cut bud and flower production. Both species and individual genotypes within species were on trial for their suitability for cut flower and bud production.
Limits of space required that 20 plants per species were grown, selected from seed grown material of unknown provenance. In recognition of the small population size and potential for limited genetic variability within the trial, it was not considered to be a full trial for selection of plants for seed production, but rather an example of how a full trial might show variability and potential for genetic gains based on selection.
The results of the trial showed that while some species, such as E. tetragona and E. forrestiana subsp. dolichorhyncha, have considerable merit, others, such as E. anceps, do not, under the conditions of the trial. These species may grow differently in other climates and soil types. The trial showed the variation in morphology that may be seen in seed grown material, highlighting the need for clonal propagation of selected forms. The observed variation within species of bud (consisting of peduncle, pedicle, hypanthium and operculum) size and colour, as well as flower (stamen) colour, gives positive evidence that selection of superior genotypes is required prior to the release of eucalypt species for both floriculture and amenity horticulture, to ensure the most economically and commercially viable variety is grown (Sedgley, 1998).
The growth rates and final recorded heights and trunk diameters reflect the natural habits of each species: E. lesouefü is a single stemmed tree reaching 18 m in height; E. erythrocorys, E. forrestiana and E. stoatei also have single trunks, but generally are small trees up to 5 m in height; E. tetragona, E. leptophylla and E. youngiana can be mallees or small trees (from 3 to l1 m); and E. yalatensis and E. anceps are mallees up to 6 m. The mallee type species used in
74 Selection 4.6. this trial were as wide as they were high, with multiple trunks, thus trunk diameter for these species was generally narrower than that of the single stemmed tree species.
Information on growth rates has important implications for selection of superior genotypes of cut flower species. Tall fast growing species, such as E. lesouefíi and E. erythrocorys, will require different management and spacing compared to low growing, multi stemmed mallee types, such as E. yalatensís and E.leptophylla.
4.6. Conclusion
Species suitable for cultivation under the trial conditions were: E. erythrocorys, E. forrestiana subsp. dolichorhyncha, E.Ieptophylla, E. lesouefii, E. stoatei, E. letragona, E. yalatensis and
E. youngiana.
Superior individuals were identified of some species: plants numbered 2.2,2.3,3.I and 5.2 from E. forrestiana subsp. dolichorhynchai plants numbered 1.3, 1.4, 2.3, 2.4, 3.1, 3.3, 4.1,
4.4 and 5.1 from E.lesouefi¡; and plants numbered 1.2,2.1,3.1 and 5.4 from E. tetragona.
75
E-'f lrrhree Gem'RAPD analvsis 5.1.
Chapter Five
Eucølyptus'IJrrbrae Gem' : Parentage determination through RAPD analysis
5.1. Abstract
The parentage of the ornamental hybrid Eucalyptus 'Urrbrae Gem' was investigated using
RAPD-PCR, and analysed using hierarchical and non-hierarchical distance methods. The
female parent was known to be E. erythronema var. erythronema,blt previous opinion placed
the male parent as either E. stricklandii or E. gomphocephala, based on adult morphological
characters. Samples of DNA from different individuals within each species were amplified
with six different 10-mer primers to produce RAPD fragments. These were used to generate a
UPGMA dendrogram based on similarity, and an ordination derived by multi-dimensional
scaling (MDS) and a minimum spanning tree (MST) to show the relative dissimilarities
between the individuals tested. The dendrogram divided the samples into three clusters, with
the hybrid slightly closer to E. stricklandii than to E. gomphocephala. The MDS ordination
and MST placed the hybrid between E. erythronema vat. erythronema and E. stricklandii,
supporting E. stricklandii as the male parent.
5.2. Introduction
E. 'Urrbrae Gem' is a spontaneously occurring interspecific hybrid (Kelly, 1969; Gardner,
C.4., 1979). The single specimen was discovered in 1936 by the then head gardener, F. A.
Cousins, growing within seed collected from a young E. erythronema Turcz. var. erythronema specimen (Gardner, unpublished, 1987). The seedling differed . in leaf morphology, so Cousins kept it separate and planted it in the Waite Arboretum. The male
76 6Urrbrae ^8. Gem' RAPD analvsis 5.2.
parent was identified as E gomphocephala DC by L. Pryor (Parks and Gardens, Canberra),
based on observation of bud, flower, leaf and fruit morphology (Gardner, unpublished, 1987).
This was later amended to E stricklandii Maiden, with characters such as leaf size and shape,
flowering times, fruit peduncle and pedicel shapes and operculum size and shape, and
proximity to pollen donors being specifically noted as diagnostic (Pryor, unpublished, 1992).
The presence of pith glands in E 'Urrbrae Gem' was considered another diagnostic feature by
Brooker (unpublished, 1991), as they are absent in E. gomphocephala and present in both E
erythronema and E. stricklandü (Brooker and Kleinig, 1990).
E 'Urrbrae Gem' is now a medium sized tree with a single trunk of varying colour, and a
wide-open canopy of glossy green leaves. The specimen is over 60 years old, and still
growing well, despite the close proximity of a mature Bunya Bunya Pine. The bark varies
from white to pinkish brown, depending on the time of year. The spectacular clusters of
reddish blossoms appear in early summer and remain through to late summer. The umbels
have up to seven greenish-yellow buds, appearing up to 18 months before anthesis. Open
pollinated seed from the tree is viable, however, seedlings generally exhibit poor form and
low vigour. A number of seedlings from E. 'Urrbrae Gem' have been planted in the Waite
Arboretum over the last 50 years, however, only one adult specimen remains. Aerial rooting
was attempted in 1958, apparently with no success (Gardner, unpublished, 1987), as was
cutting propagation and tissue culture in l99l (Mc Laughlin, 1991).
The study to identify the parents was problematic, as previous studies of adult morphology
had indicated that both E. stricklandii and E. gomphocephala shared adult morphological
characters with the hybrid. Furthermore, due to the length of time involved, none of the original parent material was available for analysis. Representative specimens from each
species involved were required to generate DNA furgerprints indicative of the genetic makeup
77 E- ¡llrr]rrae Gemt RAPD analvsis 5-3.1.
of each for comparison to the hybrid. Other eucalypt species gro.'wing nearby at the time were
discounted as possible pollen donors/parent species, either on the basis of their floral and leaf
morpholog), or due to their flowering period not overlapping with that of E. 'Urrbrae Gem' or
E. erythronema. The ornamental appeal of the tree makes it an ideal candidate for
horticultural development, but prior to registering it under the International Convention of the
Union of New Plant Varieties (UPOV), both parents should be known. A thorough study of
the hybrid, the female parent species and both potential male parent species, was required to
clarify paternal lineage prior to further development.
Recently, random amplified polymorphic DNA (RAPD) (Williams et a1.,1990) has been used
in a number of eucalypt studies, including investigating genetic distance (Chen and De
Filippis, 1996), pedigree analysis (Vaillancourt et aL,1998), generating genetic linkage maps
(de Silva and Grattapaglia, 1997; Brondani and Grattapaglia, 1997), and identification of
parentage of hybrids (Rossetto et aI., 1997). In view of the superiority of E. 'Urrbrae gem'
for ornamental horticulture over the known or putative parents, this study aimed to use RAPD
analysis to determine parentage. The parents could then be included in the University of
Adelaide Eucalyptus improvement program (Wirthensohn et a1.,1999).
5.3. Materials and Methods
5.3.1. Plant material
The original female parent tree and the possible male parent trees had been removed from the
Arboretum prior to the commencement of the present study so that none of the original parent material was available for DNA fingerprinting. Alternative specimens from different provenances were sourced to be representative of the species involved. The original female parent specimen, planted in 1931, was removed from the Arboretum in 1983. The possible
78 E- rllrrbrae Gemt RAPD anelvsis 5-3-1-
male parent specimens of E. gomphocephala in the Arboretum were located nearby
(Figure5.l) and were both planted in 1928. Others were planted in 1934 and 1937. The
majority died in the 1940s, with the last in 1996 (Table 5.1). E. stricklandii was also present
close to the female parent, and was planted in 1931 (Figure 5.1) and removed in 1969. Thus
none of the original parent material was available for DNA fingerprinting. Representative
trees from each of E erythronema var. erythronema (five trees), E. stricklandii (five trees), E
gomphocephala (fle trees), E. erythronema var. marginata (three trees) and E. 'Urrbrae
Gem' (one tree) were selected for DNA extraction. The 19 individuals used were sourced
from the Waite Arboretun¡ the Waite Campus plantings and the Monarto Woodland, and are
listed in Table 5.2. Taxonomy follows the informal classification of Pryor and Johnson
(1971) and all species are from subgenus symphyomyrtus sect. Bisectaria.
Table 5.7. Eucalyprøs species located near the female parent of E. 'Urrbrae Gem', Waite Arboretum, 1955. Applies to Figure 5.1.
Number Species Planted Removed t867 E 'Urrbrae Gem' t936 157 E. calophylla t928 r72 E. calophylla t928 178 E. calophylla 1928 198 E. calophylla 1928 42 E. camaldulensis original 28 E. cladocalyx 1915 4t E. cladocalyx 1915 27 E. conica 193 l 104 E. erythronema var. erythronema t93t 1983 23 E. diversiþlia t928 188 E. ficiþlia 1928 JJ E. gomphocephala 1934 1940 36 E. gomphocephala t934 t942 38 E. gomphocephala t937 l96l 44 E. gomphocephala 1928 1942 48 E. gomphocephala t928 t996 636 E. leucorylon original 34 E. maculatq 1928 30 E. megacarpa 1928 40 E. moluccana 1928 32 E. sideroryIon 46 E. siderorylon 43 E. stricwandii l93l 1969 26 E. terminalis t928
79 .8. ¡Urrbrae Gem'RAPD analvsis 5.3.1.
Figure 5.1. The V/aite Arboretum (1955): section of arboretum showing layout of plantings near the female parent of E. 'Urrbrae Gem'. Apptes to Table 5'1.
E. erythronema E. gomPhocePhala :ü, f. stiicklandit Other Eucalypløs species Right side is Claremont Avenue, running East-West.
80 E. 'Ilrrbrae Gemt RAPD analvsis 5-3-2-
Table 5.2. Individuals used in the E 'Urrbrae Gem' analysis. Taxonomy follows the informal classification of Pryor and Johnson (1971) and all species are from subgenus Symphyomyrtus, sec. Bisectaria.
Species Code # Study Specimen # Loca- Seed source; tion provenance E. 'Urrbrae Gem' Fl FI MSA r867 WaA WaA tree 104; unknown E. 'Urrbrae Gem' open pollinated FIA SA 67 WaA WaA ree 1867; unknown Series Er¡hronemae E. erythronema var. marginata Eem2 M A 24 WaA A. Southcott, W.A.; unknown E. erythroncma vaÍ. marg,nata Eem3 MS 7.2 MoW unknown; unknown E. erythronema var. marginata Eem4 MSA 1841A WaA unknown; unknown E. erythronema var. erythronema Eee5 MSCPA 7.t MoW unknown; unknown E. erythronema var. erythronema Eee6 MA 7,4 MoW unknown; unknown E. erythronema var. erythronema EeeT MCPSA 7.2 MoW unknown; unknown E. erythroncma var. erythronentt Eee8 MCPSA 7.3 MoW unknown; unknown E. e ry thronema v ar. e ryt hronema Eee9 MA 7,4 MoW unknown; unknown Series Grossae E. stricHandii Es10 MCPSA t614 WaA F. Law Smittr, S.A.; unknown E. stricmandii Esl I MSA 2B V/aI unknown; unknown E. stricHandíí Esl2 MSA 2C WaI unknown; unknown E. sticHandii EsI3 MSA 3X WaI unknown; unknown E. sticHandii Esl4 MSA 3Y WaI unknown; unknown Series Cornutae 'Woods E. gomphocephala Egl5 MCPSA 1611 WaA & For, S.A.; unknown E. gomphocephala E916 M 8.1 MoW unknown; unknown E. gomphocephala EsIT MSA 8.2 MoW unknown; unknown E. gomphocephala Es18 M 8.3 MoW unknown; unknown E. gomphocephala Egl9 MSA 8.4 MoW unknown; unknown Series Reduncae E. gardneri Ega2Ù A 2025 WaA W.A. For. Dept.; unknown E. gardneri Ega2l A 10.4 Mo\ù/ unknown; unknown M = specimens used in RAPD analysis (Chapter 5) CP = specimens used in controlled pollination (Chapter 6) S = specimens used in seedling morphology (Chapter 6) A = specimen used in adult morphology (Chapter 6).
5.3.2. DNA extraction
Young, fully expanded leaf material was collected, petioles and midribs discarded, and the laminae immediately stored at -20oC until required (up to 4 weeks). DNA was extracted using the method of Doyle and Doyle (1990), modified as described by Steenkamp et al.
81 .Irrhrqe E- 'f êcrnt RÀPf) snnlvsis -5-3^2-
(1994) and Wirthensohn et aL (1999). Approximately 2.0 g of frozen leaf lamina was ground to a fine powder in liquid nitrogen in a chilled mortar and pestle. The ground leaf tissue was mixed in 9 ml pre-warmed3Vo CTAB containing l%o mercaptoethanol and 4Vo PVP-40T, and incubated at 65oC for thirty minutes, with inversions every ten minutes. After incubation, the tubes were placed on ice and extracted once with twice volume of choloform/isoamyl alcohol
124:l w:vl by gently mixing for thirty minutes on a spinning wheel, followed by centrifugation (swinging rotor type) at 3000 rpm for thirty minutes at room temperature. The upper aqueous phase was transferred to a clean tube using a wide bore pipette, a 213 volume ice-cold isopropanol was added to precipitate the DNA, and the tubes were stored at -20"C for sixty minutes to enhance precipitation. The mix was centrifuged at 4000 rpm for twenty minutes (room temperature) to collect DNA. The upper aqueous phase was removed, and the
DNA pellet washed for sixty minutes in 5 mL wash buffer [76Vo ethanol and 10 mM NII¿Ac] until clean, centrifuged again for ten minutes at 4000 rpm (room temperature) then the wash buffer removed. One mL cold TE buffer [0 mM Tris-HCl, 1 mM EDTA (pH 7.40] was added to the DNA, which was allowed to dissolve prior to adding 2 ¡tL of 10 mg/ml RNAse and the DNA solution incubated at37"C for 30 minutes. The solution was transferred to a 50 mL plastic tube,2 mL TE buffer and 1 mL 7.5M NH¿Ac added and placed on ice for twenty minutes (to precipitate proteins), then centrifuged at 10 000 rpm for twenty minutes at 4"C.
The upper aqueous phase was transferred to a clean, sterile glass corex tube and 2 volumes of ice-cold ethanol added, mixed slowly, placed on ice for twenty minutes, then centrifuged at
8000 rpm at 4"C for 10 minutes, allowing the DNA to collect on the sides of the tube. The ethanol was poured off, and the tube inverted and allowed to air dry. The DNA was dissolved in 1 mL TE buffer and placed in 1.5 mL plastic centrifuge tubes for storage at -20"C until required. DNA concentration was estimated by visual assessment of band intensities, compared to olive (Olea europea) genomic DNA of known concentration (Mekuria et aI.,
1999) and adjusted to 20 ngl¡L. DNA samples, plus the olive DNA, were placed on a I.ïVo
82 E- 'Urrbrae Gemt RAPI) analvsis 5.3-3.
agarose gel (Seakem GTG FMC BioProducts) in I x TBE buffer (Sambrook et a\.,1989) and
run for thirty minutes at 80 milliamp. Gels were stained with ethidium bromide for twenty
minutes and photographed under UV light with Polaroid 667 film. The DNA bands were
compared to the intensities of the bands of known concentrations, and each sample diluted to
20 nglrnl.
5.3.3. RAPD analysrs
The optimised PCR reactions consisted of 1 x Taq buffer (Gibco-BRL, Gaithersburg), 3 mM
MgCl2, 200 pM each of dGTP, dATP, dCTP, dTTP (Promega, Madison), I pM lO-mer
oligodeoxy ribonucleotide primer (Operon Technologies, Alameda), 1 unit Taq polymerase
(Gibco-BRL, Gaithersburg), 0.5 FL T¿ Gene 32 Protein (Pharmacia Biotech, Upsala) and 20
ng of genomic DNA in a 25 pL volume. One drop of PCR grade paraffin oil was overlaid
each on mixture. DNA amplification was performed on a PTC-100rM Programmable Thermal 'Watertown), Controller (MJ Research, following the protocol used by Wirthensohn et al.
(1999): initial denaturation at 94oC for two minutes, followed by 4l cycles of 94oC for one
minute, 36oC for one minute, 72oC two for minutes, and a final extension at 72oC for fle
minutes.
DNA amplification fragments were separated by electrophoresis on 2Vo agarose gels (Seakem
GTG FMC BioProducts, Rockland) using 1 x TBE buffer (Sambrook et al. 1989). Gels were
stained with ethidium bromide and photographed under UV light with Polaroid 667 film. A
negative control of all PCR reagents, but omitting DNA, was added in each run to test for
contamination.
Eighty primers were evaluated for their suitability in a pilot survey (series OPA, OPB, OPC
and OPD, Operon Technologies, Alameda) with one representative DNA sample for each of
83 .8. 'Urrbrae Gem' RAPD analvsis 5.3.4. the three species groups. From this survey, six primers were selected that showed clear polymorphisms and gave reproducible banding patterns (OPD-06 ACCTGAACGG, OPD-10
GGTCTACACC, OPD-11 AGCGCCATTG, OPD-14 CTTCCCCAAG, OPD-16
AGGGCGTAAG, OPD-19 CTGGGGACTT). The selected primers were tested on all 19 samples using duplicate PCRs and two gel runs of each separate PCR to test for reproducibility. Bands that were consistently amplified over all reactions were scored.
The gels were scanned directly under UV light using CreamrM for WindowsrM Version 1
Revision A (Kem-En-Tec A/S, 1995) and the image saved in WindowsrM bitmap format for analysis using DNA Simdex 2.0 (Archer,1996). Within lanes on the gel, band mobilities were calculated relative to the mobility of the 600 bp band marker from the DNA ladder (Ready-
LoadrM 100 bp DNA Ladder, Gibco BRL, Gaithersburg) to compensate for differences in absolute mobilities within and between gels. RAPD fragments were scored as present (1) or absent (0) for all individuals, and the data recorded in a binary matrix.
5.3.4. Hierarchical distance analysis
Genetic similarities among all pairs of individuals was estimated using the hierarchical distance method of simple matching coefficient, SMC, (Sokal and Michener, 1958). Cluster analysis was performed on the estimated similarity using the unweighed pair group method with arithmetic average (UPGMA) and the resulting clusters were expressed as a dendrogram using the SAHN algorithm in the computer program NTSYS-pc (Exeter Software) (Rohlf,
1993).
A similarity matrix was calculated using the simple matching coeffrcient (SMC). The SMC algorithm is preferred over Nei's genetic distance index (Nei and Li, 1979) as the group of individuals from the three different species are highly heterozygous, and with most RAPD
84 l1- rlJrrhree Gemt RAPD analvsis 5.3.4- markers, heterozygous individuals cannot be distinguished from homozygous dominant individuals (Bartolozzi et al., 1998). The similarity coefficients from the simple matching algorithm were used to cluster individuals (SAHN) using UPGMA, and a dendrogram constructed. This method has been used to study genetic variation in garlic (Bradley et al.,
1996), beans (Skroch et a1.,1995) and collards (Farhnam, 1996); also to study intra and inter- specific variation in Lens (Abo-elwafa et a1.,1995); and for identification and characterisation of woody perennial cultivars of almond (Bartozzi et al., 1998), peach (Warburton and Bliss,
1996), grapevine (Loureiro et a1.,1998) and citrus (Federici et a1.,1998). SMC has also been used in fingerprinting studies of Eucalyptus globul¿¿s clones (Nesbitt et al., 1997) and
Eucalyptus nitens ramets (Vaillancourt et a1.,1998).
5.3.5. Non-hierarchical distance analysis
The PATN computer analysis program (Belbin, 1994) was used for the non-hierarchical distance multivariate analysis with multidimensional scaling. An association matrix between the objects (ASOs) was fnst calculated using Gower's metric, a standardised Manhattan
Metric, which generated a matrix showing dissimilarity between individuals or operational taxonomic units (OTUs). Gower's metric, when used on presence/absence data, corresponds to the simple matching coefficient (Belbin, 1994), and considers a (0,0) as evidence of homology, and provides nearly equivalent information to the (1,1) comparisons.
Multi-dimensional scaling (MDS) takes the matrix of ASO dissimilarities and orders the individuals in two or three dimensions such that it is the ordering of the input dissimilarities and dissimilarities in the 2 or 3 dimensional ordination space which is maintained. Semi- strong hybrid multi-dimensional scaling (SSH MDS) was used to ordinate ASO dissimilarities. SSH MDS aims to find the minimum value of stress (i.e. distortion of the points away from their original interdistance dissimilarity, and a value less than 20Vo is
85 ^8. 'Urrbrae Gem'RAPD analysis 5.3.5.
considered satisfactory (Belbin, 1994). The stress level was recorded, and the lowest
dimensionality with stress less than 20Vo adopfed. The resulting ordination points were
plotted as a 3 dimensional scatter plot (Sigmaplot@ 4.0 for Windows@, SPSS), to enable
clearer visualisation of the groups of individuals in relation to each other.
Multidimensional scaling (MDS) and ordination of RAPD data enables visualisation of
potential patterns of grouping. Many studies have shown that ordination methods involving
hybrids generally place the hybrid between clusters of individuals containing the parent
species (see McDade, 1997), and when using MDS and ordination, hybrids generally fall
between their parents. Principal component analysis (PCA), based on similarity coeffrcients,
is the most common method used, and is documented on studies of beans (Skroch et al.,
1995), plum cultivars (Ortiz et al., 1997) and lentils (Sharma et al., 1995). PCA is also
documented for use on eucalypts: Sale et aL (1996) studied differentiation between two
Eucalyptus species (8. risdonii and E. amygdalina) and their hybrids, Costa de Silva and
Grattapaglia (1997) studied the relatedness of E. urophylla elite clones, and Nesbitt et aI.
(1995) studied variation between different subspecies of E. globulus. However, PCA is
affected by non-linear or heterozygous data (Shi, 1993) and Belbin (1994) states that SSH is
preferred to principal component analysis and other multi-dimensional scaling programs.
Nicolle and Conran (1999) used SSH to study variation in the E. flocktoniae complex and
Cayzer et al. (1999) used it to revise Bursaria (Pittosporaceae).
Minimum Spanning Tree (MST) analysis is an option of PATN that enables the generation of
a set of lines (from the ASO matrix) that minimally interconnect all the objects in the analysis by their relative distances in multidimensional space (provided that all objects have at least one connection, there are no circuits or loops and the network or tree is minimal length)
(Belbin, 1994). MST is complementary to ordination, and individuals that are linked directly
86 Æ .f lnnlrrqo êamt PÀPÍl qnqlwcic 5 /. 1
are more similar than those not directly linked, or those linked by connection. Because the
numbers are 'distances', not actual lengths, they can only be used to indicate links and
connections, instead of definite directions or real scales. The ASO matrix was used to
generate MST linkages between all objects in the analysis.
5.4. Results
5.4.1. RAPD analysis
PCR, using six primers on 19 individual samples of DNA, produced a total of 561 RAPD
fragments of which 27.5Vo were monomorphic, 57.4Vo were polymorphic and l5.2Vo unique
to the different species groups (Table 5.3). As this study examined three different species groups, monomorphic bands were those present in all individuals within a group and polymorphic were those bands present in at least one, but not all, individuals within a group.
Unique bands were those present in at least one individual in a group and not present in any other groups. Only four bands (L.gEo) were present in all individuals rested. With the exception of E. 'Urrbrae Gem', all other taxa showed similar numbers of monomorphic, polymorphic and unique bands. Further observation of the data matrix indicated that most bands were present in combinations that included E. erythronema var. erythronema and E. erythronema var. marginata, and either E. stricklandii or E. gomphocephala, or both. Bands coÍrmon to E. stricklandii and E. gomphocephala rarely appeared. E. 'Urrbrae Gem' showed
60 scorable bands; of these 22 were present in all other species, 14 were present in the hybrid and both varieties of E. erythronema, and three were unique to the hybrid. Eight bands were common to the E. 'Urrbrae Gem', both varieties of E. erythronema and E. stricklandii. Tbuee bands were contmon to the E. 'Urrbrae Gem' and E. stricklandü only. Two bands were conìmon to the E. 'Urrbrae Gem' and E. gomphocephala. Five bands were common to the E.
'Urrbrae Gem', E. stricklandii and E. gomphocephala but neither variety of E. erythronema.
87 E. 'Urrbrae Gem' RAPD analvsis 5.4.2.
Table 5.3. Species used in the RAPD analysis. Pooled primer data showing the number of monomorphic, polymorphic and unique bands for each group.
Species Grp # frees in Monomorphic Polymorphic Unique samples bandsb bands" bandsd E 'Urrbrae Gem'u I I ffi 3
E. erythronema var. marginata 3 5 26 80 L7
E. erythrorcma var. erythronerna 2 J 25 87 t2
E. strtcHandii 4 5 17 90 27
E. gomphocephala 5 5 26 65 26
total l9 r54 322 85
" E. 'Urrbrae Gem' is a single individual, thus all bands belong to each category. b Monomorphic bands: present in all individuals in a group. " Polymorphic bands: present in some individuals in a group. o Unique bands: present in one group only.
Bands common to E. stricklandii and E. 'Urrbrae Gem' occurred at approximately 620 bp for primer OPD-ll and at450 bp and 510 bp for primer OPD-14 (Plate 5.1). Other primers,
OPD-10 and OPD-19, are shown on Plate 5.2.
5.4.2. Hierarchical distance analysis
The dendrogram showed three clusters (Figure 5.2). The five genotypes of E gomphocephala formed a cluster distinct from all other genotypes with a genetic similarity of 65Vo to the other
two clusters. The five genotypes of E stricklandü also formed a cluster with a similarity of
70Vo to the third cluster that contained the five genotypes of .E erythronemo, var. erythronema,
three of E erythronemavat. marginata and E. 'Urrbrae Gem'.
Within the genotypes of E gomphocephala, the maximum genetic distance was about 187o,
suggesting that this species has a relatively narrow gene pool in the areas from which the
samples were collected. Four of the samples of E. stricklandü also show a relatively small degree of genetic variability, although one that was collected from the Waite Arboretum
showed a25Vo genetic distance from the others. In contrast, E. erythronema showed high
88 E.'Urrbrae Gem'RAPD analvsis 5.4.1
A 1 2 3 4 5 6 7 I 9 10 111213141516 17 1819 201
1500 bp $rt---- 1500 bp ¿1.- -Çrirr- Ça .- --=--ll- 620 bp 620 -.----O- rll¡r¡roÓf 600 -
B 12 34 5 6 78 9101112 131415 16 17 181920 1
1500 bp 1500 bp
600 bp 600 bp 510 510 450 450
Plate 5. L Agarose gels of RAPD primers A: OPD-I1 and B: OPD-14' Lane 1: 100 bp ladder, Lane 2: E. 'Ijrbrae Gem', Lane 3-5 : E. etythronetna vat. marginata 2 to 4, Lane 6-10 : E. erythronentct vat. erythronen'ta 5 to 9, Lane 11-15 : E. striclclandii IO to 15, Lane 16-20 : E. gomphocephala 16 to 20,Lane 2l : 100 bp ladder.
89 .8. 'Urrbrae Gem'RAPD analysis 5.4.1"
A 1 2 3 4 5 6 7 I 9 1011121314151617181920 1
¡E:! !
1500 bp 1500 bp F
600 bp 600 bp ÈiDÉ
B 1 2 3 4 5 6 7 I I 10111213141516171819201
L I i,f 3 3 33 f t 1..
- t- 1 500 bp 1500 bp - -- --3==t
600 bp 600 bp - -
Plate 5.2. Agarose gels of RAPD primers A: OPD-10 and B: OPD-19' Lane 1: 100 bp ladder, Lane 2: E. 'Urrbrae Gem', Lane 3-5 : E. erythronemd Var. margirtata 2 to 4, Lane 6-10 : E. erythronema var. erythronenx¿7 5 to 9, Lane 11-15 : E. stricklandii I0 to' 15, Lane 16-20: E. gomphocephala 16 to Z},Lane2L:100 bp ladder.
90 R^ 'f lrrhree Gemt RAPD analvsis 5.4.3. genetic diversity. The three genotypes of var. marginata showed a maximum genetic distance of 327o. Within the genotypes of E. erythronema vaÍ. erythronema, Eeel was distinct at a similarity of 737o, whereas the other four were more closely linked at greater than 807o similarity. E. 'Urrbrae Gem' clustered with the eight genotypes of E. erythronema, with a genetic similarity of 74Vo, and was placed closer to E. stricklandii thanto E. gomphocephala.
5.4.3. Non-hierarchical distance analysis
The SSH ordination of all specimens into three dimensions (Figure 5.3) showed five closely associated groups of points- E. 'Urrbrae Gem' (one),8. erythronemavN. erythronema (five),
E. erythronema var. marginata (three), E. stricklandii (five), and E. gomphocephala (fle).
The stress level for the diagram is 0.0849, or 8.5Vo, which is lower than the maximum acceptable value of 20Vo.
The MST analysis connects the individuals to their most similar neighbour, with the MST values indicating the relative 'distances' between individuals, and the ordering of the connection indicating individuals with the most similarity. The first connection of E.
'Urrbrae Gem' was to E. stricklandii,with a MST value of 0.2133, and the second connection
was to E. erythronemavaÍ. erythronema, with a MST value of 0.2417.
From these values, it appeared that E. stricklandii and E. 'Urrbrae Gem' were more similar
than E. 'Urrbrae Gem' and E. erythronema vaÍ. erythronema. The E. gomphocephala cluster
linked to E. stricklandii, with a MST value of 0.2559, indicating it was dissimilar to E
'Urrbrae Gem'. The average within group MST values were 0.1923 for E. erythronema var.
erythronema and E. erythronema yar . marginata, 0. I 801 for E. stricklandií and 0. 1 1 85 for E
gomphocephala. While within group MST values were high in the former and lower in the
latter, they were still less than the between group MST values, indicating that the clusters are
91 .8. 'Urrbrae Gem'RAPD analvsis 5.4.3.
Figure 5.2. Dendrogram of RAPD data showing genetic similarity between E, 'Urrbrae Gem' (EUGem), E. erythronema vaÍ. erythronema (Eee), E. erythronema vaÍ. marginata (Eem), E stricklandit (Es) and E. gomphocephala (Eg), using six primers, the simple matching coefficient and UPGMA clustering.
EUGem
Eem2
EeeZ
Eee4
Eee3
Eee5
Eem 3
Eeel
Eem I Esl
Es2
Es3
Es4
Es5 Egl
Ee2
Eg3
Ee4
Eg5
0.s 0 0.6 3 0.75 0.8 8 I .00 Coefficien t
92 F 'flrllrrqe llcmt RAPD nnnlvsis 5-4-3.
Figure 5.3. MDS ordination with MST linkages of RAPD data from six primers, for .E-. 'Urrbrae Gem', E. erythronema vaÍ. erythronema, E. erythronema vat. marginqta, E. str icklandii and E. gomphocephala.
2
I
X U)â
0
-l 2
t $o\ xps
O E. erythronemavar. erythronema O E. erythronemavar.marg¡nata I E. stricldandii A E. gomphocephala O E'Unbrae Gem' E-'I]rrbrae Gem' RAPD analvsis 5.5. clearly distinct.
5.5. Discussion
This study has shown that E. stricklandii was the probable male parent of E. 'Urrbrae Gem, based on analysis of RAPD data by UPGMA clustering and multidimensional scaling. The data generated from the RAPD analysis were analysed using two distance methods: hierarchical, using the simple matching coefficient (SMC) and UPGMA to produce dendrograms, and non-hierarchical, multi-dimensional scaling (MDS) and minimum spanning tree (MST) to produce ordinations and linkages. Distance methods use similarity (or distance) values that summarise character data using pairwise comparisons, either between taxa or characters. Such methods have long been employed in systematics and other biological disciplines to summarise data and compare taxa (McDade, 1997).
The RAPD analysis produced few bands common to all species tested, with most bands being
monomorphic, polymorphic or unique to species groups. The hybrid showed a number of
bands common to E. erythronema (both varieties) and E. stricklandii, and relativley few
common to E. stricklandii only, with three unique bands. These three bands may have been
present in the banding patterns for the actual parent plants, but not others for that species. The
variation in banding patterns within each species, and the fact that the actual parent
individuals of the hybrid could not be located, could explain the presence of unique hybrid
bands and the low level of common bands between the hybrid and the parent species. Other
factors that could explain the presence of band unique to the hybrid are the reassortment of
sequences during hybridisation and the presence of PCR artifacts (Lamboy, 1994).
The dendrogram constructed for the similarity data shows three separate clusters, with each of
94 .8. 'Urrbrae Gem'RAPD analvsis 5.5. the species groups being clearly distinguished. The hybrid, E. 'Unbrae Gem', clustered with individuals from the known female parent species. Hybrids are frequently intermediate between parents, and a study by McDade (1997) noted that distance methods are consistent in placing hybrids between parents, or close to the most similar species. McDade (1997) also showed that the hierarchical distance method using UPGMA will place a hybrid in the cluster with one or both of the known parental species. This analysis indicated that E. 'Urrbrae Gem' was more similar to E. stricklandii than to E. gomphocephala.
Further confirmation rwas sought, using a non-hierarchical method of analysis, so the RAPD data were analysed by multivariate analysis, with SSH and showing MST linkage, to generate
a three dimensional ordination of the ASO matrix. The semi-strong hybrid multidimensional
ordination allowed greater visualisation of the clusters of individuals. The three dendrogram
operational taxonomic units groups are clearly visible, with E. erythronema further separating
into the two varieties - var. erythronema and var. marginata, and all individuals for the E.
stricklandii and E. gomphocephala groups clustering into their respective taxa. E. 'Urrbrae
Gem' was placed between the female parent species and E. stricklandü. The MST values
show that E. 'Urrbrae Gem' was directly linked to E. stricklandii first, then to E. erythronema
var. erythronema, and was not directly linked to E. gomphocephala.
The hypothesis for this study was that the male parent of '8. Urrbrae Gem' either E
stricklandii or E. gomphocephala. The placement of the hybrid with the female parent - E
erythronema var. erythronema - in the case of the UPGMA clustering method, and the
placement of the hybrid between the female parent and E. stricklandii, in the case of the
multidimensional scaling ordination, supports E. stricklandii as the most probable male
paront.
95 E- 'IIrrbrae Gemt RAPD analvsis -5-ll- 'Welsh Since the discovery of RAPD-PCR by Williams et aI. (1990) and and McClelland
(1990), the technique has been used in the determination of genetic variation and linkage
maps between species and cultivars from arcnge of plant genera, including Eucalyptus.
It has been noted frequently that RAPD-PCR is not without its limitations in systematic
studies, such that there a¡e inherent problems in the use of RAPDs as a diagnostic tool for
determining relationships between species, populations and hybrids. There is the inference
that RAPD analysis should not be used on its own; and that topographies generated, through
analyses such as UPGMA, should be compared with alternative topographies. It has also been suggested that alternative methods (such as isozymes or RFLPs) or alternative diagnostic methods (such as morphological character analysis) might be useful to support RAPD results
(Arnold and Emms, 1998). Gillies and Abbott (1998) argue that RAPDS are not useful indistinguishing between distantly related genera, only in very similar ones. This said, there are inherent problems with all DNA sequencing techniques (Wolf and Liston, 1998) and
RAPDs were deemed a sufficiently descriptive technique to determine the parental status of
E. 'Urrbrae Gem', given that every effort was made to remove experimental error, and that the species involved were related closely. Further studies to support these data were carried out on adult morphological characters, as well as manipulated hybridisations and seedling morphological character analysis (Chapter 6).
5.6. Conclusion
The conclusion to this study is that RAPD data, when analysed with hierarchical and non- hierarchical distance methods, placed the interspecific hybrid E. 'lJr:lbrae Gem' between E stricklandä and the female parent species, E. erythronema var. erythronema, strongly suggesting that E. stricklandü is the male parent of E. 'Urrbrae Gem'.
96 .8. rUrrbrae Gem t mornholosical analvsis
Selection at Species Selection for Floral Criteria Species level Gene Pool - Wild and Cultivated populations
for Floral Criteria Selection at Genotype Selection Genotype Level
Hybrid Production through C ontrolled Pollination
Hybrid/Parent Identification/Confirmation
Superior Development of Propagation Methods Genotype
Postharvest Testing
PBR Registration
Multiplication and Release to Industry
Ornamental eucalypt breeding cycle flow chart.
Areas covered by this chapter are highlighted. E. 'Urrbrae Gem' morphological analysis 6.1.
Chapter Six (U Euc ølyptus rrbrae Gem' : Paren tage determination through morphological analysis
6.1. Abstract
Eucalyptus'Urrbrae Gem' is an attractive amenity tree which is believed to be a natural Fl
hybrid with E. erythronema var. erythronema the known female parent. Previous
observations based on adult morphology led to suggestions that either E. gomphocephala or
E. stricklandü could be the male parent. Multivariate analysis of 54 adult morphological
characters placed E 'Urrbrae Gem' between E. erythronema yar. erythronema and E.
stricklandü, with minimum distance values of 0.21 and 0.27 respectively. Controlled
pollinations between E. erythronema var. erythronema and both E stricklandii and E.
gomphocephala were undertaken, and the resulting seedlings were compared to each other, to
open pollinated seedlings of the putative parent species, and to open pollinated seedlings of
the Fl and 2"d generation E 'Urrbrae Gem' trees. Based on 14 morphological characters,
seedlings clustered into eight groups, with E. erythronema vaÍ. erythronema x E. stricklandii
seedlings most similar to open pollinated seedlings of the Fl and 2"d generation E 'Urrbrae
Gem' trees. In contrast, E. erythronema vff. erythronema x E. gomphocephala seedlings
clustered into a number of groups, with some close to E. erythronema var. erythronema
seedlings and others to E. gomphocephala open pollinated seedlings. The results indicated that E. stricklandü is the male parent of E. 'Urrbrae Gem'.
97 R- 'f lrrhrqe êernt ânâlvsis 6-2- 6.2. Introduction
The aim of this study was to determine which of E. stricklandii or E. gomphocephala was the
most likely male parent of E. 'Urrbrae Gem' based on morphological characters The study
was conducted in two parts; firstly, adult morphological characters of the candidate species
were measured and compared with the hybrid; and secondly, seedlings generated from
controlled pollinations between the known female parent and each of the possible male
parents, were compared with open pollinated seedlings of each of the parent species, as well
as open pollinated seedlings from E 'Urrbrae Gem' and the 2"d generation E. 'Urrbrae Gem' tree.
6.3. Materials and methods
6.3.1. Plant Material
Mature flowering trees of the Fl (one) open pollinated 2nd generation (one) E 'Urrbrae Gem',
E' erythronema var. marginata (three), E. erythronema var. erythronema (five), E. stricklandii trees (five), E. gomphocephala (three) and E. gardneri (two), were sourced from the Waite Arboretum, the Waite Campus grounds and the Monarto Woodland. Individuals from each taxa are listed in Table 5.1. E. gardneri was included as it is a closely related species to E. erythronemavar. erythronema, providing a reference for the degree of difference that might be expected between related species.
Species descriptions - adapted from Brooker and Kleinig (1990)
E' erythronemaTurcz. var. erythronema (Red flowered mallee) (Plate 3.4) is a small mallee, of the eastern end of the southern wheatbelt of Western Australia. The trunk varies in colour, from white to purplish to red, with an open canopy of glossy green narrowly lanceolate leaves. The inflorescences are usually 7-flowered, with down-curved peduncles and long
98 E. 'Urrbrae Gem' mornholosical analvsis 6.3.1.
curved pendulous pedicels, hypanthium obconical, and operculum conical. Flowers can be
bright red to creamy white, occurring from October to January. E. erythronema, vaÍ.
marginata (Benth.) Domin (plate 3.3) is closely related to E. erythronema Turcz. var.
erythronemø, differing in the slightly more northern distribution, consistently 3-flowered
umbels, slightly ribbed buds and fruit, and the flared rim at the top of the hypanthium. The
buds of both species appear on the tree several months before flowering, and the previous
season's fruit falls when mature,
E. gomphocephala DC. (Tuart) (Plate 3.6) is a small to medium tree of the western limestone
'Western coastal dunes and subcoastal plains of south west Australia. The bark is rough and dark grey on most of the tree, with branchlets having smooth yellow bark. The leaves are glossy, light green, lanceolate to falcate, and thin. The inflorescences are usually 7-flowered, with broad, flat peduncles and short stout pedicels. The buds are mushroomed shaped and yellow, with the hemispherical operculum slightly wider than the hypanthium. The creamy flowers open from January to April. Buds appear a few months prior to flowering, and the fruit remains on the tree for a few seasons when mature.
E. stricklandiil:|'Iaiden (Strickland's Gum) (Plate 3.6) occurs around the goldfields of Western
Australia. The bark is dark grey to black, rough, being flaky or hard and tessellated on the lower part of the trunk, variable from white to grey over copper above, with shiny, reddish or yellow, glaucous branchlets. The leaves are lanceolate, glossy, green and thick.
Inflorescences are 7-flowered, with a broad, flattened peduncle and little or no pedicel, operculum conical, hypanthium campanulate The yellow buds open to show yellow - green flowers from November to March. Buds appear many months prior to flowering, and fruit remain for many months when mature.
99 R- 'f lrrhrne Gemt analvsis 6.3.1.
E. gardneri Maiden (Blue Mallet) (Plate 3.2) is a mallee of scattered distribution in south west \ryestern Australia. The trunk has smooth light coloured bark, with imperfectly shedding flakes, and the adult leaves are dull, intensely blue-green to purptish. The inflorescences are more than 7-flowered, with a flattened peduncle. The yellow-red buds are small and horn- shaped, with pale-yellow green flowers opening in March to November.
E 'Urrbrae Gem' (Plate 6.1) has a smooth trunk of varying colours similar to E. erythronema var. erythronema, shedding seasonally. The open canopy consists of narrow lanceolate to falcate shining dark green leaves. The inflorescences contain up to seven yellowish buds, each with a conical operculum and an obconical to campanulate hypanthium with a slight lip, a flattened peduncle and a 3-sided short pedicel. The buds appear up to 18 months prior to flowering and open from late spring to early surtmer, with pink-red stamens.
100 .E. tUrrbrae
i,
\
F
\ t
Plate 6.1. Flowers of E. 'Urrbrae Gem' (photo courtesy of Dr. J. Gardner).
101 E- 'f lrrhrne (]emt analvsis 6.3.2-
6.3.2. Adult morphology
Sixty binar], continuous or ordered multistate characters were measured on fresh material for
each tree (Table 6.1.). One hundred measurements were made for each character; ranging from 20 measurements from five individuals from each species, or 5 x 20 measurements from sub-samples of the single available individual (in the case of the Fl and 2"d generation specimens of E 'Urrbrae Gem').
6.3.3. Controlled pollination
The controlled pollination method followed that of van Wyk (1977) and Moncur (1995).
Pollen was collected from single trees of E. stricklandii and E. gomphocephala flowers prior to anthesis at operculum lift. Flowers at this stage were cut from the tree and taken to the laboratory. The anthers \ryere removed with forceps and spread out on hlter paper over silica gel for 24-48 hours to promote dehiscence. After drying, the pollen and anthers were removed from the stamens and placed in 1.5 ml plastic tubes and labeled. The tubes were placed inside larger vials containing I cm silica gel and stored at -20"C until required. A small amount of pollen was placed in another 1.5 rnl vial for regular use and stored at 4"C between pollinations. This 'working' pollen was replaced every ten days from stored reserves.
Viability of the pollen was checked after 12 months storage by germination on Agar medium for 24 hours; all samples showed a minimum of 50Vo viability.
E. erythronema var. erythronemø flowered from August to November, with peak flowering in
October. Controlled pollination took place over four weeks during this peak flowering period. Flower buds at anthesis were emasculated by removing all anthers; all flowers on the tree at this stage were emasculated, and those already open were removed. Pollen was applied with a small paint brush to the stigma, and the umbel tagged. This step was repeated every five days, with every emasculated flower having repeated pollinations, until all flowers in the
102 E. 'Urrbrae Gem' moroholosical analvsis 6.3.4. umbel had been pollinated at least twice. Pollinated flowers were not isolated , as additional empirical information could be gathered on the interaction and competition between applied pollen and pollen applied through natural vectors on emasculated flowers. Information on the effectiveness of the controlled pollination technique without bagging was also sought, in light of recent work by Harbard et al. (1999). Pollinated flowers were not randomised on the tree.
Capsules were harvested eight months after pollination and stored separately in paper bags.
They were placed in a warm dry room at 30oC until open (up to four weeks). Seed was removed from each capsule, counted and weighed, then stored in a cool dry dark place until planting. A total of two hundred seeds were randomly selected from different capsules from each plant for each cross: seeds were soaked for 24 hours in 3000 ppm gibberellic acid to aid germination (Ostrowska et al., 1998), then four seeds planted per 25 mm pot, containing soil
(sand and pinebark in a l:2 ratio) and covered with a thin layer of vermiculite, and the forty pots within each tray covered with plastic to retain humidity. The plastic was removed when the majority of seeds in the tray had germinated. Dates of germination were recorded, and the shape of the cotyledons noted seven days after emergence when both cotyledons were free from the seed coat. Seedlings were grown in a glasshouse during Spring, under conditions of natural daylength and temperatures of l8+3'C to 25+3"C. Liquid fertiliser (Aquasol@) was applied at fortnightly intervals from 21 days after germination. Seed from parent species was sourced from commercial seed companies and supplemented with open pollinated seed from the actual parent trees.
6.3.4. Seedling morphology
The surviving seedlings from both crosses were measured at three months of age for 17 binary, continuous or ordered multistate characters at the fifth fully expanded leaf pair after the cotyledons (Table 6.1). Sixty-nine seedlings from E erythronema var. erythronema x E.
103 E. 'Urrbrae Gem' moroholosical analvsis 6.3.5. stricklandii (EeEs), and 93 from E erythronema vaÍ. erythronema x E. gomphocephala
(EeEg) were measured, along with 65 open pollinated seedlings from each of the parent species, with the exception of E. erythronema var. erythronema and E. erythronema var. marginata, for which 45 and 20 were measured, respectively. Open pollinated seed from the
Fl and 2nd generation trees of E. 'Urrbrae Gem' was included in the analysis. As the pollinated flowers were not isolated and the female trees were growing in a multi-species environment, the possibility existed that seed from the controlled crosses and open pollinated seed may have contained contaminants such as intraspecific crosses (selfs or outcrosses) or interspecific crosses with other species.
6.3.5. Data Analysrs
The PATN analysis package (Belbin, 1994) was used to analyse the morphological character data for both adults and seedlings using hierarchical (UPGMA) and non-hierarchical
(multidimensional scaling, minimum spanning tree and principal canonical correlation) analysis to determine the relationships between the hybrid E. 'Urrbrae Gem' and the putative parent species. The morphological characters were standardised using Gower's metric, a standardised Manhattan Metric (see Chapter 5).
In addition to allowing dendrogram groups to be identif,red and characterised, the Group
Definition option of PATN (GDEF) enables large numbers of individuals to be averaged into groups of equally dissimilar individuals, and the means of these groups re-analysed to provide a more succinct picture of the overall pattern. This option was used on the seedling morphology study, due to the large numbers of seedlings measured. The groups statistics option (GSTA) was used to interpret each dendrogram group in terms of characters, with the
Kruskal-Wallis,Fl-statistic used as a non-parametric analysis of variance to determine whether the ranked groups means are significantly different from one another for each character
to4 -8. 'Urrbrae Gem' moroholosical analvsis 6.3.5.
(Nicolle and Conran, 1999).
Principal Canonical Correlation (PCC) analysis was used to determine whether there were significant correlations between the semi-strong hybrid ordination OTU coordinates and the individual characters scores for these OTUs. This enabled the ordination to be interpreted in terms of the characters causing the clusters and for clusters/taxa to be defined more clearly.
The correlation coefficient (l) for each cha¡acter against the spread fo the OTUs in the ordination space was calculated, and the significance of the correlation was Bonferroni adjusted to allow for the multiple comparisons (Nicolle and Conran, 1999). When a character is significantly correlated to the MDS coordinate for the individual, that character will be descriptive for that taxon.
105 E úf lrrhrae f]crnt rnnrnhnloqienl nnalvsis ll-3-5-
Table 6.1. Characters used in phenetic analysis (seedling and adult) with codes.
Seedling - continuous LLE Iæaf length (mm) LWI Iæaf width (mm) LPL læaf petiole length (mm) Seedling - binary LEA I,eaf concolorous (0) discolorous (l) LSM Leaf smoothness smooth (0) rough (1) LPS Leaf petiol atelsessile sessile (0) petiolate (1) LPO Leaf position opposite (0) alternate (1) STE Stem single (0) branching (1) SSM Stem smoothness smooth (0) rough (1) OGP Oil glands prominent inconspicuous (0) prominent (1) Seedling - ordered multistate LSH I-eaf shape lanceolate (0) falcate (1) elliptic (2) ovate (3) LST læaf tip shape acute (0) mucronate (1) mucronulate (2) obtuse (3) LSB Iæaf base shape attenuate (0) oblique (1) obtuse (2) cordate (3) LSW læaf surface wax shining (0) dull (1) glaucous (2) LCO Leaf colour light green (0) green (1) blue green (2) SSH Stem shape square (0) angular(1) round (2) LMP Iæaf margin proximity close (0) medium (1) far (2) Adult inflore scence - continuous HLE Hypanthium length (mm) FIWI Hypanthium width (mm) OLE Operculum length (mm) owr Operculum width (mm) PLE Peduncle length (mm) PWI Peduncle width (mm) PDE Peduncle depth (mm) PELE Pedicel length (mm) PEWI Pedicel width (mm) PEDE Pedicel depth (mm) Adult fruit -continuous FDI Fruit diameter (mm) FLE Fruit length (mm) FPL Fruit peduncle length (mm) Continued Table 6.1, Continued FPW Fruit peduncle width (mm) FPD Fruit (mm) FPEL Fruit pedicel length (mm) FPEV/ Fruit pedicel width (mm) FPED Fruit pedicel depth (mm)
Adult mis c ellane ous - c ontinuous NBU Number of buds in umbel LAL Leaf petiole angle o left LAR læaf petiole angle o right LWI Iæaf width (mm) LLE Læaf length (mm) LPL Iæaf petiole length (mm) LAT Leaf lamina thickness (mm)
Continued
106 F 6f Trrhrne (lemt mornholosical analvsis 6.3.5.
Table 6. 1. continued Adult fruit - binam PCU Peduncle curvature erect (0) pendulous (l) Adult inflorescence - binam BAN Bud angular not angular (0) angular (1) BSM Bud smooth smooth (0) warty (1) HYR h¡,panthium ring absent (0) present (1) OAP Operculum apex shape rounded hemispherical (0) acutely conical (1) OAT Operculum attachment abutting (0) enclosing (1) OBS Operculum base shape not overhanging (0) overhanging (1) osc Operculum base scar wider at base (0) wider at apex (l) Adult miscellancous - binam LPS Leaf petiolate/sessile sessile (0) petiolate (1) LPO I-eaf position opposite (0) alternate (1) PEC I-eaf petiole colour orange (0) red (1) SSW Stem smooth/warty smooth (0) warty (l) sco Stem colour orange (0) red (1) PGS Pith glands stem absent (0) present (l) PGF Pith-like glands at base of fruit absent (0) present (l) OGP Oil glands inconspicuous (0) prominent (1) Adult inflorescence - ordcred multistate BWA Bud wax shiny (0) dull (1) glaucous(2) HSH Hypanthium shape obconical (0) slightly campanulate (1) campanulate (2) OSH Operculum shape beaked (0) conical (1) horn-shaped (2) hemispherical BCO Bud colour yellow-green (0) orange (1) darkred (2) brown (3) PSH Peduncle shape absent (0) flattened (1) round (2) PESH Pedicel shape absent (0) flattened (1) round (2) Adult .fruit - ordered multistate FSH Fruit shape conical (0) slightly campanulate (1) campanulate (2) FWA Fruit wax shiny (0) dull (l) glaucous (2) FRR Fruit ribbing smooth (0 slight ribbing (l) strong ribbing (2) VPO Valve position shrunken (0) level (1) exserted (2) DPO Disc position descending (0) level (1) ascending (2) FCO Fruit colour green (0) orange (1) redbrown (2) brown (3) Adult miscellaneous - ordered multistate LSH Leaf shape lanceolate (0) falcate (1) elliptic (2) ovate (3) LSB Iæaf base attenuate (0) oblique (1) obtuse (2) cordate (3 LST Leaf tip acute (0) mucronate (1) mucronulate (2) obtuse (3) LSW Iæaf surface wax shining (0) dull (1) glaucous (2) LCO Leaf colour light green (0) green (1) blue gteen (2) STW Stem wax shining (0) dull (1) glaucous (2) FLC Flower colour red (0) pink red (1) cream (2) greenish yellow (3)
107 .8. 'Urrbrae Gem' morpholoeical analvsis 6.4.1.
6.4. Results
6.4.I. Adult morphology analysis
Sixty morphological characters were measured, although leaf shape (LSH), leaf base shape
(LSB), leaf tip shape (LST), leaf surface wax (LSW), leaf petiolate/sessile (LPS) and leaf position (LPO) were found to be invariant and were removed, leaving 54 characters for analysis. For simplicity, the five representative points for the Fl and open pollinated 2"d generation E. 'Urrbrae Gem' trees were averaged after full analysis, resulting in one point for each existing tree on the dendrogram and the ordination.
The UPGMA dendrogram (Figure 6.1) separated the four species, two sub-species and Fl and open pollinated2"d generation E. 'Urrbrae Gem' into five clusters. Individuals within clusters were very similar to each other. The Fl and open pollinated 2"d generation E. 'Urrbrae Gem' were most closely associated with each other in cluster 1, and then with cluster 2, including the varieties of E erythronemø. Cluster 3 contained E. gardneri, cluster 4 E. stricklandii and cluster 5 E. gomphocephala. Neither putative parent was associated closely with the Fl and open pollin ated 2"d generation E. 'Urrbrae Gem' in the dendrogram.
The semi-strong hybrid ordination of all specimens into two dimensions (Figure 6.2) follows the dendrogram with five clusters of points. The Fl and open pollinated 2nd generation E.
'Urrbrae Gem' were placed centrally and relatively close to each other (MST value 0.13). The
E. erythronema vaÍ. erythronema and E. erythronema var. marginata groups were connected via MST value 0.07, with the former close to the open pollinated 2nd generation E 'Urrbrae
Gem'(MST value 0.21), Both possible male parent species are represented as outlying groups, both closest to the F1 E 'Urrbrae Gem', but with E. stricklandii closer than E gomphocephala (MST distances of 0.27 and 0.34 respectively). E. gardner¡ was an outlying group nearest the F2 (MST 0.28). The stress value for the ordination is 10.37o, well within
108 E. 'Urrbrae Gem' morpholosical analvsis 6.4.1. the 20Vo distortion level generally deemed to be acceptable (Belbin, 1994). Additional analysis of group means showed that the centroid MST linkages connect the Fl and 2"d generation E. 'Urrbrae Gern" to the female parent species group (MST 0.30), to the E. stricklandä group (MST 0.39), to the E. gardneri group (MST 0.40) and finally to the E. gomphocephala group (MST 0.42), further clarifying the connections between groups.
PCC analysis of all adult characters shows clea¡ correlations between the plotted OTUs and taxon groups and the characters used to derive them (Figure 6.3.). Table 6.2 lists all adult characters with their correlation coefficients and significance levels. All taxon groups are almost equidistant from the origin, indicating extremes of very high or very low values for those morphological characters that define them. The female parent, E. erythronema, shared two unique characters, peduncle shape (PSH) and peduncle curvature (PCU), while E erythronema var. marginata showed one unique character, that of hypanthium ring (HYR).
Neither the Fl or open pollinated 2nd generation E 'Urrbrae Gem' showed any unique characters. E. stricklandii had powdery wax present on all young stems, and the characteristics of pedicel shape, length, width and depth (PESH, PELE, PEWI, PEDE) and fruit pedicel width and depth (FPEW, FPED). E. gomphocephala lacked bud angularity
(BAN), had shiny fruit (FWA), an operculum enclosing the hypanthium (OAT), a rounded hemispherical operculum apex (OAP), an overhanging operculum base (OBS) and an operculum scar widest at its base (OSC). All of these characters were unique to the species.
E. gardneri is defined by absence of pith-like glands at the base of the flower (PGF), and comparatively small values for hypanthium width (HWI), operculum width (OWI), fruit diameter and length (FDI, FLE) and fruit peduncle depth (FPD). The Fl E 'Urrbrae Gem' and both possible male parents all shared warty buds (BSM), the Fl E. 'Urrbrae Gem' and E. stricklandü both possessed green fruit (FCO) and warty stems (SSW), and the Fl E 'Urrbrae
Gem' and E. gomphocephala shared orange petiole colour (PEC). The F1 and 2nd generation
109 F aI f¡rlr¡oa nnrnhnlnoinql qnqlvsis 6-4-1
E. 'Urrbrae Gem" and E. strícklandil trees shared the character of dull bud wax (BWA) with each other and E. gardneri. There were no characters that were uniquely common to both the
F1 and FZ E. 'Urrbrae Gem' trees and either variety of the female parent, although fruit pedicel depth (FPED), leaf width (LIVI) and leaf petiole length (LPL) were similar and very different from the putative male parents. The continuous characters measured for the Fl hybrid E. 'Urrbrae Gem' were most often intermediate in value between E. erythronema (both varieties) a¡¡d E. stricklandii, however, as the continuous characters measured for E. gomphocephala were of values close to that of the Fl, the significance of many of these characters were lost. Key descriptive characters, such as bud and fruit colour, showed strong similarities between the hybrid and E' stricklandii.
EUGFl
EUG2n I EeeL Eee2 Eee3 Eee4 Eee5 EemL Eem2 Eem3 EgaI 3 Esa2 I EsL Es3 Es5 -T_r_l Es4 I Es2 4 EsL Es3 t Eg2 -T I I I TI 6 0 030 1- 0. t-855 0.3409 0 .496 2 0.651 0.8070
Figure 6.1. UPGMA dendrogram of 20 adult individuals measured. Scale represents standardised Manhattan distance (Gower metric) based on 54 characters measured (Table 6.2). The dendrogram shows five clusters: cluster 1 - Fl E. 'Urrbrae Gem' (EUGFI) and 2"d generation E. 'Urrbrae Gem' (EUG2n); cluster 2 - E. erythronema var. erythronema (Eee trees l-5) and E. erythronema var. marginata (Eem trees 1-3); cluster 3 - E. gardneri (Ega trees l-2); cluster 4 - E. stricklandil (Es trees l-5); and cluster 5 - E. gomphocephala (Eg trees 1-3),
110 F 'fIrr.hrqc (]ernt mnrnhnlosienl nnnlvsis 6-4-l-
2
\
õl
0 .t) H o/ o a
-1
,t 0 2
MDSAxis I
t E. erythronetnyar. erythronema O E. erythronema\u. nwrgineta @ E. gardneri t E. snicklandii A E. gomphocephala O Fl ,E. 'Unbrae Gem- O 2nd generationE 'Unbrae Gem
Figure 6.2. Semi-strong hybrid multidimensional scaling of 2l individuals into their five taxonomic groups based on 54 adult characters, Distances and linkages are results from a minimum spanning tree analysis of the data.
111 .8. 'Urrbrae Gem' morphological analysis 6.4.1.
infl oresccnce chôraclers
PE SH OL¿
.9 ÞE IE ÞsH ô aco ô"f" ptfi?' o¡r HLE " -1
-2 -1 0 M DS Ax¡s I
fruil chârâcle¡s
.2
ô
FPÉo t"'t"" -1 FDr FlE
-2 0 M DS Axis I
" L$o
.9 ooP 2-q!
ô Lw I sco tlE.Lrf.L
-1
-l 0 MDS Ax¡s I
Figure 6.3. Principal canonical correlation plot for the SSH ordination (Figure 6.2) based on the adult characters measured - inflorescence, fruit, and miscellaneous, all vector lengths 1, Character codes are those detailed in Table 6.1., italics indicate characters not significantly correlated with the SSH OTU coordinates, characters from Table 6.2,
rt2 Table 6.2. Adult morphological characters (Table 6.1.) of E. 'Urrbrae Gem' and putative relatives (from Table 5.1), with correlation coefficient" (r2) o of the individual character vectors in the ordination space, Kruskal-Wallis É1 statisticb and probabntty (P). Measurements in mm or presented as range (mean), when 12 >0.66 significant at P <0.05, NA = not applicable as character invariant and not included in analysis.
Character Fl E 'Urrbrae F2Ë.'Urrbrae E.erythronema E.erythronema E.stri¿klandü E.gomphocephab E.gardneri ¿ H P
Hypanthium length 9.6-ls.l (l 1.9) 6.6-9.6 (8.2') s.4-9.9 (7.3) 6.5-10.5 (8.4) r0.8-16.9 (13.0) 8.7-t2.9 (t0.4) 3.6-s.s (4.4) 0.81 16.24 0.0027 Hypanthium width 9.4-12.8 (l l.l) 't.4-9.9 (8.9) 7.9-10.3 (8.8) 7.8-t2.8 (r0.2) 9.8-13.8 (l 1.6) 9.3-12.9 (l1.3) t.8-2.s (2.3) o.75 13.39 0.0082 Operculum lengrh 8.6-12.4 (10.9) 7.1-10.6 (10.1) r0.6-14.9 (12.1) 10.7-16.6 (13.1) 10.6-17.9 (13.2) to.9-t3.s (t2.2) 9.7-16.6 (13.3) 0.22 6.2t 0.1838 Operculum width 8.6-12.4 (10.9) 8.1-10.2 (9.3) 7.8-10.9 (8.9) 6.7-11.3 (9.5) '7.3-13.5 (tt.2) 12.9-16.2 (r4.5) r.6-2.3 (2.r) 0.92 15.69 0.0035 Peduncle length l1.8-28.0 (21.6) t2.s-21.3 (t7 .3) 9.9-23.2 (t8.1) 13.8-25.6 (19.8) r5.6-30.9 (20.9) ts.6-32.8 (2r.1) 9.8-19.8 (1s.6) 0.67 9.99 0.0405 Peduncle width 3.3-10.1 (5.4) 2.6-s.t (3.6) t.s-3.8 (2.2) r.7-3.4 (2.s) 6.9-15.8 (l l.l) 5.2-10.0 (8.3) r.9-3.s (2.9) 0.95 16.77 0.0021 Peduncle depth 3.8-5.8 (4.7) 2.3-3.s (2.8) 1.8-3.0 (2.3) r.74.O (2.s) 3.1-s.2 (3.7) 4.0-5.5 (4.6) 1.2-2.O (1.6) 0.89 t4.59 0.0056 Pedicel length 2.04.3 (3.4) 4.2-7.9 (6.3) 7.6-14.5 (12.1) 8.7-16.5 (14.1) 0 1.0-2.1 (1.7) 3.2-s.6 (4.3) 0.95 t7.59 0.0015
Pedicel width 2.34.6 (3.9) 2.1-s.l (3.3) 1.74.4 (2.8) t.9-3.7 (2.6) 0 3.64.2 (s.0) 1.0-1.8 (1.3) 0.86 17.75 0.0014
Pedicel depth 2.8-3.8 (3.3) 2.s-3.2 (2.9) 1.9-3.O (2.3) t.9-2.8 (2.2) 0 4.04.9 (4.4) 1.0-1.8 (1.3) 0.84 r 7.80 0.00r 3
Fruit diameter rt.2-12.9 (r2.3) 10.8-r 3.0 (l 1.9) t2.r-t3.8 (13.2) 10.3-15.0 (r2.7) 12.6-16.9 (14.4) 13.0-16.0 (14.7) 4.44.3 (s.2) 0.74 15.41 0.0039
Fruit length l 1.5-13.5 (12.8) 10.3-12.1 (l 1.5) tr.s-r4.2 (r2.4) 10.8-16.4 (14.5) 14.5-18.8 (16.3) l3.l-18.0 (15.4) 6.0-8.0 (7.1) 0.77 15.95 0.0031 Fruit @uncle 16.6-2s.3 (21.9) 14.8-20.8 (17.8) 12.6-24.0 (ts.7) 15.4-2s.9 (18.4) r7.t-32.t (23.7' 14.0-29.2 (20.6) 12.8-17.3 (r5.2) 0.81 13.85 0.0078 length Fruit peduncle 3.4-7.s (s.2) 3.24.9 (4.r) 2.4-3.8 (3.3) 254.2 (3.4) 7.8-16.9 (tr.7) 3.2-9.9 (8.8) 2.8-3.6 (3.2) 0.92 16.37 0.0026 width Fruit peduncle 2;t4.3 (3.5) 2.64.2 (3.3) 2.3-3.s (2.s) 2.44.0 (3.t) 2.84.s (4.7) 6.8-12.4 (8.7) 1.3-2.0 (1.6) 0.86 16.41 0.0025 depth Fruit@icel length 2.04.6 (3.3) 4.1{.9 (5.5) 7.2-l 1.9 (8.8) 4.s-2s.9 (r3.8) 0 0.2-t.2 (0.6) 2.6-3.6 (3.o) 0.90 t'?.60 0.0015 Fruit pedicet width 3.2{.s (4.1) 3.4-5.5 (4.1) 3.2-5.1 (3.8) 3.s4.3 (4.4) 0 6.4-9.s (7.9) t.s-2.r (r.7) 0.87 t7.76 0.0014 Fruit pedicel de¡h 2.64.7 (3.s) 3.0-3.9 (3.3) 2.2-3.s (3.2) 2.9-s.t (3.6) 0 5.3-8.7 (6.9) t.4-1.7 (t.6) 0.86 l7.l I 0.0018 Iæafpetiole angle s0-90 (75.2) 60-90 (76.4) 50-90 (79.5) 60-90 (7s.7) 60-90 (71.3) s0-90 (72.6) 50-90 (76.8) 0.72 9.99 0.0407 læaf petiole angle 90-130 (r04.8) 90-120 (103.6) 90-130 (100.s) 90-120 (104.3) 90-120 (108.7) 90-130 (107.4) 90-130 (103.2) 0.69 9.18 0.0569
Iæaf width rt.8-2o.4 (14.9) 9.7 -17 .0 (t2;t) 8.9-r'7.5 (12.4) 6.2-17.6 (8.8) 22.0-s0.3 (33.0) 17.6-30.9 (22.4) l 1.0-18.4 (14.9) 0.94 15.70 0.0035 Continued Table 6.2. Continued Character Fl E.'Urrbrae F2 E.'Urrbrae E.erythronemø E.erythronema E.strickbndü E.gomphocephab E.gardneri r' H P Gem' Gem' var. Leaf length 90.0-r2s.0 (106.3) 67.0-l 15.0 (90.2) 44.2-77.9 (60.5) 49.8-87.2 (69.0) 103.2-192.1 (150.8) 84.0-1s2.0(l 18.5) 55.9-92.5 (74.2) 0.89 16.81 0.0017
læafpetiole length t2.t-21.2 (ts.2) 6.0-13.3 (10.3) 4.9-12.2 (7.0) 5.1-10.6 (8.4) 2s.143.7 (33.6) 13.0-23.0 (18.1) 7.e-r7.5 (12.0) o.92 17.31 0.0017 [,e¿f lamina 0.34.s (0.43) 0.34.4 (0.37) 0.34.5 (0.38) 0.1{.4 (0.26) 0.44-7 (0.s5) 0.24.5 (0.31) 0.24.3 (0.23) 0.57 15.43 0.0039 thickness (4.8) Number ofbuds in 3-7 l-7 (3.6) r-s (2.6) 14 (2.s\ 3-7 (s.7) t-7 (3.9) 7-tt (9.6) 0.83 16.81 0.0021 umbel Bud shape angular angular angular angular angular not angular angular 0.82 19.00 0.0008 Bud surface warty smooth smooth smooth wafy warty smooth 0.94 17.08 0.0019 Hypanthium ring absent absent absent present absent absent absent 0.52 19.00 0.0008 Operculum apex acutely conical acutely conical acutely conical acutely conical acutely conical rounded acutely conical 0.83 19.00 0.0008 shape hemispherical Operculum abutting abutting abutting abutting abutting enclosing abutting 0.83 19.00 0.0008 attachment Operculum base not overhanging not overhanging not overhanging not overhanging not overhanging overhanging not overhanging 0.83 19.00 0.0008 shape Operculum base wider at apex wider at apex wider at apex wider at apex wider at apex wider at base wider at apex 0.83 r9.00 0.0008 scar læaf petiolate/sessi I e peúolate petiolate petiolate petiolate petiolate petiolate petiolate NA Iæaf position alternate altemate alternate altemate altemate altemate altemate NA
Leafpetiole colour orange orange red red red orange orange o.26 19.00 0.0008
Stem smootlr/warty warty smooth smooth smooth warty smoolh smooth 0.96 17.08 0.0019 Stem colour red orange red red rd orange orange 0.24 16.74 0.0022 Pith glands present stem prcsent Present present Present absent absent 0.28 19.00 0.0008 Pith-like glands at present pf€sent present present present present absent 0.67 19.00 0.00û8 base offruit Oil glands prominent prominent prominent prominent inconspicuous inconspicuous inconspicuous 0.87 19.00 0.0008 Bud wax dull dull shiny shiny dull shiny dull 0.92 19.00 0.0008 Hypanthium shape slightly campanulate slightly obconical obconical campanulate campanulate obconical 0.99 r9.00 0.0008 campanulate Operculum shape conical conical conical conical conical hemispherical horn-sha@ 0.55 19.00 0.0008 Continued Table 6.2. Continued Character Fl E.'Urrbrae F2 E.'Urrbrae E. erythronema E. erythronema E. stricklandü E.gomphocephala E.gørdneri ¿ H P Gem' Gemt var. erythronema var. marsinata Bud colour yellow-green yellow-green dark red dark red yellow-green orange yellow-green o.92 19.00 0.0008
Peduncle shape flattened flattened round round flattened flattened flattened 0.89 19.00 0.0008
Pedicel shape flattened flanened flanened flanened absent flattened round 0.62 19.00 0.0008 Peduncle curvature erect erect pendulous pendulous erect erect erect 0.89 19.00 0.0008 Fruit shape slightly campanulate slightly conical conical campanulate campanulate obpyriform 0.84 19.00 0.0008 campanulate Fruit wax dull dull dull dull dull shiny dull 0.83 19.00 0.0008 Fnrit ribbing slight slight slight strong slight slight none 0.61 19.00 0.0008 Valve position exserted level level level exserted exserted shn¡nken 0.89 19.00 0.0008
Disc position descending descending descending descending ascending level descending 0.91 19.00 0.0008 Fruit colour green green red brown red brown green orange brown o.79 19.00 0.0008 tæaf shape lanceolate lanceolate lanceolate lanceolate lanceolate lanceolate lanceolate NA læaf base attenuate attenuate attenuate attenuate attenuate attenuate attenuate NA Iæaf tip mucronulate mucronulate mucronulate mucronulate mucronulate mucronulate mucronulate NA læafsurface wax dull dull dull dull dull dull dull NA Leafcolour green green gfeen green blue green green blue green 0.96 19.00 0.0008 Stem wax dull dull dull dull glaucous dull dull o.82 r9.00 0.0008 Flower colour pink red pink red red red greenish creÍùn greenish 0.89 17.45 0.0016
Correlation coefficient" : showing the fit (r2¡ of the individual character vectors in the ordination space.
Kruskal-Wallis Èl statisticb : the non parametric analysis of variance to determine whether the ranked group means are significantly different from one another for each character. P .f Trrhrqo f]crnt rnnrnholnoieel annlvsis 6^4-2-
6.4.2. Controlled pollination seed data
A total of 161 E. erythronema vaÍ. erythronema flowers were pollinated with E. stricklandii
pollen resulting in a 34.2Vo pollination success rate. This compares ,with the 23.7Vo
pollination rate success rate lor E. erythronema var. erythronema x E. gomphocephala qoss
(Table 6.3.), with an average of 8.6 seeds (E'. stricklandü) and 4.4 (8. gomphocephala) seeds produced for every flower pollinated. Mean seed weight for each cross was similar, and, on
average, weighed less than seed from either male parent or the female parent. From the 200
seeds planted for each cross, 69 E. erythronema yar. erythronema x E. stricklandii (34.5Vo
germination rate) and 93 E. erythronema var. erythronema x E. gomphocephala (46.5Vo
germination rate) seedlings were measured. All cotyledon leaves of open pollinated parent
species seedlings were Y shaped, and were divided into three types representing each parent
species: E. erythronema vaÍ. erythronemø cotyledons were thin, E. stricklandü were medium
and E. gomphocephalawere wide. Cotyledons of seedlings from controlled pollinations bore
strong resemblance to male parent species cotyledons in all cases.
6.4.3. Seedling morphology
Seventeen characters were measured initially, with leaf surface wax (LSVD, leaf petiolate or sessile (LPS) and stem single or branching (STE) recorded as uniform and subsequently deleted, leaving 14 characters for analysis.
Open pollinated E. erythronema seedlings, three months after germination, had petiolate, alternating, medium sized lanceolate leaves, with acute tips and attenuate bases. The leaf surface was rough and concolorous, with prominent oil glands. Open pollinated E stricklandü seedlings, of the same age, had large, alternating, lanceolate to ovate leaves, with
116 Table 6.3. Seed data from E. erythronema, var. erythronema x E. stricklandii and. E. erythronema val. erythronema x E. gomphocephala controlled, pollinations and open pollinations of putative parent species.
female male # flrs # # Eo caps. VofTrs total # mean # mean # mean # min # max # mean mrn max Poll. caps caps harv. poll. seed seeds per seeds per seeds per seed seed weight weight weight harv with with prd. flr poll." caps cap with per cap per cap seed per seed (mg) seed seedb seed" ha¡v.d seeds" Eee 7.A. l3 4 2 50.00 15.39 18 1.39 4.5 9.0 9 9 0.402 0.377 0.426 Es3 ll 0 0 0.00 0.00 0 0.00 0.0 0.0 0 0 0.000 0.000 0.000 Eee 7.2.1 Eg' 132 53 35 66.O4 26.52 66s 5.04 12.55 19.0 3 55 0.337 0.209 o.597 Es3 r12 78 49 62.82 43.75 1305 11.65 16.73 26.63 4 7l 0.316 0.200 0.532 Eee 7.3.1 Eg' 45 18 8 44.45 17.78 150 3.34 8.34 18.75 4 37 0.332 0.201 0.48s Es3 38 22 6 27.27 15.79 83 2.18 3.77 13.83 2 36 0.343 0.249 0.415 total Eg' 190 75 45 60.00. 23.68. 833 4.39* ll.ll- 1g.52' 3 55 0.357 0.201 0.597 total Es3 t6l 100 55 55.00. 34.16' 1388 9.62. 13.88' 25.36' 4 7t 0.334 0.200 0.532 Eee 7.4.1 open 65 278 4.27 4.28 o.436 Eee7.2.L open 75 ttTt 15.61 24.04 0.40r Eee7.3.l 84 276 3.29 3.6s 0.448 224 I725 7 0.428 open 40 2.27 open 40 1.08 a # capsules with seed / # flowers pollinated x 100/l b # caps with seeds / capsule harvested x 100/l c total number of seeds / total flowers pollinated d total number ofseeds / capsules harvested e total number of seeds / total capsules harvested with seed I E. erythronema var. erythronema Plot 7 tree A,2 or 3 2 E. gomphocephala 3 E. stricklandii mean .8. 'Urrbrae Gem' morpholosical analvsis 6.4.3. petioles, mucronate tips and obtuse or oblique bases. The leaves were discolorous, smooth, blue green with variably conspicuous oil glands. The leaves of open pollinated E gomphocephala seedlings were petiolate, alternate and ovate, with mucronate tips, however the bases were cordate rarely obtuse. The small leaves were discolorous, smooth, light green with inconspicuous oil glands. The leaves of seedlings from open pollinated Fl and 2nd generation E 'Urrbrae Gem' trees were variable, with characters similar to either E erythronema var. erythronema or E. stricklandíi. Seedling characters from both controlled crosses were intermediate between parent species (Plate 6.2), with the main diagnostic features between the taxa studied leaf length and width (Table 6.4).
Seventeen characters were measured with leaf surface wax, leaf petiolate or sessile and stem single or branching recorded as uniform and deleted from the analysis leaving 14 characters
(Table 6.4). The UPGMA dendrogram (Figure 6.4) separated the seedlings into eight clusters based on 20 group means of similar individuals. All E. erythronema vaÍ. erythronema x E. stricklandä seedlings and the majority of the E. erythronema var. erythronema x E. gomphocephala seedhngs clustered separately from the maternal species (Tables 6.5).
The seedling semi-strong hybrid ordination (Figure 6.5) showed the relative positions of the eight clusters from the dendrogram in two dimensions (stress = I9.4Vo), with connecting lines representing minimum spanning tree linkages between clusters. Cluster l, containing six E. erythronema var. erythronema x E. stricklandü seedlings, connected to cluster 2 at 0.15, which included the majority of E. erythronema yar. erythronema x E. stricklandü seedlings
(54) and one open pollinated Fl E. 'Urrbrae Gem' seedling. Cluster 2 connected to cluster 3 at 0.19, which comprised ó5 open pollinated seedlings of E. stricklandü and three E. erythronema var. erythronema x E. gomphoceplrølø seedlings. Cluster I also connected to cluster 4 at 0.15, which contained 64 and 65 open pollinated seedlings of Fl and 2"d
118 .8. 'Urrbrae Gem' moroholosical analvsis 6.4.3.
Plate 6.2. Seedling leaves of A: a) open pollinated E. erythronema var. erythronema,b) E. erythronema var. erythronema x E. stricklandü and c) open pollinated E. strícklandii, and B: a) open pollinated E. erythronema vat. erythronema,b) E. erythronema var. erythronemax E. gomphocephala and c) open pollinated E. gomphocephala,three months after germination. Top two rows show lower surfaces, lower two rows show upper surfaces. Bar represents 1 cm.
119 Table 6.4. Seedling morphological characters of E. 'Urrbrae Gem' (from Table 6.1) and putative relatives with correlation coefficientu (r2), Kruskal- o Wallis Il statisticb and probability (P). Measurements in rnm or presented as range (mean), r' >0.61 significant at P<0.05, NA = not applicable as character invariant and not included in analysis.
Characters Fl E. 'Urrbrae Gem' F2 E. 'Urrbrae Gem' E. erythronema va.. E.erythronema E. stricklandü E.erythronemavt. E.gomphocephala f HP open pollinated open pollinated erythronema and var. vaterythronema open pollinated erythmnema x open pollinated seedlings seedlings marginataopen x E-stricklnndü seedlings E.gomphoceplult seedlings pollinated seedlines Iæaf shape lanceolate or falcate or lanceolate lanceolate lanceolate or elliptic lanceolate or ovate lanceolate or ovate ovate 0.92 l1.58 0.0090 ovate or ovate t-eaf tip acute acute acute mucronate, mucronate acute or mucronate mucronate 0.87 5.52 0.1372 sometimes acute or obtuse Leaf base attenuate, sometrmes attenuate, sometimes attenuate attenuate or oblique obtuse or oblique attenuate or oblique obtuse or cordate 0.80 s.99 0.1I t7 obuse obtuse or obtuse or obtuse Iæaf length 30.5-105.0 (61.5) 4s.6-129.0 (79.3) 38.1-89.5 (60.3) 57.0-128.0 (80-7) 63.5-t24.3 (92.4) 27.O-tr2.s (49.9) 15.3-72.3 (53.0) o.73 8.05 0.0450 Iæaf width 8.1-37.4 (21.r) 12.5-37.2 (22.2) 9.8-39.s (18.7) 22.s-57.9 (37.6) 34.0:73.5 (47.6) 13.0-s0.0 (28.3) t7 .5{.3.2 (40.7) 0.81 10.67 0.0137 læafsurface wax dull dull dull dull dull dull dull NA I¡¿f surfaces concolorous concolorous concolorous concolorous discolorous discolorous discolorous 0.56 5.38 0.1461 Leaf smoothness smooth rough rough smooth smooth smooth smooth 0.55 4.49 o.2t35 Iæaf petiolate/sessile petiolate petiolate petiolate petiolate petiolate petiolate petiolate NA Iæafpetiole length s.2-ts.2 (8.6) 4.9-20.1 (9.3) s.6-21.6 (8.2) 5.7-17.0 (10.s) 8.6-23.2 (r5.5) 3.2-ts.2 (8;t) 8.4-21.5 (13.5) o.77 12.40 0.0061 læafposition alternate altemate altemate alternate, sometimes altemate alternate, sometimes alternate o.76 7.03 0.0709 opposite opposite Leafcolour blue green blue green green blue green blue green green light green or green 0.55 5.56 0.t239 læaf margin prominence close, medium or far medium or far medium or far medium or far far or medium medium or far far 0.53 3.20 o.3617 Stem shape angular squaf€ squarc angular angular angular round or angular 0.55 r6.83 0.0008 Stem single single single single single single single NA Stem smoothness smooth or rough smooth or rough smooth or rough not smooth not smooth smooth or rough smooth 0.60 6.02 0.1 108 Oil glands prominent prominent prominent prominent inconspicuous inconspicuous rnconsP¡cuous inconspicuous 0.70 19.00 0.0003 or prominent or Drominent or orominent
Correlation coefficient" : showing the fit (r2¡ of the individual character vectors in the ordination space
Kruskal-Wallis FI statisticb : the non parametric analysis of variance to determine whether the ranked group means are significantly different from one another for each character. E- 'f lrrbrse Gemt mornholosical analvsis ll-4-3-
EeEs 1 .EeEs, F1 9 EeEs 10 EeEg, Es 11 EeEg, Es \2 EeEs, F1 2 F1 3 F2 4 EUG2n 5 EeEg 6 EeEg 7 EeEg ó 5 EeEg 1,3 Eee 74 Eee, Eem 15 Eee, Eem 16 EeEg 77 EeEg 18 EeEg 20 Eg 19)
I I 0.089 3 0.197 2 0.3052 0.413 0.521 0.629
Figure 6.4. UPGMA dendrogram representing 486 seedlings of E. erythronema var. erythronema x E. strícklandii (EeEs), E. erythronema var. erythronema x E._gomphocephala (EeEg), open pollinated E. erythronema var. erythronema (Eee), open pollinated E. erythronema var. marginata (Eem), open pollinated E. stricklandü (Es), open pollinated E. gomphocephala (Eg), open pollinated Fl E. 'Urrbrae Gem' (Fl) and open pollinated, 2"d generation E. 'Urrbrae Gem' (EUG2n). The dendrogram identifies eight clusters (l-8) based on 20 group (in parentheses) means. Scale represents standardised Manhattan distance (Gower metric) based on 14 characters (Table 6.4).
Table 6.5. Number of seedlings of E. 'Urrbrae Gem' and putative relatives in each dendrogram cluster (Figure 6.4).
Cluster 12345678 E. erythronema var. erythronema 45 open pollinated seedlings
E. erythronema vat. marginata 1 20 open pollinated seedlings Fl E. 'Urrbrae Gem' 64 open pollinated seedlings 2"d generation E. 'Urrbrae Gem' 65 open pollinated seedlings E. erythronema yaÍ. erythronema 6 54 9 x E. stricklandii E. stricklandii 65 open pollinated seedlings E. erythronema vaÍ. erythronema 3 1622 70 x E. gomphocephala E. gomphocephala 65 open pollinated seedlings
t2t E- .rlrrhrae Gemt mornholosical analvsis 6.4.3. generation E. 'Urrbrae Gem' respectively, plus the remainder of the E. erythronema var. erythronema x E. -stricklandii seedlings (9).
The majority of E. erythronema var. erythronema x E. gomphocephala seedbngs (70) were in cluster 8, with the 65 open pollinated seedlings from E. gomphocephala. Cluster 8 connected to cluster 5 at 0.26, which contained 16 E. erythronema var. erythronema x E. gomphocephala seedltngs. This group connected to cluster 4 at 0.20. The remaining two E. erythronema var. erythronema x E. gomphocephala seedltngs were separated as cluster 6, which was connected to cluster 2 at 0.33. Cluster 7 included the open pollinated seedlings of
E. erythronema vars. erythronema and marginata, and was connected to cluster 4 at 0.20.
Principal canonical correlation analysis of seedling characters (Figure 6.6) showed significant correlations between most taxon groups and characters (Table 6.4). There were no def,rning characters for the hybrid seedling groups, but the open pollinated Fl E. 'Urrbrae Gem' were more similar to the E. erythronema vaÍ. erythronema x E. stricklandü seedlings than to any other group. Leaves of the seedlings produced by controlled pollination were intermediate in appearance between those of the open pollinated seedlings of the pa.rents at the same age.
The results of this study indicate that progeny from a controlled cross between E.
erythronema var. erythronema and E. stricklandii are more similar to the open pollinated
seedlings from the Fl and 2nd generation E 'Urrbrae Gem', than to the progeny of a cross
between E. erythronema vat. erythronema and E. gomphocephala. It is possible to identify
hybrids between these species from differences between seedling leaves at the cotyledonary
leaf stage and at three months of age.
122 E- 'fTrrhrse (]emt mornholosical analvsis ó.4.3.
2
I
0.1 o.t X /. 0 ,/ 0.33 rt) 0.15 à l/ AA -l 8¡ A
-2 a -l 0 2
MDSAxis I
I E. erythronema vú. erythronewra xE. shicklandii ffi Fl .8. 'Urrbrae Gern' open pollinated seedlings tr E. stricWandil open pollinated seedlings o 2nd generation E 'lJrrbrae Gorn' open pollinated seedlings A E. erythronemavñ. erythronema x E. gomphocephala O E. erythronemavü. erythronema andvar. marginnta open pollinæed seedlings A E. gomphocephala open pollinatod seedlings
Figure 6,5. SSH Multidimensional scaling of 20 groups means of 486 seedlings using all juvenile characters. Distances and linkages are results from a minimum spanning tree analysis of the data.
123 .8. 'Urrbrae Gem' morphological an4lysis 6.4.3.
2
OGP
ôl LPO X 0 .... ssM.. (^ o rre l!n$
LPL LSH LSTISa LW LEA
a ,| -1 0 2
MDS Axis I
Figure 6.6. Principal canonical correlation plot for the semi-strong hybrid ordination (Fig. 6.5) based on the seedling characters measured, all vector lengths l. Character codes are those detailed in Table 6.1., characters listed in Table 6.4.
.=' i
IA E- 'Ilrrhrae Gem' moroholoeical analvsis 6.5.
6.5. Discussion
Interspecific hybridisation within the genus Eucalyptus reflects the hierarchy of taxonomic affinities (Griffin et a\.,1988). Natural, or spontaneous, hybridisation does occur, particularly in areas where the geographical barriers to hybridisation have been removed, however the occurrence of such hybrids is often poorly documented (Griffin et al., 1988; Hopper, 1995).
Several suspected Fl hybrid adult trees have been studied in detail, by examining adult morphological characters and seedling morphological characters of progeny (Pryor, 1950;
Hopper et aL, 1978) as well as investigating chemical traits (Grayling and Brooker, 1996).
Natural hybrids can occur as single individuals some distance from the male parent (Ashton and Sandiford, 1988), single individuals near the junction of two species (Potts and Reid,
1983), or as hybrid swarms at the junction of the parent species' range (Potts and Reid, 1985).
The effect of areas such as arboreta on the potential for spontaneous hybridisation has not yet been documented, but as species are removed from their natural distribution and planted near others normally geographically isolated, the rate of hybridisation may increase. Manipulated hybridisation is a tool used frequently in tree improvement progtams, and is quite successful in eucalypts (Eldridge et a|.,1993).
In a review of interspecific hybridisation patterns n Eucalyptøs, Griffin et al. (1988) listed five interspecific hybrids involving two of the species investigated in this study. Naturally occurring crosses between E. erythronema vaÍ. erythronema and E. eremophila (from sect.
Bisectaria ser. Erythronemae and sect. Bisectaria ser. Occidentales), E. gomphocephala and
E. cornuta (from sect. Bisectaria ser. Cornutae and sect. Bisectaria ser. Cornutae), and E. gomphocephala and E. wandoo (from sect. Bisectaria ser. Cornutae and sect. Bisectaria ser.
Reduncae) were recorded in Eucalist (Chippendale and Wolfe, 1984). Hybrids occurring as manipulated or spontaneous were also reported by Griffin et aI. (1988), between E erythronema vaÍ. erythronema and E. torquata (from sect. Bisectaria ser. Erythronemae and
L2s F- .f lrrhrne êemt mornhnlooical ânâlvsis ll.5- sect. Dumaría ser. Torquatae) (Chippendale and Wolf, 1984; Gardner, 1979), E. gomphocephala and E. cornuta (from sect. Bisectaria ser. Comutae) (Jacobs 1979), and E gomphocephala and E. occidentalis (fromsect. Bisectaria ser. Cornutae and sect. Bisectaria ser. Occidentales) (Jacobs, 1979). The final species, E. stricklandii (sect. Bisectaria ser.
Grossae) had no reported hybrids from that study.
Determination of the parentage of the spontaneous Fl hybrid, E 'Urrbrae Gem', followed some of the criteria for defining a putative eucalypt hybrid (Hopper et aI., 1978: Hopper,
1995). The criteria included evidence of intermediate features (morphological, anatomical and physiological) between the putative hybrid and the parent species, and a close resemblance between suspected and manipulated hybrids; and of secondary consideration, phenotypic segregation and impaired reproductive capabilities of hybrids relative to the paternal individuals (i.e. F2 breakdown).
To investigate the criteria of intermediate morphology and hybrid resemblance, 60 adult and
17 seedling characters were measured. Morphological characters are frequently used to identify species and hybrids. In eucalypts, morphological characters have been used to investigate differences between species, within species, between provenances, complexes and intergrades, as well as to study geographical variation within species. A diverse range of characters is measured from cotyledons and leaves (from seedlings and adults), inflorescences, buds and fruit, as well as the bark and overall tree habit (Doran and Burgess,
1993; Jordan et aI., 1993: Chappill and Ladiges, 1996). In some cases environmental attributes are taken into account, such as associated soil type and rainfall data (Nicolle and
Conran, 1999), as are characters such as leaf waxes (Hallam and Chambers 1970), trichomes
(Ladiges 1984) and oil composition (Dunlop et al., 1997). For identification of Fl hybrids, the characters measured are often specific to the species involved. For example,Hopper et al.
L26 ,8. 'I]rrbrae Gem' moroholosical analvsis 6.5.
(1978) determined the relationship between E. preissiana and E. buprestium and thetr
possible hybrid E. chrysantha using adult morphological characters specifically selected to
provide the best species discrimination.
The results of the MDS analysis of adult characters placed the Fl E. 'Unbrae Gem' centrally
on the ordination axis, and most closely associated with the open pollinated 2"d genention E
'Urrbrae Gem'. The two varieties of the female parent species were linked very closely to
each other and less closely to the open pollinated 2"d generation E 'Urrbrae Gem'. This
indicates that the analysis was robust enough to show the difference between two varieties,
even though they possessed few differing morphological characters, and that the open pollinated 2nd generation population was segregating back to the female parent, indicating possible back crossing to the original female parent species. The two postulated male parent
species were both equally close to the Fl E 'Urrbrae Gem', however, the MST values
showed that E. stricklandü was closer to the Fl E. 'Urrbrae Gem' than E gomphocephala,
with additional analysis of group means confirming this.
On the assumption that a hybrid will often exhibit intermediate morphology (Hopper et al.
1978; Hopper, 1995), hybrids should be placed between clusters of individuals from the parental species in an ordination plot. McDade (1997) discussed MDS and similar non- hierarchical graphical methods, and their use in hybrid parental analysis, noting that a hybrid will be placed either with one of the parents or between them, and that calculation of a distance matrix should enable the identification of the closest (parent) taxa. Our study placed
E. 'Urrbrae Gem' directly between E. erythronema val. erythronema and E. stricklandii, with which it shares the two shortest MST distances, indicating that it is intermediate between them. The positions and MST distances of the remaining species (8. gomphocephala and E gardneri) to the Fl, are not as close and thus these are less likely parental candidates.
127 F 'TTrrÌ¡rne êernt rnnrnhnlnoienl ennlvsis 6^5-
The PCC analysis for adult characters showed that there are few characters uniquely shared by the hybrid and either of the possible male parents, and that the Fl and 2nd generation hybrids have intermediate scores for nearly all of the characters measured. The fact that E. stricklandä was only marginally closer to the Fl E. 'Urrbrae Gem' than E. gomphocephala, may explain the original confusion in opinion on the identity of the male parent.
The criteria of secondary consideration; that is phenotypic segregation and impaired reproductive capabilities of hybrids relative to the paternal individuals (i.e. F2 breakdown), were studied on an empirical level only due to limited numbers of plants. The 2nd generation
E. 'Urrbrae Gem' tree used throughout the study is the single surviving adult specimen of a number of seedlings from E. 'Urrbrae Gem' (Fl) to be planted in the Waite Arboretum over the last 50 years. While open pollinated seed from the Fl and 2nd generation E. 'Urrbrae Gem is viable, seedlings generally exhibit poor form, increased phenotypic segregation and low vigour. This was observed whilst germinating open pollinated seed from both the Fl and 2nd generation E. 'Urrbrae Gem" during this study.
Controlled or manipulated hybridisation has been used in numerous studies investigating hybridisation in eucalypts, to refute or support the identity of a hybrid through field studies and progeny tests of natural and manipulated hybrid combinations (Pryor, 1950, 1951a,
1951b, 1952, 1954, 1956, 1957). The standard technique of controlled pollination for eucalypts varies with researchers, but generally, the flowers are ernasculated prior to anthesis, bagged to exclude external pollen, pollinated manually some days later, and left to develop.
Research has found that emasculation of flower buds can discourage pollinators such as bees
(Hodgson, 1976), bagging can have a detrimental effect on the survival of the pollinated flowers (Pryor, l95la), and that successful pollination can be achieved with neither bagging
(Tibbits, 1989; Beardsell et aI., 1979) or emasculation (Beardsell ¿l aL, 1979). Flowers in
t28 F 6f lrrhrqp êernt rnnrnholnqieel enâlvsis 6-5- this study were emasculated byt not bagged in order to observe the proficiency of controlled pollination without isolation.
Pollen and pistil physiology also play an important part in hybridisation success, and should be considered in the pollination protocol. Pollen from different species varies in its level and rate of germination and tube growth (Potts and Marsden-Smedley, 1989). Pollen grains may take up to two days to germinate if the stigma is not receptive (Hodgson, 1976), and individual grains may germinate at different rates (Heslop-Harrison and Heslop-Harrison,
1985). In addition, there is variability between species in time to stigma receptivity after anthesis, from two days for E. macarthurii, E. cinerea and E. occidentalis (Davis, 1968) to 13 days for E. urnigera (Savva et aI., 1988). Cauvin (1988) demonstrated that cutting of the style, to remove the stigma, could have applications to breeding programs, as it meant that it was no longer necessary to wait for stigma receptivity prior to pollination. Recent work by
Harbard et al. (1999), where the style is cut longitudinally, was found to increase seed set in
E. globulus. In both cases, masculation, cutting and pollination could be completed in one step; the chances of contamination were reduced; and the problem of the length of style in relation to pollen tube length could be overcome. This last point has been considered to be the cause of some previous failed interspecific hybridisation attempts between long styled and short styled species, as it was suggested that pollen tube length is correlated to style length.
These include E. caesia x E. sideroxylon and E. caesia x E. leucoxylon (Beardsell et aL,
1979), E. caesia x E. pulveruIenta (Pryor, 1956) and E nitens x E. globulzs (Tibbits, 1988;
1989; Gore et al., 1990) where the reciprocal was successful. In each case, the cross was successfi¡l only when the style from the male species flower was longer than that of the female species. In both cases in this study, the style and stigma of E. erythronema yaÍ. erythronema are very similar in length and size to those of E. stricklandii and E. gomphocephala.
t29 E. 6Urrbrae Gemt mornholosical analvsis 6.5.
Controlled pollination between E. erythronema yar. erythronema and E. stricklandii produced more seed per flower pollinated that the cross between E. erythronema vaÍ. erythronema and
E. gomphocephala. The seed collection data indicated that there may be more of an effect of female tree, rather than male species, on seed production, indicating maternal inheritance.
Maternal inheritance in interspecific hybridisation of eucalypts has been identified (Tibbits,
1989). However, the cotyledon shapes of the seedlings for each cross were similar to the cotyledon shapes of the open pollinated seedlings of the respective male parent. Large numbers of seedlings were obtained for measurement, despite seedling mortality rates of 65 and 53Vo for E erythronema var. erythronema and E. stricklandii, and E. erythronema vaÍ. erythronema and E. gomphoc ephala, respectively.
In the present study, progeny from controlled pollinations between E. erythronema var. erythronema and E. stricklandü, and E. erythronema yar erythronema and E. gomphocephala" were measured for a range of seedling leaf and stem characters three months after germination. E. erythronema var. erythronema x E. stricklandü seedlings were intermediate between the parent species, with morphology similar to that of the open pollinated seedlings of Fl and 2nd generation E. 'Urrbrae Gem'. The majority of the E. erythronema vaÍ. erythronema x E. gomphocephala seedlings grouped with open pollinated E. gomphocephala seedlings, supporting their hybrid status, and isolating them from the open pollinated seedlings of Fl and 2nd generation E. 'Urrbrae Gem'. A small number of E. erythronema vaÍ erythronema x E. gomphocephala seedlings was placed very close to the open pollinated seedlings of Fl and 2nd generation E. 'Urrbrae Gem'. This may reflect early seedling variability, or may be due to past hybridisation events within this cross fertile group of species. It is notable that large numbers of hybrid seedlings were obtained, despite seedling mortality rates of 65 and 537o for E. erythronema var. erythronema x E. stricklandii and E. erythronema v ar. erythronema x E. g omphoc ephala respectively.
130 E- 'f Tnhrne (lemt mornholosical analvsis 6-6-
6.6. Conclusion
This study, through comparison of adult and seedling morphological characters, has strongly suggested that E stricklandü is the probable male parent of the hybrid E 'Urrbrae Gem'. The adult morphological characters study placed the hybrid intermediately between clusters of E erythronema yar. erythronema and E. stricklandlf seedlings. Morphological character analysis of seedlings from controlled pollinations indicated similarities between E. erythronema vaÍ. erythronema x E. stricklandir hybrid seedlings and the open pollinated seedlings from the Fl and 2nd generation trees of E. 'Urrbrae Gem', while seedlings from crosses between E erythronema var. erythronema x E. gomphocephala are not similar. This study has also shown that adult and seedling morphology studies are valuable tools in plant improvement programs.
They are cheap to conduct and can be applied to large numbers of plants. The data can be used to indicate profitable hybridisation matrices for the generation of novel lines for ornamental horticulture.
131 Mucrocarpae
Selection at Species Selection for Floral Criteria - and Cultivated Species level Gene Pool Wild populations
Selection for Floral Criteria Selection at Genotype Genotype Level
Hybrid Production through C ontrolled Pollination
H ybrid/Parent Identification/Confirmation
Superior Development of Propagation Methods Genotype
Postharvest Testing
PBR Registration
Multiplication and Release to Industry
Ornamental eucalypt breeding cycle flow chart.
Areas covered by this chapter are highlighted. Møaroearnnc 7-l-
Chapter Seven
Interspecifïc hybridisation : E. macrocørpø, E. pyriformis and E. youngiøna
7.1. Abstract
Interspecific hybridisation between three Eucalyptus species with horticultural merit was undertaken to investigate the likelihood of success of such crosses. E. macrocarpa, E. pyriformis and E. youngiana are closely related species from Western Australia, with slightly differing geographic distributions. Crosses were conducted between species with all combinations producing fertile seed. There was no effect of male parent, with interspecific crosses as successful as intraspecific crosses, but there were difference in fertility between female trees within a species. Hierarchical and non-hierarchical analysis of each cross indicated a high degree of interspecific hybridisation, with the majority of seedlings measured clustering separately from either parent. The 166 seedlings from the interspecific cross seed, when measured for a range of leaf and stem characters at three different nodes, show strong evidence of intermediacy between parents, with I.2Vo grouped with the male parent,94.6Vo clustered between the parent species, and the remaining 4.27o with the female parent. The success of this program in producing both large numbers of seed and large numbers of hybrids indicated that it is possible that these species will hybridise naturally in the same location. It also demonstrated the potential of controlled pollination, between closely related species of similar floral morphology, as a method to produce hybrids for further evaluation.
132 Mna¡oanrno¿ 7-2-
7.2. Introduction
The international cut-flower and nursery industries are constantly searching for new and novel
products. A number of Eucalyprøs species show floricultural potential, with coloured buds of
varying sizes and shapes, which open to flowers of a range of colours. Three such species
with potential are E. macrocarpaHook (Plate. 7.1), E pyriformis Turcz. (Plate 7.1) and E.
youngiana F. Muell. (Plate 7.1), alI of which have buds and flowers greater than 40 mm in
diameter, ranging in colour from yellow to deep red, flowering from early spring to late
summer. These species also show merit as garden plants, as they are small growing trees or
mallees, with interesting tree forms, long flowering seasons and attractive seed capsules.
All three taxa occur naturally in Western Australia: distributions of E. macrocarpa and E. pyriþrmis range from Geraldton to Perth, with E pyriformis more northerly. E. youngiana
occurs further east, from near Kalgoorlie to the north western part of South Australia (Figure
7.r).
E. macrocarpa, E. pyriþrmis and E youngiana are closely related taxonomically, in E. series
Curviptera (Chippendale, 1988), also known as Subgenus Symphyomyrtus, Section
Bisectaria, Series Macrocarpae (Pryor and Johnson, 1971). A later revision (Brooker and
Hopper, 1993) divided E. ser. Curviptera (Chippendale, 1988) into two subseries - subser.
Inflexae (stamens inflexed) and subser. Curviptera (stamens erect, oblique or variously
curved). Natural hybrids involving these species have been reported between E. macrocarpa
and E. pyriþrmis, E. pyriformis and E. youngiana and E. drummondii and E. macrocarpa (a
very rare and almost sterile hybrid known as E. x carnabyi) (Griffin et aI., 1988; Hopper,
1995) and it has been suggested that natural hybridisation between these species may occur
where their distributions overlap.
133 Møcrocaroae 7.2-
The technique of manipulated hybridisation, or controlled pollination, can be a useful tool in
plant breeding to generate hybrids that may show improved characteristics, and the possibility
exists to combine species that are incapable of breeding through geographical or temporal
isolation (Tibbits, 1988). Manipulated hybridisation has also been used to test the probability
of natural hybridisation (Pryor, 1950, l95l , 1952, 1954, 1956, 1957).
All three species have characters that make them suitable for floriculture or garden plants, however, each also has undesirable characters. E. macrocarpa has sessile buds, flowers and leaves, as well as being slow growing with poor form, but the large red flowers and heavily glaucous leaves are very attractive. E. pyriformis has pendulous buds and flowers, but has a range of bud and flower colours and sizes as well as good tree form. E. youngiana has good vigour and form, as well as a range of flower colours, but has a tendency to hold fruit for a long time, resulting in heavy branches that frequently break. The combination of desirable characters, such as flower colour, glaucousness, tree form and vigour, in seedlings from crosses between these species, may enhance their ornamental merit, resulting in a plant more commercially viable than its parents.
The aim of this study was to determine the likelihood of success of controlled hybridisations between these three species, and to generate hybrids for evaluation for ornamental merit.
134 Macrocarpae 7,2.
I
Plate 7.1. A: E. macrocarpa flower; B: E. pyriformis flower; C: E. youngiana flower. Bar: 1cm,
135 Mn¡¡nanrnnc 7^2-
Figure 7.1. Map of natural distribution of E. macrocarpa, E. pyriformis and
E. youngiana.
) n. macroca,rpa, ' E. pyriformis I r. youngiana
p
o
\
0ù
L36 Macrocamae 7.3.1.
7.3. Materials and methods
7.3.1. Plant Material
The fourteen adult plants used are listed in Table 7.1. Plants were sourced from the Waite
Arboretum and the Monarto Woodland. Plants were selected for their different bud and flower colours. Interspecific crosses between E. macrocarpa, E. pyriþrmis and E. youngiana were made, as well as intraspecific crosses. Open pollinated capsules were collected from these trees for comparison with seed from controlled pollinations.
7.3.2. Controlled pollination
The pollination technique follows that of van Wyk (1977) and Moncur (1995), and described in detail in Chapter 6, section 6.3.3. Equal amounts of pollen from different plants of the same species were mixed for use, reducing the chance of pollinations with low fertility or related pollen. Between four and forty flowers per tree per cross were pollinated on each tree of E. pyriformis, depending on the number of flowers available, with all flowers pollinated and pollinations distributed evenly between crosses. All flowers on E. macrocarpd were pollinated, up to fourteen per cross, with pollinations distributed evenly between crosses. Up to forty flowers were pollinated for each cross on each E. youngiana tree with all other flowers being removed. Flowers were emasculated but not isolated, nor were they randomised to the tree (see section 6.3.3.). Capsules were harvested 8-10 months after pollination, and each capsule was placed separately in a paper bag. The capsules were stored in boxes over silica gel, in a dry room at 30'C until open (c. 6 weeks). Seed was extracted and counted, and seedlots of individual capsules weighed. Seeds were planted and grown as described in Chapter 6, section 6.3.3., with 200 seeds randomly selected for each cross, dates of germination recorded and seedlings grown on under glasshouse conditions. In addition, seed of the parent species from various commercial seed sources was grown for comparison; this proved to be highly variable.
137 Møcrocaroae 7.3-3-
7.3.3. Seed data analysis
Differences in the mean number of seeds produced per flower pollinated, mean seed weight for all seeds of that cross and germination rates were calculated and compared using a randomised complete block design, general analysis of variance and least significant difference where appropriate (Genstat 5 Release 4.2.).
Table 7.1. Plants used in controlled pollination matrix from subgenns Symphyomyrtus, sect. Bisectaria, ser. Macrocarpae (Pryor and Johnson, l97I).
Numberu Codeb Location" Useo Characters" Seed source E. macrocarpa 1632 Eml WaA FM SM; large red flowers, glaucous WA Forestry leaves Dept., 1952 t952 \üaA M SM; small to medium red flowers, Nind., 1963 glaucous leaves 1848 WaA M SM; small red flowers, glaucous Eastern leaves Park, 1963 1838 WaA M SM; red flowers, glaucous leaves Seed Index At26t,1962 Nind, RPSr S
E. pyriformis t926A Ep1 WaA FM SM; red flowers, green buds Seed from 19264, WaA r813C Ep2 WaA FM ST; yellow flowers, green buds Woods and Forest, 196l 8.9.2 Ep3 MoW F SM; yellow flowers, green buds unknown 8.9.4 Ep4 MoW F SM; yellow flowers, purple buds unknown 8.27.2 Ep5 MoW F SM; peach flowers, purple buds unknown Nind S E. youngiana 8.54.2 Ey2 MoW FM ST; red flowers unknown 8.54,4 Ev4 MoW FM ST; peach flowers unknown 8.s4.5 Ev5 MoW F ST; red flowers unknown 8.54.7 Ev7 MoV/ F ST; yellow flowers unknown 8.s4.8 MoW M ST; yellow flowers unknown 1569 WaA M ST; yellow flowers D. Symons 1984 Nind, RPS S Number: number of plant in plantation b Code: number given to plant in crossing program (female tees only) ' Location: WaA = Waite Arboretum, MoW = Monarto Woodland o Use: useof plantsin program (F=femaleparent, M=maleparent, S = seedling morphologyonly) " Cha¡acter: SM = small mallee (to 4 m, multiple trunk), ST = small tree (to 4 m, single trunk), desirable character of plant. r Seed source: Nind = Nindethana seeds W.4., RPS= Royston Petrie seeds N.S.W.
138 Macrocømac 7.3.4.
7.3.4. Seedling morphology
All surviving seedlings from controlled pollinations were measured three months after germination for thirteen leaf characters on fully expanded leaves at the 3'0, 5* and 7û nodes above the cotyledons and for three stem characters (Table 7.2). In addition, open pollinated seed from the female parent trees and seed from commercial sources was grown to provide a representative sample of seedling morphology of each species. Between 40 and 80 seedlings, selected at random from each parent species depending on survival rates, were measured also at three months after germination. Data were averaged to generate five representative individuals for each species, and these individuals used in the analysis for comparison with the seedlings from the controlled pollinations. The characters of leaf petiole/sessile (LPS) and stem structure (STE) were removed from all crosses prior to analysis as they were identical for all individuals measured.
Table 7.2. Characters and codes used in phenetic analysis (seedling), measured at nodes 3, 5 and 7, three months after germination.
Code Character Description LSH# Leaf shape linear (0) lanceolate (1) elliptic (2) ovate (3) orbicular (4)" LST# Leaf tip acuminate (0) acute (1) mucronate (2) obtuse (3) LSB# Leaf base attenuate (0) obtuse (l) cordate(2) LLE# Leaf length (mm) LWI# Leaf width (mm) LSW# Leaf surface wax shining (0) dull (1) glaucous (2) very glaucous (3) LEA# Leaf sides concolorous (0) discolorous (l) LSM# Leaf surface smooth (0) not smooth (l) LPS# Leaf attachment sessile (0) petiolate (l) LPL# Leaf petiole length (mm) LPO# Leaf position opposite (0) alternate (l) LCO# Iæaf colour light green (0) green (1) grey green (2) blue green (3) dark green (4) LMI# Leaf margin smooth (0) partially jagged (1) jagged (2) STE# Stem arrangement single (0) branching (1) SSH# Stem shape round (0) angular (1) SSM# Stem su¡face smooth (0) not smooth (1) "linear (L:41W) lanceolate (2W 139 Maaroaarnac 7-4-7- 7.3.5. Data analysis The PATN multivariate analysis package (Belbin, 1994) was used to analyse the morphological character data for the seedlings, using hierarchical (Gower UPGMA) and non- hierarchical (multidimensional scaling, minimum spanning tree and principal canonical correlation) analysis to determine the relationships between the seedlings and their parents. 7.4. Results 7.4.1. Controlled pollination seed data 7.4.1.1. Between species comparison All controlled pollinations between the three species produced seed (Table 7.3). The lowest number of seeds per capsule was produced from the intraspecific cross, and for E. macrocarpa and E. youngiana the highest number of seeds was produced from the open pollinated cross. No obvious trends were revealed when comparisons of mean number of seeds per flower pollinated to mean weight of seeds (mg) (Figure 7.2) and percent germination of seed to mean seed weight (Figure 7.3) were represented graphically. There were no significant differences between the male species in the mean number of seeds produced per flower pollinated, or percent seed germinated. There were differences between female parent species in mean seed weight (Table 7.3) with E. macrocarpa having the heaviest seed (3.10 mg), E. pyriformis the lightest (2.2I mg) and E. youngiana seed intermediate (2.52 mg). 140 Table 7.3. Summary of flower, capsule, seed and seedling data for interspecific, intraspecific and open pollination of E. macrocarpa, E. pyriformis and, E. youngiana. Female species Cross Number Number Total Mean number Number Number Number of of seed of seeds per seed of of seeds of flowers capsules flower weight seeds seeds germinated seedlings pollinated with seed pollinated (ms) planted serminated measured E. macrocatpa E. pyriformis l0 4 187 18.7 3.26 186 11 5.9 5 E. macrocarpa E. youngiana 14 5 230 t6.4 2.90 200 36 18.0 20 E. macrocarpa E. macrocarpa (intra) 14 3 69 4.9 3.41 ø 20 31.3 16 E. macrocarpa open l0 8 409 40.9 2.86 200 53 26.5 E. pyriformis E. macrocarpa ll0 55 I45l 13.2 2.28 200 62 31.0 36 E. pyriformis E. youngiana 100 56 2029 20.3 2.18 200 66 33.0 28 E. pyriformis E. pyriformis (intra) 88 73 1450 13. I 2.29 200 63 31.5 28 E. pyriformis open 54 30 903 16.7 2.16 200 85 42.5 E. youngiana E. macrocarpa 135 8l 1330 9.9 2.51 200 74 37.0 39 E. youngiana E. pyriformis 128 68 I24t 9.7 2.57 200 8l 40.5 38 E. youngiana E. youngiana (intra) l2O 67 1076 9.0 2.40 200 16 8.0 6 E. youngiana open 80 76 3124 39. I 2.51 200 63 31.5 E. macrocarpa 20.5 3.10 20.4 E. pyriformis 16.5 2.2t 34.5 E. youngiana 15.9 2.52 29.3 L.S.D. 23.30 o.292 t 17.7t total number of seed from all capsules of that cross divided by the total number of flowers pollinate for that cross. 2 weight of all seeds in a capsule divided by the number of seeds in that capsule. 3 number of seeds germinated divided by the number of seeds planted, multiplied by 100. IMean number of seeds per flower pollinated ÔMe¿n seed weiSht (ffn) 45 0.004 .u 40 0.0035 o d O .E ls a 0.003 Àô a ¡o 5.b o 0.0025 g rÉ tao q 1{ aata å B. 'õ -d 0.002 ¡ Bzo o o 0.0015 o bls Ê ,o od E 0.001 tr ir l0 Go E5 0.0005 0 0 ñ 01" .{nso* 1, Figure T.2.Meannumber of seeds per flower pollinated and mean seed weight (mg) IMean seed weight (Sm) O Percent seeds germinated 0.004 45 40 0.0035 35 0.003 30 0,0025 tr .9 25s E Þ0 o o 0.002 o È 20 e !o c) 0.0015 l5 0.001 10 0.0005 5 0 0 *."'5.t$$"55î:i.,.;$îi'''tï:.'i'.*::-'l'þ.* Figure 7.3. Comparison of mean seed weight (mg) and percent germination of seed Macrocama¿ 7.4.1 7.4.1.2. Within species comparison E. macrocarpa as female parent V/hile four E macrocarpd trees were available as pollen donors, only one was available for use as a female due to limited flowering. A total of 38 flowers on this tree were hand pollinated, using pollen from E. pyriformis, E. youngiana, or E. macrocarpa. Open pollinated seed was collected from capsules from the previous flowering season. The data are summarised in Table 7.4, with no statistical analysis possible as only one tree was available as a female parent. Data showed that open pollinated flowers produced the greatest number of seeds per flower pollinated, the two interspecific crosses produced similar amounts, while the intraspecific cross produced less. Weight of seeds from each cross was not greatly different. For hand pollinated flowers, fewer than 50Vo of flowers pollinated produced seed, regardless of the cross. Of the seeds planted, germination percentages for each cross were very low, particularly in the E. macrocarpa x E. pyriþrmis cross, where only 11 seeds out of 186 germinated and only five survived to three months. Highest germination rates were recorded following open pollination. E. pyriformis as female parent There were no significant differences between male species on the mean number of seeds produced per flower hand pollinated, with open pollination the least successful cross (Table 7.5). Mean seed weight and percent seeds germinated were not affected by male species (Table 7.5), but were affected by female trees, as were the number of seeds per flower pollinated. E. pyriformis 8.21.2. (Ep5) produced the most seed, the heaviest seed and the second highest germination rate of the five female trees tested, while E. pyriformis 8.9.2. (Ep3) produced the least seed, the lightest seed and the second lowest germination rate. 143 Table 7.4.Etrect of male parent on controlled pollination of E macrocarpu Cross Female Number Number Total Mean number of seed Number Number Percent Number of tree of of seed seeds per flower weight of of seeds seed seedlings flowers capsules pollinated (mg) seeds germinated germinateda 3 months after 2(ranqe) pollinated with seed (ran planted E. pyriformis Eml l0 4 t87 18.7 (s-88) 3.26 (2.76-4.23) 186 ll 5.9 5 E. youngiana Eml 14 5 230 t6.4 (22-84) 2.90 (2.10-3.96) 2OO 36 18.0 20 E. macrocarpa Eml 14 3 69 4.9 (8-47) 3.41(2.63-3.08) & 20 31.3 l6 - controlled intraspecific Open Eml 10 8 409 40.9 (20-77) 2.86 (3.16-3.63) 2W 53 26.s 46 I total number of seed from all capsules of that cross divided by the total number of flowers pollinate for that cross. 2 Range of number of seeds in capsules for that cross minimum to maximum 3 - weight of all seeds in a capsule divided by the number of seeds in that capsule. 4 number of seeds germinated divided by the number of seeds planted, multiplied by 100/1. Table 7.5. Effect of male parent on controlled pollination of E pyriformis. Superscripts denote significant differences in a column at P<0.05, numerical superscripts as for Table 7 -3- tMean Cross Female Number Number Total number 'Mean Number Number Percent Number of tree of of seed of seeds/flower seed weight of of seeds seedlings 3 flowers capsules pollinated (mg) seeds seeds germinated3 months after 2(range) pollinated with seed (range) planted germinated germination E. macrocarpa Epl 4 2 29 7.3 (s-24) 2.ts (2.12-2.17) l6 6 37.5 4 Ep2 12 8 127 10.6 (2-31) 2.04 (1.72-232) 32 7 21.9 3 Ep3 38 t7 198 s.2 (r-s3) 1.86 (1.58-2.54) 48 6 12.5 3 Ep4 t6 6 262 r6.4 (7-88) 2.51(1.94-3.37) 24 10 4t.7 9 Ep5 40 22 835 20.9 (1-88) 2.83 (2.0r-3.6t) 80 33 4t.3 t7 Total ll0 55 t45t 12.1" (l-88) 2.28" (1.58-3.61) 2W 62 31.0" 36 E. youngiana Epl 4 2 63 15.8 (16-47) 2.ro (1.91-2.29) 8 3 37.5 2 Ep2 11 1l 23r 21.0 (10-46) 2.23 (1.82-3.12) 40 26 65.0 t4 Ep3 31 l0 193 6.2 (t-s3) 2.O8 (t.48-2.71) 32 5 15.6 I Eú t4 6 334 23.9 (rt-s3) t.s9 (t.29-2.62) 24 I 4.2 I Ep5 40 27 1208 30.2 (t-tt7) 2.84 (1.26-4.07) 96 31 32.3 l0 Total 100 56 2029 19.4^ (t-tt7) 2.18" (t.26-4.07) 200 66 30.9" 28 E. pyriformis Epl 7 5 t23 17.6 (10-36) 2.19 (1.89-2.s3) 28 8 28.6 J - controlled Ep2 5 3 16 3.2 (8-8) 2.33 (2.31-2.3s) 8 4 50.0 I intraspecific Ep3 t9 lt 4 o.2r (4-4) 1.18 (1.10-1.28) 4 0 0.0 0 Ep4 t7 t6 354 20.8 (r-sz) 2.7r (2.18-3.s8) 68 5 7.4 5 Ep5 40 38 9s6 239 (t-39) 3.03 (2.r3-4.s7) 92 46 50.0 t9 Total 88 73 1453 t3.l^ (t-52) 2.29^ (r.t0-4.57) 200 63 27.2^ 28 Open Epl 9 6 135 ls.0 (4-s0) 2r2 (1.7O-2.s4) 20 9 45.0 I Ep2 10 6 42 4.2 (r-32) 227 (2.06-2.40) t2 8 66.7 4 Ep3 l1 I I 0.1 (l-1) 1.18 (1.18-1.18) 8 5 62.5 4 Ep4 7 2 43 6.1(20-23) 2.36 (2.t7-2.s4) 16 1 6.3 0 Ep5 l7 15 682 10.1 (1-110) 2.88 (2.t1-3.s9) 144 62 43.t 22 Total 54 30 903 7.1b (1-110) 2.16" (1.18-3.59) 200 85 44.7" 31 L.S.D. 11.1 0.47 24.9 Table 7.6. Effect of male patent on pollination of E. youngiana. Superscripts denote significant differences in a column at P<0.05, numerical superscripts as for Table 7.3. lMean Cross Female Number Number Total number of Mean seed Number of Number Percent Number of tree of of seed seeds per flower weight seeds of seeds seedlings 3 flowers capsules pollinated (mg)' planted seeds germinated3 months after 2(ranse) pollinated with seed (ranse) serminated sermination E. macrocarpa Eyz 36 l1 494 13.7 (5-13l) 2.63 (2.16-2.97) 40 4 10.0 0 Ev4 30 t9 105 3.5 (l-16) 3.U (1.99-3.6s) 28 2t 75.0 7 Ev5 32 24 203 6.3 (l-33) 2.90 (t.U-4.r2) 48 34 70.8 2t Ev7 37 27 528 14.3 (1-105) 1.47 (0.58-1.98) 84 15 17.9 10 Total 135 8l 1330 9.9b (1-l3l) 2.51" (0.58-4.12) 2OO 74 43.4^ 38 E. pyriformis Ey2 36 l1 652 18.1 (11-133) 2.6s (2.26-3.23) Ø 5 7.8 0 Ev4 30 28 173 5.8 (l-14) 3.13 (1.98-3.96) 68 38 s5.9 15 Ev5 32 t7 3& tr.4 (t-94) 3.00 (2.29-4.69) 36 34 94.4 2I Ev1 30 l2 52 r.7 (r-43) 1.49 (r.os-1.97) 32 4 12.5 3 Total r28 68 t24r 9.7b (1-133) 2.57^ (1.05-4.69) 2æ 81 42.7" 39 E. youngiana Ev2 37 28 63s 17.2 (4-6t) 2.47 (1.8r-3.26) tt2 ) 1.8 0 - controlled Ev4 35 24 208 s.e (1-s8) 2.20 (0.98-3.t2) 40 2 5.0 0 intraspecific Ev5 t5 9 223 r4.9 (3-51) 3.35 (0.37-4.60) 48 t2 25.0 6 Ev1 33 6 l0 0.3 (l-3) r.s1 (r.æ-2.42) 0 0 0.0 0 Total r20 67 r076 9.ob (1-61) 2.40^ (0.37-4.60) 2m t6 9.0" 6 Open Ev2 20 19 635 12.2 3.59 48 20 4r.6 13 Ev4 20 20 208 35.0 2.54 48 7 t4.6 2 Ev5 20 l8 223 84.4 2.26 48 t7 35.4 3 Ev1 20 t9 l0 24.8 r.65 s2 19 36.5 10 Total 80 76 1076 39.1' 2.51" 200 63 32.tr 28 L.S.D. 27.0 0.74 36.8 lla¡s¡¡nmno A1 E. youngiana asthe female parent There was no significant effect of male species on the mean number of seeds produced per flower hand pollinated, with open pollinated flowers producing most seed (Table 7.6). Mean seed weight and percent seeds germinated were affected by female tree, with E. youngiana 8.54.7 (Ey7) producing the lightest seed with low germination (Table7.7). Table 7.7. Comparison of female parent on controlled pollination of E. macrocarpa, E. pyriþrmis and E. youngiana. Superscripts denote significant differences in a column at *P<0.05. Female species Female hee 'Mean number 'Mean seed Percent of seeds per weight per flower seeds flower pollinated oollinated (me) germinated3 E. macrocarpa Eml 20.5 3.10 20.4 E. pyriformis Epl 13.9'b 2.r4" 37.P Ep2 g.gb" 2.22h" 50.9u Ep3 2.9" l.5gd 22.6b Ep4 l6.g'b 2.29b 14.gb Ep5 21.3u 2.go^ 4r.7ù L.S.D. 7.7 0.52 27.8 E. youngiana E\2 15.3" 2.83^ 15.3b Ev4 12.5u 2.73u 37.6^ Ev5 29.2u 2.ggu s6.4u Ev7 10.3u 1.54b 16.7b L.S.D 26.9 o.74 36.8 All E. macrocarpa 20.5 3.10 20.4 E. pyriformis 16.5 2.21 34.5 E. youngiana 15.9 2.52 29.3 L.S.D 23.3 0.29 t7.7 numbered superscripts as for Table 7.3 Anova table Appendix 1.2. 147 Møaroaarnac 7-4-2^ 7.4.2. Seedling morphology analysis 7.4.2.1. Summary of all crosses Seedling morphological characters measured for all crosses are described in Tables 7.8, 7.9 and 7.10: considerable differences in characters, such as leaf surface wax, petiole length, leaf length and width, were recorded, and indicated potential hybrid status of the seedlings. Characters that are distinctive for taxa in a cross are noted. Seed from a variety of different commercial seed sources were grown as the parent seedling comparators, and the variation displayed within the intraspecifc crosses of E. macrocarpa and E. youngianø reflects the naturally high levels of variability within these species. In the case of E. pyriformis, commercially sourced seed proved inviable, so comparisons were made with open pollinated seed from the female parent trees, resulting in less apparent variation within the intraspecific cross. The results of the hierarchical and non-hierarchical analyses for each of the nine crosses are summarised in Table 7.11., with 96Vo of seedlings from interspecific crosses clustering separately from the female parent. Numbers of seedlings measured for each cross ranged from five for E. macrocarpa x E. pyriþrmis to 39 for E. youngiana x E. pyriformis, and reflect the germination rates of seeds planted for each cross. The Gower UPGMA dendrogram for each interspecific cross produced three main clusters. These clusters represented the female species, the male species, and the Fl hybrid seedlings. In three srosses, some seedlings linked with either the male or female parent species. The low values of dissimilarity on each dendrogram show the relatively high similarities between each species and their seedlings. The dendrogranìs for the E. macrocarpa and E. pyriformis intraspecific crosses did not show any distinctive clusters, while the E. youngiana intraspecific cross showed two distinct clusters. t48 Table 7.8. Seedling morphological characters of seedlings from E macrocarpa and putative parents. Measurements in mm or presented as range bCharacters (mean). "Characters unique or highly descriptive for that taxon according to PCC (Figs 7.7, 7.10, 7.13). significantly correlated' (r2) to variation amoungst individuals (Table 7.12). Cha¡acters E. macrocarpa opn E. pyriformis opn E, youngiana opn E. macrocarpa E. macrocarpø E. macrocarpa pollinated seedlings pollinated seedlings pollinated seedlings x E. pyriformis x E. youngiana x E. macrocarpa e d LSH3 lanceolate ovate, rarely elliptic lanceolate, ovate, elliptic lanceolate, elliptic elliptic or ovate lanceolate b LST3 acute or obtuse acute or obtuse acute or obtuse acute or obtuse acute or obtuse acute or obtuse b ò LSB3 attenuate attenuate or obtuse attenuate or obtuse attenuate attenuate or obtuse attenuate LLE3 26.t-s5.7 (36.8)' 19.0-52.3 (38.8r 20.242.2 (32.s) 24.84t.6(34.7) 24.344.9 (34.4) 26.s44.6 (3s.7) LWI3 9.2-22.8 (r4.2) tL.341.7 (22.Ð'b 8.r-23.4 (14.6)ab 7;t-r3.4 (tt.4) 7.6-22.6 (16.2) 6.6-18.4 (r4.0) ó b b LSW3 glaucous or very glaucous dull, rarely gtaucous dull, rarely shining dull or glaucous dull or glaucous glaucous or very glaucous LEA3 concolorous or discolorous discolorous, rarely concolorous " discolorous discolorous discolorous discolorous LSM3 smooth smooth smooth smooth smooth smooth LPS3 petiolate petiolate petiolate petiolate petiolate petiolate LPL3 2.34.3 (4.r)' 2.8-10.9 (4.8) 2.5-8.1 (5.1)" 3.24.3 (4.s) 2.34.1(3.6) 0.54.1 (3.0) LPO3 opposite opposite, rarely al ternate opposite opposiæ opposite opposiæ ó LCO3 grey green or dark green " dark green, rarely green green, blue green or dark dark green dark green dark green, rarely grey geen g.een o b LMI3 partiallyjagged smooth, partially jagged smooth smooth orpartiallyjagged smooth orpartiallyjagged partiallyjaggedorjagged orjagged " orjagged ú LSH5 elliptic or ovate ovate, sometimes elliptic ovate elliptic or ovate elliptic ovate b LST5 acute, mucronate or obtuse obtuse, acute or obtuse, acute or obtuse acute or obtuse acute, mucronate or obtuse mucronate mucronate b ó LSB5 attenuate attenu ate, rarely obtuse attenuate or obtuse attenuate or obtuse attenuate or obtuse or attenuale ò or cordate cordate LLE5 3t.7-71.2 (43.Tù 22.8-74.0 (4'1.2)' 22.6-7s.8 (43.8)' 33.446.9 (4r.3) 34.3{.2.s (46.8) 26.6-s7.7 (46) LWr 13.342.8 (23.Ð6 19.8-62.7 (37.3)" 13.3-52.7 (27.Ð"b 13.8-32.t (22.7) t8.447.s (31.9) tt.440.s (27.s) b LSW5 very glaucous " dull, sometimes glaucous dull, rarely shining dull or glaucous dull or glaucous dull, glaucous or very glaucous ù LEA5 concolorous or discolorous or concolorous discolorous, rarely discolorous concolorous or concolorous or discolorous b discolorous concolorous b discolorous I b LSM5 smooth smooth, rarely not smooth smooth smooth. rarely not smooth smooth, rarely not smooth smooth LPS5 petiolate petiolate petiolate petiolate petiolate petiolate Continued Table 7.8. Continued Characters E. macrocarpa opn E. pyriformis open E. youngiana opn E. macrocarpa E. macrocarpa E. macrocarpa pollinated seedlings pollinated seedlings pollinated seedlings x E. pyriformís x E. youngiana x E. mncrocarpa LPL5 r.34.6 (4.Ðh 2.7-t2.0 (7.O)^Þ 2.1-14.5 (6.Ðù 2.9-s.3 (4.s) 2.r-7.3 (4.6) 1.04.5 (3.9) LPO5 opposite ó alternate or opposite alternate or opposite " opposite alternate or opposite opposite LCO5 grey geen green or dark green, sometimes dark green' light green or dark green green, sometimes light or green, grey green or dark green, grey green, or dark green dark green gfeen LMI5 partiallyjagged ja b partial ly gged, smooth smooth partially jagged partiallyjagged partially jagged, smooth or orjagged " orjagged b jaggd LSHT ovate ovate elliptic or ovate ovate ovate ovate LSTT mucronate b b or obtuse' acute or mucronate acute, mucronate or obtuse acute or mucronate acuminate, acute or mucronate or obtuse mucronate LSBT attenuate or obtuse ô ó ¡ obtuse obtuse, rarely attenuate attenuate or obtuse attenuate, obtuse or attenuate or obtuse cordate LLE? 28.9-85.3 (sz.Ðh l6.s-87.5 (57.1f 33.0-80.0 (56.2)' 4s.741.3 (s3.3) 33.4-82.s (62.8) 37.6-76.3 (63.0) (34.Th LWIT 24.845.3 18.565.9 (43.ofb 15.6-44.8 (30.3r 26.643.3 (3s.4) 23.8-s4.6 (39.91 22.349.0 (4O.3) b b LSWT very glaucous " dull or glaucous dull or shiny dull glaucous very glaucous LEA,I concolorous concolorous or discolorous concolorous or discolorous concolorou s or discolorous concolorous or discolorou s concolorous LSMT ó b smooth smooth or not smooth smooth not smooth smooth or not smooth smooth LPST petiolate petiolate petiolate petiolate petiolate petiolate LPLT 0.1{.1 (3.4)b 3.1-13. l (8.4)"b 3.9-l?.5 (9.6)"b t.0-7.3 (3.4) 0.2-8.3 (5.0) 1.04.3 (2.s) LPOT ó ó opPosrte alternate, sometimes opposite altemate opposite opposite or alternate opposite LCOT grey green b b green, rarely light green green, blue green or grey green gleen or grey green green or grey green dark green LMIT partiallyjagged partially jagged b b or smooth smooth partially jagged orjagged partialty jagged smooth or partially jagged orjagged " b b SSHT angular round round angular angular angular SSMT b b not smooth " smooth or not smooth smooth smooth or not smooth smooth or not smooth not smooth Correlation coefFrcient* : showing the fit (r2) ofthe individual character vectors in the ordination space. Table 7.9. Seedling morphological characters of seedlings from E pyriformis and putative parents. Measurements in mm or presented as range (mean). bCharacters "Characters unique or highly descriptive for that taxon according to PCC (Figs 8.16, 8.I9,8.22). significantly correlated. (r2) to individuals. Characters E macrocarpaopn E. pyriþrmis opn E. youngiana open E. pyriformis x E. pyriformís E. pyriformís pollinated seedlings pollinated seedlings pollinated seedlings E. macrocarpa x E. youngiana x E. pyriformís Þ ¡b LSH3 lanceolate ovate, rarely elliptic lanceolate, ovate, elliptic elliptic or ovate lanceolate, elliptic or ovate lanceolate, elliptic or ovate b b b LST3 acute or obtuse acute or obtuse acute or obtuse acute or obtuse acute or obtuse acute or obtuse b b b T^SB3 a(enuate attenuate or obtuse attenuate or obtuse attenuate or obtuse attenuate or obtuse attenuate or obtuse LLE3 26.1-s5;t (36.8) r9.0-s2.3 (38.8)',b 20.242.2 (32.Ðb 14.944.O (32.4) 2r.oóo.s (36.7) 15.741.7 (34.9) LWI3 9.2-22.8 (t4.2) 1r.341.7 (22.Ð^h 8.1-23.4 (14.6)b 6.9-28.0 (17.6) 8.1-39.4 (r8.5) 7.5-33.6 (2r.t) ó LSW3 glaucous or very glaucous dull, rarely glaucous dull, rarely shining dull, sometimes glaucou s dull dull, rarely glaucous LEA3 concolorous or discolorous, rarely discolorous ù discolorous, rarely discolorous, discolorous, discolorous concolorous concolorous rarely concolorous LSM3 smooth smooth smooth smooth, rarely not smooth smooth smooth LPS3 petiolate petiolate petiolate petiolate petiolale petiolate LPL3 2.34.3 (4.1) 2.8-10.9 (4.8)b 2.5-8.1 (5.1)b 1.8-7.1 (3.5) 2.r-8.1(4.7) t.6-7.9 (4.8) LPO3 opposite opposite or alternate " opposite opposite or alternate opposite or altemate opposite or alternate b ù LCO3 grey green or dark green dark green, rarely green green, blue green or dark green green, grey green or dark green dark green, rarely green dark green, rarely green b LMI3 partialty jagged or jagged " smooth, partially jagged smooth smooth, partially jagged or smooth or partially jagged smooth or partially jagged orjagged' jaged b LSH5 elliptic or ovate ovate, sometimes elliptic ovate " ovate, rarely orbicular ovate, rarely lanceolate ovate, rarely orbicular b b LST5 acute, mucronate or obtuse obtuse, acute or mucronate obtuse, acute or mucronate " acute, rarely obtuse acute, rarely mucronate or acute, mucronate or obtuse obtuse b LSB5 attenuate attenuate, rarely obtuse or cordate attenuate or obtuse obtu se, rarely attenuate obtuse, rarely attenuate or obtuse, rarely attenuate cordate LLE5 3t.7-71.2 (43;t) 22.8-74.0 (47.Ðù 22.6-7s.8 (43.Ðh 31.7-70.1 (45.8) 28.4-82.r (51.4) 28.0-71.2 (49.2) LWI5 13.342.8 (23.2)b 19.842.7 (373)"h 13.3-52.7 (27.21b 23.442.9 (34.3) 16.8-56.8 (35.0) 22.644.7 (38.9) ú b LSW5 ver5r glaucous dull sometimes glaucous dull rarely shining " dull or glaucous shining dull, rarely glaucous ò LEA5 concolorous discolorous or concolorous discolorous, discolorous or concolorous discolorous discolorous, sometimes b b or discolorous rarely concolorous concolorous ó ¡ LSM5 smooth smooth, rarely not smooth smooth smooth, rarely not smooth smooth, rarely not smooth smooth LPS5 petiolate petiolate petiolate petiolate petiolate petiolate LPL5 t.34.6 (4.Ðb 2:1-r2.0 (7.q^b 2.1-14.5 (6.5)b 2.8-ll.l (4.8) 4.3-13.6 (8.1) 2.1-13.8 (6.8) Continued Table 7.9. Continued Characters E macrocarpa o n E. pyriformis open E. youngiana open E. pyriformis x E. pyriformís E. pyríformß pollinated seedlings pollinated seedlings pollinated seedlings E. macrocarpa tE.youngíann x E. pyríformis LPO5 opposite alternate or opposite alternate or opposite opposite, rarely altemate alternate or oPposite altemate or opposite b LCO5 grey green or dark green green, sometimes light green or light green or dark green green, grey green or dark green, rarely green green or dark green dark green b dark green b LMI5 partially jagged or jaggeí ^ partially jagged, rarely smooth smooth partially jagged, rarely smooth partially jagged, sometimes partially jagged, rarely smooth orjagged " orjagged smooth orjagged tsHT ovate " ovate elliptic or ovate ovate, rarely elliptic ovate, rarely elliptic ovate b LSTT mucronate or obtuse acute or mucronate acute, mucronate or obtuse acuminate, acute or obtuse acute, sometimes obtuse acute, or obtuse LSBT attenuate or obNse obtuse " obtu se, rarely attenuate obtuse, rarely attenuate or obtuse, rarely attenuate or obtuse, rarely attenuate cordate cordate LLET 28.9-8s.3 (52.2) 16.5-87.5 (57.1)"b 33.0-80.0 (56.2)b 27.1-98.s (s6.3) 35.8-89.6 (56.7) 42.5:78.5 (56.7) LWIT 24.845.3 (34.7) 18.s65.9 (43.O)', 15.644.8 (30.3)b 17.4-52.4 (37.9) 26.842.4 (37.6) 20.7-58.0 (41.8) ó b tswT very glaucous dull or glaucous "b dull or shiny dull, glaucous or very glaucous dull, sometimes glaucous dull or glaucous b b LEAT concolorous concolorous or discolorous " concolorous or discolorous concolorous or discolorou s concolorous or discolorous concolorous or discolorous b LSMT smooth smooth or not smooth " smooth smooth or not smooth smooth smooth LPST petiolate petiolate petiolate petiolate petiolate petiolate LPLT o.o5.l (3.4)b 3.1-13. l (8.4) 3.9-17.5 (9.6)b 0.5-12.0 (4.5) 5.7-t6.2 (t0.2) 4.0-13.6 (8.8) b ó LPOT opposite altemate, sometimes opposite alternate " altemate or opposite altemate, rarely opposite alternate, sometimes opposite * LCOT grey green green, rarely light green green, blue green or gre€n or grey green green, rarely dark green green, rarely dark green dark green s o LMT7 partiallyjagged orj"gg"d partiallyjagged or smooth' smooth partially jagged or jagged partially jagged, rarely partially jagged, rarely smooth smooth orjagged orjagged ó SSHT angular round round angular round round, rarely angular ó SSMT not smooth smooth or not smooth smoolh not smooth, rarely smooth smooth, rarely not smooth smooth, rarely not smooth Correlation coef,Flcient' : showing the fit (r2) ofthe individual character vectors in the ordination space. Table 7.10. Seedling morphological characters of seedlings from E. youngiana and putative parents. Measurements in mm or presented as range uCha¡acters bCharacters (mean). unique or highly descriptive for that taxon according to PCC (Figs 8.25, 8.28, 8.31). significantly correlated. (r2) to individuals. Characters E macrocatpa opn E. pyriformis open E. youngiana opn E. youngiana E. youngiana E. youngiana xE- x LSH3 lanceolate ovate, rarely elliptic lanceolate, ovate, elliptic lanceolate, elliptic or ovate ovate, rarely elliptic or ovate, rarely lanceolate lanceolate b b b LST3 acute or obtuse acute or obtuse acute or obtuse acute or obtuse acute, rarcly mucronate or acute, rarely obtuse obtuse b b ¡ LSB3 attenuate attenuate or obtuse attenuate or obtuse attenuate, rarely obtuse attenuate or obtuse attenuate, rarely obtuse LLE3 26.1-55.7 (36.8)b r9.0-52.3 (38.8fb 20.242.2 (32.s) r9.540.8 (29.3) t6.943.9 (31.2) 29540.6 (25.9) LWI3 9.2-22.8 (14.2)ù tt.34t.7 (22.9)'.b 8.1-23.4 (t4.Oh 7.9-2r.t (t2.4) s.2-32.4(19.O) t7.r-25.6 (20.3) ó LSW3 glaucous or very glaucous dull dull, rarely shining dull dull dull LE,d3 concolorous or discolorous discolorous, rarely concolorous " discolorous " discolorous discolorous discolorous LSM3 smooth smooth smooth smooth smooth smooth LPS3 petiolate petiolate petiolate petiolate petiolate petiolate LPL3 2.34.3 (4.r) 2.8-10.9 (4.8f 2.s-8.1 (s.r)''b 2.74.1 (4.2) 2.9-.6 (5.2) 4.9-8.r (6.4) b LPO3 oPposrte opposite, rarely altemate opposite opposite or altemate opposite, rarely alternate opposite, rarely alternate LCO3 grey geen or dark green dark green, rarely green green, blue green or dark green dark green, rarely green dark green, rarely green dark green, rarely green * LMI3 partially jagged or j"gg"d smoottr, partially jagged smooth smooth or not smooth smooth or not smooth smooth orjagged b b LSH5 elliptic or ovate ovate, sometimes elliptic ovate " ovate ovate, rarely lanceolate ovate b LST5 acute, mucronate or obtuse obtuse, acute or mucronate obtuse, acute or mucronate acute, r¿rrely mucronate or acute, rarely mucronate or mucronate, rarely acute obtuse obtuse b LSB5 altenuate attenuate, rarely obtuse or cordate attenuate or obtuse ' attenuate or obtuse attenuate, rarely obtuse obtuse LLE5 31.7-7t.2 (43.Tb 22.8-74.O (47.Ðú 22.6:75.8 (43.Ðh 27.948.9 (36.7) 24.443.6 (4r.9) 41.841.3 (s2.t) LWr 13.342.8 (23.Ðù t9.842.7 (373)ú t3.3-52.7 (27.2)b 14.3-34.r (24.r) 15.447.1 (30.7) 22.742.O (33.1) ó b LSW5 very glaucous dull, sometimes glaucous dull, rarely shining dull, glaucous or very dull or glaucous dull, rarely shining glaucous LEA5 concolorous or di scolorous or concolorous discolorous, rarely discolorous discolorous discolorous discolorous b concolorous u LSM5 smooth smooth, rarely not smooth smooth smooth smooth smooth LPS5 petiolate petiolate petiolate petiolate petiolaæ petiolate Continued Table 7.10. Continued Characters E. macrocatpa opn E. pyríformis open pollinated E. youngiana open pollinated E. youngiana x E. youngiana x E. youngianø pollinated seedlings seedlings seedlings E. macrocarpa E. pyriformis x E. youngiana LPL5 1.3{.6 2.7-12.O (7 2.1-t4.5 2.t-7.4 (4.9) 3.0-13.9 (7.6) 6.8-10.9 (9.7) b b ó LPO5 opposite alternate or opposite alternate or opposite opposite, rarely altemate altemate, rarely opposite alternate, rarely opposite b LCOs grey green or dark green green, sometimes dark or light or dark green grey green or dark green, dark green, sometinres dark green light green " rarely light green green or grey green * LMI5 partially jagged or j"gg"d partially jagged, sometimes smooth partially jagged, rarely partially jagged, rarely smooth b smooth orjagged smooth smooth LSHT ovate ovate elliptic or ovate ovate, rarely elliptic ovate ovate ù b LSTT mucronate or obtuse acute or mucronate acute, mucronate or obtuse acute or mucronate acute, sornetimes mucronate mucronate, sometimes acute or obtuse b LSBT aûenuate or obtuse obtuse " obtuse, rarely attenuate "b obtuse, rarely attenuate obtuse obtuse LLET 28.9-85.3 (52.2)b l6.s-87.5 (s7.rF 33.0-80.0 (56.2)b 34.1-75.r (54.8) 32.t-89.s (s2.3) 45.8{5.4 (58.4) LWIT 24.845.3 (34.Tù l8.s{s.9 (43.0r 15.644.8 (303)b 21.0-s4.3 (3s.7) 2s.4-s6.9 (38.3) 27.340.3(34.8) ò LSWT very glaucous dull or glaucous dull or shining very glaucous, rarely dull or dull orglaucous shining or dull glaucous b b LEAT concolorous concolorous or discolorous concolorous or discolorous discolorous, rarely concolorous discolorous, sometimes concolorous concolorous LSMT smooth " smooth or not smooth smooth smooth, rarely not smooth smooth smooth LPST petiolate petiolate petiolate sessile or petiolate petiolate petiolate LPLT o.o5.l (3.4)b 3.1-13. l (8.4r 3.9-17.5 (9.6)b 3.3-15.0 (6.2) 4.9-15.9 (10.0) 8.0-14.8 (l1.5) b LPOT opposite alternate, sometimes opposite " altemate " opposite or alternate altemate opposite ó LCOT grey green green, rarely light green green, blue green or grey green, rarely dark green green, grey gf€en or blue green or dark green b dark green dark green b LMIT partiallyjagged partially jagged or smooth smooth partially jagged, jagged, ó rarely partially sometimes smmth orjagged smooth orjagged smooth b SSHT angular round round angular round round ó SSMT not smooth smooth or not smooth " smooth smooth, rarely not smooth smooth smooth Correlation coefñcient': showing the f,rt (r2) ofthe individual character vectors in the ordination space. Macrocørnae 7-4-2- Table 7.11. Summary of information from UPGMA dendrograms, MDS ordination and MST distance values for all nine crosses. S = seedlings, M = male, F = female. Female E. mac E. mac E.pyr E.pyr E. you E. you E. mac E, pyr E, you Male E. pyr E. you E. mac E. you E. mac E.pyr E. mac E. pyr E. you #S 5 20 36 28 38 39 16286 measured # Dendrogram J 3 3 3 3 3 112 clusters #Ssin 0 0 2 5 0 0 16286 F cluster #Sin 0 0 0 2 I 0 000 M cluster # Sin 5 20 34 2T 37 39 000 separate cluster # MDS 3 3 3 3 3 3 scattered scattered 2 bunch" SSH stress 8.07o 15.970 T8,IVO 15.4Vo 18.2Vo I7.3Vo I7.5Vo 7.2Vo 7.2Vo Mean MST of 0.1224 0.1041 0.1564 0.1884 0.t022 0.1014 0.rM0 0.1665 0.1825 F cluster b MST distance 0.2578 0. I 888 0.1966 0.t493 0.1348 0.1755 FtoS" S link #3 #t4 #10 many #32 #7 many many many to Fd' Mean MST of 0.1540 0.1044 0.0854 0.1052 0.t617 0.1110 M cluster " MST M distance 0.3358 0.2017 0.t677 0.1330 0.1749 0.2054 to Sr # S links #2 #7 #18 #28 #r9 #tr to M cluster s Mean MST of 0.1837 0.1458 0.1607 0,t233 0.r3t7 0.1078 0.1848 0.1562 0.2986 S clusterh u 'bunch' refers to a discrete cluster of similar individuals b Mean of all MST values connecting female parent individuals " MST link connecting female bunch to seedlings o' Seedlings (code number from dendrogram) linking to female (code number from dendrogram) " Mean of all MST values connecting male parent individuals 'MST link connecting male bunch to seedlings s Seedlings (code number from dendrogram) linking to male (code number from dendrogram) n Mean of all MST values connecting seedlings 155 Macrocarnae 7.4.2. The semi-strong hybrid MDS ordinations for each interspecific cross reflected the equivalent UPGMA dendrogram clusters, with seedlings placed intermediately between parents. In the cases of the intraspecific crosses, the points were scattered across the plot, with no apparent pattern. In most cases, the minimum spanning tree connections between seedlings were variable, connecting individuals seemingly at random, with rarely more than one connection to either parent, while all female or male parent seedlings connect to each other. Mean MST values connecting female parent individuals to each other ranged from 0.1014 to 0.1884 (with an average of 0.1409), mean MST values connecting male individuals ranged from 0.0854 to 0.1617 (average of 0.1203) and mean MST values'connecting seedlings ranged from 0.1337 to 0.2986 (average 0.1658). The mean MST values connecting seedlings to females and seedlings to males of 0.1869 and 0.2031, respectively, show that although there is some variation within parent or seedlings groups, the variation between groups of parents and seedlings is, on average, greater that the variation within these groups. The PCC results for each cross are summarised in Table 7.12, with the correlation coefficient 1r2¡ for each cross. The invariant nature of some characters for particular crosses was also noted, where the character was the same for each individual measured and so was not descriptive. The intraspecific crosses, E. macrocarpa x E. macrocarpa and E. youngiana x E. youngiana. showed the highest number of invariant characters, 11 and 16 respectively, indicating there was little variation within the seedlings for these crosses. The interspecific cross, E. pyriformls x E. macrocarpa, showed no invariant characters. Characters that were found to be highly descriptive for most crosses were leaf width (LWI), leaf length (LLE), leaf tip shape (LST), leaf surface wax (LSW), leaf petiole length (LPL) and leaf margin shape (LMr). 156 Møcrocarnae 7.4.2- Descriptions of each character for all crosses are given in tables 7.8,7.9 and 7.10, where the significantly correlated characters for each cross are noted, as are the characters unique or highly descriptive for each cross from the PCC ordinations. The PCC ordinations allow for identification of the characters causing the clusters, and the clearer definition of clusters/taxa. Table 7.12. Summary gf variant (Y) and invariant (-) characters and significance (*) from correlation coefficient 12. AU measurements at nodes 3, 5 and 7 (e.g. LSH3 = LSH at node 3). Characters LPS(3, 5, 7) and STE (3, 5, 7) removed prior to analysis as invariant for all individuals. EmxEm 0.74 0.54 0.62 0.46 0.52 0.s6 0.4 0.44 0.78 LSH3 Y* Y,k Y* Y Y't Y¡k Y* Y* LST3 Y Y Y* Y:l' Y'ß Y'ß Y* Y¡ß Yr< LSB3 Y* Y:r. Y*, Ytc Y{< Y', Y{, Y LLE3 Y Y Y Y Yr. Y* Y¡t. Y:t Y LWI3 Y{< Yr. Y Y Y:f Y* Y* Y{' Yr, LSW3 Y{, Yr< Y* Y* Y Y:t. LEA3 Y Y Y Y Y Y* Y Y LSM3 Y LPL3 Y Y Y Y Y'l' Y* Y Y Yr, LPO3 Y Y Y Y Yr< Y Y LCO3 Y* Y't Y Y'f Y Y:ß Y Y LMI3 Y Y* Y Y Y{, Y Y't Y* I. LSH5 Y Yr. Y Y{. Y Y Y Y¡l' LST5 Y Y{< Yr< Y* Y Y{. Y* Y Y LSB5 Y* Y* Y Y:l' Y Y Y Y* LLE5 Y Y Y* Y Yi' Y*' Y{' Y'C ;. L\vI5 Yf, Y* Y:l' Yr, Y* Y* Y'l' Y* Y* LSW5 Y* Y* Y Y*' Y Y{< Y,I, Y*c LEA5 Yx Y't. Y* Y* Y* Y* Y Y:ß l LSM5 Y Y{< Y'|'< Y LPL5 Y;k Y* Y{, Y* Y', Y*< Y Y'ß Y'ß LPO5 Y* Y Y* Y't Y Y*' Y* Y* LCO5 Y Y Y Y Y* Yx Y Y* Y LMI5 Y*< Y'k Y Y Y{< Y Y't Y* LSHT Y Y Y Y Y Y LSTT Y{, Y* Y Y Y:f, Y Y{' Y* Y LSBT Ytc Y Y* Y Y Y Y Yi' LLET Y Y Y*, Y Y'ß Yr< Y Y* Y* LIWIT Y{< Y Y,l, Y Yt' Y Y Y'l' Y'lc LSWT Y* Y*, Y Y{, Y{< Y't' Y Y* Y{' LEAT Y Y Y¡r, Yr< Y Y't Y* LSMT Y* Yx Y Y* Y LPLT Y* Y'ß Y* Y{' Y*, Y Y Y'l' Y'l' LPOT Y:* Y* Y'¡ Y Y* Y Y*r LCOT Y't Y Y*c Y* Y:t Y Y{, Y Y* LMIT Ytc Y* Y Yr. Y Y Yi' Y't SSHT Y:ß Y¡k Yt' Y Y:¡ SSMT Yx Y:t Yr< Y Y Y Y* ts7 Macrocaroa¿ 7.4.2. 7.4.2.2. Summary of each cross E. macrocarpa x E. pyriþrmis The dendrogram (Figure 7.4) grouped the five seedlings from this cross in a separate cluster from the female and male parent seedlings, and the MDS ordination plot placed them in the same distinct groups with the seedling cluster between the parents and closer to the mother (Figure 7.5). The MST values for the connections between the seedlings and the female and male parent seedlings were 0.2578 and 0.3358, respectively, and the mean MST value within the seedling cluster was 0.1387, indicating that while there was some variation between seedlings, and considerable variation between the seedlings and both parents, the seedlings were most similar to each other, then most similar to the female parent. PCC analysis (Figure 7.6.) identified characters, such as LLE, LWI, LSW, LMI and LCO, as distinctive of either the male or female parent taxa. Leaf surface wax (LSW) and variations in length (LLE) and width (LlM) were highly distinctive from an early age (Ptate7.2). E. macrocarpa x E. youngiana Both the UPGMA dendrogram (Figure 7.7) and the MDS ordination (Figure 7.8) showed the twenty seedlings clustering into one group, distinctly separate from, but intermediate between, either parent. The MST values linking the seedlings to the female and male seedlings were 0.1888 and 0.2017 respectively, and the mean MST value within the seedling cluster was 0.1458, indicating that although there was some variation within the seedlings and between the seedlings and the parents, the seedlings are most similar to each other then most similar to the female parent. PCC analysis (Figure 7.9.) identified characters, such as LLE, LWI, LSW, LMI, LPL and LSB, as distinctive of either the male or female parent taxa. Again, leaf surface wax (LSW) and variations in length (LLE) and width (LIVI) were highly distinctive from an early age (Plate 7 .2). 158 Macrocarna¿ 7-4-2- E. macrocarpa x E. macrocarp The dendrogram (Fig 7.10) showed no distinct clustering and no obvious structure. The MDS ordination (Fig 7.11) scattered the individuals across the plot, intermingling E.macrocarpa open pollinated seedlings with seedlings. The MST value connecting seedlings and female parents was O.1432 and the mean MST value between seedlings was 0.1458. This indicated that the variation within the seedling cluster was the same as the variation between the seedlings and the female parent species. Descriptive characters were difficult to define, as this was an intraspecific cross (Figure 7.L2.). E. pyriformis x E. macrocarpa The UPGMA dendrogram (Figure 7.13) of this cross placed 33 of the seedlings in a cluster, one on its own, with the remaining three clustered with the female parent group. The MDS ordination (Fig 7.1a) showed the points scattered across the plot, with the majority of the seedlings placed between the parent species groups. The MST values linking the seedlings to the female and male seedlings were moderate, 0.1966 and 0.1677 respectively, with the seedlings connected more closely to the male. The mean MST value within the seedling cluster, 0.1607, indicated some variation within the cluster. Three seedlings, EpxEm3, EpxEml4 and EpxEml9, were shown close to E. pyriformis. The MST connections place these seedlings with the female parent, E. pyriformis. PCC analysis (Figure 7.15.) identified characters, such as LLE, LWI, LSW, LMI, LCO, LPL and LSB, as distinctive of either the male or female parent taxa. Again, leaf surface wÐ( (LS\Ð and va¡iations in length (LLE) and width (LIYI) were highly distinctive from an early age (Plate 7.3). E. pyriformis x E. youngiana The UPGMA dendrogram (Figure 7.16) placed five seedlings in a cluster with the female parent, most on their own, and two clustered with the male parent group. The MDS 159 Macrocøroac 7.4.2. ordination (Fig 7.17) showed the points scattered across the plot, with the majority of the seedlings placed between the parent species. There were many MST points of connection between the seedlings and the female parent, and the five seedlings from the dendrogram were connected directly to E. pyriformis. The male parents were connected to the seedlings by a value of 0.1330, with the two seedlings that clustered with E. youngiana in the dendrogram connected closely to EpxEy28, which connects directly to E. youngiana. The mean MST value within the seedling cluster was 0.1233. These low values and the lack of clear groupings in the MDS ordination reflect the relative similarities between the seedlings of these two species. PCC analysis (Figure 7.18.) identified characters, such as LLE, LWI, LMI, LCO, LPL and LSB, as distinctive of E. pyriþrmis, and only LPOT and LCOT distinctive for E. youngiana. Variations in leaf shape (LSH), length (LLE) and width (L!U) were highly distinctive from an early age (Plate 7.3). E. pyriformls x E pyriformis The UPGMA dendrogram (Fig 7.19) did not clearly define any groups, and the MDS ordination (Fig 7 .20) scattered the points across the plot. The MST value linking the female parent to the seedlings was 0.2000 and the mean MST value for the seedling cluster was 0.1562, indicating some variation within the seedlings. No defining characters were evident from the PCC analysis (Figure 7.21) as this was an interspecific cross, with no distinct groups of female and seedlings individuals. E. youngianax E. macrocarpa The dendrogram (Figure 7.22) grouped the 38 seedlings one clusters, with one cluster of female and one of male seedlings. The MDS ordination (Fig 7.23) placed the seedlings between the parent groups. The MST values linking the seedling to the female and male seedlings were 0.1348 and 0.1749 respectively, and the mean MST value within the seedling 160 Mn¡¡n¡.nntnp 7-4-2- cluster was 0.1317, indicating a moderate level of variation within the seedlings and a greater similarity to the female parent than to the male. PCC analysis (Figure 7.24) identified characters, such as LSH, LSB, LWI, LSW, LEA, LPL, LMI, and LPO, as distinctive of either the male or female parent taxa. Again, leaf surface wax (LSW) and variations in length (LLE) and width (L!VI) were highly distinctive from an early age (Plate7.4). E. youngiana x E. pyriformis The dendrogram (Fig 7.25) grouped 38 seedlings into one cluster, with one cluster of E. youngiana and one seedlings and another of E. pyriformis. The MDS ordination (Fig 7.26) scattered the individuals across the whole plot, with the majority of seedlings between the parent groups. The MST values for the seedlings to the female and male groups were 0.1755 and 0.2054 respectively, and the mean MST value between the seedlings was 0.1078, indicating that the seedlings are vary similar to each other, and then more simila¡ to the female parent. PCC analysis (Figure 7+.27) identified few characters as distinctive for E. youngiana (LPO and LEA, both at node 3), while E. pyriformis was identified by such as LLE, LWI, LCO, and LPL. Seedlings of this cross differed in leaf length (LLE) and width (L!u) (Plate7.4). E. youngianax E. youngiana Only six intraspecific seedlings survived to be measured at three months, and the dendrogram (Fig 7 .28) clusters them into two groups. The MDS ordination (Figure 7 .29) refTected these groupings. The MST value connecting the seedlings with the female parent was 0.1986, and the value within the seedlings was 0.2986, indicating there to be more variation within the seedlings than between the seedlings and the parents. The PCC analysis (Figure 7.30) did not reveal any distinctive characters. 16r Mnrtoonrnna 1-â-2 EmxEpl l-) EmxEp2 ¿) EmxEp3 J,' EmxEp4 4) I EnxEp5 5) Emacl 6) Emac2 7) Enac3 8) I Emac4 e) -T Emac5 10) Epyrl- 11) Epyr2 L2l Epyr3 Epyr4 1,4) I Epyr5 1s) I I I o.0622 0.1918 0.3213 0.4509 0.5804 0.?100 Figure 7.4. Dendrogram of E. macrocarpa x E. pyriþrrn ¡s seedlings and parent seedlings. ,E'. macrocalpa (Emac), E. pyriformls (Epyr) E. macrocarpax E. pyriformls (EmxEp). Scale represents standardised Manhattan distance (Gower metric) based on variant seedling characters (Table 7, 8). O seedlings I E. macrocarpa a E. pyriformis 2 4, 0 Ú)â 2 -l 2 -2 0^ É" 0 +tr -l -2 -2 Figure 7.5. Multidimensional ordination of E. macrocarpa x E. pyriform ls seedlings and pargnts. 162 Mda¡oaarnø¿ 7-4-2- 2 LLE' IPLs 5 ¿tE, LSA! ¿r¡isBt 16xs LWtú Lt Af !POt l!^¡ twt? !9Í! lPst ."o"tlîT,, @ LSUt LA86 tstt ----1-8-9-{ ru tr tsMt -l MDS Axis I LE A' Latt s58t tcot LCOE raxlsMT I¡r6 LL'f LPO5 tcr$I: g!!-! LlBr !sw6 .¡ç¡s lawt LsEtPtå¡ls,r LL!! ¡s¡1 !¿!¿ ll 15 LS It .tSr'r1"or !w'" L2OJ -2 0 2 MDS Axis I lt¡t t!¡¡ L¡I LEAS ' tcoT s5H7 -L-!!L tco5 tsH¡ I -..-sM 7 t3&6 LLE f tPoS t¡Ê¡. Ø l:îrt"ot ' "t' rM;E+,.fs¡l !¿r-l: '""'fjål-l¿lt !-!-4{s¡¡ Lw rs Lw t3 qgr lT!t, t"o' -2 MDS Axis 2 Figure 7.6. Principal component correlation graphs of E. macrocarpa x E. pyriformis seedlings and parents. Characters in italics and underlined not significantly correlated to MDS ordinations (Table 7.8). 163 ll'ln¡tnnnmøo 1 A 7 EmxEyl L EmxEyL2 T2 I EmxEy3 EmxEylT 77 EmxEy4 4 I EnxEy6 6 EnxEyl-8 t8 EmxEyl4 14 I EmxEy9 9 EmxEy2 2 EmxEyl 6 16 -T EmxEy5 5 I EmxEyl3 AJ EmxEyL0 L0 ErnxEyT 7 I EnxEyB o EmxEyLl 1L I EmxEyl5 15 EmxEy20 ,^ EmxEyl 9 19 Emacl )1 Enac2 ut I Emac3 ¿J I Emac4 24], Emac5 251, Eyoul 26) Eyou2 21 ') ------T I Eyou5 30) -I I 281, I Eyou3 - I -l Eyou4 29\ I I I 0.0551. o.]-529 0.250't 0.3484 o .4462 0 544 0 Figure 7.7. Dendrog¡am of E. macrocarpa x E. youngiana seedlings and parent seedlings. .E macrocarpa (Emac), ð. (Eyou) E. macrocarpa x E. youngiana (EmxBy). Scale represents standardised Manhattan distance (Gower metric) based on variant seedling characters (Table 7.8), a seedlings I E.macrocarpa O E.youngiana 2 0 A ã -t I . ""þ F+o -l ¡fD¡ 4&:r I Figure 7.8. Multidimensional ordination of E. macrocarpa x E. youngiana seedlings and parent seedlings. 164 fuIacrocaroac 7.4.2. @ tsw6!st6 Itr t6 LSW ' 5Af,7 o !ÞLr Ð -!@ "'i!o,!'o, '*'1t"";. W '"ii:' t"*"'-- altt M DS Axis I : ss[7u'¡utz tsä7 s1ëhs LSM? LAWT LEAT L6W3 Lèø' x LCOI 0 . . t€Á¡ Ø LPO' lsHS LS83 H LEA6 -LS17 ÈLEt rPrs 1997 - rs'.Lwt5. --- LW t3 LsHs LPOT :¿PtJ LSE5 Àst7 iru: tst¡ -z 0 MDS Axis I gst t tsHT t0Í7 J'Jllltd*. LEA' L'H7 .4 loo! LAO6 !-!Ð ts 17 L9.9! LSt6 MDS Axis 2 Figure 7.9. Principal component correlation graphs of E. rnacrocarpa x E. youngiana seedlings and parents. Characters in italics and underlined not significantly correlated to MDS ordinations (Tabte 7.8). 165 Macrocarpae 1.4.2. Plate 7.2. Hybrid seedlings six weeks after gennination. A: a) E, macrocarpa, b) E, macrocarpa x E. pyriformis, and c) E. pyriþrmis; B: a) E. macrocarpa, b) E. macrocarpa x E. youngiana, and c) E, youngiana seedlings Bar: lcm. 166 Møcrocarnøe 7.4.2. EmxEmsl 1) Enacl L7t Emac2 18) EmxEms6 6) Emac3 1e) EmxEms2 EmxEms 3 3) I EmxEmsT 7) I EmxEmsS 8) I EmxEms9 e) EmxEms4 4l Emac4 20') I Emac5 2I\ lt EmxEms5 s) EmxEmsl0 10) EmxEmsl-l- t1) I EmxEms 1 6 16) EmxEmsl-3 13) EnxEmsl-4 14) EnxEmsL5 ls) EmxEmsl2 12) I I I I 0,1060 o .).1 42 o.2424 0.3r.06 0.3788 0.4410 Figure 7.10, Dendrogram of E, macrocarpa x E. macrocarpa seedlings and parent seedlings. E. macrocarpa (Emac), E. macrocarpa x E. mqcrocarpa (EmxEms). Scale represents standardised Manhattan distance (Gower metric) based on variant seedling characters (Table 7.8) I E. macrocarpa t seedlings 2 C) IA ã. 0 aô a 2 -l Þ$s a \ftg$ Figure 7.11. Multidimensional ordination of E. macrocarpax E. macrocarpa seedlings and parent seedlings. 167 Macrocaroa.e 7.4.2. !r,s rssr fSiE5#É ¿so lr¿¡ lPl5 rswt LWIALET IPLT LWtt - LLlr - Lß IJ LCOT ISr6 ¿ E¡, L3B' -LLIâà !r!! LCOt Lt-]4-! Lco Lgt5 -2 -7 0 MDS Axis I 2 LCOT tptT lSWt ttElg-¿ .r,. !!!-¡. @ t"u, .2 LST] LEA5 LCOl LEAs LSBT LLE 5 LSrt @."r, tTl5-- LSdS 44wt¿ L--UJ7 tw'l"' -2 0 MDS Axis I LCOT LSW 3 LPLT- LSWS LPL5 @ LUI' LSgT .2 LSTS mt5 LsTJ LEA5 IE¡J LCO t LLES LS87 ¡ry¡5 Lsg5 LSfT lsflJ LLEJ., --LS13 Ø -1f6*'rwu 0 M DS Axis 2 Figure 7.12. Pnncipal component correlation graphs of E. macrocarpa x E. macrocarpa seedlings and parents. Characters in italics and underlined not significantly correlated to MDS ordinations (Table 7.8). 168 EpxEml 1) 8pxEm2 2) EpxEm5 s) EpxEnl2 72J EpxEml 3 13) '7], EpxEm? I EPxEm20 20j 8pxEn10 10) EpxEn9 9) EpxEn35 35) BpxEñ4 4l EpxEn24 24) Epx tn3 0 30) EpxEm33 33) EpxEn6 6) EpxEml5 1s) --t_ EpxEnl6 16) I Epx Em3 6 36)_---t._-t_ EpxEnB 8) I EpxEm2 9 29]' EpxEml 1 11) EpxEm2 3 23]- opxEm2 5 25) EpxEn32 32!, I EpxEn2S 28') 8pxEn2 6 26) EpxBn2T 21) EpxEm34 34) ÉpxEm31 31) EpxEmlT 11) Epx En1 B 18) EpxEm2 1 27) I EpxEn22 22) tl Enacl 42) Emac2 43) I Enac3 44]- | Enac4 4s) Enac5 46) I EpxEm3 3) I EpxEml4 14) apyrl 37) Epyr2 3B) Bpyr4 40) Epyr3 39) I Epyr5 Al) I EpxEnl9 19) Figure r ;; ;,#:;"*:;; ,r';:;;. *"i'^)o","o;;;r,seedrings and parent seedrings E macrocarpa (Emac), E. pyrformls (Epyr), E. pyriformis xE. macrocarpa (EpxEm). Scale represents standardised Manhattan distance (Gower metric) based on variant seedling characters (Table 7.9). ¡ E, macrocarpa ç E. prriforn¡s ¡ seodlings ¡ EpxEm3,EpxEml4 ¡ EpxEml9 2 .9 å A z -2 -t 0 f" -2 s MDs^xri 2 r Figure 7.14. Multidimensional ordination of ,E pyriformis x E. macrocarpa seedlings and parent seedlings. 169 Mn¡tn¡nrnno 7-4-2- 2 LCO3 LSl5 rsJ¡ tfa-l LSH5 LPL5 Lwltsa:aro, LPoT .""¡ []E 't LWtl Lsu 7 LLEj L+E¿, ô ozss*f !3s¡¡ L9-E-L ¿{16 lco? Lswr crïèw¡ r¡q LSTJ -2 0 MDS Axis I LSff LCO a ffi LPOI 'I ls l5 LCO3 L9B3 0 rPOS LSw5LAl¡!ÉAJ !PL5 ts8t rs*L¡¿rLw 15 ,-i:.- LCO? LÉ^5 "iË". ssHT LSHS lsrr @- .T6et Lwtf -2 0 2 MDS Axis I tPoT ¿s17 LU IJ LCOS Ltr .2 LH I5 LMIT tsf5 LS83 0 tsw 3 ttSt Ø LPLS o ISr7 ttE5 LSB3 LSt3 --IsMs r*,s LEA - LCOT lLET ffi LEA 5 ' LsMJ SSHT LSHS TssM 7 !-5llzrsM -LSW -2 1 0 M DS Axis 2 Figure 7.15. Principal component correlation graphs of E. pyriformis x E. macrocarpa seedlings and parents. Characters in italics and underlined not significantly correlated to MDS ordinations (Table 7,9). 170 Macrocamae 7.4.2. EpxEyl 1) Epyr4 32]. EpxEy? 7l Epy¡5 33) Epyr2 30) Epyr3 31) EpxEy2 2) EpxEy24 24) Epyrl 29) EpxEy5 5) I EpxEy3 3) EpxEyLl 11) ÐpxEy15 1s) -l I EpxEyl0 10) EpxEyl-8 18) EpxEyl9 19) ll Epxgy2'1 2',7 ) ì EPxEy4 4) EpxEy6 6) EpxEyB 8) ------T EpxEy9 9) EpxEy20 20) -l EpxEy25 25], -T EpxEy2B 281 ttl EpxBy12 721 -----l EpxÉy1,6 16) EpxEy2l 21) EpxBy22 22) EpxEyl4 14) EpxEylT r7) ÐpxEy23 23], BpxEyl3 13) Epxqy26 26) Eyoul 34) I EyaÐ2 3s) ---T --l Eyou3 36) I Eyou4 3?) T Eyou5 38) Figure t.;;.'"eå: grarr of E. pyriformis x E. youngianq seedlings and parent seedlings. E pyriþrmis (Epyr) E. youngiana (Eyou), E. pyriformis x E. youngiana (EpxBy). Scale represents standardised Manhattan distance (Gower metric) based on variant seedling characters (Table 7.9). o E. pyrdormis, EpxEyl, EpxEY2, EpxEy5, EpxEyT, EpxEy24 a seedlings o E youngiøna EpxEyl3, EpxEy 26 ä I -l hY 0 ?' -2 -1 ÀÍ1s2 NIDS Figure 7.17. Multidimensional ordination of E. pyriþrmis x E. youngiana seedlings and parent seedlings. t7l Maclo¿arnae 7-4-2- !!#,iÑåt ."*, Ls13 ¡s¡ t Lwrt !!Er,,..."*tlJL.4 Lt-gt- t5tI ..¡1,. i"jl" t5!t fstS LS T' L2!Z LIA' lsfl LE LCO 5 -2 0 MDS Axis I ùscfJll¡'?t3 !-e{írs rw rz 5 ItES .!t^E LEA' LW 15 L29! larj Lwl! LCO6 ¿E^' tsfl!129! 'I LPO5 !M15 fsB! Lgft LLE3ssrt ¿9Wl LÑ ts ¡5 !j-q¿ !sr6 0 MDS Axis I LSHT LP O7 .-PL3 r4Ì ttt' LPLS LLEs i39J LLEtwtT tEA5 LEÀ7 Lwt5 !!!¡ LC05 t€Á3 LPot Lwì3 Àsä5 LMIS '! LPOs LSB3 LSfT !4.12 LMl3sstT LSWS ts¡t -sw? ¿547 M MDS Axis 2 Figure 7.18. Principal component correlation gaphs of E. pyriformìs x E. youngiana seedlings and parents. Characters in italics and underlined not significantly correlated to MDS ordinations (Table 7.9). 172 Møcrocaroae 7.4.2. tÐ a I g I Plate 7.3. Hybrid seedlings six weeks after germination. A: a) E. pyriformls, b) E. pyriþrmis x E. macrocarpa, and c) E. macrocarpa, B: a) E. pyriþrmis,b) E. pyriþrmis x E. youngiana, and c) E' youngiana' Bar - 1 cm. 173 Ma¿toaarnne 7-4-2- Epyrl 1) EpxApÊ5 10) EpxÉps14 19) EpxEpsl? EpxEps23 28) I EpxEps24 EpyÊ2 2T EpyrJ 3) Epyr4 4) EpxBps22 2'1) I EpxEps4 e) Epyr5 s) EpxEps6 11) EpxEps2T 32t I EpxEpsl6 27]- I EÞxEÞs1 9 24) _l _t _ EpxEpsll 16) I EpxEps 1 I 23) _t _t _ EpxEps? 72) I EpxEÞsg 13) EpxEps20 25) _l EpxEps 1 6) I EpxEps3 EpxEpsl5 20 )_ t-- t__ EpxEps9 14) | EpxEps25 30)_ t_t_ EpxEpsl0 1s) EpxEps2B 33) I EpxEpsl2 1'7 ) epxEpsl3 18) EpxEps2l 26) EpxEps2 '1) EpxEps26 31) I 0, 1100 0.r902 o .2'104 0 - 3506 0. 4 308 0 5110 Figure 7.19. Dendrogfam of E. pyriþrmis x E. pyriþrmls seedlings and parent seedlings. E pyriformis (Epyr) E. pyriþrmis x E. pyriformis (EpxEps). Scale represents standardised Manhattan distance (Gower metric) based on variant seedling characters (Table 7.9). O E. pyriþrmis y seedlings 2 Y .9 Y U) V ! 2 o YV -1 ov Þ 7 -2 H 0 \, â N{DSAúS2 Figure 7.20. Multidimensional ordination of E. pyriformis x E. pyriformis seedlings and parent seedlings. 174 Macrocarno¿ 7-4-2- ts l3: LS¡t ¿s17 'R LG O3 LCOT Ø o ----E-9!C-tLSHT LÞO5 TTEJ LCOS LSAJ LSH3 rr-å!tt ."l".ti,, rr.¡HJ1*,, -2 -2 MDS Axis I LS H' ts¡tLCOT !s¡J lsx3 'I LAOA LPL5 Ltat LPot fLE5 LPO5 !Pt3 too! ' LlEr !-q-! ¿5Wt lsl3 -2 0 MD Axis I ¿sÍt !co? !stt Lwr¡ |& LSHS tPL5 !lq-g! 0 ,n,rì,ffi'"*'a-,- 'LþU3 Lp 05 LCOI LLÉ7 LAW6 ts 15 LSl3 -2 0 MDS Axis 2 Figure 7.2I.Pnncipal component corelation graphs of E pyriformis x E. pyrifonøis seedlings and parents. Characters in italics and underlined not significantly correlated to MDS ordinations (Table 7.9). 175 Mnntoenrnac 1-[-2- EyåEn1 1) EyxEmB 8) EyxEn22 221 EyxEn2 2t EyxEn6 6) EyxEn4 4) EyxEm25 2s) I EyxEm39 3e) I I EyxE¡n7 7) EyxEn2T 2-t I ByxEn33 33) I EyxÐm3? 37) EyxEm20 20) ByxEm23 23) EyxEn36 36) EyxE¡3 3) EyxEml0 10) EyxEn32 32) Eyx En2 4 24\ EyxEnll 11) EyxE¡n12 72) Eyx En3€ 3B) EyxEnl5 1s) I EyxEn 5 s) EyxEm30 30) I EyxEm9 ql EyxEml3 13) EyxEml4 14) EyxFñ31 31) ¡ --i EyxEnf 6 15) EyxBmlT 17) I I EyxEn 1 B 18) EyxEnl 9 19) --- EyxEn2l 21) EyxEm35 35) I EyxEm2E 2B) EyxEn34 34) I EyxEn2 6 26) -T EyxEm2 9 I Byoul 40) Eyou2 4r) Eyou5 44) Eyou3 42) I Eyou4 43)_t_l Enacl 4s) Enac2 46) Enâc3 41) Enac 4 4B) Enac 5 49) I I I 0. 04 83 0.1530 0.2514 0.3625 0.4673 0,5720 Figure 7.Z2.Dendrogram of E. youngiana x E. macrocarpa seedliîgs and parent seedlings. E. youngiana (F'yol), E. macrocarpa (F;mac), E. youngiana x E. macrocarpa (Eyxt.m). Scale represents standardised Manhattan distance (Gower metric) based on variant seedling characters (Table 7. 10). O E. youngiqna I E, macrocarpa ¡ eeedlings ô: ? lr*þ -2 úDÊ Figure 7.23. Mvllidimensional ordination of E. youngiana x E. macrocarpd seedlings and parent seedlings. 176 Macrocaroo¿ 7.4.2. rrrz ¡y.,; i""ii rsifl'r LSlrL"6r .cålrt LSa5 .¡ÞEt LlE LsH¡ IEA5 IEÁ¡ i !l¿¿ Ø tstt z -|.sLSW3WÈ LEAf rs¡, lsw 5 ssfT -2 -2 MDS Axis I tsat twl! LSE3 LSH3 LSlt SSHT LSBI LEAT LSD5 't .'åfr{"Jl[, rres LswT 4Iå... fEAJ LCOT Ltr LCOS LÊA5 LSH5 LPOI ffi LCOt -2 -2 0 MDS Axis I , êu, tcflt -_.." ltrLSAT asHt LW13 lsa3 Lss5 tWlTLLETLWt5 IPOT LLE5 l1'l¡1 fEÁl t"Ëþ¿ LSf t -!jJår¿¡ LCO5 TPL5 LEA5 5 LW LSIT -2 -2 0 MDS Axis 2 Figure 7.24. Pnncipal component correlation graphs of E. youngiana x E. macrocarpa seedlings and parents. Characters in italics and underlined not significantly coffelated to MDS ordinations (Table 7. l0). t77 Macrocqrpøe 7.4.2. eyxEpl 1) 8yxEp32 31) ByxEp36 34) EyxEp2 2) EyxÉp15 14 ) EyxEp25 24) EyxBp22 27) Eyxqp2T 26) EyxEp3T 35) EyxEpl3 72) EyxEp3l 30) EyxEpl4 13) Eyx Ep2 9 2el EyxEp3 3) EyxEpl6 1s) I EyxEp5 5) I EyxEpB '1) EyxEpll 10) EyxBplT 16) EyxEp4 A) EyxEp26 25) I EyxEp3S 36) I EyxEplS 17) I EyxEpl-9 18) tl DyxEp30 I EyxEp6 6) EyxEp35 33) EyxEp40 38) EyxEp39 37) EyxEp9 8) EyxEp34 32) EyxEpl2 11) I EyxEp23 22) I ÊyxEpl-0 9) EyxEp20 EyxEp2S 2't ) EyxEp2l 20) tt ttr EyxBp24 23) Epyr2 45) EÞyr3 a6) --l Epyr4 4'7 ) I Epyr 5 48) Epyrl 44) I Eyou 1 3e) Eyou2 40) Eyou3 41) Eyou4 42) Byoù5 43) I I 0. 051 1 0,139? 0.2283 0.3168 0.4054 494 Figure 7 .25. Dendrogram of E. youngiana x E. pyriþrmls seedlings and parent seedlings. E youngiana (Eyou), E. pyriþrmls (Epyr) E. youngiana x E. pyriformis (EfxEp). Scale represents standardised Manhattan distance (Gower metric) based on variant seedling characters (Table 7. 10). a E.youngiana O seedlings Ç E. pyrilormis and ElxEpZ O OOO ¡o oo 2 0 Þo -l -2 s ¿ø" 416/ -l Figure 7.26. Mtltidimensional ordination of E. youngiana x E. pyriþrmis seedlings and parent seedlings. 178 2 L!E\¡çt, tpLt tpts rooolsá7 LPOS Lwlf ^813 L!-97 !.99-1 d ,åi5'- LzL! !-I!¡ LEÅ' LEA2 ssil7 Lt!.| Ø ßHr LST¡ tPo3 â tsYtl . LCOt tif.lÞ LEA 7 LsMT LsWS S6H7 LSWT MDS Axis I iäi¡6t"" lwrrlEs rs'! LtE3 LLE' .."", tïlto, -rtPs# LCOS 'i lslT 13 0 LEAT LS L SEf LCOT !sÌ5 LEA' ! st5 LE Aã -2 2 M DS Axis I LCOJ LEA 7 t#î.il'' LpoI LCOT LMIS LEA' 'I rco¡ !!.!-Z 0 IEA¡ LS13 rprs !I-r! o LsrT t!91 iirÌ.'.""t à LPIJ Lswt gir- ¿sd5 LSAT . -^ LSlS -q7 - -2 M DS Axis 2 Figure 7.27. Pnncipal component correlation graphs of E. youngiana x E. pyriþrmis s;dlings and parents. Characters in italics and underlined not significantly correlated to MDS ordinations (Table 7. 10). 179 Macrocaroøe 7.4.2 Plate 7.4. Hybrid seedlings six weeks after germination. A: a) E. youngiana, b) E. youngiana x E. macrocarpa, and c) E. macrocarp(t; B: a) E. youngiana, b) E, youngiana x E. pyriþrmis, and c) E. pyrifurm¿s. Bar : lcm 180 Mucrocarosc 7,4.2, EyxEysl r.) Eyoul 1l Eyou2 8) EyxEys 2 2) gyou3 9) Eyou4 10) EyxÉys3 3) EyxEys4 4) I EyxEys 6 6l Eyx Eys 5 5) I Eyou5 11) I 1 050 0.1890 o,2'130 0. 35? 0 0.4410 52 50 Figure 7.28. Dendrogram of E. youngiana x E. youngiana seedliîgs and parent seedlings. E youngiana (Eyou), E. youngiana x E. youngiana (EyxEys). Scale represents standardised Manhattan distance (Gower metric) based on variant seedling characters (Table 7.10). O E.youngiøna, EyxEysl andEyxEys2 O seedlings ) ã 0 ú)â 2 2 -1 a il4¿s Axis 2 Figure 7.29. Mttltidimensional ordination of .8. youngiana x E. youngiana seedlings and parent seedlings. 181 Macrocarpac 7.4.2. LSr3 d ls f! 'I lsrt tco3 0 lc o7 I St7 o ----Esw5 LPO5 ItEt LCOS ls9 J LSH3 rzå5t' .r.¡Hl1** !".ttÍi,, -2 -l 0 MDS Axis I .b99' L SH7 ¿s9J LS H3 LCOS LPLS LLE' LPO' LSwT LLE5 , Lwrz o LPO5 a"aaa*,, z LC O3 IPL 7 : LLET !s.I! !s4.g LST3 -2 0 MD Axis I lsrt lcot lgl¿ LSH3 .! LPL5 !9-9! 0 ,n.rr,**- t"* ta,!l o 1'È[3 Lpos Ldo3 LLET lswS t5¡5 LST3 -2 0 MDS Axis 2 Figure 7.30. Principal component correlation graphs of E. youngiana x E. youngiana seedlings and parents. Characters in italics and underlined not significantly correlated to MDS ordinations (Table 7.10). 182 Macrocaroa¿ 7.5. 7.5. Discussion E. macrocarpa, E. pyriformís and E youngiana will hybridise successfully under controlled conditions, shown by the results of this study. Analysis with hierarchical and non- hierarchical distance methods placed the majority of interspecific seedlings as distinct clusters in the Gower UPGMA dendrogram and between seedlings of both parents on the MDS ordination, thus supporting their Fl hybrid status. Analysis of seed data showed that mean number of seeds produced per flower pollinated and mean seed weight generally varied more between female plants than between crosses. Percent seedlings germinated gave an indication of the fertility of each cross or female tree, the results again showed that there was more variation within species than between. This would indicate that with these closely related species of eucalypts, the effect of interspecific crosses was less than the effect of different maternal genotypes on seed production. The effect of maternal influence over number of seed set and seed weight has been recorded in other interspecific pollination trials (Tibbits, 1989). Such an effect can have important implications in breeding programs, as plants with limited reproductive capabilities would not be used as females in a breeding program, unless they exhibited other, more desirable, characters. Seed set is also influenced by the number and size of individual buds in an umbel, while time of flowering and time to fruit maturation are influenced by bud and fruit volume (Keatley and Hudson, 1998, Primack, l9S7). All three species in the study had fewer than three large buds per umbel, and took up to eight months for fruit maturation, with generally less than 507o abscission of pollinated flowers. This compares well with E. nitens, where 30- 50Vo of flowers from interspecific crosses produced seed (Tibbits, 1989). The same study found that a higher seed set can be realised with hand pollination as opposed to pollination by natural vectors (Tibbits, 1989). 183 Macrocamae 7.5- Hierarchical and non-hierarchical analysis of each cross showed a promising trend across all crossesr in that the majority of seedlings measured (96Vo of interspecifîc crosses) did not cluster with the female parent. Such clustering of seedlings either between parent species clusters or close to male parent species clusters, is an indicator of hybrid status (McDade, 1997) as was the generally intermediate position of the seedlings in the MDS plots. A few seedlings clustered with the male species, for example, two seedlings from the E. pyriformis x E. youngiana and one from the E. youngiana x E. macrocarpa clustered with the male parent; these seedlings will be important to observe as they develop adult characters as they have the highest chance of showing combinations of maternal and paternal characters. Seedlings that clustered with the female parents are more likely to be a result of contamination with female parent pollen, rather than extremes of hybrid distribution. Principal canonical correlation revealed a number of characters that differentiated between seedlings of the three species: glaucousness, leaf length and width, petiole length, leaf margin shape and leaf colour the most descriptive for all species. E. macrocarpa was defined by high levels of glaucousness, a trait which was inherited by its seedlings, regardless of whether the species was the male or female parent, and in many seedlings hybrid status could be identified on the basis of that single character. A similar situation of identification of hybrids by single characters was discussed by Tibbits (1988) in a study of E. nitens. All parts of the E. macrocarpa plant were covered in high densities of long tubes of wax (Wirthensohn and Sedgley, 1996). Inheritance of glaucousness may be simple, requiring one or two genes (Barber, 1955; Cauvin et al, 1987)). Differing leaf length and width was descriptive for all species, as was leaf margin shape and petiole length, while leaf colour was most descriptive for seedlings from crosses with E. youngiana. Several of the twenty-five species within Subgenus Symphyomyrtus Section Bisectaria Series Macrocarpae (Pryor and Johnson, I97l) or E. ser. Curviptera subseries Curviptera (Brooker 184 Mnaroaarnac 7-5- and Hopper, 1993) bear strong similarities to E. macrocarpa, E. pyriformis and E. youngiana. E. rhodanth¿, a distinct and rare species from Watheroo, W.4., differs from E. macrocarpa in its smaller leaves and 'stalked' flowers, and was originally thought to be a hybrid between E. macrocarpa and E. pyriformis (Sampson et al., 1990). In addition, E. rameliana and E. impensa are similar to both E. macrocarpa and E. rhodantha, but without glaucous leaves and with petioles, differing from each other in leaf colour and petiole length (Brooker and Hopper, 1993). Another species of note is E. carnabyi, a very rare possible cross between E. macrocarpa and E. drummondii (a closely related species in same subseries) with shortly petiolate leaves and three to seven flowers per inflorescence (Hopper, 1995). Incidences of naturally occurring unnamed hybrids, where the distributions of the species overlap, between E. pyriformis and E. macrocarpa have also been recorded (Griffin et al., 1988; Hopper, 1995). In a study on the allozyme variation at 11 loci in 45 populations of fourteen species of E. ser. Curviptera, Sampson et aL (1999) found that while phylogenetic analysis of the molecular data supported continued recognition of several species, other species had populations displaying significant divergence in allozyme frequencies. The suggestion has also been raised that E. youngiana is a subspecies of E. pyriformis (Boomsma, 1972), however the majority of classifications place it as a sep¿uate species. E. macrocarpa, E. pyriþrmis and E. youngiana exhibit similar floral morphology: all three have large flowers, where the hypanthium may measure up to 80 mm in diameter with a depth of 40 mm, and the style may measure up to 40 mm in length. Although there is currently no information regarding the stigma morphology of these species, E. caesia, from the same section, has a tapered or blunt stigma (Boland and Sedgley, 1986), and the gross morphology of these species indicates that this type of stigma may be constant for each. The styles of each species are of similar length (30 - 40 mm), negating the possibility of unilateral failure of hybridisation due to incompatible style to pollen tube length ratio (Gore et aI., 1990). 185 Macrocaroae 7.5. Differing style to pollen tube length ratios have been reported as a possible cause of failure to hybridise in a number of studies involving Eucalyptus species (Pryor, 1956: Beardsell et al., 1979; Tibbits, 1988, 1989; Gore et al., 1990). The srudy showed rhar depsite rhe non- isolation of flowers from contaminating pollen, a relatively high percentage of seedlings were hybrids, indicating that isolation may not be necess¿uy if all remaining flowers are removed. A high level of seedüng mortality was observed in both open pollinated seed and seed produced by controlled pollination. Less than 43 Vo seed germination was recorded for any of the twelve crosses studied, and the number of seedlings surviving to three months of age ranged from 807o for E. macrocarpa x E. macrocarpa to 37.5Vo for E. youngiana x E. youngiana. Despite the high mortality, hybrid seedlings were more vigorous than parental seedlings, and there were few abnormalities observed. RAPD analysis of the hybrid seedlings and the parent plants involved in this crossing program has confirmed the data from the morphological analysis (Neaylon, 1999), and provided evidence that some of the hybrids are extremes of the hybrid distribution or more likely contaminants. Two putative natural hybrids were included in the RAPD analysis for comparison (Plate 7.5). 186 Macrocarpae 7,5, Plate 7.5. A: Putative hybrid between E. pyrifornzis and E. youngiana from the Waite Alboretum; B: Putative hybrid between E. youngiana and E. macrocarpa ftom the Waite Arboretum. x.87 Macrocøma¿ 7.6. 7.6. Conclusion The aim of this study was to determine whether E. macrocarpa, E. pyriformis and E. youngiana would hybridise successfully by controlled pollination, generating hybrids. The results indicate that the three species concerned will hybridise, producing viable seed. The 166 seedlings from the interspecific cross seed, when measured for a range of leaf and stem characters at th¡ee different nodes, show strong evidence of intermediacy between parents, with l.2Vo grouped with the male parent,94.6Vo clustered between the parent species, and the remaining 4.2Vo with the female parent. 188 Sect. Bisectariø Adn.a.to.rí¿ Selection at Species Selection for Floral Criteria Species level Gene Pool - Wild and Cultivated populations Selection for Floral Criteria Selection at Genotype Genotype Level Hybrid Production through Controlled Pollination Hybrid/P arent Identification/C onfirm ation Superior Development of Propagation Methods Genotype PBR = Registration Multiplication and Release to Industry Ornamental eucalypt breeding cycle flow chart. Areas covered by this chapter are highlighted. Secl Risectaria and Adnataria 8.1. Chapter Eight InterspecifÏc hybridisation: subgenus Symphyomyrtus, sections Bis ectaria and, Adnøtaria 8.1. Abstract Eucalyptus gillii and E. socialis were used as the female parents in a controlled pollination program using pollen collected from sixteen species with desirable characters for floriculture. The 425 seedlings produced were measured at three months for f,ifteen seedling characters, and the results analysed using hierarchical and non-hierarchical methods. The UPGMA dendrograms showed 225 seedlings from E. giUü x E. gardneri, E. gillii x E. kruseana, E. gillii x E. oldfieldü, E. gillii x E. polyanthemos, E. gillü x E. socialis, E. gillii x E. transcontinentalis, E. socialis x E. gardneri, E. socialis x E. gillii, E. socialis x E. kruseana, E. socialis x E. steedmanii and E. socialis x E. transcontinentalis clustered with neither the male nor female parent seedlings, suggesting intermediacy. The remaining 200 seedlings, from E. gillii x E. caesía, E. giUü x E. eremophila, E. gillii x E. gillii, E. gillii x E. orbiþlia, E. giUä x siderorylon, E. giIIü x websteriana, E. gillii x E. youngiana, E. socialis x E. caesia, E. socialis x E. eremophila, E. socialis x E. macrocarpa, E. socialis x E. oldfieldü, E. socialis x E. orbifolia, E. socialis x E. siderorylon, E. socialis x E. socialis, E. socialis x E. websteriana and E. socialis x E. youngiana clustered with the female parent, and could be the result of self pollination. The multivariate analysis supported the UPGMA results, with the ordination point clusters remaining consistent with the dendrogram groupings. Crosses between closely related species showed a higher degree of success than those between distant crosses, as did those between species with similar flower size. 189 Se¡l- Rìspatnrìn qnd Adnnînrin l--2- 8.2. Introduction The potential for natural hybridisation between species in the genus Eucalyptus L'Herit. has been discussed recently by Ladiges (1997), with respect to genetic isolation between subgenera, sections and some series, and also by Potts and Wiltshire (1997), regarding patterns of hybridisation and its evolutionary significance, and taxonomic, structural and physiological barriers to crossing. The topic was covered by Griffin et al. (1988), in their review of the patterns of natural and manipulated hybridisation, where it was determined that taxonomic affinity will greatly influence hybridisation success. Eucalypts may belong to groups, and Pryor and Johnson (1971) proposed a classification into a hierarchy of groups of subgenera, sections, series, subseries, superspecies, species and subspecies, with groupings based on shared diagnostic morphological characteristics of the constituent taxa. Subgenera are reproductively isolated and the likelihood of a successful cross increases with closeness of taxonomic affinity i.e. intraseries crosses have a greater chance than interseries crosses, which have a greater chance than intersectional crosses (Griffin et al., 1988). Some sections show greater affinity than others to crossing:Ellis et aI. (1991) showed that Bisectaria and Adnataria have a higher chance of pollination success than intersectional crosses between Bisectaria and Maidenaria, Exsertaría, Tingleria, Transversaria, or Dumaria. Ellis er aI. (1991) also found that wide hybridisations are more likely to fail pre-fertilisation than close hybridisations. Hybrids may exhibit heterosis (increased vigour) when compared to parents, including adaptive heterosis when the parents are at an optimal genetic distance, or abnormalities and reduced growth (dwarhng) due to inbreeding depression (with intraspecific crosses that are too close) or heterozygosity (crosses that are too distant) (Eldridge et al., 1993: Martin, 1988). This has implications for improvement through crossing programs: potential crosses must be considered not only for the combination of desirable characters, but also for taxonomic affinities and possible 190 Scoi- Ri.scatnrìn snd Adnatnrin 8-2- structural and physiological barriers to hybridisation. Controlled pollination, or manipulated hybridisation, is a coÍrmon technique used for the generation of interspecific hybrids for plant improvement and is widely used in the development of eucalypts for forestry (Potts et al., 1992; van Wyk et aI., 1989). The technique was seen as a useful method to test natural hybrids, where putative crosses were replicated manually, and the seedlings measured for morphological characters (Prryor, 1950; l95l; 1952; 1956; 1957). Seedlings from manipulated crosses often show intermediate morphology, phenetic segregation in their open pollinated seedlings, and impaired reproductive capabilities compared to the parents (i.e. F2 breakdown) (Hopper et aI., 1978; Hopper, 1995). The aim of the study was th¡ee-fold: to investigate controlled pollination as a method to produce hybrids with increased merit as ornamentals; to determine hybrid status through seedling morphological character analysis; and to assess the likelihood of successful crosses between species from different series in section Bisectaria, and between species from sections Bisectaria and Adnataria. Each species was selected for ornamental characters; specifically tree habit and vigour, floriferousness, inflorescence insertion and structure, flower size, shape and colour, leaf size, shape and colour. The taxonomic position of the species in relation to others in the program and accessibility of specimens were also important. 19r S,cet Rìret"lnrìn qn¡l Ad.nøânrìn 8-3-l- 8.3. Materials and Methods 8.3.1. Plant material The sixteen species used in the study were selected on the basis of one or more desirable ornamental characters, as well as their phylogenetic relationships to the female parent species. The species used are listed in Table 8.1, and were sourced from the Waite Arboretum and the Monarto Woodland. 8.3.2. Controlled pollination The method of controlled pollination followed that of van V/yk (1977) and Moncur (1995),and described in detail in Chapter 6, section 6.3.3. Pollen was collected from a single representative from each male parent species over a twelve month period, and stored in the freezer at -20"C until required. Pollen was tested for viability prior to pollinations (refer section 6.3.3). Three plants from each of the female parent species were used, with between 50 and 150 flowers pollinated for each cross. Each umbel was tagged, and the pollination step repeated every three days. Every emasculated flower was pollinated at least twice. Flowers were not isolated. Capsules were harvested eight months after pollination, when most were swollen and woody. Each capsule was placed separately in a paper bag, and stored at 30oC until open (c. four weeks). Seed was extracted and each seed weighed and stored separately. Seed was planted, germinated and recorded in the method described in Chapter 6 (Section 6.3.3.). 8.3.3. Seed data analysis Differences in the mean number of seeds produced per flower pollinated, mean seed weight for all seeds of that cross and germination rates were calculated . 192 Table 8.1. Species used in controlled pollinations. Based on Pryor and Johnson (1971) informal classification. Crosses to E. gihä 8. socinlis Species used as male parents Section Series Desirable characters 3' 3 E. caesia subsp. cøesla Benth. Bisectaria Macrocarpae medium pink flowers, glaucous buds, weeping small habit 4 4 E. eremophila (Diels) Maiden Bisectaria Occidentales floriferous, pink flowers 3 4 E. gardneri Maiden Bisectaria Reduncae blue foliage, highly floriferous I 2 E. gillii Maiden Bisectaria Oleosae grey foliage and buds, highly floriferous, long flowering season 4 4 E. kruseana F.Muell. Bisecta¡ia Kruseanae grey round foliage, small habit, long flowering season 3 3 E. macrocarp¿ Hook. Bisectaria Macrocarpae very large red flowers, glaucous buds, leaves and flowers 3 J E. oldfieldii F.Muell. Bisectaria Macrocarpae medium cream flowers, round buds, small habit J 3 E. orbiþlia F.Muell. Bisectaria Macrocarpae medium cream flowers, heart shaped leaves, glaucous buds 5 5 E. polyanthenos Schauer Adnataria Polyanthemae numerous small terminal flowers, grey leaves 5 5 E. pruinosa Schauer Adnata¡ia Pruinosae numerous medium cream terminal flowers 5 5 E. sideroryIon Cunn. Ex Wools Adnataria Melliodorae medium pink buds and flowers 2 I E. socialis F.Muell. ex Miq. Bisectaria Oleosae floriferous, glaucous buds, flowers and leaves 4 4 E. steedmanü C.Gardner Bisectaria Occidentales yellow square buds and flowers, small neat habit 2 2 E. transcontinentalis Maiden Bisectaria Oleosae numerous yellow buds and flowers 3 3 E. websteriana Maiden Bisectaria Macrocarpae small cream flowers, heart shaped/round leaves, glaucous buds 3 3 E. youngiana F.Muell. Bisectaria Macrocarpae large yellow - red flowers, green - red - purple buds ul intraspecific cross 2 intraseries cross 3 close interseries cross 4 distant interseries cross 5 intersectional cross SecL Bis ectøria and Adnatar¡a 8.3.4. 8.3.4. Seedling morphology Each surviving seedling from controlled pollinations was measured for fifteen continuous, binary or ordered multistate leaf and stem characters on fully expanded leaves at the 3'0,7^ and 10th nodes above the cotyledons, and all measurements were conducted three months after germination (Table 8.2). In addition, open pollinated seed from the female parent trees and open pollinated seed sourced from other populations of the species used, was grown to ensure a broad representation of seedling morphology for each species. Between 40 and 80 seedlings from each parent species were measured three months after germination for the same characters. These scores were averaged to generate ñve representative individuals for each species, which were used in the analysis for comparison with the seedlings from controlled pollinations. Only leaf data for node 7 were used, as nodes 3 and 10 provided incomplete data sets. The invariant characters were leaf surface wax (LSW), leaf concolorous or discolorous (LEA), leaf smoothness (LSM) and stem smoothness (SSM). 8.3.5. Data analysis The seedling morphological data were analysed using the PATN analysis package (Belbin, 1994) in order to determine the similarity between seedlings and parent species, using hierarchical (UPGMA) and non-hierarchical (multidimensional scaling, minimum spanning tree and principal canonical correlation) analysis. 194 Rìsoolnrin qnà Aãnnfnìn *-/l-1 Table 8.2. Characters and codes used in phenetic analysis (seedling), measured at node 3,7 and 10, three months after germination (node 7 was used in the analysis due to incomplete data sets for nodes 3 and 10). Code Characûer LSH Leaf shape linear (0) lanceolate (l) ovate (2) cordate (3) orbicular (4) LST Leaf tip acuminate (0) acute (l) mucronulate (2) obtuse (3) uuncate (4) LSB Leaf base attenuate (0) oblique (l) obtuse (2) cordate (3) LLE Leaf length (mm) LWI Leaf width (mm) LSW Leaf surface wax shining (0) dull (1) glaucous (2) very glaucous (3) LEA Leaf surfaces concolorous (0) discolorous (1) LSM Iæaf surface smooth (0) not smooth (1) LPS Iæaf stalk sessile (0) petiolate (1) LPL Leaf petiole length (mm) LPO Leaf position opposite (0) alternate (l) LCO læaf colour light green (0) green (1) gey green (2) blue green (3) dark green (4) SSH Stem shape square (0) angular (1) round (2) STE Stem single (0) branching (1) SSM Stem smooth (0) not smooth (l) 8.4. Results 8.4.1. Seed data analysis The mean number of seeds per flowerwas low for all crosses, including open pollinated flowers (Tables 8.3 and 8.4). Statistical analysis was not possible as only one female parent produced seed for both E. gillii and E. socialis. Seed weights were highly variable within crosses, however there was no correlation between seed weight and germination. There was no correlation between male parent seed weight and seed weight of crosses. All cotyledons resembled those of the female parent, with the exception of five E. giUii x E. polyanthemos seedlings whose cotyledons resembled those of E. polyanthemos, however these seedlings died before true leaves emerged. High levels of seedling mortality occurred in all E. giUii crosses soon after germination, while crosses with E. socialis as the female showed low seedling mortality. 195 Table 8.3. Summary of flower, capsule, seed and seedling data for interspecific, intraspecific and open pollination with E. giUii as the female parent. Data for one tree only, other two did not set seed. Number Number Percent Total Mean Mean seed Number Number Percent Number of Number of of of capsule seed number weight of of seeds seedlings showing seedlings flowers capsules set of seeds (mg)' seeds seedlings germinated3 similarity to: intermediate Male parent pollinated with per flower (range) planted Germ Meas. female male between seed pollinatedr parent parent parents E. caesia 119 6 5.0 6 0.05 t.4s (0.77-r.8s) 6 6 6 100.0 6 0 0 E. eremophila 130 t4 10.8 23 0.18 r.2s (0.26-r.72) 23 20 1l 87.0 11 0 0 E. gardneri r62 29 t7.9 64 0.40 1.23 (0.19-1.7r) 64 52 42 81.3 0 0 42 E. gillii (intra) 100 62 62.0 122 1.22 1.11 (0.33-1.71) r22 118 68 96.7 68 0 0 E. kruseana t28 20 15.6 66 0.52 0.98 (0.17-1.49) 66 56 38 84.8 0 0 38 E. macrocarpa 190 8 4.2 t2 0.06 0.64 (0.16-1.25) t2 5 0 41.7 0 0 0 E. oldfieldii 139 35 25.2 106 0.76 1.04 (0.09-1.61) 106 62 54 58.5 I 0 53 E. orbiþIia l4t 36 25.5 135 0.96 1.3s (0.33-2.03) r35 tt7 10 86.7 9 0 1 E. polyanthemos 100 l8 18.0 48 0.48 1.32 (0.91-1.68) 48 2T 6 73.8 0 0 6 E. pruinosa 60 0 0 0 0.00 0.00 0 0 0 0 0 0 0 E. siderorylon 118 I 0.8 1 0.09 1.30 I I I 100 1 0 0 E. socialis 110 30 37.3 9t 0.83 1.02 (0.t0-2.46) 9l 53 38 58.2 0 0 38 E. steedmanii 116 1 0.9 2 o.o2 0.24 (0.22-0.26) 2 0 0 0.0 0 0 0 E. lranscontinentalis 79 26 32.9 64 0.81 1.29 (0.30-1.Ø) 64 47 35 73.4 10 0 25 E. websteriana 136 4 2.9 t3 o.t4 0.72 (0.15-1.20) t3 8 I 61.5 I 0 0 E. youngiana 189 16 8.5 39 0.21 0.78 (0.r6-1.99) 39 t2 4 30.8 4 0 0 open 50 20 40.0 22 0.44 1.33 (0.6s-1.65) TotaI 2067 326 814 792 578 3r4 111 0 203 I total number of seed from all capsules of that cross divided by the total number of flowers pollinate for that cross. 2 weight of all se¿ds in a capsule divided by the number of seeds in that capsule. 3 number of seeds germinated divided by the number of seeds planted, multiplied by 100/1. Table 8.4. Summary of flower, capsule, seed and seedling data for interspecific, intraspecific and open pollination with E. socialis as the female parent Data for one tree only, other two did not set seed. Number Number Percent Total Mean Mean seed Number Numþr Percent Number of Number of of of capsule seed number weight of of seedlings seed seedlings showing seedlings flowers capsules set of seeds (mg)' seeds germinated3 simila¡ity to: intermediate Male parent pollinated with per flower (range) planted Germ Meas. female male seed pollinatedl parent parent E. caesia 73 20 27.4 26 0.36 0.98 (0.2s-1.45) 26 8 6 30.8 6 0 0 E. eremophila 65 l0 6.5 18 o.28 0.96 (0.19-1.30) 18 7 7 38.9 7 0 0 E. gardneri 100 3I 31.0 49 0.49 0.86 (0.12-1.33) 49 l0 5 20.4 2 0 3 E. gillii 63 10 6.3 20 0.32 0.86 (0.13-1.27) 20 5 5 25.0 I 0 4 E. kruseana 62 t7 27.4 28 0.45 t.o4 (0.2t-1.67) 28 l0 8 35.7 5 0 3 E. macrocatpa 58 3 5.2 5 0.09 0.76 (0.rs-1.1s) 5 2 2 40.0 2 0 0 E. oldfteldii 100 29 29.O 55 0.55 0.91 (0.33-1.30) 55 l2 l0 21.8 10 0 0 E. orbiþlia 43 9 20.9 t3 0.30 0.95 (0.18-1.47) 13 5 5 38.5 5 0 0 E. polyanthemos 63 28 44.4 49 0.78 0.94 (0.23-1.74) 49 1l 9 22.4 9 0 0 E. pruinosa 54 0 0.0 0 0 0.00 0 0 0 0.0 0 0 0 E. sideroryIon 90 l0 11.1 2t 0.23 t.ot (0.@-152) 2T 5 3 23.8 3 0 0 E. socialis intra 48 10 20.8 l8 0.38 1.08 (0.38-1.76) 18 11 l0 61.1 l0 0 0 E. steedmanii 25 19 76.0 38 1.52 0.90 (0.21-1.36) 38 15 9 39.5 5 0 4 E. transcontinentalis M 32 72.7 62 t.4r 0.93 (0.17-1.46) 62 T2 t2 19.4 3 0 9 E. websteriana 100 67 67.0 142 1.42 1.00 (0.2s-1.73) 142 20 18 14.2 l8 0 0 E. youngiana 78 22 28.2 4T 0.53 0.83 (0.23-1.27) 4l 2 2 4.8 2 0 0 Open 50 48 96.0 96 2.0 r.l8 (0.68-1.35) Total 1116 365 681 585 135 111 88 0 23 I total number of seed from all capsules of that cross divided by the total number of flowers pollinate for that cross. 2 weight of all seeds in a capsule divided by the number of seeds in that capsule. 3 number of seeds germinated divided by the number of seeds planted, multiplied by 100/1. Sp.at, Bíscatarín sntl Adn¿tarín A-4-2-l- 8.4.2. Seedling morphology analysis 8.4.2.1. E. SiUii as thefemale parent The results of the hierarchical and non-hierarchical analyses for each of the thirteen successful crosses with E gillii as the female parent are surnmarised in Table 8.5. Numbers of seedlings measured for each cross varied considerably (Table 8.3), from one seedling only for crosses with E sideroxylon and E. websteríana, lewer than twelve for crosses with E. caesia, E. eremophila, E. orbifolia, E. polyanthemos and E. youngiana, and between 35 and 54 for the remaining crosses. The intraspecific cross of E. gillii produced 68 seedlings. Crosses with E pruinosa, E. steedmanii and E. macrocarpa did not produce seedlings. Seedling comparisons were made with open pollinated seed from the female parent tree, resulting in less apparent variation within the intraspecific cross. On each of the thirteen dendrograms, seedlings and parent species were ¿uranged in two or three clusters. All seedlings from crosses with E. caesía (Figure 8.1), E. eremophila (Figure 8.2), E. sideroxylon (Figure 8.3), E. websteriana (Figure 8.4) and E. youngiareø (Figure 8.5) as the male parent, clustered with E. gillii. A portion of seedlings from crosses between E. gillii and E. orbifolia (9) (Figure 8.6), E. tanscontinentalis (10) (Figure 8.7) and E. oldfieldii (1) (Figure 8.8) clustered with E. gillii, with the remaining seedlings from each cross forming distinct groups, suggesting intermediacy. All seedlings from crosses with E. gardneri (Figure 8.9), E. kruseana (Figure 8.10), E. polyanthemos (Figure 8.11) and E. socialis (Figure 8.12) clustered between the female and male parent clusters, also suggesting intermediacy. The E. gillii x E. giUü cross produced the highest number of seedlings, with all seedlings clustering with the female parent (Figure 8.13). The MDS scatter plots for each cross reflected the dendrogram groupings, with seedlings from crosses with E. caesia (Figure 8.14), E. eremophila (Figure 8.15), E. sideroxylon (Figare 198 Qoot Rioooînån qnìl AÅnnlnrin ß-4-2-1 8.16), E. websteriana (Figxe 8.17) aûd E. youngiana (Figure 8.18) as the male parent, grouped with the female parent, E. giUii. Seedlings from crosses with E. gardneri (Figure 8.19), E. kruseana (Figure 8.20), E. polyanthemos (Figure 8.2I), and E. socialis (Figure 8.22) were positioned between the parents. Seedlings from crosses with E. oldfieldli (Figure 8.23), E. orbifoliø (Figure 8.24) and E. transcontinentalis (Figure 8.25) clustered either between both parents or with E. giUíi. Seedlings from E. gillii x E. gillü were randomly scattered across the plot (Figure 8.26). The stress values for all SSH analyses were between I.l7 and' 10.807o, and well below the accepted2} 7o distortion value. Minimum spanning tree analysis for the crosses between E. giUii and E. oldfieldii, E. sideroxylon and E. websterian¿, showed the female parent group linked to the male parent group, indicating that the seedlings bear little resemblance to the male parent (MST value: min. 0.5056 max. 0.6058). The other crosses linked the male parent group to the seedlings at a distance, indicating a low level of similarity between them (MST value: min.0.2979 max. 0.7340). Two exceptions were seedlings from the cross with E. socialis (MST value 0.1303), and the cross with E. tanscontinentalis (0.2258), which were linked relatively closely to the male parent species. The highest MST value linking hybrids to female parents was 0.1035, and the lowest was 0.037, indicating high levels of similarity between all seedlings and E gillii. The same data for the female parent group was used in each analysis, and the mean MST values between female individuals ranged from 0.037 to 0.0867. The mean MST value linking individuals for each male parent group was between 0.0126 and 0.0927, indicating that each group was very uniform. E. websteriana and E. youngiana were the two exceptions, with values of 0.1210 and 0.1370 respectively, indicating a moderate level of variability within each group. One individual, E. giúii x E. kruseana #l was distinct from both female (MST value 0.2248) and male p¿ìrent groups (0.4574), indicating a high degree of dissimilarity to both parent species. Principal canonical correlation analysis (Table 8.6) 199 Seet- Bi-ecelarh. cnd Adnnlorfut 8--¿l-2-l- showed most characters to be significantly correlated to the MDS ordination coordinates (Appendix 2.1: Figures 8.57 - 8.69). 200 Table 8.5. Summary of data for E. gillii: Gower UPGMA dendrogram, MDS plot and MST analysis S = seedlings from cross; M = male; F = female. Cross #Sin #Sin #Sin # MDS Mean MST MST S link Mean MST S link Mean MST #S # Dend female male s MDS SSH of female female to femaled MST of male to maleg ofS male clusters cluster cluster cluster bunch" stress bunchb to S" S F bunch" to Sr s M bunctf E. caesia 6 2 6 0 0 2 l.55Vo 0.0555 0.0380 44 0.0339 0.7340 4 4 0.0107 E. eremophila ll 2 ll 0 0 2 l.7l7o 0.0370 0.0154 many 0.0370 0.6802 6 4 E. gardneri 42 3 0 0 42 3 9.49Vo 0.0/.87 0.0967 3ll 0.0486 o.2979 t7 5 o.0267 0.0934 94 E. gillii (intra) 68 I 68 0 0 scat 7.33Vo E- kruseana 38 3 0 0 38 3 2.97Vo 0.0533 0.1035 35 2 0.0854 0.4570 I 5 0.0128 E. oldfteldii 54 2 I 0 53 3 6.4l%o 0.0778 0.0906 I 5 0.0463 0.5056 FI 5 0.0343 0.1387 3 5 FI 5 E. orbiþIia l0 J 9 0 I 3 2.8OVo 0.0s95 0.0527 l0 4 0.0709 0.521I I I 0.0628 E. polyanthemos 6 3 0 0 6 J 2.82Vo 0.0642 0.1201 5 4 0.0927 o.4633 6 3 0.0516 E. siderorylon I 2 I 0 0 2 l.l74o 0.0670 0.0079 5 6 o.0677 0.5572 FI 4 E. socialis 38 3 0 0 38 J l0.80Vo 0.0867 0.1940 4 2 0.0635 0.1303 20 4 0.0544 E- transcontinentalis 35 3 l0 0 25 3 8.377o 0.0789 0. l 150 6 4 0.0126 0.2258 ll I 0.0684 o.1235 5 5 0.1070 7 5 o.2438 t7 I E. websteriana I 2 I 0 0 2 2.l7Vo 0.0665 0.0r52 I 4 0.t2t9 0.6058 4 5 E. youngiana 4 2 4 0 0 2 4.l7Vo 0.0598 0.1392 4 4 0.1370 o.4745 I 2 0.0257 Total 3t4 lll 0 203 0.0336 2 5 " 'bunch' refers to a discrete cluster of simila¡ individuals o Mean of all MST yalues connecting female parent individuals " MST link connecting female bunch to seedlings d Seedlings (code number from dendrogram) linking to female (code number from dendrogram) " Mean of all MST values connecting male parent individuals t MST link connecting male group to seedlings s Seedlings (code number from dendrogram) linking to male (code number from dendrogram) h Mean of all MST values connecting seedlings Table 8.6. Summary of measured va¡iant (Y) and invariant (-) seedling characters (Table 8.2.) and significance (*) from correlation coefficientl 1r2¡ for bcharacters E. gihii. "Characters significantly correlated (*) if greater than (r2), calculated from PCC analysis, invariant (-) if identical for all individuals in that cross. E. eillií r^ LSH Lsr LsB LLE LwI LSw LEA LSM Lps LpL Lpro LCo ssH srE ssM Totat E. caesia 0.65 Y* _b Y{< Y* Y* Y{< Y* Y* Y{C Y'ß 9n5 E. eremophila 0.60 Y{< Y* Y* Yr. Y* Y* Y* Yr, Y'C Yr< Y* Ytc t- Y'C l4n5 E. gardneri 0.39 Y* Y{' Y¡ß Y* Yr, Y* Y'C Y{< Y* Y tut5 E. gillii intra 0.31 Y{' Yr< YTC Yr< Y{< r Y 6n5 E. kruseana o.4t Y{. Yr< Y:È Yt< Yt' Y{< Y* Y{. Y* Y* Yx t- tzlt5 E. oldfieldii 0.36 Y* Y* Y* Y* Y* Y* Yr. Yr< Y{< Y* Y* Y* t2n5 E. orbiþlia 0.59 Y* Y* Y* Y* Yx Yx Y* Y* Y* Y* 10/15 E. polyanthemos 0.Ø Ytc Yx Y" Y* Y* Y{c Y{< Y* Y* 9t15 E. siderorylon 0.76 Yr< Y{c Y{< Y* Y* Y{< Y" Ytc t- Yr< tzl15 E. socialis 0.39 Y{< Y* Yr< Y* Y* I. I. Y* Y{' Y* Y* 9lt5 E. transcontinentalis 0.39 Y{. Y* Y* Ytc Yr< Y{< Y* 7trs E. websteriana 0.76 Y* Y* Y Y* Y* Y* Y* Yr< Y{. Y{< Y* Y* rzn5 E. youngiana 0.70 Y{< Y* Yt' Y* Y{< Y* Y* Yr< Y{' Y* Y* Y{. Y{< T3I15 Correlation coefñcientt : showing the fit 1r2) of the individual character vectors in the ordination space Table 8.7. Seedling morphological characters of seedlings from crosses between E. gillii and other species. in rnm or presented as mean bcharacters.Measurements (range). "Charactðrs unique ãr highly descriptive for that taxon according to PCC (Figs 8.28-8.40), significantly correlatedt (É) to individuals. crosses shaded gardnerí E. giilii E. gillii x E. caesia E. caesia E. gillä x E. eremophila E. eremophila E. gillíixE. gardneri E. ovate or lanceolate " LSH lanceolate or ovate, ovate cordate "o lanceolate or ovate ovate or lanceolate - ovate or lanceolate ó rarely linear ù acute âcute obtuse acute or mucronulaûe acute, mucronulate LST acute, rarely obtuse or acute b or acuminate acuminate "b b b cordate, rarely obtuse obtuse or acuminate, obtuse obtuse or obtuse, oblique or LSB attenuate, obtuse or cordate b attenuate attenuate cordate "b rarely cordate 53. I LLE 27.4 29.O 46.6 30.2 39.6 29.3 (26.0-35.0) (38.640.5) (24.s-35.8) (4O.7:743)ù (26.1-28.8) "b (26.0-3 1.0) (26.047.Ðù "b 26.8 LWI 16.0 19.2 52.1 15.3 23.7 t6-3 (10.8-28.1) .b (16.s-24.s) (33.356.8)'b (l1.6-21.0) (21.6-26.Ðù (10.4-22.8) (10.941.2)ù b dull LSW dull or glaucous " dull shining dull dull dull b concolorous LEA concolorous concolorous concolorous concolorous, rarely concolorous concolorous discolorous smooth LSM smooth smooth smooth smooth not smooth smooth ô ò petiolate ó LPS sessile sessile petiolate sessile petiolate sessile LPL 0.0 0.0 I 1.9 0.0 10.0 0.0 8.4 (6.6-20.9)ù (9.4-10.8) "b (3.r-13.2)ú ú ô alternate ó LPO opposiæ opposite alternate opposlte altemate opposiæ ó green b LCO green green dark green green grey green "b blue green blue round SSH round, rarely angular round round round round round branching STE branching branching branching branching branching branching, rarely mund smooth SSM smooth smooth smooth smoolh not smooth smooth Continued Table 8.7. Continued E. giüii x E. gillii E. gillä x E. kruseana E. kruseana E. giüä x E. oldfieldü 8. oldfeldü E. gihä x E. orbifolia E. orbifolia o o orbicular, LSH lanceolateorovate lanceolate or ovate cordate or orbicular ovate or lanceolate lanceolate ovate ú rarely cordate b ó ó acute, rafely obtuse truncate or mucronulate LST acute acute, rarely obtuse or mucronulate acute or obtuse mucronulate obtuse b ù attenuate or obtuse attenuate obtuse, cordate or obtuse, cordate or LSB attenuate, oblique, obtuse attenuate, rarely acute, obtuse or cordate b attenuate attenuate or cordate obtuse 3t.4 42.3 30.1 23.9 LLE 28.0 29.6 23.7 b b (17.0-29.3) (21.3-37.5) (23.r-36.0) (22.0-26.Db (2s.1-37.8) (38.045.1) (27.5-32.0',) t7.l 16.4 18.5 30.1 LWI t4.7 16.5 24.2 b b (13.5-23.0) (19.741.8) (9.0-20.s) (9.4-20.8) (22.r-26.8)b (9.1-23.0) ( l o.l -21 .5) ù dull dull LSW dull dull dull or glaucous dull dull ù concolorous concolorous concolorous LEA concolorous concolorous discolorous concolorous smooth smooth LSM smooth smooth not smooth smooth smooth ù ù petiolate ó sessile petiolate LPS sessile sessile petiolate sessile 0.0 7.O LPL 0.0 0.0 0.9 0.0 4.7 (2.9-10.8) "b (0.7-1.5) "b (2.55.1) "b b ó opposite opposite LPO opposlte opposite opposite opposite altemate b b green, rarely grey grey green, blue green grey green, blue green blue green LCO green blue green blue green blue t grcen or green or green or grcen ù angular angular round SSH round round angular round b branching single or branching STE branching branching branching branching branching "b ô ó ù smooth not smooth SSM smooth smooth not smooth smooth not smooth Continued Table 8.7. Continued E. gää x E. polyanthemos E. polyanthemos E. gillii x E. sideroryIon E. siderorylon E. gíllü x E. socialís E- socialís lanceolate lanceolate, rarely ovate - LSH ovateorlanceolate orbicular "o lanceolate lanceolate or ovate' ovate or o acute truncate or acute "b acute acute, acute, rarely obtuse or acute LST b sometimes obtuse acuminate b b obtus€, cordate obtuse attenuate, rarely obtuse attenuate, cord¿te rârely attenuate or obtuse LSB obtuse ù or acute obtuse LLE 28.8 37.5 25.O 58.9 3l.l 31.3 (20.3-38.8) (27.0-29.s) (31.047.1) "b (43.6:76.Ðù (t2.1-2s.9) "b LWI 15.9 47.9 t2.0 21.7 18.8 12.5 (5.9-30.4) b (13.5-19.s) (31.358.6) "b (9.9-333) "b (12.1-25.9) LSW dull dull dull dull dull dull ô LEA concolorous concolorous concolorous discolorous concolorous concolorous LSM smooth smooth smooth smooth smooth smooth b ò sessile LPS sessile petiolate sessile petiolate sessile LPL 0.0 14.6 0.0 5.0 0.0 0.0 (9.0-20.0)'b (2.0-8.?) "b b ù ó opposite or altemate LPO opposite alternate opposite alternate opposiæ ó * blue green ù LCO blue green blue green green grey gr"en blue green ó square b SSH round round round angular square, sometimes angular or round ó branching b STE branching branching branching branching, rarely single branching, rarely single smooth SSM smooth smooth smooth smooth smmth Continued Table 8.7. Continued E. gillíi x E. transcontinentalis E. gillii x E. websteriana E. websteríana E.gilläxE.youngiana E.youngiana E. lranscontinentalis b ó LSH ovate, sometimes linear ovate cordate or orbicular õvate or lanceolate onutã *.o.¿utã- lanceolate, rarely linear or cordate ú ub LST obtuse, sometimes acute obtuse acute truncate or obtuse acute muc¡onulate, acute or acu¡ninate ù b LSB atteouate or cordate, obtuse obtuse obtuse, mucronulate or obtuse obtuse or attenuate ó rarely obtuse acumnate LLE 31.0 18.3 27.5 22.2 31.5 s9.9 (20.7-39.8) (16.6-2t.2\b (13.8-27.5) "b (3O.O-32.3) (33.O:72.Dh LWI 15.5 5.9 16.5 25.O 15.2 30.0 (6.8-25.3) (5.2-7.Db (15.7-33.Ðù (10.0-19.7) (1s.845.6) b ó LSW dull dull dull dull dull dull or shining LEA concolorous concolorous concolo¡ous concolorous b concolorous concolorous b LSM smooth smooth smooth smooth smooth smooth b LPS sessile sessile sessile petiolate "b sessile petiolate LPL 0.0 0.0 0.0 3.0 0.0 9.9 (1.24.Th (s.7-14.Ðh LPO opposite opposite opposite opposite or alternate b opposlte altemate b s LCO blue green, sometimes blue green light green dark green. blue green, blue green or grey green dark green, ù green, rarely green or tight green or green rarely green "b dark green ssH square, rarely angular or square o round round b round angular b round ó b STE branching branching branching branching, rarely single branching single ssM smooth smooth smooth not smooth smooth smooth SecL Bísce.î.artn entl Atlnnlørìn. 8-4-2^2- 8.4.2.2. E. socialis as thefemale parent The results of the hierarchical and non-hierarchical analyses for each of the fifteen successful crosses with E socialis as the female parent are suÍrmarised in Table 8.8. Numbers of seedlings measured for each cross ranged from two to eighteen, with most under ten (Table 8.4). The E. socialis x E. socialis cross produced ten seedlings. The cross with E pruinosa did not produce seed. Seed from a coûrmercial seed source were grown, in addition to seed from the female parent tree, as the parent seedling comparators, and the variation displayed reflects the naturally high levels of variability within this species. Dendrograms from nine of the fifteen crosses divided the seedlings into two clusters: female parent with all seedlings, and all male individuals (Figures 8.27 to 8.35). Dendrograms from crosses between E. socialis and E. gardneri (Figures 8.36), E. gíUíi (Figure 8.37), E. kruseana (Figure 8.38), E. steedmanii (Figure 8.39), and E. transcontinentalis (Figure 8.40) were divided into three clusters, with around half the seedlings for each cross clustered separately to either parent, and the other half clustered with the female parent. The male parent seedlings were always clustered together. E. socialis x E. sociahs seedlings were clustered with the female parent (Figure 8.41). The MDS plots reflected the dendrogram groupings, with nine crosses showing two groups, one containing all female parent seedlings and seedlings, the other containing all male parent seedlings (Figures 8.42 to 8.50). The five interspecific crosses between E. socialis and E. gardneri (Figure 8.51), E. gillii (Figure 8.52), E. kruseanc (Figure 8.53), E. steedmanii (Figure 8.54) and E. transcontinentalis (Figure 8.55), placed all or some of the seedlings between the parent groups. The plot of E. socialis x E. socialis (Figure 8.56), scattered the seedlings across the plot, with four groups apparent. The stress values for all SSH analyses were very low, between 3.66 and7.30Vo. 207 SecL Bisectarìa and Adnataria 8.4.2.2. The MST linkages gave finer resolution to the scatter plots. In the case of the seedlings of E socialis x E. caesia, E. socialis x E kruseana, E. socialis x E. sideroxylon, and E. socialis x E. steedmanii, the male parents were linked to E. socialis #l (MST value range 0.3582 - 0.5043). For E. socialis x E. transcontinentalis (MST value 0.24L8) and E. socialis x E. youngiana (MST value 0.4039), the male parent linked to E. socialis #2. These results indicate that the seedlings, at this stage, bear low resemblance to the male parent seedlings. The remaining crosses link between seedlings and male parent, with a high MST value (range 0.3771-0.5273). E. socialis x E. gillii (MST value 0.1976) shows the closest link between seedling #3 and E. gillii, indicating a relatively high similarity between them. The same data for the female parent group were used in each analysis, and the mean MST values between female individuals are between 0.0596 and 0.1831, indicating a moderate level of variability within the group. The mean MST value linking individuals for each male parent group is between 0.0125 and 0.0881, indicating that each group is very uniform. E. youngian¿ was the exception, with a value of 0.1502, indicating a moderate level of variability within the group. Principal canonical correlation analysis (Table 8.9) showed most cha¡acters to be significantly correlated to the MDS ordination coordinates (Appendix 2.2.: Figures 8.70 - 8.84). 208 Table 8.8. Summary data for E. socialis: UPGMA dendrogram, MDS plot and MST analysis S = seedlings from cross; M = male; F = female Cross #Sin #Sin #Sin # MDS Mean MST MST S link Mean MST S link Mean MST #S # Dend female male S MDS SSH of female female to femaled MST of male to males ofS male clusters cluster cluster cluster bunch" stress bunchb to S" S F bunch" toSr S M bunchh E. caesia 6 2 6 0 0 2 4.667o o.ts67 0.to22 allStoF 0.o372 0.48',t5 FI 2 o.0622 E. eremophila 7 2 7 0 0 2 4.37Vo 0. I 104 0.1303 allStoF 0.0125 0.5273 6 3 0.0459 E. gardneri 5 3 2 0 J 3 4.59Vo o.t257 0.1679 allStoF 0.0579 0.38r3 2 3 0.0910 E. gillii 5 3 I 0 4 3 6.427o o.t375 0.181 I allStoF 0.0656 0.1976 J 3 0.3096 E. kruseana 8 3 5 0 3 3 5.65Vo 0.183 l 0.1 158 allStoF o.0562 0.3582 FI I 0.1095 E. macrocarpa 2 2 2 0 0 2 3.66Vo o.t4.r''6 o.t43t 24 0.0830 0.3994 2 I 0. l 182 E. oldfieldii l0 2 l0 0 0 2 5.22Vo o.t457 0.0865 allStoF 0.0318 0.5498 5 5 0.1081 E. orbiþlia 5 2 5 0 0 2 3.79Vo 0.1545 0.0888 allStoF 0.0500 0.377t 2 2 o.tØ3 E. polyanthemos 9 2 9 0 0 2 4.7\Vo 0.0596 o.t233 allStoF 0.061I 0.4s26 FI 3 0.0973 E. siderorylon 3 2 3 0 0 2 5.907o 0.t3t4 o.225t allStoF 0.0533 0.5043 FI 4 0.0 E. socialis (intra) l0 I l0 0 0 4 6.50Vo 0.0 0.0 allStoF 0.0 0.0 0 0 0.0 E. steedmanii 9 3 5 0 4 J 6.O8Vo 0.0863 o.t702 allStoF 0.0881 0.4108 FI 5 0.0946 E. transcontinentalis t2 3 3 0 9 3 6.777o 0.1507 o.3073 44 0.0488 o.24t8 F2 I 0.0854 0. I 128 23 E. websteriana t8 2 180 0 2 7.30Vo 0.W02 o.t526 55 0.0295 0.4150 9 5 0.0697 0.0551 182 E. youngiana 2 2 2 0 0 2 5.37Vo 0.1138 o.1787 l3 0.1502 0.4039 F2 2 0.0 0. l 875 l5 Total ll0 890 22 " 'bunch' refers to a discrete cluster of similar individuals o Mean of all MST values connecting female parent individuals " MST link connecting female group to seedlings o Seedlings (code number from dendrogram) linking to female (code number from dendrogram) " Mean of all MST values connecting male parent individuals t MST link connecting male group to seedlings sSeedlings (code number from dendrogram) linking to male (code number from dendrogram) n Mean of all MST values connecting seedlings Table 8.9. Summary of measured variant (Y) and invariant (-) seedting characters (Table 8.2) and significance (*) from correlation coefficientt 1r2¡ for bcharacters E. socialis. "Characters significantly correlated (x) if greater than (r2), calculated from PCC analysis, invariant (-) if identical for all individuals in that cross. E. socìalis r" LSH LST LSB LLE LWI LS\ry LEA LSM LPS LPL LPO LCO SSH STE SSM Total E. caesia 0.66 Ytc Y* Y* Y,r. Y{< Y* Y* Yx Y* Yr< Y{< Y* t2n5 E. eremophila o.& Yx Y* Y1. Yt< Y* Y* Y* Y{< Y* Yr< Y{< Yr< Y* t3n5 E. gardneri 0.67 Y* Y* Y* Y,ß Y* Y{c Y{. Y* Y Y* Y* rvt5 E. gillii 0.67 Y* Y*. Y* Yr. Y* Y* Yr. Y* Y{< Y* Y* tvt5 E. kntseana 0.63 Y* Y* Y Y{< Y* Y* Yr< Y* Y Y* Y{. Y* Y* ;. t4n5 E. macrocarpa 0.75 Y Y* Y{< Y* Yr. Y* Yr< Y* Y* Y* Yr< Yr< t3n5 E. oldfieldii 0.61 Y* Yt Y* Y* Y* Y{< Y* Y* Y* Y Y* Y* Y* t3n5 E. orbiþlia 0.69 Yr< Y{. Y* Yx Y* Y Y{< Y* Y* Y Y{< Y{< t3n5 E. polyanthemos 0.61 Y* Y* Y* Yr< Y* Y{< Y* Yr< Yr< Y* Yr, Y* l. t2/15 E. sideroryIon 0.72 Y{< Y* Yr. Y* Y{< Yr< Y* Y{. Y* Yr< Y{< Yx Y* t3n5 E. socialis intra 0.59 Y* Y* Y* Y{< Y* Y* Yt Y* Y* Y* Yr. Y* t2t15 E. steedmanii 0.62 Yr< Y{c Yr. Y* Y{< Y* Y Y* Y* Y{. Yr' Y* Yx t- t4lt5 E. transcontinentqlis 0.61 Yr< Yr< Y{c Y* Y* Y Y* Y{< Y{. Y{< Y* Y* tzn5 E. websteriana o.s2 Y* Y* Y{< Y* Y* Y* Ytc Y* Y* Y Y{< Y{< Y* Y{< t4n5 E. youngiana 0.73 Y* Yt< Y* Y* Yr< Y Y* Yr< Y{' Y* Yx Y t2lt5 Correlation coefficientt : showing the fit 1r2¡ of the individual character vectors in the ordination space. Table 8.10. Seedling morphological characters of seedlings from E socialis and putative male parent species. Measurements in rnm or presented as bcharacters mean (range). "Characters unique or highly descriptive for that taxon according to PCC (Figures 8.71-8.85), significantly correlatedl (l) to individuals. Hybrid seedling crosses shaded. E. socialis E. socialis x E. caesia E, caesia E. socialis x E. eremophila E. socialis x E. gardneri E. socinlis x E. eremophila E. gardneri E. socialis LSH lanceolate, rarely ovate, rarely cordate ovate ovate or lanceolate ovate ovate or lanceolate ovate, rarely ovate ó lanceolate lanceolate ó b b LST acute acute, rarely obtuse acute acute, mucronulate or obtuse obtuse or acute acute, mucronulate obtuse, acute, rarely b obtuse or acuminate mucronulate ó b ô LSB attenuate or obtuse obtuse, rarely cordate cordate, rarely obtuse obtuse, cordate or obtuse obtuse or cordate obtuse, oblique or cordate, obliqug b attenuate attenuate rarely attenuate LLE 31.3 36.7 46.6 36.r 39.6 37.9 53. I 36.3 (zo.¡-¡t.s) * (2s549.0) (26.047.Ðb (29.3-ss-o) (38.6-40.5) "b (26.344.7) (40.7-743)ú (27.849.0) LWI 12.5 23.8 52.r 25.6 23.7 24.9 26.8 23.3 * b (s.g-¡0.¿) (12.s-29.O) (33.3{6.8) "b (19.2-37.s) (21.6-26.Ð^b (r9.7-28.O) (10.941.2) (14.6-31.5) LSW dull dull shining b dull dull b dull dull b dull LEA concolorous concolorous concolorous concolorous concolorous concolorous concolorous concolorous ub LSM smooth smooth smooth smooth not smooth smooth smooth smooth ò b b ú LPS sessile sessile petiolate sessile petiolate sessile petiolate sessile LPL 0.0 "b 0.0 l 1.9 0.0 10.0 0.0 8.4 0.0 (6.6-20.Ð^b (9.4-10.8) "b (3.1-13.2) "b ó b b LPO opposite, rarely opposite or alternate altemate opposite or altemate altemate opposite alternate opposite or altemate ó alternate ó ô b LCO blue green grey green or blue dark green green, grey gleen or grey green green, dark green or blue green green, or rarely green green blue green blue green o ú ú ó SSH quate squafe round squarc round square or angular round square orangular b b ò b STE branching single branching single or branching branching single or branching branching single or branching SSM smootlr smooth smooth smooth not smooth'b smooth smooth smooth Continued Table 8.10 Continued E. socialîs x E. gíIlii E. gillii E. socialß x E. kruseana E. socialis x E. macrocarpa E. socialis x E. oldfieldü E. kruseana E- macrocarpa E. oldfieldíi Ð b LSH ovate lanceolate or ovate, ovate or lanceolate cordate or orbicula¡ ovate ovate ovate lanceolate rarely linear b LST acute or obtuse acute, rarely obtuse or mucronulate, acute, obtuse or acute, obtus€ acute or obtuse, rarely acute or mucfonulate ó ú mucronulate acuminate obtuse or acuminate mucronulate "b acuminate b T.sB obtuse, cordate or attenuate, obtuse or cordate, obtuse or obtuse or cordate cordate obtuse or attenuate obtuse, cordate or attenuate b b attenuate cordate attenuate attenuate LLE 32.2 27.4 35.4 23.7 40.3 50;t 32.3 42.3 b (26.3-36.1) (26.r-28.Ðb (16.747.4) (22.0-26.Db (37.143.4) (33.565.8) "b (16.s41.8) (38.045.1) LWI 25.2 16.0 23.6 24.2 31.2 33.7 21.2 t6.4 b b (9.7-32.9) (10.8-28.1) (7.9-3't.t) (22.t-26.Ðb (28.1-34.2) (19.3453) "b (tt.t-29.4) (lo.l-21.5) b ó b LSW dull dull, sometimes dull dull or glaucous glaucous very glaucous dull or glaucous dull glaucous b ô LEA concolorous concolorous concolomus discolorous concolorous concolorous or concolorous concolorous discolorous LSM smmth smooth smooth not smooth smoolh smooth smooth smooth b ù b LPS sessile or petiolate sessile sessile petiolaæ sessile sessile or petiolate sessile petiolate ó b LPL 0.5 0.0 0.0 0.9 0.0 3.3 0.0 4.7 (0.0-2.0) (0.7-1.5) (0.0-6.1) (2.56.1) "b b b LPO opposite or alternate opposite opposite or alternate opposite opposite opposite opposite or altermte altemate b o b b LCO gIeen, gley green or green dark green, blue green blue green $rey green green or grey green green, grey green, blue grey green, blue green o blue green or light green green or light green or green ó SSH round, square or roun4 angular or square angular square angular ú square or angular angular ó ó angulaf rarely angular Ú ù b STE single or branching branching single or branching branching single single single branching ò b ó b SSM smooth smooth smooth not smooth smooth smoolh smooth not smooth ó Continued Table 8.10 Continued E. socialis x E. orbifolia E. socialis x E. polyanthemos E. socialis x E. siderorylon E. socialis x E. steednanü E. orbifolia E. polyanthemos E. sideroryIon E. steedmanü LSH ovate orbicular, rarely ovate or orbicular orbicular " ovate lanceolate or ovate " orbicular or ovate lanceolaæ or ovate' b cordate b LST acute, mucronulate or truncate or acute or obtuse lruncate or acute acute, mucronulate or acute, sometimes acute, or mucronulate acute, mucronulate or b b obtuse mucronulate obtuse obtuse obtuse b LSB obtuse or cordate obtuse, cordate or cordate, obtuse or obtuse, cordate attenuate or cordate attenuate, rarely attenuate, obtuse or attenuate or obtuse b b attenuate b attenuate or acute obtuse cordaæ LLE 35.2 23.9 36.0 37.5 33.3 58.9 37.5 64.2 (19.447.9) (r7.o-293)b (r9.746.O) (31.047.1) "b (22.342.0) (43.6-76.Ðú (23.64s.O) (38.6-813) "b LWI 20.1 30.1 22.3 47.9 25.1 2r.7 31.5 t4.5 b b ( l3.l -31.6) (19.7-41.8) (t3.4-25.6) (3 r .368.6) "b (18.5-30.0) (9.9-333) "b (13.s47.0) (5.0-29.5) b b b LSW dull dull dull or glaucous dutl glaucous or dull dull dull or glaucous dull or shiny ó LEA concolorous concolorous concolorous concolorous concolorous discolorous concolorous concolorous LSM smooth smooth smooth smooth smooth smooth smooth smmth or not smooth ó ó ú LPS sessile or petiolate petiolate sessile or petiolate petiolate sessile petiolate "b sessile or petiolate petiolate LPL 0.2 7.O 0.1 14.6 0.0 5.0 2.33 5-4 b (0.0-r.0) (2.9-10.8) "b (0.04.5) (9.0-20.0) (2.0-8.7)'b (0.0-7.0) (0.5-13.4) "b b b b b LPC) opposite or alternate opposiæ opposite altemate opposiæ or alternate altemate opposite or altemate alternate ò o b LCO blue green, dark green blue green blue green, light green blue green blue green grey green blue green or blue green or green or green or green g€y green b b ú ù SSH square round square or angular round square or angular angular round round ù ó STE single single or branching single or branching branching single or branching branching, rarely branching branching'b single b ú ó SSM smooth not smoolh smoolh smooth not smooth smooth smooth not smooth Continued Table 8.10 Continued E. socialis x E. transcontinentalis E. socialis x E. websteriana E. socialis x E.youngiana E. ,ransconrtnentalis E. websteriana E. youngiana b b I.sH cordate, rarely ovate linear ovate, rarely cordate or orbicular ovate ovate or cordate b lanceolate ó b LST acute, rarely obtuse acuminate, acute truncate or obtuse obtuse or acute mucronulate, acute mucronulate or or obtuse or acuminate b acuminate b LSB obtuse or cordate obtuse obtuse or cordate obtuse, mucronulate obtuse or cordate obtuse or attenuate b or acuminate ó LLE 37.3 18.3 33.3 22.2 34.4 59.9 (23.7-sO.6) (t6.6-2r.Ðb (t7.747 .0) (13.8-27.5) "b (3.4-36.3) (33.0-72.Dù LWI 26.O 5.9 24.5 25.0 23.1 30.0 (13.9-30.0) (5.2:7 Ðh (10.5-36.4) (Ls.7-33.4rú (22.7-23.s) (15.845.6) .b ó LSW dull dull dull or glaucous dull dull or glaucous dull or shining LEA concolorous concolorous concolorous concolorous b concolorous concolorous LSM smmth smooth smooth smooth smooth smooth b b LPS sessile or petiolate sessile sessile petiolate sessile petiolate b LPL 0.3 0.0 b 0.0 3.0 0.0 9.9 (0.0-2.r) (1.24.7)b (5.7-t4.Ðù b LPO alærnaæ or opposite opposite opposite opposite, rarely opposite altemate ù alternate LCO blue green, green or blue green b light green, grey green dark green, blue green, blue green dark green, light green or blue green light green or green b rarely green ù SSH angular or round squa.e b angular or round round b angular angular s ô STE single or branching branching single or branching branching, rarely single or branching single b single b SSM smooth smooth smooth not smooth smooth smooth Correlation coefficientt : showing the fit 1r2¡ of the individual character vectors in the ordination space. Secl Bísectaria and Adnøtar¡a 8.4.2.3. 8.4.2.3. Summary of crosses The number of hybrids produced for each cross varied, from no hybrids from nine and eleven out of sixteen crosses for E. gillii and E. socialis, respectively, to 53 in the cross between E. gillii and E. oldfieldil (Table 8.3 and 8.4). The phylogenetic relationships between the species affected hybridisation success, with intraseries crosses producing more hybrid seedlings than interseries and intersectional crosses (Table 8.11). Observations of the morphology of these species indicated that a higher degree of hybridisation success was realised between species with similar flower sizes than between species with dissimilar flower sizes. Flowers from the female parents E. gillü and E. socialis, and pollen donors E. kruseana, E. transcontinentalis, E. oldfieldii, E. gardneri, E. eremophila and E. orbiþIia, are between 9 and 18 mm long. Crosses between these species produced the most hybrids. The flowers of E. polyanthemos, E. sideroxylon, E. pruinosa and E. websteriana are less than 8 mm long, and only the E. gillii x E. polyanthemos cross produced hybrids. Flowers from E. caesia and E. steedmanii are between 19 and 25 mm long, flowers from E. macrocarpa and E. youngiana are greater than 25 mm long and only four hybrids were produced from these crosses, all from E. socialis x E. steedmanii. 2t5 Sect. Bisectarìa and Adnafaria 8.4.2.3. Table 8.11. Number of hybrids produced in relation to phylogenetic placement by Pryor and Johnson (I97I). Female Male Number Total of number of hybrids seedlings measured Se,ct. Bisectaria Sect. Bisectaria Ser. Oleosae E. gillii Ser. Oleosae E. gillii 0 68 E. transcontinentalis 25 35 E. socialis 38 38 Ser. Macrocarpae E. orbiþlia I l0 E. websteriana 0 I E. caesia 0 6 E. macrocarpa 0 0 E, oldfieldii 53 54 E. youngiana 0 4 Ser. Kruseanae E. kruseana 38 38 Ser. Reduncae E. gardnerii 42 42 Ser. Occidentales E. eremophila 0 11 E. steedmanü 0 0 Sect. Adnataria Pruinosae E. pruinosa 0 0 Ser. Polyanthemae E. polyanthemos 6 6 Ser. Melliodorae E. siderorylon 0 I Sect. Bisectaria Se*t. Bisectaria Ser. Oleosae E. sociølis Ser. Oleosae E. socialis 0 t0 E. transcontinentalis 9 t2 E. gillii 4 5 Set. Macrocarpae E. orbiþIia 0 5 E. websteriana 0 18 E. caesia 0 6 E. macrocørpa 0 ) E. oldfieldü 0 10 E. youngiana 0 2 Ser. Kruseanae E. kruseana 3 8 Ser. Reduncae E. gardnerii 3 5 Ser. Occidentales E. eremophila 0 7 E. steedmnnii 4 9 Sect. Adnataria Ser. Pruinosae E. pruinosa 0 0 Ser. Polyanthemae E. polyanthemos 0 9 Ser. Melliodorae E. sideroxylon 0 3 216 Seef^ zndAdnatarìa 8.5. 8.5. Discussion Naturally occurring hybrids involving the species used in this study were reviewed by Griffin et aI. (1988), with twenty-five records listed. These consisted of one intersectional cross between E. kruseana (sect. Bisectaria ser. Kruseana) and E. ovularis (sect. Dumosae ser. Dundasianae), thnteen interseries crosses (twelve from sect. Adnataria) and eleven intraseries crosses, from ser. Macrocarpae and ser. Oleosae (sect. Bisectaria), and ser, Melliodorae and ser. Polyanthemae (sect. Adnataría). The listings for recorded manipulated or spontaneous crosses are fewer (six), with five between species from different sections e.g. E. caesia (sect. Bisectaria ser. Macrocarpae) and E. pulverulenta (sect. Maidenarid ser. Viminales). and one between species from different series in the same section. In this study, the flowers were emasculated but not bagged following pollination, as bagging resulted in high levels of drop of the small flowers. Some intraspecific seedlings were produced amongst the hybrid seedlings following interspecific hybridisation, either through faulty emasculation or via insect visits. The substantial numbers of hybrid seedlings produced indicates either that contamination was low, or that these species have a high propensity for interspecific hybridis ation. Three different trees were used as female parents for each of E. giltii and E. socialis, and over 3000 flowers pollinated, however, only one tree from each species produced viable seed. This may have been due to the age of the trees: all of the E. gillü trees and two of the E. socialis trees were under six years old at the time of pollination, and although each produced large numbers of flowers, most dropped after pollination. The trees may not have been mature enough physiologically to carry high seed loads, although they carried a small number of capsules from the previous year. The E. gillü tree that produced seed was under six years old, but was much larger than the other two used. The E. socialis tree that produced seed was 22 217 SecL Bísectaria andAdnalaría 8.5. years old, a large, mature specimen. The high seedling mortality observed in seedlings from crosses with E. gillii as the female parent may have been caused by lethal gene combinations or incompatible crosses, resulting in low seedling vigour and higher disease susceptibility. The program produced over 400 seedlings from the two female parent species crossed with the sixteen male parent species. Hierarchical and non-hierarchical analysis of all seedlings, based on seedling morphology, placed approximately 50Vo intermediately between their parents on both the dendrograms and the ordination plots. The remaining seedlings were positioned with the female parents and considered to be the product of self pollination. Crosses between species from the same series, E. gillü, E. socialis and E. transcontinentalis (Ser. Oleosae), produced a high proportion of hybrid seedlings, while crosses between E. gillii and E. socialis and species from the next closest series, ser. Macrocarpae, produced few. Most species selected from ser. Macrocarpae had large flowers, and this may have contributed to the low success rate, with the exception of E. gillii x E. oldfieldii, wherc the flowers were similar in size. Crosses between E. giUii and E. kruseana, from distantly related series, produced a high proportion of hybrids, as did the crosses E. giUii x E. gardneri, E. socialis x E. kruseana and E. socialis x E. gardneri, again this may be due to similar flower sizes. Six hybrid seedlings were produced from the intersectional cross between E. gillíi (sect. Bisectaria) and E. polyanthemos (sect. Adnataria), however the majority of intersectional pollinations failed, possibly due to the relativley large genetic distance between the species. The relatively low dissimilarities between the female parent and the seedlings, shown on the dendrograms, reflects probable short term inheritance of maternal characters. As the seedlings mature and exhibit adult morphology, further clarification of hybrid status will be possible. 218 Sect. Bisectarìa andAdnalar¡a 8.6. 8.6. Conclusion Of the 246 seedlings resulting from the interspecihc controlled pollinations with E. gillii as the female parent, 43 seedlings clustered with E. gillii, and there is a high probability they are the result of self pollination. The remaining 203 seedlings, from crosses between E. gillii and E. gardneri, E. kruseana, E. oldfieldii, E. orbifolia, E. polyanthemos, E. socialis and E transcontinentalis, clustered between the female and male parent clusters, which indicated that the seedlings are morphologically intermediate, and thus suggests that the seedlings are Fl hybrids. Of the 111 seedlings resulting from the interspecific controlled pollinations with E. socialis as the female parent, 88 seedlings clustered with the female parent and there is a high probability they are the result of self pollination. The remaining 23 seedlings, from crosses between E. socialis and E. gardnert, E gillii, E. kruseana, E. steedmanii and E. transcontinentalis, clustered with neither the male nor female parent, which indicated that the seedlings are morphologically intermediate, and thus suggests that the seedlings are Fl hybrids. 219 S'ect- Bìscatarín sinò. Adnataria E. siJJ¡i dendrosrans Egil I Egil 3 Egil a Esil Esil 4 EgiEcal 6 EgiEca5 10 EgiEca¿ 9 EgiEca6 11 EgiEca2 '1 EgiEca3 8 Ecael L2 Ecae2 13 Ecae3 14 Ecae5 16 Ecae¿ I5 ll tt 0.30¿1 0.6056 0.90?0 1.2085 Figure 8.1. Dendrogram of E. gillii x E. caesia seedlings and parent seedlings; E. gillii (Egil), E. caesia (Ecae) and E. gillii x E. caesia @giEca). Scale represents standardised Manhattan distance (Gower metric) based on variant seedling characters (Table 8.6) for dendrograms 8.1 to 8.13. EgiIl 1) EgEel 0 15) EgEe3 8) EgEe9 14) EgEeS 13 EgEe11 16 T Egi 13 3 Esi 15 5 EgEe5 10 EgEel 6 Egi 12 2 Egi 14 4 )_ EgEeT L2 rl EgEe6 11 )_ EgEe4 9 EgEe2 'l Eerel L7 Eere3 19 Eere2 18 Eere4 20 Eere5 2l 0.294 0.s83 1.16 Figure 8.2. Dendrogram of E. gillii x E. eremophila seedlings and parent seedlings; E. giUii (Egil), E. eremophila (Ecae) and E. gillii x E. eremophila (EgEe). Eqil1 1) EgiI3 3) Eqi15 5) I EgxEsil 6) r_l- Eq j.12 Esi 14 4t Esidl 7t Esid2 8)_-l- Esid3 Esid5 Esid4 I tl 63 o .464 0.693 2 0.92L6 1.150 Figure 8.3. Dendrogram of E. gillii x E. siderorylon seedlings and parent seedlings; E. giUii (Egil), E. sideroxylon (Esid) and E. gillii x E. sideroxylon (EgxEsi). 220 SecL Bìsectaría and.Adnafarìa E. s¡ilíi dendrosrams Esil1 1) Esil2 2') ¡si13 3) Þgit¿ ¿) EgxEvl 6) Egil5 5) Evebl Eveb¿ 10) Eveb5 11) Eveb2 8) Eseb3 I 0152lt 0.2442 0.¿?31rtt 0.7027 0.9310 1.160 Figure 8.4. Dendrogram of E. gillii x E. websteriana seedlings and parent seedlings; E. giUii (Egil), E. websteriana (Eweb) and E. gillii x E. websteriana (EgxBw). Egit 1 1 Esi 13 3 Egi 15 5 _t_ EgxEyol 5 EqxEyo2 7 rl_ Esil2 2 Esil¿ ¿ EgxEyo3 I EgxEyo4 9 Eyoul 10 EYou5 1¿ EYou2 Eyou3 tyou¿ I 0.22r90 ¿3 0.8 r.01 Figure 8.5. Dendrogram of E. gillii x E. youngiana seedlings and p¿uent seedlings; E. giUü (Egil), E. youngiana (Eyou) aîd E. gillii x E. youngiana (EgxByo). Egil1 1) Esil2 2t Esil4 ¿) EgEorl0 1s) Esil3 3) Esil5 EgEor3 8) EgEor¿ EgEor9 1¿) t_ EgEor5 10) _t EgEorS 11) rr- EgEorS 13) I EgEorl 6) EgEor? 72) _t_ Eggot2 'tl Eorbl 16) Eorb3 18) Eorb5 20t Eorb4 19) -T Eorb2 1'71 tt 0.0 264 0,523 o.'t82 1 1.0410 1.3 Figure 8.6. Dendrogram of E. gillii x E. orbifolia seedlings and parent seedlings; E. gillü (Egil), E. orbiþlia (Eorb) and E. gillii x E. orbifolia (EgEor). 221 S,cot Rico¡lnìn qndl A¡Inalnrìn E- øíIlìi dendrosrams Esill ( Egil2 ( Egil¿ ( Esil3 ( Esil.5 ( EgxEc6 ( 11) EgxEE9 ( L4 EgxEEl { EgxEE2 ( 1l EqxEt4 ( 9 EgxEt 3 ( I )_ EgxE t.5 ( 10 )-l- Eqx¡87 ( L2 EgxECl0 ( 15 )_ EgxEcB ( 13 EgxEÈ.11 ( 16 EgxE!26 ( 31 EgxEtl 2 ( T1 EgxEE2 { ( 29 ( EgxEÈ2 7 32 I EgxEc 1 7 ( EgxEt2l ( 26 EgxEE22 ( 2'l ìl EsxEÈ 3 1 ( 36 )-l EgxEt 1 6 ( 21 EgxEÈ18 ( 23 EsxEEl3 ( 18 EqxEÈ19 ( 24 EgxEE3 0 ( 35 EgxEE2 9 ( 34 EgxEE2 3 ( 28 EgxEr33 ( 38 EgxEEl 4 ( 19 EgxEÈ2 0 ( 25 EgxEEl5 ( EgxEt25 ( 30 EgxEt.3 { ( 39 EgxEt.28 ( 33 EgxEÈ3 5 ( ¿0 = EgxEt32 ( Etral ( Ebra2 ( 42 Etra5 ( EEra3 ( 43 EEra4 ( 44 / 0 0060 0. 1830 0 .3500 0.53?0 0.?1¿0 0.8910 Figure 8.7. Dendrogram of E. gillii x E. transcontinentalis seedlings and parent seedlings; E. giUii (Egil), E. transcontinentalis (Etra) and E. giIIü x E. transcontinentalis (EgxEt). 222 9,ool Risorl¡Ån qnll Adnntnrìn E- øìIlìí. dendrnqrems Esil1 1 E9il3 3 Esil5 5 EgEoll 6 EsiI2 2 Egil¡l EgEoI2 7 EgEo110 15 EgEol 2 6 31 EgEoL2 0 25 EgEoIl 5 20t _ EgEo132 37''_ EgEol3 s EgEol4 9 EgEol{ 6 51 EgEol? L2 EgEoI 5 10 EgEol 16 2L EgEo12? 32 EgEol3 I ¡13 EgEol¿3 48 EgEo151 56 EgEo13 1 36 EgEol 5¿ 59 I EqEoIl9 24 EgEoI3 6 ¿1 EgEo134 39 l EgEol3 7 t2 EgEo140 ¿5 EgEoI4S 53 I EgEol.5 3 58 EgEol 6 11 EgEoI I 13 EgEol 13 18 EgEo19 1{ EgEo13 3 38 EgEo145 50 EgEol2 ¿ 29 EgEol2 1 26 EgEo123 28 EgEo139 44 EgEol52 57 EgEol50 55 EgEol30 35) EgEol1¿ 19) EgEo11? 221 EgEol¡¡1 ¿6) EgEol2 I 33) EgEol¿7 52t I EgEoll 1 16) EgEoL22 21t T- EgEol 1 I 23t _t EgEol¿2 41t EgEo12 5 30) il EgEol 12 17) EgEo12 9 3¿) EgEol¿ 9 5¿) T EgEo135 ¿0) EqEol¿ 4 ¿9) Eoldl Eo1d2 61 Eold3 Eold4 Eold5 I I 0 .340't 0. 680 1.0204 1.360 L.7 Figure 8.8. Dendrogram of E. giUü x E. oldfieldli seedlings and parent seedlings; E. giUii (Egil), E. oldfieldi¡ (Eold) and E. gillü x E. oldfieldil (EgEol). 223 Secl Bisectarìn and.Adnatarìa E. eillü dendrosrans Egill 1 Esil3 3 _t Esi15 5 _t_l_ Esi12 Egil.¿ 4 r-l EgEga'7 r2 EqEga2l 26 EgEgå20 25 EgEga2S 33 EgEgal9 24 )_ EgEga23 28 )_ EgEgaS 13)_ EgEga2T EgEga29 3¿ EgEgall 16 EqEga3l 36) EgEgal 6 EgEga3 8)_ EgEga36 ¿1) I EgEgal3 EgEgalS lit I EgEga2¿ EgEga33 33ìl EgEgô4 9)_ EgEga9 1¿r I Egag¿1¿ rgi I EgEga6 11) _ EgEga22 2'jt I EgEga25 ¡ol I EgEga34 3e) | EgEga35 40) I EgEgal5 zol I EgEga30 EgEga32 EgEgal2 EgEqal6 EqEgal0 EgEga2 EgEga5 EqEga26 EgEgalT EgEga3S EgEga3? EgEqa¿2 EgEga39 EgEga¿0 EàEqa¿1 Egarl ¿8) Egar3 50) _t Egat2 1¡9)_ Egar4 s1) I Egar5 s2t I Itlt 0.000 50 2224 0.4443 0. 6662 0 . 8881 1.1 Figure 8.9. Dendrogram of E. gillü x E. gardn¿ri seedlings and parent seedlings; E. gillii (Egil), E. gardneri (Egar) and E. giIIü x E. gardneri (EgEga). 2A Seet- Biscatarìn sind. Adnøtøría E. s¡llü dendrosrams Esi l1 EsiI2 Esil3 Egil¿ -I Esi15 EgEkl +l E9Ek2 EgEk31 EsEk3 EgEk29 34 I EgEk5 (10 EgEk24 129 EqEk35 (40 T EgEk6 (11 EgEk12 {17 _r EgEk32 137 ll_ EgEk¿ (9 EsEkl? 122 _l EgEkS (13 _t EgEk2? 132 il EgEk30 (3s) EgEk33 (38) TI EgEkT lt2l EgEk26 ( 31) EgEk15 t2Lt EgEk11 (16) EgEk38 (431 EsEk15 120 EgEk19 124 EgEk3¿ (39 EgEk18 123 EgEk23 (28 EgEk20 125 I EgEk9 (14 )_ EgEk14 (19 EgEk21 126 ìt EgEk10 (15 )_ EgEk3 6 (41 )l EsEk13 (18 )_ EqEk22 127 )l EgEk25 (30 EgEk37 142 EgEk28 (33 Ekrul (4¿ )_ Ekru5 (¿8 )-l- Ekru3 146 rl Ekru2 (¿s Ekru{ 141 _t_ I 001 0 0 .286 0.5't2 1.1 1.430 Figure 8.10. Dendrogramof E. gilliixE. kruseand seedlings and parent seedlings; E. giUii (Egil), E. kruseanø (Ekru) and E. gillii x E. kruseana (EgEk). Egil 1 Esil2 Esi 13 Esi15 Es j.l4 EgEpol EgEpo3 EgEpo2 EgEpo5 EgEpo4 EgEpo6 Epol 1 I2 Epol2 13 Epol 3 !4 EpoL5 _l Epol 4 s)_ I 0,25'1 0 .493 0.9 Figure 8.11. Dendrogram of E. gillii x E. polyanthemos seedlings and parent seedlings; E. gillii (Egil), E. polyanthemos (Epol) and E. gillii x E. polyanthemos (EgBpo). 225 S.col Ríto¡ln¡ìn qnà Aànntnrín E oíIIü ¡lendrnorn¡ns Egttl 1 Egil3 3 )_ Esil5 5 )-l- a;gLr2 2 ) Egi11¡ 4 ) EgxEsl 5l_ EgxEsS 13)-l- EgxEs2 1l- I EgxEe3 s)_l I EgxEsl2 1r)_l_l_ I EgxEs9 1aì I I EgxEsl0 1si_T_l_l E xEs¿ 9 EqxEB25 30 Egxa,e2l 32 EgxEs29 EgxEs32 37 EgxEs 3 0 35)_ EgxEsl4 19 EgxEÊ37 42 EgxEEl5 20 EgxEsl6 2t EgxEEl? 22 ìr EgxEsl9 2!t EgxEs35 ¿0) EgxE621 261 lr EgxEs36 ¿1) EgxEs2 3 2et EgxEa26 31) EgxEB2S 33) EgxEa 3 3 38) EgxEs3l 36) lr EgxEB 5 10) EgxEB 6 11) EgxEBT L2l EgxEs13 18) EgxEsll 16) EgxEs 1 I 231 EgxEB20 251 _T_ EgxEs2¿ 29t I EgxEs3¿ 39) _tl EgxEE3S {3) _il_t_ EgxEs2 2 211 I E6oc1 4{) Eaoc3 ¿6) EEoc2 ¿5) Eaoc4 Eaoc5 tt 0 D .4253 0 .5661 0.707 Figure 8.12. Dendrogram of E. gillü x E. socialds seedlings and parent seedlings; E. giUii (Egil), E. socialis (Esoc) and E. gillii x E. socialis (EgxEs). 226 S,ect^ Bì.soatarìn c.nd Adnalaria E. süä dendrosrams Esill 1) Egils50 55) Egi1s15 20t Egl1s1¡7 52t Egile3? a2t EgilsST 62t Esi1s58 53) lr Esí1s10 1s) Egi1B17 22t Eqi1s19 2Ð -l EgiIs56 61) Tl_ EgiIB3l 36) EqilB¿ 0 ¿s) Egils{ 9 5¿ EgI lÊ5 11 EgilsS 13) Egi1s13 EgiIs2l 26)_ EgiIs2 6 31)_l EgiIê55 eol_l_ EgiIs33 38)_l BgiIB¿ I 53) ll EgiIs60 EgiIÊ63 :åìilll Egi13 3 Eqi 1s5 10 Egils1 6 Egils2 1 EgiI5 5 Esil2 2 Egils3 I Egi ls4 9 Egi 1s9 r4 Egil,s54 69 Egils2¡¡ 29 Egl 1611 16 Egil63 5 40 Egil67 t2 EgilBl6 2r Egilsl2 r7 EglIs3 6 ¿1 EsilE2 I 33 Egi Is14 19) Eg j.Is2 0 25t EgilB6l 66) Egi 1s3 0 3s) Egils¿2 471 Egils3 9 44t Egi1B62 671 Egilsl I 23t-L Egi1s52 5'7 Egils{3 ¿8 Egils23 28 Egi 1s54 59 Egi 1s3 2 31 l EgiIs2? Egils2 9 34 Egils53 58 Egils4 ¿ 49 Egi1s51 56 Egi 1s¿5 50 tsgl rs¿ ) 30 EgiIs¿ 1 46 Egils3 I ¿3 Egils5 9 6{ Egils22 2'l Esil4 r¡ Egil.s4 6 51 Esi 1s3 7 39 tl 00 0.¿ 0,6672 0.8886 1.110 Figure 8.13. Dendrogram of E. gillii x E. gillii seedlings and parent seedlings; E. gillii (Egil) and E. gillii x E. giUii (Egils). 227 Sccf o¡¡l Á¡fnnlaÅn F s¡Iliì ll¡fllS nr¡linqfinn nlofs 'i 0 Ê -1 -3 1 -3 -1 -2 NtJ"? !Ð$ a Ë. silht 1 E, caesla O Seedlings Figure 8.14. Multidimensional ordination of E. gillii x E. caesia seedlings and parents. a E.çiilii I E. eremophila O Seedlings 3 1 Øô 3 1 -3 -l -2 ÀaDs Axis -3 2 Figure 8.15. Multidimensional ordination of -E'. gillii x E. eremophlla seedlings and parents. Se.c.t- BìsectarÍa g'nd Adnatafia E. píllíí MDS ordination nlots a Egillti I E, siderorylon O Seedlings 3 t (, Øo J , -3 -1 -2 À¿DS¿*rb¿ .J Figure 8.16. Multidimensional ordination of E. gillii x E. sideroxylon seedlings and parents. o E. gillíi T E, websteriana A Seedlings .g{ Ø Ê -1 -3 -3 MDS Axis 2 Figure 8.17. Multidimensional ordination of E. gillii x E. websteriana seedlings and parents. Sect- Bìsectaña ønd Adnalarìø E. süì¡ MDS ordination olots o E. giUil I E, ynngiana O Seedlings 3 2 ä (t) 0 ô Ë -l a -3 -3 -2 -3 ìiDs ¡"ß2 Figure 8.18. Multidimensional ordination of E. gillii x E. youngianø seedlings and parents. a E. gtilü I E. gørdnerl O Seedlings 3 4 ô E -l t\ -1 ÀfDS4¡¿sz '3 Figure 8.19. Multidimensional ordinatiorl of E. gillii x E. gardnerl seedlings and parents. 230 S,p¡t- IlìscataÅa nnd Adnahrta E. pillü MDS ordination nlots o E. gillii T E, lotseana o Seedlinç 3 2 Ø E a -l -3 -3 MDSAxis I Figure 8.20. Multidimensional ordination of E. gillii x E. kruseana seedlings and parents. O E. gillii ¡ E. polyanthemos O Seedlings 3 Ø 0 z -l 2 -3 -3 À4DS 4r¡.¡ 2 Figure 8.21. Multidimensional ordination of E. gillii x E. polyanthemos seedlings and parents. 231 See.t- Bìsectarìa ¡nd Adnatoria E. síllìì MDS ordination nlots a E. g¡ilii I E, socialls ô Seedlings J il I 0 H -1 -3 n -J -t ¡oitl ìfe'g --t Figure S.22.Mritidimensional ordination of E. giUiixE. socialls seedlings and parents. a E. gillii ¡ E. oldfeldii o Seedlings J x 0 Øo E -J -3 t-I ÞJ]t2 -J .fes Figure 8.23. Multidimensional ordination of E. gillii x E. oldfieldü seedlings and parents. 232 Í¡ot. Ríso¿aarín snd Adnota¡ìa E. eíIlì¡ MDS ordination olots a E. gillii I E, orbiþlia ./i\ Seedlings 3 â. -3 Ìø84&b/ J Figure 8.24. Multidimensional ordination of E. gillii x E. orbiþlla seedlings and parents. o E. gíIlil ¡ E. ûanscontinentdlis o Seedlings 3 2 .Ðx A 2 -3 MDS -3 Ax¡.s 2 Figure 8.25, Multidimensional ordination of E. giltii x E. trqnscontinentalls seedlings and parents. 233 Secf. Bi.s¿cf¿rìa nnd Adnølaña E. eilli¡ MDS ordination nlots a E. gillit O Seedlings a ã ûI 0 -l -3 -1 ìaàs¿¡r;¿ -3 Fígure 8.26. Multidimensional ordination of E. gillii x E. gillii seedlings and pa^rents. 234 SecL Bìscctarìn anll AdnatarÍa E. socialis dendrosrams ESXECZ 1 EsxEcl 11 EsxEc4 3 E6xEc5 4 Esoc2 1 Esoc{ 9 EsxEc6 5 EsxEc3 2 Esoc3 I _t_ Esocl 6 E9oc5 10 Ecael I2 Ecae5 16 Ecae2 I Ecae3 14 Ecae¿ 15 .2501tttt 0.4926 0.?350 0.9175 t,220 Figure 8.27. Dendrogram of E. socialis x E. caesia seedlings and parent seedlings; E. socialís (Esoc), E. caesia (Ecae) and E. socialis x E. caesia (EsxEc). Scale represents standardised Manhattan distance (Gower metric) based on variant seedling cha¡acters (Table 8.9) for dendrograms 8.27 to 8.4I. Esocl 1) Esoc5 s) Esoc2 Esoc3 3) Esoc¿ ¿) EsxEe? L2t E6xEe3 8t EsxEe6 11) ÈEXEez 1l EsxEe5 10) EsxEe¿ 9) EsxEel 6) --t__ Eerel 13) Ee!e3 15) Eere2 14) Eere4 16 )_ Eere5 url 0053 o.22 o.614 0-89? 1.1 Figure 8.28. Dendrogram of E. socialis x E. eremophila seedlings and parent seedlings; E. socialis (Esoc), E. eremophila (Eerc) and E. socialis x E. eremophila (EsvBe). EsxÉn1 (1 ESXh2 l2 Esoc2 (4 Esoc3 (s _t_ Esoc 4 (6 l- Esoc 1 (3 Esoc5 l7 Emac I (8 Ànac 5 l12 Þnac3 ( 10 Þnac2 ( 9 Ênac4 ( 11 ) tt 0.016 0.1 0.3429 0.5060 0.569 0. 832 Figure 8.29. Dendrogram of E. socialis x E. macrocarpa seedlings and parent seedlings; E. socialis (Esoc), E. macro c arpa (Emac) and E. socialis x E. macrocarpa (EsxEm). 235 Sp.ot- Rì.scatnrín qndl A¡Inntnrìn E- socí¿,lìs dendrosrams EExEoll 1)_ EsxEol I al_l EsxEol 6 6)_ E6oc4 r¿l_l_ E6xEo 1 3 3) EsxEol.? 'tl_ EsxEo I 2 EsxEol9 9 EsxEol¿ 4 E9oc2 r2 Esoc3 13 EsxEol5 5 EsxEol0 10 Esocl 11 Eaoc5 15 Eo1d1 16 Eold2 17)_ Eold3 1sr I EoId4 rg i_T EoId5 20t _ I 0. 0195 mt0.2731 0.527A 0.7818 1.0359 L.2 Figure 8.30. Dendrogram of E. socialis x E. oldfieldi¡ seedlings and parent seedlings; E. socialis (Esoc), E. oldfieldi, (Eold) and E. socialis x E. oldfieldri (EsxEol). Esx¡or1 Esoc3 EsxEor3 Esoc4 _t_ EEoc2 I EÊxEor2 EsxEor¿ E6xEor5 s) Esocl 6) Esoc5 10) Eorbl 11) Eorb3 13) Eorb5 15) Tl- Eorb4 1¿) l------T Eorb2 12t TTIII 0. 0111 0.20].7 0. 3923 0 .5828 0 .1114 0.96¿ Figure 8.31. Dendrogram of E. socialis x E. orbifolia seedlings and parent seedÏngs; E. socialis (Esoc), E. orbiþlia (Eorb) and E. socialis x E. orbifoliø (EsxEor). EsxEsi 1 1 EsxEsi2 2 ESXEST I 3 Esoc2 5 Esoc3 6 Esoc4 'l Esoc 1 4 Esoc5 I Esidl 9 Esid3 11 Esid5 13 t_ Esid4 T2 Esid2 10 -t- 02 0.239 0.454 0.6 0.8 1.10 Figure 8.32. Dendrogram of E. socialis x E. sideroxylon seedlings and parent seedlings; E socialis (Esoc), E. sideroxylon (Esid) and E. socialis x E. sideroxylon (EsxEsi). 236 SecL Bísectarìn enrl Adnafnrìn E. socìnlis dendrosrams EExEpl 1 EsxEp3 3 t_ Esoc3 1a ) l_ Esoc2 11 )_l E6oc4 13 r-tl EsxEp¿ EsxEp5 5 E6xEp9 9 Esocl 10 EsxEp2 )_ Esoc5 L4 )_l_ EsxEp6 5 )_ EsxEÞ7 1 )-l EsxEpS 8) Epo11 15 Epol 2 Epol 3 Epol4 EÞo15 ttt 235 0 .4515 0 .6644 0 .8'7'12 Figure 8.33. Dendrogram of E. socialis x E. polyanthemos seedlings and p¿uent seedlings; E. socialis (Esoc), E. polyanthemos (Epol) and E. socialis x E. polyanthemos (EsxEp). EsxEwl 1) EExEw3 3) ESXEVII 11) EsxEìwl0 101 EsxEv2 2t ESXEWIð 18) E6xEv14 1¿) E6xEw15 15) EsxEvl6 16) EsxEw6 6) EsxEvlT 1?) Esoc2 201 Esoc4 22',| Esoc3 2Lt E6xEw9 9) EsxEwl3 13) EsxEw4 4t EsxEw5 s) EsxEwl2 L2l EsxEw? '1) EsxEwS 8) Esocl 19) Esoc 5 23) Ewebl 241 Ðweb4 2'1) Eweb5 28t Eweb2 25l Eweb3 2 6)_ I I lt 0.027t 0 .255? 0.¿84 o .7r28 0. 9414 1.1?00 Figure 8.34. Dendrogram of E. socialis x E. websteriana seedlings and parent seedlings; E. socialis (Esoc), E. websteriana (Eweb) and E. socialís x E. webstertana (EsxEw). EsxEyl 1) Esoc2 4) Esoc3 s) Esoc4 Þ, Esocl 3) Esoc5 1t EsxEy2 2t Eyoul 8) Eyou5 2't Eyou2 9) Eyou3 0) Eyou4 1) Tlt 0. 0546 0 .2rr5 0.368 0 0.6 0.83 Figure 8.35. Dendrogram of E. socialis x E. youngíana seedlings and parent seedlings; E socialis (Esoc), E. youngíareø (Eyou) aîd E. socialis x E. youngíana (EsxBy). 237 Ricoetnrìn qndl Ad.nntøria E- socínlìs dendroprams Esocl 1 EBoc 5 5 Esoc2 2 EBoc3 3 Esoc¿ ¡¡ EsxEga¿ 9 EsxEgal 6 I EsxEga2 7 EsxEga3 8 E6xEga5 10 Egarl 11 Egar2 l2 Egar¿ 1{ l-r- Egar3 13 Egar5 15 0.01 0.18 0.532 0 .703 0.8?5 Figure 8.36. Dendrogram of E. socialis x E. gardn¿ri seedlings and parent seedlings; E. socialis (Esoc), E. gardneri (Egar) and E. socialis x E. gardnerl (EsxEga). E6xEgil 1) Esi11 11 Eqi 13 13 )_ Eqi15 15 )l- Egi 12 L2 )_ Egi 1{ 14 )l- tssxtsglz 2 tssxEg15 5 EsxEgi3 3 )_ Esoc4 9 )-l EsxEgi4 4 1 Esoc3 8 E6oc1 Esoc 5 -t 0 2'13 0.50¿ 0.620 Figure 8.37. Dendrogram of E. socialis x E. gillií seedlings and parent seedlings; E. socialis (Esoc), E. giUü (Egil) and E. socialis x E. gillii (EsxEgi). EsxEkl 1 EsxEk3 3 EExEk2 2 EsxEk4 4 EsxEk6 6 EsxEk5 5 EsxEkT 't Esoc2 10 Esoc3 11 -T Esoc4 72 rl- Esocl 9 Esoc5 13 EsxEkB I Ekrul 14 Ek¡u5 18) Ekru3 16) Ekru2 15) Ekru4 1?) 0 .021 Figure 8.38. Dendrogram of E. socialis x E. kruseana seedlings and parent seedlings; E. socialis (Esoc), E. kruseana (Ekru) and E. socialis x E. kruseareø (EsxEk). 238 Secl Bisectarin and.Adnøtaria E. socíalís dendrosrams EsxE6 E1 1 Esoc¡l 13 EsxEsC8 8 EsxEsÈ9 9 _t_ Esoc2 11 Esoc3 L2 EsxEst'2 :T-l- EsxEst3 3 _t_ Esocl 10 E9oc5 1¿ ------r I EsxEsE{ r¡ EsxEsÈ.5 EsxEsE6 6 _T EsxEaET 1 t_l_ EsÈe1 15 EsEe5 19 EsÈe2 16 EsEe3 t1 EEÈe¿ I8 0.19 0.3?3 0 .90 Figure 8.39. Dendrogram of E. socialis x E. steedmanii seedlings and parent seedlings; E. socialis (Esoc), E. steedmanii (Este) and E. socialis x E. steedmanll @sxEst). EaxEEl 1) EsxE!2 2t EsxEÈ3 3) Eaoc2 14) E6oc¿ 16) Esoc3 1s) Esocl 13) Esoc5 L1l EsxE!6 6) EsxEtg 8) EsxEE? EsxEtl0 10) EsxEÈ12 L2t EsxEÈ9 EsxEÈ11 11) EsxEt¿ 4) EsxE!5 5) Etral 18) Etra2 19) Etra5 22t Etra3 201 Etra¿ 2Lt 0 0.161 0 0.?98 Figure 8.40. Dendrogram of E. socialis x E. transcontinentalis seedlings and parent seedlings; E. socialis @soc), E. transcontinentalis (Etra) and E. socialis x E. t rans c ontinent alis (EsxEt). ESXESI EsxEsS ESXESv Esoc2 t- Esoc3 Esoc4 EsxEs3 -l- ESXESIU EsxEs¿ EsxEs5 EsxEs? EsxEs6 Esocl Esoc5 EsxEs2 o,t{'t o.2 0.3 o .411 0 .58? Figure 8.41. Dendrogram of E. socialis x E. socialis seedlings and parent seedlings; E. socialis (Esoc), and E. socialis x E. socialis (EsxEs). 239 Seef- qn¡l Ádnnlntín. E^ soaÍalìs MIIS ordinntion nlots a E. socialis t E. caesia o Seedlings 3 Òâ -l 3 -3 hox,t, -3 Figure 8.42. Multidimensional ordination of E. socialis x E. caesia seedlings and parents. o E. socialis I E. eremophila o Seedlìngs 3 å q 3 1 g -3 -3 MDS A¡is 2 Figure 8.43. Multidimensional ordination of E. socialis x E. eremophila seedlings and parents, 240 Secf on¡l ÁÃnnlntîn E snrìnlfu MI)S nrdinnfion nlots o E. socialis I E. macrocarpø o Seedlings 0 A 2 Ê â -l MDS Axis 2 Figure 8.44. Multidimensional ordination of E. socialis x E. macrocarpa seedlings and parents. O E. socialis t E. oldfieldii O Seedlings a û 0 A -l t ÞJ}" -1 *"*0, -2 Figure 8.45. Multidimensional ordination of .8. socialis x E. oldfieldil seedlings and parents' 241 Sect^ qnd Áãnnlnrín E- soci¿,lís MDS ordination olots O E. soeialis I E, orbifolia O Seedlings n 4 â 2 -1 ilfDS¿xr¡¿ Figure 8.46. Multidimensional ordination of ,8. socìalis x E. orbiþlla seedlings and parents. a E. socialis I E. polyanthemos O Seedlings , ûô z -1 ùros Axis 2 Figure 8.47, Multidimensional ordination of .E'. socialis x E. polyanthemos seedlings and parents. 242 S,oot Ricoolorin qnd Á¡Innlnrìn E- s¿,eí.nlìs MDS nrdintfion nlofs o E. socialis I E. sideroxylon o Seedlings a O¡ 'A ,o I ô¡ -l -2 -l l"'1É? -2 }lfDS Figure 8.48. Multidimensional ordination of E. socialis x E. sideroxylon seedlings and parents. o E. socialis I E. websletiana Seedlings 2 * û 2 ÞJ06 -t t4Òs A*i" -2 2 Figure E.49. Multidimensional ordination of E. socialis x E. websteriana seedlings and parents. 243 ÍeeJ- lìíse,ctarÍa c.nd Adnataria E. socir,lís MDS ordination nlots O E. socíalis I E.youngiana O Seedlings ,9 a 2 1o -t o{ -1 > -2 MDSAxis I Figure 8.50. Multidimensional ordination of E. socialis x E. youngiana seedlings and parents. a E, socialis T E. gardneri o S€€dlings 2 'É 0 .t)â 0 -1 rvIDS 4¡rì ¡ Figure 8.51. Multidimensional ordination of E. socialis x E. gardneri seedlings and parents. 244 Sp¡f qn¡l .ÁÃnnlnsin H snaìnlìa MrlS nr¡linqfinn nlofs o E. sociatis I E, gillii (2 Seadlings 2 ä â 2 -2 -l l¿DS¿¡r.c ¿ Figure 8.52. Multidimensional ordination of E. socialis x E. gillii seedlings and parents. I E. htseana a E. socialis O Seedlings 1 û o -1 -t A¡risl MDS Figure 8.53. Multidimensional ordination of E. sociøl¡s x E. kruseana seedlings and parents. 245 S,cot Rìso¡lnrìn øn¡l Á¡Innln¡ìn F.- soe.ìalís MDS nrdinetion nlots O E. socialis I E. steedmanii O Seedlings t I â .Aâ 2 -l a 0 -l -l ,È*t 'p- -2 Figure 8,54. Multidimensional ordination of E. socialis x E. steedmanii seedlings and parents. a E. socialis I E, ilanscontinentalis O Seedlings ã Òo 2 0 l'' -l "B' MDS Axis 2 Figure 8.55. Multidimensional ordination of ^8. socialis x E. transcontinentalis seedlings and parents, 246 Seof- Risp¿l¿Åa qlnd Adnalaña E. soeløIis MDS ordinrtlon nlots O E, socioüs ê Soedlings ä û Ê .t 4 ll4D,S4¡"¿ Figure 8.56. Multidimensional ordination of E. socialis x E. socialis seedlings and paxents. 247 Postharvest 9.1. Chøpter Nine Postharvest vase life of four flowering Eucølyptus species 9.1. Abstract The effects of sucrose, citric acid and cold storage were assessed on flowering stems of Eucalyptus tetragona, E. youngiana, E. forestiana subsp. forrestiana and E stoatei. Continuous application of 0.5 to 5.0Vo sucrose in the vase solution reduced vase life of E. tetragona flowers, from >13 days for the reverse osmosis water control to <10 days; vase life of E. youngianawas not affected by sucrose. Pulsing with 0.5 to lÙVo sucrose, in conjunction with cold dry storage at 3oC for one to two weeks, had no effect on vase life of E. tetragona flowers, which had a subsequent vase life of 1l-12 days. Preliminary trials with E forrestiana subsp. forcestiana and E. stoatei indicate that pulsing with sucrose at 2.0 to 5.0Vo may be beneficial to vase life. Sucrose, at any concentration, pulsed or in holding solution, did not increase flower opening after harvest of E tetragona or E. youngiana. Citric acid had no effect on vase life of any species tested. Significant differences in vase life were found between plants within a species; the implications of this for future testing, selection, and breeding programs are discussed. 248 Postharvest 9.2. 9.2. Introduction Some species from the genus Eucalyptus are recognised on world floriculture markets as foliage filler crops, but little attention has been paid to the flowers and buds of eucalypts, which have potential as features in floral ¿urangements (Sedgley, 1998). There are a number of species that may prove suitable, and research is required into production, postharvest handling and varietal selection. The eucalypt flower is distinctive, as it has no visible sepals and petals when open. The stamens, often brightly coloured, are a significant component of the showy display of the flower, while the sepals and petals have evolved to become fused to form the cap, or operculum, which covers the flower buds. The cap is shed at anthesis, enabling the inflexed filaments to unfold radially and the anthers to dehisce, releasing pollen (Eldridge et a1.,1993). In some species the operculum may be coloured and attractive, as may the hypanthium. Inflorescences are either axillary or terminal, with one to many flowers. The lack of sepals and petals, the clustering of flowers in an inflorescence, and the progressive opening of flowers from the base to the apex of the shoot, will influence the response of the stem to postharvest treatments. Studies to date have focussed on the production and postharvest care of eucalypt foliage, indicating low levels of sucrose may be beneficial to vase life (Wirthensohn et aI., 1996; Jones et al., 1994), although levels of I07o caused leaf browning and damage (Jones et aI., 1994; Jones and Sedgley, 1993). Dry storage at cool temperatures (1-5'C) is possible for up to 30 days with no reduction in vase life, however, higher storage temperatures reduce vase life significantly (Wirthensohn et aI., 1996; Jones et al., 1994; Jones et aI., 1993; Forrest, l99l). Antimicrobial treatments have an effect on vase life, depending on the substance and plant material. Jones et al. (1993) concluded that vase life of E. gunnii was not increased by 249 Postharvest 9-2- five different antimicrobial compounds, whereas vase life of E. crenulata increased with the addition of 5 mg/L BCDMH (1-bromo-3-chloro-5,5-dimethylhydrantoin, l0 mg/L available chlorine) with no positive effect by 50 mgll. sodium dichloroisocyanuric acid (DICA), 250 mg[L benzalkonium chloride, 250 mg Hydroxy-quinoline-citrate (8HQC/L) or 50 mg[L n- alkyl dimethyl ethylbemyl ammonium chloride (Physan-2Ù) with water as the control. Sucrose pulsing (between 0.57o and 107o sucrose) for 24 hours has no significant effect on the vase life of a range of eucalypt foliage species (Wirthensohn et aI., 1996; Jones et al., 1993; Jones et al.1994). Research on flowers of similar Australian native species, such as Grevillea and Acacia, indicate that a 37o Sucrose holding solution may increase vase life of some Grevillea genotypes, but higher sucrose concentrations damage leaves (Ligawa et a1.,1997). Acacia cut flower stems require a flower opening solution immediately after harvest, containing Agral, aluminum sulphate and l7o sucrose, supplied as a cool pulse to enhance flower opening (Jones and Horlock, 1997). The pH of the holding solution should be low, about 3 to 4, and may reduce microbial activity (Halevy and Mayak, 1981). The pH is generally lowered by the addition of citric acid, which significantly increased the vase life of Acacia amoena flowering stems (Williamson and Milburn, 1995). The inclusion of citric acid in rehydration solutions for flowers such as Acacia, Protea and Helicona, amongst others, is recommended (Sacalis, 1993). There is considerable variation across species and genotypes, and the potential of hybrids and superior genotypes to significantly increase vase life is apparent (V/irthensohn et aI., 1996), highlighting the importance of selection and rigorous standardised testing of all suitable species. Selection of genotypes can be made on the basis of postharvest performance. Anecdotal evidence suggests that the flowers of eucalypts have physical characteristics that 250 Prstharvest 9-2- relate to different methods of pollination. One example is the secretion of nectff during anthesis, to encourage nectar feeding birds to the flower, which may not be desirable in a cut flower. The recording and study of such characteristics is important, and should be considered during selection at both species and genotype level. In contrast to eucalypt foliage production, where plants are kept juvenile to produce suitable material (Wirthensohn et aI., 1996), the production of Eucalyptus buds and flowers requires the plants reach adulthood as early as possible. Buds and flowers should also be terminal, with little or no shoot growth above the buds, and flowers should continue to open after harvest. A selection and crossing program was commenced in l99l at the University of Adelaide 'Wirthensohn (Ellis el aI., l99l; et al., 1999) and continues to identify and develop Eucalyptus species for cut flower and bud production, in addition to cut foliage. One species identified with potential for cut flower and bud production is E. tetragona (Plate 9.14). E. tetragona is a medium shrub with opposite grey leaves and square stems, covered in a thick layer of wax (Wirthensohn and Sedgley, 1996). The seed capsules, produced after flowering, are also well accepted on world cut flower markets. Flower buds appear approximately 6 weeks before the flower season (November to January). Buds are small and white, in umbels of 3 in every leaf axil, opening to a small cream flower (Brooker and Kleinig, 1990). The white stems and flowers of E. tetragona, in combination with the time of flowering, indicate the potential of this species for the Christmas market, and interest by the industry is apparent (Jones and Sedgley, 1993). Other species identified with potential for the cut flower market are E. youngiana (Plate 9.1B), E. forcestiana subsp. forrestiana (Plate 9.lC), E. forrestiarea subsp. dolichorhynchaBrooker, and E. stoatei (Plate 9.1D). E. youngianahas very large flowers (from yellow to deep red) during Spring and Summer, while E. forrestiarzø subsp. forcestiana, 251 Postharvest 9.3.1. { ¡nL' T Plate 9.1. Species used in postharvest trials. A.: E. tetragona;B: E. youngiana; C: E.forrestianct subsp.forrestiana;D: E' stoatei .Bat: 1cm. 252 Postharvest 9.3.1. E. forrestiana subsp. dolichorhyncha and E. stoatei have bright red, pendulous buds throughout the year. 'We conducted our study to establish the optimal postharvest holding conditions of E. tetragona, investigating continual holding in sucrose solutions, sucrose pulsing and cold storage to simulate transport to distant markets and their effect on flower opening and vase life. E. youngiana, was trialed with continual sucrose, while E. stoatei and E. fonestiana were also tested on a preliminary basis. 9.3. Materials and Methods 9.3.1. Plant material Eucalyptus tetragona material was harvested from six trees between 2.5 and 4.5 years of age in the Laidlaw Plantation. Plants were grown from seed of unknown provenance, purchased 'W.4. from Nindethana Seed Company, Plants were grown under conditions of summer irrigation and quarterly fertiliser applications. Material from E youngiana (six trees), E forrestiana subsp. forrestiana (one tree) and E stoatei (two trees) was harvested from 20-25 years old trees in the Monarto Woodland. Trees were grown from seed of unknown provenance, under natural conditions with no supplementary irrigation or fertilisers. 9.3.2. Harvest Stems, between 15-40 cm, were harvested from all species when the first l-2 flowers had opened. They were cut from the tree, left dry for up to two hours, and brought to the laboratory as quickly as possible. The lower leaves were removed, the stems recut (diagonal cut removing lower 10-20 mm of stem) and placed in RO water until allocation to treatments. 253 Postharvest 9.3.3. 9.3.3. Vase life of flowers and leaves The number of flowers on each stem was recorded at harvest, after pulsing and then daily as one of 4 stages: I - not open, 2 - operculum lift, 3 - flower fully open,4 - flower wilt or stamen drop. Vase life of flowers was considered terminated when more than 507o of the flowers were at stage 4. The 'percentage of flowers open' for each stem was calculated as the maximum number of flowers at stage 2 and 3 throughout the assessment period, divided by the total number of flowers on that stem. The general appearance and colour of the leaves was recorded daily, and vase life of leaves was considered terminated when more than 50Vo of leaves showed desiccation and browning. Fresh weight and water uptake of each stem were measured daily, allowing the calculation of the frst day of negative weight gain, indicating the onset of vascular blockage. Solutions were renewed daily as no biocide was used. 9.3.4. Experimental Treatments 9.3.4.1. Continual sucrose Stems of E. tetragona and E. youngiana were placed in solutions containing Reverse Osmosis (RO) water (pH 6 to 7), RO water + 0.05 glL citnc acid (CA) (AnalaR, BDH Chemicals, Australia) (pH 3.5), 0.57o sucrose (AR Bulk, AJAX Chemicals, Australia)+ RO water + CA (pH 3.5), 1.07o sucrose + RO water + CA (pH 3.5), 2.0Vo sucrose + RO water + CA (pH 3.5) or 5.0Vo sucrose + RO water + CA (pH 3.5). Vase life was assessed, at 22oC in a controlled temperature environment, with 12 hour day/night cycles under standard fluorescent lights (5.5 ¡rmol.m-2.s-l). 9.3.4.2. Sucrose pulsing and cold dry storage Stems of E. tetragona, E. forrestiana subsp. foruestiana and E. stoatei were pulsed in solutions containing RO water (pH 6 to 7), RO water + citric acid (CA) (pH 3.5), 0.5vo sucrose + RO water + CA (pH 3.5), 2.0Vo sucrose + RO water + CA (pH 3.5) or I0.0Vo 254 Prrstharvest 9-3-5- sucrose + RO water + CA (pH 3.5) for 24 hours at 22"C. After pulsing, the stems were placed in cardboard boxes that were lined with a layer of newspaper then a layer of plastic sheet (100 pm), and placed in cold, dry storage at 3oC for 0, 7 or 14 days. After pulsing and storage, the stems were recut (diagonal cut to remove bottom 10-20 mm), placed in RO water and kept at 22"C in a controlled temperature environment, with 12 hours day/night cycles under standard fluorescent lights (5.5 pmol.rn'.s-t¡, for vase life measurement. 9.3.5. Statistical Analysis The statistical designs (Randomised Complete Block Design) were: la) continual sucrose (E tetragona) - five plants (as replicates) with six sucrose treatments and three stems per treatment [5 x 6 x 3 = 90 stems] with stems per plant randomly assigned to sucrose treatments; lb) continual sucrose (8. youngiana) - six plants (as replicates) with five sucrose treatments and three stems per treatment [6 x5 x 3 = 90 stems] with stems per plant randomly assigned to sucrose treatments; and2) sucrose pulsing and cold storage (8. tetragona) - ttree storage periods for six plants (as replicates) with five sucrose treatments and three stems per treatment[3x6x5x3=270stems]withstemsperplantrandomlyallocatedtopulsesand storage periods. Trials with E forrestiana and E. stoatei were set up as design 2) with three storage treatments, five sucrose treatments and three stems per treatments, but only one and two plants per species were available. General Analysis of Variance (ANOVA) was used to determine the effect of the treatments on the criteria measured. Where no significant effect of treatment was found, analysis for the effect of plants was undertaken (One-way ANOVA). The data was tested using Genstat 5 Release 4.1. (Pc/Windows NT, 1997, Lawes Agricultural Trust, Rothamsted Experimental Station) with L.S.D. and Power Analysis used where appropriate (Appendix 1.3.). 255 Pmtharvest 9.4.1. 9.4. Results 9.4.1. Continual Sucrose Maximum vase life of E. tetragona flowers and leaves was 13 days and 17 days respectively, with no effect of citric acid (Table 9.1). Vase life was reduced with continual sucrose exposure but the effect was largely independent of concentration (Figure 9.1). Continual high sucrose (5.0Vo) also induced leaf margin browning, and hastened desiccation. No difference due to sucrose was evident in the percentage flowers opening after harvest (Figure 9.2) or time to weight loss after harvest (Figure 9.3). Differences between plants were evident (Table 9.2), with plant 2 exhibiting the longest vase life of flowers and leaves, 12 and 16 days respectively (Figure 9.4). The most flowers opened after harvest on plant 2 (Figure 9.5). Time to weight change is not a good indicator to predict life of stems from different plants, as plant 2 showed negative weight gain after three days, while Plant 3, with the next longest vase life, showed negative weight gain after hve days (Fig 9.6). Visual observations of the stem did not reveal any detrimental features of the species, with tittle nectar production, insignificant pollen release and limited stamen drop. The maximum vase life of flowers and leaves of E. youngiana was ll.1 and 13.5 days, respectively, with no effect of sucrose treatment or acidification of vase solution (Table 9.1., Figure 9.7). There was no effect of sucrose on the percentage flowers opening after harvest (Figure 9.8) or day of weight change after harvest (Figure 9.9). Further analysis indicated that significant differences between plants were evident (Table 9.2), with plant 2 having the longest vase life (Figure 9,10) and longest time to negative weight change (Figure 9.ll), but least percent flowers opening after harvest (Figure 9.13). Plants 3,4 and 5 had the shortest vase life and fastest day to weight change, but the highest percentage of flowers opening after harvest. Plant 6 showed similar vase life and 56.7Vo flowers open after harvest. Undesirable features recorded for E youngiana included the secretion of considerable quantities of nectar 256 Prxtharvest 9.4.1- from each flower during the fnst three days after opening, and the dropping of large quantities of pollen during the same period. Table 9.1. Effect of continual sucrose on vase life of flowers and leaves, flowers opening after harvest and time to negative weight change for Eucalyptus tetragona and E. youngiana stems. Vase life Flowers open Time to negative (days)Y after harvest weight change Flowers Leaves (vo) (days) E. tetragona control (pH 6-7) 13.3" r7.5^ 79.7u 3.2^ control + CA' (pH 3.5) ltJù 16.3" 80.7" 4.lu 0.57o sucrose + CA (pH 3.5) 10.2b" 13. lb 73.ru 3.gu 1.07o sucrose + CA (pH 3.5) 9.5b" I 1. lb" 66.3u 4.Ou 2.07o sucrose + CA (pH 3.5) 8.1" 10.2" 54.0u J.J^^A 5.07o sucrose + CA (pH 3.5) g.g' 10.1" 62.3u 3.gu E. youngiana control (pH 6-7) g.lu 12.7u 69.2^ 2.7^ control + CA" (pH 3.5) g.3u 12.4^ 48.4u J.J^^ø 0.57o sucrose + CA (pH 3.5) g.4u 12.2u 77.9u 4.0u 1.07o sucrose + CA (pH 3.5) 11.1u 13.5u 53.9" 3.3u 2.07o sucrose + CA (pH 3.5) r0.7u 12.7^ 56.4^ 3.gu v Data are averages of three stems from each of five plants (as replicates) with six treatments for E. tetragona, or averages of three stems each of six plants (as replicates) with five treatments for E. youngiana. Different superscripts indicate significant differences in a column at P<0.05. ' CA = citric acid Anova tables Appendix 1.3 257 Prntharvest 9-4-l- Table 9.2. Effect of plant on vase life of flowers and leaves, flowers opening after harvest and time to negative weight change in continual sucrose trials for E. tetragona and E. youngiana stems. Vase life Flowers open Time to negative (days)Y after ha¡vest weight change Flowers l.eaves (7o) (days) E. tetragona Plant I 9.7' 1 1.9b" 40.9" 2.go Plant 2 12.3u 15.7u 92.0" 3.4b Plant 3 I 1.9'b 14.6', 6g.3b 5.4^ Plant 4 10.lb" 13.6',b 90.0u 4.6u Plant 5 8.4" 9.6" 54.4h" 2.3" E. younginna Plant I g.5o' 15.5" 41.50" 2.70 Plant 2 11.4" 15.3" 35.2b" 5.7^ Plant 3 7.4" 10.3" 70.0'b 2.7b Plant 4 g.4ub" 10.9' 92.5u 2.gb Plant 5 g.2ub" 10.9" 79.8u 2.gb Plant 6 tt.2ù 13.4b 56.7ù" 3.7b Y Data are averages of 18 stems from each of five plants (as replicates) for E. tetragona, 01 avetzges of 18 stems each of six plants (as replicates) for E. youngiana. Different superscripts indicate significant differences in a column at P<0.05. Anova tables Appendix 1.3 258 20 l8 Iflowers 16 lle¿ves l4 12 €to I 6 4 2 0 control CA + conkol 0.50á suc¡ose 1.00lo suorose 2.00lo suorose 5 0olo suo¡ose Figure 9.1. Vase life (day$ of E. tetragona tvtth sucrose 100 90 80 T T T 70 60 oÉ o 50 8. 40 30 20 10 0 control CA + oontrol 0.50lo sucrose 1.00lo suo¡ose 2.00á suo¡ose 5.00lo sUcrose Figure 9.2. Percent flowers opening of E. tetragona with sucrose 6 5 T T 4 l_ I .s>1 I T , I 0 control CA * conhol 0 5olo suorose 1.0oá sucrose 2,070 sucrosç 5.07o sucrose Figure 9.3, Time to negative weight change of E. teilagona with sucrose l8 lflowers 16 lleaves t4 t2 10 .8 8 6 4 2 0 I t 3 4 5 plant Figure 9.4. Vase life (days) of E. þtrqgona plants with sucrose 120 100 80 il o o 60 T & 40 20 n I ) -t 4 5 plant Figure 9.5. Percent flowers openingof,E tetragona plants with sucrose 'l 6 5 å ¡ 4 I .do J T ù Å 2 I I I I I I I 0 I I I ) .J 4 5 plant Figure 9.6. Time to negative weight change of E. tetragona plants with sucrose t6 lflowers lleaves 14 T l2 ¡ J. ¡ ï l0 I II tã'8 6 4 ) 0 control CA*control 0 .50á sucrose LOolo suoorse 2.00lo suorose treatment Figure 9.7. Vase life (day$ of E. youngiand \¡/ith sucrose 100 90 T 80 T '10 60 õ o 50 8. 40 30 20 10 0 conhol CA*oontrol 0.50lo sucrose 1.07o sucorse 2 0oZ sucrose heatnent Figure. 9.8. Percent flowers opening of E. youngiana with sucrose 5 4.5 T 4 T T 3.5 T 3 I- - €z.s -J- I - ) I I I I 1.5 I I II 1 I T I I I I I I 0.5 I I 0 conhol CA+cont¡ol 0.50á suorose L07o sucorse 2.00á sucrose teatment Figure 9.9. Time to negative weight change of E. youngiana with sucrose Iflowers 18 lleaves l6 14 t2 10 € 6 4 2 0 J 4 5 6 I 2 plant Figure 9.10. Vase life (days) of E. youngiana plarús with sucrose 120 100 T 80 ï Êo T o 60 o o. T 40 20 0 2 J 4 5 6 plant Figure 9.11. Percent flowers open of ¿. youngians plants withsucrose 7 6 T 5 t 4 €d T T 3 I ) tl tl I 0 I I I I ,) 1 3 4 5 6 plant Figure 9.12. Time to negative weight change of E. youngiana plants with sucrose Postharvest 9-4-2- 9.4.2. Sucrose pulsing and cold storagg There was no effect of citric acid or sucrose pulsing on any of the criteria measured on stems of E. tetragona that were subsequently held in water (Table 9.3). Vase life ranged from 10.5 to 11.6 days for flowers and 15.8 to 17.2 days for leaves (Figure 9.13). Percentage of flowers opening after harvest was between 83 and 897o (Figure 9.14), and time to negative weight change was 9.9 to 10.3 days (Figure 9.15). There was no significant interaction between sucrose pulsing and cold storage on any of the criteria measured. Cold storage for 7 days extended the vase life of E. tetragona leaves adding 1.3 days, induced 7.2Vo more flowers to open after harvest and extended time to negative weight change by 1.4 days. Cold storage for 14 days, when compared to no storage, added 0.6 days vase life, induced 4.4Vo more flowers to open and extended time to negative weight change by 1.6 days. Further analysis determined there to be differences between plants of E. tetragona in all criteria measured (Table 9.4). Plant 2 had the longest vase life of flowers and leaves (Figure 9.16), 14 and 20 days respectively, and longest time to negative weight change, 1l days (Figure 9.17), but less than 787o flowers opening after harvest (Figure 9. I S). The other plants were variable in their response, but worthy of note are plants 6 and 8, each with over 96Vo flowers opening after harvest, but with less than eleven days vase life of flowers. Pulsing with sucro se at l07o induced marginal blueing on the leaves; however, this disappeared within 24 hours of the stem being placed in RO water. 263 Postharvest 9.4.2. Table 9.3. Effect of sucrose pulsing and cold storage on vase life of flowers and leaves, flowers opening after harvest and time to negative weight change for Eucalyptus tetragona stems. Vase life Flowers open Time to negative (days)Y after ha¡vest weight change Flowers Leaves (vo) (days) sucrose control (pH 6-7) 10.5" 15.9" 87.7^ g.g^ control + CA'(pH 3.5) 11.6u t7.t^ 99.3" 10.3u 0.57o sucrose + CA (pH 3.5) 11.6u 16.6u 93.0u 10.0" 2.07o sucrose + CA (pH 3.5) 11.0" 17.2^ 97.0" 10.1u 10.07o sucrose + CA (pH 3.5) ll.4u 16.6u 93.9" lo.2^ cold storage 0 days at 3oC rt.4^ 16.0u g23b 9.10 7 days at 3'C tt.4u n3b gg.5u 10.5u 14 days at 3oC 10.9u 16.6"b 86.7ú 10.7" Y Data are averages of three stems each of six plants (as replications) with five sucrose x three cold heatments. Different superscripts indicate significant differences in a column at p<0.05. ' CA = citric acid Anova tables Appendix 1.3 Table 9.4. Effect of plant on vase life of flowers and leaves, flowers opening after harvest and time to negative weight change in sucrose pulsing trial for Eucalyptus tetragona stems. Vase life Flowers open Time to negative (days)' after harvest weight change Flowers læaves 7o (days) Plant2 14.0u 20.0^ 79.70 11.0" Plant 5 11.3b 15.7" 793b 10.0b' Plant 6 9.0" 13.5d 96.gu 9.7b" Plant 7 l l,3b L6.6" 793b g.gb" Plant 8 10.4b lg.2b 97.gu 10.6'b Plant 9 Í.4b 15.9" g3.gb 9.2" z Data a¡e averages of 18 stems each of six plants (as replications). Different superscripts indicate signihcant differences in a column at p<0.05. Anova tables Appendix 1.3 264 4 Frl Frl oo 0a days 6' days É Pe¡cent E t {{æææco00þ\o\o\o I o @ 6caoNSO\æON5O\ (D ÔNèÔæON5AæO -È¡.) p O N o\ æ ¡.) p yt !r' è .(^) conbol oontol @H 6- oontol @H 6- È @H6- oE i) 7) aÞ¡ t) () @ (D tD o ô' (Ð o o- 0a contol + CAz È Þ oontrol f CAz {(¡ oontrol+ CAz Ø (pH 3 5) Ø (pH 3.5) (pH 3 s) (D o oHl Þ(D (D{ F: @ (\ oFà 0 57o srrcrose * 0.50% sucrose 0,502 sucrose * C) !t: cA (pH 3.5) + CA (pH 3.s) ñ cA (pH 3.s) â¡ (! 0a (D È o 06 ÞÞ, € 2.00lo suorose 2.00/o sr¡crose * ì=l 2-0olo sucrose * ID (\ cA (pH 3.s) + cA (pH 3.5) CA(pH 3.5) 3 C)ä o ñ Ø tlØ cJt Þ 10,0olo sucrose o 10.0% sucrose I0.0olo sr¡crose Ø Ê- € + cA (pH 3-s) (l + CA (pH 3.5) c) + cA (pH 3.5) Þ o Þ- Ø À Ø o cJ o o I o ê¡ Ø è ùa o 0 days at 3oC @ 0 days at 3'C o 0 days at 3oC A¡ ot êr Þ- @ c) (Ì Þ. Ø 7 days at 3"C 7 days at 3oC 7 days at 3'C ot 0a(! TI 14 days at 3"C 14 days at 3"C 14 davs at 3"C gE2; 25 lFlowers ILeaves 20 15 € l0 5 0 I ) 3 4 5 6 plant Figure 9.16. Vase life (dayÐ of E. tetragolrq plants with sucrôse pulsing and cold storage 120 I 100 1 80 T 1 o o 60 o 40 20 0 I ) -t 4 5 6 plant Figure 9.17. Percent flowers open of E tetragonq plants with sucrose pulsing and cold storage t4 12 I å T 10 - I I I I - >\ d 6 I I I 4 I I I ) I I I 0 I I 1 ) 3 4 5 6 plant Figure 9. 1 8 . Time to negative weight change of E. tutragona plants with sucrose pulsing and cold storage Postharvest 9-5. 9.4.3. Observations of other species As only one plant was available of E. forrestiareø subsp. forrestiana. and only two plants for E. stoatei, these results can be used only to indicate potential effects of sucrose pulsing and cold storage on vase life. Sucrose pulsing with 2.0 Vo and 5.0Vo extended vase life to 12.8 and 12.1 days, respectively, compared to 10.3 days in RO water. No interaction between pulsing and cold dry storage was observed, and the latter did not affect vase life for E. stoatei, but there was a possible effect of sucrose pulsing on vase life, with a vase life of 18.6 days for those stems pulsed with 5Vo sucrose compared ro 12.9 days in RO water and 13.3 days in RO water + citric acid. Cold storage for 7 days increased vase life by three days. Overall, these two species show acceptable vase life characters, and warrant further testing. 9.5. Discussion The four flowering species studied varied in their response to postharvest treatments. Continual sucrose reduced vase life of E. tetragonaby three to five days, compared to the RO water control, but this was not concentration dependent. The exogenously supplied sucrose had deleterious effects, such as browning and desiccation of leaves, at levels of 5.0Vo. These results are in contrast to the pulsing trials, where sucrose pulsing, with concentrations as high as 107o, had no effect on vase life and did not induce leaf browning. This difference could be due to the constant uptake of sucrose in the continual sucrose, resulting in a constantly high level within the plant, compared to the pulse, where a high concentration is taken up once only and thereafter only water is taken up. The high concentration, as a continuous supply, may do more damage on a cellular level, similar to the effect on rose leaves (Markart and Harper, 1995) than the single uptake of a high concentration. Neither continual sucrose nor sucrose pulsing increased the percentage of flowers open after harvest or cold storage, and had no effect on time to weight change. These results were reflected in the continual 267 9.5. sucrose trials with E. youngiana. Tnals with E forrestiana subsp. forrestiana and E. stoatei indicate that there may be a positive effect of sucrose pulsing in prolonging vase life, but these results are only preliminary and further testing is required. Sucrose in solution, at varying concentrations, is used to prolong vase life and enhance flower opening of some flowering species. The presence of sucrose enables the plant to replace depleted endogenous carbohydrates, utilised during and after storage (Halevy and Mayak, 1981). It improves the water balance of the stem by triggering the closure of stomata resulting in a reduction of water loss; the sugar also accumulates in the flowers, increasing their osmotic concentration, and improving their ability to absorb water and maintain turgidity (Halevy and Mayak, 1981). However, sucrose does not benefit all species, in some cases the presence of sucrose is deleterious to the quality of the stem and overall vase life. The flower is not the only part of the flower to be considered the leaves are just as important in assessing vase life. In many cases the concentration of sugars and other substances used in solutions for 'pulsing' 'bud and opening' of flowers is determined by the sensitivity of the foliage to these ingredients and not by the optimal effect on flower development and longevity (Halevy and Mayak 1979)' Markart and Harper (1995) found that the amount and speed of crisping of leaves of roses was positively related to the amount of sucrose in the preservative solution. Jones et (7993) al. found that sucrose increased the vase life of immature cut foliage stems of Eucalyptus species, and commented that exogenous sucrose may have caused an improvement in the development of immature leaf buds and maintenance of turgor in leaves. Solution uptake rates were not affected by sucrose concentration (data not shown). There is considerable difference in time to weight change between the two trials, for E tetragona between three and four days for continual sucrose and nine and ten days for sucrose pulsing. This cannot be attributed to sucrose treatment, as each trial included controls of RO water and 268 Postharvest 9.5. RO water with citric acid, both of which reflected the time to weight change of the other treatments. Both trials were conducted on material collected in mid December of consecutive years, negating the possible effect of time of year. It is possible that the preharvest condition of the plants affected this, and raises questions regarding the influence of production methods on vase life. The addition of citric acid, or similar acid, to the holding solutions may increase longevity by reducing microbial population levels, or by increasing the flow of water through the stem (Halevy and Mayak, 1981). In these trials, acidification of the water by addition of citric acid had no discernible effect on vase life. Macroscopic observation of the solution did not reveal any evidence of increased microbial activity in the RO water (pH 6-7) compared to the acidified solutions. Water was replaced daily to reduce microbial activity, however microbial growth was apparent on the stems of E. youngiana, E. forrestiarca subsp. forrestiana and E. stoatei, with no correlation to sucrose treatment or solution pH level. There is no obvious stem wax on any of these three species. At no stage, even at the end of the trial, was there evidence of microbial growth on the stem of E. tetragona. This may be caused by the high levels of wax on the surface of the stem. The precise physiological significance of stem wax is not known, but could be similar to that of epicuticular wax, which has been reported to act as a water repellent, in plant pathogen resistance and as an insect deterrent (Wirthensohn and Sedgley, 1996). The vase life for E. tetragona flowers stored at 3oC for 7 or 14 days was the same as that for flowers with no storage at all, indicating that this length of cold storage is not detrimental to vase life. Although statistically significant differences were recorded for each of the cold storage periods for the criteria of vase life of leaves, percent flowers opening after harvest and time to negative weight change, the limiting factor was the life of the flowers. The 269 Postharvest 9-5. preliminary trials for E. forrestiand subsp. fotestiana and E. stoatei indicated some effect of sucrose pulsing and cold storage on vase life, with higher concentrations of sucrose extending vase life by several says, and 7 day cold dry storage also extending vase life by several days. Cold temperatures slow the metabolic processes of the stem, enabling flowering material of some plants to be stored for some time with no reduction in vase life. The stem should be harvested at an optimal stage for storage - one that will allow flowers to continue to develop afterwards. Some flowers, such as rose, gladioli and iris, are picked at the bud stage, while others, such as carnation, chrysanthemum, and gerbera, do not develop once picked, and thus are picked at a later stage (Goszczyflska & Rudnicki l98S). Observations of the four species studied in these trials indicated that the flowers will continue to open up to seven days after harvest or storage, but only if they were sufficiently developed. The majority of E forrestiana subsp. forrestiana and E. stoatei flowers did not open after harvest or storage. Both E. tetragona and E. youngiana exhibited considerable differences between plants. For example, E. tetragona plant 2 was identical in both trials and showed the longest vase life in both of them. Plant 5 was identical in both trials, and in both trials showed medium vase life of flowers. Visual observations of these plants showed very similar morphology, with minor variation in flower size and number of flowers per stem. The six plants tested for E youngiana showed variation in vase life of flowers, from seven to eleven days. The most noticeable variation was shown in the percentage of flowers opening after harvest, where values ranged from 35 to 82Vo. V/ith the exception of plant 1, those plants with the longest vase life had the lowest number of flowers opening after harvest, and the greatest time to negative weight change, indicating a possible positive correlation between flower opening and rate of senescence. The flowers of E. youngiana are very large, possibly 270 Pmtharvest 9.5. exacting a heavy toll on the physiological reserves of the cut stem when open and releasing nectar, resulting in faster senescence, as opposed to those stems whose flowers stay closed and static. It was determined that while pre-harvest health of the plant and time of harvest may have an effect on subsequent vase life, genotype may have a greater effect. Improvement of postharvest longevity is possible through testing a range of genotypes and selection of those with a long vase life for further development and breeding. It is likely that vase life is influenced by a combination of a number of heritable components, as was found in gerbera (Wernett et al., 1996). Eucalypt breeding and selection to date has focussed on phenotypic quantitative and qualitative traits associated with forestry production, but the technique could be used to breed and select for genotypes with superior floral attributes and postharvest life. The reproductive cycle of eucalypts is common to all species, but varies in length and time of year, between and within species. The cycle consists of six stages: inflorescence formation, formation of inflorescence initials (macroscopic appearance), development of flower buds from pin to cylindrical to plump, operculum shed and flowering, capsule development and seed dissemination. This cycle may start when the tree is one year old, but commonly commences in the third or fourth year after germination (Eldridge et al., 1993). The cycle may take less than 12 months, from emergence of inflorescence initials to maturation of seed, to five years, depending on species, location and season (Florence, 1996). Trees may have buds, flowers and capsules at all stages on the tree at one time, so a knowledge of this cycle is important in production systems to ensure a harvestable crop each year. Inflorescences consist of a number of buds grouped in umbels, with the number of buds in an umbel varying from l, 3,5,7 or 11, depending on species. The opening, or operculum shedding, of each individual flower in an umbel may take over seven days, and the spread of flowering on a tree may take from one to ten months. Once the operculum is shed and the 271 Postharvest 9.6. stamens fully reflexed, it may take between two and fourteen days for the stigma to become receptive to pollen, so in most cases an individual flower may be in 'bloom' for over two weeks. There have been no published reports specifically relating to the effect of pollination on flower life of eucalypts, although a study by Savva et al. (1988) recognised that treatments preventing pollination appear to delay anther senescence for E. urnigera.In contrast, Sturt's Desert Pea, once pollinated, begins to senesce, and is completely perished in three days. This has necessitated extreme care in postharvest handling procedures and the possible development of pollen sterile varieties (Kirby, 1996). The lack of this kind of information for eucalypts highlights the need for a better understanding of the reproductive biology and its effects on postharvest vase life, handling and treatments. 9.6. Conclusion The results for E. tetragona indicate enormous potential for this species as a cut bud and flower crop. The species has a long vase life, and even after cold storage, the flowers continue to open and develop normally, with no detrimental features such as copious nectar production or stamen drop. There is variation within the species, allowing for selection and breeding of superior genotypes to improve on the current vase life, as well as for selection of different leaf and flower sizes. Trials with E youngiana indicate a suitable vase life of flowers, but problems such as nectar production and stamen drop detract from the commercial potential of the species. There is variation within the species, and this should be explored to select those with smaller flowers, less nectar production and a longer vase life. Preliminary data for E. forrestiana subsp. forrestiana and E. stoatei indicate that both cold storage and sucrose pulsing may increase vase life. However, the percentage of flowers opening after harvest was very low in both cases, indicating that more studies on timing of harvest and optimal bud opening treatments are required to assess the optimal postharvest vase life and 272 Postharvest 9.6. requirements of these species. 273 General Discussion Chapter 10 General Discussion The project aimed to improve species from the Eucalyptus genus for floriculture and for garden or pot plants, by addressing three aspects of plant improvement: species and genotype selection; interspecific hybridisation and hybrid identification; and post harvest physiology and treatments. Within most eucalypt species, plants exhibit variation in morphology; with differences in the size, shape and colour of buds (operculum and hypanthium), flowers (stamens), leaves, stems and fruit. Such variability is an asset to breeders, as there is an increased chance for improvement, but is not desirable in commercial plantations, as it leads to lack of consistent supply. Eucalyptus plants suitable for ornamental horticulture (floriculture and amenity planting) have certain morphological features, principally large, colourful, ornate buds and flowers. Leaves, stems and fruit are of secondary importance, but must complement the flowers and buds. Plants from selected species are assessed in trials, to facilitate comparisons of growth rate, flowering and pest resistance. From these trials, plants exhibiting fast growth, exceptional flowers and high levels of disease resistance, are considered superior and suitable for further assessment. Further assessment requires quantification of the response of each plant to cultivation techniques such as pruning, postharvest treatments and clonal propagation. Plants identified with desired characters can be used in breeding programs. 274 General Discussion Of the thirty Eucalyptus species studied in this work, 17 have potential for development, primarily for floriculture. These are E. caesia, E. erythrocorys, E. erythronema, E. forrestiana, E. gillii, E. kruseana, E. Iesouefii, E. macrocarpa, E. orbiþlia, E. oldfieldii, E. pruinosa, E. pyriþrmis, E. stricklandii, E. stoatei, E. tetragona, E. websteriana and E. youngiana. An additional23 species with potential for development were identified but not studied. These are E. burracoppinensis, E. campaspe, E. clelandíi, E. cocinna, E. coronata, E. corrugata, E. crucis, E. drummondii, E. eudesmoides, E. ewartina, E. kingsmillii, E. lane- poolei, E.leptopoda, E. megacarpa, E. nutang E. orymitra, E. pachyphylla, E. pimpiniana, E. preissiana, E. rameliana, E. rhodantha, E. synandra, and E. tetraptera. All species listed have medium to large ornate buds and colourful or contrasting flowers. Selection at genotype level facilitates the identification of plants exhibiting superior characters, in the areas of flowering, plant form and plant health. Twenty plants from nine species were subjected to selection at this level through growth trials, where rates of growth and development were determined and compared, both within and between species. Seventeen plants from E. fonestiana subsp. dolichorhyncha, E. lesouefii and E. tetragona were selected for further assessment for plant response to cultivation, clonal propagation and postharvest testing. The majority of remaining genotypes have some desirable features and should be included in intra or interspecific breeding programs. The use of manipulated hybridisation to generate hybrids with superior characters to either parent is a common and reliable technique in ornamental plant breeding, as well as in eucalypt breeding for forestry. Interspecific hybridisation has been adapted as the method of choice for ornamental eucalypts, as the majority of major cut flower lines have hybrid pedigree, one of the most spectacular examples is the vast range of new cultivars which resulted from new methods to hybridise orchids. Hybrid plants, generated through controlled pollination, can be 275 General Discr¡ssion identified quickly and parental status confirmed through measurement and analysis of seedling morphological characters and DNA fingerprints. Those plants with intermediate characters can be identified as early as three months after germination, enabling the culling of those with limited hybrid value. The possibility exists that those plants identified as hybrids will exhibit superior floral characters to either parent under cultivation conditions. This opens the way to quick assessment of the hybrids for floral characters that will lead on to development as ornamentals. The use of DNA fingerprinting to clarify parentage, as demonstrated in chapter 5 and noted in chapter 7, will enable each unique hybrid to be defined, opening the way to registration as a cultiva¡. The study of the putative natural hybrid, E. 'Urrbrae Gem', used adult and seedling morphology, as well as DNA fingerprinting, to clarify parentage, with all three methods identifying the same species as the male parent. The ability to generate hybrids through controlled pollination was investigated in three studies involving a number of different Eucalyptas species. The first study aimed to reproduce E. 'Urrbrae Gem' by crossing the known female parent species with both possible male parents; the second crossed three closely related species with similar morphology; while the third crossed 16 species from different series within the genus, with differing floral morphology. The results of these trials suggested that while taxonomic relatedness was an important consideration, as some species were simply too distant to hybridise successfully, a further factor to be taken into account when developing pollination matrices was the comparative size of the flowers of both male and female species. Species with similar sized flowers hybridised more successfully than those which differed. This problem has been noted in other studies with Eucalyplus, however, it may be overcome with alterations to the pollination technique, including physical manipulation of the style to reduce style length. 2t6 General Discussion While E. 'Urrbrae Gem' could be commercialised now with the development of appropriate clonal propagation techniques, the seedlings generated in this study must be grown to the flowering stage for evaluation. In the future, the development of molecular ma¡kers, for characters such as flower colour, disease tolerance or waxiness, could accelerate the selection process by identification of potential superior genotypes at the seedling stage. This could be achieved by either bulked segregant analysis (Michelmore, et aI., 1991) or by mapping of the genome. Maps of some eucalypts species have been developed already (Brondani and Grattapaglia, 1999; Bundock and Vaillancourt, 1999; Shepherd et al., 1999), and it may be possible to adapt these maps for use with ornamental species. Once a superior genotype or hybrid has been identified, determination of the response of the genotype to postharvest treatments, cultivation techniques, such as pruning, and to clonal propagation must be undertaken. Observations of stems and flowers after harvest, and their response to various treatments, will facilitate the development of optimal treatments for maximum vase life and postharvest flower opening. The addition of sucrose to vase solutions and cold storage were treatments that extend vase life of a number of different plants, however, their effect on Eucalyprus buds and flowers was not known. By assessing the response of flowering stems of different plants to a variety of sucrose concentrations, the role of such additives to postharvest vase life can be determined. This study found that sucrose is not necessary to prolong vase life of some species, nor to enhance flower opening after harvest, while for others it may have a slightly beneficial effect. Cold storage does not have a detrimental effect on vase life, and may in fact enhance flower opening with some species. The undertaking of this trial over two consecutive years on some of the same plants from a species has shown that the pre-harvest condition and genotype of the plant may have a greater effect on vase life than any 277 Generel Discrrssinn postharvest treatment. Investigation of a greater number of species is necessary prior to development of a standard procedure for all flowering eucalypt stems. Trail consignments of stems to distant markets will test the postharvest procedures and also consumer acceptability of new lines. Va¡ious cultivation practices are used in production of cut flower stems of eucalypts, and depend on the climate of the growing area, with practices such as spacing, supplementary irrigation and weed control varying with location. The effect of pruning and harvesting on the future cropping potential of plants is unknown and requires investigation. Eucalyptus material available for commercial plantations is grown from seed sourced from natural and cultivated populations, with no control over the parentage. The outcrossing nature of the genus means that seedlings from such populations are variable, often lacking the superior qualities of the source tree. The variability leads to problems with management of commercial plantations, and ultimately to problems with the continual supply of quality product. The supply of quality product is foremost in the minds of producers, and species or cultivars that produce poorly will be quickly discarded. The solution to the problem of variability is the development of viable methods of clonal propagation. Clonal propagation is widely used for ornamental plants, and successful protocols have been established for some Eucalyptus species desired for forestry. The exploration of these protocols for use on ornamental eucalypts, and the development of quick, cost-effective methods for their propagation, is vital to the ongoing task of producing ornamental eucalypts. The careful selection and thorough testing of both natural genotypes and a¡tificially generated hybrids is vital for the development of Eucalyptus as ornamentals. 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Zietkiewicz,8., Rafalski, A. and Labuda, D. (1994). Genome fingerprinting by Simple Sequence Repeat (SSR) - Anchored Polymerase Chain Reaction Amplification. Genomics 20,176-183. 300 Annendix 1.1. Appendix 1.L Anova tables for section 4.4.1. (Table 4.1.) Final úee height General Analysis of variance - Variate: final height Source of variation d.f. s.s. m.s. v.r. F pr. block stratum 4 3276279 819070 3.85 block.*Units* sfratum species 9 48382609 5375845 25.26 <0.001 Residual 170(16) 36172871 2t2782 Total 183(16) 85507764 Tree height Erowth rate General Analysis of variance - Variate: growth rate Source ofvariation d.f. s.s. m.s. v.r. F pr. block stratum 4 3311.8 827.9 3.6s block.*Units* stratum species 9 40787.7 4532.0 19.99 <0.001 Residual 170(16) 38547.1 226.7 Total 183(16) 80814.8 Final tree trunk diameter General Analysis of variance - Variate: hnal diameter Source of variation d.f. S.S, m,s. v.r. F pr. block stratum 4 1343.7 335.9 2.55 block.*Units* s[atum species 9 10009.0 tt2.t 8.46 <0.001 Residual 170(16) 22357.4 r31.5 Total 183(16) 33173.8 Tree trunk erowth rate General Analysis of variance - Va¡iate: growth rate Source ofvariation d.f. s.s. m.s, v.r. F pr. block stratum 4 1.3098 0.3275 2.27 block.*Units* stratum species 9 10.5888 1.t765 8.16 <0.001 Residual 170(16) 24.5r78 0.r4/'2 Total 183(16) 35.8191 Note: Each of the analyses showed minimal differences between plants within species, and high species level differences meant that no fi.uther information could be gathered by including another level in the analysis. 301 Aonendix 1.2. Appendix 1.2 Anova tables for section 7.3.3 (Tables 7 .4,7 .5,7 .6 andT .7). All species cannot be analysed due to insufficient replication for all crosses (only one female for E. macrocarpa). E. macrocarpa as female parent species - only one female plant so no analysis possible E. pyriformis as female parent species Mean number of seeds per flower pollinated General Analysis of variance - Va¡iate: mean number of seeds Source of variation d.f. s.s. m.s. v.r, F pr. female sEatum 4 786.12 t96.53 7.80 female.*Units* stratum male 3 383.76 r27.92 5.07 0.017 Residual t2 302.54 25.2r Total t9 1472.42 Mean weight of seeds per flower pollinated General Analysis of variance - Variate: mean seed weight Source ofvariation d.f. s.s. m.s. v.r. F pr. female sftatum 4 0.353E-05 0.8838-06 7.71 female. *Units* stratum male 3 0.700E-07 0.233F-07 0.20 0.892 Residual 12 0.138E-05 0.115E-06 Total 19 0.498E-05 Percent seeds germinated General Analysis of variance - Variate: percent seed germinated Source ofvariation d.f. s.s. m.s. v.r. Fpr female sratum 4 3386.3 846.6 2.60 female. *Units* stratum male 3 892.9 297.6 0.91 0.463 Residual 12 3908.4 325.7 Total 19 8187.6 E. youngiana as female parent species Mean number of seeds per flower pollinated General Analysis of variance - Variate: mean number of seeds Source ofvariation d.f. s.s. m.s, v.r, F pr. female stratum 3 871.2 290.4 1.03 female. *Units* stratum male 3 2633.3 877.8 3.10 0.082 Residual 9 2548.8 283.2 Total 15 60s3.3 302 Mean weight of seeds per flower pollinated General Analysis of variance - Variate: mean seed weight Source of variation d.f. s.s. m.s. v.r. F pr. male stratum 3 0.608E-07 0.203E-06 0.09 male.*Unitsx stratum Female 3 0.6088-07 0.162E-05 7.57 0.008 Residual 9 0.193E-07 o.2t5E-07 Total 15 0.6878-05 Percent seeds germinated General Analysis of variance - Va¡iate: percent seed germinated Source ofvariation d.f. s.s. m.s. v.r, F Pr. male stratum 3 3285.6 t095.2 2.07 male.*Units* statum female 3 4553.1 1517.7 2.87 0.096 Residual 9 4766.4 529.6 Total 15 12605.1 303 Aooendix 1.3. Appendix L.3 Anova tables for section 9.4. (Tables9.1,9.2,9.3 and9.4) E. youngiana: effect of treatment on Flower vase life General Analysis of variance - Variate: days Source ofvariation d.f. S.S. m.s. v.r. Fpr Plant stratum 5 r79.t22 3s.824 2.40 Plant.sucrose stratum sucrose 4 t32.067 33.0r7 2.21 0.104 Residual 20 298.6n 14.930 t.97 Plant.sucrose.steml stratum 60 454.667 7.578 Total 89 106/.456 Leaf vase life General Analysis of variance - Variate: days Source ofvariation d.f. S. S, m.s. v,r. F pr. Plant stratum 5 4t4.767 82.953 15.99 Plant.sucrose stratum sucrose 4 18.400 4.600 0.89 0.490 Residual 20 r03.733 5.1 87 r.67 Plant. sucrose.stem 1 sfratum ó0 186.000 3.100 Total 89 722.9N Percent flowers open after harvest General Analysis of variance - Variate: days Source of variation d.f. S.S, m.s. v.r. F pr Plant stratum 5 29417 5883 2.57 Plant.sucrose stratum sucrose 4 10236 2559 1 l2 0.377 Residual 20 45868 2293 1 18 Plant.sucrose.stem I sFatum 60 TT6137 r936 Total 89 201658 Time to negative weight change General Analysis of variance - Variate: days Source of variation d.f. S.S. m,s, v.r F pr. Plant sfratum 5 108.89 21.6t8 8.47 Plant.sucrose stratum sucrose 4 18.844 4.7tl 1.85 0.160 Residual 20 5r.022 2.551 0.80 Plant.sucrose.steml stratum 60 192.000 3.200 Total 89 369.956 304 Äooendix 1.3. E. tetragona; effect of treatment on Flower vase life General Analysis of variance - Variate: days Source of variation d.f. s.s. m.s. v.r. Fpr Plant shatum 5 108.89 2r.6t8 8.47 Plant.sucrose stratum sucrose 4 18.844 4.7r1 1.85 0.160 Residual 20 5t.022 2.55r 0.80 Plant.sucrose.steml sfratum 60 192.000 3.200 Total 89 369.9s6 Leaf vase life General Analysis of variance - Variate: days Source ofvariation d.f. s.s. m.s. v.r. Fpr Plant stratum 5 414.767 82.953 15.99 Plant.sucrose stratum sucrose 4 18.400 4.600 0.89 0.490 Residual 20 r03.733 5.187 1.67 Plant.sucrose.stem 1 stratum 60 186.000 3.100 Total 89 722.900 Percent flowers open after harvest General Analysis of variance - Variate: days Source of variation d.f. s.s. m.s. v.r. F pr. Plant stratum 5 294r7 5883 2.57 Plant.sucrose stratum sucrose 4 t0236 2s59 r.t2 0.377 Residual 20 45868 2293 1.18 Plant. sucrose. stem I stratum 60 rr6t37 t936 Total 89 201658 Time to negative weight change General Analysis of variance - Variate: days Source of variation d.f. S.S. m.s. v.r F pr. Plant shatum 5 108.89 2r.618 8.47 Plant.sucrose stratum sucrose 4 18.844 4.711 1.85 0.160 Residual 20 51.022 2.551 0.80 Plant.sucrose.stem 1 stratum 60 192.000 3.200 Total 89 369.9s6 30s Aonendix 1.3. E. tetragona: effect of plant on Flower vase life One-Way Analysis of variance - Variate: days Source ofvariation d.f. S.S. m.s. v.r. F pr. Plant 4 234.71 58.68 5.61 <0.001 Residual 85 889.78 10.47 Total 89 t124.49 I-eaf vase life One-Way Analysis of variance - Variate: days Source ofvariation d.f. S.S, m.s. v.r. F pr. Plant 5 4t2.71 103. l8 7.17 <0.001 Residual 85 1223.tt 14.39 Total 89 1635.82 Percent flowers open after harvest One-Way Analysis of variance - Variate: days Source ofvariation d.f. S.S, m.s. v.r. F pr. Plant 5 36209.5 9052.4 to.& <0.001 Residual 85 72296.8 850.6 Total 89 108506.4 Time to negative weight change One-Way Analysis of variance - Variate: days Source ofvariation d.f. s.s. m.s. v.r F pr. Plant 5 t14.489 28.622 18.01 <0.001 Residual 85 135.1 1 1 1.509 Total 89 249.600 E. youngiana; effect of plant on Flower vase life One-Way Analysis of variance - Variate: days Source ofvariation d.f. s.s. m.s. v.r F pr. Plant 5 r79.12 35.82 3.40 0.008 Residual 84 885.33 r0.54 Total 89 to6/'.46 Leaf vase life One-Way Analysis of variance - Variate: days Source ofvariation d.f. s.s. m.s. v.r. F pr. Plant 5 4r4.767 82.953 22.61 <0.001 Residual 84 308.133 3.668 Total 89 7229æ 306 Annendix 1.3- Percent flowers open after ha¡vest One-Way Analysis of variance - Variate: days Source ofvariation d.f. s.s. m.s. v.r F pr. Plant 5 294r7 5883 2.87 0.019 Residual 84 r7224r 2050 Total 89 201658 Time to negative weight change One-Way Analysis of variance - Variate: days Source of variation d.tì S.S. m.s. v.r. F pr. Plant 5 108.089 2r.618 6.93 <0.001 Residual 84 26t.867 3.117 Total 89 369.956 E. tetragona: effect of treatment on Flower vase life General Analysis of variance - Variate: days Source ofvariation d.f. s.s. m,s. v.r. F pr. Plant stratum 5 597.707 rr9.54t t2.40 Plant.*Units* stratum Sucrose 4 42.126 r0.531 1.09 0.361 Cold 2 17.474 8.737 0.91 0.405 Sucrose.Cold 8 34,452 4.306 0.45 0.892 Residual 250 2409.459 9.638 Total 269 3IOI.2L9 Leaf vase life General Analysis of variance - Variate: days Source of variation d.f. S.S. m.s. v.r. Fpr Plant stratum 5 rt23.896 224.779 24.48 Plant.*Units* stratum Sucrose 4 70.237 17.559 r.91 0.109 Cold 2 74.O07 37.004 4.03 0,019 Sucrose.Cold 8 44.030 5.504 0.60 0.778 Residual 250 229s.104 9.180 Total 269 3607.2'74 Percent flowers open after harvest General Analysis of variance - Variate: days Source ofvariation d.f. s,s. m,s. v.r. Fpr Plant stratum 5 t't715.2 3543.0 13.84 Plant.xUnits* sftatum Sucrose 4 1538.4 384.6 1.50 0.202 Cold 2 2363.6 1181.8 4.62 0.011 Sucrose.Cold 8 25s3.9 3r9.2 1.25 0.272 Residual 250 63978.4 255.9 Total 269 88149.6 307 Annendix 1.3- Time to negative weight change after harvest General Analysis of variance - Variate: days Source ofvariation d.f. S.S. m,s. v.r. F pr. Plant shatum 5 90.033 18.007 3.91 Plant.*Units* stratum Sucrose 4 6.681 r.670 0.36 0.835 Cold 2 r34.467 67.233 14.59 <0.001 Sucrose.Cold 8 23.830 2.979 0.65 0.738 Residual 250 rr52.356 4.609 Total 269 1407.367 E. tetragona : effect of plant on Flower vase life One-Way Analysis of variance - Variate: days Source ofvariation d.f. s.s. m.s. v.r. F pr. Plant 5 597.707 119.541 r2.6t <0.001 Residual 264 2503.5tr 9.483 Total 269 3r0r.2r9 Iæaf vase life One-Way Analysis of variance - Variate: days Source ofvariation d.f. s.s, m.s. v.r. F pr. Plant 5 1123.896 224.779 23.90 <0.001 Residual 2@ 2483.378 9.407 Total 269 3607.274 Percent flowers open after harvest One-Way Analysis of variance - Variate: days Source ofvariation d.f. s.s. m.s. v.r. F pr. Plant 5 r7715.2 3543.0 13.28 <0.001 Residual 264 7M34.4 266.8 Total 269 88149.6 Time to negative weight change One-Way Analysis of variance - Variate: days Source of variation d.f. S.S. m.s. v.r F pr. Plant 5 90.033 18.007 3.61 0.004 Residual 264 r3t7.333 4.990 Total 269 1407.367 308 Annandiv I 1 2 I I I l*' ILE LSB I tpttCO I |f,Q rsr 0 L LSW -2 -2 0 2 M DS Axis I I I I I tsH t, rþ$or p s LPI I I ILE ts8 -2 0 2 MDS Ax¡s I I I I I sF ttPot"" I L____ LIE tsE -2 0 MDS Ax¡s 2 Figure 8.57. Principal canonical correlation graphs of E. gillü x E. caesiø seedlings and parents. Characters in italics and underlined not significantly correlated to MDS ordinations (Table 8.7). 309 Annendix 2-1 - ILE LPS LCO tsM srE LP! 9SM t9H tsT L tsb lE^ -2 -1 0 MDS Aris I I I I ."L I lE^ rV[br lPs tPl I tsM tPo 0 [email protected] J- s9M tat LLE -2 0 MDS Axis I tsa LEA LSH LW I LPS LST .2 LPL ã 0 tæ slE SSM LLE -z -2 0 M DS A¡is 2 Figure 8.58. Principal canonical conelation graphs of E. gillii x E. eremophila seedlings and parents. 31.0 Á.nncndiw î 1 LCO fst I .2 LSH LPS r A I ssr tw I rbs I I I 0 MDS Axis I I I I ffil* IL LFS I !P! I l___ o LWI lco la", .1" LsH I I I I 0 MDS A¡ls I I I I.ú ü ."I l:r .2 I 0 L :ê tg? LSB t6 -l 0 MDSAlis2 Figure 8.59. Principal canonical correlation graphs of E. gillii x E. gardn¿ri seedlings and parents. Characters in italics and underlined not significantly correlated to MDS ordinations (Table 8.7). 31r Ännandiv 2 1 Ê.t-E L5H L L!E LS8 0 MDS A¡li ¡ râå ,2 tsH 0 L ¡I sl ttE -2 0 MDS Axts I tsE L sa SIÉ LSI tw I ffE 0 M DS Ax¡s 2 Figure 8.60. Principal canonical correlation graphs of E. giUü x E. gillü seedlings and parents. Characters in italics and underlined not significantly correlated to MDS ordinations (Table 8.7). 312 Ä nnanrliv I 1 I I I qw r tEþ I !PS ItEl ssü t88 lE^ .? 0 L tco tst -l 0 MDS Axis I ILE LCO I lsH 1*, L LPL tPrÊ ¡ lst SsM LS8 1 0 MDS Axis I ttE LCO tsH '? +- o rþIw tPs rEA tst ssM tsa -2 .2 0 MDS A¡is 2 Figure 8.61. Principal canonical correlation graphs of E. gillii x E. kruseqn4 seedlings and parents. 313 A nnen¡f iv I 1 2 LSB LSH SH 0 L LOO TPS LPO SIE rPt ss$ L5l tLE 0 2 MDS Axis I LSH tPs tPo stE ssM LPLtsr 3SH L ê LIE LCO -2 -2 0 M DS ,{x¡s I SH LPS LPO LSI SfE SSM LPT ss .2 0 L o rlE LS6 -l MDS Ax¡¡ 2 Figure 8.62. Principal canonical correlation graphs of .8. gillii x E. oldfieldli seedlings and parents. 314 Annendix 2.1. sfE tPs ssM .2 .+. L6H LSI tsB tco ttE -2 0 MDS Axis I 'r LSE ssM I I LCO IE LSI -2 0 MDS Ar¡s I I I I tsH LSS '*l rPs I ssMtPr .2 I _ JE_ L lco IE lst .I 0 M DS Arls 2 Figure 8.63. Principal canonical correlation graphs of E gillií x E. orbifolia seedlings and parents. 315 Annendix 2-1- LIE ts8 t3H lco rPt L rFs tst -l -2 -t 0 M DS Aris I LP5 l5l tco lPl !PO L tÊs tsH l*' aaa -2 -l 0 MDS Axis I tPs tst LCO tgB LS LLE -2 0 MDS Ax¡s 2 Figure 8.64. Principal canonical correlation graphs of E. giUii x E. polyanthemos seedlings and parents. 316 Aonendix 2.1- 2 a 0 LLE L -t 0 MDs Axls I SI LtE TEÀ TPS tPo ssx LCO o _ l_ _rsH I I rw¡ rpl I38 rrtI I I -2 -l 0 MDS A¡¡s I sl E rtE tE^ tPo sslH LCO I lsH 0 L I .",1 t""f"I ¡ I 0 M DS Ax¡s 2 Figure 8.65. Principal canonical correlation graphs of E. gillii x E. sideroryIon seedlings and parents. 5L7 Ànnanâiv I 1 tPo l6f tco .2 I 0 L tsH I ttE saH rsF i SIE -l I I I -2 -2 -l 0 MDS Aris I 2 I I I rsþ !sa 3ë5r .9 0 ô slE ILE 0 MDS Ax¡s I LAB LSH ssH LST 0 srE ! LLE 0 MDS A¡¡s 2 Figure 8.66. Principal canonical correlation graphs of E. gillü x E. socialls seedlings and parents. 318 Annendix 2.1. .2 LSt ssH __ _ _ _rc_q L o ttE LEð SH -2 2 2 M DS Arls I ItE 0 L.Er i" L ô - lco - -. -. tsB -2 0 2 M DS Aris I ssH ILE .2 SH ô tco !s8 -t 0 MDSAris2 Figure 8.67. Principal canonical correlation graphs of E. giIIü x E. transcontinentalis seedlings and parents. 319 Ànnen¡liw ? 1 I I I N, I LÞo I .2 I 0 _l__ ê I ItË LPI .jf '"8{' '"" 0 MDS Axis I TgI LEÁ5t& rtÊõ r ¡s 0 ô : sfE LLE -2 0 M DS Ari6 I tsl LEA lPs ssM L.SH .9 0 L o tco I sff LLE I I I i -2 0 M DS Axis 2 Figure 8.68. Principal canonical correlation graphs of E. gillii x E. websteriana seedlings and parents. Characters in italics and underlined not significantly correlated to MDS ordinations (Table 8.7). 320 Annendix 2-l- .¡rrco L3P I Ipo tat .t E l-at ô 1.", : LôH ILE lPl I I I I I .2 0 2 M DS Axls I I I I LrE LPL LPq LE^ LSW ssH sfE 0 M DS Ax¡s I *fÈ tE9 o !sB ttèw ssH STE 0 MDSA¡is2 Figure 8.69. Principal canonical correlation graphs of E. gihii x E. youngiana seedlings and parents. 3Zt Annendix 2.2. t6 ILE slE LFOLbI6H tså !sr LPS L-- o -l 0 2 M DS Aris I LCO LPS srE L$s¡ LSW : LsHLw I ls8 TLE i LPO LST -2 -2 0 M DS Axis I I I I Lcl LPS STÊ sbll 0 : LSB o ---Lsl4---l-----ktll LLE Lsr LPo -l 0 M DS A¡is 2 Figure 8.70. Principal canonical correlation graphs of E. socialis x E. caesia seedlings and parents. 322 Annendix 2-2- ) Its r SSM LSM S5H I SIE TPL LLE I 0 L o tPs LCO taa .2 0 MDS Aris I tsw Le! LPS LCO ssx sTE tPo ¡"sS LsB LTE d 0 L LW I 151 .l -7 0 M DS Axis I LPS IPL LTE slE fsM LCO ssH ssM tsB .2 L o tsl 0 M DS Ax¡s 2 Figure 8.71. Principal canonical correlation graphs of E. socialis x E. eremophíIa seedlings and parents. 323 Annendix 2^2^ I I I I LFs LPL LPA¡ t" L6w Lr. ' I Ls8 _J__ - -ssH - -rc-l tsT -2 -l 0 2. MDS Aris I tps lP9-pr 0 L s6H I LSB ttE I tco L¡r tsr I I I .2 -2 -l 0 M DS Ax¡s I ? LSW LPI I LPOLPL 0 asH LSB LLE J+T LJçO 0 a MDSAxis2 Figure 8.12. Principal canonical correlation graphs of E. socialis x E. gardneri seedlings and parents. 324 Annendix 2.2. I I I ssH I I rilr slE 0 LSW LLE LST LSH LSg LCO 0 2 MDS Ax¡s I ts8 LS15¡g t9w "* - -TL LlE I rwr rs' I r 'T' I I 0 2 MDS Axls I tsg LCO tst LIE tsH LPL -2 0 MDS Axis 2 Figure 8.73. Principal canonical correlation graphs of E. socialis x E. gillii seedlings and parents. 325 Annendiv 2-2- 2 PO 5TF tw I rtE J- _ !9o-- Lsw - - tE^ LTL I ast ."!" I LSI I I I -t 0 2 MDS Ar¡s I 2 tsH *'it ltE 8S¡ ssM ILE STE IEÄ "rì" r*L ô I -l -l 0 2 MDS A¡is I I I I @ lcar @ 3SH ILE tE^ slE 0 -2 0 MDS A¡¡s2 Figure 8.74. Principal canonical correlation graphs of E. socialis x E. kruseana seedlings and parents. Characters in italics and underlined are not significantly correlated to MDS ordinations (Table 8. l0). 326 Annendix 2-2 llrSTE PS tsa 0 ssH ssM ,tÍ'." * LLE -2 -2 0 MDS Axis I sst LLE .Z 0 ô tsa d 0 MDS Axis I ssN ssM LPS LLE rij 0 MDSÁxis2 Figure 8.75. Principal canonical correlation graphs of E. socialis x E. macrocarpø seedlings and parents. Characters in italics and underlined are not significantly correlated to MDS ordinations (Table 8.7). 327 Annendix 2.2. I ssH BTE I LLE LPs I @ ,2 st 0 L SE I LSH I -l 0 MDS Axis I slE ssM[er 0 t- ô = tst Lt€ @ -2 0 MDS Axis I rps sftl _a L "."d1 ê rblw ILAPo -2 0 MDS Ax¡s2 Figure 8.76. Principal canonical correlation graphs of E. socialis x E. oldfieldii seedlings and parents. Characters in italics and underlined are not significantly correlated to MDS ordinations (Table 8. l0). 328 Appendix 2.2. ,.'¿ LslH SIE LLÊ *1 I M DS Aris I L9W LSa -2 q LPO LPS LST STE ["" LsH LPL I "ru I I MDS Axis I ¿t_w .2 _'- ô -Lø - -.# LPS I sTE tsr I ssH ssMI rsx reLl I I -2 -2 0 2 M DS Àxis 2 Figure 8.77. Principal canonical correlation graphs of E. socialis x E. orbifuIia seedlings and parents. Characters in italics and underlined are not significantly correlated to MDS ordinations (Table 8. l0). 329 Anoendix 2.2. 2 LPS LCO L9BW LLE asH [€.H 0 slE -2 -2 0 MDS Àxis I I I I b'e ssH I r¡ìtH srE lwtl __PrL tco tt€ r$êw -2 0 M DS Axis I ssf,PLQt L¡TI IE lwl L rL q{qilú -2 0 MDSAxis2 Figure 8.78. Principal canonical correlation graphs of E. socialis x E. polyanthemos seedlings and parents. 330 Annendix 2-2. tco S TEL sE 0 -E¿F¡ ô I LL I sþ -¡ I I I -2 0 MDS Axis I LSH LSB .2 LSW LSt 0 Lr.c-a- I slE tPs LPO LLE "[" LdÆL 0 M DS Ax¡s I I I I 'r' I tsB I Lwr Lsw I Lsl 0 I srE ssH l" LLE ItE I I i I 0 MDSAxls2 Figure 8.79. Principal canonical correlation graphs of E. socialis x E. sideroxylon seedlings and parents. 331 Annendix 2-2. 2 I I I t9E tgo rLw !3H ."1 | ... tPo ssH LSf -2 0 MDS A¡¡8 ¡ 2 STE LSB .2 ,.l". tco f ssH o rs[Fr I LPO ,Ir ,*1, LLE i I I -2 0 1 MDS Axis I ! I I slE I tsB I tPt ,2 ssH uco LPS tsr Ê o L 6H LLE -2 0 MDS Ax¡s 2 Figure 8.80. Principal canonical correlation graphs of E. socialis x E. socialis seedlings and parents. 332 Annen¡liv 2-2- LAS i"#, srE Lrs.r. ssH !co L6M 0 L LAT as [re -l -2 0 MDS Arls I LST L9H L9A ssB 0 L I rp$Pr SlEILE I SSi tPO rrtI i I 0 M DS Axts I tsf tsH tsB .E LCO SSH 0 L ê LPt!PS -LW - LLE slE ssÍ LPO fsM -1 -2 0 2 MDS Axis 2 Figure 8.81. Principal canonical correlation graphs of E. socialis x E. steedmanii seedlings and parents. 333 Annendix 2.2. I I rl\I ssH ø ,9 LCO STE LSf -2 -2 0 M DS A¡is I I I I tî tt LLE ts B I 's SH I rf{ L _ra L PT tco - srE LSr -2 0 M DS Àxis I ssH tsH ,a LST 0 ¡lE ô LLE ¿ r-s¡¿ tsB stE 0 MDS Axis 2 Figure 8.82. Principal canonical correlation graphs of E. sociqlis x E. transcontinentalis seedlings and parents. Characters in italics and underlined are not significantly correlated to MDS ordinations (Table 8.10). 334 Annenrliv 2-2- s5 LS8 { ô ¿ 129 tl{E¡qo slE -t 0 MDS A¡is I 2 fåf i;¡ I LPS 129 LI lcol6B stE LLE -2 0 MDS Axls I ,r" ,rrl s3{ rEl rPl ssH lPs I Lrc I tco L LSg sTE lw I ttE .2 0 2 MDS Axis 2 Figure 8.83. Principal canonical correlation graphs of E. socialis x E. websteriana seedlings and parents. Characters in italics and underlined are not significantly correlated to MDS ordinations (Table 8. 10). 335 Annendix 2-2- tPs gg6tPtLPo L SSH tCO tsB tsH lsr ¡lÁ 0 MDS Aris I E6H .2 L taa tPo lco ''.| LLE lsH -2 0 MDS A¡is I 2 ssH .2 0 L a LS8 LCO LPO 9fE LPL LPS LIE LSH -¡ 0 2 MDS Axis 2 Figure 8.84, Principal canonical correlation graphs of E. socialis x E. youngiana seedlings and parents. Characters in italics and underlined are not significantly correlated to MDS ordinations (Table 8. 10). 336 Anoendix 3.1. Appendix 3.L - Presentations 17ú October,1997 Research update to Scholarship donors and interested parties Urrbrae House, V/aite Agricultural Research Institute "Improvement of Ornamental Eucalypts" 1lú February, 1999 Summary of research for Playford Memorial Trust Scholarship Horticulture donors and other interested parties Plant Research Centre Auditorium, Waite Agricultural Research Institute 'Improvement of Ornamental Eucalypts" 16ú April, 1999 5ü Australian Wildflower Conference Melbourne, 14ú to 17ü April, 1999 "Development of Ornamental Eucalypts" 21't April, 1999 Adelaide Botanic Gardens Guides Adelaide Botanic Gardens "Improvement of Ornamental Eucalypts" 2"d June,1999 Departmental Seminar Plant Research Centre Auditorium, Vy'aite Agricultural Research Institute "Improvement of Ornamental Eucalypts" 2nd June, 1999 Interdepartmental Seminar Department of Environmental Biology, North Terrace "Improvement of Ornamental Eucalypts" 337 Ännendix 3-2- Appendix3.2. - Posters Delaporte, K.L., Collins, G., Conran, J., Klieber, A. and Sedgley, M. (1999). Improvement of Ornamental Eucalypts. l4'h National AFPGA Conference, Hahndorf, South Australia, 20-22 August, 1999. Delaporte, K.L., Conran, J. and Sedgley, M. (2000). Hybridisation in Eucalyptus ser. Curviptera; a morphological study. Flowers 2000: The first Australian Flower Conference, Tumbi-Umbi, New South Wales, 2-6 August, 2000. Appendix 3.3. - Papers Delaporte, K.L., Klieber, A. and Sedgley, M. (2000). Postharvest vase life of two flowering Eucalyptus species. Postharve st B iolo gy and Technolo gy 19, 1 I 1 - I 86. Delaporte, K.L., Conran, J. and Sedgley, M. (2000). Hybridisation in Eucalyptus ser. Curviptera: a morphological study. ln Flowers 2000: The first Australian Flower 'Worrall), Conference (Ed: R. Tumbi-Umbi, New South Wales, 2-6 August, 2000. Delaporte, K.L., Collins, G., Conran, J. and Sedgley, M. (2000). Molecular analysis of an interspecific ornamental Eucalypt hybrid for parental identification. Euphytica (in press). Delaporte, K.L., Conran, J. and Sedgley, M. (2000). Morphological analysis to identify the pollen parent of an ornamental interspecific hybrid Eucalyptus. Scientia Horticulturae (in press). Delaporte, K.L., and Sedgley, M. (2000). Ornamental Eucalypts.' Selection within species for cut bud and flower production. Australian Plants (in press). Delaporte, K.L., Collins, G., Conran, J., Klieber, A. and Sedgley, M. (1999). Development of Ornamental Eucalypts. Proceedings of the 5ú Australian Wildflower Conference, Melbourne, 14ú to 17û April, 1999. pp:51-52. Delaporte, K.L., Conran, J. and Sedgley, M. (2000). Interspecific hybridisation between three closely related Eucalyptus species: E. macrocarpd, E. youngiana and E. pyriformis. ( In preparation) Delaporte, K.L., Conran, J. and Sedgley, M. (2000). Interspecific hybridisation within Eucalyptus (Myrtaceae); subgenus Symphyomyrtus, sections Bísectaria a¡d Adnataria. (In preparation). 338