1

Screening for newly generated polyploidy in trees using chromosome 1

counts and leaf traits 2

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Running head: Screening for polyploidy in trees 5

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Harshi K. GamageA, Malcolm LamontB, Christopher QuinnB, Peer M. SchenkA, Susanne SchmidtA,C 8 9 A School of Integrative Biology, The University of Queensland, Brisbane QLD 4072, Australia. 10 B Arbour Technologies Pty. Ltd., 981 Mount Tamborine, Oxenford Road, Wongawallan, QLD 4210, 11

Australia. 12 C Corresponding author: email Susanne.Schmidt@uq.edu.au 13

14 15 16

Key words: angiosperms, chromosomes, gymnosperms, leaf morphology, polyploidy, stomatal 17

aperture, trees 18

2

Abstract 19

Trees have a slow breeding cycle which makes them vulnerable to rapid environmental change. 20

Artificially induced polyploidy has many applications including increased yield and enhanced 21

environmental resilience, and we have recently developed a novel procedure for polyploidisation of 22

trees. Here we report early results of this world-first polyploidisation technique using vegetative tissue 23

of angiosperm and gymnosperm trees. We aimed to optimise a technique for chromosome counts and 24

to compare indirect ploidy assessment techniques for rapid screening of Australian and exotic tree 25

species and newly generated lines. For each of the 16 studied species, one diploid parent and putative 26

polyploid clone (termed clone in the following) was compared. Further, we tested the response of 27

diploid parents and three clone lines of Elaeocarpus angustifolius to soil water limitation. Chromosome 28

counts confirmed that the Acacia crassicarpa clone is a newly generated tetraploid (parent n=26, clone 29

n=52). Techniques for chromosome counting have to be refined for the other species. Stomatal aperture 30

lengths of diploid parents and clones were mostly similar, including A. crassicarpa, but were 31

substantially larger in clones than parents of Dysoxylum muelleri and three species in the 32

Araucariaceae. In all species, leaves of clones were larger and mostly thicker, and had higher 33

chlorophyll and soluble protein contents than leaves of diploids. Under mild water limitation in 34

controlled growth conditions, two E. angustifolius clone lines grew taller and produced a larger root 35

collar diameter than diploid parents, an early indication for improved tolerance of polyploids to lower 36

water availability. We conclude that foliar traits could be useful for rapid screening for polyploidy, and 37

that ploidy levels have to be confirmed via chromosome counts before taxa-specific screening tools can 38

be applied. 39

3

Introduction 40

Over the past 100 years the global average temperature has increased by approximately 0.6 °C and 41

temperatures are projected to increase by 1.1 to 6.4 °C during the 21st century; and altered rainfall 42

patterns, drought periods and heat waves are predicted for future decades (IPCC 2007). Although 43

organisms have responded to climatic changes throughout evolutionary history, a primary concern for 44

wild species is the current rapid rate of change (Root et al. 2003). Particularly vulnerable to climate 45

change are species with a slow breeding cycle. 46

Polyploidy, the presence of more than two sets of chromosomes per nucleus in diploid organisms, 47

provides genetic novelty and has shaped plant evolution (Adams and Wendel 2005; Comai 2005). 48

Numerous studies have demonstrated faster growth and greater environmental resilience of polyploid 49

plants compared with diploids (Levin 1983; Ramsey and Schemske 2002; Rothera and Davy 1986). 50

Polyploidy can result in extensive genomic rearrangements, including exchanges between genomes and 51

gene loss as well as molecular and physiological adjustments (Soltis and Soltis 1993; Wendel 2000; 52

Adam and Wendel 2005). As a result, polyploid plants can have greater diversity in metabolites and 53

metabolic pathways (Dhawan and Lavania 1996) and can maintain greater phenotypic homeostasis of 54

fitness-related traits allowing adaptation to marginal or extreme environments (Levin 1983; Thompson 55

and Lumaret 1992). 56

While breeding for polyploidy is less common in forestry, polyploid shrubs and herbs are widely used 57

in agriculture and horticulture (Leitch and Bennett 1997; Väinölä 2000; Osborn 2003; Shoemaker et al. 58

2006). Advantages of polyploids over diploids include increased resistance to parasitism or herbivory, 59

increased yield, and adaptation to specific growing conditions (Levin 1983; Dhawan and Lavania 1996; 60

Ramsey and Schemske 2002). 61

Despite the numerous potential advantages of polyploidy, artificial polyploidisation in trees is rare. 62

While polyploidisation is comparatively easily accomplished with colchicine treated seeds, it is 63

extremely difficult to achieve with vegetative tissues (Blakesley et al. 2002). Yet only polyploidisation 64

4

of elite trees ensures that desired germplasm forms the basis of new lines. Recently, a new procedure 65

has been developed in Australia for routine polyploidisation of trees via somatic embryogenesis which 66

produces clonal polyploid trees. For identification of newly generated polyploid trees, ploidy levels 67

have to be confirmed at different growth stages, and rapid screening tools are required for identification 68

and assessment of polyploid lines. 69

Chromosome counts in root tip squashes are commonly used for ploidy assessment (Beck et al. 2003a). 70

However, it can be difficult to identify polyploidy in species with many small chromosomes (Beck et 71

al. 2003a). Chromosome counting is time-consuming and not easily achieved in all tree species 72

(Przywara et al. 1988). While flow cytometry is a rapid and powerful technique for DNA quantification 73

(Shapiro 2004; Beck et al. 2005), contradictory results have been obtained by researchers (Greilhuber 74

and Obermayer 1997; Price et al. 2000). Further, a high content of phenolics, tannins and oils prevents 75

successful use of flow cytometry in certain taxa including Myrtaceae and Rutaceae (Morgan and 76

Westoby 2005). Alternative methods for ploidy assessment include measurements of pollen grain 77

diameter (Cohen and Yao 1996), stomatal aperture length (Li et al. 1996; Przywara et al. 1998; Beck et 78

al. 2003a,b), and chloroplast numbers in stomatal guard cells (Beck et al. 2005). While stomatal 79

properties have widely been applied for ploidy determination, combined assessment of leaf 80

morphology, anatomy, and physiology has rarely been used. 81

We aimed to optimise chromosome counting techniques for the study species and to assess leaf traits as 82

rapid screening tools for ploidy assessment. Lastly, we carried out a pilot study of diploid parent and 83

three clone lines of Elaeocarpus angustifolius across a soil moisture gradient in the glasshouse to 84

determine if putative polyploids have greater tolerance to low soil moisture levels. 85

86

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5

Methods and Materials 90

Plant material 91

Diploid parent plants and putative polyploid clones in the tree genera Acacia, Agathis, Araucaria, 92

Bischofia, Dysoxylum, Elaeocarpus, Eucalyptus, Flindersia, Gmelina, Pongamia, Pinus, and Toona 93

were studied (Table 1). Clones were generated via somatic embryogenesis using shoot tips of seedlings 94

purchased from a nursery in North Queensland, Australia. Diploid parents were treated identical to 95

clones and were generated from tissue culture except that the polyploidisation procedure was not 96

applied. Diploid parents and clones were grown either hydroponically in temperature controlled growth 97

rooms (23 day and 22.5 ºC night for 16 h day length, light intensity 1000 µmol m-2 s-1) or in the field 98

(Mt. Tamborine, South East Queensland, Australia) (Table 1). Across study species, plant height 99

ranged from 40 to 150 cm. One diploid parent and clone was examined for each species because these 100

plants were the first ones generated with the new polyploidisation procedure. 101

102

Chromosome counting 103

Root tips of 0.5 cm length were harvested from hydroponically grown plants and pretreated with 0.04 104

% 8-hydroxyquinoline at room temperature for 4 h. Six root tips were used for each diploid parent and 105

clone. The material was fixed in absolute ethanol and glacial acetic acid (3:1) for 72 h at room 106

temperature and stored in 70 % ethanol at 4 ºC. Samples were washed and hydrolysed in 0.25 N HCl 107

for 10 min at room temperature. The distal 1-1.5 mm root tips were cut and digested in a mixture of 108

cellulase and pectolyase (5:1, Yakult Pharmaceutical Ind. Co. Ltd., Japan) for approximately 1.5 h at 109

37 ºC. Digested root tips were washed and spread onto a glass slide, air dried, and stained with DAPI 110

(2, 4-diamidino-2-phenylindole, Australian Laboratory Services Pty. Ltd., Sydney). Chromosome 111

counts in individual protoplasts were performed at x1000 magnification using a compound light 112

microscope with excitation wave lengths of 330-380 nm and digital camera (Nikon Eclipse E600, 113

6

Japan; Spot digital camera, Diagnostic Instruments, Inc. USA). At least six protoplasts were counted 114

for A. crassicarpa parent and clone. 115

116

Leaf morphology and stomatal properties 117

One youngest fully expanded leaf from each parent and clone was examined for leaf morphology, 118

stomatal properties, chlorophyll and soluble protein content. Examined leaves were representative for 119

other leaves of the plants. Leaf area was measured using a leaf area meter (Li-3000C, Li-Cor Inc., 120

Lincoln, Nebraska, USA) and leaves were oven dried at 85 ºC to constant weight. Specific leaf area 121

was calculated as the ratio of leaf area to leaf dry mass. Leaf sections (1 cm2) were taken in the middle 122

portion of the lamina. Each leaf section was incubated at 50 ºC in 5 % w/v sodium hydroxide to clear 123

leaf pigments. Sections were stained with 0.5 % w/v aqueous toluidine blue solution and mounted on a 124

viewing slide (Ashton et al. 1999). For each section, 18 stoma aperture lengths were measured in six 125

different fields of view on the abaxial side of the leaf. Stomata per unit area of abaxial leaf surface were 126

counted in six different fields of view on the same leaf peel at x400 magnification with a light 127

microscope and attached digital camera. Stomatal aperture length measurement and stomata counts 128

were performed on digital images using Spot Advance software (Spot software BV, version 4, 129

Amsterdam, The Netherlands). For determining total leaf chlorophyll content (chlorophyll a and b), 130

three leaf discs of 3 mm diameter were taken from base, center, and tip from the youngest fully 131

expanded leaves. Leaf discs were immediately frozen in liquid nitrogen and were ground with buffered 132

80% w/v aqueous acetone (Porra et al. 1989). Samples were centrifuged and chlorophyll extracted. 133

Absorbance of chlorophyll a and b was measured at 663 nm and 646 nm. Total chlorophyll content was 134

calculated according to Porra et al. (1989). For measuring soluble protein content of leaves, three leaf 135

discs of 3 mm in diameter were taken from base, centre and tip from the same leaves that were used for 136

chlorophyll measurements. Leaf discs were immediately frozen in liquid nitrogen and were ground 137

with buffered phosphate solution (Bradford 1976). Soluble protein was determined using the Biorad 138

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Protein kit (BioRad laboratories, Inc., Hercules, CA, USA) with bovine serum albumen (BSA) as the 139

protein standard. 140

141

Water stress experiment 142

Elaeocarpus angustifolius plants were used as three clone lines and replicates were available. One 143

diploid parent and three clone lines were used (n=3 for parent and each clone line). Plants were 30-40 144

cm high and were grown in 7 liter plastic pots with Californian potting mix. Plants were grown in a 145

naturally lit glasshouse at the University of Queensland, Brisbane, Australia, from June to Sept 2007. 146

Plants were exposed to three moisture regimes: (1) ever moist soil condition in which plants were 147

watered daily to 100 % soil moisture, corresponding to 38-40 % volumetric soil moisture content 148

(VSM), (2) moderately dry soil conditions with water applied every second day to 90 % soil moisture 149

and 33-35 % VSM, and (3) dry soil conditions with watering every third day to 70 % soil moisture and 150

26-28% VSM. Volumetric soil moisture content was measured using a portable soil moisture meter 151

(MPM 160 moisture probe meter, ICT International Pty Ltd., Australia) inserted through a hole at the 152

bottom of the pots. The comparatively mild water stress treatments were applied because E. 153

angustifolius is sensitive to very low soil moisture levels. Height and root collar diameter were 154

measured for each plant at the start and end of the experiment. 155

156

Statistics 157

ANOVA (general linear model) was used to test for differences in growth of E. angustifolius parent 158

and clones across the soil moisture gradient using MINITAB Version 12 (Minitab Inc., PA, USA). 159

Data were log transformed prior to analysis to meet the assumption of ANOVA. We tested for 160

differences in growth among moisture treatment, genotype (parent, clones) and the interaction between 161

treatment and genotype. Interactions that were significant were further tested between parent and each 162

clone within a treatment using a two sample t-test. 163

8

Results 164

Chromosome counts of A. crassicarpa protoplasts confirmed that the clone is tetraploid (parent n=26, 165

clone n=52) (Fig. 1). The chromosome counting procedure gives robust and repeatable results for A. 166

crassicarpa. Chromosome spreads for Pongamia, Bischofia, Elaeocarpus and Toona visualised 167

individual chromosome but clumping of chromosomes prevented accurate quantification of 168

chromosomes (data not shown), and the method requires further refinement. Roots from field grown 169

plants could not be used for chromosome counting, rather, white root tips of hydroponically grown 170

plants provided the most promising material. 171

Putative polyploid clones and the confirmed tetraploid A. crassicarpa clone had leaf areas 12 to 164 % 172

larger than respective diploid parents (Fig. 2, Table 2). Eucalyptus robusta had the greatest differences 173

with a 643 % larger leaf area of the clone compared with the parent. Compared with parents, clones had 174

mostly thicker leaves (3 to 52 % lower specific leaf area), with the exception of clones of Eucalytpus 175

microcorys, Bischofia bourjotiana and Pongamia pinnata which had thinner leaves than parents (Table 176

2). Clones had mostly similar stomatal aperture lengths as diploid parents, except clones of Dysoxylum 177

muelleri and gymnosperms Agathis robusta, Araucaria cunninghamii, and A. bidwilli which had 178

substantially greater stomatal aperture lengths than parents (Figs. 3 and 4). Stomatal density was higher 179

in the four Eucalyptus and B. javanica clones than parents (Fig. 4). Clones which were examined for 180

chlorophyll and soluble protein content had somewhat higher chlorophyll and soluble protein contents 181

than parents (Table 2). 182

The water stress experiment showed that significant effects of plant height and root collar diameter 183

occurred between treatments and genotypes, but no treatment-genotype interaction was observed 184

(Table 3). Clone line two and three performed significantly (P<0.05, t-test) better than the diploid 185

parent in wet and moderately dry soil conditions (data not shown). 186

187

188

9

Discussion 189

A new procedure was recently developed in Australia for polyploidisation of tree species via somatic 190

embryogenesis. Such procedure holds great promise for rapid breeding of trees because elite trees, 191

rather than seeds of unknown genetic composition, can serve as the source of genetic material for 192

breeding. Polyploidisation is promising for rapid domestication of forest trees and for improvement of 193

plantation species. Rapid breeding tools for plantation trees are vital for adaptation to new 194

environmental conditions, and domestication of native trees holds much promise for forestry which 195

considers ecosystem services such as preservation of local biodiversity (Lamb et al. 2005). 196

We quantified chromosomes in root tip cells and confirmed that the Acacia crassicarpa clone is a 197

newly generated tetraploid. Further optimisation of the chromosome counting technique is required for 198

the other species studied here. Putative polyploid clones and the confirmed tetraploid clone had larger 199

leaves than parent plants, and clones of 11 species had thicker leaves than diploid parents. There is 200

general agreement that polyploidy results in larger organs and cells, the so-called gigas effect (Levin 201

1983; Ramsey and Schemske 2002; Przywara et al. 1988; Mishra 1997). A pronounced increase in 202

stomatal aperture size was observed in clones of the three Araucariaceae species and in Dysoxylum 203

muelleri, but not in the other species. Comparing several clones of Agathis robusta we found that 204

clones had consistently significantly larger and thicker leaves and larger stomata than the diploid parent 205

(Gamage et al., unpublished data), lending confidence to the results here. Clones of all studied 206

Eucalyptus species and Bischofia javanica had markedly higher stomatal density than diploids. Leaves 207

of E. grandis, E. microcorys and B. javanica clones were thinner than leaves of diploids, indicating that 208

taxa specific trade-offs between leaf thickness, stomatal size and density may occur. In the Dysoxylum 209

muelleri clone, larger stomatal aperture was associated with lower stomatal density, similar to what has 210

been described for other polyploids (Li et al. 1996; Beck et al. 2003a,b). Di- and tetraploid Acacia 211

mearnsii had stomatal aperture lengths and stomatal densities of 27.2 and 40.2 µm, and 22.1 and 10.3 212

10

stomata per unit leaf area, respectively (Beck et al. 2003a), while in our study di- and tetraploid A. 213

crassicarpa had similar stomatal properties. 214

Most clones had greater chlorophyll and protein content than diploids on a leaf area basis. Similarly, 215

polyploidisation increased cell size and chloroplast numbers per cell, and resulted in greater 216

chlorophyll content (Beck et al. 2003b; Joseph et al. 1981; Mathura et al. 2006). In grasses, chlorophyll 217

and soluble protein content increased significantly from tetra- to decaploid (Warner et al. 1987; Joseph 218

et al. 1997). However, in Atriplex confertifolia chlorophyll content remained constant, while soluble 219

protein content increased with increasing ploidy level (Warner and Edwards 1989), indicating that 220

polyploidy causes taxa specific effects. 221

We tested if polyploidy causes greater tolerance to low soil moisture in the putative polyploid clones of 222

Elaeocarpus angustifolius. As plants will undergo further testing, only non-destructive measurements 223

were performed. Height growth was similar in clones and diploids in the lowest water supply, but two 224

of the three clone lines grew significantly taller and greater in root collar diameter in high and medium 225

water supply than the diploid. The results are an early indication that putative polyploidy in E. 226

angustifolius may not confer the ability to tolerate low soil water levels but that growth at high and 227

intermediate water supply is enhanced. There is support for the notion that polyploidy enhances 228

drought tolerance. For example, tetraploid Lolium perenne remained green and continued to grow 229

longer than diploids during hot dry summers (Wit and Speckmann 1955), and polyploid Betula 230

papyrifera grew in warm and dry environments while diploids favored wetter and cooler sites (Li et al. 231

1996). Thicker leaves of polyploids can provide a structural basis for better growth under mild water 232

stress (Ashton and Berlyn 1994; Li et al. 1996), as well as reduced transpiration (Tal and Gardi 1971). 233

In conclusion, we have a degree of confidence that the new polyploidisation procedure successfully 234

generates polyploid trees, and tetraploidy was indeed confirmed for A. crassicarpa. Chromosome 235

counts have to confirm whether clones of the other study species are polyploids or have increased 236

replicates of individual chromosomes (polysomy). Clones exhibit novel traits which could convey 237

11

advantages in different environments. While leaf area, chlorophyll and soluble protein content of all 238

putative polyploid clones and the confirmed tetraploid clone were elevated in all studied species, other 239

examined traits including specific leaf area, stomatal aperture length, and stomatal density varied. We 240

conclude that for rapid screening of polyploids and identification of elite clones, taxa specific traits 241

have to be considered. 242

243

Acknowledgements 244

We thank Dr Natalie Piperidis from BSES Ltd. at Mackay for help with developing the chromosome 245

counting technique and Prof Peter Gresshoff from the ARC Centre of Excellence for Integrated 246

Legume Research at the University of Queensland for access to microscope facilities. 247

248

References 249 Adams KL and Wendel JF (2005) Polyploidy and genome evolution in plants. Plant Biology 8, 135-250

141. 251 Ashton PMS, Berlyn GP (1994) A comparison of leaf physiology and anatomy of Quercus (section 252

Erythrobalanus-Fagaceae) species in different light environments. American Journal of Botany 253 81, 589-597. 254

Ashton PMS, Yoon HS, Thadani R, Berlyn GP (1999) Seedling leaf structure of New England 255 Maples (Acer) in relation to light environment. Forest Science 45, 512-519. 256 Beck SL, Dunlop RW, Fossey A (2003a) Stomatal length as a measure of ploidy level in black 257 wattle, Acacia mearnsii (de Wild). Botanical Journal of the Linnean Society 141, 177-181. 258 Beck SL, Dunlop RW, Fossey A (2003b) Evaluation of induced polyploidy in Acacia mearnsii 259 through stomatal counts and guard cell measurements. South African Journal of Botany 69, 260 563-567. 261 Beck SL, Visser G, Dunlop RW (2005) A comparison of direct (flow cytometry) and other indirect 262 (stomatal length and chloroplast numbers within stoma) techniques as a measure of ploidy 263 level in black wattle, Acacia mearnsii (De Wild). South African Journal of Botany 71, 359- 264 363. 265 Blakesley D, Allen A, Pellny TK, Roberts AV (2002) Natural and induced polyploidy in Acacia 266 dealbata Link. and Acacia mangium Wild. Annals of Botany 90, 391-398. 267 Bradford MM (1976) A rapid and sensitive method of the quantification of microgram quantities 268

of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248-254. 269 Cohen DJ, Yao L (1996) In vitro chromosome doubling of nine Zantedeschia cultivars. Plant Cell 270 and Tissue Organ Culture 47, 43-49. 271 Comai L (2005) The advantages and disadvantages of being polyploid. Nature Review Genetics 6, 272 836-846. 273 Dhawan OP, Lavania UG (1996) Enhancing the productivity of secondary metabolites via induced 274 polyploidy: a review. Euphytica 87, 81-89. 275

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Greilhuber J, Obermayer R (1997) Genome size and maturity group in Glycine max (soybean). 276 Heredity 78, 547-561. 277 Intergovermental Panel on Climate Change (2007) Fourth Assessment Report Climate Change 2007: 278 The physical science basis, IPCC, Switzerland. http://ipcc.-wg1.ucar.edu/wg1/wg1-report.html 279 Joseph MC, Randall DD, Nelson CJ (1981) Photosynthesis in polyploid tall Fescue. II Photosynthesis 280 and ribulose-1, 5–bisphosphate carboxylase of polyploid tall fescue. Plant Physiology 68, 281 894-898. 282 Lamb D, Erskine PD, Parrotta JA (2005) Restoration of degraded tropical forest landscapes. Science 283 310, 1628-1632. 284 Leitch I J, Bennett MD (1997) Polyploidy in angiosperms. Trends in Plant Science 2, 470-476. 285 Levin DA (1983) Polyploidy and novelty in flowering plants. American Naturalist 122, 1-25. 286 Li W, Berlyn GP, Ashton PM (1996) Polyploids and their structural and physiological 287 characteristics relative to water deficit in Betula papyrifera (Betulaceae). American Journal of 288

Botany 83, 15-20. 289 Mathura S, Fossey A, Beck SL (2006) Comparative study of chlorophyll content in diploid and 290

tetraploid black wattle (Acacia mearnsii). Forestry 79, 381-388. 291 Mishra MK (1997) Stomatal characteristics at different ploidy levels in Coffea L. Annals of Botany 80, 292

689-692. 293 Morgan HD, Westoby M (2005) The relationship between nuclear DNA content and leaf strategy in 294 seed plants. Annals of Botany 96, 1321-1330. 295 Osborn TC, Pires JC, Birchler JA, Auger DL, Chen ZJ, Lee H, Comai L, Madlung A, Doerge RW, 296 Porra RJ, Thompson WA, Kriedemann PE (1989) Determination of accurate extinction coefficients 297 and simultaneous equations for assaying chlorophylls a and b extracted with four different 298 solvents: verification of the concentration of chlorophyll standards by atomic absorption 299 spectroscopy. Biochimica et Biophysica Acta 975, 384-394. 300 Przywara L, Pandey KK, Sanders PM (1988) Length of stomata as an indicator of ploidy level in 301 Actinidia deliciosa. New Zealand Journal of Botany 26, 79-82. 302 Price HJ, Hodnett G, Johnston JS (2000) Sunflower (Helianthus annuus) leaves contain compounds 303 that reduce nuclear propidium iodide fluorescence. Annals of Botany 86, 929-934. 304 Ramsey J, Schemske DW (2002) Neopolyploidy in flowering plants. Annual Review of Ecology and 305

Systematics 33, 589-639. 306 Rothera SL, Davy AJ (1986) Polyploidy and habitat differentiation in Deschampsia espitosa. New 307 Phytologist 102, 449-467. 308 Root TL, Price JT, Hall KR, Schneider SH, Rosenzweig C, Pounds JA (2003) Fingerprints on global 309 warming on wild animals and plants. Nature 421, 57-60. 310 Shapiro H (2004) Practical flow cytometry, 4th edition. New York, Wiley-Liss. 311 Shoemaker RC, Schlueter J, Doyle JJ (2006) Paleopolyploidy and gene duplication in soybean and 312 other legumes. Current Opinion in Plant Biology 9, 104-109. 313 Soltis DE, Soltis PS (1993) Molecular data and the dynamic nature of polyploidy. Critical Review. 314 Plant Science 12, 243-273. 315 Tal M, Gardi I (1971) Physiology of polyploid plants: Water balance in autotetraploid and diploid 316 tomato under low and high salinity. Physiologia Plantarum 38, 257-261. 317 Thompson JD, Lumaret R (1992) The evolutionary dynamics of polyploid plants-origins, establishment 318 and persistence. Trends in Ecology and Evolution 7, 302-307. 319 Väinölä A (2000) Polyploidisation and early screening of Rhododendron hybrids. Euphytica 112, 320 239-244. 321 Warner DA, Ku MSB, Edward GE (1987) Photosynthesis, leaf anatomy, and cellular constituents in 322 the polyploid C4 grass Panicum virgatum. Plant Physiology 84, 461-466. 323

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Warner DA, Edwards GE (1989) Effects of polyploidy on photosynthetic rates, photosynthetic 324 enzymes, contents of DNA, chlorophyll and sizes and number of photosynthetic cells in the 325 C4 dicot Atriplex confertifolia. Plant Physiology 91, 1143-1151. 326 Wendel JF (2000) Genome evolution in polyploids. Plant Molecular Biology 42, 225-249. 327 Wit F, Speckmann GJ (1955) Tetraploid Westerwolths rye grass. Euphytica 4, 245-253. 328 329

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Table 1. Studied tree species which have undergone induced polyploidisation. Native describes 331

Australian plants. 332

333

Botanical name Common name Family Origin Growth conditions 334

335

Angiosperms 336

Acacia crassicarpa Northern wattle Mimosaceae Native Hydroponics 337 Bischofia javanica Javanese cedar Euphorbiaceae Native Hydroponics 338 Dysoxylum muelleri Miva mahogany Ebenaceae Native Field 339 Elaeocarpus angustifolius Blue quandong Eleocarpaceae Native Hydroponics 340 Eucalyptus dunnii White gum Myrtaceae Native Hydroponics 341 E. grandis Flooded gum Myrtaceae Native Hydroponics 342 E. microcorys Tallow wood Myrtaceae Native Hydroponics 343 E. robusta Swamp mahogany Myrtaceae Native Hydroponics 344 Flindersia bourjotiana Silver ash Rutaceae Native Field 345 Gmelina leichhardtii White beech Verbenaceae Native Field 346 Pongamia pinnata Honge Fabaceae Introduced Hydroponics 347 Toona ciliata Red cedar Meliaceae Native Hydroponics 348 Gymnosperms 349 Agathis robusta Kauri pine Araucariaceae Native Hydroponics 350 Araucaria bidwillii Bunya pine Araucariaceae Native Field 351 A. cunninghamii Hoop pine Araucariaceae Native Hydroponics 352 Pinus radiata Radiata pine Pinaceae Introduced Hydroponics 353

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Table 2. Leaf traits of diploid parents and putative polyploid clones. One diploid parent and one clone 354

per species were studied. na = data not available. A. cunninghamii and P. radiata were not analysed for 355

leaf properties. *confirmed tetraploid. 356

Single leaf Specific leaf Leaf chlorophyll Leaf soluble protein 357 area (cm2) area (cm2g-1) content µg cm-2) content (µg cm-2) 358 Acacia crassicarpa 359 Parent 46.14 103.70 27.81 14.3 360 Clone* 105.47 71.46 38.43 19.6 361 Agathis robusta 362 Parent 31.47 122.92 27.08 24.5 363 Clone 82.46 58.69 47.46 28.4 364 Araucaria bidwillii 365 Parent 2.5 108.2 na na 366 Clone 4.6 100.9 na na 367 Bischofia javanica 368 Parent 44.53 181.01 21.51 19.2 369 Clone 112.94 313.71 24.38 26.9 370 Dysoxylum muelleri 371 Parent 41.0 380.1 na na 372 Clone 69.2 288.1 na na 373 Elaeocarpus angustifolius 374 Parent 110.07 193.79 28.21 32.4 375 Clone 136.57 164.74 29.74 46.3 376 Eucalyptus grandis 377 Parent 21.89 135.97 28.20 15.2 378 Clone 39.43 160.28 31.66 26.0 379 E. dunnii 380 Parent 23.26 200.51 23.53 18.3 381 Clone 38.56 126.00 27.69 28.9 382 E. microcorys 383 Parent 15.67 208.96 18.53 23.0 384 Clone 25.87 213.81 27.86 49.0 385 E. robusta 386 Parent 26.87 165.84 28.32 27.4 387 Clone 172.89 69.38 29.33 28.5 388 Flindersia bourjotiana 389 Parent 131.8 80.4 na na 390 Clone 148.1 74.5 na na 391 Gmelina leichhardtii 392 Parent 77.0 232.6 na na 393 Clone 105.1 209.8 na na 394 Pongamia pinnata 395 Parent 19.8 274.7 na na 396 Clone 45.6 362.3 na na 397 Toona ciliata 398 Parent 61.94 288.09 15.24 26.1 399 Clone 125.87 222.78 16.68 30.9 400

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Table 3. F-statistics for nested ANOVA for plant height and root collar diameter increment. 401

Treatments are fixed factors (three water regimes: wet, moderately wet, moderately dry, see methods 402

for details). Genotypes include one parental line and 3 putative polyploidy lines. Df, degree of freedom, 403

degree of significance, * P<0.05,**P<0.01. 404

405

Df Height increment Root collar diameter 406 increment 407

Treatments 2 3.4* 3.2* 408

Genotype 3 5.3** 2.9* 409

Treatments x Genotype 6 ns ns 410

411 412

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413 414 415 416 417 418 419 420 421

422

423

424

425

426

427

428

429

430

431

432

Figure 1. Fluorescence microscopic photographs (converted to black and white) of chromosomes of A. 433

crassicarpa (A = diploid parent, 2n = 26; B = clone, 4n = 52). Scale bar = 10 µm. 434

A B

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435 Figure 2. Leaves of diploid parents (left) and putative polyploid clones (right). A=A. crassicarpa*, 436

B=B. javanica, C=D. muelleri, D=E. angustifolius, E=E. dunnii, F=E. grandis, G=E. microcorys, H=E. 437

robusta, I=F. bourjotiana, J=G. leichhardtii, K=P. pinnata, L=T. ciliata, M=A. robusta, N=A. 438

cunninghamii, O=A. bidwillii, P=P. radiata. *confirmed tetraploid. Scale bar = 10 µm. 439

A CB D

E G

I K L

M O P

F H

J

N

18

440 441

442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479

Figure 3. Stomatal aperture size of diploid parents (left) and putative polyploid clones (right). A=A. 480

crassicarpa*, B=B. javanica, C=D. muelleri, D=E. angustifolius, E=E. dunnii, F=E. grandis, G=E. 481

microcorys, H=E. robusta, I=F. bourjotiana, J=G. leichhardtii, K=P. pinnata, L=T. ciliata, M=A. 482

robusta, N=A. cunninghamii, O=A. bidwillii, P=P. radiata. Arrows indicate the wax plugs in conifers. 483

*confirmed tetraploid. Scale bar = 20 µm. 484

A B C

D E F

G H

J K

I

L

M N

O P

19

485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524

525

Figure 4. Mean stomatal aperture length and stomatal density for parents (open bars) and putative 526

polyploid clones (closed bars). Bars and standard deviations describe measurements within one 527

examined leaf per plant. One diploid parent and one putative polyploid clone were studied for each 528

species. A. crassicarpa is a confirmed tetraploid. 529

Sto

mat

al d

ensi

ty (n

umbe

r/mm

2 )

0

100

200

300

400

500

600

700

Sto

mat

al a

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ngth

(µm

)

0

10

20

30

40StandardsClones

i i i s i

A. c

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P. r

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. rob

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. cun

ning

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