Freezing Impact on Cone Dehiscence, Samara Release and Seed Germination in Casuarina Cunninghamiana (Casuarinaceae)

Botany

Freezing impact on cone dehiscence, samara release and seed germination in Casuarina cunninghamiana (Casuarinaceae)

Journal: Botany

Manuscript ID cjb-2021-0031.R2

Manuscript Type: Note

Date Submitted by the 31-Mar-2021 Author:

Complete List of Authors: Riley, Ian; Niğde Ömer Halisdemir University, Department of Plant Production and Technologies Saygi, A.; Niğde Ömer Halisdemir University, Department of Plant ProductionDraft and Technologies Keyword: abiotic stress, cold tolerance, freezing damage, reproductive biology

Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue? :

1 Freezing impact on cone dehiscence, samara release and seed germination in Casuarina

2 cunninghamiana (Casuarinaceae)

3 Ian T. Riley, and A. Hayriye Saygı 4 Department of Plant Production and Technologies, Faculty of Agricultural Science and 5 Technologies, Niğde Ömer Halisdemir University, Niğde, Turkey 6 Corresponding author: Ian T. Riley, [email protected] 7

9 ORCID: Ian Timothy Riley- https://orcid.org/0000-0002-3592-0785

10 ORCDI: A. Hayriye Saygı - https://orcid.org/0000-0003-0956-1704 11 Draft

25 Abstract: Freezing, as a climatic extreme, can contribute to patterns of plant distribution and

26 this might operate through impacts on mechanisms of seed release. Therefore, the impact of freezing on samara release and seed germination in infructescences (cones) of Casuarina 27 cunninghamiana was assessed. Cones at field moisture content were frozen (22 h) and thawed 28 (2 h) though 0 to 5 cycles. Freezing impaired cone dehiscence and samara release (<1% 29 samaras released with ≥2 freezing cycles) and reduced germination from samaras frozen 30 while still in the cone (30 to 50% loss in total germination with 1 to 5 freezing cycles, 31 respectively). Seed germination from a sample of air dried samaras was only mildly impacted 32 (10% drop in total germination with 5 freezing cycles). This vulnerability of C. 33 cunninghamiana to freezing damage, particularly samara release, appears to be a novel

34 finding for woody perennials with fruiting structures retained in the canopy during winter, and

35 a potential contributory factor in speciesDraft persistence and invasiveness.

36 Key words: abiotic stress, cold tolerance, freezing damage, reproductive biology

37 Introduction

38 Climate and weather extremes, including heat, cold, drought, wind and solar radiation, are

39 well recognized as major determinants of plant distribution (Grace 1987). Freezing, causing

40 ice formation within plant tissues, can damage many organs with consequences for both plant

41 survival and distribution (Pearce 2001). The long term consequences for distribution will

42 operate, to varying degrees, through the impact of cold on reproductive biology include

43 diaspore production, release, dispersal, and seed viability and germination, as well as seedling establishment. Although there is considerable information on the effects of frost and cold 44 stress on fruiting and seedlings (Grace 1987 and many others since), the impact on diaspore 45 release has not been a focus of investigation. 46 Plants that produce fruiting structures that retain seeds within the canopy for varying 47 periods until conditions stimulate diaspore release through dehiscence can be serotinous, 48

49 weakly serotinous or facultatively serotinous, and during those periods these fruiting

50 structures can be exposed to freezing temperatures. The conifers of the Northern Hemisphere are commonly found at high altitudes and latitudes where subzero temperatures occur, and 51 although there have been no comprehensive studies, it appears (and is logical) that cone 52 freezing does not prevent their cones opening to release seeds, at least in most species (e.g., 53 Lev-Yadun 1995; Sharpe and Ryu 2015). Even in non-serotinous conifers, unopened 54 (immature) cones can overwinter on the tree and be exposed to freezing (e.g., Pinus sylvestris; 55 Hannerz et al. 2002; Tapias et al. 2004). Fire frequency has been a major driver for the 56 evolution of serotiny (Lamont et al. 2020), so similarly, freezing frequency could be a driver 57 of freezing tolerance of fruiting structures. Conversely, where subzero weather events are

58 infrequent, it is possible that both serotinous and non-serotinous fruiting structures are prone

59 to cold damage impairing diaspore release.Draft Also, even if release is not impaired, the seeds

60 they contain might not be freezing tolerant.

61 Casuarina cunninghamiana (Casuarinaceae), the subject of this study, is a sheoak species

62 with a wide natural distribution from 20 to 1000 m altitude, extending from 12 to 38° S,

63 which includes areas where subzero temperatures are common with up to 50 frosts per year

64 and temperatures as low as -8°C (Doran and Turnbull 1997 and Fig. S1). However, the cold

tolerance of C. cunninghamiana has had limited investigation. Provenances representative of 65 a wide range of climatic conditions in its native range were assessed for frost damage when 66 grown in California, USA, with most tolerant to -8°C under dry conditions but severely 67 damaged at lower temperatures (-13°C) (Merwin et al. 1995). Similarly, little is know about 68 the cold tolerance of other members of this family, but many occur in areas of Australia that 69 are subject to a moderate frequency of subzero temperatures [Fig. S2, and compare maps in 70 Riley (2019) with Australian plant hardiness zones in Dawson (1991) available at 71 www.anbg.gov.au/gardens/research/hort.research/zones.html]. This study determined the 72

73 effect of freezing on samara release and seed germination in C. cunninghamiana for two

74 purposes: (1) to further understand the nature of the recently proposed mechanism of samara release in sheoaks (Riley 2020), and (2) predict the possible contribution of subzero weather 75 conditions in limiting secondary spread of C. cunninghamiana as a potentially invasive 76 species (Riley and Korkmaz 2019). 77 Materials and Methods 78 Plant samples 79 About 1 kg of cones were collected (6 November 2019) from the lower branches of a 80 single C. cunninghamiana tree on Çukurova University campus, Adana Province, Turkey 81 (37°03’28.4” N, 35°21’24.7” E; no permit required). Cones, other than those used for the

82 freezing experiment (described below), were allowed to air dry for more than 2 weeks at room

83 temperature and samaras collected, storedDraft in a sealed container at room temperature.

84 Samara release

85 To test the effect freezing-thawing cycles on samara release, within 24 h of collection, 102

86 fresh cones of representative size were arbitrarily placed in six 17-well (10 ml) trays, five of

87 which were frozen at -20°C. The remaining tray was kept at room temperature as an unfrozen

88 control (0 freezing cycles). Over the next 5 days, the trays were thawed for 2 h at room

temperature (in a single layer on a bench) and all but one tray refrozen to give 1 to 5 freezing 89 cycles (22 h frozen and 2 h thaw). After the final thaw, the trays (uncovered) were kept at 90 room temperature in a closed cupboard for over 2 weeks for the cones to air dry. The cone and 91 any released samaras were transferred to a Petri dish for examination and the cone dropped 5 92 times from a height of 50 mm to dislodge any loosely retained samaras. The number of fertile 93 whorls (with 3 or more developed carpels) and the number of carpels per whorl were 94 recorded, and the number of potentially fertile carpels was estimated as the product of these 95 two values. Released and retained samaras were recorded; in all cases the wings of retained

97 samaras were visible because bracteoles partially open even when dehiscence was incomplete

98 (Fig. 1). These data confirmed that the subsamples to which the treatments were applied were statistically homogenous (Table S1) and were consistent with the previously reported 99 morphometrics (Riley and Korkmaz 2019). To determine if failed release could be remediated 10 0 by rehydration and subsequent air drying, six cones from each treatment were submerged in 10 1 water (24 h at 5°C to avoid stimulating germination) then allowed to air dry at room 10 2 temperature and the assessments detailed above repeated. 10 3 Germination 10 4 One hundred samaras (5 replicates of 20 samaras) from the bulk samaras (air dry) were 10 5 frozen through 0 to 5 cycles as for the cones. Germination was tested on moist peat at ~25°C

10 6 and constant light (suitable conditions for C. cunninghamiana; Turnbull and Martensz 1982) 10 Draft 7 in complete blocks. Germination was recorded weekly for 2 weeks. Also, to determine if 10 freezing had affected the germination of seeds retained within cones that had undergone 8 10 freezing cycles, five replicates of 20 released samaras (0 to 1 cycles) and single cones with 9

11 retrained samaras (2 to 5 cycles) were placed on peat (as above) in complete blocks. The 0

11 cones were placed laterally and embedded to about 20% of the width of the cone. 1

11 Germination was recorded weekly for 3 weeks. 2 Statistical analysis 11 3 Data analysis was performed with R ver. 3.6.3 (R Core Team 2020). Least squares linear 11 4 and logarithmic regression was conducted for cone morphometrics, samara release and seed 11 5 germination data, as appropriate, after checking the data for skewness and kurtosis, and visual 11 6 examination for outliers using box plots (no observed data were missing or rejected). The 11 7 model formula used removed the effect of blocking. 11 8 Results 11 9

12 0

12 Dehiscence of bracteoles and release of samaras were both affected by freezing (Figs 1 and 1

12 2A, and Table S2). A single freezing cycle reduced samara release from over 98% to about 2 69%. For two or more freezing cycles, samara release was <1% with two cycles falling to no 12 3 release with five cycles. The failure to release samaras appears to have been, in part, due to 12 4 the failure of bracteoles of frozen cones to open widely upon drying (Fig. 1). However, the 12 5 degree of dehiscence seemed potentially sufficient (Fig. 1) to allow more samara release than 12 6 observed, but the samaras remained tightly held at their proximal end and had not risen far 12 7 above the bracteoles as previously described (Riley 2020), and could not be removed with 12 8 forceps. In all cases, the bracteoles opened sufficiently to reveal the presence of samaras in 12 9 fertile florets. When the cones were rehydrated the bracteoles in most cases fully reclosed

13 0 (Fig. 3), although not fully where the wing of retained samaras extended slightly beyond the 13 Draft 1 bracteoles (particularly with one freezing cycle). After subsequent air drying, some retained 13 samaras were released (Fig. 2B). However, this single rehydration-drying cycle did not 2 13 facilitate release of the few samaras retained in unfrozen cones, with these samaras being 3

13 mostly in carpels that failed to develop normally. However, rehydration-drying of cones that 4

13 had only been frozen once resulted in release of 71% of the retained samaras (a combined 5

13 release of 91%). The effect of rehydration-drying diminished logarithmically (Fig. 2B) for 6 cones that had undergone more than one freezing cycle. By five freezing cycles, rehydration- 13 7 drying resulted in 6% of retained samaras being released, and given that no samaras were 13 8 released before rehydration, this represents the total samara release. 13 9 Germination of seeds in samaras from the bulk sample was only slightly affected by 14 0 freezing cycles (Fig. 2B). There was slight but significant (p = 0.037) decline in germination 14 1 after 1 week; without freezing, germination (fitted value) was about 57% and with five 14 2 freezing cycles it fell to 47%. After 2 weeks (data not shown), these values were 59 and 46%, 14 3 respectively, so there was little additional germination and no indication that freezing had

14 4

14 delayed germination. However, the results were distinctly different for the germination of 5

14 seeds from cones that had been frozen (Fig. 4). Unlike the samaras in the first germination 6 test, in which air dried samaras were frozen, seeds within in frozen cones would have been at 14 7 field moisture content (perhaps only losing a small proportion of moisture with each freezing 14 8 cycle). After 1 week (Fig. 4A), germination of unfrozen seeds was about 67%, and this did 14 9 not change over the 3 weeks (Fig. 4B,C). However, with one freezing cycle there was a large 15 0 drop in germination to around 40%, which again did not change over the 3 weeks (Fig. 4). For 15 1 freezing cycles 2 to 5, insufficient samaras were released for germination testing, so the data 15 2 presented in Fig. 4 for these treatments are germination from samaras retained within cones. 15 3 After 1 week (Fig. 4A), there was only a single germinant from retained samaras but this

15 4 increased after 2 and 3 weeks to give 6 and 15% germination, respectively, from cones frozen 15 Draft 5 twice (Fig. 4C). After five freezing cycles, germination after 2 and 3 weeks in retained 15 samaras was only 3 and 6% (Fig. 4C). For two freezing cycles after 3 weeks, the extrapolated 6 15 germination of released samaras was 15%, the same as the observed value for the retained 7

15 samaras (Fig. 4C). 8

15 Representative examples of germination from samaras retained in cones are shown in Fig. 9

16 5. Mostly germinants emerged from carpels closest to the substrate. Where the cotyledons did 0 not release easily, the hypocotyls became swollen and twisted, a response which might aid 16 1 cotyledon escape from the only partially open bracteoles. Although saprophytic fungal 16 2 colonization of all cones occurred rapidly (within a week and before emergence of any 16 3 seedlings), no pathogenic colonization of the seedlings occurred. So apart from the 16 4 mechanically induce malformation, the seedlings were healthy. 16 5 Discussion 16 6 The key finding from this study was that samara release from C. cunninghamiana cones 16 7 was substantially impaired by a single freezing cycle and prevented by two or more freezing

16 8

16 cycles. Rehydration remediated this effect to some extent but decreasingly so with more 9

17 freezing cycles. Although, the germination of air-dry samaras was only mildly impacted by 0 freezing, there was a clear decline in germination from samaras frozen at field moisture 17 1 content within cones even with one freezing cycle. These results indicate that the mechanism 17 2 of bracteole dehiscence and samara release, and the seed viability, are vulnerable to repeated 17 3 freezing events. Therefore, although this species occurs naturally in areas with temperature 17 4 dropping to -8°C (Doran and Turnbull 1997), it appears that diaspore release and seed 17 5 viability in C. cunninghamiana are not tolerant of freezing. It is possible that the provenance 17 6 of the material used in this study was not in the colder part of the natural range of C. 17 7 cunninghamiana so this might not apply to accessions from those areas. Alternatively, even

17 8 with frosty conditions in those areas, cones in the canopy are unlikely to freeze. Extreme cold 17 Draft 9 in these areas is likely to be associated with temperature inversions and with mature C. 18 cunninghamiana growing to 20 to 35 m (Doran and Turnbull 1997), the cones in the upper 0 18 canopy are unlikely to freeze. Therefore, it is likely that tolerance to extreme cold has not 1

18 evolved in C. cunninghamiana given the lack of selection pressure in Australia. 2

18 Flowering in C. cunninghamiana extends from autumn to spring producing cones that 3

18 remain on the tree for less than 12 months before releasing samaras (Boland et al. 1996 cited 4 in CABI 2019). Samara release mostly occurs in autumn or spring depending on the access of 18 5 the tree to water (Woolfrey and Ladd 2001), so there appears to be wide phenotypic plasticity 18 6 in this species and phenotypic data covering its extensive range have apparently not been 18 7 systematically collected. Nevertheless, it appears that unopened cones are held in the crown 18 8 over winter and these could potentially be exposed to damage by extreme cold. However, in 18 9 the Australian context, such weather events are rare, and unlikely to impact population 19 0 recruitment and persistence in the native range of C. cunninghamiana. 19 1

19 2

19 Plants have mechanisms that can prevent ice nucleation (with the lower limit around - 3

19 40°C) but, when it occurs, damage is mostly due to dehydration leading to the death of living 4 tissue (Pearce 2001). However, in the frozen cones, impaired dehiscence was most likely to 19 5 have operated through structural damage to senescing fibrous tissues within the cone caused 19 6 by internal ice nucleation rather than a process of simple dehydration and death of cells. 19 7 Dehydration, both slow air drying and rapid oven drying, causes the bracteoles of sheoak 19 8 cones to dehisce (Turnbull and Martensz 1982), so tissue dehydration by ice formation is 19 9 unlikely to be the mechanism. In this work, freezing was to -20°C, well below temperatures 20 0 sheoaks would experience in Australia, so this ensured that ice crystal formation would occur 20 1 and reveal any potential vulnerably in the mechanism of cone dehiscence, which might not

20 2 have been evident by gross symptomatology with freezing at warmer temperatures. With 20 Draft 3 repeated freezing events, extracellular freezing will increase due to the presence of water 20 drawn from the cells during the in the previous freezing event (Pearce 2001), so it is 4 20 reasonable to expect repeated freezing cycles will increase damage, as observed, with the 5

20 second freezing cycle effectively preventing samara release. This damage prevented full 6

20 dehiscence of bracteoles and, presumably, impaired incurving of the lower bracteole margins 7

20 (Riley 2020). Clearly additional work is needed to determine the threshold temperature for 8 freezing damage with a range of subzero temperatures including those that occur in the extant 20 9 range of sheoaks, as well as, anatomical studies to determine the nature of the structural 21 0 damage to the fibrous tissues that impairs samara release. 21 1 Freezing tolerance of Australian flora in general, and C. cunninghamiana in particular, 21 2 could reasonably be assumed to low because only a small area of the continent has more than 21 3 10 days with temperatures falling below -5ºC (Fig. S1). Also, in Australia, radiation frost, 21 4 which occurs close to the ground, is the most common (Bureau of meteorology, Australian 21 5 Government, www.bom.gov.au/climate/map/frost/what-is-frost.shtml), so most shrubs and

21 6

21 trees are unlikely to be severely affected by freezing, or placed under significant selection 7

21 pressure for freezing tolerance. A number of sheoaks, including C. cunninghamiana, are now 8 globally distributed in tropical and subtropical climates, and considered invasive (Potgieter et 21 9 al. 2014), but none have been reported to have naturalized in areas with extended cold 22 0 periods. Although, cold as a determinant of climatic suitability for Australia flora could 22 1 reasonably be assumed to mostly likely be a consequence of vegetative rather than 22 2 reproductive sensitivity. 22 3 Although seeds in air dried samaras were not particularly sensitive to freezing, seeds at 22 4 field moisture content within the frozen cones were. Sheoak seeds (Casuarina equisetifolia, as 22 5 species on average from warmer climates that C. cunninghamiana) has been successfully

22 6 stored frozen for 2 years (Jones 1967), so the freezing tolerance of air-dry seeds is known. 22 Draft 7 However, in the field, sensitivity of seeds within cones or shed samaras on the ground cannot 22 be predicted by this, as their moisture content will be higher and they could be exposed to 8 22 repeated freezing cycles. So the freezing sensitivity of seeds could reasonably be assumed to 9

23 also contribute to plant climatic adaptation (Grace 1987). 0

23 An unexpected finding of this study was the ability of seeds in retained samara to 1

23 germinate and establish from within a cone (Fig. 5). Samaras retained within cones was 2 recently postulated as an alternative, albeit minor, means of sheoak dispersal (Riley 2020). It 23 3 is therefore feasible that in a water dispersed context, which is likely for C. cunninghamiana 23 4 (Woolfrey and Ladd 2001) and other facultatively or weakly serotinous Casuarina spp. that 23 5 grow adjacent to waterbodies, shed cones might disperse differently and at a different times to 23 6 samara, so could contribute to the distribution of the species. 23 7 This study has touched upon some aspects of freezing tolerance of plants that have not 23 8 been widely researched and are new observations for an Australian sheoak (C. 23 9 cunninghamiana). However, this initial investigation was not broad, so future work could

24 0

24 consider how these findings apply to different provenances of C. cunninghamiana or different 1

24 species in the Casuarinaceae. 2 In conclusion, cone dehiscence, samara release and seed viability in one specimen of C. 24 3 cunninghamiana were found to be vulnerable to freezing damage, which is consistent with the 24 4 presumed low selection pressure for freezing tolerance in its native range. Also, research on 24 5 the effect of freezing on release diaspores from infructescences of woody perennials appears 24 6 to be novel and so might be productively applied to taxa (such as in the conifers) from 24 7 climatic zones that range from no to high selection pressure for freezing tolerance. 24 8 Acknowledgements 24 9 Eniola Ajibola Olowu and Kaddijatou Jawneh are thanked for their help in sample

25 0 collection and support to AHS in the completion of her thesis project. AHS was supported on 25 Draft 1 a scholarship provided by the Ayhan Şahenk Foundation. 25 References 2 25 Boland, D.J., Moncur, M.W., and Pinyopusarerk, K. 1996. Review of some floral and 3

25 vegetative aspects to consider when domesticating casuarina. In Recent Casuarina 4

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26 113–130. doi:10.1111/j.1469-8137.1987.tb04686.x. 6 Hannerz, M., Almqvist, C., and Hörnfeldt, R. 2002. Timing of seed dispersal in Pinus 26 7 sylvestris stands in central Sweden. Silva Fenn. 36: 757–765. doi:10.14214/sf.518. 26 8 Jones, L. 1967. Effects of storage at various moisture contents and temperatures on seed 26 9 germination of silk oak, Australian pine, and Eucalyptus spp. Southeastern Forest 27 0 Experiment Station, USDA Forest Service, Asheville, NC, USA. 27 1 Lamont, B.B., Pausas, J.G., He, T., Witkowski, E.T.F., and Hanley, M.E. 2020. Fire as a 27 2 selective agent for both serotiny and nonserotiny over space and time. Crit. Rev. Plant Sci. 27 3 39: 140–172. doi:10.1080/07352689.2020.1768465.

27 4 Lev-Yadun, S. 1995. Living serotinous cones in Cupressus sempervirens. Int. J. Plant Sci. 27 Draft 5 156: 50–54. doi:doi.org/10.1086/297228. 27 Merwin, M.L., Martin, J.A., and Westfall, R.D. 1995. Provenance and progeny variation in 6 27 growth and frost tolerance of Casuarina cunninghamiana in California, USA. For. Ecol. 7

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2931 agroecosystem improvement in semi-arid areas with a focus on Central Anatolia, Turkey. 04 Front. Agric. Sci. Eng.: [Epub ahead of print] 16 pp. doi:10.15302/J-FASE-2019270. 2931 15 Riley, I.T. 2020. Infructescence and samara morphometrics and potential mechanism of 2931 26 samara release in Allocasuarina and Casuarina (Casuarinaceae). Aust. J. Bot. 68(2): 108- 2931 37 118. doi:10.1071/BT19153. 2931 48 Riley, I.T., and Korkmaz, L.N. 2019. Identity of the Casuarina sp. in Turkey. Turk. J. Weed 2931 59 Sci. 20(2): 159–168. 2932 60 Sharpe, M., and Ryu, S.R. 2015. The moisture content and opening of serotinous cones from 2932 71 lodgepole pine killed by the mountain pine beetle. For. Chron. 91: 260–265.

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33 Figure captions 7

33 Fig. 1. Air dried cones of Casuarina cunninghamiana following 0 to 5 cycles of freezing (- 8 20°C for 22 h) and thawing (room temperature for 2 h), and air drying (2 weeks). (A-F) 0 to 5 33 9 cycles. All samaras were released from dried unfrozen cones (A), most were released after 1 34 0 freezing cycle (B) and none were released after 2 to 5 freezing cycles (C-F). 34 1

34 2 Fig. 2. (A) Release of samaras (percentage of total samara released) from air dried cones of 34 3 Casuarina cunninghamiana following 0 to 5 cycles of freezing (-20°C for 22 h) and thawing 34 4 (room temperature for 2 h), and air drying (2 weeks). (B) Release of retained samaras 34 5 (percentage of retained samaras released) from the same cones after subsequent rewetting (24

34 6 h at 5°C) and air drying (2 weeks). Linear regression: ln(germination + 1) = 5.28 - 0.848 ×

34 2 Draft 7 cycle, R = 0.742, p < 0.001. C, Germination (after 1 week) of seeds in C. cunninghamiana 34 samaras following the same 0 to 5 cycles of freezing and thawing (from bulk samaras from 8 34 cones collected at the same time as those in A and B). Linear regression: germination = 57.0 - 9

35 2.11 × cycle, R2 = 0.488, p = 0.037. Box plots are for quartiles with mean as the larger closed 0

35 circle and outliers as small open circles. 1

35 2 Fig. 3. Rehydrated cones of Casuarina cunninghamiana that had undergoing 0 to 5 cycles of 35 3 freezing (-20°C for 22 h) and thawing (room temperature for 2 h), and air drying (2 weeks). 35 4 (A-F) 0 to 5 cycles. Bracteoles reclosed, or almost so, in most cases and regreened. In cones 35 5 that had undergone one freezing-thawing cycle (B), partially projecting samara wings 35 6 prevented full reclosure of some bracteoles. 35 7

35 8 Fig. 4. Germination over 3 weeks (A, B and C) of seed in samaras released (cycles 0 and 1) 35 9 from or retained (cycles 2 to 5) within cones of Casuarina cunninghamiana following 0 to 5

36 0

cycles of freezing (-20°C for 22 h) and thawing (room temperature for 2 h), and air drying (2

weeks). Germination of seed in samaras following a single freezing cycle was partially

suppressed. Germination of seed having underdone two or more freezing cycle was delayed

and limited (see Fig. 5), but with some germination across all freezing cycles. Week 3 linear

regressions: released samaras, germination = 67 - 26 × cycle, R2 = 0.820, p = 0.020; retained

samaras, germination = 20.2 - 2.88 × cycle, R2 = 0.733, p = 0.007. Box plots are for quartiles

with mean as the larger closed circle and outliers as small open circles.

Fig. 5. Representative examples of germination of seed in samaras retained within cones of

Casuarina cunninghamiana following two or more cycles of freezing (-20°C for 22 h) and

thawing (room temperature for 2 h), followed by air drying (2 weeks). (A) Germination was

mostly from samaras in close contact withDraft the peat substrate. (B) Where the cotyledons did

not release easily, the hypocotyls became swollen and twisted, a response which might aid

cotyledon release from the bracteoles.

A B C

D E F

2 mm Draft

Fig. 1. Air dried cones of Casuarina cunninghamiana following 0 to 5 cycles of freezing (-20°C for 22 h) and thawing (room temperature for 2 h), and air drying (2 weeks). (A-F) 0 to 5 cycles.

All samaras were released from dried unfrozen cones (A), most were released after 1 freezing cycle (B) and none were released after 2 to 5 freezing cycles (C-F).

100 A

Release (%) 20

100 B

Release (%) 20

100 C

60 40 Draft

Germinatin (%) 20

012345 Freezing cycles

Fig. 2. (A) Release of samaras (percentage of total samara released) from air dried cones of

Casuarina cunninghamiana following 0 to 5 cycles of freezing (-20°C for 22 h) and thawing

(room temperature for 2 h), and air drying (2 weeks). (B) Release of retained samaras (percentage

of retained samaras released) from the same cones after subsequent rewetting (24 h at 5°C) and air

drying (2 weeks). Linear regression: ln(germination + 1) = 5.28 - 0.848 × cycle, R2 = 0.742, p <

0.001. C, Germination (after 1 week) of seeds in C. cunninghamiana samaras following the same

0 to 5 cycles of freezing and thawing (from bulk samaras from cones collected at the same time as

those in A and B). Linear regression: germination = 57.0 - 2.11 × cycle, R2 = 0.488, p = 0.037.

Box plots are for quartiles with mean as the larger closed circle and outliers as small open circles.

A B C

D E F

2 mm Draft

Fig. 3. Rehydrated cones of Casuarina cunninghamiana that had undergoing 0 to 5 cycles of freezing (-20°C for 22 h) and thawing (room temperature for 2 h), and air drying (2 weeks). (A-F)

0 to 5 cycles. Bracteoles reclosed, or almost so, in most cases and regreened. In cones that had undergone one freezing-thawing cycle (B), partially projecting samara wings prevented full reclosure of some bracteoles.

<− − Released samaras − −> 80 A

40 <− − − − − − − − Retained samaras in cones − − − − − − − −>

20 Week 1 0

80 B

20 Germinatin (%) Week 2 0

80 C

60 40 Draft 20 Week 3 0

012345 Freezing cycles

Fig. 4. Germination over 3 weeks (A, B and C) of seed in samaras released (cycles 0 and 1) from

or retained (cycles 2 to 5) within cones of Casuarina cunninghamiana following 0 to 5 cycles of

freezing (-20°C for 22 h) and thawing (room temperature for 2 h), and air drying (2 weeks).

Germination of seed in samaras following a single freezing cycle was partially suppressed.

Germination of seed having underdone two or more freezing cycle was delayed and limited (see

Fig. 5), but with some germination across all freezing cycles. Week 3 linear regressions: released

samaras, germination = 67 - 26 × cycle, R2 = 0.820, p = 0.020; retained samaras, germination =

20.2 - 2.88 × cycle, R2 = 0.733, p = 0.007. Box plots are for quartiles with mean as the larger

closed circle and outliers as small open circles.

A B

10 mm

Fig. 5. Representative examples of germination of seed in samaras retained within cones of

Casuarina cunninghamiana following two or more cycles of freezing (-20°C for 22 h) and thawing (room temperature for 2 h), followedDraft by air drying (2 weeks). (A) Germination was mostly from samaras in close contact with the peat substrate. (B) Where the cotyledons did not release easily, the hypocotyls became swollen and twisted, a response which might aid cotyledon release from the bracteoles.