Incubation under fluctuating temperatures reduces ...

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Bradford (1996), this is a functional definition of seed dormancy. ... values (Bradford and Somasco, 1994; Kebreab and. Murdoch, 1999 .... The coefficients of determination (r 2) of the .... (2000) for C. acanthoides, and Forcella and Wood (1986).
Seed Science Research (2005) 15, 89 – 97

DOI: 10.1079/SSR2005199

Incubation under fluctuating temperatures reduces mean base water potential for seed germination in several non-cultivated species Roberto Huarte1* and Roberto L. Benech-Arnold2 1

Ca´tedra de Fisiologı´a Vegetal, Facultad de Ciencias Agrarias, Universidad Nacional de Lomas de Zamora, Ruta 4 Kilometro Z. Llaudllol. CP (1836), Repu´blica Argentina; 2IFEVA, Ca´tedra de Cerealicultura, Facultad de Agronomı´a, CONICET/Universidad de Buenos Aı´res, Avenida San Martı´n 4453, CP (1417), Ciudad de Buenos Aı´res, Repu´blica Argentina

Abstract Seeds of Carduus acanthoides, Cynara cardunculus, Cirsium vulgare, Brassica campestris, and Sisymbrium altissimum were incubated at a range of decreasing osmotic potentials (Co) under fluctuating temperatures or the median temperature of the fluctuation cycle. Fluctuating temperatures promoted total seed germination in water and at reduced osmotic potential. Total germination was reduced as the Co decreased. However, this trend was smallest under fluctuating temperatures, signalling a higher tolerance of seeds to reduced osmotic potential. Effects of osmoticum and temperature were modelled with the hydrotime model. The parameters estimated from the model, the hydrotime constant (uH), the mean base water potential Cb(50) and its standard deviation (sCb) gave good descriptions of germination time courses. For all species, incubation under fluctuating temperatures shifted Cb(50) values downwards without modifying their distribution substantially. This accounted for the greater tolerance of germination to reduced Co under fluctuating temperatures. To confirm that these effects were mediated by temperature fluctuations per se, the behaviour of C. acanthoides and C. cardunculus incubated at the minimum, the mean and the maximum temperature of the fluctuation cycle was also analysed. Constant maximum and minimum temperatures of the cycle did not stimulate germination, nor did they shift Cb(50) towards more negative values. The hydrotime model provides a physiologically based quantitative

*Correspondence Fax: þ54 11 4282 0233 Email: [email protected]

description for germination promotion due to fluctuating temperature. Keywords: hydrotime model, fluctuating temperatures, Carduus acanthoides, Cynara cardunculus, Cirsium vulgare, Brassica campestris, Sisymbrium altissimum

Introduction Fluctuating temperatures terminate seed dormancy in various species (Thompson and Grime, 1983; Probert, 1992). Numerous reports reveal that incubation under fluctuating temperature regimes enhances germination in comparison to that observed under constant temperature regimes (Ekstam et al., 1999; BenechArnold et al., 2000). Responses to fluctuating temperatures are consistent among seeds of many species, but the physiological mechanisms underlying such responses are still unknown. One initial step towards understanding the physiological basis of seed response to fluctuating temperatures could be the comparison of parameters derived from the hydrotime model (Gummerson, 1986). The hydrotime model relates the time to germination to the difference between the actual water potential (C) and the threshold or base water potential (Cb) for germination to occur. The hydrotime model may be described as follows: uH ¼ ½C 2 Cb ðgÞtg

ð1Þ

where Cb(g) is the threshold or base water potential below which germination of fraction, g, of the seed population will be prevented; uH is the accumulated hydrotime (expressed in MPa h or MPa d) required from imbibition to radicle emergence; and tg is the germination time for the corresponding fraction, g, of seeds. As uH is a constant and C is determined by the

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environment surrounding the seeds, differences in t among seeds are based on the distribution of Cb within the seed population (Bradford, 1990). It has been shown that the Cb value generally varies among seeds in a population according to a normal distribution that can be characterized by the median Cb(50) and standard deviation (sCb) in Cb(g) values among seeds (Bradford, 1995). Germination time courses in different C can be described by the probit equation (Bradford, 1990): probitðgÞ ¼ ½C 2 ðuH =tg Þ 2 Cb ð50Þ=sCb

ð2Þ

The value of uH quantifies the germination speed, which varies according to the species evaluated and the physiological status of the seeds. The sCb value estimates the germination uniformity of a seed population, while Cb(50) indicates sensitivity to C. For a given uH, a seed population with a more positive value will germinate more slowly than a population with a more negative Cb(50) value. The fraction of the population whose Cb is higher than the C of the incubation medium will be completely prevented from germination. If a fraction of the seed population has a Cb higher than 0 MPa, then its germination will be prevented even in pure water. According to Bradford (1996), this is a functional definition of seed dormancy. The Cb concept has physiological implications. Its value is related to the ability of the embryo to overcome restraints to its growth (imposed either by the conditions on the embryo itself or those of the surrounding tissues). This ability is susceptible to modification by different endogenous (i.e. dormancy level of the seed population) or exogenous (temperature, light, exogenously applied hormones) factors (Ni and Bradford, 1993; Bradford, 1995). Factors that stimulate germination result in decreased Cb(50) (i.e. more negative values), whereas those that inhibit germination tend to increase Cb(50) (i.e. less negative values). For example, dormancy alleviation through dry after-ripening or exogenous application of gibberellins (GAs) results in a decrease of Cb(50) (Ni and Bradford, 1993; Christensen et al., 1996). On the other hand, germination inhibitors such as abscisic acid (ABA) shift Cb(50) values upward (Ni and Bradford, 1992). Similarly, the progressive inhibition of germination at supra-optimal temperatures was accompanied by a progressive increment of Cb(50) values (Bradford and Somasco, 1994; Kebreab and Murdoch, 1999; Alvarado and Bradford, 2002). Within this framework, it could be hypothesized that germination promotion produced by fluctuating temperatures is the result of a displacement of Cb(50) to more negative values. The objective of this study was to obtain a quantitative description of the responses to temperature fluctuations in several

non-cultivated species. Specifically, we attempted to determine whether incubation under fluctuating temperatures promotes changes in the Cb(50) within seed populations, relative to those observed in seeds incubated under constant temperatures. Materials and methods Seeds Mature Brassica campestris (L.) and Carduus acanthoides (L.) seeds were collected during March 2000 at the INTA Balcarce Experimental Station (latitude 378450 S, longitude 588150 W). Cirsium vulgare (Savi) Ten., Cynara cardunculus (L.) and Sisymbrium altissimum (L.) seeds were gathered at the same location during 2001 (first set of experiments). C. acanthoides and C. cardunculus seeds were also collected near Lomas de Zamora, Argentina (348480 S, 588310 W) during March 2003 and January 2004, respectively (second set of experiments). After initial cleaning, seeds were kept in paper bags at room temperature (20 ^ 28C) before use. Germination tests Six and four replicates of 50 seeds each of B. campestris and C. acanthoides, respectively, were incubated in germination chambers at constant (258C) or fluctuating (20/308C; 12/12 h) temperatures. Five replicates of 25 seeds of C. cardunculus, C. vulgare and S. altissimum were incubated at 158C or 20/108C (12/12 h). In addition, three replicates of 50 seeds of C. acanthoides were incubated in germination chambers at constant temperatures of 20, 25 and 308C, and under a fluctuating temperature of 20/308C (12/12 h). Similar experiments were conducted with C. cardunculus seeds incubated at constant temperatures of 10, 15 and 208C and a fluctuating temperature of 20/108C (12/12 h). In all cases, seeds were incubated in darkness, and Petri dishes were covered with plastic film to prevent evaporation. Seeds were placed on two filter papers in 9-cm Petri dishes containing 5 ml of distilled water or polyethylene glycol (PEG) 8000 solutions (Anedra, Buenos Aı´res, Argentina) prepared according to the Michel equation (Michel, 1983). Separate solutions were prepared to give equivalent C at each temperature. For incubation at fluctuating temperatures, the PEG concentration calculated for the corresponding average temperature was used (i.e. for the regime 20/108C the same solution as that prepared for 158C was used, and for the 20/308C regime the solution prepared for use at 258C was used). A subtle variation in the osmotic potential of the solution is to be expected according to fluctuation of temperatures within the cycle. For instance, for an alternation

Fluctuating temperatures reduce mean base water potential of 20/108C, at 208C the potential, calculated according to the Michel equation, will be 2 0.291 MPa instead of 2 0.3 MPa, and at the minimum cycle temperature (108C) the potential will be 2 0.308 MPa. A vapour pressure osmometer (Model C 5200; Wescor Inc., Logan, Utah, USA) calibrated against NaCl solutions was used to corroborate the resulting Co. The values of Co used were 0 (distilled water), 20.3, 20.5, 2 0.8 and 2 1.11 MPa. For C. cardunculus and C. acanthoides, seed responses to 2 0.15 MPa were also evaluated. Seeds incubated in water or PEG solutions were transferred to fresh solutions after the first 24 h and then after 6 d, to maintain a constant C. Germination was scored during 14 consecutive days (except for C. cardunculus seeds incubated at 108C, which were kept in the germination chambers for one more week). Visible radicle protrusion indicated completion of germination and those seeds were removed. Data analysis Data analyses to determine the values of the model parameters were conducted using repeated probit regression analysis, as described previously by Bradford (1990, 1995). Results Fluctuating temperatures promoted total germination of C. cardunculus, B. campestris and C. acanthoides at 0 MPa (Fig. 1F, I and J). In contrast, C. vulgare and S. altissimum seeds incubated at 0 MPa germinated equally well under both thermal regimes (Fig. 1G and H). A reduction of Co delayed and lowered total germination (Fig. 1A–J); however, higher germination percentages were observed under fluctuating temperatures in comparison to those recorded under constant regimes. These results show that fluctuating temperatures reduced seed sensitivity to reduced water potentials. Germination data recorded for each species were analysed using the hydrotime model to quantify changes in water relations parameters produced by incubation under the fluctuating thermal regime. Data were converted into probit values and plotted against Cb ðgÞ ¼ C 2 ðuH =tg Þ, using different values of uH until the best fit was obtained. The resulting parameters were used to predict seed germination time courses according to equation (2), converting probit values back into germination percentages. Because of the good match obtained between experimental and predicted data, the parameters derived from the hydrotime model were used to compare the effects of the incubation thermal regime on the water relations in seeds of all the species studied here. In all cases, fluctuating temperatures shifted Cb(50) to more negative values (Table 1), consistent

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with the higher total germination observed at reduced C under fluctuating temperatures. With the exception of C. vulgare and B. campestris, the downwards displacement in Cb(50) was not accompanied by a substantial modification of its standard deviation (sCb) relative to that obtained under constant temperatures (Table 1). A consistent increase in uH values under fluctuating temperatures was observed (Table 1). The coefficients of determination (r 2) of the probit regressions ranged from 0.67 to 0.96 (Table 1). The response of each seed population to the thermal regime of incubation can be illustrated by plotting the normal distribution of Cb(g) using the Cb(50) and its sCb values. In all species evaluated, the Cb(50) distribution shifted to more negative values when incubation was conducted under fluctuating temperatures (Fig. 1K– O). To rule out the possibility that the promotion of total germination was mediated by the minimum or maximum temperatures of the fluctuation cycle, instead of temperature fluctuations per se, seeds from two species (C. acanthoides and C. cardunculus), representing responsiveness to 20/308C or 20/108C, were further tested for germination under osmotic stress by incubating them not only under the stimulatory fluctuating regime and the average between the extremes, but also at the minimum and maximum temperatures of the fluctuation cycle. At 0 MPa, fluctuating temperatures promoted total germination of C. cardunculus seeds (Fig. 2D). C. acanthoides seeds achieved similar total germination in all thermal treatments, with the exception of 308C, where a sharp reduction of total germination was scored (Fig. 3A –D). As expected, a reduction in Co gradually delayed and reduced total germination, but this effect was particularly noticeable at 108C (the minimum temperature of the fluctuation cycle) for C. cardunculus, and at 308C (the maximum) for C. acanthoides seeds (Figs 2A and 3C, respectively), although in the latter case, even germination in distilled water was restricted. Again, fluctuating temperatures enhanced total germination at reduced osmotic potential (Figs 2D and 3D). These results confirm that the promotion of germination was caused by temperature fluctuation per se and not by any of the temperatures comprising the fluctuation cycle. Germination data recorded for each species and thermal treatments were analysed using the hydrotime model. In all cases, incubation under fluctuating temperature shifted Cb(50) values downwards (Figs 2 and 3, E–H). Similar Cb(50) values were determined after incubation under the different constant temperatures used, except for C. acanthoides seeds incubated at 308C, where the Cb(50) was a positive value. This is consistent with the drastic reduction in total germination percentages observed at this temperature, even when incubation was performed

R. Huarte and R.L. Benech-Arnold

20/10 C

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Figure 1. Germination time courses at constant temperatures (A– E) or fluctuating temperatures (F –J) at a range of water potentials. The symbols are the experimental data, and the curves are the time courses predicted by the hydrotime model at each C, based upon the parameters of Table 1. Distributions of base water potentials [Cb(g)] according to the thermal treatment are also shown (K –O).

Fluctuating temperatures reduce mean base water potential Table 1. Parameters of the hydrotime model characterizing germination of seeds incubated under constant and fluctuating temperature regimes. The hydrotime model was used to estimate the median base water potential [Cb(50)], the standard deviation of the base potential among seeds (sCb) and the hydrotime constant (uH), based upon germination time courses at a range of water potentials. The coefficients of determination (r 2) indicate the fractions of total variation accounted for by the model

C. vulgare C. cardunculus S. altissimum B. campestris C. acanthoides

T (8C)

Cb(50) (MPa)

sCb (MPa)

uH (MPa d)

r2

15 20/10 15 20/10 15 20/10 25 20/30 25 20/30

20.72 21.24 20.34 21.17 20.33 20.6 20.02 20.55 20.28 20.65

0.1 0.22 0.14 0.15 0.38 0.33 0.38 0.58 0.47 0.44

2.2 4.2 1.8 5.8 1.4 2 0.4 1 2.1 3.5

0.95 0.77 0.95 0.77 0.67 0.90 0.96 0.90 0.85 0.93

in distilled water. Only slight variation in sCb was observed in C. cardunculus. On the other hand, in C. acanthoides considerable variation in sCb among thermal treatments was registered (Figs 2 and 3, E–H). As in the first set of experiments, an increase in the uH value under fluctuating temperatures was observed (Figs 2 and 3, E –H). The r 2 of the probit regressions ranged from 0.83 to 0.93. The response of each seed population to the thermal regime of incubation was illustrated by plotting the normal distribution of Cb(g) using the Cb(50) and sCb values (Figs 2 and 3, E–H).

Discussion In several species, the exit from dormancy is completed only after the seeds have been exposed to fluctuating temperatures. The ecological significance for this requirement has been related to the possibility of detecting canopy gaps as well as depth of burial (Benech-Arnold et al., 2000). As expected, fluctuating temperatures promoted total seed germination in relation to that observed at constant temperature. This is in agreement with previous results of Soriano et al. (1963) for B. campestris, Kruk and Benech-Arnold (2000) for C. acanthoides, and Forcella and Wood (1986) for C. vulgare. In contrast, our C. vulgare results under fluctuating temperatures were inconsistent with those published by Groves and Kaye (1989); the higher incubation temperatures used in their studies (20/ 308C) or a higher dormancy level in their seeds might account for these differences. Incubation under fluctuating temperatures markedly reduced seed sensitivity to lower C (Figs 1–3), as higher total

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germination was observed at the same C when the incubation was carried out under fluctuating temperatures. Fluctuating temperatures shifted Cb(50) toward more negative values. This shift would explain the greater tolerance of germination to the otherwise inhibitory effect of reduced C. In addition, the displacement of Cb(50) has physiological implications, since it would suggest that fluctuating temperatures stimulate germination through an enhancement of the embryo’s capacity to overcome either physical or environmental restraints. To rule out the possibility that any of the components (i.e. maximum or minimum temperature) of the fluctuating cycle (rather than fluctuating temperatures per se) were responsible for the physiological changes, we analysed the seed population behaviour under those constant temperatures, and in no case was an effect on Cb(50) similar to that caused by fluctuating temperatures observed (Figs 2, 3). Moreover, some of the component constant temperatures were clearly inhibitory for germination (i.e. incubation of C. acanthoides at 308C). The physiological mechanisms underlying seed responses to fluctuating temperatures are largely unknown, although one report proposes that fluctuating temperatures stimulate germination of immature, dormant sorghum caryopses by reducing embryo sensitivity to abscisic acid (ABA) (Benech-Arnold et al., 1995). As Cb(50) becomes more negative, germination can take place under a wider range of Co. Bradford (1995) stated that if the Cb(50) shifts downwards, both the total percentage and the germination rates may increase. However, just the increased total germination was observed here, as an increase in uH under fluctuating temperatures offset the more rapid germination rate expected from a reduction in Cb(50). The increment in uH could be due to the time required by fluctuating temperatures to exert a promoting effect. For example, Sorghum halepense seeds require about 20 cycles for maximum response to fluctuating temperatures (Benech-Arnold et al., 1990) and Phragmites australis needs three cycles (Ekstam et al., 1999). There is evidence for this in Fig. 3D, where the germination patterns resulting from incubation under fluctuating temperatures were biphasic. The maximum temperature of the cycle was clearly inhibitory, and it apparently took 6 d for the promotive effect of cycling temperatures to become evident. This behaviour could be due to the presence of two sub-populations of seeds; the data analysis considered only the whole population and, therefore, represents an average response. Incubation at different constant temperatures did not modify Cb(50) values for C. cardunculus (10, 15 and 208C) and C. acanthoides (20 and 258C). However, for the latter an upwards shift in Cb(50) values was observed when the seeds were incubated at 308C.

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R. Huarte and R.L. Benech-Arnold Cynara cardunculus 10 C E

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Figure 2. Hydrotime analysis of germination of Cynara cardunculus seeds under different thermal treatments at a range of water potentials. Germination time courses are shown in the left panel for each temperature condition, with the symbols representing the experimental data and the curves showing the time courses predicted by the hydrotime model at each C. Distributions of base water potentials [Cb(g)] at each incubation temperature, and the parameters of the hydrotime model that describe germination, are shown in the right panels for each temperature condition {median base water potential [Cb(50)], the standard deviation of the base potential among seeds (sCb) and the hydrotime constant (uH)}. The coefficients of determination (r 2) indicate the fraction of total variation accounted for by the model.

Fluctuating temperatures reduce mean base water potential

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Figure 3. Hydrotime analysis of germination of Carduus acanthoides seeds under different thermal treatments at a range of water potentials. Germination time courses are shown in the left panel at each temperature condition, with the symbols representing the experimental data and the curves showing the time courses predicted by the hydrotime model at each C. Distributions of base water potentials [Cb(g)] at each incubation temperature and the parameters of the hydrotime model that describe germination are shown in the right panels for each temperature condition {median base water potential [Cb(50)], the standard deviation of the base potential among seeds (sCb) and the hydrotime constant (uH)}. The coefficients of determination (r 2) indicate the fractions of total variation accounted for by the model.

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Similar observations were reported previously for lettuce (Bradford and Somasco, 1994; Dutta and Bradford, 1994) and potato (Alvarado and Bradford, 2002) seeds at supra-optimal temperatures, and for Orobanche aegyptiaca seeds when incubation was performed at sub- or supra-optimal temperatures (Kebreab and Murdoch, 1999). It should be noted that the C. cardunculus populations used in the two sets of experiments were not the same; therefore, neither can population parameters derived from the hydrotime analysis be expected to be the same. Moreover, the dormancy level of each population was probably different and, consequently, sensitivity to fluctuating temperatures might be also different (Batlla et al., 2003). This would explain why the Cb(50) shifted to a different extent as a result of exposure to fluctuating temperatures in each set of experiments (Table 1 versus Fig. 2H). Another germination-initiating factor, such as GA, was also reported to produce a downward displacement of Cb(50) in tomato seeds (Ni and Bradford, 1993). Therefore, it appears that factors that govern changes in dormancy of seed populations (chilling, after-ripening), and those that terminate it (fluctuating temperatures, GA), operate through a reduction of Cb(50). As expressed by Dutta and Bradford (1994), these dormancy-breaking agents may act through a general mechanism involving changes in sensitivity to environmental Co. With the exception of C. vulgare and B. campestris (Table 1) and C. acanthoides (Fig. 3F and H), no modifications in the sCb were observed as a result of incubation under fluctuating temperatures. Therefore, fluctuating temperatures may act mainly by shifting the Cb(50) to lower values, with little effect on variation in Cb values among seeds in the population. This means that the effect of fluctuating temperatures would be equal for all seeds and did not act differentially on different fractions of the seed population. The reduction observed in Cb(50) as a result of incubation under fluctuating temperatures would mean a widening of the range of hydric situations permissive for germination in seeds naturally exposed to fluctuating temperature conditions. This capacity could compensate for the lower water availability (lower water potential) of the uppermost soil layer caused by environmental factors, such as wind and sun radiation, that desiccate this soil layer faster than deeper layers or soil beneath a canopy. The results presented here also indicate that attempts to model field germination using this approach must consider the shift in Cb(50) and uH values under naturally occurring temperature fluctuations. The hydrotime model provided a quantitative explanation for the promotion of germination observed in several species under fluctuating

temperature treatments, due to a reduction of Cb(50) values of the seed populations. The data reported in this work support previous studies indicating that breaking seed dormancy shifted mean base water potentials to more negative values, allowing the seeds to germinate at a wider range of water potentials.

References Alvarado, V. and Bradford, K.J. (2002) A hydrothermal time model explains the cardinal temperatures for seed germination. Plant, Cell and Environment 25, 1061 –1069. Batlla, D., Verges, V. and Benech-Arnold, R.L. (2003) A quantitative analysis of seed responses to cycle-doses of fluctuating temperatures in relation to dormancy: development of a thermal time model for Polygonum aviculare L. seeds. Seed Science Research 13, 197–207. Benech-Arnold, R.L., Ghersa, C.M., Sa´nchez, R.A. and Insausti, P. (1990) Temperature effects on dormancy release and germination rate in Sorghum halepense (L.) Pers. seeds: a quantitative analysis. Weed Research 30, 81 –89. Benech-Arnold, R.L., Kristof, G., Steinbach, H.S. and Sa´nchez, R.A. (1995) Fluctuating temperatures have different effects on embryonic sensitivity to ABA in Sorghum varieties with contrasting pre-harvest susceptibility. Journal of Experimental Botany 46, 711– 717. Benech-Arnold, R.L., Sa´nchez, R.A., Forcella, F., Kruk, B.C. and Ghersa, C.M. (2000) Environmental control of dormancy in weed seed banks in soil. Field Crops Research 67, 105– 122. Bradford, K.J. (1990) A water relations analysis of seed germination rates. Plant Physiology 94, 840– 849. Bradford, K.J. (1995) Water relations in seed germination. pp. 351– 396 in Kigel, J.; Galili, G. (Eds) Seed development and germination. New York, Marcel Dekker. Bradford, K.J. (1996) Population-based models describing seed dormancy behaviour: implications for experimental design and interpretation. pp. 313– 339 in Lang, G.A. (Ed.) Plant dormancy: Physiology, biochemistry and molecular biology. Wallingford, CAB International. Bradford, K.J. and Somasco, O.A. (1994) Water relations of lettuce seed thermoinhibition. I. Priming and endosperm effects on base water potential. Seed Science Research 4, 1 – 10. Christensen, M., Meyer, S.E. and Allen, P.S. (1996) A hydrothermal time model of seed after-ripening in Bromus tectorum L. Seed Science Research 6, 155– 163. Dutta, S. and Bradford, K.J. (1994) Water relations of lettuce seed thermoinhibition. II. Ethylene and endosperm effects on base water potential. Seed Science Research 4, 11 – 18. Ekstam, B., Johannesson, R. and Milberg, P. (1999) The effects of light and number of diurnal temperature fluctuations on germination of Phragmites australis. Seed Science Research 9, 165– 170. Forcella, F. and Wood, H. (1986) Sequential flowering of thistles (Cynareae, Asteraceae) in southern Australia. Australian Journal of Botany 34, 455– 461.

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Received 28 June 2004 accepted after revision 7 February 2005 q CAB International 2005