The role of temperature in the regulation of dormancy and germination of two related summer-annual mudflat species

Aquatic Botany 79 (2004) 15–32

The role of temperature in the regulation of dormancy and germination of two related summer-annual mudflat species Markus Brändel∗ University of Kiel, Ecology Research Center, Landscape Ecology, Olshausenstr. 40, 24098 Kiel Germany Received 14 November 2002; received in revised form 29 August 2003; accepted 16 November 2003

Abstract Dormancy and germination requirements were investigated in seeds (achenes) of the closelyrelated annuals Bidens cernua and Bidens tripartita (Asteraceae). They showed clear differences in their temperature requirements for germination, their dormancy and seed longevity in soil. In B. cernua seeds, primary dormancy was relieved when stratified at 3, 8, and 12 ◦ C, as seeds germinated to a maximum of more than 80% (3 ◦ C and 8 ◦ C) and 20% (12 ◦ C), while temperatures of 15 ◦ C and 18 ◦ C had no effect. Secondary dormancy was induced after 20 weeks at 12 ◦ C. Temperatures between 3 and 18 ◦ C were effective in relieving dormancy in B. tripartita seeds as they germinated to more than 60% regardless of stratification temperature. With prolonged incubation time, dormancy was induced at 18 ◦ C. Both species exhibited an annual dormancy cycle. B. cernua seeds came out of dormancy in spring at temperatures <15 ◦ C and dormancy was induced in summer (>15 ◦ C). In late summer, seeds were completely dormant before temperatures of <7 ◦ C led to a relief of secondary dormancy. B. tripartita seeds came out of both primary and secondary dormancy when ambient temperatures were <12 ◦ C in autumn. At temperatures >7 ◦ C in spring and summer, dormancy was induced. Nevertheless, B. tripartita seeds could germinate (>40%) during the whole year at high and fluctuating temperatures. B. tripartita had the potential to accumulate a persistent seed bank, while buried B. cernua seeds were all dead after 20 months. Germination tests with dry-stored seeds showed that a fluctuating-temperature amplitude of ≥8 ◦ C (mean 22 ◦ C) led to a significantly higher germination (40%) of B. tripartita seeds, while B. cernua seeds did not germinate even at high amplitudes. Stratified B. tripartita seeds germinated

∗ Tel.: +49-431-8801229; fax: +49-431-8804083. E-mail address: [email protected] (M. Brändel).

0304-3770/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aquabot.2003.11.008

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at high constant temperatures >21 ◦ C, while B. cernua seeds showed a nearly absolute requirement for fluctuating temperatures. © 2004 Elsevier B.V. All rights reserved. Keywords: Bidens; Dormancy cycle; Seed germination; Temperature

1. Introduction The role of temperature in determining the germination time is a dual one as (i) it influences germination directly and (ii) regulates dormancy (Bouwmeester and Karssen, 1992,1993; Probert, 2000). In summer annuals, low winter temperatures relieve dormancy, while high summer temperatures induce it (Baskin and Baskin, 1988). As seeds come out of dormancy they germinate first at high temperatures, and after subsequent additional release, the required minimum temperature decreases until the widest temperature range for germination, corresponding to maximum dormancy relief is attained (Baskin and Baskin, 1988). The induction of dormancy is accompanied by an increase in the required minimum temperature until seeds germinate only at high temperatures, or fail to germinate at all (Baskin and Baskin, 1988; Probert, 2000). The cyclic changes in dormancy recur annually, until the remaining seeds either germinate or die. The existence of dormancy cycles has been shown for some mudflat species (e.g. Baskin et al., 1993a,b; Bouwmeester and Karssen, 1992; Milberg, 1994; Schütz, 1997). However, in some wetland species, dormancy cannot be induced once seeds came out of dormancy and non-dormant seeds can germinate during the whole growing season when environmental conditions are favorable for germination, e.g. light (Baskin and Baskin, 1988; Baskin et al., 1993a,b; Jensen, 2001; Milberg, 1994). Temperature requirements for germination, and those for changes in dormancy differ. Dormancy can be broken at temperatures that prevent germination, and temperatures which favor germination can also induce dormancy (Bouwmeester and Karssen, 1992). Germination of wetland plants is directly affected by the minimum and maximum temperature requirements for germination, and the amplitude of daily temperature fluctuations. Fluctuating temperatures often have a promotive effect on germination of wetland species (Thompson and Grime, 1983). The main aspect of almost all studies have been to explore the changes in germination temperature requirements of seeds over the year and, so far, only little attention has been paid to the temperatures that cause changes in dormancy. Even laboratory studies examining the effect that temperatures have on changing the dormancy are rare, and that too focused only on very few species (Totterdell and Roberts, 1979). The combination of burial experiments along with experiments under controlled laboratory conditions, to examine threshold temperatures for relief and induction of dormancy in seeds, is largely missing. The exceptions are those of: Bouwmeester and Karssen (1992,1993) examining Sisymbrium officinale and Polygonum persicaria; Baskin et al. (1995) studying B. polylepis; and, Brändel and Schütz (2003) examining Verbena officinalis. The aim of the present study was to identify temperature ranges or thresholds for the relief and induction of dormancy of seeds both in the field and in the laboratory. Furthermore,

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I was interested in finding out whether the field and laboratory temperatures responsible for both dormancy processes coincide. The main aspect was to examine if fixed threshold temperatures for the release and induction of dormancy exist, as surmised by Bewley and Black (1994), and Egley and Duke (1985). As habitat differences may be responsible for the differences in dormancy levels and germination requirements between the species of a single genus, e.g. those from wetlands and those from more typical of drier sites (Schütz and Rave, 1999), two clearly related species (Bidens cernua L. and Bidens tripartita L.) occurring in the same habitat were chosen for the present study. Both species have primary dormant seeds (Hogue, 1976; Rollin, 1965). To identify how these mudflat species may differ in their germination requirements and dormancy, following points were examined. (i) The germination response to fluctuating temperature; (ii) the range of temperature suitable for germination; (iii) effective temperatures for relief and induction of dormancy; (iv) the annual dormancy cycle; and (v) the viability and longevity of seeds in the soil. 2. Material and methods 2.1. Study species Both B. cernua and B. tripartita (Asteraceae) are annual species growing on moist, nutrient-rich muddy soils or muddy-sandy soil in ditches, brooks or pond banks, mostly near villages or on moist farmland. Characteristically their growing sites have seasonally changing water levels. The geographical distribution of B. tripartita extends from North Africa and Eurasia to Australia, while B. cernua is distributed in Europe, Asia and North America (Hegi, 1964). They are pollinated by insects and B. tripartita is additionally self-pollinated (Oberdorfer, 1994). Achenes of B. cernua weighed 1.12 mg and those of B. tripartita 2.80 mg. 2.2. Seed collection and storage Freshly matured achenes (hereafter referred to as seeds) of both species were collected from one respective population, in the autumn of 2000 in the valley of the river Eider near Kiel, northern Germany (54◦ N10◦ E). Seeds were air dried after collection, cleaned, and stored at room temperature (15–20 ◦ C, 40–60% RH) until used. Seeds were stored for 4 weeks for the stratification experiment, 8 weeks for the burial experiment, 7 months for the test of the effect of diurnal fluctuating temperatures, and 14 months for the optimum temperature experiment. An effect of after-ripening was not detectable after 7 months of dry storage as indicated by no increase in final germination percentages. 2.3. Germination tests The germination tests were carried out in incubators (Rubarth Apparatebau, Hannover, Germany) equipped with a warm fluorescent light (Philips TL 20 W/29 RS) providing a photon flux density (PFD) of approximately 30 ␮mol m−2 s−1 , with a R:FR ratio of about

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14.5 and a daily 12 h photoperiod. Petri dishes with seeds for the treatments in darkness were wrapped with a double layer of aluminum foil. Seeds to be tested were placed in 9 cm diameter petri dishes on filter paper (Schleicher & Schüll, no. 595) moistened with de-ionized water. Three replicates of approximately 50 seeds were used. Petri dishes of the light treatments were inspected weekly for germinated seeds, those of the dark treatments every second week; protrusion of the radicle was the criterion for germination. Germinated seeds of the dark treatments were counted under filtered light (‘bright blue’- and ‘light red’-filter, LEE-filter, Andover, UK). Germination tests at constant temperatures were terminated after 4 weeks. Since there was some germination beyond 4 weeks at 15/5 ◦ C, these tests were terminated only after 6 weeks. Non-germinated seeds were pinched with forceps to see if the embryos were white and firm, revealing that they were alive. If the seed content was soft and brown, they were considered dead and excluded from the calculation of germination percentages (Baskin and Baskin, 1998). 2.4. Stratification experiment Approximately 900 seeds of both species were placed on fine nylon-mesh tissue in 35 plastic trays (9 cm diameter), moistened with de-ionized water, and wrapped immediately with a double layer of aluminum foil. Seven trays were kept in cooled incubators at 3, 8, 12, 15 ◦ C, and 18 ◦ C, respectively. After 2, 4, 8, 12, 16, 20, and 28 weeks, one tray from each incubator (i.e. each temperature regime) was retrieved and the germinability of the containing seeds was tested at 8, 15, 25 ◦ C, and 15/5 ◦ C in light, and at 25 ◦ C and 15/5 ◦ C additionally in darkness. 2.5. Burial experiment In November 2000, 66 bags of fine nylon-mesh tissue, each containing approximately 240 seeds of one species, were buried at 5 cm depth in 9 cm diameter plastic pots, filled with a mixture of 2:1 loam and sand and with drainage holes at the bottom. Each pot contained three bags of both species. They were sunk into the ground up to the rim at the edge of a canopy of deciduous shrubs and trees in the experimental garden of the University of Kiel. Temperature was monitored at 5 cm depth every half an hour during the study period with a temperature sensor (Pt 100) connected to a data-logger (Squirrel 1200, Grant Intruments, Cambridge, Great Britain). The retrieval of the buried seeds commenced in March 2001 and ended in September 2002. The intervals between the retrievals were 2 weeks in the spring of 2001 and 4 weeks from autumn 2001 to September 2002. At each retrieval, one pot was exhumed and transferred to the laboratory. Bags were exhumed under filtered light. The seeds of each bag were distributed into nine petri dishes and tested at 15, 25 ◦ C, and 15/5 ◦ C in both light and darkness. 2.6. Diurnal fluctuating temperatures The effect of diurnal temperature fluctuations on germination was tested at different amplitudes in a thermogradient incubator (Rubarth Apparatebau, Hannover, Germany),

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described in detail by Ekstam and Bengtsson (1993). Seeds were sown at nine different positions representing daily alternating temperatures of 30/14, 29/15, 28/16, 27/17, 26/18, 25/19, 24/20, 23/21 ◦ C and a constant temperature of 22 ◦ C. Temperatures were applied 11 h per day with 1 h of cooling and warming. Three replicates were made with approximately 50 seeds per position. The experiment was carried out twice with dry-stored seeds, first giving 12 h of warm fluorescent light (Philips TL 20 W/29 RS) providing a photon flux density (PFD) of approximately 25 ␮mol m−2 s−1 and an R:FR ratio of about 14.5 at the higher temperature and secondly in darkness. The experiment was carried out additionally with seeds stratified for 4 months at 5 ◦ C, but germination was tested only in light. Seeds tested in light were inspected weekly for germination while seeds of the dark treatment were inspected at the end of the experiment after 8 weeks. 2.7. Optimum germination temperature Because of the limited capacity of the thermogradient incubator to adjust a temperature range above 25 ◦ C, the experiment was carried out twice with two temperature ranges, one from 3 ◦ C to 21 ◦ C and another from 24 ◦ C to 40 ◦ C. Dry-stored seeds were stratified for 10 weeks at 5 ◦ C prior to the experiment using three replicates of approximately 350 seeds. The seeds of one replicate were transferred to seven positions (with 50 seeds each) representing firstly 3, 6, 9, 12, 15, 18 ◦ C, and 21 ◦ C, and secondly 24, 27, 30, 33, 36, 38 ◦ C, and 40 ◦ C. Germinated seeds were counted every second day for 4 weeks. 2.8. Statistical analysis A logistic regression model using the SAS-procedure Logistic (SAS, 1996) was performed to analyze the influence of the pre-treatments (stratification temperature, -period) and of the germination temperatures on germination. Treatments where the variance of all replicates was zero (nil germination) had to be excluded from the model (SAS, 1996). Because of the absence of germination of B. cernua seeds at 25 ◦ C, the performance of one model that included all data of the stratification experiment failed. Consequently, two models were made, one including the data (i) of B. tripartita tested at 15/5 ◦ C and 25 ◦ C, and another (ii) of B. cernua tested at 15/5 ◦ C. In these models, ‘germination event per petri dish’ was the response variable, and ‘stratification temperature’, ‘stratification period’, and for the first model only ‘test temperature’, were included as explanatory variables (factors). Overdispersion, a phenomenon often observed in germination data (SAS, 1996), was corrected with the Williams method. Because of low or nil germination in darkness, no calculations were made for such germination test conditions. Final germination events per replicate (response variable) at the various germination treatments (explanatory variables) in the optimum germination experiment and in the experiment of fluctuating temperatures were contrasted using the contrast statement within the logistic function of SAS (1996).

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Fig. 1. Final germination percentages (means ± S.E. if >5%) of stratified seeds (28 weeks at 5 ◦ C) of B. cernua at a mean temperature of 22 ◦ C and a range of temperature amplitudes in light. Different letters above the bars indicate significant differences (P < 5%). Dry-stored seeds did not germinate by any of the treatments (data not shown).

3. Results 3.1. Effect of fluctuating temperatures Dry-stored B. cernua seeds did not germinate in any of the treatments, while stratified seeds showed signficantly higher germination (≥80%) at amplitudes ≥8 ◦ C. At lower amplitudes germination was below 25% (Fig. 1). Dry-stored seeds of B. tripartita germinated well at high daily temperature amplitudes in light (Fig. 2). Germination was highest (98%) at the largest amplitude of 16 ◦ C corresponding to a daily fluctuation of 30/14 ◦ C. Daily fluctuations of 29/15 ◦ C and 28/16 ◦ C were also effective for a high germination response (90% and 75%, respectively), whereas germinability at a temperature of 27/17 ◦ C was significantly lower and reached only 43%. Amplitudes below 8 ◦ C had only a small effect or none on germination. Stratified seeds germinated to nearly 100% regardless of the amplitude of temperature fluctuations. No germination occurred in darkness. 3.2. Optimum temperature B. cernua seeds did not germinate at any of the constant temperatures. B. tripartita seeds showed clear germination preferences at a range of temperatures between 21 ◦ C and 36 ◦ C (Fig. 3). The optimum temperature for germination was 33 ◦ C (82%), maximum temperature was 36 ◦ C, and the lowest temperature for germination (>10%) was 21 ◦ C. At lower temperatures (>9 ◦ C) only very few seeds germinated.

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a

100

a

b

Germination (%)

80

c

60

40

e

e

d e

e

22/22

23/21

24/20

25/19

20

d

0 26/18

27/17

28/16

29/15

30/14

Temperatures (˚C)

Fig. 2. Final germination percentages (means ± S.E. if >5%) of freshly matured seeds of B. tripartita at a mean temperature of 22 ◦ C and a range of temperature amplitudes in light. Different letters above the bars indicate significant differences (P < 5%). Very few seeds germinated in darkness (data not shown). Stratified B. tripartita seeds germinated to nearly 100% in all treatments (data not shown).

3.3. Stratification experiment B. cernua seeds showed higher germination only at the fluctuating temperature (15/5 ◦ C) in light (Fig. 4), after stratification between 3 ◦ C and 12 ◦ C. The results of the logistic regression showed a significant influence of the factors ‘stratification temperature’ and ‘stratification period’ (Table 1). There was also a significant interaction between both factors,

Fig. 3. Final germination percentages (means ± S.E. if >5%) of B. tripartita seeds stored for 10 weeks at 5 ◦ C and incubated at temperatures between 3 ◦ C and 40 ◦ C in light. Different letters above the bars indicate significant differences (P < 5%). Very few seeds germinated in darkness (data not shown).

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Fig. 4. Germination of stratified B. cernua seeds at 15/5 ◦ C in light. Final germination percentages (means±S.E. if >5%) of freshly matured and stratified B. cernua seeds incubated at 15/5 ◦ C in light. Seeds were stratified between 2 and 28 weeks at different temperatures. Very few seeds germinated at 8, 15, 25 ◦ C, and 15/5 ◦ C in darkness (data not shown).

mainly due to differences in germination between seeds stratified >8 ◦ C for more than 8 weeks. Most effective for dormancy relief were stratification temperatures of 3 ◦ C and 8 ◦ C. Less effective was a temperature of 12 ◦ C, while stratification at 15 ◦ C and 18 ◦ C had no or only a slight effect on germination (Fig. 4). The influence of the stratification temperatures became apparent at the beginning of the 4th week (at 3 ◦ C), and after the 8th week (at 8 ◦ C and 12 ◦ C). Maximum germination (99% and 90%) was reached in seeds stratified at 3 ◦ C and 8 ◦ C for 28 weeks (Fig. 4). Seeds stratified at 12 ◦ C showed highest dormancy relief

Table 1 Results of the reduced logistic regression model applied to the germination data Parameters

d.f.

Wald chi-square

P

B. cernua Stratification temperature Stratification period Stratification temperature × stratification period

3 5 15

134.1 60.5 403.4

<0.0001 <0.0001 <0.0001

1 4 6 6 4 24

182 216.6 172.1 81.2 35.9 165.9

<0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001

B. tripartita Test temperature Stratification temperature Stratification period Test temperature × stratification period Test temperature × stratification temperature Stratification temperature × stratification period

Data obtained for B. cernua in light at 15/5 ◦ C after stratification at 3, 8, 12, and 15 ◦ C for 8, 12, 16, 20, and 28 weeks and for B. tripartita in light at 15/5 ◦ C and 25 ◦ C, after stratification at 3, 8, 12, 15, and 18 ◦ C for 2, 4, 8, 12, 16, 20, and 28 weeks.

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Fig. 5. Germination of stratified B. tripartita seed tested at different temperatures in light and darkness. Final germination percentages (means ± S.E. if >5%) of freshly matured and stratified B. tripartita seeds incubated at (a) 15/5 ◦ C in light, (b) 15/5 ◦ C in darkness, (c) 25 ◦ C in light and (d) 25 ◦ C in darkness. Seeds were stratified between 2 and 28 weeks at different temperatures. Very few seeds germinated at 8 ◦ C and 15 ◦ C (data not shown).

after 16 weeks, but after 16 weeks of stratification germinability decreased considerably from 29 down to 8%, indicating an induction of dormancy. All stratification temperatures were effective in breaking primary dormancy in B. tripartita seeds when exposed to light at 25 ◦ C and 15/5 ◦ C (Fig. 5a and c). Only a minor proportion of seeds germinated in darkness and only when stratification temperatures were ≤12 ◦ C. All factors (stratification temperature, -period, test temperature) and their interactions had a highly significant effect on germination (Table 1). Seeds tested at 15/5 ◦ C in light, germinated between 40% (at 18 ◦ C) and 75% (at 3 ◦ C) even after 2 weeks of stratification. A stratification at 3 ◦ C and 8 ◦ C were equally effective in releasing primary dormancy with a maximum germination of 99% (Fig. 5a). A stratification temperature of 12 ◦ C was only slightly less effective, while stratification at 15 ◦ C and 18 ◦ C resulted in a remarkably lower germination. After an initial decrease of dormancy in seeds stratified at 18 ◦ C within the first 12 weeks, an induction of dormancy was apparent after 16 weeks of stratification as germination decreased from 65% (after 12 weeks) to 35% (after 16 weeks). Seeds tested at 15/5 ◦ C in darkness germinated to a higher proportion (>20%) only when stratified at 3 ◦ C and 8 ◦ C for 28 weeks (Fig. 5b). At a test temperature of 25 ◦ C in light, seeds showed a dormancy relief after more than 8 weeks of stratification (Fig. 5c). A stratification at 8 ◦ C and 12 ◦ C was equally effective in

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breaking primary dormancy for any stratification period. Seeds stratified at 3 ◦ C required 16 weeks of stratification to show high germination (80%). The maximum germination after stratification at 3, 8, and 12 ◦ C was reached after 28 weeks (>95% germination), and after 20 weeks at 15 ◦ C (80% germination). An induction of secondary dormancy was apparent in seeds stratified at 18 ◦ C as germination fell from 50% (16th week) to below 5% (20 weeks). At 25 ◦ C in darkness, germination was only slightly influenced by a stratification at 15 ◦ C and 18 ◦ C (Fig. 5d). Stratification temperatures of 8 ◦ C and 12 ◦ C had almost similar effects. But after 28 weeks of stratification at 12 ◦ C, an induction of dormancy was apparent as germinability decreased from 45% (20 weeks) to 0% (28 weeks). Highest germination (50%) was reached after a stratification of 20 weeks at 8 ◦ C and 12 ◦ C, and of 28 weeks at 3 ◦ C. 3.4. Burial experiment Both species exhibited dormancy cycles with a dormancy relief during the first winter and spring. An induction of secondary dormancy during late summer and autumn, and a relief of secondary dormancy in late autumn and winter of the following year was noted (Figs. 6 and 7). Only in B. tripartita seeds was dormancy re-induced in the summer of 2002. There were considerable differences in germination at the different retrieval dates between the test conditions (Figs. 6 and 7). 3.4.1. B. cernua Seeds incubated at 25 ◦ C in light, and at 15/5 ◦ C in light and darkness, exhibited the most marked dormancy cycles (Fig. 6). At 25 ◦ C in darkness, and at 15 ◦ C in light and darkness, only slight fluctuations accompanied by a low germinability (<20%) occurred. Dormancy was relieved during the winter 2000/2001 indicated by the high germinability (>90%) of seeds exhumed in spring (2001) and tested at 15/5 ◦ C in light. Dormancy was induced at ca. 15 ◦ C in August and September. When temperatures fell <12 ◦ C in November 2001, dormancy relief commenced and proceeded until March 2002 when germination was about 80%. Exhumed seeds tested at 25 ◦ C in light showed high germination (>25%) during May and July 2001. Both dormancy relief and induction occurred at ca. 15 ◦ C. All exhumed seeds were dead when exhumed in April 2002 and after June 2002. 3.4.2. B. tripartita In exhumed B. tripartita seeds, changes in dormancy throughout the test period was observable at all test temperatures (Fig. 7). Germination of exhumed seeds tested at 15/5 ◦ C in light was 100% in spring and early summer 2001, until dormancy was induced in August at ca. 15 ◦ C (80% germination). In December 2001, temperatures <3 ◦ C resulted in a relief of dormancy as seeds germinated almost completely. At 15/5 ◦ C in darkness, germination of exhumed seeds remained below 25% until December 2001, when temperatures decreased below 3 ◦ C and germination increased up to 90%. Germination remained high (>50%) until spring 2002 when dormancy was induced at temperatures >12 ◦ C. In January 2002, exhumed seeds showed a decrease followed by an increase of germination at temperatures below 4 ◦ C.

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Fig. 6. Germination of exhumed B. cernua seeds. Final germination percentages (means±S.E. if >5%) of B. cernua seeds incubated at 15/5, 25 ◦ C, and 15 ◦ C in light and darkness for 4 weeks (for the constant temperatures) and 6 weeks (for the fluctuating temperature), following 5–19 months of burial in an experimental garden. Temperatures given are the daily mean temperatures at a depth of 5 cm at the burial site.

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Fig. 7. Germination of exhumed B. tripartita seeds. Final germination percentages (means ± S.E. if >5%) of B. tripartita seeds incubated at 15/5, 25 ◦ C, and 15 ◦ C in light and darkness for 4 weeks (for constant temperatures) and 6 weeks (for the fluctuating temperature), following 5–23 months of burial in an experimental garden. Temperatures given are the daily mean temperatures at a depth of 5 cm at the burial site.

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At a test temperature of 25 ◦ C in light, a continuous dormancy relief over 3 months in spring (mean temperature <12 ◦ C) was observable until in June 2001 germination was 100%. In July, germination decreased within 2 weeks from 100% to 39% at temperature around 15 ◦ C. In October, the temperature decreased below 3 ◦ C and germination increased during the winter 2001/2002 and stayed at >80%, until in September 2002 germination decreased to nearly 0% at temperatures >16 ◦ C. Germination of exhumed seeds tested at 25 ◦ C in darkness was below 15% until the winter 2001/2002. In January 2001, dormancy was relieved at 3 ◦ C. Until June 2002, at temperatures between 1.5 ◦ C and 8 ◦ C, extreme fluctuations in germination of exhumed seeds were apparent. In July 2002, dormancy was induced at temperatures above 15 ◦ C. The annual course of germination of exhumed seeds tested at 15 ◦ C was similar in light and darkness. However, germination in darkness was always lower than in light. Exhumed seeds did not germinate before January 2002 (temperature 3 ◦ C) when germination showed a peak (78% and 38% in light and in darkness, respectively). At low temperatures of 1.5 ◦ C, dormancy was induced in January 2002, and relieved in February at a temperature of 4 ◦ C. When temperatures increased to 8 ◦ C in May and June 2002, germination decreased and remained <10% until the end of the experiment. Exhumed seeds lost dormancy, when tested at 15 ◦ C in light and darkness and at 25 ◦ C and 15/5 ◦ C in darkness, only in the winter and early spring of 2002. That is to say, a relief of secondary dormancy in B. tripartita seeds was accompanied by a partial release of the light requirement and a wider temperature window for germination.

4. Discussion 4.1. Influence of temperature on germination Both Bidens species responded positively to fluctuating temperatures. The requirement seems to be obligate in B. cernua, while seeds of B. tripartita were able to germinate even at high constant temperatures (>21 ◦ C). However, in the burial experiment, exhumed seeds of B. cernua were able to germinate at a constant 25 ◦ C in light. A requirement for fluctuating or high temperatures for germination was found by Thompson and Grime (1983) to be highly incident in species of wetland habitats and disturbed grounds. The promotive effect of fluctuating temperatures was regarded as a sensoring mechanism for gaps in the vegetation cover (Pons, 1989), for depth of burial (Thompson and Grime, 1983), or for inundation of the soil (Probert, 2000). In both Bidens species, germination is prevented after shedding in autumn and in winter by low temperatures and low temperature fluctuations. The onset of temperature conditions favorable for germination, in late spring and early summer, coincides with a decrease in the water levels of ponds, marshes, and streams. With falling water levels, bare areas of mud are revealed, highly favorable for the establishment of both fast-growing (personal observation) Bidens species when the exposed ground experiences high temperatures, large daily fluctuations in temperature, and an increasing level of light.

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The sensitivity of B. cernua and B. tripartita seeds to daily fluctuating temperatures, and the requirement of high temperatures, seems to be the main mechanism ensuring that seeds germinate when conditions are suitable for seedling establishment, just as Thompson and Grime (1983) imagined for wetland species in general. 4.2. Influence of temperature on the level and course of dormancy 4.2.1. Annual course of dormancy Freshly matured seeds of B. cernua were dormant, while freshly matured seeds of B. tripartita were conditionally dormant. However, the level of primary dormancy in seeds of B. tripartita can be regarded as sufficient to avoid germination after shedding, since temperatures in autumn are too low to trigger germination. While the loss of dormancy of freshly matured B. tripartita proceeds rapidly within 2–4 weeks (at temperatures between 3 ◦ C and 18 ◦ C, tested at 15/5 ◦ C), seeds of B. cernua require several weeks (at temperatures <12 ◦ C, tested at 15/5 ◦ C) to become non-dormant. When primary dormancy was relieved, seeds of B. tripartita had a nearly absolute light requirement for germination, while seeds of B. cernua could germinate additionally in darkness. If germination was prevented during late spring and early summer, secondary dormancy was induced due to high ambient temperatures, which were similar for both species and remained above 12 ◦ C. While seeds of B. cernua were dormant during September, seeds of B. tripartita entered conditional dormancy and are thus capable of germination during the whole growing season in light, provided that higher temperatures are present. Baskin et al. (1993a) proposed that species from unpredictable habitats must be able to germinate whenever conditions become non-limiting, even if only small plants can be developed and only few seeds can be produced. With decreasing temperatures in autumn, dormancy relief occurred first in B. cernua when temperatures fell below 12 ◦ C, and in the case of B. tripartita when the temperature fell below 4 ◦ C. The extremely fluctuating germinability of B. tripartita seeds exhumed in the winter 2001/2002, and tested at 15/5 ◦ C and 25 ◦ C in darkness and at 15 ◦ C in light and darkness (Fig. 6), was probably caused by inundations that occurred during the trial (exhumations of 25 January 2002 and 5 April 2002), which led to an induction of secondary dormancy as described by Benvenuti and Macchia (1997) for B. tripartita. Seeds relieved from primary dormancy, and those relieved from secondary dormancy, differed in their germination response: as fluctuating temperatures were essential for seeds of B. cernua if secondary dormancy was to be relieved, and a light requirement for seeds of B. tripartita vanished in the second year of burial. Seeds of B. cernua died in the summer of the second year of burial until the experiment end. Similar results were also found by Benoˆıt (1991). In contrast, B. tripartita has the potential to accumulate a persistent seed bank, as the viability of seeds remained high until the end of the experiment. 4.3. Temperatures for relief and induction of dormancy The seed dormancy cycle of B. cernua and B. tripartita resembles the most than any other wetland species of the temperate region. Dormancy was induced in early summer

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(May–July) and relieved in November/December (e.g. Milberg, 1994; Baskin et al., 1995). Consequently, it appears probable that changes in the state of dormancy are caused by similar temperatures, but they may differ in ecological or phylogenetic groups (Schütz, 1997). Nevertheless, little is known about the temperature conditions leading to a change in the level of dormancy (Bewley and Black, 1994). Bewley and Black (1994) reported concerning a threshold temperature for the relief and induction of dormancy of approximately 15 ◦ C for barley, lettuce and wheat. Even Egley and Duke (1985) gave an upper temperature limit of 15 ◦ C for dormancy loss. In the present study, the minimum temperature for an induction of dormancy was 12 ◦ C for B. cernua and 7 ◦ C for B. tripartita. While a maximum temperature for a relief of dormancy could be identified for B. cernua at 12 ◦ C, all experimental stratification temperatures (3–18 ◦ C) were effective in relieving primary dormancy in seeds of B. tripartita. The results of the burial and laboratory experiment of effective temperatures coincided for both species (Table 2). Low ambient temperatures in the field (burial experiment) already caused a relief of dormancy before higher temperatures had being reached. In seeds of both Bidens species, ambient temperatures for a relief and an induction of dormancy overlap. Hogue (1976) found that in seeds of B. cernua, tested at 25/15 ◦ C in light, an incubation temperature of 2 ◦ C was effective in both relieving and inducing dormancy, an observation supporting the assumption of an overlap of the effective temperatures changing dormancy. The effectiveness of low temperatures in inducing dormancy was also found in Rumex crispus (Totterdell and Roberts, 1979) and in P. persicaria (Bouwmeester and Karssen, 1992). The assumption, that physiological processes and states (Bewley and Black, 1994) which cause a relief and an induction of dormancy occur at different temperatures, seems to be invalid for this species. Identification of the physiological processes behind changes of

Table 2 Comparison of temperatures effective in relieving and inducing dormancy in the laboratory and in the burial experiment indicated by a change in germination percentages of more than 20% Treatment

15 ◦ C in light

Species

Dormancy relief (◦ C)

Dormancy induction (◦ C)

Laboratory

Laboratory

Field None <3

B. cernua B. tripartita

None None

15/5 ◦ C in light

B. cernua B. tripartita

3–12 3–18

<12 <7

12 18

>15 >16

15/5 ◦ C in darkness

B. cernua B. tripartita

None 3–8

<11 <3

None None

>15 >12

25 ◦ C in light

B. cernua B. tripartita

None 3–18

<15 <12

None 18

>15 >12

25 ◦ C in darkness

B. cernua B. tripartita

None 3–12

None None

>16

None <3

None None

Field

Results are shown separately for both Bidens species and for the various treatments.

None >7

None

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dormancy will clarify the influence of different temperatures on dormancy and will improve our understanding of the annual course of dormancy. 4.4. Comparison of dormancy behavior of Bidens species Within the genus Bidens, species differ in their state of primary dormancy after shedding: B. frondosa (Brändel, in press), B. leavis (Leck et al., 1994), B. odorata (Corkidi et al., 1991), B. polylepis (Baskin et al., 1995), and B. vulgare (Maguire and Overland, 1959) are dormant; B. pilosa (Sahoo and Jha, 1997) is weakly dormant; and B. bipinnata (Dakshini and Aggarwal, 1974), and B. radiata (Rollin, 1975) are non-dormant. But even within one inflorencense, exist differences in the dormancy characteristics of B. bipinnata (Dakshini and Aggarwal, 1974), B. gardneri (Felippe, 1990), B. pilosa (Forsyth and Brown, 1982; Rocha, 1996), and B. odorata (Corkidi et al., 1991). Seeds from the center of the inflorescense germinated more readily than seeds from the periphery (Felippe, 1990; Forsyth and Brown, 1982). Seeds of B. laevis (Leck et al., 1994), B. pilosa (Forsyth and Brown, 1982; Reddy and Singh, 1992), B. polylepis (Baskin et al., 1995) and B. radiata (Rollin, 1975) germinated better at high temperatures (approximately, mean >25 ◦ C), than at low temperatures (approximately, mean <15 ◦ C), as shown for B. tripartita in the present study. The optimum temperature of about 33 ◦ C is similar in seeds of B. radiata (Rollin, 1975) and B. tripartita. The present data give no indication of a general promotive effect of fluctuating temperatures on germination within the genus Bidens. B. laevis shows an absolute light requirement for germination (Leck et al., 1994), while the above-mentioned Bidens species can germinate at high temperatures even in darkness. Similar to B. cernua, freshly matured seeds of B. laevis need a stratification period of 8 weeks at 5 ◦ C to show a germinability of over 15% (Leck et al., 1994). B. polylepis seeds needed a stratification period of 6 weeks at 5 ◦ C or 15/6 ◦ C for germination above 50% (Baskin et al., 1995). While stratification at 22/10 ◦ C was less effective in releasing primary dormancy (maximum germination 50%), a temperature regime of 35/20 ◦ C was ineffective in relieving dormancy. Interestingly, in the results of Baskin et al. (1995), they found an induction of dormancy after 12 weeks of stratification at 22/10 ◦ C. The current data on the germination requirements and temperatures effecting the level and course of dormancy of species within the genus Bidens is insufficient to make general conclusions. Further comparative studies are necessary in which environmental influences on the motherplant are excluded in order to clarify in which way the differences in dormancy and germination requirements shown by different species are adaptations to the local environment or are genetically fixed in each Bidens species.

Acknowledgements I am grateful to W. Schütz and K. Jensen for discussion, and comments on the manuscript, and G. Rave for statistical advice. I also thank A. Kobarg, L. Rasran and K. Vogt for their assistance in the experiments and for collecting the seeds. The study was funded by the Deutsche Forschungsgesellschaft (DFG, Schu 1221/3-1).

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