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Germination responses of diploid Butomus umbellatus to light, temperature and flooding
Flora (2003) 198, 37–44 http://www.urbanfischer.de/journals/flora
Germination responses of diploid Butomus umbellatus to light, temperature and flooding Zdenka Hroudová* and Petr Zákravsk´y Institute of Botany, Academy of Sciences of the Czech Republic, Pru˚honice, Czech Republic Submitted: Feb 18, 2002 · Accepted, in revised form: Nov 5, 2002
Summary The germination of seeds of diploid Butomus umbellatus was studied to determine the favourable conditions for germination and whether seed germination may represent a limitation within the sexual reproductive cycle of this cytotype. The need to stratify the seeds and the response of the seeds to environmental conditions during germination were studied. The influence of light/darkness, temperature, day length, and flooding/aerobic conditions was tested. Seed germination was enhanced in the winter by cold wet stratification, which indicates prevailing seed germination in the spring. Seeds germinated best in light, at relatively high temperatures (20°–30°C), in long days. Seeds were able to germinate when flooded; in most cases, flooding supported germination. In field habitats, the optimum conditions for the germination of B. umbellatus seeds may be found on an open emerged bottom saturated with water or under a shallow water layer. The process of germination does not represent a high-risk stage in the sexual reproduction of this cytotype of B. umbellatus. The time of germination (late spring or summer months) contributes to seedling mortality due to the competition of adult plants or other early-germinating species. Key words: emergent macrophytes, sexual reproduction, seeds, ploidy level
Introduction Ploidy level may play an important role in the ecological properties of plant species and their adaptability to new habitats. Most polyploid taxa have ecological tolerances, which are quite different from those of their diploid progenitors (Levin 1983). Polyploids were found to be more resistant to pests and pathogens, or other kinds of stress, and appeared to be stronger competitors, with greater emphasis on vegetative reproduction (Tal 1980; Levin 1983). On the other hand, lowered fertility is more frequently observed in polyploids (namely in autopolyploids – Stebbins 1971), while diploids are usually fully fertile, and able to spread by seeds. This is the case also of Butomus umbellatus L. The results of former investigations (Krahulcová & Jarolímová 1993) have shown that seed production in Butomus umbellatus depends on ploidy level; triploids are self-incompatible, while diploids are self-compatible. Consequently, a more frequent occurrence of
diploids is to be supposed due to their seed reproduction ability. However, triploids were found prevailing in the Czech and Slovak Republics (Hroudová & Zákravsky´ 1993 b). This indicates that the sexual reproduction of diploids is not as successful in compensating for the efficiency of the other progressive ecological properties of triploids (intensive vegetative spreading, vigorous growth, resistance to eutrophication – for comparison of both cytotypes see Hroudová et al. 1996), which increase the adaptability of triploids to a wider scale of habitat conditions and enable them to find suitable habitats more frequently. In Central Europe, B. umbellatus occurs predominantly in shallow water reservoirs, or along stream shores, in habitats with a fluctuating water level. Water level dynamics is the crucial factor influencing all other habitat conditions, and an adaptation to changing water level may be of primary importance for the success of seed reproduction. In this study, conditions which occur under a changing aerobic/submerged environment (flooding in winter season, or in the spring) were simu-
* Corresponding author: Zdenka Hroudová, Institute of Botany, Academy of Sciences of the Czech Republic, 252 43 Pru˚honice, Czech Republic, e-mail: [email protected] 0367-2530/03/198/01-037 $ 15.00/0
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lated. We tested the need of stratification to break seed dormancy, the influence of flooding or aerobic conditions on dormant seeds during wintering and on seeds during germination, and the influence of light or darkness, day length, and temperature on germination. We tried to find, which environmental factors dependent on water level changes may affect the germination of seeds of diploid B. umbellatus, and whether the phase of seed germination may represent a high-risk stage within its sexual reproductive cycle.
Material and methods Plant material Sampling a satisfactory amount of seeds was the main methodological problem. While the development of populations of Butomus umbellatus fluctuates from year to year depending on water level, the fertility of the populations is irregular and unpredictable. For this reason we could not collect seeds from several localities at the same time and use them for all tests. We have kept in mind that the seeds from one locality do not fully represent all diploids. Seeds used for germination tests originated from two localities: a) the central reservoir “Prostřední zdrž” of the Novoml´ynské dam reservoirs in South Moravia, alt. 165 m; and b) the Ostr´y fishpond near Kolence village in South Bohemia, alt. 425 m. The seeds from South Moravia were used for a dormancy test in 1982–1983, and the seeds from South Bohemia were used for all other tests in 1985 and 1991. The population of diploid Butomus umbellatus on the shore of the central reservoir in South Moravia was totally destroyed by the dam repairs made in 1983–1984, and thus we had to find a new locality suitable for collecting seeds. Seed production of B. umbellatus in the Ostr´y fishpond fluctuated from year to year depending on the water level dynamics and the weather course in a given year; in some years, the plants remained sterile. Thus it was not possible to use the seeds collected at the same time for all tests, and to perform all tests in the same year.
Laboratory tests Test of seed dormancy a) one part of seeds sampled in August 1982 was tested for germination immediately after ripening (4 days after sampling); b) the other part of seeds sampled in August 1982 was tested for germination after the following pretreatment: seeds were stratified during winter in conditions simulating overwintering in natural habitats. Sampled seeds were stored dry in paper bags in laboratory conditions (18–20 °C) till October, when they were placed in an experimental garden to be stratified. Seeds were kept in water in polyethylene bottles, and put under water at a depth of about 0.3 m in a 38
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reservoir in an experimental garden in Pru˚honice. In April 1983, seeds were transferred in a refrigerator, and at the end of April, the germination tests were started. Seeds were tested for germination in a thermostat, under a 8/16 hours light/darkness regime (with a fluorescent lamp of 15 W as a light source) and constant temperature 20 °C. In both cases, a) and b) (and also in the following tests), the seeds germinated under aerobic conditions (on filter paper continually saturated by water in Petri dishes – 100 seeds per dish, 300 seeds in total), and in water (seeds put in glass beakers – 100 seeds per beaker of 250 ml volume filled by 150 ml of distilled water, 300 seeds in total). The number of seeds germinating was counted every day, and germinated seeds were removed. The seeds were considered as germinating when a radicle emerged. The experiments lasted for about one month, until the number of germinated seeds stabilized (no newly germinating seeds were found).
Influence of light conditions on germination Seeds sampled in 1990 were stratified for the winter season in water in natural conditions in the experimental garden, similarly as in test 1. In the spring of 1991, the seeds were tested for germination at a constant temperature of 25 °C, in aerobic conditions, and in water (see test 1), at constant light (in thermostat with fluorescent lamp of 15 W), or constant darkness.
Influence of day length – alternating temperatures and light/darkness conditions Seeds sampled in 1990 were stratified for the winter season in water in natural conditions in the experimental garden, similarly as in test 1. In the spring of 1991, the seeds were tested for germination in aerobic conditions and in water in thermostat (see test 1). Two treatments were tested: a) 16 hours 25 °C, light/8 hours 10 °C, darkness (long day); b) 8 hours 25 °C, light/16 hours 10 °C, darkness (short day).
Influence of different constant temperatures combined with aeration or submergence during stratification and germination Seeds collected in 1984 were stratified during the winter season either in water (see test 1), or in aerobic conditions, simulating an emerged bottom; seeds closed in nylon bags were placed 1–2 cm below the soil surface in the experimental garden. In the spring of 1985, seed germination was tested at constant temperatures of 5°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45 °C, and alternating light regimes of 8 hours light/16 hours darkness, in water, and in aerobic conditions (see test 1).
Statistical treatment Differences in frequency of germinated and non-germinated seeds were tested by a log-linear analysis for goodness of fit. The program SOLO (BMDP Statistical Software 1991) was used for statistical analysis.
Results
Influence of different constant temperatures combined with aeration or submergence during stratification and germination
Test of seed dormancy Fresh seeds germinated significantly less in comparison with the stratified seeds. Wintering under water undoubtedly had a positive effect on breaking the dormancy of seeds. A water environment also significantly supported germination (Tabs. 1, 2).
Influence of light or darkness Germination was significantly enhanced by constant light ; in addition, the significant positive influence of a water environment on the germination of seeds appeared both in light and in darkness (Tabs. 1, 2).
No seeds germinated at 5°, 10°, and 45°C. The first germinating seeds appeared at 15 °C; this may be considered as the threshold temperature needed to start germination. The germination optimum was in most cases found between 20° and 30 °C. In some cases, seeds germinated even at 40 °C (mainly when stratified in water and germinated in aerobic conditions) – see Fig. 1. Within a temperature range of 20° to 30 °C, the germination medium was found to have a significant effect: aerobic conditions enhanced germination. The stratified environment significantly influenced germination at marginal temperatures (15°, 35°, and 40 °C – see Tab. 3); in this case, wintering in water enhanced germination (Fig. 1).
Influence of day length (alternating light and temperature conditions)
Discussion
The effect of day length on germination was strongly significant in interaction with the germination medium: when the seeds were flooded, the conditions of long day enhanced the germination, while in short-day conditions the germination strongly decreased. When the seeds germinated in aerobic conditions, the conditions of short day were somewhat less adverse (Tabs. 1, 2). Overall, the seeds germinated best in water in conditions of long day.
The germination of B. umbellatus seeds was found to be conditioned by cold winter stratification, in accordance with the data by Muenscher (1936) [sec. Baskin & Baskin (1998)], Lukina & Papchenkov (1999), Eckert et al. (2000) and our former results (Hroudová 1980). Although in some cases fresh seeds are able to germinate, no autumn seedlings were found (Lukina & Papchenkov 1999). These results indicate that this species is a spring germinator, similarly as was found in
Table 1. Total numbers of germinated seeds of Butomus umbellatus as influenced by the stratification of seeds, light or darkness, and day length. Germination values represent means of three replicates. For details of test condition see Methods. test
1. seed dormancy
stratification conditions
germination light regime
germination temperature regime
in air
mean germination (%) in water
without stratification stratified in water
light 8 hours darkness 16 hrs light 8 hours darkness 16 hrs
constant 20 °C
12.0
16.3
constant 20 °C
67.0
77.0
2. influence of in water light or darkness in water
constant darkness constant light
constant 25 °C constant 25°C
2.3 43.0
39.3 90.0
3. influence of day length
light 16 hours darkness 8 hrs light 8 hours darkness 16 hrs
25 °C 16 hours 10 °C 8 hours 25 °C 8 hours 10 °C 16 hours
33.0
79.0
44.7
5.7
in water in water
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Table 2. Effects of pre-treatment (stratification), constant light of darkness, and day length on the germination of B. umbellatus seeds combined with water conditions of germination (seeds submerged in water or under aerobic conditions). The significance of effects was tested using a log-linear analysis for goodness of fit. Bold letters denote significant effects. test
effect
d.f.
Partial
Marginal
chi-sqr
prob
chi-sqr
prob
1. seed dormancy
A. stratification (stratified/fresh seeds) B. water conditions of germination C. germination AB AC BC ABC
1 1 1 1 1 1 7
0.00 0.00 23.04 3.28 442.68 9.57 472.23
1.0000 1.0000 0.0000 0.0700 0.0000 0.0020 0.0000
0.00 0.00 23.04 0.00 439.40 6.29 0.22
1.0000 1.0000 0.0000 1.0000 0.0000 0.0121 1.0000
2. influence of light or darkness
A. light/darkness B. water conditions of germination C. germination AB AC BC ABC
1 1 1 1 1 1 7
0.00 0.00 19.31 79.50 344.47 302.11 589.94
1.0000 1.0000 0.0000 0.0000 0.0000 0.0000 0.0000
0.00 0.00 19.31 0.00 267.96 222.60 3.56
1.0000 1.0000 0.0000 1.0000 0.0000 0.0000 0.8285
3. influence A. long/short day of day length B. water conditions of germination C. germination AB AC BC ABC
1 1 1 1 1 1 7
0.00 0.00 42.82 0.17 120.80 1.69 431.59
1.0000 1.0000 0.0000 0.6830 0.0000 0.1934 0.0000
0.00 0.00 42.82 0.00 120.63 1.52 266.45
1.0000 1.0000 0.0000 1.0000 0.0000 0.2196 0.0000
Fig. 1. Germination of Butomus umbellatus seeds at constant temperatures of 10° to 40 °C, combined with different conditions during winter stratification and during germination. w–a: stratification in water, germination in aerobic conditions; a–a: stratification in soil, germination in aerobic conditions; w–w: stratification and germination in water, a–w: stratification in soil, germination in water. Values represent total numbers of germinated seeds in per cent (means of three replicates). For details of test conditions see Methods. 40
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Table 3. Effects of different stable temperatures and water regimes during pre-treatment (seeds stratified either in soil or under water) combined with water conditions of germination (seeds submerged in water or under aerobic conditions) on the germination of B. umbellatus seeds. The significance of effects was tested using a log-linear analysis for goodness of fit. Bold letters denote significant effects. d.f. – degree of freedom temperature
effect
d.f.
Partial chi-sqr
Marginal prob
chi-sqr
prob
15°C
A. stratification B. water conditions of germination C. germination AB AC BC ABC
1 1 1 1 1 1 7
0.00 0.00 709.97 0.02 8.77 2.59 752.45
1.0000 1.0000 0.0000 0.8912 0.0031 0.1078 0.0000
0.00 0.00 709.97 0.00 8.76 2.57 4.14
1.0000 1.0000 0.0000 1.0000 0.0031 0.1091 0.7633
20°C
A. stratification B. water conditions of germination C. germination AB AC BC ABC
1 1 1 1 1 1 7
0.00 0.00 487.97 0.03 0.94 45.43 534.36
1.0000 1.0000 0.0000 0.8523 0.3320 0.0000 0.0000
0.00 0.00 487.97 0.00 0.91 45.40 0.06
1.0000 1.0000 0.0000 1.0000 0.3411 0.0000 1.0000
25°C
A. stratification B. water conditions of germination C. germination AB AC BC ABC
1 1 1 1 1 1 7
0.00 0.00 340.96 0.00 0.07 10.57 353.38
1.0000 1.0000 0.0000 0.9807 0.7860 0.0012 0.0000
0.00 0.00 340.96 0.00 0.07 10.56 1.78
1.0000 1.0000 0.0000 1.0000 0.7869 0.0012 0.9708
30°C
A. stratification B. water conditions of germination C. germination AB AC BC ABC
1 1 1 1 1 1 7
0.00 0.00 393.93 0.00 0.00 12.13 407.34
1.0000 1.0000 0.0000 1.0000 1.0000 0.0005 0.0000
0.00 0.00 393.93 0.00 0.00 12.13 1.27
1.0000 1.0000 0.0000 1.0000 1.0000 0.0005 0.9891
35°C
A. stratification B. water conditions of germination C. germination AB AC BC ABC
1 1 1 1 1 1 7
0.00 0.00 578.99 0.02 34.33 0.74 622.20
1.0000 1.0000 0.0000 0.8852 0.0000 0.3883 0.0000
0.00 0.00 578.99 0.00 34.31 0.72 8.15
1.0000 1.0000 0.0000 1.0000 0.0000 0.3950 0.3192
40°C
A. stratification B. water conditions of germination C. germination AB AC BC ABC
1 1 1 1 1 1 7
0.00 0.00 1428.26 0.25 20.09 20.09 1468.54
1.0000 1.0000 0.0000 0.6202 0.0000 0.0000 0.0000
0.00 0.00 1428.26 0.00 19.84 19.84 0.35
1.0000 1.0000 0.0000 1.0000 0.0000 0.0000 0.9998
many species of temperate sedges (Carex sp.) (Schütz & Rave 1999). The probability of seedling survival is connected closely to the seasonal timing of germination (Harper 1977). Dormancy prevents germination if the
climatic conditions favourable for germination may arrive, but at a time of the year when it can be expected that the seedlings will not survive (Vleeshouwers et al. 1995). Seed dormancy appears to be highly functional FLORA (2003) 198
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for the survival of B. umbellatus: although favourable conditions for seed germination may occur in the time of seed ripening and shedding on temporarily emerged pond bottoms the seedlings of B. umbellatus are hardly able to survive flooding in the following winter season. Fluctuations in water level are typical for habitats of this species; flooding to high water level is more frequent in the winter season, so the need of cold stratification may be considered as an adaptation to habitats with fluctuating water level in temperate regions. The positive influence of light on the germination of B. umbellatus seeds indicates their sensitivity to irradiation. This feature was found for B. umbellatus also by Lukina & Papchenkov (1999), and is similar to many other wetland plants inhabiting sites near shorelines, with changing littoral/limosal/terrestrial conditions, e.g., Litorella uniflora (Arts & van der Heijden 1990), Oenanthe aquatica (Hroudová et al. 1992), and many species of temperate sedges (Schütz 1999; Schütz & Rave 1999; Busch 2001). Light requirement by seeds is considered to be a mechanism preventing germination when seeds are buried in the soil, covered by leaf litter or by a muddy layer, or flooded by a high water level (Pons 1992; Schütz & Rave 1999). This means that seeds of Butomus umbellatus can germinate after water recedes – on an emerged bottom, or on the soil surface under a very shallow water layer. Some other environmental factors can also influence the response of a seed to light (Pons 1992). The germination of B. umbellatus seeds in darkness was supported by flooding, which would make it possible for them to germinate to some extent even under a higher water level or in mud layer. A relatively high temperature is optimal for seed germination of Butomus umbellatus, and the upper temperature limit is high. Requiring a relatively high temperature for seed germination was found to be a common feature of many wetland plants (Grime et al. 1981; Hroudová et al. 1988); it was considered as an adaptation to germination in summer months when the water level recedes, and also as a mechanism, which prevents germination under a deep water level. In the case of B. umbellatus, this is in agreement with light requirements. Cold wet stratification supported seed germination in marginal temperatures, and thus seems to be a mechanism that modifies the temperature range of germination. This is in agreement with findings that the period of chilling reduces the requirements for other environmental factors affecting germination (Probert 1992). Alternating temperatures evidently are not necessary for the germination of B. umbellatus seeds as it may be seen from comparison with high germination percentage attained in some tests under a constant temperature regime (Tab. 1). The duration of the light period does not 42
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appear to be a decisive factor as well – seeds are able to germinate well under a short light period, but with constant temperature. The favourable effect of long-day conditions may be attributed to a higher temperature attained during day period, which is evident also from strong reduction of seed germination in water environment where water temperature increased slowly. The percentage of germinated seeds differed between some tests although the same treatments were used (compare test 1 in Tab. 1 and Fig. 1). This difference could be caused either by the different physiological properties of the seeds belonging to different genotypes or by the environmental conditions in natural habitats during ripening and/or wintering of seeds: The seed germination was generally low in the test 4 (Fig. 1), when the seeds ripened during cold and wet summer in 1984 (Fig. 2).
Fig. 2. Weather course during growing seasons and following winter seasons in the years, when the seeds of Butomus umbellatus used for tests of germination were sampled and stratified. Total monthly precipitations and mean monthly temperatures are given (the data from Agrometeorological Bulletin for the years 1982/83, 1984/85 and 1990/91) from climatological station of Velké Pavlovice (South Moravia) in 1982/83, and climatological station of Tábor (South Bohemia) in 1984/85 and 1990/91.
From the results of the present study we can conclude that the conditions suitable for germination of seeds of B. umbellatus in natural habitats may occur when the temperature of the environment does not sink below 15 °C (or sinks only for a short period), and day temperatures reach 20°–25 °C (or even more). Such climatic conditions may occur from late May to June: in the Czech Republic, mean daily temperature of 15 °C is attained in the altitudinal range of occurrence of B. umbellatus from 20.5. to 25.6. (Götz 1966). Shifting the seed germination of B. umbellatus to the early summer months is disadvantageous for seedlings, which were found to be small and slowly growing in the first phase of their development (Hroudová & Zákravsk´y 1993 a), and may be competitively suppressed by fastgrowing species that germinated in early spring, or by adult plants of B. umbellatus. This may be the reason that this species is able to establish successfully in new localities on open emerged shores without vegetation, but repeatedly not within established populations of adult plants (initial seedling recruitment sensu Eriksson 1989, 1992). This corresponds with the results of isozyme analyses: in the Czech and Slovak Republics, natural populations of diploids were found to be genetically homogeneous in spite of their high seed production, which documents their clonal origin. On the other hand, inter-populational genetical variability was found relatively high, and isozyme analyses indicated gene flow among populations of diploids (Kirschner et al. in press.). The colonization of a new distant locality by diploid B. umbellatus was observed recently on the shore of Rozkosˇ dam reservoir (NE Bohemia) (Krahulec & Krahulcová pers. com.). The optimum conditions for the germination of diploid B. umbellatus seeds may be found on emerged bottoms saturated by water, or on bottom surfaces covered by a shallow water layer. Lukina & Papchenkov (1995, 1999) found the depth to 10 cm suitable for seedling establishment of B. umbellatus. Seed production may be high – on average 7030 seeds per umbel (n = 30) in the Velk´y Dubovec fishpond (South Bohemia) were sampled by Hroudová (1980) on 13. 7. 1972, from a diploid population. 6148 seeds per umbel (ranging from 3831 to 9010) and two populations sterile, not related to ploidy levels, were reported by Eckert et al. 2000). From the circumstances of germination and the amount of seed production it is concluded that the process of germination does not represent a high-risk stage in the sexual reproduction of diploid B. umbellatus.
Acknowledgements Our sincere thanks are due to F. Krahulec and L. Moravcová for reading the manuscript and for their valuable critical comments on the text, to Janice Forry for language revision, and to
E. Zamazalová for technical assistance. The work was partly supported by the Grant Agency of the Czechoslovak Academy of Sciences (Grant no. 60543), and by the project of the Institute of Botany of ASCR (AV0Z 6005908).
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Probert, R. J. (1992): The role of temperature in germination ecophysiology. – In: Fenner, M. (ed.): Seeds. The ecology of regeneration in plant communities. C.A.B. International, Wallingford. 285–325. Schütz, W. (1999): Germination responses of temperate Carex-species to diurnally fluctuating temperatures – a comparative study. – Flora 194: 21–32. Schütz, W. & Rave, G. (1999): The effect of cold stratification and light on the seed germination of temperate sedges (Carex) from various habitats and implications for regenerative strategies. – Plant Ecol. 144: 215–230. Stebbins, G. L. (1971): Chromosomal evolution in higher plants. – London. Tal, M. (1980): Physiology of polyploids. – In: Lewis, W. H. (ed.): Polyploidy. Biological relevance. Plenum, New York. 61–75. Vleeshouwers, L. M.; Bouwmeester, H. J. & Karssen, C. M. (1995): Redefining seed dormancy: an attempt to integrate physiology and ecology. – J. Ecol. 83: 1031–1037.
Germination responses of diploid Butomus umbellatus to light, temperature and flooding Zdenka Hroudová* and Petr Zákravsk´y Institute of Botany, Academy of Sciences of the Czech Republic, Pru˚honice, Czech Republic Submitted: Feb 18, 2002 · Accepted, in revised form: Nov 5, 2002
Summary The germination of seeds of diploid Butomus umbellatus was studied to determine the favourable conditions for germination and whether seed germination may represent a limitation within the sexual reproductive cycle of this cytotype. The need to stratify the seeds and the response of the seeds to environmental conditions during germination were studied. The influence of light/darkness, temperature, day length, and flooding/aerobic conditions was tested. Seed germination was enhanced in the winter by cold wet stratification, which indicates prevailing seed germination in the spring. Seeds germinated best in light, at relatively high temperatures (20°–30°C), in long days. Seeds were able to germinate when flooded; in most cases, flooding supported germination. In field habitats, the optimum conditions for the germination of B. umbellatus seeds may be found on an open emerged bottom saturated with water or under a shallow water layer. The process of germination does not represent a high-risk stage in the sexual reproduction of this cytotype of B. umbellatus. The time of germination (late spring or summer months) contributes to seedling mortality due to the competition of adult plants or other early-germinating species. Key words: emergent macrophytes, sexual reproduction, seeds, ploidy level
Introduction Ploidy level may play an important role in the ecological properties of plant species and their adaptability to new habitats. Most polyploid taxa have ecological tolerances, which are quite different from those of their diploid progenitors (Levin 1983). Polyploids were found to be more resistant to pests and pathogens, or other kinds of stress, and appeared to be stronger competitors, with greater emphasis on vegetative reproduction (Tal 1980; Levin 1983). On the other hand, lowered fertility is more frequently observed in polyploids (namely in autopolyploids – Stebbins 1971), while diploids are usually fully fertile, and able to spread by seeds. This is the case also of Butomus umbellatus L. The results of former investigations (Krahulcová & Jarolímová 1993) have shown that seed production in Butomus umbellatus depends on ploidy level; triploids are self-incompatible, while diploids are self-compatible. Consequently, a more frequent occurrence of
diploids is to be supposed due to their seed reproduction ability. However, triploids were found prevailing in the Czech and Slovak Republics (Hroudová & Zákravsky´ 1993 b). This indicates that the sexual reproduction of diploids is not as successful in compensating for the efficiency of the other progressive ecological properties of triploids (intensive vegetative spreading, vigorous growth, resistance to eutrophication – for comparison of both cytotypes see Hroudová et al. 1996), which increase the adaptability of triploids to a wider scale of habitat conditions and enable them to find suitable habitats more frequently. In Central Europe, B. umbellatus occurs predominantly in shallow water reservoirs, or along stream shores, in habitats with a fluctuating water level. Water level dynamics is the crucial factor influencing all other habitat conditions, and an adaptation to changing water level may be of primary importance for the success of seed reproduction. In this study, conditions which occur under a changing aerobic/submerged environment (flooding in winter season, or in the spring) were simu-
* Corresponding author: Zdenka Hroudová, Institute of Botany, Academy of Sciences of the Czech Republic, 252 43 Pru˚honice, Czech Republic, e-mail: [email protected] 0367-2530/03/198/01-037 $ 15.00/0
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lated. We tested the need of stratification to break seed dormancy, the influence of flooding or aerobic conditions on dormant seeds during wintering and on seeds during germination, and the influence of light or darkness, day length, and temperature on germination. We tried to find, which environmental factors dependent on water level changes may affect the germination of seeds of diploid B. umbellatus, and whether the phase of seed germination may represent a high-risk stage within its sexual reproductive cycle.
Material and methods Plant material Sampling a satisfactory amount of seeds was the main methodological problem. While the development of populations of Butomus umbellatus fluctuates from year to year depending on water level, the fertility of the populations is irregular and unpredictable. For this reason we could not collect seeds from several localities at the same time and use them for all tests. We have kept in mind that the seeds from one locality do not fully represent all diploids. Seeds used for germination tests originated from two localities: a) the central reservoir “Prostřední zdrž” of the Novoml´ynské dam reservoirs in South Moravia, alt. 165 m; and b) the Ostr´y fishpond near Kolence village in South Bohemia, alt. 425 m. The seeds from South Moravia were used for a dormancy test in 1982–1983, and the seeds from South Bohemia were used for all other tests in 1985 and 1991. The population of diploid Butomus umbellatus on the shore of the central reservoir in South Moravia was totally destroyed by the dam repairs made in 1983–1984, and thus we had to find a new locality suitable for collecting seeds. Seed production of B. umbellatus in the Ostr´y fishpond fluctuated from year to year depending on the water level dynamics and the weather course in a given year; in some years, the plants remained sterile. Thus it was not possible to use the seeds collected at the same time for all tests, and to perform all tests in the same year.
Laboratory tests Test of seed dormancy a) one part of seeds sampled in August 1982 was tested for germination immediately after ripening (4 days after sampling); b) the other part of seeds sampled in August 1982 was tested for germination after the following pretreatment: seeds were stratified during winter in conditions simulating overwintering in natural habitats. Sampled seeds were stored dry in paper bags in laboratory conditions (18–20 °C) till October, when they were placed in an experimental garden to be stratified. Seeds were kept in water in polyethylene bottles, and put under water at a depth of about 0.3 m in a 38
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reservoir in an experimental garden in Pru˚honice. In April 1983, seeds were transferred in a refrigerator, and at the end of April, the germination tests were started. Seeds were tested for germination in a thermostat, under a 8/16 hours light/darkness regime (with a fluorescent lamp of 15 W as a light source) and constant temperature 20 °C. In both cases, a) and b) (and also in the following tests), the seeds germinated under aerobic conditions (on filter paper continually saturated by water in Petri dishes – 100 seeds per dish, 300 seeds in total), and in water (seeds put in glass beakers – 100 seeds per beaker of 250 ml volume filled by 150 ml of distilled water, 300 seeds in total). The number of seeds germinating was counted every day, and germinated seeds were removed. The seeds were considered as germinating when a radicle emerged. The experiments lasted for about one month, until the number of germinated seeds stabilized (no newly germinating seeds were found).
Influence of light conditions on germination Seeds sampled in 1990 were stratified for the winter season in water in natural conditions in the experimental garden, similarly as in test 1. In the spring of 1991, the seeds were tested for germination at a constant temperature of 25 °C, in aerobic conditions, and in water (see test 1), at constant light (in thermostat with fluorescent lamp of 15 W), or constant darkness.
Influence of day length – alternating temperatures and light/darkness conditions Seeds sampled in 1990 were stratified for the winter season in water in natural conditions in the experimental garden, similarly as in test 1. In the spring of 1991, the seeds were tested for germination in aerobic conditions and in water in thermostat (see test 1). Two treatments were tested: a) 16 hours 25 °C, light/8 hours 10 °C, darkness (long day); b) 8 hours 25 °C, light/16 hours 10 °C, darkness (short day).
Influence of different constant temperatures combined with aeration or submergence during stratification and germination Seeds collected in 1984 were stratified during the winter season either in water (see test 1), or in aerobic conditions, simulating an emerged bottom; seeds closed in nylon bags were placed 1–2 cm below the soil surface in the experimental garden. In the spring of 1985, seed germination was tested at constant temperatures of 5°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45 °C, and alternating light regimes of 8 hours light/16 hours darkness, in water, and in aerobic conditions (see test 1).
Statistical treatment Differences in frequency of germinated and non-germinated seeds were tested by a log-linear analysis for goodness of fit. The program SOLO (BMDP Statistical Software 1991) was used for statistical analysis.
Results
Influence of different constant temperatures combined with aeration or submergence during stratification and germination
Test of seed dormancy Fresh seeds germinated significantly less in comparison with the stratified seeds. Wintering under water undoubtedly had a positive effect on breaking the dormancy of seeds. A water environment also significantly supported germination (Tabs. 1, 2).
Influence of light or darkness Germination was significantly enhanced by constant light ; in addition, the significant positive influence of a water environment on the germination of seeds appeared both in light and in darkness (Tabs. 1, 2).
No seeds germinated at 5°, 10°, and 45°C. The first germinating seeds appeared at 15 °C; this may be considered as the threshold temperature needed to start germination. The germination optimum was in most cases found between 20° and 30 °C. In some cases, seeds germinated even at 40 °C (mainly when stratified in water and germinated in aerobic conditions) – see Fig. 1. Within a temperature range of 20° to 30 °C, the germination medium was found to have a significant effect: aerobic conditions enhanced germination. The stratified environment significantly influenced germination at marginal temperatures (15°, 35°, and 40 °C – see Tab. 3); in this case, wintering in water enhanced germination (Fig. 1).
Influence of day length (alternating light and temperature conditions)
Discussion
The effect of day length on germination was strongly significant in interaction with the germination medium: when the seeds were flooded, the conditions of long day enhanced the germination, while in short-day conditions the germination strongly decreased. When the seeds germinated in aerobic conditions, the conditions of short day were somewhat less adverse (Tabs. 1, 2). Overall, the seeds germinated best in water in conditions of long day.
The germination of B. umbellatus seeds was found to be conditioned by cold winter stratification, in accordance with the data by Muenscher (1936) [sec. Baskin & Baskin (1998)], Lukina & Papchenkov (1999), Eckert et al. (2000) and our former results (Hroudová 1980). Although in some cases fresh seeds are able to germinate, no autumn seedlings were found (Lukina & Papchenkov 1999). These results indicate that this species is a spring germinator, similarly as was found in
Table 1. Total numbers of germinated seeds of Butomus umbellatus as influenced by the stratification of seeds, light or darkness, and day length. Germination values represent means of three replicates. For details of test condition see Methods. test
1. seed dormancy
stratification conditions
germination light regime
germination temperature regime
in air
mean germination (%) in water
without stratification stratified in water
light 8 hours darkness 16 hrs light 8 hours darkness 16 hrs
constant 20 °C
12.0
16.3
constant 20 °C
67.0
77.0
2. influence of in water light or darkness in water
constant darkness constant light
constant 25 °C constant 25°C
2.3 43.0
39.3 90.0
3. influence of day length
light 16 hours darkness 8 hrs light 8 hours darkness 16 hrs
25 °C 16 hours 10 °C 8 hours 25 °C 8 hours 10 °C 16 hours
33.0
79.0
44.7
5.7
in water in water
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Table 2. Effects of pre-treatment (stratification), constant light of darkness, and day length on the germination of B. umbellatus seeds combined with water conditions of germination (seeds submerged in water or under aerobic conditions). The significance of effects was tested using a log-linear analysis for goodness of fit. Bold letters denote significant effects. test
effect
d.f.
Partial
Marginal
chi-sqr
prob
chi-sqr
prob
1. seed dormancy
A. stratification (stratified/fresh seeds) B. water conditions of germination C. germination AB AC BC ABC
1 1 1 1 1 1 7
0.00 0.00 23.04 3.28 442.68 9.57 472.23
1.0000 1.0000 0.0000 0.0700 0.0000 0.0020 0.0000
0.00 0.00 23.04 0.00 439.40 6.29 0.22
1.0000 1.0000 0.0000 1.0000 0.0000 0.0121 1.0000
2. influence of light or darkness
A. light/darkness B. water conditions of germination C. germination AB AC BC ABC
1 1 1 1 1 1 7
0.00 0.00 19.31 79.50 344.47 302.11 589.94
1.0000 1.0000 0.0000 0.0000 0.0000 0.0000 0.0000
0.00 0.00 19.31 0.00 267.96 222.60 3.56
1.0000 1.0000 0.0000 1.0000 0.0000 0.0000 0.8285
3. influence A. long/short day of day length B. water conditions of germination C. germination AB AC BC ABC
1 1 1 1 1 1 7
0.00 0.00 42.82 0.17 120.80 1.69 431.59
1.0000 1.0000 0.0000 0.6830 0.0000 0.1934 0.0000
0.00 0.00 42.82 0.00 120.63 1.52 266.45
1.0000 1.0000 0.0000 1.0000 0.0000 0.2196 0.0000
Fig. 1. Germination of Butomus umbellatus seeds at constant temperatures of 10° to 40 °C, combined with different conditions during winter stratification and during germination. w–a: stratification in water, germination in aerobic conditions; a–a: stratification in soil, germination in aerobic conditions; w–w: stratification and germination in water, a–w: stratification in soil, germination in water. Values represent total numbers of germinated seeds in per cent (means of three replicates). For details of test conditions see Methods. 40
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Table 3. Effects of different stable temperatures and water regimes during pre-treatment (seeds stratified either in soil or under water) combined with water conditions of germination (seeds submerged in water or under aerobic conditions) on the germination of B. umbellatus seeds. The significance of effects was tested using a log-linear analysis for goodness of fit. Bold letters denote significant effects. d.f. – degree of freedom temperature
effect
d.f.
Partial chi-sqr
Marginal prob
chi-sqr
prob
15°C
A. stratification B. water conditions of germination C. germination AB AC BC ABC
1 1 1 1 1 1 7
0.00 0.00 709.97 0.02 8.77 2.59 752.45
1.0000 1.0000 0.0000 0.8912 0.0031 0.1078 0.0000
0.00 0.00 709.97 0.00 8.76 2.57 4.14
1.0000 1.0000 0.0000 1.0000 0.0031 0.1091 0.7633
20°C
A. stratification B. water conditions of germination C. germination AB AC BC ABC
1 1 1 1 1 1 7
0.00 0.00 487.97 0.03 0.94 45.43 534.36
1.0000 1.0000 0.0000 0.8523 0.3320 0.0000 0.0000
0.00 0.00 487.97 0.00 0.91 45.40 0.06
1.0000 1.0000 0.0000 1.0000 0.3411 0.0000 1.0000
25°C
A. stratification B. water conditions of germination C. germination AB AC BC ABC
1 1 1 1 1 1 7
0.00 0.00 340.96 0.00 0.07 10.57 353.38
1.0000 1.0000 0.0000 0.9807 0.7860 0.0012 0.0000
0.00 0.00 340.96 0.00 0.07 10.56 1.78
1.0000 1.0000 0.0000 1.0000 0.7869 0.0012 0.9708
30°C
A. stratification B. water conditions of germination C. germination AB AC BC ABC
1 1 1 1 1 1 7
0.00 0.00 393.93 0.00 0.00 12.13 407.34
1.0000 1.0000 0.0000 1.0000 1.0000 0.0005 0.0000
0.00 0.00 393.93 0.00 0.00 12.13 1.27
1.0000 1.0000 0.0000 1.0000 1.0000 0.0005 0.9891
35°C
A. stratification B. water conditions of germination C. germination AB AC BC ABC
1 1 1 1 1 1 7
0.00 0.00 578.99 0.02 34.33 0.74 622.20
1.0000 1.0000 0.0000 0.8852 0.0000 0.3883 0.0000
0.00 0.00 578.99 0.00 34.31 0.72 8.15
1.0000 1.0000 0.0000 1.0000 0.0000 0.3950 0.3192
40°C
A. stratification B. water conditions of germination C. germination AB AC BC ABC
1 1 1 1 1 1 7
0.00 0.00 1428.26 0.25 20.09 20.09 1468.54
1.0000 1.0000 0.0000 0.6202 0.0000 0.0000 0.0000
0.00 0.00 1428.26 0.00 19.84 19.84 0.35
1.0000 1.0000 0.0000 1.0000 0.0000 0.0000 0.9998
many species of temperate sedges (Carex sp.) (Schütz & Rave 1999). The probability of seedling survival is connected closely to the seasonal timing of germination (Harper 1977). Dormancy prevents germination if the
climatic conditions favourable for germination may arrive, but at a time of the year when it can be expected that the seedlings will not survive (Vleeshouwers et al. 1995). Seed dormancy appears to be highly functional FLORA (2003) 198
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for the survival of B. umbellatus: although favourable conditions for seed germination may occur in the time of seed ripening and shedding on temporarily emerged pond bottoms the seedlings of B. umbellatus are hardly able to survive flooding in the following winter season. Fluctuations in water level are typical for habitats of this species; flooding to high water level is more frequent in the winter season, so the need of cold stratification may be considered as an adaptation to habitats with fluctuating water level in temperate regions. The positive influence of light on the germination of B. umbellatus seeds indicates their sensitivity to irradiation. This feature was found for B. umbellatus also by Lukina & Papchenkov (1999), and is similar to many other wetland plants inhabiting sites near shorelines, with changing littoral/limosal/terrestrial conditions, e.g., Litorella uniflora (Arts & van der Heijden 1990), Oenanthe aquatica (Hroudová et al. 1992), and many species of temperate sedges (Schütz 1999; Schütz & Rave 1999; Busch 2001). Light requirement by seeds is considered to be a mechanism preventing germination when seeds are buried in the soil, covered by leaf litter or by a muddy layer, or flooded by a high water level (Pons 1992; Schütz & Rave 1999). This means that seeds of Butomus umbellatus can germinate after water recedes – on an emerged bottom, or on the soil surface under a very shallow water layer. Some other environmental factors can also influence the response of a seed to light (Pons 1992). The germination of B. umbellatus seeds in darkness was supported by flooding, which would make it possible for them to germinate to some extent even under a higher water level or in mud layer. A relatively high temperature is optimal for seed germination of Butomus umbellatus, and the upper temperature limit is high. Requiring a relatively high temperature for seed germination was found to be a common feature of many wetland plants (Grime et al. 1981; Hroudová et al. 1988); it was considered as an adaptation to germination in summer months when the water level recedes, and also as a mechanism, which prevents germination under a deep water level. In the case of B. umbellatus, this is in agreement with light requirements. Cold wet stratification supported seed germination in marginal temperatures, and thus seems to be a mechanism that modifies the temperature range of germination. This is in agreement with findings that the period of chilling reduces the requirements for other environmental factors affecting germination (Probert 1992). Alternating temperatures evidently are not necessary for the germination of B. umbellatus seeds as it may be seen from comparison with high germination percentage attained in some tests under a constant temperature regime (Tab. 1). The duration of the light period does not 42
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appear to be a decisive factor as well – seeds are able to germinate well under a short light period, but with constant temperature. The favourable effect of long-day conditions may be attributed to a higher temperature attained during day period, which is evident also from strong reduction of seed germination in water environment where water temperature increased slowly. The percentage of germinated seeds differed between some tests although the same treatments were used (compare test 1 in Tab. 1 and Fig. 1). This difference could be caused either by the different physiological properties of the seeds belonging to different genotypes or by the environmental conditions in natural habitats during ripening and/or wintering of seeds: The seed germination was generally low in the test 4 (Fig. 1), when the seeds ripened during cold and wet summer in 1984 (Fig. 2).
Fig. 2. Weather course during growing seasons and following winter seasons in the years, when the seeds of Butomus umbellatus used for tests of germination were sampled and stratified. Total monthly precipitations and mean monthly temperatures are given (the data from Agrometeorological Bulletin for the years 1982/83, 1984/85 and 1990/91) from climatological station of Velké Pavlovice (South Moravia) in 1982/83, and climatological station of Tábor (South Bohemia) in 1984/85 and 1990/91.
From the results of the present study we can conclude that the conditions suitable for germination of seeds of B. umbellatus in natural habitats may occur when the temperature of the environment does not sink below 15 °C (or sinks only for a short period), and day temperatures reach 20°–25 °C (or even more). Such climatic conditions may occur from late May to June: in the Czech Republic, mean daily temperature of 15 °C is attained in the altitudinal range of occurrence of B. umbellatus from 20.5. to 25.6. (Götz 1966). Shifting the seed germination of B. umbellatus to the early summer months is disadvantageous for seedlings, which were found to be small and slowly growing in the first phase of their development (Hroudová & Zákravsk´y 1993 a), and may be competitively suppressed by fastgrowing species that germinated in early spring, or by adult plants of B. umbellatus. This may be the reason that this species is able to establish successfully in new localities on open emerged shores without vegetation, but repeatedly not within established populations of adult plants (initial seedling recruitment sensu Eriksson 1989, 1992). This corresponds with the results of isozyme analyses: in the Czech and Slovak Republics, natural populations of diploids were found to be genetically homogeneous in spite of their high seed production, which documents their clonal origin. On the other hand, inter-populational genetical variability was found relatively high, and isozyme analyses indicated gene flow among populations of diploids (Kirschner et al. in press.). The colonization of a new distant locality by diploid B. umbellatus was observed recently on the shore of Rozkosˇ dam reservoir (NE Bohemia) (Krahulec & Krahulcová pers. com.). The optimum conditions for the germination of diploid B. umbellatus seeds may be found on emerged bottoms saturated by water, or on bottom surfaces covered by a shallow water layer. Lukina & Papchenkov (1995, 1999) found the depth to 10 cm suitable for seedling establishment of B. umbellatus. Seed production may be high – on average 7030 seeds per umbel (n = 30) in the Velk´y Dubovec fishpond (South Bohemia) were sampled by Hroudová (1980) on 13. 7. 1972, from a diploid population. 6148 seeds per umbel (ranging from 3831 to 9010) and two populations sterile, not related to ploidy levels, were reported by Eckert et al. 2000). From the circumstances of germination and the amount of seed production it is concluded that the process of germination does not represent a high-risk stage in the sexual reproduction of diploid B. umbellatus.
Acknowledgements Our sincere thanks are due to F. Krahulec and L. Moravcová for reading the manuscript and for their valuable critical comments on the text, to Janice Forry for language revision, and to
E. Zamazalová for technical assistance. The work was partly supported by the Grant Agency of the Czechoslovak Academy of Sciences (Grant no. 60543), and by the project of the Institute of Botany of ASCR (AV0Z 6005908).
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