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Germination variability and the effect of various pre-treatment on germination in the perennial spurge Euphorbia nicaeensis All.
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Flora 201 (2006) 633–641 www.elsevier.de/flora
Germination variability and the effect of various pre-treatment on germination in the perennial spurge Euphorbia nicaeensis All. E. Narbonaa,, P.L. Ortizb, M. Aristab a
Departamento de Ciencias Ambientales, Universidad Pablo de Olavide, Ctra. Utrera Km 1, 41013 Sevilla, Spain Departamento de Biologı´a Vegetal y Ecologı´a, Universidad de Sevilla, Apdo. 1095. 4180 Sevilla, Spain
b
Received 7 November 2005; accepted 1 February 2006
Abstract Patterns of seed germination of the perennial spurge Euphorbia nicaeensis were studied in three populations in southwestern Spain. We investigated the variation in seed viability and germination among individuals, and among populations over 2 consecutive years. We also studied if diverse factors such as temperature, acid scarification, darkness or caruncle loss affect the germination of the seeds. Interindividual and interpopulation variability in seed viability was found. E. nicaeensis seeds are nondormant, so differences in interpopulation viability translate into differences in final germination rates. The germination percentage of the seeds from each population was similar in the 2 years studied. The effects of diverse factors were homogeneous in the two populations studied. Darkness has no effect on seed germination, and ecarunculate seeds germinated in the same proportions as carunculate seeds; this could allow the seeds to germinate in the chambers of deserted anthills. Acid scarification significantly reduced the germination percentage in only one of the populations, but over 50% of the seeds germinated, which could allow herbivores to act as occasional dispersing agents. The seeds that were preheated at 100 1C for 1 and 5 min germinated in the same proportions as the control group. The seeds that were preheated at 120 1C for 5 min displayed a significant decrease in germination, but the percentage was over 40% for both populations, indicating that the seeds could still germinate after the passage of a fire. r 2006 Elsevier GmbH. All rights reserved. Keywords: Acid scarification; Caruncle; Heat pre-treatment; Interpopulation variability; Interyear variability; Seed viability
Introduction Variation in seed germination capacity of a species can occur at least at three levels: within individuals (Gutterman, 1992; Silvertown, 1984), within populations (Hardin, 1984; Pe´rez-Garcı´ a, 1997; Venable and Lawlor, 1980) or among populations (Angosto and Matilla, 1993; Hacker et al., 1984; McGee and Marshall, Corresponding author. Fax: +954349151.
E-mail address: [email protected] (E. Narbona). 0367-2530/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.flora.2006.02.004
1994; Nordborg and Bergelson, 1999). Interpopulation variability can be linked with latitude, altitude, moisture and soil nutrients, temperature, type and density of the ecosystem’s plant cover and the degree of disturbance of the location (Baskin and Baskin, 1998). However, differences in germination capacity are not always related to the ecological characteristics of each population (Bevington, 1986; Martin et al., 1995) and several studies have confirmed that this variability between populations could be at least partly due to genetic factors (Hacker et al., 1984; Van der Vegte, 1978).
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Leaving aside intraspecific variability, several different environmental factors can affect the germination capacity of the seeds of a species. In Mediterranean ecosystems, fire is a common disturbance (Moreno et al., 1998) and two types of plants have been described according to their response to fire: seeder and resprouter species (Keeley, 1991). Generally, seeders die after the fire and the seeds germinate in the first rainy season following the fire, since their dormancy is easily interrupted by thermal shock or by substances leached from burnt plant material. In laboratory conditions, the seeds of these species need to be subjected to a heat pretreatment for 1–10 min at 100–120 1C in order to be able to germinate (Bell et al., 1993; Keeley, 1991; Pereiras et al., 1985; Tarrega et al., 1992; Valbuena et al., 1992), but excessive and prolonged heat exposure (X120 1C for several minutes) causes a significant decrease in germination (Keeley et al., 1985; Parker, 1987). Resprouters, on the other hand, following a fire, elongate the dormant buds on the lower knots of the stem, generally underground (Keeley, 1991), and do not usually form seed banks (Parker and Kelly, 1989). Within the Euphorbiaceae family, the genus Euphorbia has the largest number of species (approx. 1000–1600) and a widespread distribution, especially in subtropical and warm areas (Steinmann and Porter, 2002; Webster, 1994). Most studies about germination in Euphorbia have been carried out on annual species (Brenchley and Warington, 1930; Brecke, 1995; Capon and Van der Asdall, 1967; Heit, 1942; Kigel et al., 1992; Kivilaan and Bandurski, 1981; Krueger and Shaner, 1982; Van der Rooden et al., 1970; Sen and Chatterji, 1968; Voigt, 1977; Washitani and Masuda, 1990). However, only a few perennial species have been studied, such as Euphorbia caducifolia, E. characias and E. esula (Best et al., 1980; Go´mez and Espadaler, 1997; Selleck et al., 1962; Sen and Chatterji, 1966). E. nicaeensis All. subsp. nicaeensis is a perennial, herbaceous spurge with a circum-Mediterranean and Central Europe distribution and it prefers calcareous soils (Benedı´ et al., 1997; Smith and Tutin, 1978). This species grows in dry and sunny places between 100 and 1800 m, in degraded stages of Mediterranean Quercus forests (Benedı´ et al., 1997). E. nicaeensis is functionally andromonoecious, and the pistillate cyathia are situated predominantly at higher levels of the inflorescence (Narbona et al., 2002). Flowering time is in early summer and the seed dispersal takes place in late summer. This species produces an average of 117 capsules per individual (Narbona, 2002) and each capsule have three seeds, each one weighing between 4 and 8 mg (Narbona et al., 2005). E. nicaeensis is a diplochorous species: it has a primary explosive dispersal system (Narbona et al., 2005) and a secondary dispersal system by ants, since its seeds have a caruncle (Molinier and Mu¨ller, 1938). E. nicaeensis seeds are
transported by myrmecochorous and granivorous ants in various parts of the Mediterranean basin (Go´mez and Espadaler, 1994; Narbona, 2002; Wolff and Debussche, 1999) and they are nondormant (Al-Samman et al., 2001). In this study, we examined the germination variability of E. nicaeensis seeds and their ability to germinate under diverse environmental conditions. The specific objectives of this study were as follows: (1) investigate if there are differences in the viability and germination of seeds from different populations, (2) find out if there are differences in germination capacity over 2 consecutive years and (3) investigate if different factors such as temperature, darkness or caruncle loss affect seed germination.
Methods Site selection Three populations of E. nicaeensis were studied in south-western Spain: Sierra de Aracena, La Camilla and Puerto de las Palomas. These populations are relatively close (maximum 163 km between Sierra de Aracena and Puerto de las Palomas), but they were selected because they show important differences in environmental conditions (soils, altitude and rainfall). The Sierra de Aracena population is situated in the province of Huelva (371530 N, 61330 W), at an altitude of 700 m on Cambric decalcified limestones. Vegetation consists of a cultivated woodland of Castanea sativa Mill. with shrub species such as Viburnum tinus L., Daphne gnidium L. or Phlomis purpurea L. This population experiences a mean annual temperature of 14.5 1C and a mean annual rainfall of 1025 mm. La Camilla population is in the Sierra de Grazalema Natural Park (Province of Cadiz, 361470 N, 51240 W), at an altitude of 800 m on Jurassic dolomites. Vegetation consists of dense scrub with abundant trees. Most of the trees species are Quercus ilex L. and Ceratonia siliqua L.; the scrub layer comprises Juniperus oxycedrus L., J. phoenicea L., Pistacia lentiscus L. and Ulex baeticus Boiss. This population experiences a mean annual temperature of 15.3 1C and a mean annual rainfall of 966 mm. The Puerto de las Palomas population is also in the Sierra de Grazalema Natural Park (361470 N, 51220 W), but at an altitude of 1200 m on Jurassic marly limestones. Vegetation consists of dense scrub dominated by Berberis hispanica Boiss. and Reuter, Erinacea anthyllis Link, Lavandula lanata Boiss. and Vella spinosa Boiss. This population grows under a mean annual temperature of 12.9 1C and a mean annual rainfall of 1963 mm. The 2 years studied, 1999 and 2000, were similar in temperatures but 1999 was a dry year with only the 70% of the mean rainfall.
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Intra- and inter-population variability in seed viability Seed viability was estimated in 10 plants from Aracena, 10 from La Camilla and 5 from Puerto de las Palomas. From each of these plants, 75 seeds were collected and a tetrazolium test was performed (Grabe, 1970). The seeds were collected from the third and fourth braching levels of inflorescence (see Narbona et al., 2002, for more details), and preliminary analyses demonstrated that there are not significant differences in germination between these two levels (Narbona, unpubl. results).
Interpopulation and interannual variability in seed germination In order to discover if the germination capacity of seeds varies between populations and years, seeds from 15 to 30 plants were collected in each population. Given that this species has also vegetative reproduction, we selected plants separated more than 2 m from each other, throughout two 100 m transects. This gathering process took place during the summers of 1999 and 2000, except in Puerto de las Palomas where they were only collected in 1999. The seeds were sown each year in December, using three batches replicates with 25 seeds each one. These seeds were placed in Petri dishes on permanently moist Whatman #1 filter paper and they were left to germinate in a chamber room at temperatures of between 17 and 21 1C (night–day). Preliminary tests have shown that this temperature range is optimum for germination. The period of light per day was 11 h for the entire length of the experiment. The dishes were checked every 1–3 days and the number of germinated seeds noted. The dishes were randomly arranged and the layout was changed each revision day to avoid pseudoreplication problems (Hurlbert, 1984). Seeds with a radicle that had emerged more than 0.2 mm were considered to have germinated. At the end of each count, the germinated seeds were removed. The trials lasted 40 days, but the dishes continued to be checked for 3 further weeks, to confirm that no more germination had occurred (Baskin and Baskin, 1998). A tetrazolium test was performed on nongerminated seeds to check their viability (Cheplick and Sung, 1998).
Seed germination variability with different pre-treatments and conditions In August 1999, 1380 seeds were collected from 20 plants in Aracena and 1200 seeds from 18 plants in La Camilla. The selection of the plants was made just as described in the previous section. The seeds from each population were mixed and kept in paper envelopes, in
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the dark, and at laboratory temperatures, until December 1999. A batch of seeds that were left untreated was considered the ‘‘control’’ group. In addition to the control group, other tests were carried out in which the seeds were subjected to one of the treatments described below. Germination was assessed as described above. (A) Germination following low temperature shock. In order to assess the influence of low temperatures on the germination of E. nicaeensis seeds, they were subjected to +4 1C for 1 month (Baskin and Baskin, 1998). The treatment was begun immediately before the seeds were sown. (B) Germination following high temperature shock. Before they were sown, the seeds were subjected to temperatures of 100 1C for 1 and 5 min, and 120 1C for 5 min. These treatments aimed to simulate the conditions that occur in the soil following a fire in the Mediterranean area (Trabaud, 1979). (C) Sulphuric acid scarification. In order to simulate the seeds passing through the digestive tract of animals, they were immersed in concentrated sulphuric acid (96%) for 5 min and then washed in abundant water before sowing. (D) Elimination of the caruncle. To discover whether or not the caruncle has an effect on germination, the seeds with removed from the caruncle, using tweezers, simulating the action of an ant’s jaws (Pacini, 1990). (E) Darkness. In order to determine whether the seeds are capable of germinating in darkness, a batch from the Aracena population was placed on Petri dishes and wrapped in two layers of aluminium foil and then left to germinate. The dishes were checked every 5 days with a low-intensity green light since some seeds are incapable of germinating in darkness but can do if they receive a small amount of white light (Baskin and Baskin, 1998).
Statistical analysis With the data obtained from the previous tests, the mean germination percentage was calculated along with mean germination time (t50). The latter refers to the number of days taken for 50% of total germination to be achieved (Bewley and Black, 1985). Prior to data analysis, the variable ‘‘mean germination percentage’’ was transformed when it did not fulfil the requirements of normality and homogeneity of variances, using the arcsine of the square root (Zar, 1999). The variable ‘‘t50’’ was transformed using logarithm or square root (Zar, 1999). The homogeneity of variances was verified using the Levene test (Day and Quinn, 1989; StatSoft, 1999). To investigate whether the distribution of the
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data fit the normal function, the Kolmogorov–Smirnov goodness-of-fit test was used with the Lilliefors correction (StatSoft, 1999). The intra- and inter-population variability of seed viability was analysed using a nested ANOVA where the individual factor was nested in the population factor and both were considered random. In order to analyse interpopulation and interannual variability, the germination percentages and t50 were analysed using a random model two-factor ANOVA. The germination percentages and t50 of the treatments were analysed using a two-factor ANOVA, considering the treatment as a fixed factor and the population as random. The effect of darkness was controlled with the control group using a single-factor ANOVA, since this treatment was only used on the population from Aracena. In order to compare the percentage of viable seeds with the total germination percentage, both in the control group and all the treatment groups, a single-factor ANOVA was used. When the ANOVA showed significant differences, the means of groups were compared using post-hoc tests (Zar, 1999). LSD tests were used when the variance between groups was homogenous and planned comparisons were to be made; Dunnett tests were used when the treatment was compared with the control group (Day and Quinn, 1989).
particularly between plants within each population (F 22;50 ¼ 16:70, po0:0001). The average viability percentage varied between 9272.7% in La Camilla and 66.777.20% in Puerto de las Palomas (Fig. 1). The seeds from Aracena displayed intermediate viability (72.377.75%) but its individuals displayed the greatest variability, between 27.3% and 98.7% (Fig. 1).
Interpopulation and interannual variability in seed germination
Results
In each population, the germination percentage did not vary significantly between the 2 years; however, for each year there were differences between populations (Table 1). In 1999, the mean germination percentage in the populations from Aracena and Puerto de las Palomas was similar, 6474.3% and 6374.7%, respectively (p ¼ 0:881, LSD test; Fig. 2); in La Camilla, on the other hand, the germination percentage was much higher: 8973.0% (p ¼ 0:0012 and 0:0017, respectively, LSD test). In the year 2000, differences were also found between the populations from Aracena and La Camilla in the germination percentages of the seeds (7577.6% and 9871.1%; p ¼ 0:003, test LSD; Fig. 2). The t50 varied between 12.470.8 for Aracena and 13.371.8 for La Camilla in 1999 and between 9.570.4 for Aracena and 10.171.1 for La Camilla in 2000 (Fig. 2). Therefore, for t50, the significant differences occurred between years and not between populations (Table 1).
Intra- and inter-population variability in seed viability
Seed germination variability with various pretreatments and conditions
The viability of the E. nicaeensis seeds was variable between the populations (F 2;50 ¼ 4:79, p ¼ 0:019) and
The average germination percentage of the control batches was 64% in Aracena and 89% in La Camilla
100
VIABILITY (%)
80
60
40
20
0
1 2 3 4 5 6 7 8 9 10 Aracena
1 2 3 4 5 6 7 8 9 10 La Camilla
1 2 3 4 5 P. Palomas
Fig. 1. Viability percentages (means7standard errors) of E. nicaeensis seeds of different plants from three populations.
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Table 1. ANOVA results comparing germination percentages and mean times of germination (t50) of E. nicaeensis seeds between years and populations df
Year Population Year population Error
1 2 1 15
SS
F
p
SS
F
p
336 2752 4 1300
84.12 344.08 0.05
0.069 0.038 0.832
2.347 32.410 0.059 64.303
550.93 19.86 0.01
0.027 0.156 0.911
16
1999 2000
100 80
12
60 40
10 8 6 4
20 0
1999 2000
14 t50 (DAYS)
GERMINATION (%)
t50
Germination (%)
2 Aracena
La Camilla P. Palomas
0
Aracena
La Camilla P. Palomas
Fig. 2. Germination percentages and mean times of germination (t50) of E. nicaeensis seeds from three populations and 2 consecutive years. Columns represent the means and bars represent the standard errors.
Table 2. Germination percentages and mean times of germination (t50) of E. nicaeensis seeds from Aracena and La Camilla populations after several treatments and the significance level of post-hoc Dunnett test comparing each treatment with the control Aracena
Control 4 1C 1 month 100 1C 1 min 100 1C 5 min 120 1C 5 min Sulphuric acid Without caruncle Darkness
La Camilla
Germination (%)
t50
Germination (%)
t50
64.074.3 50.773.5 n.s. 53.3712.2 n.s. 45.372.7 n.s. 44.074.0 50.777.4 n.s. 56.0712.2 n.s. 61.374.6 n.s.
12.470.8 10.171.6 n.s. 9.470.5 15.271.9 n.s. 16.771.2 8.370.9 9.971.0 n.s. 14.971.0 n.s.
89.073.0 81.373.5 n.s. 81.371.3 n.s. 78.772.7 n.s. 53.373.5 69.374.8 81.371.3 n.s.
13.371.8 7.970.2 12.570.4 n.s. 12.671.2 n.s. 17.170.6 16.671.8 n.s. 10.770.5 n.s.
Means7standard errors are represented. n.s. ¼ not significant. Po0:05.
(Table 2). In the control batch from La Camilla, all the viable seeds germinated (p40:8), whereas in Aracena, 17% of the viable seeds remained ungerminated (F 1;4 ¼ 8:75, p ¼ 0:025). The germination percentages of the seeds following the different treatments were statistically different to the control batch (F 6;30 ¼ 5:05, p ¼ 0:035). There were also differences between populations (F 1;30 ¼ 66:27, po0:001), but the germination percentage following the treatments varied in the same way in both populations (F 6;30 ¼ 1:17, p ¼ 0:35; Table 2).
Low temperatures slightly decreased both the germination percentage and the t50, although this effect was only significant in the t50 of Aracena (Table 2, Fig. 3a and c). Germination was similarly not affected in either of the two populations by the high-temperature treatments of 100 1C for 1 and 5 min, although t50 decreased significantly in Aracena (Table 2, Fig. 3b and d). At temperatures of 120 1C for 5 min, germination was over 40% in both populations, but was significantly less than in the control batch (Table 2, Fig. 3b and d); t50 increased significantly in both
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80 60 40
Control 4 ºCx1month Sulfuric Acid 5 min
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80 60 40 Control 4 ºCx1month Sulfuric Acid 5 min
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30
35
D
80 60 40
Control 100 ºCx1min 100 ºCx5min 120 ºCx5min
20 0
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20 25 DAYS
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35
Fig. 3. Temporal distribution of germination of E. nicaeensis seeds from Aracena (A and B) and La Camilla (C and D) subjected to different treatments. Bars represent standard errors of the mean.
populations (Table 2). Scarification with sulphuric acid caused a lower germination percentage than the control batch both in the population from Aracena and from La Camilla (Table 2; Fig. 3a and c), but only in the latter there were significant differences in relation to the control batch (Table 2). Paradoxically, the t50 decreased in Aracena and increased in La Camilla, but this variation was only significant in Aracena (Table 2). The seeds from which the caruncle was artificially removed displayed similar germination percentages to the control seeds both in the population from Aracena and La Camilla (Table 2). In this treatment, the t50 was not significantly different to that from the control batch in either of the populations (Table 2). Finally, E. nicaeensis seeds do not need light to germinate, since the seeds that were left in darkness obtained a germination percentage and t50 similar to the control batch (Table 2).
Discussion The viability of the E. nicaeensis seeds was highly variable between populations and individuals. The seeds of this species are nondormant (Al-Samman et al., 2001), so the differences in viability found between the populations translated into differences in the total germination
percentage. Interpopulation variability in germination is a common fact in other species (Baskin and Baskin, 1973; Hardin, 1984; Martin et al., 1995; Pe´rez-Garcı´ a, 1997; Van der Vegte, 1978) and might correspond to genetic factors (Hacker et al., 1984; Van der Vegte, 1978) as well as environment variations during seed ripening (Gutterman, 1992; Meyer and Allen, 1999). In E. nicaeensis, the germination percentage of the seeds from each population was similar for the 2 years studied; hence the differences between populations remained for 2(?) consecutive years. These results indicate that the germination capacity of the seeds in each population is probably at least partly due to genetic factors (Qaderi and Cavers, 2002). However, this does not rule out the possibility that such differences are caused by the different environment characteristics (soil, rainfall, temperature) of the three populations (Gutterman, 1992). Therefore, we agree with other authors (such as Qaderi and Cavers, 2002; Pe´rez-Garcı´ a et al., 2003) that studies of seed germination from a single population of a species must be interpreted with caution since other populations probably have a different germination capacity. On the other hand, seeds collected from various individuals of the same population also display differences in seed germinability, which supports the idea that the genotype of the individuals also has an important influence on seed germination. The seeds sown in the dark displayed the same germination capacity as in light. This behaviour has also
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been found for E. heterophylla and E. esula (Best et al., 1980; Brecke, 1995); however, in E. corollata, E. maculata and E. supina germination is favoured by luminosity conditions (Baskin and Baskin, 1988; Kivilaan and Bandurski, 1981; Krueger and Shaner, 1982; Voigt, 1977). The seeds that are collected by ants often remain underground, in ant chambers (Beattie, 1985; Espadaler and Go´mez, 1996; Espadaler et al., 1997; Pacini, 1990); hence the capacity to germinate in darkness can be an advantage. The capacity of E. nicaeensis seeds to germinate in total darkness permits them to do so in ant chambers, which would be important for this species since most of their seeds are actively collected by ants (Narbona, 2002). With regards to fire response, E. nicaeensis has the ability to resprout after a fire (Narbona, obs. per.) and their seeds do not possess dormancy mechanisms that can be broken by thermal shock. However, seeds of this species present a certain tolerance to fire, since many of them survived the extreme temperature treatments they were exposed to in this experiment. Thus, even if E. nicaeensis seeds do not possess an ability to adapt to fire sensu Keeley (1991), many of the buried seeds maintain their capacity to germinate, since at a depth of 2.5 cm, the temperature is not normally above 100 or 120 1C (Trabaud, 1979). Furthermore, the seeds found in ant chambers would not be exposed to high temperatures and could germinate once the autumn and winter rains arrive (fire-escape hypothesis), which would be an adaptive advantage of the myrmecochory (Bond and Slingsby, 1983). The elimination of the caruncle from the E. nicaeensis seeds had no effect on germination. In relation to this, important differences have been found in the behaviour of different species; in some, germination only occurs after the caruncle or oleosome is removed; in others, seeds germinate in similar percentages with or without the caruncle; and in others the elimination of the caruncle provokes an increase in germination (Go´mez and Espadaler, 1997; Lisci et al., 1996; Slingsby and Bond, 1985). The caruncle is the seed’s reward for myrmecochorous species; these seeds are transported to the ant nest where the caruncles are eaten by the ants (Beattie, 1985; Culver and Beattie, 1978; Stiles, 1992). However, on several occasions effective seed dispersal occurs even though the caruncle has not been removed: accidental dispersal, seeds robbed by ants from other ants, partial removal of the caruncle, etc. (Espadaler et al., 1997; Go´mez and Espadaler, 1997, 1998; Ho¨lldobler and Lumsden, 1980; Pudlo et al., 1980). In these circumstances, the possibility that seeds can germinate with the caruncle, as is the case with E. nicaeensis, would be an advantage. The seeds of this species are actively collected both by myrmecochorous ants and granivorous ants in the populations from Aracena and La Camilla (Narbona, 2002).
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Most E. nicaeensis seeds resisted scarification with sulphuric acid. Acid scarification usually breaks physical dormancy, since it destroys the testa (Brant et al., 1971), but prolonged exposure times can damage the embryo (Maeda and Pereira, 1987). In nature, acid scarification occurs when seeds are eaten by herbivores or frugivores and therefore pass through their digestive tract (Janzen, 1983). Olson et al. (1997) observed that sheep are important consumers of E. esula inflorescences and seeds, and estimated that a sheep can excrete between 2 and 141 seeds a day, of which between 5% and 24% are still viable. In E. nicaeensis, the consumption of the fruit by goats has been observed (Narbona, obs. pers.) but the viability of the excreted seeds is unknown. However, the fact that a high percentage of the seeds of this species remain viable after acid scarification suggests that goat or other ungulates could act as occasional dispersal agents. In conclusion, E. nicaeensis seeds can germinate both in light and darkness and both with and without the caruncle; this enables them to germinate in a variety of situations (buried vs. surface, conservation vs. loss of caruncle) that can occur following their handling by myrmecochorous or granivorous ants in their natural habits. Furthermore, a proportion of the seeds maintain their germination capacity after fire and herbivory, both of which are very common phenomena in the Mediterranean basin.
Acknowledgements This work was supported by a grant of the Programa de Ayuda a los Grupos de Investigacio´n (Junta de Andalucı´ a, RNM204) and by the Natural Park Sierra de Grazalema (Proyecto Pinsapar). We are grateful to Salvador Talavera for perceptive comments on the manuscript.
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Flora 201 (2006) 633–641 www.elsevier.de/flora
Germination variability and the effect of various pre-treatment on germination in the perennial spurge Euphorbia nicaeensis All. E. Narbonaa,, P.L. Ortizb, M. Aristab a
Departamento de Ciencias Ambientales, Universidad Pablo de Olavide, Ctra. Utrera Km 1, 41013 Sevilla, Spain Departamento de Biologı´a Vegetal y Ecologı´a, Universidad de Sevilla, Apdo. 1095. 4180 Sevilla, Spain
b
Received 7 November 2005; accepted 1 February 2006
Abstract Patterns of seed germination of the perennial spurge Euphorbia nicaeensis were studied in three populations in southwestern Spain. We investigated the variation in seed viability and germination among individuals, and among populations over 2 consecutive years. We also studied if diverse factors such as temperature, acid scarification, darkness or caruncle loss affect the germination of the seeds. Interindividual and interpopulation variability in seed viability was found. E. nicaeensis seeds are nondormant, so differences in interpopulation viability translate into differences in final germination rates. The germination percentage of the seeds from each population was similar in the 2 years studied. The effects of diverse factors were homogeneous in the two populations studied. Darkness has no effect on seed germination, and ecarunculate seeds germinated in the same proportions as carunculate seeds; this could allow the seeds to germinate in the chambers of deserted anthills. Acid scarification significantly reduced the germination percentage in only one of the populations, but over 50% of the seeds germinated, which could allow herbivores to act as occasional dispersing agents. The seeds that were preheated at 100 1C for 1 and 5 min germinated in the same proportions as the control group. The seeds that were preheated at 120 1C for 5 min displayed a significant decrease in germination, but the percentage was over 40% for both populations, indicating that the seeds could still germinate after the passage of a fire. r 2006 Elsevier GmbH. All rights reserved. Keywords: Acid scarification; Caruncle; Heat pre-treatment; Interpopulation variability; Interyear variability; Seed viability
Introduction Variation in seed germination capacity of a species can occur at least at three levels: within individuals (Gutterman, 1992; Silvertown, 1984), within populations (Hardin, 1984; Pe´rez-Garcı´ a, 1997; Venable and Lawlor, 1980) or among populations (Angosto and Matilla, 1993; Hacker et al., 1984; McGee and Marshall, Corresponding author. Fax: +954349151.
E-mail address: [email protected] (E. Narbona). 0367-2530/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.flora.2006.02.004
1994; Nordborg and Bergelson, 1999). Interpopulation variability can be linked with latitude, altitude, moisture and soil nutrients, temperature, type and density of the ecosystem’s plant cover and the degree of disturbance of the location (Baskin and Baskin, 1998). However, differences in germination capacity are not always related to the ecological characteristics of each population (Bevington, 1986; Martin et al., 1995) and several studies have confirmed that this variability between populations could be at least partly due to genetic factors (Hacker et al., 1984; Van der Vegte, 1978).
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Leaving aside intraspecific variability, several different environmental factors can affect the germination capacity of the seeds of a species. In Mediterranean ecosystems, fire is a common disturbance (Moreno et al., 1998) and two types of plants have been described according to their response to fire: seeder and resprouter species (Keeley, 1991). Generally, seeders die after the fire and the seeds germinate in the first rainy season following the fire, since their dormancy is easily interrupted by thermal shock or by substances leached from burnt plant material. In laboratory conditions, the seeds of these species need to be subjected to a heat pretreatment for 1–10 min at 100–120 1C in order to be able to germinate (Bell et al., 1993; Keeley, 1991; Pereiras et al., 1985; Tarrega et al., 1992; Valbuena et al., 1992), but excessive and prolonged heat exposure (X120 1C for several minutes) causes a significant decrease in germination (Keeley et al., 1985; Parker, 1987). Resprouters, on the other hand, following a fire, elongate the dormant buds on the lower knots of the stem, generally underground (Keeley, 1991), and do not usually form seed banks (Parker and Kelly, 1989). Within the Euphorbiaceae family, the genus Euphorbia has the largest number of species (approx. 1000–1600) and a widespread distribution, especially in subtropical and warm areas (Steinmann and Porter, 2002; Webster, 1994). Most studies about germination in Euphorbia have been carried out on annual species (Brenchley and Warington, 1930; Brecke, 1995; Capon and Van der Asdall, 1967; Heit, 1942; Kigel et al., 1992; Kivilaan and Bandurski, 1981; Krueger and Shaner, 1982; Van der Rooden et al., 1970; Sen and Chatterji, 1968; Voigt, 1977; Washitani and Masuda, 1990). However, only a few perennial species have been studied, such as Euphorbia caducifolia, E. characias and E. esula (Best et al., 1980; Go´mez and Espadaler, 1997; Selleck et al., 1962; Sen and Chatterji, 1966). E. nicaeensis All. subsp. nicaeensis is a perennial, herbaceous spurge with a circum-Mediterranean and Central Europe distribution and it prefers calcareous soils (Benedı´ et al., 1997; Smith and Tutin, 1978). This species grows in dry and sunny places between 100 and 1800 m, in degraded stages of Mediterranean Quercus forests (Benedı´ et al., 1997). E. nicaeensis is functionally andromonoecious, and the pistillate cyathia are situated predominantly at higher levels of the inflorescence (Narbona et al., 2002). Flowering time is in early summer and the seed dispersal takes place in late summer. This species produces an average of 117 capsules per individual (Narbona, 2002) and each capsule have three seeds, each one weighing between 4 and 8 mg (Narbona et al., 2005). E. nicaeensis is a diplochorous species: it has a primary explosive dispersal system (Narbona et al., 2005) and a secondary dispersal system by ants, since its seeds have a caruncle (Molinier and Mu¨ller, 1938). E. nicaeensis seeds are
transported by myrmecochorous and granivorous ants in various parts of the Mediterranean basin (Go´mez and Espadaler, 1994; Narbona, 2002; Wolff and Debussche, 1999) and they are nondormant (Al-Samman et al., 2001). In this study, we examined the germination variability of E. nicaeensis seeds and their ability to germinate under diverse environmental conditions. The specific objectives of this study were as follows: (1) investigate if there are differences in the viability and germination of seeds from different populations, (2) find out if there are differences in germination capacity over 2 consecutive years and (3) investigate if different factors such as temperature, darkness or caruncle loss affect seed germination.
Methods Site selection Three populations of E. nicaeensis were studied in south-western Spain: Sierra de Aracena, La Camilla and Puerto de las Palomas. These populations are relatively close (maximum 163 km between Sierra de Aracena and Puerto de las Palomas), but they were selected because they show important differences in environmental conditions (soils, altitude and rainfall). The Sierra de Aracena population is situated in the province of Huelva (371530 N, 61330 W), at an altitude of 700 m on Cambric decalcified limestones. Vegetation consists of a cultivated woodland of Castanea sativa Mill. with shrub species such as Viburnum tinus L., Daphne gnidium L. or Phlomis purpurea L. This population experiences a mean annual temperature of 14.5 1C and a mean annual rainfall of 1025 mm. La Camilla population is in the Sierra de Grazalema Natural Park (Province of Cadiz, 361470 N, 51240 W), at an altitude of 800 m on Jurassic dolomites. Vegetation consists of dense scrub with abundant trees. Most of the trees species are Quercus ilex L. and Ceratonia siliqua L.; the scrub layer comprises Juniperus oxycedrus L., J. phoenicea L., Pistacia lentiscus L. and Ulex baeticus Boiss. This population experiences a mean annual temperature of 15.3 1C and a mean annual rainfall of 966 mm. The Puerto de las Palomas population is also in the Sierra de Grazalema Natural Park (361470 N, 51220 W), but at an altitude of 1200 m on Jurassic marly limestones. Vegetation consists of dense scrub dominated by Berberis hispanica Boiss. and Reuter, Erinacea anthyllis Link, Lavandula lanata Boiss. and Vella spinosa Boiss. This population grows under a mean annual temperature of 12.9 1C and a mean annual rainfall of 1963 mm. The 2 years studied, 1999 and 2000, were similar in temperatures but 1999 was a dry year with only the 70% of the mean rainfall.
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Intra- and inter-population variability in seed viability Seed viability was estimated in 10 plants from Aracena, 10 from La Camilla and 5 from Puerto de las Palomas. From each of these plants, 75 seeds were collected and a tetrazolium test was performed (Grabe, 1970). The seeds were collected from the third and fourth braching levels of inflorescence (see Narbona et al., 2002, for more details), and preliminary analyses demonstrated that there are not significant differences in germination between these two levels (Narbona, unpubl. results).
Interpopulation and interannual variability in seed germination In order to discover if the germination capacity of seeds varies between populations and years, seeds from 15 to 30 plants were collected in each population. Given that this species has also vegetative reproduction, we selected plants separated more than 2 m from each other, throughout two 100 m transects. This gathering process took place during the summers of 1999 and 2000, except in Puerto de las Palomas where they were only collected in 1999. The seeds were sown each year in December, using three batches replicates with 25 seeds each one. These seeds were placed in Petri dishes on permanently moist Whatman #1 filter paper and they were left to germinate in a chamber room at temperatures of between 17 and 21 1C (night–day). Preliminary tests have shown that this temperature range is optimum for germination. The period of light per day was 11 h for the entire length of the experiment. The dishes were checked every 1–3 days and the number of germinated seeds noted. The dishes were randomly arranged and the layout was changed each revision day to avoid pseudoreplication problems (Hurlbert, 1984). Seeds with a radicle that had emerged more than 0.2 mm were considered to have germinated. At the end of each count, the germinated seeds were removed. The trials lasted 40 days, but the dishes continued to be checked for 3 further weeks, to confirm that no more germination had occurred (Baskin and Baskin, 1998). A tetrazolium test was performed on nongerminated seeds to check their viability (Cheplick and Sung, 1998).
Seed germination variability with different pre-treatments and conditions In August 1999, 1380 seeds were collected from 20 plants in Aracena and 1200 seeds from 18 plants in La Camilla. The selection of the plants was made just as described in the previous section. The seeds from each population were mixed and kept in paper envelopes, in
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the dark, and at laboratory temperatures, until December 1999. A batch of seeds that were left untreated was considered the ‘‘control’’ group. In addition to the control group, other tests were carried out in which the seeds were subjected to one of the treatments described below. Germination was assessed as described above. (A) Germination following low temperature shock. In order to assess the influence of low temperatures on the germination of E. nicaeensis seeds, they were subjected to +4 1C for 1 month (Baskin and Baskin, 1998). The treatment was begun immediately before the seeds were sown. (B) Germination following high temperature shock. Before they were sown, the seeds were subjected to temperatures of 100 1C for 1 and 5 min, and 120 1C for 5 min. These treatments aimed to simulate the conditions that occur in the soil following a fire in the Mediterranean area (Trabaud, 1979). (C) Sulphuric acid scarification. In order to simulate the seeds passing through the digestive tract of animals, they were immersed in concentrated sulphuric acid (96%) for 5 min and then washed in abundant water before sowing. (D) Elimination of the caruncle. To discover whether or not the caruncle has an effect on germination, the seeds with removed from the caruncle, using tweezers, simulating the action of an ant’s jaws (Pacini, 1990). (E) Darkness. In order to determine whether the seeds are capable of germinating in darkness, a batch from the Aracena population was placed on Petri dishes and wrapped in two layers of aluminium foil and then left to germinate. The dishes were checked every 5 days with a low-intensity green light since some seeds are incapable of germinating in darkness but can do if they receive a small amount of white light (Baskin and Baskin, 1998).
Statistical analysis With the data obtained from the previous tests, the mean germination percentage was calculated along with mean germination time (t50). The latter refers to the number of days taken for 50% of total germination to be achieved (Bewley and Black, 1985). Prior to data analysis, the variable ‘‘mean germination percentage’’ was transformed when it did not fulfil the requirements of normality and homogeneity of variances, using the arcsine of the square root (Zar, 1999). The variable ‘‘t50’’ was transformed using logarithm or square root (Zar, 1999). The homogeneity of variances was verified using the Levene test (Day and Quinn, 1989; StatSoft, 1999). To investigate whether the distribution of the
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data fit the normal function, the Kolmogorov–Smirnov goodness-of-fit test was used with the Lilliefors correction (StatSoft, 1999). The intra- and inter-population variability of seed viability was analysed using a nested ANOVA where the individual factor was nested in the population factor and both were considered random. In order to analyse interpopulation and interannual variability, the germination percentages and t50 were analysed using a random model two-factor ANOVA. The germination percentages and t50 of the treatments were analysed using a two-factor ANOVA, considering the treatment as a fixed factor and the population as random. The effect of darkness was controlled with the control group using a single-factor ANOVA, since this treatment was only used on the population from Aracena. In order to compare the percentage of viable seeds with the total germination percentage, both in the control group and all the treatment groups, a single-factor ANOVA was used. When the ANOVA showed significant differences, the means of groups were compared using post-hoc tests (Zar, 1999). LSD tests were used when the variance between groups was homogenous and planned comparisons were to be made; Dunnett tests were used when the treatment was compared with the control group (Day and Quinn, 1989).
particularly between plants within each population (F 22;50 ¼ 16:70, po0:0001). The average viability percentage varied between 9272.7% in La Camilla and 66.777.20% in Puerto de las Palomas (Fig. 1). The seeds from Aracena displayed intermediate viability (72.377.75%) but its individuals displayed the greatest variability, between 27.3% and 98.7% (Fig. 1).
Interpopulation and interannual variability in seed germination
Results
In each population, the germination percentage did not vary significantly between the 2 years; however, for each year there were differences between populations (Table 1). In 1999, the mean germination percentage in the populations from Aracena and Puerto de las Palomas was similar, 6474.3% and 6374.7%, respectively (p ¼ 0:881, LSD test; Fig. 2); in La Camilla, on the other hand, the germination percentage was much higher: 8973.0% (p ¼ 0:0012 and 0:0017, respectively, LSD test). In the year 2000, differences were also found between the populations from Aracena and La Camilla in the germination percentages of the seeds (7577.6% and 9871.1%; p ¼ 0:003, test LSD; Fig. 2). The t50 varied between 12.470.8 for Aracena and 13.371.8 for La Camilla in 1999 and between 9.570.4 for Aracena and 10.171.1 for La Camilla in 2000 (Fig. 2). Therefore, for t50, the significant differences occurred between years and not between populations (Table 1).
Intra- and inter-population variability in seed viability
Seed germination variability with various pretreatments and conditions
The viability of the E. nicaeensis seeds was variable between the populations (F 2;50 ¼ 4:79, p ¼ 0:019) and
The average germination percentage of the control batches was 64% in Aracena and 89% in La Camilla
100
VIABILITY (%)
80
60
40
20
0
1 2 3 4 5 6 7 8 9 10 Aracena
1 2 3 4 5 6 7 8 9 10 La Camilla
1 2 3 4 5 P. Palomas
Fig. 1. Viability percentages (means7standard errors) of E. nicaeensis seeds of different plants from three populations.
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Table 1. ANOVA results comparing germination percentages and mean times of germination (t50) of E. nicaeensis seeds between years and populations df
Year Population Year population Error
1 2 1 15
SS
F
p
SS
F
p
336 2752 4 1300
84.12 344.08 0.05
0.069 0.038 0.832
2.347 32.410 0.059 64.303
550.93 19.86 0.01
0.027 0.156 0.911
16
1999 2000
100 80
12
60 40
10 8 6 4
20 0
1999 2000
14 t50 (DAYS)
GERMINATION (%)
t50
Germination (%)
2 Aracena
La Camilla P. Palomas
0
Aracena
La Camilla P. Palomas
Fig. 2. Germination percentages and mean times of germination (t50) of E. nicaeensis seeds from three populations and 2 consecutive years. Columns represent the means and bars represent the standard errors.
Table 2. Germination percentages and mean times of germination (t50) of E. nicaeensis seeds from Aracena and La Camilla populations after several treatments and the significance level of post-hoc Dunnett test comparing each treatment with the control Aracena
Control 4 1C 1 month 100 1C 1 min 100 1C 5 min 120 1C 5 min Sulphuric acid Without caruncle Darkness
La Camilla
Germination (%)
t50
Germination (%)
t50
64.074.3 50.773.5 n.s. 53.3712.2 n.s. 45.372.7 n.s. 44.074.0 50.777.4 n.s. 56.0712.2 n.s. 61.374.6 n.s.
12.470.8 10.171.6 n.s. 9.470.5 15.271.9 n.s. 16.771.2 8.370.9 9.971.0 n.s. 14.971.0 n.s.
89.073.0 81.373.5 n.s. 81.371.3 n.s. 78.772.7 n.s. 53.373.5 69.374.8 81.371.3 n.s.
13.371.8 7.970.2 12.570.4 n.s. 12.671.2 n.s. 17.170.6 16.671.8 n.s. 10.770.5 n.s.
Means7standard errors are represented. n.s. ¼ not significant. Po0:05.
(Table 2). In the control batch from La Camilla, all the viable seeds germinated (p40:8), whereas in Aracena, 17% of the viable seeds remained ungerminated (F 1;4 ¼ 8:75, p ¼ 0:025). The germination percentages of the seeds following the different treatments were statistically different to the control batch (F 6;30 ¼ 5:05, p ¼ 0:035). There were also differences between populations (F 1;30 ¼ 66:27, po0:001), but the germination percentage following the treatments varied in the same way in both populations (F 6;30 ¼ 1:17, p ¼ 0:35; Table 2).
Low temperatures slightly decreased both the germination percentage and the t50, although this effect was only significant in the t50 of Aracena (Table 2, Fig. 3a and c). Germination was similarly not affected in either of the two populations by the high-temperature treatments of 100 1C for 1 and 5 min, although t50 decreased significantly in Aracena (Table 2, Fig. 3b and d). At temperatures of 120 1C for 5 min, germination was over 40% in both populations, but was significantly less than in the control batch (Table 2, Fig. 3b and d); t50 increased significantly in both
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Fig. 3. Temporal distribution of germination of E. nicaeensis seeds from Aracena (A and B) and La Camilla (C and D) subjected to different treatments. Bars represent standard errors of the mean.
populations (Table 2). Scarification with sulphuric acid caused a lower germination percentage than the control batch both in the population from Aracena and from La Camilla (Table 2; Fig. 3a and c), but only in the latter there were significant differences in relation to the control batch (Table 2). Paradoxically, the t50 decreased in Aracena and increased in La Camilla, but this variation was only significant in Aracena (Table 2). The seeds from which the caruncle was artificially removed displayed similar germination percentages to the control seeds both in the population from Aracena and La Camilla (Table 2). In this treatment, the t50 was not significantly different to that from the control batch in either of the populations (Table 2). Finally, E. nicaeensis seeds do not need light to germinate, since the seeds that were left in darkness obtained a germination percentage and t50 similar to the control batch (Table 2).
Discussion The viability of the E. nicaeensis seeds was highly variable between populations and individuals. The seeds of this species are nondormant (Al-Samman et al., 2001), so the differences in viability found between the populations translated into differences in the total germination
percentage. Interpopulation variability in germination is a common fact in other species (Baskin and Baskin, 1973; Hardin, 1984; Martin et al., 1995; Pe´rez-Garcı´ a, 1997; Van der Vegte, 1978) and might correspond to genetic factors (Hacker et al., 1984; Van der Vegte, 1978) as well as environment variations during seed ripening (Gutterman, 1992; Meyer and Allen, 1999). In E. nicaeensis, the germination percentage of the seeds from each population was similar for the 2 years studied; hence the differences between populations remained for 2(?) consecutive years. These results indicate that the germination capacity of the seeds in each population is probably at least partly due to genetic factors (Qaderi and Cavers, 2002). However, this does not rule out the possibility that such differences are caused by the different environment characteristics (soil, rainfall, temperature) of the three populations (Gutterman, 1992). Therefore, we agree with other authors (such as Qaderi and Cavers, 2002; Pe´rez-Garcı´ a et al., 2003) that studies of seed germination from a single population of a species must be interpreted with caution since other populations probably have a different germination capacity. On the other hand, seeds collected from various individuals of the same population also display differences in seed germinability, which supports the idea that the genotype of the individuals also has an important influence on seed germination. The seeds sown in the dark displayed the same germination capacity as in light. This behaviour has also
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been found for E. heterophylla and E. esula (Best et al., 1980; Brecke, 1995); however, in E. corollata, E. maculata and E. supina germination is favoured by luminosity conditions (Baskin and Baskin, 1988; Kivilaan and Bandurski, 1981; Krueger and Shaner, 1982; Voigt, 1977). The seeds that are collected by ants often remain underground, in ant chambers (Beattie, 1985; Espadaler and Go´mez, 1996; Espadaler et al., 1997; Pacini, 1990); hence the capacity to germinate in darkness can be an advantage. The capacity of E. nicaeensis seeds to germinate in total darkness permits them to do so in ant chambers, which would be important for this species since most of their seeds are actively collected by ants (Narbona, 2002). With regards to fire response, E. nicaeensis has the ability to resprout after a fire (Narbona, obs. per.) and their seeds do not possess dormancy mechanisms that can be broken by thermal shock. However, seeds of this species present a certain tolerance to fire, since many of them survived the extreme temperature treatments they were exposed to in this experiment. Thus, even if E. nicaeensis seeds do not possess an ability to adapt to fire sensu Keeley (1991), many of the buried seeds maintain their capacity to germinate, since at a depth of 2.5 cm, the temperature is not normally above 100 or 120 1C (Trabaud, 1979). Furthermore, the seeds found in ant chambers would not be exposed to high temperatures and could germinate once the autumn and winter rains arrive (fire-escape hypothesis), which would be an adaptive advantage of the myrmecochory (Bond and Slingsby, 1983). The elimination of the caruncle from the E. nicaeensis seeds had no effect on germination. In relation to this, important differences have been found in the behaviour of different species; in some, germination only occurs after the caruncle or oleosome is removed; in others, seeds germinate in similar percentages with or without the caruncle; and in others the elimination of the caruncle provokes an increase in germination (Go´mez and Espadaler, 1997; Lisci et al., 1996; Slingsby and Bond, 1985). The caruncle is the seed’s reward for myrmecochorous species; these seeds are transported to the ant nest where the caruncles are eaten by the ants (Beattie, 1985; Culver and Beattie, 1978; Stiles, 1992). However, on several occasions effective seed dispersal occurs even though the caruncle has not been removed: accidental dispersal, seeds robbed by ants from other ants, partial removal of the caruncle, etc. (Espadaler et al., 1997; Go´mez and Espadaler, 1997, 1998; Ho¨lldobler and Lumsden, 1980; Pudlo et al., 1980). In these circumstances, the possibility that seeds can germinate with the caruncle, as is the case with E. nicaeensis, would be an advantage. The seeds of this species are actively collected both by myrmecochorous ants and granivorous ants in the populations from Aracena and La Camilla (Narbona, 2002).
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Most E. nicaeensis seeds resisted scarification with sulphuric acid. Acid scarification usually breaks physical dormancy, since it destroys the testa (Brant et al., 1971), but prolonged exposure times can damage the embryo (Maeda and Pereira, 1987). In nature, acid scarification occurs when seeds are eaten by herbivores or frugivores and therefore pass through their digestive tract (Janzen, 1983). Olson et al. (1997) observed that sheep are important consumers of E. esula inflorescences and seeds, and estimated that a sheep can excrete between 2 and 141 seeds a day, of which between 5% and 24% are still viable. In E. nicaeensis, the consumption of the fruit by goats has been observed (Narbona, obs. pers.) but the viability of the excreted seeds is unknown. However, the fact that a high percentage of the seeds of this species remain viable after acid scarification suggests that goat or other ungulates could act as occasional dispersal agents. In conclusion, E. nicaeensis seeds can germinate both in light and darkness and both with and without the caruncle; this enables them to germinate in a variety of situations (buried vs. surface, conservation vs. loss of caruncle) that can occur following their handling by myrmecochorous or granivorous ants in their natural habits. Furthermore, a proportion of the seeds maintain their germination capacity after fire and herbivory, both of which are very common phenomena in the Mediterranean basin.
Acknowledgements This work was supported by a grant of the Programa de Ayuda a los Grupos de Investigacio´n (Junta de Andalucı´ a, RNM204) and by the Natural Park Sierra de Grazalema (Proyecto Pinsapar). We are grateful to Salvador Talavera for perceptive comments on the manuscript.
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