Folia Geobot (2015) 50:275–282 DOI 10.1007/s12224-015-9224-x
Temperature-related effects on the germination capacity of black locust (Robinia pseudoacacia L., Fabaceae) seeds Claudia Giuliani & Lorenzo Lazzaro & Marta Mariotti Lippi & Roberto Calamassi & Bruno Foggi
Received: 18 July 2014 / Accepted: 23 July 2015 / Published online: 12 September 2015 # Institute of Botany, Academy of Sciences of the Czech Republic 2015
Abstract Germination is a key trait promoting species invasiveness and has become a major goal in invasion ecology. In this study, the effects of different scarification methods and temperature regimes on seed germination performance of the invasive exotic tree Robinia pseudoacacia L. were investigated. Ripe seeds were gathered from six collection sites in Tuscany (central Italy). Mechanical, chemical and thermal scarifications and no pretreatment were applied; seeds were incubated in a range (9–21°C) of constant temperature regimes. The scarification treatments proved highly important for the germination performance of seeds in term of both final response and germination rate in time, with mechanical scarification being the most effective treatment. Temperature significantly affected the germination rate and the mean germination time of the mechanically scarified seeds. The cumulative germination rate significantly decreased with the reduction of the temperature, with the highest value in the 18–21°C regimes (ca 98%), and the lowest in the 9–12°C regimes (ca 60%). The mean germination time slightly decreased with increasing temperature, but was substantially similar between 18°C and 21°C. Our data indicate that the temperature affected the onset of seed germination and that the lowest temperature tested delayed germination. These plastic germination responses, as well as the high germination capacity at
C. Giuliani (*) : L. Lazzaro : . Mariotti Lippi : R. Calamassi : B. Foggi Department of Biology, University of Florence, via G. La Pira, 4, I-50121 Florence, Italy e-mail:
[email protected]
lower temperature, may result in the spread of the propagule germination over a large time interval, contributing to the invasiveness of this species. Keywords dormancy . Fabaceae . germination . invasive plants . scarification
Introduction The North-American black locust (Robinia pseudoacacia L., Fabaceae, Papilionoideae) has been introduced worldwide as a commercially important multipurpose tree because of its adaptability to environmental stresses, its rapid growth and the hardness of its wood (Jung et al. 2009). Currently, it is considered one of the most problematic invaders on the Old Continent (Kleinbauer et al. 2010). The species is extensively naturalized in the British Isles and in Central, Southern and Eastern Europe, ascending to 800–1,000 m a.s.l. and having its distribution optimum in sub-Mediterranean to warm continental climates (Cierjacks et al. 2013). In Italy its rapid expansion in northern and central regions (Celesti-Grapow et al. 2009) is causing the progressive decline of native forests with adverse effects on biodiversity at the species, ecosystem and landscape levels (Benesperi et al. 2012). In its native and introduced ranges, natural regeneration of black locust is primarily vegetative; however, this species also relies on sexual reproduction with beepollinated hermaphrodite flowers and prolific seed
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production and dispersal (Surles et al. 1989; Giovanetti and Aronne 2013). Seed ripening takes place in late summer and early autumn, and pods are shed from September to April. Pods containing seeds remain on the crown throughout winter; they can open on the tree or can be dispersed in the surroundings and also far away from the mother plants (Morimoto et al. 2010). Therefore, R. pseudoacacia forms both short-term aerial seed banks (Masaka and Yamada 2009) and long-term soil seed banks, which can survive for at least 40 years (Hille Ris Lambers et al. 2005). Regeneration from seeds is hindered by physical dormancy (Baskin and Baskin 2001) imposed by a waterimpermeable seed coat (Baskin 2003; Van Assche et al. 2003). Moreover, seeds with various levels in dormancy may be produced, depending on the weather conditions during winter (Masaka and Yamada 2009). While clonal regeneration has long been studied in detail (Jung et al. 2009), only recently there has been renewed interest in investigating the role of sexual reproduction in the invasion success of this species (Masaka and Yamada 2009), focusing on the balance between the two regeneration strategies. Seedlings originating from dispersed seeds have been proved to play a central role in the establishment of new populations in northern Japan (Morimoto et al. 2010). Of course, the success or failure of regeneration from seeds depends on their ability to germinate under different environmental conditions such as temperature, water availability and light (Pinna et al. 2014). These factors may be extreme, so their effects can be crucial in both the survival and the establishment of seedlings in the introduced range (Ferreras and Galetto 2010; Meiado et al. 2010). Therefore, given that regeneration from seeds is now considered an important determinant in the successful invasion pattern of black locust (Masaka and Yamada 2009) and that the role of abiotic factors is essential to understand the germination responses of dispersed seeds (Baskin and Baskin 2001), seed dispersal and germination may be crucial management concerns. Moreover, in Europe information on seed regeneration is lacking or is insufficient for accurate management, and germination requirements are not completely clarified. It is therefore a priority to analyse seed germination characters under varying conditions in order to contribute to a comprehensive knowledge
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of the bioecology of black locust in its introduced range, also in the perspective of a global change scenario (Luna et al. 2012) expected to increase considerably the geographical area potentially invaded by R. pseudoacacia (Kleinbauer et al. 2010). In this framework, the present study aimed at assessing and comparing the seed germination capacity of R. pseudoacacia under different thermal regimes, providing new data on the sexual reproduction of black locust for its accurate management. In addition, since scarification processes are crucial in determining seed germination performances in many species of the Fabaceae (Baskin and Baskin 2001), a preliminary test was also conducted to assess the possible effects of different scarification methods on seed germination capacity and germination time.
Materials and methods Seed collection Mature dry seeds of R. pseudoacacia were collected at the time of natural dispersal from September to October of the years 2011 and 2012 from six invasive populations in Tuscany (central Italy, Table 1). Seeds were collected from pods fallen on the ground and stored in paper bags at the temperature of 4°C for 30 days before starting the experiments. Scarification methods A preliminary test with different scarification methods was conducted in order to select the most efficient treatment Table 1 Details of seed collection sites in Tuscany (central Italy). Site
Coordinates
Altitude (m a.s.l.)
Pianosa Island
N42°35.166′, E10°05.900′
20
Elba Island (Marciana)
N42°47.498′, E10°10.752′
330
Scarperia, Omomorto (Florence) Luco di Mugello (Florence) Pistoia Piteccio
N44°02.090′, E11°22.410′
633
N43°59.619′, E11°23.609′
278
N44°00.224′, E10°53.971′
392
Pistoia San Vito
N43°56.670′, E10°50.855′
375
Temperature effects on the germination of black locust
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curvature. Germinated or damaged seeds were counted daily and removed from the dishes at each census.
with regard to both germination capacity and germination time. Four treatments were assessed using seeds from three provenances (Pianosa, Luco di Mugello and Pistoia Piteccio): control without scarification, mechanical scarification with a razor blade, hot water bath at the temperature of 90°C with two levels of soak time durations (20 and 30 minutes) and acid soak with H2SO4 for 90 minutes. Two replicates of twenty seeds per population and per scarification treatment were employed in the preliminary tests. Although the low number of replicates weakens the statistical significance of the scarification method results, they represented an important indication for selecting the most efficient treatment to be applied in this study. As a result of the preliminary test, mechanical incision of the integument was employed for the scarification of the seeds from all the provenances.
Data analysis Two main response variables were investigated, Mean Germination Time (MGT) and Germination Rate (GR). MGT was calculated using the formula cited by Ellis and Roberts (1980), and GR was calculated for each day using Pieper's (1952) formula: X MGT ¼ GR ¼
ðntÞ
N
ðt 1 n1 Þ þ ðt 2 n2 Þ þ ðt 3 n3 Þ þ …ðt i ni Þ; N
where t is the number of days since the beginning of the germination test, n is the number of germinated seeds in each counting day at time t and N is the total number of germinated seeds. For the data analysis, generalized linear mixed models (GLMM) were used with the population of origin as a random factor. For the data analysis of the preliminary germination tests with different scarification methods, a GLMM was fitted studying the GR on the 30th day as response variable with the scarification method (SM) as the explanatory variable with fixed effect and the source population (POP) as a factor with a random effect. Multiple a posteriori comparisons of means through the Tukey contrasts were used to investigate differences in the cumulative GRs under different scarification methods. GR variation in time, from the 1st day up to the 30th day (every 3 days), was studied adopting a repeated measurement ANOVA design in the
Germination tests Seed germination responses were evaluated at different constant temperature treatments: 9°C, 12°C, 15°C, 18°C and 21°C. Replicates of 25 seeds each per provenance and temperature treatment were scarified individually with a razor blade to allow them to keep water and quickly germinate (Table 2). Therefore, seeds were placed in 9 cm diameter Petri dishes on filter paper moistened with distilled water and incubated in a thermostated chamber in continuous darkness for 30 days. No disinfectants were applied to the seeds since we have no information about their effects on the germination response. Radicle protrusion and geotropic curvature were the criteria for germination; in the present study, germination is defined as the time when the radicle tip emerged 1 cm or more from the seed coat plus geotropic
Table 2 Number of replicates of 25 seeds each per population/temperature treatment combination used in the germination tests. Population
Temperature treatment T 9°C
T 12°C
T 15°C
T 18°C
T 21°C
Total
Pianosa
8
4
8
8
8
Elba Marciana
8
8
8
4
4
32
Scarperia
8
8
8
8
8
40
Luco
8
8
8
8
8
40
PT Piteccio
8
8
8
8
8
40
PT San Vito Total
36
4
8
8
8
8
36
44
44
48
44
44
224
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performed using the ‘multicomp’ package version 1.3-0 (Hothorn et al. 2008), and all graphs were drawn using ‘ggplot2’ version 0.9.3.1 (Wickham 2009).
framework of GLMMs using POP as a random effect factor aiming to test the interaction between the two main terms SM and Time. For the results of germination tests at different temperatures, MGT and GR were analysed by means of GLMMs with temperature treatment (TT) as the main factor with fixed effect and source population (POP) as a factor with random effect. In cases of significant differences, multiple a posteriori comparisons of means through Tukey contrasts were used to investigate differences between the temperature treatments. GR on the 30th day was studied as the final response to the different temperature treatments; its variation in time, from the 1st day up to the 30th day (every 3 days) was studied in a repeated measurement ANOVA design in the framework of GLMMs using POP as random effect term aiming to test the interaction of TT and Time. In all the analyses, a normal distribution of residuals was adopted to set the models. GR values were transformed with the use of an angular transformation, aiming to normalize the residuals as usually done in case of ratios (Zar 1999). P < 0.05 was considered as the significance threshold in this study. All analyses were carried out in R version 3.1.0 (R Core Team 2014). GLMMs were performed using the ‘lme4’ package version 1.1-6 (Bates et al. 2014) which is well suitable for managing unbalanced crossed designs. While it does not produce P values, they were estimated using the ‘lmerTest’ package version 2.0-6 (Kuznetsova et al. 2014) using the Satterthwaite approximation to estimate the degrees of freedom for the denominator. Tukey’s post hoc comparison were
Results Pre-treatment effects As result of the preliminary test, the scarification methods turned out to be highly important for the germination performance of seeds in terms of both their final response and GR over time (Table 3). After 30 days, the seeds treated with different scarification methods displayed different GRs, while no germination was recorded in the control (Table 4). Mechanical scarification with a razor blade led to the highest rates of germinated seeds (close to 100 %). GRs slightly higher than 50 % were observed in seeds treated with hot water bath at 90°C for 30 minutes and with H 2SO 4 soaking for 90 minutes. GR slightly exceeded 25 % after hot water bath at 90°C for 20 minutes (Table 4). In the case of repeated measurement ANOVA, the significance of the interaction between the factors Time and SM highlighted that GR changed in time differently according to the different scarification method employed (Table 3). In addition, under mechanical scarification GR increased quickly, reaching a germination plateau (close to 100 %) after a few days whereas for the other methods germination was slower and delayed in time (Fig. 1).
Table 3 ANOVA table for the performed Generalized Linear Mixed Models (GLMM). Models 1 and 2 refer to the preliminary tests on scarification methods whereas models 3, 4 and 5 refer to germination tests under different temperature treatments (TT). Cumulative GR – germination rate on the 30th day; SM – scarification method; MGT – mean germination time; Num. D.f. – numerator degree of freedom; Denom. D.f. – denominator (residual) degrees of freedom. Model
Factor
Num. D.f.
Sum Sq.
Mean Sq.
F value
Denom. D.f.
P value
1 Cumulative GR ~ SM
SM
4
17,981
4,495.1
155.21
25.00
< 0.05
2 GR ~ SM * Time
SM
4
41,115
10,278.8
447.37
24.99
< 0.05
Time
10
65,788
6,578.8
286.33
249.99
< 0.05
Time: SM
40
33,330
833.2
36.27
249.99
< 0.05 < 0.05
3 MGT ~ TT
TT
4
1,716
429.0
107.78
215.32
4 Cumulative GR ~ TT
TT
4
30,104
7,525.9
106.38
214.46
< 0.05
5 GR ~ TT * Time
Time
10
1,313,445
131,345.0
3,306.20
2,190.00
< 0.05
4
43,233
10,808.0
274.20
214.80
< 0.05
5,414.0
137.40
2,190.00
< 0.05
TT Time: TT
40
21,654.2
Temperature effects on the germination of black locust Table 4 Cumulative germination rate (germination rate at the 30th day) of Robinia pseudoacacia seeds with different scarification methods. Mean real values ± SE are given. Values marked with different superscript letters (a, b, c, d) are significantly different at P < 0.05 after the post hoc test. Values are the results of 30 days after seed incision. Scarification method
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The course of germination differed in response to different temperatures (Table 3 and Fig. 2): At lower temperatures germination started later and reached its maximum later than under higher temperatures.
Germination rate at the 30th day [mean ± SE]
Discussion Control
0±0a
90°C_20min
0.28 ± 0.03 b
90°C_30min
0.54 ± 0.04 c
H2SO4 _90min
0.53 ± 0.02 c
Incision
0.92 ± 0.02 d
Temperature effects Changes in temperature significantly affected GR and MGT of R. pseudoacacia scarified seeds, as indicated by the GLMM results (Table 5). The MGT slightly decreased with increasing temperatures (P < 0.05), but was substantially similar between 18°C and 21°C (Table 5). A delay in germination was more evident at 9°C and 12°C in comparison to the other temperatures (Table 5). MGT varied from 13.76 days for the 9°C treatment up to 6.44 days for the 21°C treatment. At the end of the experiment, GR significantly decreased with the reduction of the temperature (P < 0.05). The highest GR was obtained under the 18–21°C regimes, with no significant difference between these temperatures, and the lowest under the 9–12°C regimes (Table 5). At the higher temperature, i.e. 21°C, cumulative GR was 98 % compared to 62 % at the lower temperature, i.e. 9°C (Table 5). Fig. 1 Seed germination curves of Robinia pseudoacacia seeds over 30 days under different scarification methods (SM). Standard error bars are included where appropriate.
The germination performance of black locust seeds after different scarification pre-treatments and under a wide range of constant temperatures was assessed. Preliminary mechanical, chemical and thermal scarification treatments showed differences in both the cumulative germination percentage and the germination response in time, corroborating previous studies (Chapman 1936; Singh et al. 1991; Paris and Cannata 1992; Basbag et al. 2010). Our germination experiments confirm the high germination ability of Robinia seeds after scarification (heat or cold shock, burning, sulfuric acid treatment and seed piercing, as recently reported in the literature, with GRs up to and over 95 % (Masaka and Yamada 2009; Morimoto et al. 2010). However, these authors found no significant differences in GRs among scarification treatments, while the preliminary tests performed in this research suggested that mechanical scarification provides the highest germination percentages, as already observed in many other species of the Fabaceae (Baskin and Baskin 2001). In addition, we found that different thermal regimes significantly affected the seed germination response, with lower mean germination time and greater GR under the higher temperatures. However, the cumulative germination percentage was fairly high also under the lowest temperature treatment (about 60 % at 9°C).
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Table 5 Mean germination time and cumulative germination rate of Robinia pseuacacia seeds for different constant temperature treatments in the dark. Mean values ± SE are given. Values marked with different super script letters (a, b, c, d) are significantly different at P < 0.05 after the post hoc test. Values are the results of 30 days after seed incision. Temperature treatment
Mean germination time means ± SE
Germination rate at the 30th day means ± SE
9°C
13.79 ± 0.39 a
0.62 ± 0.02 a
11.75 ± 0.36
b
0.66 ± 0.02 b
9.05 ± 0.32
c
0.89 ± 0.01 c
18°C
6.94 ± 0.17
d
0.91 ± 0.01 d
21°C
6.43 ± 0.18 d
0.95 ± 0.01 d
12°C 15°C
With respect to temperatures, all the provenances responded positively over a wide temperature interval (9–21°C), characterizing the germination performances of highly invasive species (Ferreras and Galetto 2010; Moravcová et al. 2006; Wainwright and Cleland 2013). Indeed, the cumulative GRs obtained in this study were similar to values reported for other invasive Fabaceae in Italy (Meloni et al. 2013). In addition to GR, another parameter (MGT) was considered in the seed germination responses to temperature. Both parameters can contribute substantially to our understanding of seed germination processes and seedlings recruitment of R. pseudoacacia in natural conditions, which are influenced by many environmental conditions. Significant differences in seed germination were found among the temperature treatments. For GR, the favourable temperature range was 18–21°C, with no significant difference between these temperatures; however, the GR at 21°C (close to 100 %) was the highest
Fig. 2 Seed germination curves of Robinia pseudoacacia seeds over 30 days under different temperatures treatments (TT). Standard errors are given where appropriate.
among the different treatments. We therefore considered 21°C as the optimal germination temperature for R. pseudoacacia because all the parameters evaluated in the present study were favoured by this treatment, resulting in the highest germination percentage and lowest MGT (6.44 ± 0.645 days). Our data indicate that the temperature affects the onset of seed germination and that the lowest temperature tested delayed germination (Fig. 2). These traits, as well as the high germination capacity at lower temperature, may result in the spread of propagule germination over a large time interval, contributing to the invasiveness of this species. This temperaturedependent physiological behaviour is ecologically important to the species by contributing in the formation of a heterogeneous persistent soil seed bank, which may potentially lead to an irregular GR over time (Masaka et al. 2010; Gioria et al. 2012). This would be an adaptive characteristic, since an irregular germination, distributed along a longer period of time increases the probability that at least some of the seeds will germinate and establish themselves under more favourable environmental conditions (de Lima et al. 1997). The evidence that no non-scarified seeds germinated throughout the duration of our lab experiments highlights the importance of scarification processes in this species. Germination could be increased by many factors in natural conditions, such as abrasion by soil particles, microbial and fungal actions, temperature changes or chemical scarification as passage through the digestive tract of an animal (Baskin and Baskin 2001). In the areas under study, many of those factors may be involved in mechanical scarification of the seeds, such as wide seasonal temperature fluctuations.
Temperature effects on the germination of black locust
In addition, signs of predator-mediated scarification in the field, i.e. gnawing by rodents, have been observed in the areas of study (C. Giuliani, pers. obs.). The mechanical scarification of the seeds in the present study actually removed the differences in the thickness of the seed integument. The polymorphism in seed physical dormancy observed in R. pseudoacacia seeds also at individual tree level (Morimoto et al. 2010), resulting in heterogeneous percentages of germination, may be reliant on this anatomical diversity. However, due to the lack of anatomical knowledge, this source of variation deserves future study. Endogenous development of water impermeability of the seed coat is often related to seed maturation (Baskin and Baskin 2004). Therefore, because R. pseudoacacia seeds used in the present study were collected in September-October, when the pods were already brown and dry, the seeds were mature and physically-dormant at the time of collection. Nevertheless, Morimoto et al. (2010) stated that freezing rather than dry conditions influence the induction of physical dormancy in R. pseudoacacia seeds. This induction of physical dormancy is a surprising result because a freezing or chilling treatment generally enhances the break of seed dormancy (Morimoto et al. 2010). Indeed, several R. pseudoacacia seeds experienced extreme cold during winter because some seed pods remain in the crowns, thus functioning as an aerial seed bank (Masaka et al. 2010). Several authors have reported that seeds of a majority of pioneer species remain viable but dormant in the soil (Boring and Swank 1984; de Lima et al. 1997). This agrees with ecological characteristics of R. pseudoacacia that is in fact a pioneer species. However, the role of germinative behaviour of the soil seed bank in regulating this pattern of distribution remains to be further explored in Italy. Besides the germination capacity, the success or failure of regeneration from seeds depends on the different resistance of seedlings to environmental stresses (Klimeš et al. 1997). Because R. pseudoacacia is a typical pioneer species, seedlings cannot survive under a forest canopy (Boring and Swank 1984). In the areas under study, more seedlings were found on the paths or on bare ground created by a disturbance than in interior woodlands (pers. obs.). Indeed, seeds are able to germinate in the shade, but seedlings mortality is higher under a closed canopy (Kowarik 2011). Therefore, seedling establishment is best under high light conditions such as in forest gaps, where there is less competition (Cierjacks et al. 2013).
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In conclusion, our results confirm the high germination capacity of black locust seeds under different temperatures and their potential germination over a large time interval. These plastic germination responses indicate that regeneration from seeds, besides the dominant clonal growth, likely confers further potential for invasiveness. On the other hand, the highly variable reproductive patterns that may be found under natural conditions enable R. pseudoacacia to spread rapidly worldwide (Masaka and Yamada 2009). Since R. pseudoacacia shows growing populations in many places of northern and central Italy, future management plans should focus on the seed bank (both soil and aerial) dynamics and on seedling establishment. Finally, it is worth relating our results to upcoming global change scenarios, since R. pseudoacacia is expected to expand its range by both invading new sites and increasing its population size in already occupied areas (Kleinbauer et al. 2010). The warmer climate may promote sexual reproduction of black locust, in turn increasing its ability to spread and to adapt to new environmental conditions facilitated by increased genetic variability, which may cause further problems. Acknowledgements The research grant to Dr. C.G. came from the Regione Toscana QuiT Project No. 21313 (POR-FSE 2007– 2013). The authors wish to thank Dr. Renato Benesperi for his help with seed collection and Prof. Laura Maleci Bini and Francesca Cappelletti for their help with germination tests. Thanks are also due to the associate editor and to an anonymous reviewer for their helpful comments and suggestions greatly improving our manuscript.
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