Effect of seed mass and number of cotyledons on seed germination after heat treatment in Pinus sylvestris L. var. iberica Svob

Aim of study: We investigated the combined effect of seed mass and number of cotyledons on the seed germination of Pinus sylvestris var. iberica (Iberian Scots pine) in simulated fire conditions. Material and methods: We used 3,600 fresh seeds extracted from 158 cones obtained from 10 pine trees located at the Biological Station of the Complutense University (Guadarrama mountains, Madrid region). All the seeds were individually weighed and assigned to one of the two following seed mass classes: class I (1.6-12.5 mg) and class II (12.6-145.0 mg). Germination capacity (GC) and mean germination time (MGT) were studied in combined experiments of four different temperatures (100°, 125°, 150° and 175°C) and two exposure times (1 and 5 minutes) together with a control (no treatment). Four replicates of 50 seeds each were used for each treatment and hydrated daily for 14 days to germinate under constant illumination. The number of cotyledons was counted in seedlings. Main results: Germination was depressed at above 125°C for 5 min. GC and MGT were negatively related, and were influenced by temperature and exposure time. Seed mass was found to have a significant effect on GC at some moderate heat treatment but not on MGT. The number of cotyledons was positively correlated to seed mass but there was not found correlated with germination after seed heat treatments. Research highlights: In the case of the Iberian Scots pine, higher seed mass mitigate the negative effects of temperature on seed germination after moderate heat treatment simulating fire.

ce on early seedling growth in Scots pine (Wennström et al., 2002). The number of cotyledons is also related to a maternal effect due to their relation with seed mass (Squillace, 1964). In Scots pine, the higher the number of cotyledons, the greater the growth height (Reich et al., 1994). The number of cotyledons has been positively related to seed size and hence to early plant survival in pine species (Buchholz, 1946;Isik, 1985) other than Scots pine, of which the same behaviour could be expected. Southern European populations of Scots pine have heavier seeds than central and northern populations (Reich et al., 1994). It is therefore important to know whether these maternal factors (seed mass and number of cotyledons) are an advantage against fire in southern European populations where fire is one of the key stressors. The interaction between seed size-number of cotyledons and germination in a range of thermal-shock temperatures simulating fires in Scots pine is unknown.
Our working hypothesis is that seed size and number of cotyledons influence the germination of seeds after being subjected to temperatures simulating fire conditions. The specific objectives address the following questions: a) how does the combined effect of seed mass and heat influence germination? and b) how does the combined effect of number of cotyledons and heat influence germination?

Seed source
Scots pine cones were collected in the Biological Field Station of the Complutense University located in the foothills of Guadarrama Mountains (Madrid region, 1,200 m a.s.l., 40°54' N, 3°52' W). In November 2011 we selected at least 10 cones each -before opening-from 10 dominant, healthy wild trees of similar size, obtaining 158 cones as a result from which 3,600 seeds were selected.
The area is flat with a negligible difference in altitude. The plant landscape is mainly a semi-natural forest consisting of patches of young Quercus pyrenaica Willd. mixed with stands of Iberian Scots pine (Castoldi & Molina, 2012). Pinus sylvestris trees reach an average height of 22 m and an average diameter at breast height of 48 cm. Open woodlands of Fraxinus angustifolia Vahl and subhumid meadows of Agrostis castellana Boiss. & Reuter occur in wetter soils. The area has undergone numerous burns as the consequence of prescribed slash-pile fire management (1,708 slash-pile burns carried out in the last ten years, A. Canencia, pers. comm.). The climate in the study area is sub-Mediterranean, with 895 mm of mean annual precipitation and a mean annual temperature of 10.1°C (Elías Castillo & Ruiz Beltrán, 1977). Soils correspond to brown soils on silicate rocks (Guerra et al., 1966). Soils are developed mainly on glandular gneisses and the average of pH soils in the area is 7.5 (Castoldi, unpublished data).

Experimental design
The seeds were previously removed from the cones by placing in a dry air oven at 45°C for 24 hours. We extracted the seeds from the cones manually and removed the seed wings. We used fresh seeds (taken directly from the cone and not from the ground) collected 23 days before the experiment without any prior selection in order to reproduce natural forest conditions. All the seeds were individually weighed with a precision balance and assigned to one of two seed mass classes (class I and class II, Table 1) defined for the bimodal distribution found in the data. Seed mass distribution was normal for each class (class I: 1.6-12.5 mg, mean and SD: 8.9 ± 0.1; class II: 12.6-145.0 mg, mean and SD: 15 ± 0.3). Since the correlation between seed weight and seed-coat weight in pines is highly significant (Yeatman, 1966) we considered only seed weight in our experiment. Combinations of four different temperatures (100°, 125°, 150°and 175°C) and two exposure times (1 and 5 minutes) were studied together with a control (no treatment). Treatments were the following: 1) 100°C, 1min, I class; 2) 100°C, 1 min, II class; 3) 100°C, 5 min, I class; 4) 100°C, 5 min, II class; 5) 125°C, 1 min, I class; 6) 125°C, 1 min, II 484 E. Castoldi and J. A. Molina / Forest Systems (2014)  class; 7) 125°C, 5 min, I class; 8) 125°C, 5 min, II class; 9) 150°C, 1 min, I class; 10) 150°C, 1 min, II class; 11) 150°C, 5 min, I class; 12) 150°C, 5 min, II class; 13) 175°C, 1 min, I class; 14) 175°C, 1 min, II class; 15) 175°C, 5 min, I class; 16) 175°C, 5 min, II class; 17) control, I class; 18) control, II class. These temperatures were chosen because they are commonly used in Scots pine germination studies to reproduce natural fire conditions (Habrouk et al., 1999;Escudero et al., 1997;Núñez & Calvo, 2000). Four replicates of 50 seeds each were used for each treatment and placed on Petri dishes. Seeds were placed on filter papers moistened with purified water. Germination was run at a constant temperature of 20°C and 28% HR (air) and 22°C and 56% HR (germination tables) under constant illumination of 20 µE m-2s-1 (fluorescent lamp F 40 W/33 RS cool white light). Germination was checked daily at the same hour for 14 days. This period is considered sufficient since it has been reported that 98% of Scots pine seeds germinate in the first five days under constant light and temperature -20°C- (Nygren, 1987). Come's criterion (1970) was followed to determine the germinated seeds: we considered that a seed had germinated when its radicle could clearly be observed outside the tegument, and its size was equal to the size of the seed. Seeds that were considered germinated were removed and the number of cotyledons counted in order to study the relation between it and seed size (length and width). After the experiment we investigated the un-germinated seeds by the cutting test and divided them into filled or empty seeds in order to study seed viability. Secondary fungi contamination was che-cked by recording the percentage of fungi cover in the Petri dishes the 4 th , 8 th and the 14 th day and allocated to seven classes (Table 1). Fungi were taxonomically determined up to the genus level (Seifert et al., 2011). All germination tests and seed weightings were performed in the Seed Laboratory at the Swedish University of Agricultural Sciences, Department of Southern Swedish Forest Research Centre, Alnarp (Sweden).

Statistical analysis
Germination capacity (GC) and mean germination time (MGT) were calculated for each treatment as follows: GC (%) = (N°germinated seeds/total N°of seeds sown) * 100; MGT (days) = Σ (n i * i )/N, where n i is the number of seed germinated on day i and N is the total number of seeds germinated along the study period (Bewley and Black, 1994).
Data were checked for normality or transformed if necessary, and ANOVA was performed to test the effects of heat and seed mass on germination (both GC and MGT). A Student's t-test was applied to identify statistically significant differences in GC and MGT between the control and the treated seeds. One-way ANOVA was performed to determine signif icant differences between heated and control seeds, among heat treatments, between seed mass classes and between heated and control seeds in fungi second contamination. Three-way ANOVA was used to consider the temperatures (H), exposure times (T) and seed mass (S) together. Spearman's coefficient was used to relate the GC and MGT variables and number of cotyledons and seed size (length and width used as independently variables). A generalized regression model (GRM) with stepwise procedure was performed to investigate the interactions between seed mass and temperature and exposure time. Spearman's rank correlation coefficient and linear regression analysis were used to relate seed size to number of cotyledons. Student's t-test was used to compare the number of cotyledons in seedlings after heat treatment and in control. Statistical analyses were done using SPSS 13.0 and STATISTICA software.

Results
Over the total sowed seeds the 15% germinated. On cutting the un-germinated seeds, we found the 85.8 % were full. Thus the majority of the seed material was healthy and failed to germinate due to the treatments. Treated seeds showed the highest GC at 100°C for 1 min (53%), and decreased with higher temperatures and longer exposure times (H-100 T-5; H-125 T-1) (Fig. 1). GC was depressed at above 125°C for 5 min. There were signif icant differences in GC between treatments and controls and among the treatments (in both cases p < 0.001). GC was negatively influenced by temperature (F 2,23 = 12.61, p < 0.001) and exposure time (F 2,23 = 26.91, p < 0.001). The less aggressive heat treatment (100°C, 1 min) already shows significant differences in GC in comparison to the control (F 1,22 = 17.31, p < 0.001). The seed mass had a significant effect on GC when considering heat treatment at 100°C for 5 minutes (F 1,6 = 9.375, p = 0.02) and the con-trol (F 1,6 = 23.45, p = 0.003). In contrast, seed mass had no signif icant effect on GC when considering heat treatment at 100°C for 1 minute (F 1,6 = 1.652, p = 0.246), or when all the treatments were considered together (F 1,24 = 1.14, p = 0.29).
The f irst seedlings were observed in the control 4 days after sowing, with the highest seedling counts seen after 5 days. Table 2 shows that the lowest MGT was observed in the control, with no exposure to heat. MGT was influenced by temperature (F 2,23 = 27.59, p < 0.001) and exposure time (F 2,23 = 54.79, p < 0.001). MGT differences between all heat treatments and the control were not supported statistically (F 1,70 = 1.82, p = 0.181) but they were when considering the less aggressive heat treatments of 100°C for 1 min (F 1,14 =74.12, p < 0.001) and 100°C for 5 min (F 1,14 = 993.08, p < 0.001). The difference in MGT between the heat treatment of 125°C for 1 min and the control was not significant (F 1,14 = 1.06, p = 0.321). MGT varied among the different heat treatments (p < 0.001). The seed mass had no significant effect on MGT (F 1,24 = 0.008, p = 0.93). Generalized Regression Model showed that there are not significant interactions between seed mass, temperature and exposure time in MGT (Table 3). The stepwise procedure kept the two first variables in the model (T and H). The germination capacity (GC) and mean germination time (MGT) were negatively correlated, meaning that lower MGT corresponds to a high GC (r = -0.9143, p < 0.001, Fig. 2).
Descriptive statistics of cotyledons number, seed width and length for treated and control seeds are shown in Table 4. The number of cotyledons in 14-dayold seedlings was positively correlated to seed size (r = 0.358, p < 0.001). The number of cotyledons was 486 E. Castoldi and J. A. Molina / Forest Systems (2014) 23 (3): 483-489 Figure 1. GC of Iberian Scots pine seeds in response to heat treatments and control. Labels correspond to seed mass classes (I and II class) and exposure minutes (1 or 5 minutes). Values are mean ± SD. Differences between treatments and control are significant for all treatments, except for the first one (100°C, 1 min, I seed mass class, first column; p = 0.199).
Heat treatments that avoid germination capacity are not shown with the exception of the treatment 125°C, 5 min.  positively correlated to seed length (r = 0.1203, p = 0.005) and to seed width (r = 0.0949, p = 0.002). This result was confirmed by a linear regression analysis between seed size and the number of cotyledons that was highly signif icant (F = 13.927, β = 0.358, p < 0.001). The number of cotyledons in seedlings after heat treatment did not differ significantly from the number of cotyledons in the control seeds (p = 0.1034).

Discussion
As expected from the background literature our results showed that germination is depressed by temperature and long exposure. A temperature range between 120°C and 150°C has been reported in failed GC of Scots pine seeds from Spain (Habrouk et al., 1999; Effect of seed mass and number of cotyledons after heat treatment on germination in Pinus sylvestris 487  Escudero et al., 1997;Núñez & Calvo, 2000). Our results showed that heat treatments at 125°C for 5 min on healthy Iberian Scots pine seeds resulted in null germination. Moreover, long exposure (5 min) notably decreases GC even at the lowest temperature studied in this work (100°C). The slight differences seen by authors in the temperature threshold for germination can be explained by experimental conditions such as light time exposure or the seed provenance. Significant delays have been reported in germination time after heat treatments (Escudero et al., 1997) increasing the MGT both temperature and exposure time (Habrouk et al., 1999) Our results support this pattern and show that temperature and exposure time affect MGT even at the lowest temperature shock of 100°C in our experiment. Previous studies on the influence of seed mass on germination in different pine species show contradictory results. Some report a positive correlation (Simak & Gustafsson, 1954;Debain et al., 2003;Tíscas & Lucas, 2010), whereas other studies show no effect (Mikola, 1985;Zaborovskii, 1966;Parker et al., 2006;Bladé & Vallejo, 2008). In Spanish Scots pines, seed mass has been positively correlated with germination (Castro, 1999). This agrees with our results on Iberian Scots pine showing that seed mass is positively related to germination when seeds are unheated. Furthermore, seed mass still positively influences GC in moderately heated seeds, which still retain the ability to germinate. Seed mass is also positively related to the number of cotyledons (Reich et al., 1994) as our results also support. Thus it could be inferred that seeds with a higher number of cotyledons should have a better performance when germinating under moderate heat. However, due to the nature of the data this cannot be probed.
Southern European populations of Scots pine are known to have heavier seed mass (Reich et al., 1994) -as our results confirm-and a higher number of cotyledons in relation to central and northern Scots pines. Since fire is one of the most important perturbations under the Mediterranean climate, these correlated characters could confer a maternal advantage against this stressor in low-intensity fires.

Acknowledgments
Funding for this work was provided by a grant from the Fundación General Complutense to E. Castoldi. We thank Professor P.C. Odén, M. Tigabu, P. Savadogo and A. Mariscal at the Seed Laboratory at the Swedish University of Agricultural Sciences, Department of Southern Swedish Forest Research Centre, Alnarp (Sweden) for their help. A special thanks to M. Morganti for statistical analysis and T. Ruibal for identifying fungal taxa. We thank the editor and the two anonymous reviewers for their constructive comments which helped us improve the manuscript, and Prudence Brooke Turner for the English edition.