Evaluation of Different Geographic Provenances of Silver Fir ( Abies alba ) as Seed Sources, Based on Seed Traits and Germination

: The evaluation of the diversity of silver ﬁr ( Abies alba Mill.) populations and the reproductive traits of the trees are of great importance for the conservation of genetic resources and forest management. Therefore, important reproductive characteristics of A. alba from seven Romanian provenances, considered as different geographical populations, were evaluated. Signiﬁcant differences between the provenances were observed for the female cones, seed morphology, and germination. Due to the relatively low germination of silver ﬁr seeds, germination tests were conducted to identify treatments that can stimulate the germination capacity. Thus, the seed germination capacity was determined using four different stimulation treatments and the data were compared with those of untreated seeds, designed as the control. Considerable differences were recorded not only depending on the seed provenances, but also regarding the treatments applied to stimulate germination (Atonik biostimulator, scariﬁcation, acetone, H 2 SO 4 ). The biostimulator seed treatment gave the highest germination percentage, while sulfuric acid caused the lowest germination. The research also revealed that not all the forest seed sources provide high-quality reproductive material. Furthermore, for some of the seed resources, even the germination stimulation treatments did not result in adequate germination. The ﬁndings are pertinent and valuable for identifying suitable forest populations as seed sources, as well as for their use in silver ﬁr reforestation programs.


Introduction
Climate change is a major challenge that the world is currently facing, and it will continue to lead to major global changes [1][2][3].Current predictions of future climate change have a high degree of uncertainty, and their potential effects on forests are causing increasing concerns.Climate change, directly or indirectly, affects tree growth and forest productivity [4][5][6].Changes in temperature and precipitation, as well as the increase in carbon dioxide levels, also have effects on the vegetative state of trees and their biological processes, influencing even the most complex forest ecosystems in various ways.Furthermore, the loss of biodiversity constitutes one of the major problems recently caused by climate change [4,7,8].Thus, due to the expected unfavorable environment shift, the natural growing areas for most species will be affected, and trees, like other plants, react in different ways to these changes [9].Some forest species may exhibit high phenotypic plasticity, and existing populations may tolerate or adapt to environmental changes [10,11].With temperatures and precipitation predicted to change, forest species, including conifers, may become increasingly threatened [12,13].Consequently, it is considered necessary to adopt appropriate measures in the management of forests in order to reduce the damage caused by climate change and the possible adjustments in the geographical distribution of species [14 -18].Foresters play a crucial role as responsible stewards and custodians of forest resources essential to the balance of the environment and life on Earth [19][20][21].In order to improve the current situation of the forests and to counter the risks looming over the forest stock in the future, research must be focused on a better knowledge of plant responses to abiotic stress, and the genetic improvement of trees must be complemented by appropriate reforestation techniques and forest management [22][23][24].In this sense, forest genetic resources are very important and must be used pragmatically and efficiently in the future, constituting an asset and a cornerstone for sustainable forest management [25][26][27][28][29]. Climate changes have major effects on the natural range of species distribution, forests being under severe pressure in this regard due to biotic and abiotic factors [30][31][32].Depending on the forecasts provided in the evolution of climate factors and the capacity of species to adapt to climate changes, the development of forest resources and their sustainable management become national policies of great importance.
Abies alba Mill. is one of the most valuable conifers in Europe, having important ecological, economic, and soil protection functions and a wide distribution in the mountainous areas of Europe, including the Carpathians [33].Growing interest in the species, along with the recognition that silver fir has been subjected to inappropriate silvicultural measures, due to a lack of understanding of its ecology, have spurred recent studies from various disciplines, which have yielded new insights into the ecology, structure, and dynamics of silver fir populations [34][35][36].In addition, the direct seeding of silver fir, as a method of artificial regeneration with a long tradition in forestry management, has recently been reconsidered in the context of the conversion of spruce stands in central Europe [37].However, plant regeneration depends on good seed germination (high percentage) and seedling quality, regardless of the method of generative reproduction used in afforestation and reforestation [38].Even under favorable environmental conditions, mature, viable seeds frequently fail to germinate due to dormancy.
The presence of a seed coat stops water and oxygen from reaching and activating the seed or hindering seed expansion, whereas embryo dormancy is induced by the seed's state prohibiting germination [39].There are significant differences between tree species in terms of the causes of seed dormancy; however, a straightforward stratification treatment can reduce the dormant period of silver fir seeds.Stratification is the process of soaking and chilling seeds prior to sowing, which mimics the natural conditions under which seeds spend the winter on damp and cold soil [32,40].In silver fir seeds, ethereal oils stored in the seed coat, such as terpenes, induce dormancy, which typically lasts for one winter.For stored seeds to germinate, a six-week period of wet-cold stratification is required, and autumn sowing ensures effective germination [32].The process of germination holds significant importance for the plant life cycle, as it serves as the initial stage for the creation of a new generation.Given the fragile state of the emerging seedling, germination represents a moment of heightened vulnerability [38].
It is well known that most forest tree species do not have significant seed production annually, this being influenced by different internal and external factors, causing seeding to be abundant only in some years, at certain time intervals.Consequently, improving the methods of seed production is of great interest on conifer plantations [41].In the case of silver fir, the periodicity of seed production is abundant every 4-5 years in seed source trees, while it is reduced to 2-3 years on plantations [42].The external integuments of silver fir seeds are rich in essential oils that have a germination-inhibiting effect; thus, silver fir is considered a species with a low germination capacity (25%-35%) [43,44].Due to this fact, it is necessary to stimulate the germination capacity of the seeds through different techniques.In Romania, there is no standard method for silver fir, but technical guidelines provide recommendations for the production, processing, and conditioning of forest seeds, respectively the production of forest reproductive material (FRM), including sowing in autumn, in order to prevent the reduction in the germination percentage, which can decrease by even 50%, by preserving the seeds until the following spring [42].
To break seed dormancy, different physical or chemical stimuli act on the seeds, or the seed integument must be damaged; in natural conditions, this damage occurs due to the action of different factors, such as low temperatures (frosts), high temperatures (fires) [45,46].Currently, different methods are used to scarify the seeds, the mechanical scarification method being one of the most used [47][48][49].Biostimulators like Atonik and Cropmax are widely used for different species, as they improve both seed germination and seedlings' rooting [50].Biostimulators positively influence most of the vital processes in plants, and even amplify the germinative energy of seeds, ensuring the obtainment of seedlings that sprout faster and more uniformly [50].As with other species, germination stimulation methods must be correlated with the ecological and biological particularities of the species [51,52].
Starting from these premises, the current study aimed to evaluate several A. alba provenances in Romania to identify potential genetic resources of forestry interest with appropriate characteristics, and to obtain saplings for use in afforestation and reforestation programs.Furthermore, the performances of five germination stimulant treatments in raising the germination percentage of silver fir seeds were investigated, with the intention of obtaining appropriate reproductive materials.In order to obtain useful information for increasing the efficiency of the work of obtaining breeding material for silver fir, the interactions between the different geographical origins (i.e., genetic and ecological variation) and the treatments carried out to stimulate seed germination were also considered.

Geographical Locations of the Provenances
Cones were collected from seven geographically different areas of silver fir in Romania, from natural seed stands.Consequently, the cones and seeds used as biological material in the study belong to seven provenances, originating in seven District Forest areas (further named as provenances).With the exception of two provenances, the rest were representative forest stands attested in the Romanian Gene Reserved Forests and Seed Stands, included in the National Catalog of Forest Genetic Resources and Forest Reproductive Materials [53].The provenance, county, code, production unit (UP), and management unit (u.a.) data from each stand from the provenances of the seeds are presented in Table 1.The geographical locations for the seven provenances of A. alba studied in Romania are represented in Figure 1.All the provenances originated in areas of the Romanian Carpathians, with five of the provenances in the northwestern part of the country, one in the northern part, and one in the central part.

Characterization of Cones and Seeds of A. alba
Cones were collected in a mixture from at least ten adult trees per provenance, with favorable growth, wood quality, vegetation, and health characteristics; thereafter, seeds were stored at a constant temperature of 20 oratory.The following characteristics were determined and processed as mean values: the cone length (cm), cone diameter (cm), cone mass (g), number of seeds per cone, seed length, which included the wing (cm), seed width (cm), and seed mass with and without the wing (g) (Figure 2a).The precision balance was used to determine the mass, and specialized software (Digimizer Image Analysis Software version 6.3.0,MedCalc, Ostend, Belgium) was used to measure the length (Figure 2b).

Characterization of Cones and Seeds of A. alba
Cones were collected in a mixture from at least ten adult trees per provenance, with favorable growth, wood quality, vegetation, and health characteristics; thereafter, seeds were stored at a constant temperature of 20 °C at the ICHAT, UASVM Cluj-Napoca laboratory.The following characteristics were determined and processed as mean values: the cone length (cm), cone diameter (cm), cone mass (g), number of seeds per cone, seed length, which included the wing (cm), seed width (cm), and seed mass with and without the wing (g) (Figure 2a).The precision balance was used to determine the mass, and specialized software (Digimizer Image Analysis Software version 6.3.0,MedCalc, Ostend, Belgium) was used to measure the length (Figure 2b).

The Studied Germination Parameters
The seed germination capacity was analyzed after the seeds were empirically tested for their viability by immersion in water.Only the seeds from the bottom of the bowls were used in the five different treatments (Table 2).The fir seeds were soaked in sulfuric acid solution for 10 min.
Atonik is a biostimulant that amplifies the germination capacity of seeds and accelerates the emergence of plants and their uniform growth [50].It contains 0.2% sodium orthonitrophenolate, 0.3% sodium paranitrophenolate, and 0.1% sodium 5-nitroguaiacolate.To assess the germination, all seeds were watered every two days with tap water and kept under similar conditions in the laboratory, at a temperature of 20 • C. Seeds were put in glass Petri dishes to germinate, on filter paper.Four replicates, with 50 seeds per replicate, for each combination, considering each population (provenance) and each treatment, were investigated.In order to identify the germination speed, observations were made on days 4, 7, 10, 14, 21, and 28, for all provenances and treatments.

Data Analysis
Following the verification of the normality of the data, the ANOVA test with a single factor was performed to determine whether there were real differences in the means obtained for the characteristics of the silver fir cones and seeds based on provenance.A similar method was used for the germination of the seeds, depending on the origin and the treatments to stimulate the germination, but applying the ANOVA with two factors.If statistically significant values between the means were registered, then Duncan's Multiple Range Test (Duncan MRT) was applied as a post hoc test (p < 0.05) to identify the differences between the means.In all cases, the results are presented as the mean values of the analyzed characteristics + SEMs (standard errors of the means).
Multivariate analysis, namely principal component analysis (PCA), was performed on the data.Seven different provenances of A. alba were analyzed using the multivariate principal component analysis graph of Past software 4.09 [63].The analysis also included the main characteristics of the cones and seeds, as well as the seed germination according to five distinct treatments to improve the germination capacity.A dendrogram was also constructed using this program, with the distances between the origins and attributes determined using Euclidean distances.The data were normalized, or standardized, for the multivariate analysis by scaling the attribute values to place them numerically in the same range/scale (0-1) and, hence, give them the same relevance.

The Main Characteristics of Silver Fir Cones Depending on Provenances
The main characteristics of the cones depending on the geographical provenances of A. alba are presented in Figure 3, as means and standard errors of the means.

Data Analysis
Following the verification of the normality of the data, the ANOVA test with a single factor was performed to determine whether there were real differences in the means obtained for the characteristics of the silver fir cones and seeds based on provenance.A similar method was used for the germination of the seeds, depending on the origin and the treatments to stimulate the germination, but applying the ANOVA with two factors.If statistically significant values between the means were registered, then Duncan's Multiple Range Test (Duncan MRT) was applied as a post hoc test (p < 0.05) to identify the differences between the means.In all cases, the results are presented as the mean values of the analyzed characteristics + SEMs (standard errors of the means).
Multivariate analysis, namely principal component analysis (PCA), was performed on the data.Seven different provenances of A. alba were analyzed using the multivariate principal component analysis graph of Past software 4.09 [63].The analysis also included the main characteristics of the cones and seeds, as well as the seed germination according to five distinct treatments to improve the germination capacity.A dendrogram was also constructed using this program, with the distances between the origins and attributes determined using Euclidean distances.The data were normalized, or standardized, for the multivariate analysis by scaling the attribute values to place them numerically in the same range/scale (0-1) and, hence, give them the same relevance.

The Main Characteristics of Silver Fir Cones Depending on Provenances
The main characteristics of the cones depending on the geographical provenances of A. alba are presented in Figure 3, as means and standard errors of the means.For the cone length (Figure 3a), values between 7.98 cm (Valea Bistrei) and 13.70 cm (Valea Morii) were recorded.The provenances Somes , ul Rece, Budescu, and Gârda Seacă did not show significant differences between them.For the cone diameter, the lowest values were recorded in the Valea Morii provenance (3.85 cm), while the highest value was noted in the Gârda Seacă provenance (6.2 cm); despite these mean values noted, Gârda Seacă did not register statistically significant differences from the Avrig and Budescu provenances (Figure 3b).The highest value of the number of seeds/cones was recorded in the Gârda Seacă provenance, which was 268 seeds/cones (Figure 3c), and for the cone mass, the same provenance presented superior values with statistically significant differences compared to the other provenances, recording an average of 46.43 g (Figure 3d).The lowest values for these characteristics, with statistically significant differences, were recorded in Sohodol for the number of seeds in a cone (112) and Budescu for the cone mass (30.46 g).

The Main Characteristics of Seeds Depending on Provenances
Among the seven provenances of Romanian silver fir, significant differences were registered for the main characteristics of the seeds (Figure 4).For the seed length, the Valea Morii provenance recorded the highest values, with an average of 2.54 cm, showing statistically significant differences compared to all the other fir provenances.The highest value of the seed width (thickness) was recorded for the Valea Bistrei provenance (0.87 cm), while the Budescu and Sohodol provenances recorded the lowest values, both with 0.78 cm.There were no statistically significant differences between the other provenances (Somes , ul Rece, Avrig, Valea Morii, and Gârda Seacă).The seeds with the highest mass among all the geographical resources were recorded for the Gârda Seacă provenance (Figure 4c shows the average values of the seeds with wings).For the cone length (Figure 3a), values between 7.98 cm (Valea Bistrei) and 13.70 cm (Valea Morii) were recorded.The provenances Someșul Rece, Budescu, and Gârda Seacă did not show significant differences between them.For the cone diameter, the lowest values were recorded in the Valea Morii provenance (3.85 cm), while the highest value was noted in the Gârda Seacă provenance (6.2 cm); despite these mean values noted, Gârda Seacă did not register statistically significant differences from the Avrig and Budescu provenances (Figure 3b).The highest value of the number of seeds/cones was recorded in the Gârda Seacă provenance, which was 268 seeds/cones (Figure 3c), and for the cone mass, the same provenance presented superior values with statistically significant differences compared to the other provenances, recording an average of 46.43 g (Figure 3d).The lowest values for these characteristics, with statistically significant differences, were recorded in Sohodol for the number of seeds in a cone (112) and Budescu for the cone mass (30.46 g).

The Main Characteristics of Seeds Depending on Provenances
Among the seven provenances of Romanian silver fir, significant differences were registered for the main characteristics of the seeds (Figure 4).For the seed length, the Valea Morii provenance recorded the highest values, with an average of 2.54 cm, showing statistically significant differences compared to all the other fir provenances.The highest value of the seed width (thickness) was recorded for the Valea Bistrei provenance (0.87 cm), while the Budescu and Sohodol provenances recorded the lowest values, both with 0.78 cm.There were no statistically significant differences between the other provenances (Someșul Rece, Avrig, Valea Morii, and Gârda Seacă).The seeds with the highest mass among all the geographical resources were recorded for the Gârda Seacă provenance (Figure 4c shows the average values of the seeds with wings).

The Germination of Silver Fir Seeds
The germination of the seeds (%) fluctuated strongly, depending on both the geographical provenance and the germination stimulation treatment (Figure 5).
In the general framework of the study, large differences were recorded between the mean values of the calculated seed germination in the interactions between the treatments and provenances (T × P), with oscillations between 5% and 60% (Figure 5a).

The Germination of Silver Fir Seeds
The germination of the seeds (%) fluctuated strongly, depending on both the geographical provenance and the germination stimulation treatment (Figure 5).
The germination of the seeds (%) fluctuated strongly, depending on both the geographical provenance and the germination stimulation treatment (Figure 5).
In the general framework of the study, large differences were recorded between the mean values of the calculated seed germination in the interactions between the treatments and provenances (T × P), with oscillations between 5% and 60% (Figure 5a).
The variation limits of the seed germination depending on the geographical provenance, regardless of the treatment, were between 26% (Sohodol) and 48% (Gârda Seacă) (Figure 5b).Depending on the unilateral influence of the treatments applied to the seeds, regardless of the provenance, the lowest percentage of germination (12.9%) was recorded when applying the H2SO4 treatment, and the highest percentage (8.6%)was recorded when applying Atonik (Figure 5c).Within each interaction (T × P), provenance or treatment marked, significant differences between the means are illustrated with different letters (Duncan's Multiple Range Test, α < 0.05).

The Germination Indices
The main germination indices of the A. alba seeds show differences according to the germination stimulation treatments and geographical origins (Table 3).The treatment with Atonik induced the highest value of the germination percentage (GP) index (%), which varied between 40% for the Sohodol provenance and 60% for the Garda Seacă provenance.The highest values for the GI were recorded in Valea Bistrei for the control seeds (GI = 1.3) and in the Garda Seacă provenance for the seeds treated with Atonik (GI = 1.3).
The speed of emergence (SE) oscillated between 11.1 and 100.0, and the coefficient of germination speed (CRG) oscillated between 2.3 and 10.3.Finally, the SVI (seedling vigor index) had values between 0.29 (H2SO4/Sohodol) and 7.57 (Atonik/Gârda Seacă).In the general framework of the study, large differences were recorded between the mean values of the calculated seed germination in the interactions between the treatments and provenances (T × P), with oscillations between 5% and 60% (Figure 5a).
The variation limits of the seed germination depending on the geographical provenance, regardless of the treatment, were between 26% (Sohodol) and 48% (Gârda Seacă) (Figure 5b).Depending on the unilateral influence of the treatments applied to the seeds, regardless of the provenance, the lowest percentage of germination (12.9%) was recorded when applying the H 2 SO 4 treatment, and the highest percentage (8.6%)was recorded when applying Atonik (Figure 5c).

The Germination Indices
The main germination indices of the A. alba seeds show differences according to the germination stimulation treatments and geographical origins (Table 3).The treatment with Atonik induced the highest value of the germination percentage (GP) index (%), which varied between 40% for the Sohodol provenance and 60% for the Garda Seacă provenance.
The highest values for the GI were recorded in Valea Bistrei for the control seeds (GI = 1.3) and in the Garda Seacă provenance for the seeds treated with Atonik (GI = 1.3).The speed of emergence (SE) oscillated between 11.1 and 100.0, and the coefficient of germination speed (CRG) oscillated between 2.3 and 10.3.Finally, the SVI (seedling vigor index) had values between 0.29 (H 2 SO 4 /Sohodol) and 7.57 (Atonik/Gârda Seacă).
According to the data presented in Figure 6, the post hoc analysis highlighted similarities between some germination indices, calculated as mean values over the entire experience with seven provenances of silver fir.Some similarities visible in the graphs were also confirmed statistically via the Duncan MRT test.For example, the germination stimulation treatments had identical effects in terms of the germination percentage (GP) and germination index (GI) (Figure 6a,b).For both indices, Atonik determined the highest values and H 2 SO 4 determined the lowest values.Extreme values, positive in the case of Atonik and negative in the case of H 2 SO 4 , were also registered for the coefficient of germination speed (CRG) and seedling vigor index (SVI).also confirmed statistically via the Duncan MRT test.For example, the germination s ulation treatments had identical effects in terms of the germination percentage (GP) germination index (GI) (Figure 6a,b).For both indices, Atonik determined the highest ues and H2SO4 determined the lowest values.Extreme values, positive in the cas Atonik and negative in the case of H2SO4, were also registered for the coefficient of ger nation speed (CRG) and seedling vigor index (SVI).
In Figure 6d,e, the shapes of the graphs are relatively close for the two indices, w differences were recorded in terms of the significances for the seed scarification treatm and the use of acetone.The most obvious differences between the germination ind were noted for the speed of emergence (SE) (Figure 6d).For this index, Atonik determi the lowest value and the treatment with H2SO4 determined the highest value, followed seed scarification.

Multivariate Analysis
The principal component analysis (PCA) illustrated in Figure 7 provides a synth presentation of the dimensionality of the data set represented by the variables by the m phological characteristics of the cones and seeds from seven Romanian provenance silver fir, and the germination of the seeds according to five germination stimulation tr ments.The first component of the PCA, or PC1, accounted for 54.0% of the total measu variation, while the second component, or PC2, accounted for 23.5% by finding new v ables that were linear functions of the original variables.
The PCA highlights the opposite locations of the provenances of Gârda Seacă Sohodol and the very large distance between them.On the contrary, the Avrig Budescu populations (lower right quadrant) are arranged in common quadrants and close distance.The Someșul Rece and Valea Bistrei provenances are found in a sim situation (but in the upper left quadrant).It is also noticeable that all the morpholog characteristics of the cones and seeds are to the right of the O-Y axis, but some are in upper quadrant and others in the lower one.In the same quadrants are also the sca values of the seed germination from the five types of germination stimulation treatme with a relatively low dispersion.In Figure 6d,e, the shapes of the graphs are relatively close for the two indices, while differences were recorded in terms of the significances for the seed scarification treatments and the use of acetone.The most obvious differences between the germination indices were noted for the speed of emergence (SE) (Figure 6d).For this index, Atonik determined the lowest value and the treatment with H 2 SO 4 determined the highest value, followed by seed scarification.

Multivariate Analysis
The principal component analysis (PCA) illustrated in Figure 7 provides a synthetic presentation of the dimensionality of the data set represented by the variables by the morphological characteristics of the cones and seeds from seven Romanian provenances of silver fir, and the germination of the seeds according to five germination stimulation treatments.The first component of the PCA, or PC1, accounted for 54.0% of the total measured variation, while the second component, or PC2, accounted for 23.5% by finding new variables that were linear functions of the original variables.
The PCA highlights the opposite locations of the provenances of Gârda Seacă and Sohodol and the very large distance between them.On the contrary, the Avrig and Budescu populations (lower right quadrant) are arranged in common quadrants and at a close distance.The Somes , ul Rece and Valea Bistrei provenances are found in a similar situation (but in the upper left quadrant).It is also noticeable that all the morphological characteristics of the cones and seeds are to the right of the O-Y axis, but some are in the upper quadrant and others in the lower one.In the same quadrants are also the scaled values of the seed germination from the five types of germination stimulation treatments, with a relatively low dispersion.
The PCA highlights the opposite locations of the provenances of Gârda Seacă and Sohodol and the very large distance between them.On the contrary, the Avrig and Budescu populations (lower right quadrant) are arranged in common quadrants and at a close distance.The Someșul Rece and Valea Bistrei provenances are found in a similar situation (but in the upper left quadrant).It is also noticeable that all the morphological characteristics of the cones and seeds are to the right of the O-Y axis, but some are in the upper quadrant and others in the lower one.In the same quadrants are also the scaled values of the seed germination from the five types of germination stimulation treatments, with a relatively low dispersion.Figure 8 depicts the geographical provenances and morphological characteristics of the cones and seeds, as well as the germination of the seeds subjected to the five types of stimulation treatments, based on the grouping of the variables and their levels of similarity.The tree diagram validates the dispersion results, both in terms of the pattern of cluster formation and the similarity or distance levels of the clusters containing the investigated silver fir provenances.
The distance from the rest of the Gârda Seacă population is shown in the vertical grouping for origin (also underlined by the PCA).It also confirms the close relationship between the Avrig and Budescu provenances, as well as between the Someșul Rece and Valea Bistrei provenances.
In the horizontal cluster, two subclusters are differentiated, the top one with only two branches, on which the seed width and cone mass are placed, and the bottom one with multiple subclusters.The cone length and seed length are found to be grouped in a single subcluster (the bottom one), whereas the cone diameter is found on a distinct branch of the most consistent subcluster.Within this, the expected closeness between the masses of the winged and wingless seeds is confirmed.The number of seeds in the cone is displayed on a nearby branch.The seed germination is arranged according to the stimulation treatment on subclustered subordinates, and it is completed with a common subcluster in which the acetone and scarification treatments are tightly packed.The homogeneity of Gârda Seacă for the analyzed attributes is most visible in the heatmap's vertical plane, trending toward the maximum in most cases.On the contrary, notable homogeneity is seen in the opposite direction, towards a lack of correlation, at the Sohodol provenance.The distance from the rest of the Gârda Seacă population is shown in the vertical grouping for origin (also underlined by the PCA).It also confirms the close relationship between the Avrig and Budescu provenances, as well as between the Somes , ul Rece and Valea Bistrei provenances.
In the horizontal cluster, two subclusters are differentiated, the top one with only two branches, on which the seed width and cone mass are placed, and the bottom one with multiple subclusters.The cone length and seed length are found to be grouped in a single subcluster (the bottom one), whereas the cone diameter is found on a distinct branch of the most consistent subcluster.Within this, the expected closeness between the masses of the winged and wingless seeds is confirmed.The number of seeds in the cone is displayed on a nearby branch.The seed germination is arranged according to the stimulation treatment on subclustered subordinates, and it is completed with a common subcluster in which the acetone and scarification treatments are tightly packed.The homogeneity of Gârda Seacă for the analyzed attributes is most visible in the heatmap's vertical plane, trending toward the maximum in most cases.On the contrary, notable homogeneity is seen in the opposite direction, towards a lack of correlation, at the Sohodol provenance.
Statistically significant associations were found by calculating the Pearson correlation coefficients between the examined characteristics of the cones, seeds, and seed germination (G%) depending on the stimulation treatments (Table 4).All statistically validated correlations were positive, and no significant negative correlations were found between the pairs of investigated variables.No close correlations were detected between the morphological characteristics of the cones (length and diameter) and those of the seeds and seed germination, with only one exception.This was represented by the unexpected correlation between the cone diameter and seed germination following treatment with sulfuric acid (r = 0.90 **).
The number of seeds in the cone and the mass of the seeds were correlated with each other, but they were also correlated with most of the analyzed variables.It was found that they were closely correlated with the germination percentage of the seeds that were subjected to stimulation treatments with Atonik, scarification, acetone, and H 2 SO 4 .An interesting result was that the germination percentage of the untreated seeds (without germination stimulation) was not significantly influenced by the number of seeds in the cone or the masses of the winged or non-winged seeds.Correlations for the seed germination (G%) depending on the applied treatments were recorded as follows: acetone-scarification, H 2 SO 4 -acetone (p < 0.001); acetone-control/untreated (p < 0.01); scarification-Atonik, H 2 SO 4 -scarification (p < 0.05).

Discussion
Improving the seed quality of silver fir seeds is challenging, and the germination capacity varies substantially between seed lots, ranging from 5% to 80%.Because of this, the generation of seedlings is expensive and ineffective, especially in container nurseries [64].According to the findings of this investigation, geographical provenances influence the morphological characteristics of the cones and seeds of A. alba, with the average values for these characteristics displaying considerable amounts of variation.Within the provenances investigated, the limits for the traits of the cones varied a lot both within the provenances and between the provenances, and a similar variability was also found for the traits of the seeds.According to the literature, large seeds tend to have a higher germination percentage, mainly because they are assumed to contain more resources to support such an intensive, energy-intensive biological process [65].Furthermore, larger or heavier seeds may be able to produce better and healthier seedlings [66].However, although the seed mass has a strong correlation with the germination efficiency, plumper seeds do not always germinate faster than small seeds [67,68].In our study, Garda Seacă had the heaviest seeds and ensured good germination in all the germination treatments.The thickest seeds of the Valea Bistrai provenance also conferred a good germination percentage.The seed characteristics can influence the dispersal distance, which is a crucial element in the genetic structure of forest tree populations.The seed distribution is inversely correlated to the seed weight, but this shortcoming may be partially compensated for by the silver fir seed wing length [69].
Germination was influenced by the seed morphology, genotype, and environmental conditions in the area of provenance [57,70].Furthermore, various treatments applied to the seeds to increase their germination capacity can be beneficial for obtaining a high percentage of seedlings [71][72][73][74].Different germination performances are possible because of the environmental heterogeneity that exists in the origins of the silver fir within the Romanian provenances [22].Seeds collected in sites characterized by different environmental parameters, such as distinct populations (provenances), display a noteworthy difference in their germination dynamics [75], and this aspect should always be considered when procuring biological material.
For all the germination stimulation techniques, the seed germination varied among the tested provenances.The germination percentage was not increased by the sulfuric acid treatment.Such a result may be the result of the acid medium's harmful effects, which probably damaged the embryo, not only the teguments of the seeds.Several studies have demonstrated that using sulfuric acid was more effective than other treatments for different forest species [47,57,65].To break the seed dormancy, the tegument must be deteriorated; in natural conditions, this 'damage' occurs as a result of the action of many elements on the seed tegument, such as low temperatures (frosts), high temperatures (fires), and so on [45,46].The scarification of the silver fir seeds failed to produce a high percentage of germination in our experimental conditions, but, in contrast, Atonik, a biostimulator used to stimulate both seed germination and seedling rooting [50], significantly improved the germination for all the silver fir provenances.Atonik is frequently used to improve seed germination and vegetative growth by activating plant cell metabolism and thereby improving the plant growth and yield [76]. Atonik's role in stimulating root growth helps the plant to be more active in absorbing nutrients and it thus stimulates vegetative growth [77][78][79].
Germination indices can provide information of interest for forecasting the percentage of germinated seeds, the absolute number of seeds germinating over time, etc.The reliability of germination indices is influenced by various genetic and environmental variables, as well as by the interaction of numerous factors that influence the biology and physiology of seeds [60,80,81].Cold stratification and temperature, as well as their interaction, significantly influenced the seed germination percentage (GP) and mean germination time (MGT) in Abies marocana [82].Predictions regarding the final germination or germination time can be useful for making economic decisions for the management of reproductive material, as well as from the perspective of modeling and fundamental research [83,84].In our study, the speed of emergence was the most noticeable variation across the germination indices, with Atonik having the lowest value and H 2 SO 4 having the highest, followed by seed scarification.
Multivariate analysis, including PCA, dendrograms, and correlations between variables, provide valuable information regarding the identification of viable seed sources and are fruitful [85] for the silver fir propagation and regeneration desideratum.The close correlations identified between some morphological elements of the seeds and germination can be effectively used in silver fir growth as indirect selection indices, or in the forestry practice of producing valuable biological material for afforestation and reforestation [86,87].PCA and hierarchical clustering data provide information about the possibility of identifying the most valuable seed sources among those evaluated, as well as models for further investigations.The results are also of interest because forest sources of certified seeds do not always ensure a quality biological material, and they should be reviewed and reconfirmed.

Conclusions
The current investigation conducted an evaluation of the seed characteristics and germination, including germination tests, for different silver fir provenances from geographical regions in Romania.The germination responses of A. alba seeds from distinct populations may be attributed to the varying environmental factors of the collection sites.The different populations of silver fir analyzed based on important reproductive traits exhibited varying levels of phenotypic plasticity, as well as differences in the seed characteristics, germination, and subsequent seedling growth.In our study, the treatment with the biostimulator Atonik significantly improved the germination for all the silver fir provenances.Among the provenances evaluated, Gârda Seaca demonstrated good seed germination and can be recommended as a significant seed resource, as well as for potential future breeding programs.These evaluations could enhance the silvicultural strategy of the species and help to identify potential valuable seed sources.Additionally, distinct treatments that can stimulate the germination capacity of A. alba were identified, which could have beneficial implications for silver fir afforestation programs.

Figure 1 .
Figure 1.The geographical locations of the seven provenances of silver fir (Abies alba) in Romania and the main peculiarities of each area.Figure 1.The geographical locations of the seven provenances of silver fir (Abies alba) in Romania and the main peculiarities of each area.

Figure 1 .
Figure 1.The geographical locations of the seven provenances of silver fir (Abies alba) in Romania and the main peculiarities of each area.Figure 1.The geographical locations of the seven provenances of silver fir (Abies alba) in Romania and the main peculiarities of each area.

Figure 2 .
Figure 2. Analyses conducted: (a) extraction of seeds from cones and morphological analyses of cones and seeds; (b) measurements of cones and seeds, using Digimizer software, version 6.3.0.Figure 2. Analyses conducted: (a) extraction of seeds from cones and morphological analyses of cones and seeds; (b) measurements of cones and seeds, using Digimizer software, version 6.3.0.

Figure 2 .
Figure 2. Analyses conducted: (a) extraction of seeds from cones and morphological analyses of cones and seeds; (b) measurements of cones and seeds, using Digimizer software, version 6.3.0.Figure 2. Analyses conducted: (a) extraction of seeds from cones and morphological analyses of cones and seeds; (b) measurements of cones and seeds, using Digimizer software, version 6.3.0.
germinated seeds Total number o f seeds × 100; GI (germination index): GI = Number o f germinated seeds Days f rom the f irst control + . . .+ Number o f germinated seeds Days f rom the f irst control ; SE (speed of emergence): SE = Number o f germinated seeds in the f irst day o f germination Number o f germinated seeds in the last day o f germination × 100; CRG (coefficient of germination speed):

Figure 4 .
Figure 4.The main characteristics of the seeds depending on the geographical provenances of A. alba, as means ± SEMs (respectively, F value and p-value, ANOVA test): (a) seed length (F = 3.2, p = 0.041); (b) seed width (F = 3.2, p = 0.041); (c) seed mass (F = 4.7, p = 0.010).Different letters within means indicate statistically significant differences for the respective trait, at a significance level of p < 0.05 (Duncan test).

Figure 4 .
Figure 4.The main characteristics of the seeds depending on the geographical provenances of A. alba, as means ± SEMs (respectively, F value and p-value, ANOVA test): (a) seed length (F = 3.2, p = 0.041); (b) seed width (F = 3.2, p = 0.041); (c) seed mass (F = 4.7, p = 0.010).Different letters within means indicate statistically significant differences for the respective trait, at a significance level of p < 0.05 (Duncan test).

Figure 6 .
Figure 6.The influence of the seed germination stimulation treatments on the germination ind calculated as the means of the seven provenances of silver fir (means ± SEMs): (a) germination centage (GP); (b) germination index (GI); (c) speed of emergence (SE); (d) coefficient of germina speed (CRG); (e) seedling vigor index (SVI).For each index, significant differences between m are illustrated with different letters (Duncan's Multiple Range Test, α < 0.05).

Figure 6 .
Figure 6.The influence of the seed germination stimulation treatments on the germination indices, calculated as the means of the seven provenances of silver fir (means ± SEMs): (a) germination percentage (GP); (b) germination index (GI); (c) speed of emergence (SE); (d) coefficient of germination speed (CRG); (e) seedling vigor index (SVI).For each index, significant differences between means are illustrated with different letters (Duncan's Multiple Range Test, α < 0.05).

Figure 7 .
Figure 7. Principal component analysis (PCA) plot, based on the cone and seed traits analyzed from seven Romanian sources of silver fir (A.alba), and seed germination (G%) in five germination stimulation treatments (control; Atonik; scarification; acetone; H 2 SO 4 ) applied to the seeds.

Figure 8
Figure 8 depicts the geographical provenances and morphological characteristics of the cones and seeds, as well as the germination of the seeds subjected to the five types of stimulation treatments, based on the grouping of the variables and their levels of similarity.The tree diagram validates the dispersion results, both in terms of the pattern of cluster formation and the similarity or distance levels of the clusters containing the investigated silver fir provenances.

Forests 2023 , 20 Figure 7 .
Figure 7. Principal component analysis (PCA) plot, based on the cone and seed traits analyzed from seven Romanian sources of silver fir (A.alba), and seed germination (G%) in five germination stimulation treatments (control; Atonik; scarification; acetone; H2SO4) applied to the seeds.

Figure 8 .
Figure 8. Multivariate analyses for the studied characteristics: hierarchical clustering, paired-group (UPGMA) algorithm, and Euclidean similarity index of the cones, seeds, and germination, respectively, of the seven geographical provenances of silver fir (A.alba).

Figure 8 .
Figure 8. Multivariate analyses for the studied characteristics: hierarchical clustering, paired-group (UPGMA) algorithm, and Euclidean similarity index of the cones, seeds, and germination, respectively, of the seven geographical provenances of silver fir (A.alba).

Table 1 .
The provenances (populations) of Abies alba in Romania that generated the biological material for the study, represented by cones and seeds.

Table 2 .
The five treatments performed to stimulate seed germination.

Table 3 .
The main germination indices of A. alba seeds depending on the germination stimulation treatments and geographical provenances.

Table 3 .
The main germination indices of A. alba seeds depending on the germination stimulation treatments and geographical provenances.

Table 4 .
Pearson correlations between the pairs of characteristics analyzed and seed germination (G%) depending on stimulation treatments (below the diagonal) and the p-values of the correlations (above the diagonal).Correlations were calculated from the mean values of each of the 13 characteristics.