Characteristics of the seeds and cones of Serbian spruce - indicators of species endangerment

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Understanding the characteristics of cones and seeds, along with the analysis of intra- and inter-population variability, is crucial for identifying the reasons for this decline and implementing in situ and ex situ conservation measures to preserve this species. During 2022/23 (autumn/spring), cones were collected from 111 trees of this species. The trees are distributed across seven natural populations and one urban population in Bosnia and Herzegovina. Immediately after collection, the cones were measured, processed, seeds were extracted, and germination tests were conducted. Significant differences among the tested trees and populations were found for all measured cone traits. The germination results indicate very low seed viability, averaging 57,03% for all tested trees, with the percentage of empty seeds at 26,85%. Significant differences in germination were also observed at the population level, ranging from 20,40% to 81,14%, with the percentage of empty seeds ranging from 8,10% to 59,60%. The results suggest that endangered populations with a small number of trees have significantly smaller cones, a higher percentage of empty seeds, and lower germination rates. Such a low germination rate in natural populations of Serbian spruce indicates the need for urgent actions to protect this species and assist its natural regeneration (in situ), as well as to establish plantations outside its natural range (ex situ conservation). genetic variability endangered populations cones seeds germination Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The survival, or more precisely, the regeneration of a plant species involves seed production and dispersal, germination, and the successful development of seedlings. Each species has its unique germination requirements, which can be considered adaptations for maximum survival in an unstable and unpredictable environment (Fenner 1985; Iralu et al. 2019). Seed germination and seedling development are critical phases in the life cycle that determine the population dynamics of a species and ultimately influence community development, structure, and survival (Chen and Xie 2007; Baeten et al. 2009; Grossnickle and Ivetić 2017). This phase is characterized by exceptionally high mortality rates and intense natural selection (Leck et al. 2008; Grossnickle 2018). Consequently, the seedling phase is often considered a "bottleneck" in the life history of a species (Kolb and Barsch 2010; Grossnickle 2016). A significant number of plant species worldwide are becoming endangered because processes such as seed dispersal, germination, and seedling development and survival are hindered or limited (Colin and Linda 2002; Gulias et al. 2004). The Intergovernmental Panel on Climate Change (IPCC) estimates that roughly 20–30 percent of vascular plants are at risk of extinction due to a temperature increase of 2–3°C (Parry et al. 2007). Even modest losses in biodiversity could cause consequential changes in ecosystem services (Seppälä et al. 2009). Studying the causes of population decline in endangered plant species, their ecological adaptability, and their ability to avoid threats, followed by formulating effective measures to protect their gene pool and population sizes, has become a crucial issue in biodiversity conservation research worldwide (Fernández and Tapias 2022; Xu et al. 2022; Xu and Zang 2023). Certainly, the emergence, survival, and final establishment of seedlings are largely influenced by habitat characteristics (Bazzaz 1991; Mataruga et al. 2012), a phenomenon particularly pronounced in the Serbian spruce (Pintarić 1969; Dinić 1990). Species like the Serbian spruce, with small populations and unique genetic constitutions (Mataruga et al. 2020) that exchange genes only through pollen (Aleksić et al. 2022), are particularly vulnerable to extinction due to reduced fitness resulting from inbreeding depression and random demographic and ecological effects (Hensen and Wesche 2006).Today, the importance of contemporary ecological factors limiting the growth of endangered plant species populations is poorly understood, and there is limited knowledge about the management actions required for their maintenance. Consequently, there are few scientific guidelines regarding which activities should be prioritized. For conservation purposes, it is crucial to understand the seed germination and seedling formation traits that limit endangered species in their spread, increase in numbers, or survival even at low population sizes (Yates and Broadhurst 2002). The Serbian spruce ( Picea omorika /Pančić/Purkyně) is a tertiary relict found only in the mid-course of the Drina River canyon, within a narrow range from 19°09' − 44°07' N (Tisovljak site) to 19°46' − 43°20' N (Mileševa site). All sites are located at altitudes ranging from 750 to 1550 meters on very steep, north-facing slopes (Mataruga and Milanović 2020). This species is categorized as endangered by the IUCN (Mataruga et al. 2011) and is protected by the governments of Bosnia and Herzegovina and the Republic of Serbia. The surviving populations are distributed in a fragmented landscape in very small sizes, often comprising only a few mature trees, typically several dozen and rarely around a thousand individuals. The problem of natural regeneration in these populations is recognized, with young seedlings almost non-existent unless a catastrophic event such as a fire, avalanche, or landslide occurs. Genetic and developmental characteristics have been studied to uncover the causes of its endangerment (Aleksić and Geburek 2010; Mataruga et al. 2020), with slow natural regeneration being the main limitation for its survival (Mataruga and Milanović 2020). The seed germination and seedling formation traits remain unclear concerning processes of regeneration, stability, migration, and distribution. The quality aspects and the impact of different treatments on seed germination have received little attention in this species so far (Cvjetković 2011). Object Research and Methodology In 2022, an abundant yield of Serbian spruce cones was observed in a significant number of natural populations. Cones were collected in the autumn of 2022 by climbing trees, ensuring the identification of each maternal (test) tree – half sib lines. Seeds were collected from a total of 111 trees across seven natural and one urban population. For each tree, coordinates were recorded, basic dendrometric data were taken (diameter, height, crown width, and presence of any abiotic damage), and the trees were marked in the field. Seven spatially isolated natural populations: Tisovljak, Panjak, Tesla, Veliki Stolac, Grad, Šarena bukva, and Radomišlja were included out of the 26 described in Bosnia and Herzegovina. The most endangered and habitat-specific populations with cone yield were chosen following previous recommendations (Mataruga and Milanović, 2020). In early spring 2023, cones were also collected from mature trees in the urban area of Banja Luka city. Spatial distribution (Fig. 1 ) and basic data on the populations from which seeds were collected are presented in Table 1 , and the coordinates of each test tree are in Appendix Table 1. Immediately after collection, the total mass of all collected fresh cones from each tree was measured, followed by the measurement of mass, length, and diameter at the widest part for 12 randomly selected cones from each tree. The cones were then placed in a BCC drying chamber at ± 48°C for 72 hours. After drying, the seeds were extracted from the cones, and the dry mass of the cones and the dry mass of the seeds were measured. After manual seed deburring, impurities were separated from the seeds using the BCC gravity separator. Seed purity was determined according to ISTA guidelines (Gordon et al. 1991). Following this, 4 x 100 seeds were weighed to calculate the absolute mass (mass of 1.000 seeds) and the number of seeds per kilogram for each test tree. Seed purity, absolute mass, and the number of seeds per kilogram were analyzed using samples from five randomly selected test trees in each originating natural population (a total of 35 test trees). The seeds were then stored in a refrigerator at 4 ± 1°C. In the spring of 2023 (specifically from April 11 to May 2, 2023), the seeds were placed on a germination table, known as the "Copenhagen table" or "Jacobsen table" (Germinationtable GR10), which provides a constant temperature and accurately controlled moisture level, ensuring all seed samples receive the same treatment. Four sets of 100 seeds were placed on filter paper in constant contact with water at a temperature of 21°C. A total of 101 trees out of 111 were tested, as not all trees from which cones were collected had sufficient seeds for testing. The number of normally germinated seeds was counted on days 7, 14, and 21 from the start of the test. On the final day, the number of normally germinated seeds, full but ungerminated seeds, and empty seeds were recorded. Seeds were considered normally germinated when the root had broken through the seed coat and grown to half the seed's length. Four parameters: germination percentage (GP), germination energy (GE), number of empty seeds (ES), and number of full but ungerminated seeds (FS) were calculated as follows: GP (%) = (total number of germinated seeds on the 21st day × 100) / total number of seeds set for germination GE (%) = (number of seeds germinated on the 7th day × 100) / total number of seeds set for germination ES (%) = (total number of empty seeds × 100) / total number of seeds set for germination FS (%) = (number of full but ungerminated seeds × 100) / total number of seeds set for germination For statistical analyses, Microsoft Excel Version 16.83 for Mac was used; descriptive statistics were performed using Statistica 7.0. Analysis of variance (ANOVA) and Duncan's test were employed to demonstrate the significance of differences, with significance levels set at p < 0.01. The interdependence of seed traits (size, mass, and moisture) and germination dynamics, both within and between populations, was assessed using Principal Component Analysis (PCA) in the R program. Table 1 Characteristics of locations where cones were collected Population Longitude E Latitude N Altitude [m] Area [ha] Estimated number of adult trees [pieces] Offspring Number of trees from which seeds were collected [pieces] Tisovljak 19,091117 44,072907 980–1080 2,6 100 Rare 17 Šarena bukva 19,192886 44,019238 1000–1090 1,0 150 Rare 10 Grad 19,219963 44,005645 1100–1220 4,4 300 Rare 11 Panjak 19,149446 43,998802 1270–1330 0,6 30 None 9 Tesla 19,134806 43,927498 970–1110 2,6 50 Sporadic 11 V. Stolac 19,282496 43,922926 1050–1580 43,8 10.000 Sporadic 21 Radomišlja 18,606057 43,459431 850–1395 44,1 2.000 Sporadic 8 Banja Luka 17,198876 44,764852 160–190 - - None 24 Results Mass of Cones and Seeds On average, 840,34 grams of cones (or 747,03 grams in dry weight) were collected from a single tree, yielding 28,34 grams of seeds with wings and 14,06 grams without wings (see Appendix Table 1). The smallest amount of cones was collected from tree Veliki Stolac_16–68,16 grams fresh weight (or 59,18 grams in dry weight), and the largest amount from tree Radomišlja_3–1.985,54 grams in fresh weight (or 1.741,54 grams in dry weight). The highest amount of seeds was extracted from Tisovljak_1 (82,08 grams with wings or 48,22 grams without wings), and from Radomišlja_3 (69,32 grams with wings or 50,21 grams without wings). On average, the smallest seeds were collected in the Tesla and Panjak populations, with more than 600.000 seeds per kilogram, while the largest were at Veliki Stolac (378.000 seeds per kilogram) and slightly over 400.000 seeds per kilogram at the Grad, Šarena Bukva, and Tisovljak locations (Table 2 ). In the best-case scenario, the highest production of seeds per tree was recorded for Radomišlja_1, with approximately 21.000 seeds. This number was obtained by multiplying the absolute mass of a single seed (Table 1 ) by the mass of seeds obtained from the tree (21.877 = 435.714 x 0,05021). It is also important to note that a significant number of trees did not yield any seeds after cone extraction (cones remained closed after several days of drying), such as Tesla_1, 7, 8, 9, 10; Panjak_1, 5, 6, 9 (see Appendix Table 1 ). Table 2 Number of seeds per kilogram Population Grad Š. Bukva Tesla Panjak Radomišlja Tisovljak V. Stolac Average Number of seeds per kg 407.576 406.722 621.947 600.446 435.714 433.034 378.716 460.557 Morphometric Properties of Cones The smallest cones were found in the Tesla and Panjak populations (2,86 and 2,61 grams, respectively), while the largest cones were observed in the Grad population (5,27 grams). Banja Luka stood out with longer cones (46,9 mm), whereas the cones with the largest diameter were measured in the Radomišlja population (17,80 mm). Cone size varied significantly even among test trees. For instance, the differences in cone mass were up to 9 times greater for the Šarena Bukva_4 tree (9,51 grams) compared to the Panjak_9 tree (1,1 grams). Cone lengths ranged from 20,65 mm (Panjak_9) to 57,65 mm (Banja Luka_21), and diameters ranged from 11,24 mm (Panjak_9) to 20,29 mm (Banja Luka_21). Table 3 Mean values and standard deviation for cone properties Population Mass [gr] Length [mm] Diameter in the widest part [mm] Tisovljak 4,38 b ± 2,68 37,97 b ± 4,62 15,98 c ± 1,33 Grad 5,27 d ± 1,54 39,96 c ± 5,98 16,58 d ± 1,41 Š.bukva 4,60 c ± 2,01 38,50 b ± 6,02 16,15 c ± 2,07 Tesla 2,86 a ± 1,21 30,72 a ± 6,10 14,25 b ± 1,42 Panjak 2,61 a ± 0,98 29,78 a ± 5,56 13,75 a ± 1,60 V.Stolac 4,72 c ± 1,32 38,45 b ± 4,27 16,10 c ± 1,46 B.Luka 4,89 c ± 1,83 46,90 e ± 7,19 17,13 e ± 2,15 Radomišlja 4,82 c ± 1,20 43,38 d ± 5,01 17,80 f ± 1,48 All populations 4,37 ± 1,68 39,26 ± 7,81 16,10 ± 2,01 F pop ,p pop 58.12, p < 0.001 169.01, p < 0.001 83.97, p < 0.001 Comprehensive analysis of cone characteristics based on measured mass, length, and width reveals notable differences among populations, as summarized in Table 3 . Figure 2 depicts the distribution of test trees across these metrics. The population of Banja Luka stands out with longer cones but smaller mass compared to other populations. Conversely, the Panjak and Tesla populations consistently exhibited smaller cones across all measured characteristics. Germination Energy and Germination Rate Germination analysis revealed varying germination rates and energies across different populations of Serbian spruce. The Tesla and Panjak populations exhibited lower germination rates and energies, while the Veliki Stolac population showed the highest values in both parameters. Notably, the Banja Luka population displayed lower germination rates compared to others. Analysis of variance and Duncan's test confirmed significant differences among populations, grouping them into four homogeneous categories (Fig. 3 ). The lower germination rates in the Tesla and Panjak populations can be attributed to a higher percentage of empty seeds, although differences in the number of full but non-germinated seeds were relatively small across all populations, averaging 16,12% (Fig. 4 ). These populations also have fewer mature trees compared to others (see Table 1 ). Duncan's test confirmed significant differences, aligning with observed germination rate groupings. The lowest germination rate recorded was 4% in tree Panjak_4, whereas the highest was 100% in V. Stolac_11. Tesla_11 had the highest percentage of empty seeds at 82%, whereas 11 test trees had 0% empty seeds (see Appendix Table 1). Principal Components Analysis (PCA) for Measured Traits This analysis provides a comprehensive overview of the grouped and dispersed patterns among populations based on significant measured characteristics (Fig. 5 ). Populations primarily cluster along PC1, which explains 31,63% of the total variation and is characterized by prominent traits related to germination, seed characteristics, and maternal tree traits (see Appendix Table 1). In the positive direction of PC1, populations exhibit characteristics such as higher germination rates and energies, correlating positively with traits of maternal trees. Conversely, the negative direction of PC1 highlights characteristics related to empty seeds, negatively correlating with other measured seed traits. This component also shows grouping in the positive direction of measured cone characteristics. The Panjak and Tesla populations are notably situated in the lower left quadrant of the PCA plot, reflecting smaller cones and lower germination energy and rates. Meanwhile, the Banja Luka population stands out in the upper left quadrant, demonstrating distinct cone characteristics compared to other populations. Discussion In forest trees, reproductive dynamics and success are intricately linked to seed or fruit production, which varies significantly across demographic stages and depends heavily on adequate environmental conditions (Younginger et al. 2017; Moran and Clark 2012). Fragmentation and climate change profoundly impact seed quality and dispersal, critical prerequisites for species survival (McConkey et al. 2012). Empirical research underscores the complex relationship between seed size and germination time. Studies have shown positive correlations between seed size and germination percentage and energy (Parker et al. 2006; Jones and Reekie 2006; Upadhaya et al. 2007; Souza and Fagundes 2014; Kaliniewicz et al. 2018), negative correlations (Simons and Johnston 2000; Gomez 2004; Tiscar and Lucas 2010; Hojjat 2011), and instances where no correlation exists (Larson 1963; Vaughton and Ramsey 1998). Serbian spruce populations with smaller seeds, such as those in Tesla and Panjak with over 600.000 seeds per kilogram, exhibit poor germination rates, posing a threat to their already endangered status. This underscores the positive correlation observed between seed size and germination efficiency in Serbian spruce. Larger seeds likely store more nutrients, facilitating rapid germination and early seedling growth (Domic et al. 2020). In conclusion, understanding these relationships is crucial for conservation efforts aimed at preserving Serbian spruce populations. Enhancing seed quality and ensuring favorable germination conditions are essential strategies in mitigating the impact of environmental stressors on this endangered species. When comparing Serbian spruce seeds to those of other species, it becomes evident that while seed shape may be similar across species, significant differences exist in terms of absolute mass and moisture content. These variations are influenced by genetic factors of the parent tree and environmental conditions during seed development (Dech and Maun, 2006; Li and Li, 2016). Wyse and Hulme (2021) have documented a strong relationship between seed mass and dispersal potential in various wind-dispersed Pinus species. Their findings indicate that within species, there are generally no significant differences in seed mass, which may reduce selective pressure. In environments where resources are limited, the allocation of resources towards seed mass may prioritize seedling vigor and survival over dispersal efficiency. Pintarić's studies (1956; 1957; 1969) in the mid-20th century provided foundational insights into the germination behavior of Serbian spruce seeds. His findings indicated exceptionally high germination rates under optimal conditions. Seeds aged one year exhibited germination rates as high as 97–98%, with germination energy reaching 94% after 7 days and nearly 100% after 10 days of testing. Moreover, seeds stored at room temperature in glass bottles maintained high germination rates over extended periods (Pintarić 1970). Even seeds up to 3 years old maintained around 96% germination, demonstrating robust seed viability under controlled storage conditions. However, germination rates gradually declined with increasing seed age, with 86.9% germination observed at 6.5 years. Thus, Pintarić concluded that proper seed storage can preserve the germination capacity of Serbian spruce seeds over extended periods. However, the origins of the seeds used in Pintarić's studies were not specified, which may impact the generalizability of his findings. This gap highlights the importance of understanding seed provenance in seed germination studies to ensure the relevance of the results to specific populations or genetic lineages of the species. The studies by Tucović and Isajev (1985) indeed highlight the significant genetic variability found in Serbian spruce. They explored spontaneous and induced variability in Serbian spruce seedlings from 12 test trees in a 23-year-old planted forest. Their research emphasized significant genetic variability among the analyzed traits of maternal trees or half-sib lines. The findings underscored that genetic variability was far greater than what could be inferred from mere observations of morphological variability in parent trees. Isajev (1987) analyzed the individual variability of several trees from three planted forests of Serbian spruce in Serbia. Morphometric analysis of 1.000 cones harvested in 1981 and 1983 showed the existence of spontaneous individual variability between trees for length, width, and number of grains per cone, while the shape of the cone was significantly less variable. The range of variability for the length of cones was, on average, from 20 to 70 mm, for widths from 10 to 23 mm, while the mean number of grains varied from 20 to 113 per cone. The size of the cone in the same tree varied within 5% between different harvest years, which indicates strong genetic control of this trait (Isajev 1987). The obtained results showed that the Serbian spruce seeds from all three planted forests are characterized by very good quality (average germination rate was 90%). In conclusion, while historical studies by Pintarić and subsequent work by Tucović and Isajev have contributed valuable insights into the germination and genetic variability of Serbian spruce, there remains a need for more comprehensive studies that consider the genetic diversity and specific ecological contexts of this endangered species. Understanding these factors is crucial for effective conservation and management strategies, particularly in ensuring the successful propagation and survival of Serbian spruce in natural and managed environments. Cvjetković et al. (2013) tested the germination of seeds collected from 17 trees, specifically: 5 trees from the planted forest, 7 trees from the natural population Veliki Stolac, and 5 trees from the natural population Gostilja. Fresh seeds originating from the planted forest population showed the highest germination rate, averaging 84%. Seeds from the natural populations, germinated on average at 58,57% and 65,15%, respectively, with an average seed dormancy of 8,68 and 9,72 days (retrospective). Seeds stored in a refrigerator at ± 4°C for 6 months showed better germination results by approximately 20% compared to fresh seeds and 28% compared to fungicide (captan)-treated seeds. The higher vigor and faster germination in seeds stored for 6 months can be interpreted as a form of stratification (Cvjetković 2011). These studies indicate significant differences in the germination of seeds depending on whether they were collected in natural or planted forests. Ostojić and Dinić (2009) investigated germination under natural conditions. A series of experiments involving the sowing of Serbian spruce seeds in different habitats revealed poor seed germination and subsequent seedling failure in the first year (Dinić 1988, 1989). The authors attribute these results to the decline in tree numbers at this location - from 40 trees recorded in the early 1950s, only four remain today, as confirmed by later comparative experiments examining seed germination and seedling growth (Dinić 1990). The results highlight the sensitivity of Serbian spruce seeds and seedlings to numerous abiotic and biotic environmental factors. Factors such as early spring and autumn frosts, dense forest canopy, thick layers of litter, dense herbaceous cover in clearings, and soil overheating during summer dry periods significantly impact seed germination (Ostojić and Dinić 2009). Arguments explaining the observed failure in the natural regeneration of Serbian spruce are primarily attributed to frequent fires (Aleksić et al. 2022), limited availability of suitable habitats, and strong competition with other species (Tucic and Stojković 2001; Ostojić 2005). Recent studies also suggest possible dispersal limitations due to poor genetic connectivity, even at small spatial scales (Aleksić and Geburek 2010; Mataruga et al. 2020). However, genetic diversity monitoring between two generations of Serbian spruce did not reveal negative effects on the genetic distribution of the young generation, despite only 25% of adult trees from the same population contributing to its formation, and 66% of pollen arriving from unknown sources (Aleksić et al. 2022). These findings underscore the interdependence between the number of adult trees and seed quality, which forms the basis for the poor natural regeneration and survival of this species in critical habitats. The weak germination of seeds from endangered populations with a small number of adult trees further supports the existence of mechanisms to prevent inbreeding in this species (Mataruga and Milanović 2020). Conclusion There are significant differences in the properties of cones and seeds, as well as the characteristics of seed germination among the tested trees and populations of Serbian's spruce (seven natural and one urban population). Seed germination was quite low and significantly lower compared to earlier results, which can be attributed to the climatic conditions that prevailed during the harvest year, but also to the fact that for the first time seeds were collected and tested from endangered populations with a very small number of adult trees. The biological characteristics of seeds, primarily small seeds and small absolute mass, as well as the ambient environment of the habitat where populations of this species survive, cause a significant loss of viable seeds and seriously affect the regeneration and maintenance of populations. This may be one of the main reasons why Serbian spruce is endangered in natural habitats, but thrives well in all others. The number of trees and their spatial arrangement is an important property in defining the size of cones and seeds, as well as the germination itself, that is, the quality of seeds at the population level. It is precisely the threatened populations with the smallest number of trees, as well as the populations with spatially distant trees, that have seeds with the least germination. This is in favor of the existence of a natural mechanism of blocking self-fertilization (inbreeding) in this species. Therefore, in order to protect the surviving populations and enable them to recover and spread, attention should be paid to the protection and recovery of plant communities and habitats of this species. At the same time, translocation and conservation can be carried out in areas with better preserved vegetation, higher humidity, flatter terrain and loose soil in their distribution areas, thus increasing the efficiency of restoration and conservation of the gene pool of this species. Declarations Acknowledgment The authors thank the Ministry of Agriculture, Forestry and Water Management in the Government of the Republic of Srpska for financial support; the Public Enterprise "Šume Republike Srpske" for logistical support in the field, and the representatives of the company "Arborist Banja Luka" for the cooperation and successful implementation of the cone collection. Author contributions Milan Mataruga and Branislav Cvjetković contributed to the study conception, design material preparation and data collection. Statistic analysis were performed by Borut Bosančić. The first draft of the manuscript was written by Milan Mataruga and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. References Aleksić MJ, Geburek T (2010) Mitochondrial DNA reveals complex genetic structuring in a stenoendemic conifer Picea omorika [(Panč.) Purk.] caused by its long persistence within the refugial Balkan region. 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Biological Conservation, 146(1):1- 3 Moran EV, Clark JS (2012) Causes and consequences of unequal seedling production in forest trees: a case study in red oaks. Ecology 93 (5):1082–1094 Ostojić D (2005) Ekološki činioci prirodnog održavanja i obnove cenopopulacija Pančićeve omorike u NP Tara. (Dissertation, Faculty of Forestry, University of Belgrade (in Serbian with English Abstract). Ostojić D, Dinić A (2009) Eksperimentalna fitocenološka ispitivanja prirodnog obnavljanja omorike ( Picea omorika /Pančić/Purkynĕ) u Nacionalnom Parku Tara, Šumarstvo, 23-35 Parker WC, Noland TL, Morneault AE (2006) The effects of seed mass on germination, seedling emergence, and early seedling growth of eastern white pine ( Pinus strobus L.). New For. 32:33-9. Parry M, Parry ML, Canziani O, Palutikof J, Van der Linden P, Hanson C (eds) (2007) Climate change 2007-impacts, adaptation and vulnerability: working group II contribution to the fourth assessment report of the IPCC, vol 4. Cambridge University Press, Cambridge. Pintarić K (1956) Kakav uticaj ima močenje sjemena Pančićeve omorike na proces klijanja. Narodni šumar. Sarajevo. Br. 9-12:378-380 Pintarić K (1957) Uticaj starosti sjemena i djelovanje svjetla na proces klijanja kod sjemena Pančićeve omorike Picea omorika Panč. Radovi Poljoprivredno-šumarskog fakulteta Univerziteta u Sarajevu. God II, Br. 2:89-104 Pintarić K (1969) Sjetva sjemena Pančićeve omorike ( Picea omorica Panch.) u šumskom rasadniku. Narodni šumar: “20 godina industrije za preradu drveta Bosne i Hercegovine (1945-1965)”: 467-470 Pintarić K (1970) Konzerviranje sjemena Pančićeve omorike ( Picea omorika Panč.) u hermetički zatvorenim posudama i uticaj starosti na klijavost. Šumarstvo 24:3–12. Seppälä R, Buck A, Katila P (2009) Adaptation of forests and people to climate change. IUFRO world series volume 22, Helsinki, p:224 Simons AM, Johnston MO (2000) Variation in seed traits of Lobelia inflata (Campanulaceae): sources and fitness consequences. Am J Bot 87:124–132 Souza ML, Fagundes M (2014) Seed size as key factor in germination and seedling development of Copaifera langsdorffii (Fabaceae). Am J Plant Sci 5:2566–2573 Tíscar P, Lucas M (2010) Seed mass variation, germination time and seedling performance in a population of Pinus nigra subsp. salzamannii . For Syst 19:344–353 Tucic B, Stojkovic B (2001) Shade avoidance syndrome in Picea omorika seedlings: a growth-room experiment. J. Evol. Biol. 14 (3):444–455. https://doi.org/10.1046/ j.1420-9101.2001.00291.x Tucović A, Isajev V (1985) Spontana i indukovana varijabilnost klijavaca omorike iz semenskog objekta „Bela zemlja“ u regionu Titovo Užice. Glasnik Šumarskog fakulteta, Serija A, Šumarstvo 64:47-65 Upadhaya K, Pandey H.N, Law P.S. (2007) The effect of seed mass on germination, seedling survival and growth in Prunus jenkinsii Hook.f. & Thoms. Turk. J. Bot. 31:31–36 Vaughton G, Ramsey M (1998) Sources and consequences of seed mass variation in Banksia marginata (Proteaceae). J Ecol 86:563–573 Wyse SV, Hulme PE (2021) Limited evidence for a consistent seed mass-dispersal trade-off in wind-dispersed pines. J. Ecol. 109, 284–293. https://doi.org/10.1111/1365-2745.13477 Xu J, Xiao P, Li T. et al. (2022) Research Progress on endangered plants: a bibliometric analysis. Biodivers Conserv 31:1125–1147. https://doi.org/10.1007/s10531-022-02392-y Xu Y, Zang R (2023) Conservation of rare and endangered plant species in China. iScience, 26, 106008. https://doi.org/10.1016/j.isci.2023.106008 Yates CJ, Broadhurst LM (2002) Assessing Limitations on Population Growth in Two Critically Endangered Acacia Taxa, Biological Conservation, Vol. 108, No. 1:13-26. doi:10.1016/S0006-3207(02)00084-8 Younginger BS, Sirova D, Cruzan MB, Ballhorn DJ (2017) Is biomass a reliable estimate of plant fitness? Appl. Plant Sci. 5 (2):1600094. https://doi.org/10.3732/apps.160009410.3732/apps.1600094.s1. Additional Declarations No competing interests reported. Supplementary Files AppendixTable1.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4754725","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":337606458,"identity":"6bd00baf-f114-4aae-8a81-11bfe9388e36","order_by":0,"name":"Milan Mataruga","email":"data:image/png;base64,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","orcid":"","institution":"University of Banja Luka","correspondingAuthor":true,"prefix":"","firstName":"Milan","middleName":"","lastName":"Mataruga","suffix":""},{"id":337606459,"identity":"711ad18d-27b3-4869-8a79-92c1c521cb33","order_by":1,"name":"Borut Bosančić","email":"","orcid":"","institution":"University of Banja Luka","correspondingAuthor":false,"prefix":"","firstName":"Borut","middleName":"","lastName":"Bosančić","suffix":""},{"id":337606462,"identity":"e72b29c0-ad43-403c-b931-33fb37c05e5a","order_by":2,"name":"Branislav Cvjetković","email":"","orcid":"","institution":"University of Banja Luka","correspondingAuthor":false,"prefix":"","firstName":"Branislav","middleName":"","lastName":"Cvjetković","suffix":""}],"badges":[],"createdAt":"2024-07-17 08:30:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4754725/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4754725/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":62138781,"identity":"5f5661b3-ba63-471e-b757-478763995b58","added_by":"auto","created_at":"2024-08-09 16:48:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":231577,"visible":true,"origin":"","legend":"\u003cp\u003eSpatial distribution of populations in BiH\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4754725/v1/90cdad075fa03b671d7620f0.png"},{"id":62138784,"identity":"a45e379d-47ca-4d16-acbf-8c1dac8fcc07","added_by":"auto","created_at":"2024-08-09 16:48:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":148576,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of test trees based on cone mass, length, and width\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4754725/v1/d98afe85833d73950b3da8a1.png"},{"id":62138779,"identity":"2b45b867-9b20-42ef-a822-8b12eec44faf","added_by":"auto","created_at":"2024-08-09 16:48:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":58613,"visible":true,"origin":"","legend":"\u003cp\u003eSeed Germination Dynamics Across Tested Populations [%]\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4754725/v1/92c2cfa177b8cfbd44d18eca.png"},{"id":62140393,"identity":"72ac3a69-da09-4618-b9cb-8aad90b94377","added_by":"auto","created_at":"2024-08-09 17:04:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":21675,"visible":true,"origin":"","legend":"\u003cp\u003eNumber of germinated, full but non-germinated, and empty seeds [%]\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4754725/v1/9151bb5a2a01ef0ac467bad7.png"},{"id":62140394,"identity":"0b09e13f-1601-42e6-afd9-796127f3416d","added_by":"auto","created_at":"2024-08-09 17:04:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":127565,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal vomponents analysis for all tested traits\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4754725/v1/8e3742a78ee61c261b23b86e.png"},{"id":67135948,"identity":"49afd365-31fa-4f54-8a62-d66be8b94914","added_by":"auto","created_at":"2024-10-21 13:39:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1011986,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4754725/v1/b20967e3-c9b5-42d6-bf7b-27934a457146.pdf"},{"id":62139842,"identity":"d964c0cd-bf58-43f3-96e7-d8a09c59d3d0","added_by":"auto","created_at":"2024-08-09 16:56:26","extension":"docx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":45754,"visible":true,"origin":"","legend":"","description":"","filename":"AppendixTable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4754725/v1/b7b9acb52b2d24c33b5936da.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Characteristics of the seeds and cones of Serbian spruce - indicators of species endangerment","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe survival, or more precisely, the regeneration of a plant species involves seed production and dispersal, germination, and the successful development of seedlings. Each species has its unique germination requirements, which can be considered adaptations for maximum survival in an unstable and unpredictable environment (Fenner 1985; Iralu et al. 2019). Seed germination and seedling development are critical phases in the life cycle that determine the population dynamics of a species and ultimately influence community development, structure, and survival (Chen and Xie 2007; Baeten et al. 2009; Grossnickle and Ivetić 2017). This phase is characterized by exceptionally high mortality rates and intense natural selection (Leck et al. 2008; Grossnickle 2018). Consequently, the seedling phase is often considered a \"bottleneck\" in the life history of a species (Kolb and Barsch 2010; Grossnickle 2016).\u003c/p\u003e \u003cp\u003eA significant number of plant species worldwide are becoming endangered because processes such as seed dispersal, germination, and seedling development and survival are hindered or limited (Colin and Linda 2002; Gulias et al. 2004). The Intergovernmental Panel on Climate Change (IPCC) estimates that roughly 20\u0026ndash;30 percent of vascular plants are at risk of extinction due to a temperature increase of 2\u0026ndash;3\u0026deg;C (Parry et al. 2007). Even modest losses in biodiversity could cause consequential changes in ecosystem services (Sepp\u0026auml;l\u0026auml; et al. 2009). Studying the causes of population decline in endangered plant species, their ecological adaptability, and their ability to avoid threats, followed by formulating effective measures to protect their gene pool and population sizes, has become a crucial issue in biodiversity conservation research worldwide (Fern\u0026aacute;ndez and Tapias 2022; Xu et al. 2022; Xu and Zang 2023).\u003c/p\u003e \u003cp\u003eCertainly, the emergence, survival, and final establishment of seedlings are largely influenced by habitat characteristics (Bazzaz 1991; Mataruga et al. 2012), a phenomenon particularly pronounced in the Serbian spruce (Pintarić 1969; Dinić 1990). Species like the Serbian spruce, with small populations and unique genetic constitutions (Mataruga et al. 2020) that exchange genes only through pollen (Aleksić et al. 2022), are particularly vulnerable to extinction due to reduced fitness resulting from inbreeding depression and random demographic and ecological effects (Hensen and Wesche 2006).Today, the importance of contemporary ecological factors limiting the growth of endangered plant species populations is poorly understood, and there is limited knowledge about the management actions required for their maintenance. Consequently, there are few scientific guidelines regarding which activities should be prioritized. For conservation purposes, it is crucial to understand the seed germination and seedling formation traits that limit endangered species in their spread, increase in numbers, or survival even at low population sizes (Yates and Broadhurst 2002).\u003c/p\u003e \u003cp\u003eThe Serbian spruce (\u003cem\u003ePicea omorika\u003c/em\u003e /Pančić/Purkyně) is a tertiary relict found only in the mid-course of the Drina River canyon, within a narrow range from 19\u0026deg;09' \u0026minus;\u0026thinsp;44\u0026deg;07' N (Tisovljak site) to 19\u0026deg;46' \u0026minus;\u0026thinsp;43\u0026deg;20' N (Mileševa site). All sites are located at altitudes ranging from 750 to 1550 meters on very steep, north-facing slopes (Mataruga and Milanović 2020). This species is categorized as endangered by the IUCN (Mataruga et al. 2011) and is protected by the governments of Bosnia and Herzegovina and the Republic of Serbia. The surviving populations are distributed in a fragmented landscape in very small sizes, often comprising only a few mature trees, typically several dozen and rarely around a thousand individuals. The problem of natural regeneration in these populations is recognized, with young seedlings almost non-existent unless a catastrophic event such as a fire, avalanche, or landslide occurs. Genetic and developmental characteristics have been studied to uncover the causes of its endangerment (Aleksić and Geburek 2010; Mataruga et al. 2020), with slow natural regeneration being the main limitation for its survival (Mataruga and Milanović 2020). The seed germination and seedling formation traits remain unclear concerning processes of regeneration, stability, migration, and distribution. The quality aspects and the impact of different treatments on seed germination have received little attention in this species so far (Cvjetković 2011).\u003c/p\u003e"},{"header":"Object Research and Methodology","content":"\u003cp\u003eIn 2022, an abundant yield of Serbian spruce cones was observed in a significant number of natural populations. Cones were collected in the autumn of 2022 by climbing trees, ensuring the identification of each maternal (test) tree \u0026ndash; half sib lines. Seeds were collected from a total of 111 trees across seven natural and one urban population. For each tree, coordinates were recorded, basic dendrometric data were taken (diameter, height, crown width, and presence of any abiotic damage), and the trees were marked in the field. Seven spatially isolated natural populations: Tisovljak, Panjak, Tesla, Veliki Stolac, Grad, Šarena bukva, and Radomišlja were included out of the 26 described in Bosnia and Herzegovina. The most endangered and habitat-specific populations with cone yield were chosen following previous recommendations (Mataruga and Milanović, 2020). In early spring 2023, cones were also collected from mature trees in the urban area of Banja Luka city. Spatial distribution (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and basic data on the populations from which seeds were collected are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, and the coordinates of each test tree are in Appendix Table\u0026nbsp;1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eImmediately after collection, the total mass of all collected fresh cones from each tree was measured, followed by the measurement of mass, length, and diameter at the widest part for 12 randomly selected cones from each tree. The cones were then placed in a BCC drying chamber at \u0026plusmn;\u0026thinsp;48\u0026deg;C for 72 hours. After drying, the seeds were extracted from the cones, and the dry mass of the cones and the dry mass of the seeds were measured.\u003c/p\u003e \u003cp\u003eAfter manual seed deburring, impurities were separated from the seeds using the BCC gravity separator. Seed purity was determined according to ISTA guidelines (Gordon et al. 1991). Following this, 4 x 100 seeds were weighed to calculate the absolute mass (mass of 1.000 seeds) and the number of seeds per kilogram for each test tree. Seed purity, absolute mass, and the number of seeds per kilogram were analyzed using samples from five randomly selected test trees in each originating natural population (a total of 35 test trees). The seeds were then stored in a refrigerator at 4\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C.\u003c/p\u003e \u003cp\u003eIn the spring of 2023 (specifically from April 11 to May 2, 2023), the seeds were placed on a germination table, known as the \"Copenhagen table\" or \"Jacobsen table\" (Germinationtable GR10), which provides a constant temperature and accurately controlled moisture level, ensuring all seed samples receive the same treatment. Four sets of 100 seeds were placed on filter paper in constant contact with water at a temperature of 21\u0026deg;C. A total of 101 trees out of 111 were tested, as not all trees from which cones were collected had sufficient seeds for testing. The number of normally germinated seeds was counted on days 7, 14, and 21 from the start of the test. On the final day, the number of normally germinated seeds, full but ungerminated seeds, and empty seeds were recorded. Seeds were considered normally germinated when the root had broken through the seed coat and grown to half the seed's length.\u003c/p\u003e \u003cp\u003eFour parameters: germination percentage (GP), germination energy (GE), number of empty seeds (ES), and number of full but ungerminated seeds (FS) were calculated as follows:\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eGP (%) = (total number of germinated seeds on the 21st day \u0026times; 100) / total number of seeds set for germination\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eGE (%) = (number of seeds germinated on the 7th day \u0026times; 100) / total number of seeds set for germination\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eES (%) = (total number of empty seeds \u0026times; 100) / total number of seeds set for germination\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eFS (%) = (number of full but ungerminated seeds \u0026times; 100) / total number of seeds set for germination\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eFor statistical analyses, Microsoft Excel Version 16.83 for Mac was used; descriptive statistics were performed using Statistica 7.0. Analysis of variance (ANOVA) and Duncan's test were employed to demonstrate the significance of differences, with significance levels set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.01. The interdependence of seed traits (size, mass, and moisture) and germination dynamics, both within and between populations, was assessed using Principal Component Analysis (PCA) in the R program.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCharacteristics of locations where cones were collected\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePopulation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLongitude E\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLatitude N\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAltitude [m]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eArea [ha]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eEstimated number of adult trees [pieces]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eOffspring\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNumber of trees from which seeds were collected [pieces]\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTisovljak\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e19,091117\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e44,072907\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e980\u0026ndash;1080\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2,6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eRare\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eŠarena bukva\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e19,192886\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e44,019238\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1000\u0026ndash;1090\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1,0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eRare\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGrad\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e19,219963\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e44,005645\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1100\u0026ndash;1220\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4,4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eRare\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePanjak\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e19,149446\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e43,998802\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1270\u0026ndash;1330\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0,6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTesla\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e19,134806\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e43,927498\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e970\u0026ndash;1110\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2,6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSporadic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eV. Stolac\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e19,282496\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e43,922926\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1050\u0026ndash;1580\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e43,8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSporadic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRadomišlja\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e18,606057\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e43,459431\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e850\u0026ndash;1395\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e44,1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSporadic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBanja Luka\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e17,198876\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e44,764852\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e160\u0026ndash;190\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMass of Cones and Seeds\u003c/h2\u003e \u003cp\u003eOn average, 840,34 grams of cones (or 747,03 grams in dry weight) were collected from a single tree, yielding 28,34 grams of seeds with wings and 14,06 grams without wings (see Appendix Table\u0026nbsp;1). The smallest amount of cones was collected from tree Veliki Stolac_16\u0026ndash;68,16 grams fresh weight (or 59,18 grams in dry weight), and the largest amount from tree Radomišlja_3\u0026ndash;1.985,54 grams in fresh weight (or 1.741,54 grams in dry weight). The highest amount of seeds was extracted from Tisovljak_1 (82,08 grams with wings or 48,22 grams without wings), and from Radomišlja_3 (69,32 grams with wings or 50,21 grams without wings).\u003c/p\u003e \u003cp\u003eOn average, the smallest seeds were collected in the Tesla and Panjak populations, with more than 600.000 seeds per kilogram, while the largest were at Veliki Stolac (378.000 seeds per kilogram) and slightly over 400.000 seeds per kilogram at the Grad, Šarena Bukva, and Tisovljak locations (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In the best-case scenario, the highest production of seeds per tree was recorded for Radomišlja_1, with approximately 21.000 seeds. This number was obtained by multiplying the absolute mass of a single seed (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) by the mass of seeds obtained from the tree (21.877\u0026thinsp;=\u0026thinsp;435.714 x 0,05021). It is also important to note that a significant number of trees did not yield any seeds after cone extraction (cones remained closed after several days of drying), such as Tesla_1, 7, 8, 9, 10; Panjak_1, 5, 6, 9 (see Appendix Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eNumber of seeds per kilogram\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePopulation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGrad\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eŠ. Bukva\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTesla\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePanjak\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRadomišlja\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eTisovljak\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eV. Stolac\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eAverage\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber of seeds per kg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e407.576\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e406.722\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e621.947\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e600.446\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e435.714\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e433.034\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e378.716\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e460.557\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eMorphometric Properties of Cones\u003c/h2\u003e \u003cp\u003eThe smallest cones were found in the Tesla and Panjak populations (2,86 and 2,61 grams, respectively), while the largest cones were observed in the Grad population (5,27 grams). Banja Luka stood out with longer cones (46,9 mm), whereas the cones with the largest diameter were measured in the Radomišlja population (17,80 mm). Cone size varied significantly even among test trees. For instance, the differences in cone mass were up to 9 times greater for the Šarena Bukva_4 tree (9,51 grams) compared to the Panjak_9 tree (1,1 grams). Cone lengths ranged from 20,65 mm (Panjak_9) to 57,65 mm (Banja Luka_21), and diameters ranged from 11,24 mm (Panjak_9) to 20,29 mm (Banja Luka_21).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMean values and standard deviation for cone properties\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePopulation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMass [gr]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLength [mm]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDiameter in the widest part [mm]\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTisovljak\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4,38\u003csup\u003eb\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;2,68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e37,97\u003csup\u003eb\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;4,62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15,98\u003csup\u003ec\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1,33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGrad\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5,27\u003csup\u003ed\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1,54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e39,96\u003csup\u003ec\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;5,98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16,58\u003csup\u003ed\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1,41\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eŠ.bukva\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4,60\u003csup\u003ec\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;2,01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e38,50\u003csup\u003eb\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;6,02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16,15\u003csup\u003ec\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;2,07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTesla\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2,86\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1,21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30,72\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;6,10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14,25\u003csup\u003eb\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1,42\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePanjak\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2,61\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0,98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29,78\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;5,56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13,75\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1,60\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eV.Stolac\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4,72\u003csup\u003ec\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1,32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e38,45\u003csup\u003eb\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;4,27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16,10\u003csup\u003ec\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1,46\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB.Luka\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4,89\u003csup\u003ec\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1,83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e46,90\u003csup\u003ee\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;7,19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17,13\u003csup\u003ee\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;2,15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRadomišlja\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4,82\u003csup\u003ec\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1,20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e43,38\u003csup\u003ed\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;5,01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17,80\u003csup\u003ef\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1,48\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAll populations\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4,37\u0026thinsp;\u0026plusmn;\u0026thinsp;1,68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e39,26\u0026thinsp;\u0026plusmn;\u0026thinsp;7,81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16,10\u0026thinsp;\u0026plusmn;\u0026thinsp;2,01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF\u003csub\u003epop\u003c/sub\u003e,p\u003csub\u003epop\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e58.12, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e169.01, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e83.97, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eComprehensive analysis of cone characteristics based on measured mass, length, and width reveals notable differences among populations, as summarized in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e depicts the distribution of test trees across these metrics. The population of Banja Luka stands out with longer cones but smaller mass compared to other populations. Conversely, the Panjak and Tesla populations consistently exhibited smaller cones across all measured characteristics.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eGermination Energy and Germination Rate\u003c/h2\u003e \u003cp\u003eGermination analysis revealed varying germination rates and energies across different populations of Serbian spruce. The Tesla and Panjak populations exhibited lower germination rates and energies, while the Veliki Stolac population showed the highest values in both parameters. Notably, the Banja Luka population displayed lower germination rates compared to others. Analysis of variance and Duncan's test confirmed significant differences among populations, grouping them into four homogeneous categories (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe lower germination rates in the Tesla and Panjak populations can be attributed to a higher percentage of empty seeds, although differences in the number of full but non-germinated seeds were relatively small across all populations, averaging 16,12% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These populations also have fewer mature trees compared to others (see Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Duncan's test confirmed significant differences, aligning with observed germination rate groupings.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe lowest germination rate recorded was 4% in tree Panjak_4, whereas the highest was 100% in V. Stolac_11. Tesla_11 had the highest percentage of empty seeds at 82%, whereas 11 test trees had 0% empty seeds (see Appendix Table\u0026nbsp;1).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003ePrincipal Components Analysis (PCA) for Measured Traits\u003c/h2\u003e \u003cp\u003eThis analysis provides a comprehensive overview of the grouped and dispersed patterns among populations based on significant measured characteristics (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Populations primarily cluster along PC1, which explains 31,63% of the total variation and is characterized by prominent traits related to germination, seed characteristics, and maternal tree traits (see Appendix Table\u0026nbsp;1). In the positive direction of PC1, populations exhibit characteristics such as higher germination rates and energies, correlating positively with traits of maternal trees. Conversely, the negative direction of PC1 highlights characteristics related to empty seeds, negatively correlating with other measured seed traits. This component also shows grouping in the positive direction of measured cone characteristics. The Panjak and Tesla populations are notably situated in the lower left quadrant of the PCA plot, reflecting smaller cones and lower germination energy and rates. Meanwhile, the Banja Luka population stands out in the upper left quadrant, demonstrating distinct cone characteristics compared to other populations.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn forest trees, reproductive dynamics and success are intricately linked to seed or fruit production, which varies significantly across demographic stages and depends heavily on adequate environmental conditions (Younginger et al. 2017; Moran and Clark 2012). Fragmentation and climate change profoundly impact seed quality and dispersal, critical prerequisites for species survival (McConkey et al. 2012). Empirical research underscores the complex relationship between seed size and germination time. Studies have shown positive correlations between seed size and germination percentage and energy (Parker et al. 2006; Jones and Reekie 2006; Upadhaya et al. 2007; Souza and Fagundes 2014; Kaliniewicz et al. 2018), negative correlations (Simons and Johnston 2000; Gomez 2004; Tiscar and Lucas 2010; Hojjat 2011), and instances where no correlation exists (Larson 1963; Vaughton and Ramsey 1998). Serbian spruce populations with smaller seeds, such as those in Tesla and Panjak with over 600.000 seeds per kilogram, exhibit poor germination rates, posing a threat to their already endangered status. This underscores the positive correlation observed between seed size and germination efficiency in Serbian spruce. Larger seeds likely store more nutrients, facilitating rapid germination and early seedling growth (Domic et al. 2020). In conclusion, understanding these relationships is crucial for conservation efforts aimed at preserving Serbian spruce populations. Enhancing seed quality and ensuring favorable germination conditions are essential strategies in mitigating the impact of environmental stressors on this endangered species.\u003c/p\u003e \u003cp\u003eWhen comparing Serbian spruce seeds to those of other species, it becomes evident that while seed shape may be similar across species, significant differences exist in terms of absolute mass and moisture content. These variations are influenced by genetic factors of the parent tree and environmental conditions during seed development (Dech and Maun, 2006; Li and Li, 2016). Wyse and Hulme (2021) have documented a strong relationship between seed mass and dispersal potential in various wind-dispersed \u003cem\u003ePinus\u003c/em\u003e species. Their findings indicate that within species, there are generally no significant differences in seed mass, which may reduce selective pressure. In environments where resources are limited, the allocation of resources towards seed mass may prioritize seedling vigor and survival over dispersal efficiency.\u003c/p\u003e \u003cp\u003ePintarić's studies (1956; 1957; 1969) in the mid-20th century provided foundational insights into the germination behavior of Serbian spruce seeds. His findings indicated exceptionally high germination rates under optimal conditions. Seeds aged one year exhibited germination rates as high as 97\u0026ndash;98%, with germination energy reaching 94% after 7 days and nearly 100% after 10 days of testing. Moreover, seeds stored at room temperature in glass bottles maintained high germination rates over extended periods (Pintarić 1970). Even seeds up to 3 years old maintained around 96% germination, demonstrating robust seed viability under controlled storage conditions. However, germination rates gradually declined with increasing seed age, with 86.9% germination observed at 6.5 years. Thus, Pintarić concluded that proper seed storage can preserve the germination capacity of Serbian spruce seeds over extended periods. However, the origins of the seeds used in Pintarić's studies were not specified, which may impact the generalizability of his findings. This gap highlights the importance of understanding seed provenance in seed germination studies to ensure the relevance of the results to specific populations or genetic lineages of the species.\u003c/p\u003e \u003cp\u003eThe studies by Tucović and Isajev (1985) indeed highlight the significant genetic variability found in Serbian spruce. They explored spontaneous and induced variability in Serbian spruce seedlings from 12 test trees in a 23-year-old planted forest. Their research emphasized significant genetic variability among the analyzed traits of maternal trees or half-sib lines. The findings underscored that genetic variability was far greater than what could be inferred from mere observations of morphological variability in parent trees. Isajev (1987) analyzed the individual variability of several trees from three planted forests of Serbian spruce in Serbia. Morphometric analysis of 1.000 cones harvested in 1981 and 1983 showed the existence of spontaneous individual variability between trees for length, width, and number of grains per cone, while the shape of the cone was significantly less variable. The range of variability for the length of cones was, on average, from 20 to 70 mm, for widths from 10 to 23 mm, while the mean number of grains varied from 20 to 113 per cone. The size of the cone in the same tree varied within 5% between different harvest years, which indicates strong genetic control of this trait (Isajev 1987). The obtained results showed that the Serbian spruce seeds from all three planted forests are characterized by very good quality (average germination rate was 90%).\u003c/p\u003e \u003cp\u003eIn conclusion, while historical studies by Pintarić and subsequent work by Tucović and Isajev have contributed valuable insights into the germination and genetic variability of Serbian spruce, there remains a need for more comprehensive studies that consider the genetic diversity and specific ecological contexts of this endangered species. Understanding these factors is crucial for effective conservation and management strategies, particularly in ensuring the successful propagation and survival of Serbian spruce in natural and managed environments.\u003c/p\u003e \u003cp\u003eCvjetković et al. (2013) tested the germination of seeds collected from 17 trees, specifically: 5 trees from the planted forest, 7 trees from the natural population Veliki Stolac, and 5 trees from the natural population Gostilja. Fresh seeds originating from the planted forest population showed the highest germination rate, averaging 84%. Seeds from the natural populations, germinated on average at 58,57% and 65,15%, respectively, with an average seed dormancy of 8,68 and 9,72 days (retrospective). Seeds stored in a refrigerator at \u0026plusmn;\u0026thinsp;4\u0026deg;C for 6 months showed better germination results by approximately 20% compared to fresh seeds and 28% compared to fungicide (captan)-treated seeds. The higher vigor and faster germination in seeds stored for 6 months can be interpreted as a form of stratification (Cvjetković 2011). These studies indicate significant differences in the germination of seeds depending on whether they were collected in natural or planted forests.\u003c/p\u003e \u003cp\u003eOstojić and Dinić (2009) investigated germination under natural conditions. A series of experiments involving the sowing of Serbian spruce seeds in different habitats revealed poor seed germination and subsequent seedling failure in the first year (Dinić 1988, 1989). The authors attribute these results to the decline in tree numbers at this location - from 40 trees recorded in the early 1950s, only four remain today, as confirmed by later comparative experiments examining seed germination and seedling growth (Dinić 1990). The results highlight the sensitivity of Serbian spruce seeds and seedlings to numerous abiotic and biotic environmental factors. Factors such as early spring and autumn frosts, dense forest canopy, thick layers of litter, dense herbaceous cover in clearings, and soil overheating during summer dry periods significantly impact seed germination (Ostojić and Dinić 2009).\u003c/p\u003e \u003cp\u003eArguments explaining the observed failure in the natural regeneration of Serbian spruce are primarily attributed to frequent fires (Aleksić et al. 2022), limited availability of suitable habitats, and strong competition with other species (Tucic and Stojković 2001; Ostojić 2005). Recent studies also suggest possible dispersal limitations due to poor genetic connectivity, even at small spatial scales (Aleksić and Geburek 2010; Mataruga et al. 2020). However, genetic diversity monitoring between two generations of Serbian spruce did not reveal negative effects on the genetic distribution of the young generation, despite only 25% of adult trees from the same population contributing to its formation, and 66% of pollen arriving from unknown sources (Aleksić et al. 2022). These findings underscore the interdependence between the number of adult trees and seed quality, which forms the basis for the poor natural regeneration and survival of this species in critical habitats. The weak germination of seeds from endangered populations with a small number of adult trees further supports the existence of mechanisms to prevent inbreeding in this species (Mataruga and Milanović 2020).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThere are significant differences in the properties of cones and seeds, as well as the characteristics of seed germination among the tested trees and populations of Serbian's spruce (seven natural and one urban population). Seed germination was quite low and significantly lower compared to earlier results, which can be attributed to the climatic conditions that prevailed during the harvest year, but also to the fact that for the first time seeds were collected and tested from endangered populations with a very small number of adult trees. The biological characteristics of seeds, primarily small seeds and small absolute mass, as well as the ambient environment of the habitat where populations of this species survive, cause a significant loss of viable seeds and seriously affect the regeneration and maintenance of populations. This may be one of the main reasons why Serbian spruce is endangered in natural habitats, but thrives well in all others.\u003c/p\u003e \u003cp\u003eThe number of trees and their spatial arrangement is an important property in defining the size of cones and seeds, as well as the germination itself, that is, the quality of seeds at the population level. It is precisely the threatened populations with the smallest number of trees, as well as the populations with spatially distant trees, that have seeds with the least germination. This is in favor of the existence of a natural mechanism of blocking self-fertilization (inbreeding) in this species.\u003c/p\u003e \u003cp\u003eTherefore, in order to protect the surviving populations and enable them to recover and spread, attention should be paid to the protection and recovery of plant communities and habitats of this species. At the same time, translocation and conservation can be carried out in areas with better preserved vegetation, higher humidity, flatter terrain and loose soil in their distribution areas, thus increasing the efficiency of restoration and conservation of the gene pool of this species.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank the Ministry of Agriculture, Forestry and Water Management in the Government of the Republic of Srpska for financial support; the Public Enterprise \"Šume Republike Srpske\" for logistical support in the field, and the representatives of the company \"Arborist Banja Luka\" for the cooperation and successful implementation of the cone collection.\u0026nbsp;\u003c/p\u003e\n\u003ch4\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/h4\u003e\n\u003cp\u003eMilan Mataruga and Branislav Cvjetković contributed to the study conception, design material preparation and data collection. Statistic analysis were performed by Borut Bosančić. The first draft of the manuscript was written by Milan Mataruga and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e"},{"header":"References ","content":"\u003col\u003e\n\u003cli\u003eAleksić MJ, Geburek T (2010) Mitochondrial DNA reveals complex genetic structuring in a stenoendemic conifer \u003cem\u003ePicea omorika \u003c/em\u003e[(Panč.) Purk.] caused by its long persistence within the refugial Balkan region. Plant Syst Evol 285:1\u0026ndash;11\u003c/li\u003e\n\u003cli\u003eAleksić MJ, Mataruga M, Daničić V, Cvjetković B, Milanović Đ, Vendramin GG, Avanzi C, Piotti A (2022) High pollen immigration but no gene flow via-seed into a Genetic Conservation Unit of the endangered \u003cem\u003ePicea omorika\u003c/em\u003e after disturbance, Forest Ecology and Management, Volume 510:1-12. https://doi.org/10.1016/j.foreco.2022.120115\u003c/li\u003e\n\u003cli\u003eBaeten L, Jacquemyn H, van Calster H, van Beek E, Devlaeminck R, Verheyen K Hermy M (2009) Low Recruitment across Life Stages Partly Accounts for the Slow Colonization of Forest Herbs, Journal of Ecology, Vol. 97, 1:109-117. doi:10.1111/j.1365-2745.2008.01455.x\u003c/li\u003e\n\u003cli\u003eBazzaz FA (1991) Habitat selection in plants. 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Biodivers Conserv 31:1125\u0026ndash;1147. https://doi.org/10.1007/s10531-022-02392-y\u003c/li\u003e\n\u003cli\u003eXu Y, Zang R (2023) Conservation of rare and endangered plant species in China. iScience, 26, 106008. https://doi.org/10.1016/j.isci.2023.106008\u003c/li\u003e\n\u003cli\u003eYates CJ, Broadhurst LM (2002) Assessing Limitations on Population Growth in Two Critically Endangered Acacia Taxa, Biological Conservation, Vol. 108, No. 1:13-26. doi:10.1016/S0006-3207(02)00084-8\u003c/li\u003e\n\u003cli\u003eYounginger BS, Sirova D, Cruzan MB, Ballhorn DJ (2017) Is biomass a reliable estimate of plant fitness? Appl. Plant Sci. 5 (2):1600094. https://doi.org/10.3732/apps.160009410.3732/apps.1600094.s1.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"genetic variability, endangered populations, cones, seeds, germination","lastPublishedDoi":"10.21203/rs.3.rs-4754725/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4754725/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA significant amount of existing research indicates a persistent trend of declining populations and trees of the Serbian spruce (\u003cem\u003ePicea omorika\u003c/em\u003e/Pančić/Purkyně), a tertiary relict, rare, endangered, and protected species. Understanding the characteristics of cones and seeds, along with the analysis of intra- and inter-population variability, is crucial for identifying the reasons for this decline and implementing \u003cem\u003ein situ\u003c/em\u003e and \u003cem\u003eex situ\u003c/em\u003e conservation measures to preserve this species.\u003c/p\u003e \u003cp\u003eDuring 2022/23 (autumn/spring), cones were collected from 111 trees of this species. The trees are distributed across seven natural populations and one urban population in Bosnia and Herzegovina. Immediately after collection, the cones were measured, processed, seeds were extracted, and germination tests were conducted.\u003c/p\u003e \u003cp\u003eSignificant differences among the tested trees and populations were found for all measured cone traits. The germination results indicate very low seed viability, averaging 57,03% for all tested trees, with the percentage of empty seeds at 26,85%. Significant differences in germination were also observed at the population level, ranging from 20,40% to 81,14%, with the percentage of empty seeds ranging from 8,10% to 59,60%.\u003c/p\u003e \u003cp\u003eThe results suggest that endangered populations with a small number of trees have significantly smaller cones, a higher percentage of empty seeds, and lower germination rates. Such a low germination rate in natural populations of Serbian spruce indicates the need for urgent actions to protect this species and assist its natural regeneration (in situ), as well as to establish plantations outside its natural range (ex situ conservation).\u003c/p\u003e","manuscriptTitle":"Characteristics of the seeds and cones of Serbian spruce - indicators of species endangerment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-09 16:48:21","doi":"10.21203/rs.3.rs-4754725/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e83cfb2f-0248-4473-9518-a26b98165887","owner":[],"postedDate":"August 9th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-10-21T13:39:18+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-09 16:48:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4754725","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4754725","identity":"rs-4754725","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}