Cone and seed variability in Serbian spruce – indicators of population endangerment

A significant amount of existing research indicates a persistent trend of declining populations and trees of the Serbian spruce (Picea omorika/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 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).

High levels of genetic diversity, pronounced genetic structure and limitations to gene flow in Serbian spruce, a rare and endangered tree species from the refugial Balkan region, point towards a “one population-one unit” strategy for assembling a network of Genetic Conservation Units (GCUs) for its dynamic conservation. On the other hand, genetic information also permits to prioritize populations for conservation based on their contribution to genetic diversity and differentiation. Serbian spruce, Picea omorika (Panc.) Purk., is a rare, IUCN red-listed European conifer endemic to the Balkan region. Its current rigid conservation (without any intervention allowed in ~ 30 remnant populations) and the extant network of Genetic Conservation Units (four natural populations and three planted stands from the western part of the species range, in the Republic of Srpska, Bosnia and Herzegovina, RS-BH) might be ineffective in preserving the species’ genetic diversity. To facilitate implementation of dynamic conservation of Serbian spruce by re-assessing the number and size of remnant populations in RS-BH and updating genetic knowledge on these understudied western Serbian spruce populations. Comprehensive field survey in RS-BH, genotyping 689 individuals from 14 western populations with ten highly informative nuclear EST-SSRs and analytical methods for prioritizing populations for conservation based on their contribution to the geographical structuring of genetic diversity. The genetic diversity of western Serbian spruce populations (Ae = 2.524, HE = 0.451) is comparable with what was found for eastern ones; they are highly genetically differentiated (Hedrick’s G’ST = 0.186; Jost’s D = 0.097) and comprise ten distinct gene pools. Effective population size is often ≥ 15. As much as 14% of alleles is not preserved in the extant GCUs established in natural populations. Eight populations positively contribute to within-population genetic diversity, four to genetic differentiation, and two are globally important in terms of diversity and differentiation. Although wildfires may contribute to admixture of different gene pools, re-establishment from seeds from extirpated populations has likely prevailed in studied populations. A larger network of GCUs is required for the dynamic conservation of western Serbian spruce populations. A “one population-one unit” strategy, with 14 GCUs, would represent the safest approach to conserve species extant genetic variation in this part of the species range. Nonetheless, a strategy to prioritize populations for conservation based on their contribution to allelic diversity has been put forward. Given the rapid global warming and peculiarities of Serbian spruce distribution, habitat and life history traits, conservation measures based on a rigorously designed GCU network are urgent for its rescue and survival.

Serbian spruce (Picea omorika (Pančić) Purk.) is a rare and endangered tertiary relict and endemic species, with restricted and fragmented natural range in Serbia and Bosnia and Herzegovina, mainly around the mid-course of the Drina river. Since the middle of the 19th century, its natural range declines constantly, followed by a decline in the number of mature individuals. The decline of this forest species is slow and mainly attributed to poor regeneration and low competing ability. Given the foreseen worsening of the climate in forthcoming decades, this decline can only accelerate. In recent years, dieback related to drought has been observed as response to extreme weather events suggesting that Serbian spruce will face difficulties in adapting to climate change within its natural range. However, successful use of Serbian spruce in Central and Northern Europe indicates potentially large adaptive potential of this species which, along with the high genetic variability, outweigh the limited morphological variation, self-fertilization, and limitations related to the restricted natural range in the first place, and, indicates possible directions of migration in the second place. In this paper, current conservation actions are discussed, and strategies for the species survival in a changing environment are suggested. Since migration and adaptation are the least likely responses of this species to climate change, measures such as assisted migration may be the only strategy which will enable persistence of Serbian spruce. Current conservation programs, limited to in-situ actions, need to be supplemented with ex-situ actions and strategies. In the worst case scenario, i.e. for species such as Serbian spruce which are unable to migrate and/or adapt to changing climate, the most suitable sites should be identified and colonized in order to prevent extinction in the near future.

Genetic-physiological approach was used in the study of the quality of Serbian spruce seed (Picea omorika /Pancic/Purkyn?), collected in the populations on the left bank of the river Drina. The seed originated from the three populations that represent the overall ecological and productive conditions of Serbian spruce populations in Bosnia and Herzegovina. Two natural populations were selected: Veliki Stolac and Gostilj, and one planted forest population in Srebrenica. The cones were collected in late autumn 2009 and early spring 2010. They were collected from five trees from Srebrenica and Gostilj, and seven trees from Veliki Stolac. Immediately after they were collected, the cones and seeds were processed and germination tests were done. Germination, germination viability and germination dynamics were studied in three categories of seed treatment: 1) control unit (just processed seed), 2) seed stored for six months at 0-4 ?C, 3) seed stored for seven months at 0-4? C and treated with fungicide after five months of storing. The number of germinated seed was observed and noted on the third, fourth, fifth, seventh, tenth, fourteenth, twenty-first and twenty-eighth day. There were significant differences in germination dynamics at the population level and the level of treatment during the first couple of days of germination test. Fungicide (captan) acted as an inhibitor on seed germination process. The seeds originating from the largest population of Veliki Stolac showed the best response to storing treatment with fungicide related to germination dynamics. The seed originating from Srebrenica andkept at low temperatures 0-4 ?C without treatment with fungicides showed the best result in germination. Regardless of the presence of differences in the dynamics of germination, significant differences in germination regarding different treatments on the last day of the test, were not recorded.

Serbian spruce (Piceaomorika (Pančić) Purk.) is reputedly a self-fertile and morphologically uniform species with a natural distribution restricted to a total area of less than 60 ha in central Yugoslavia. This has led to the conclusion that the species lacks genetic variation. In this study, genetic variation at 19 enzyme loci was investigated in two populations: a natural Yugoslavian population and a cultivated Finnish population. The average expected heterozygosities over 19 loci were 0.13 and 0.15 in the two populations, which are similar to those observed in other conifers. Heritabilities of four seedling traits were estimated on the basis of a progeny trial. They varied from 0.34 to 1.23, with standard deviations of 0.17–0.37. Because of maternal effects, these are probably inflated estimates, but they show that there may be considerable genetic variation in quantitative traits, as well. The observed amount of genetic variation in Serbian spruce is unexpectedly high considering its self-fertility and lack of morphological variation. We conclude that drift due to small population size has not had a large impact on the level of genetic variation in this species The lack of morphological variation observed by many authors is probably a result of selection.

Serbian spruce is an old, relict and currently rare and endangered spruce species particularly susceptible to climate change that attracts a lot of attention in the academic community since its discovery in 1875. Today, Serbian spruce is limited to the area of ~100 km2 localized around the mid-course of the Drina River in the mountainous central Balkans, at the border of Republic of Serbia and Republic of Srpska, Bosnia and Herzegovina. Within this small area, ~30 remnant populations of various sizes are scattered at north-to-northwest orientated slopes of hills. Given the current state of Serbian spruce populations as well as extreme climate events and poor natural regeneration in this species, applied “do not touch” conservation approach is severely questioned, and some alternative protection and in situ and ex situ conservation actions have been put forward. In situ actions should involve: (1) facilitation of natural regeneration by selective removal of individual competitor trees, (2) assisted natural regeneration by planting high quality seedlings throughout the species current natural range or at nearby suitable sites, and (3) assisted natural regeneration by direct seeding. Although in situ actions may provide short-term persistence of Serbian spruce, ex situ actions, i.e., assisted migration, achieved by both assisted range expansion and assisted species migration, are inevitable, since it is very likely that Serbian spruce will disappear from its natural habitats in the near future due to the rapid climate change.

Picea omorika (Pančić) Purk., (Serbian spruce) is a relic, endemic, and vulnerable conifer that remains insufficiently studied to date. To the best of our knowledge, this is the first report on the morpho-anatomical and phytochemical diversity of needles from three populations in Bosnia and Herzegovina. The length of two-year-old needles was measured with a digital caliper. The next six properties were measured based on cross-sections of the needles using a light microscope. An analysis of volatile compounds was carried out using gas chromatography coupled with mass spectrometry (GC-MS) and flame ionization detection (GC-FID). The highest values of needle traits were found in the Viogor population, with the lowest in the Tisovljak population, which was statistically confirmed. There was also a significant difference between needles from Bosnia and Herzegovina and those from Serbia. Bornyl acetate, camphene, limonene, and α-pinene were identified as the major terpene compounds. Multivariate analyses also suggested a tendency toward the separation of the Tisovljak population. A statistical comparison of three Bosnian and Herzegovinian and four Serbian populations (previously studied and published) revealed two distinct groups: (1) three Bosnian populations and the Vranjak population from Serbia, and (2) three populations from Serbia—Štula, Zmajevački Potok, and Mileševka Canyon. The general conclusions are that divergence in needle morpho-anatomy aligns with divergence in needle chemistry and that Bosnian and Herzegovinian populations are distinct from nearly all Serbian populations.

Serbian spruce (Picea omorika /Panc./ Purkyne) is rare and endangered tertiary relict and endemic species in the Balkan Peninsula. Its natural distribution is fragmented and restricted to western Serbia and eastern Bosnia and Herzegovina, mainly in the canyon of middle course of the Drina river. Since the mid-19th century, the number and size of populations have been steadily decreasing accompanied by a decline in the number of mature individuals, endangering natural regeneration is endangered. The objective of this study is to determine variability within Serbian spruce (Picea omorika /Panc./ Purkyne) natural population in the canyon of the Milesevka river on the basis of morphological and anatomical characteristics of needles and, thus, to broaden knowledge on this rare and endangered species. Twigs with needles collected from all mature individuals that represent the population were used as material for the study. The following morphological and anatomical characteristics were measured: NL- needle length; NH - needle height; NW - needle width; CT+EH - cuticle + epidermis height; HH - hypodermis height; VB - vascular bundle diameter; No of SC - number of resin channels; SC diameter - resin channel diameter. The average values of the measured characteristics are: needle length 13.23 mm; needle width 1239.93 ?m; needle height 608.63 ?m; cuticle + epidermis height 11.49; hypodermis height 17.59 ?m; vascular bundle diameter 220.64 ?m; number of resin channels 0.74; resin channel diameter 52.36 ?m. The characteristics with the highest variability are as follows: number of resin channels (161.64%); resin channel diameter (25.35%); hypodermis height (20.79%), while the needle width is a characteristic with the lowest variability (6.39%). The results of the analysis of variance showed very high statistical differences within population for all studied characteristics (p<0.01; ?=0.05). In order to make the results clearer, it is necessary to investigate the genetic diversity and population structure by analyzing various phenotypic characteristics in specially designed experiments (e.g. progeny tests) as well as by analyzing appropriate DNA markers. The studied population must be involved in continuous conservation and breeding processes, especially through appropriate forms of ex situ conservation.

Serbian spruce (Picea omorika /Panc./Purkyne) is a Balkan endemic and Tertiary relict, therefore it has an exceptional significance from the historical-geographical aspect as well as from the aspect of tree im- provement and biodiversity conservation. Pollen was collected from 24 trees in two consecutive years and an- alyzed once a month. Germination of fresh pollen and pollen stored under different temperatures (room tem- perature: 23 ±1°C, +4°C, -15°C and -20°C) by in vitro method on a medium with 10% sucrose was analyzed. Fresh pollen germination was 53.63% and 68.06% for pollen collected in first and second year, respectively. After one year of storage at -20°C pollen germination was 22.36% and 60.72%, respectively. The differences between pollen collection years, temperature treatments, storage periods and individuals were statistically significant. Since pollen germination rates of individual trees between the first and the second year showed weak positive correlation, one could conclude that germination of fresh pollen grains is under the certain in- fluence of environmental conditions. It is determined strong positive correlations between fresh and stored pollen at -20°C from both years of pollen collection.

Serbian spruce (Picea omorika [Pančić] Purk.) is a Balkan endemic coniferous species, the expansion of which is restricted by limited knowledge. This literature review paper compiles findings from 176 scientific papers and presents a summary of research results that pertain to the Serbian spruce potential in general, with a specific focus on European forests from 1951 to 2022. It summarizes the importance of its taxonomy, biological and ecological characteristics, site demands, production and silviculture, risks and pests, as well as the potential of this tree species in relation to global climate change. Serbian spruce is very resistant to the negative effect of air pollution and extreme heat waves compared to other spruce species, especially the most economically important spruce species in Europe– Norway spruce (Picea abies [L.] Karst.). Moreover, its radial growth shows highly balanced annual increments, and the density and technical parameters of the wood are comparable with Norway spruce. On the other hand, the highest weakness may be the limited genetic variability. Despite its rather limited natural range, Serbian spruce can be considered one of the most adaptable spruces to anthropogenic factors and climate change, and a valuable tree species for urban landscapes. Its production potential of wood on acidic, dry and extreme sites makes it attractive for forestry, through its introduction.

Progress in the detection of costs of phenotypic plasticity in plants

Picea omorika (Pančić) Purkyne is a well-known endemic conifer species. However, there have been few morpho-anatomical studies to date. Several years ago, preliminary population studies of P. omorika were published, but with the inclusion of additional populations, it became necessary to confirm whether the southernmost population from Mileševka Canyon, also known as var. vukomanii, (Pavlović and Matović 1994), remains distinct from the others. Two-year-old needles from the lower third of the crown were collected in late autumn from seven natural populations of P. omorika from Serbia and from the border with Bosnia and Herzegovina. The variability of ten morpho-anatomical traits and the cross-sectional anatomy of Serbian spruce needles were examined in seven natural populations in Serbia. Needle length was measured with a digital calliper, and the nine other needle traits, needle width, needle thickness, vascular bundle diameter, resin ducts diameter, number of resin ducts, distance between vascular bundle and resin duct, cuticle and epidermal thickness, hypodermal thickness, and hypodermal width, were examined under a light microscope. For most of the traits studied, the Štula population had the highest values, whereas the Bilo population had the lowest. Needles with triangular sections are most common in the southern part of Mt. Tara, while elliptical and rhomboidal sections are characteristic in populations at the edge of the distribution range in Serbia. Multivariate analyses separated populations into two main groups. Both principal component analysis and discriminant analysis indicated population overlap, while cluster analysis identified three main groups. Multivariate analysis of bioclimatic data and a correlation study of morpho-anatomical traits were also performed. Based on bioclimatic parameters, individuals in the populations from Mileševka and Štula showed the most distinct characteristics. Mantel and partial Mantel tests confirmed no correlation between the complete set of morphological traits and bioclimatic parameters, even after adjusting for regional differences.

This study is the first report on the composition and variability of essential oil in the relic, endemic, and vulnerable tree species Serbian spruce, Picea omorika, in its natural populations. In the needles of 108 trees of four natural populations, 49 components of essential oils were identified. The main compounds were bornyl acetate (29.2%), camphene (18.7%), and alpha-pinene (12.9%). Fourteen additional components had the contents of up to 0.5%: alpha-cadinol (6.1%), limonene (5.8%), santene (3.5%), (E)-hex-2-enal (2.9%), T-cadinol (2.9%), delta-cadinene (2.3%), tricyclene (2.1%), myrcene (1.6%), beta-pinene (1.2%), borneol (0.9%), germacrene D (0.9%), alpha-muurolene (0.6%), and two unidentified compounds. Population IV from Milesevka Canyon had a much higher content of bornyl acetate (42.9%). Populations I-III from Mt. Tara were more abundant in sesquiterpenes (up to 18.2%). The content of bornyl acetate, the multi-variation analyses according to seven selected components, especially the cluster analysis and genetic analysis of alpha-cadinol, which suggested the monogenic type of heredity, showed a clear differentiation of the two geographic areas, the similarity of populations I-III from the area of Mt. Tara, and the separation of the population IV from Milesevka Canyon.

In the research on the variability of the morpho-anatomical properties of the Serbian spruce (Picea omorika /PanČ./ Purkyne), we investigated seven characteristics of 2-year-old needles, collected from four natural populations in Serbia. The hypodermis of the Serbian spruce subject to our study is single layered. The needles sport 0–2 resin ducts, predominantly of the external type (touching the epidermis). The average values were as follows: 13.60 mm (needle length), 1.49 mm (needle width), 0.82 mm (needle thickness), 22.83 μm (cuticle+epidermis thickness), 17.51 μm (hypodermis thickness), 0.74 (number of resin ducts), and 51.82 μm (resin duct diameter). Needle width and thickness were the most variable characteristics. Principle component analysis visualizes an overlapping among the populations in the region of Mt. Tara and a partial separation of the population from Mileševka Canyon. Cluster analysis suggests the biggest differentiation between geographically distant populations (Štula and Mileševka Canyon). All the analyzed characteristics were compared with appropriate literature sources.

BackgroundPrimulina Hance is an emerging model for studying evolutionary divergence, adaptation and speciation of the karst flora. However, phylogenetic relationships within the genus have not been resolved due to low variation detected in the cpDNA regions. Chloroplast genomes can provide important information for phylogenetic and population genetic studies. Recent advances in next-generation sequencing (NGS) techniques greatly facilitate sequencing whole chloroplast genomes for multiple individuals. Consequently, novel strategies for development of highly polymorphic loci for population genetic and phylogenetic studies based on NGS data are needed.MethodsFor development of high polymorphic loci for population genetic and phylogenetic studies, two novel strategies are proposed here. The first protocol develops lineage-specific highly variable markers from the true high variation regions (Con_Seas) across whole cp genomes, instead of traditional noncoding regions. The pipeline has been integrated into a single perl script, and named "Con_Sea_Identification_and_PIC_Calculation". The second method assembles chloroplast fragments (poTs) and sub-super-marker (CpContigs) through our "SACRing" pipeline. This approach can fundamentally alter the strategies used in phylogenetic and population genetic studies based on cp markers, facilitating a transition from traditional Sanger sequencing to RAD-Seq. Both of these scripts are available at https://github.com/scbgfengchao/.ResultsThree complete Primulina chloroplast genomes were assembled from genome survey data, and then two novel strategies were developed to yield highly polymorphic markers. For experimental evaluation of the first protocol, a set of Primulina species were used for PCR amplification. The results showed that these newly developed markers are more variable than traditional ones, and seem to be a better choice for phylogenetic and population studies in Primulina. The second method was also successfully applied in population genetic studies of 21 individuals from three natural populations of Primulina.ConclusionsThese two novel strategies may provide a pathway for similar research in other non-model species. The newly developed high polymorphic loci in this study will promote further the phylogenetic and population genetic studies in Primulina and other genera of the family Gesneriaceae.