Mountain plants adapted to specific environments are increasingly threatened by global warming. A major concern is forced altitudinal shift, habitat fragmentation, disruption of plant-pollinator interactions, or challenges from competitors and invasive species. Numerous studies shed light on these complex dynamics and highlight the need for immediate conservation action, with maintaining the greatest possible genetic diversity in populations as one of the main goals. One such species threatened by climate change that is on the IUCN World Red List is the spring pasqueflower Pulsatilla vernalis (L.) Mill (Ranunculaceae). Our study describes recent advances and applications of nuclear microsatellite markers in the Pulsatilla subgenus, which includes both diploid and tetraploid species. To date, few studies have been conducted on the genetic structure of P. vernalis populations, and no specific markers have been developed for this purpose. In the present study, we attempted to amplify microsatellite loci for diploid P. vernalis using a cross-species procedure originally developed for tetraploid P. vulgaris. Using this procedure, eight polymorphic markers were successfully transferred to P. vernalis. The selected loci were tested on samples collected from relict lowland populations that are nearly extinct in Central Europe. Analysis of 82 individuals from three Polish lowland populations revealed that these markers were highly polymorphic, with two to 13 alleles per locus and observed heterozygosity ranging from 0.037 to 0.654. FIS coefficient values were not statistically significant for any of the loci (p>0.05) and ranged from -0.200 to 0.696. We tested how informative cross-amplified loci distinguished individuals and which loci deviated from Hardy-Weinberg Equilibrium. We conclude that the microsatellite markers tested in this study can be successfully used for population genetic studies of P. vernalis.

Background: Ash dieback, driven by the invasive fungal pathogen Hymenoscyphus fraxineus, has precipitated severe declines in Fraxinus excelsior L. populations across Europe, threatening genetic diversity and ecosystem stability. Methods: This study investigates the interplay between phenotypic vitality and genetic variation in five Polish ash stands using nuclear simple sequence repeat (nSSR) and chloroplast DNA (cpDNA) markers. Vitality assessments of 186 trees across three reserves (from Białowieża Primeval Forest and Wolica Reserve) were conducted. Results: Vitality assessments revealed a slight predominance of dying individuals (36%, 3rd degree of Roloff classification). Nuclear analyses indicated moderate to high diversity (mean HE = 0.826), significant inbreeding (FIS = 0.178, p < 0.001), and low inter-population differentiation (FST = 0.044) among all five stands. Chloroplast markers showed elevated differentiation (ΦST = 0.228, p < 0.0001), reflecting phylogeographic structure. Vitality degrees assessed in three chosen populations (Browsk FD, Hajnówka FD, and Chojnów FD) exhibited negligible genetic differentiation (nSSR FST = 0.009; cpDNA ΦST = 0.003), suggesting gene flow mitigates pathogen-induced selection. Bayesian clustering (STRUCTURE, K = 3) revealed admixture with distinct genotypes in dying trees, potentially linked to susceptibility. Conclusions: These findings underscore the resilience of ash genetic pools and advocate for selective breeding in nurseries to prevent the spread of dieback, prioritizing resistant genotypes for conservation.

Cockchafers Melolontha spp. are polyphagous root pests and outbreak species of considerable economic importance to forestry in Europe. Knowledge of genetic diversity within and between cockchafer populations is largely unexplored but could lead to more effective monitoring and management strategies of these pests. In this study, we fill several gaps in the knowledge of the genetic population structure of Melolontha melolontha and Melolontha hippocastani. Using microsatellite loci and over 200 individuals per species, we examined the genetic structure of Melolontha spp. populations collected from three primary outbreak areas in central and southeastern Poland. We demonstrated that our focal species share some common genetic patterns, such as considerable genetic diversity within subpopulations (within sampling sites) with minor effects of past bottlenecks possibly masked by current population sizes. The estimates of effective population sizes, although subject to wide credibility intervals, indicated a large number of individuals contributing to further generations of the population. The migration rates exceed the threshold of one per generation required for gene flow and maintenance of population equilibrium, indicating limited impact from forest management practices on either cockchafer species. While genetic differentiation was observed in M. melolontha populations, supported by various indices (fixation index, principal coordinate analysis, assignment tests), the M. hippocastani populations were genetically similar. This disparity may hint at the potential historical divergence between these species. In this aspect, more detailed phylogeographic and population genetic studies covering a large area of the Palaearctic distributions of both species are required. Our findings constitute one of the initial efforts aimed at delineating the genetic diversity, differentiation, population structure, gene flow, and migration patterns within Melolontha spp. This exploration is especially significant for M. hippocastani, given its completely unexplored genetic population structure.

Tuber wenchuanense ascomata (Ascomycota, Pezizales), a species originally described from Sichuan (China), were found in the Tatra Mountains in southern Poland. The purpose of this work was to (i) report and assess the first case of the holarctic natural distribution of a Tuber species, (ii) amend the original description of the species, (iii) summarize data on its host plants and (iv) describe its ectomycorrhiza. Specimens of Tuber wenchuanense from the Tatra Mountains were studied morphologically and molecularly. The ectomycorrhiza of this truffle with Picea abies was described for the first time. The distribution of T. wenchuanense, which is reconstructed based on sequences deposited in the publicly available nucleotide sequence databases, makes it the first holarctic Tuber species and the one with the northernmost habitat. In fact, its habitat is confined mainly to mountain coniferous forests and alpine and arctic tundra; although, according to known observations, the fruiting bodies of T. wenchuanense can be produced only under conifers. Based on the sequences of the internal transcribed spacer, this species appears to have low genetic variability over the entire distribution range. The phylogenetic tree showed that some of the unidentified phylotypes from the Rufum clade found by other researchers belong to T. wenchuanense. The ecological implications of these findings are discussed.

The effects of climate change are being felt on many levels. One of them are forests and the water conditions in their area. There has been a significant decline in the water table, leading to changes in forest ecosystems. Therefore, it is important to take measures to reduce the negative impact on water conditions. Tools such as mathematical models of groundwater flow can support this action. This manuscript presents the possibilities of applying the Modflow model in forest areas. The analysis confirms that the model can be successfully applied. It was shown that the model is suitable for accurate determination of changes in the amount of water in the soil. In the studied example, the increase in groundwater retention when 50 cm high dams were used was about 27% compared to the variant without dams. When 100 cm high dams were used, the increase in water retention was about 38%.

Biodiversity within composting systems involves a variety of microorganisms including nematodes. In the research, nematode populations were monitored within six simultaneously operating composting processes. These processes involved varying proportions of feedstock materials. The primary objective was to evaluate the consistency of nematode community succession patterns across the composting processes over a time of 3 months. During the study, samples were taken every month to isolate nematodes, determine the population density of the five trophic groups (per genus) and determine the dominant nematode species. It was shown that the bacterial-feeding community maintained dominance, while the fungus-feeding nematodes gradually increased in dominance as the maturation process progressed. The presence of predatory nematodes Mononchoides which were initially absent, along with the total absence of parasitic nematodes in the late stages of waste stabilization, serves as strong evidence for the reliable evaluation of the biodegradable waste processing level. Based on the obtained results, it is evident that the succession of nematode communities holds promise as a reliable method for evaluating compost maturity.

In 2017, the presence of the fly Suillia gigantea (Meigen, 1830) was noted in Poland, after many years of research related to the ecology of insects associated with the fruiting bodies of hypogeous fungi. Finally, in 2020, after further studies, the distribution of the truffle fly in Poland was confirmed. Six adults were reared from larvae inhabiting the fruiting bodies of Burgundy truffle (Tuber aestivum Vittad. (1831)). The fungi were harvested in southern Poland. Morphological and genetic analyses of the insect specimens were performed. For the first time, the DNA sequence of this fly species was isolated. This is the first record of S. gigantea in Poland, although it has already been reported from neighbouring countries. The development of S. gigantea, also known as the truffle fly, is associated with hypogeous fungi, mainly belonging to the genus Tuber P. Micheli ex F.H. Wigg. (1780). The diptera larvae develop inside the fruiting bodies. This type of damage causes great losses in truffle production. Containment of these losses is of great interest to the truffle industry. Knowledge of truffle-inhabiting insects is crucial for the development of effective methods to protect truffle crops in Europe.

Abstract Cockchafers Melolontha spp. are polyphagous root pests and outbreak species of considerable economic importance to forestry in Europe. However, knowledge about genetic diversity among and between populations of cockchafers is far from sufficient. In this study, we fill several gaps in the knowledge of genetic population structure and the intensity of gene flow between Melolontha melolontha ( Mm ) and M. hippocastani ( Mh ). Specifically, we used microsatellite markers of 421 imagines from three main outbreak areas in Poland. Our study revealed some differences in polymorphism and population structuration between the two species. Mm was characterized by higher polymorphism and some evidence of population structure, while Mh did not show population structuring, which may indicate that these two species have undergone different historical events. It is likely that only Mm experienced a reduction in population size but both species currently have high effective population sizes - likely as a result of an ongoing outbreak. A number of ~ 10 migrants per generation allows gene flow and maintains equilibrium in the population. Our study also revealed some important differences in migratory behavior between the sexes, with male cockchafers making more frequent long-distance flights. Current ecological and anthropogenic changes that alter the quantity and quality of the food base appear to favor the fecundity, development and dispersal of cockchafers. Our study adds cockchafers to the list of outbreak species with available genetic information. This information can be an important component of effective management strategies and a foundation for understanding and predicting the evolution of these species.

We sought to determine changes in the gene pool in a stand of Scots pine Pinus sylvestris subject to different thinning regimes. The pine stand under study was located in central Poland. The sim- ulations using the ForestSimulatorBWINPro involved six experimental variants representing five virtual thinning regimes, i.e. 1) low thinning of 12% intensity; 2) low thinning of 35% intensity; 3) geometric thinning; 4) selection thinning; 5) high-grading; and 6) the untreated control. We considered the impact of these regimes on stand genetic variability up to 10 years after the given treatment, by reference to eleven SSR sequences. While various changes in the level of genetic diversity of stand were forecast to arise under the different regimes, low thinning of 35% intensity was identified as the method least favourable to maintenance of the initial level of genetic variability in the stand (loss of 22 and 43% of rare alleles in nSSR and cpSSR). High-grading proved most beneficial in the short-term. The selection thinning represented a kind of 'golden mean'; it turned out to be a method that enables achieving important silvicultural goals and simultane- ously only slightly distorts the stand genetic variability. The conducted experiment with the use of ForestSimulatorBWINPro program proved that the software can be successfully used in studied on the genetic variability of trees. Because it was only a preliminary study we suggest larger resampling the populations and genotyping using a larger number of microsatellite markers.

In the old pine stands of national parks, it is possible to observe genetic processes in a state free from disturbance by humans. Studies of this type make it possible to evaluate the effectiveness of the conservation of genetic variation and its transfer between generations. The present study was conducted in the largest national forest park in Poland, located in the Central European pine area. The oldest stands of Kampinos National Park and their natural descendants were selected for detailed analyses. The main objective of the study was to compare the mother pine stand, excluded from forest management, with its progeny generations on the basis of their chloroplast DNA (cpDNA), which was used as a diagnostic tool. The results demonstrate significant genetic difference between the maternal and progeny generations of the studied sites. The degree of variation observed in the maternal generation haplotypes in the present study was found to be reduced in the next generation. A significant proportion of the genetic diversity of the studied stands was also lost in the subsequent progeny generation. The obtained results allow conclusions to be drawn about the genetic processes taking place in valuable old-growth forests.

Scots pine (Pinus sylvestris L.) is a widespread species in Eurasia, but its natural range can be significantly altered by a variety of stressors. The ability of pine to regenerate naturally is significantly reduced by its occurrence in unsuitable habitats. The processes of natural selection of pine from select habitats can be followed in stands of national parks where forestry activities are excluded. The possibility of pine regeneration is influenced by the following factors: characteristics of produced seeds, competition, and genetic characteristics. In the present study, selected factors associated with limiting the natural regeneration potential of pine were analysed. The present work generated important information related to the natural regeneration potential of pine in Central and Eastern Europe. The main objective of the analyses was to discuss the possibility of the natural regeneration of pine stands without human intervention. In addition, the genetic diversity of naturally germinating seedlings was analysed. The obtained results confirmed the high reproductive potential of pine despite the advanced age of the studied trees. The obtained seeds produced by old growth Scots pine trees had high viability, while a significant difference was observed in terms of the average number of cones per dominant tree between studied stands. Thus, the number of cones was the main element determining the regeneration potential of the stands. It should be emphasised that the number of cones did not influence the occurrence of natural regeneration. Based on the obtained results, the regeneration potential of pine stands depends mainly on the habitat and the competitive pressure. In addition, a correlation between genetic parameters and the regeneration potential of stands should be established, which may be the beginning of further research on the process discussed in this publication.

Biodiversity at the species and individual levels is one of the fundamental elements characterizing an ecosystem. It is assumed that the greater the level of biodiversity, the more tolerant the environment is to changes in external conditions. In recent years, dynamic climate change has negatively impacted the health of many forest trees across Europe, in particular Scots pine. Tree health is commonly characterized by crown defoliation. The study presented here describes and correlates crown defoliation with biodiversity indicators at the species and individual tree levels. Research was conducted in two national parks in Poland (Kampinoski and Bory Tucholskie). Since stands have been under legal protection for many years and forest management is not practiced there, stand development processes taking place there are similar to natural ones. This study provided empirical data on ecosystem response to external stresses based on species and genetic structure. The results confirm differing health of the populations, which results from, among other factors, stand age and the environmental conditions in which they grow. Pine stands in both national parks are genetically diverse but with low genetic variability. Differences in stand health are related to the number of alleles forming the genetic pool. This conclusion is supported by a high correlation coefficient for interactions between defoliation, the number of alleles, and the Shannon index for genotypes. This suggests that greater gene diversity is likely to provide a wider range of phenotypic responses to environmental change.

Molecular techniques involved in the analysis of species population variability are based mainly on the analysis of microsatellite markers. However, ongoing development of genetic techniques increasingly allows this type of research to be carried out using variation in genome SNPs, mainly using NGS technology. This significantly expands the data pool on variability of genes responsible for a number of features, e.g. population variability related to adaptation to changing climatic conditions. In this study, 20 SNP (single nucleotide polymorphism) were selected from various genes related to, for example, abscisic acid responsive protein or dehydrins. Polymorphism of these markers was analyzed with traditional Sanger sequencing methods for 96 adult trees from populations in central Poland. Haplotype variation, h, was high (0.9902) and the average number of nucleotide differences between haplotypes was 10.625. The mean observed heterozygosity (Ho) was 0.311 and the mean expected heterozygosity (He) was equal to 0.360. The obtained results confirm the usefulness of the proposed non-standardised method of analysis of genetic variability as an alternative to other common techniques.

Scots pine (Pinus silvestris), is one of the dominant species in Poland and one of the main forest tree species in northern and central Europe. This species is of great economic importance. The Scots pine is highly adaptable to changing environmental conditions. A number of ecotypes have been characterized and the formation of these ecotypes are related with development of different phenotypic characteristics; morphological, physiological, and ecological. Molecular studies, based on DNA polymorphism, have been used for more than 20 years to analyze the genetic diversity of the Scots pine population. The most popular are microsatellite markers due to the fact of wide availability and high polymorphism. However, the use of these markers is also associated with certain limitations, due to complex mutation models or high incidence of homoplasia. These features are prompting scientists to look for alternative types of markers such as, for example, SNP. In our study we conducted a comparison of the basic parameters of genetic variability of two Scots pine stands (25 and 24 trees in each) for 20 SNP markers and 4 microsatellite markers. For the 20 SNP loci the observed heterozygosity (Ho) was equal to 0.34 for both stands and the expected heterozygosity (He) was equal to 0.34 for the first stand and 0.37 for the second. No statistically significant genetic distance was observed between them. For the microsatellite markers observed heterozygosity (Ho) was 0.81 and 0.74, and the expected heterozygosity (He) was equal to 0.85 and 0.85 respectively for the stands, and similarly no statistical significant genetic distance was observed. Literature data of different genetic markers showed the higher informativeness of randomly chosen microsatellites than single nucleotide polymorphism (SNP) markers for study population differentiation. But some analyzes confirm that the appropriate number of SNP markers can be more informative for population structure inference.

Pulsatilla × hackelii Pohl. is the most common hybrid between Pulsatilla patens and Pulsatilla pratensis. It occurs in the natural environment, most often in sites together with parental forms. Pulsatilla × hackelii is morphologically intermediate between the pure Pulsatilla species. Separation of hybrid individuals from pure Pulsatilla species requires a whole set of morphological features. Completed plastid genome provides molecular evidence for the hybrid origin of Pulsatilla × hackelii. In our study, we present the—genotypic characteristics of Pulsatilla × hackelii based on microsatellite markers developed for Pulsatilla vulgaris. A specimen of the hybrid was found in the Świętokrzyskie Mountains, near Bocheniec village, in the vicinity of a forest road in a fresh mixed coniferous forest (Querco roboris-Pinetum (W. Mat. 1981) J. Mat. 1988). We amplified a set of 10 microsatellites in two multiplex PCR reactions. From the 10 analyzed loci, we successfully amplified eight. The analyzed microsatellites in population studies of three Pulsatilla species showed some species-specific alleles. In the case of the Pulsatilla × hackelii sample, we obtained PCR products in the case of 5 loci developed for P. vulgaris but successfully crossamplified in P. patens and P. pratensis. Additionally, we obtained results for two loci, which crossamplified only for Pulsatilla patens and one only for Pulsatilla pratensis. Among the investigated markers, two were monomorphic and the remaining six were polymorphic, which makes them highly informative in population studies for pasque-flower species as Pulsatilla patens, P. pratensis, and P. vernalis. The obtained results indicated that analyzed microsatellite markers could be used for hybrids identification, although there is a need for further research on a larger number of hybridized individuals.

The work detailed here in the context of the above project sought to determine changes in the gene pool (at the DNA level) in a stand of Scots pine (Pinus sylvestris L.), under the influence of various kinds of forest-tending cuts. The experimental area on which the research was focused is located in Poland’s Ostrów Mazowiecka Forest District. Genetic structure was specified using five nuclear SSR sequences and six chloroplast SSR loci, while the five thinning variants trialled were sanitation cutting, low thinning of 30% intensity, schematic thinning, selective thinning and destructive lumbering. The control variant was left untreated. It was virtual rather than real-life thinning that was pursued, using the ForestSimulator BWINPro program. Changes in the structure of the stand after a further 10 years were also simulated. The different thinning variants were shown to cause change in the gene pool and level of genetic diversity of trees in the study area. In terms of maintaining genetic variability in the stand, the least-favourable method proved to be thinning from below. Destructive selection cutting was in turn most beneficial in terms of the preservation of genetic structure, with the reduction in rare alleles being more limited than in any other analysed variant. It was with the selective thinning variant that the final number of trees, stand structure and level of genetic variation resembled the situation in the control most closely. This suggests that selective thinning provides for a rather accurate replication of processes occurring in nature.

Understanding the growth pattern of trees of various provenances in response to climatic conditions provides information on the adaptive potential of trees. Infra-annual density fluctuations (IADFs) are anatomical markers of variations in wood density within the annual wood increment. Latewood-like cells within earlywood, resulting from severe drought during spring and early summer, express the structural adaptation of the wood tissue to unfavorable climatic conditions. In this study, we used two 40-year old IUFRO provenance trials of Norway spruce located in the western and north-eastern Poland to i) determine the effect of provenance on the IADF frequency and ii) to define the climatic factors determining the formation of IADF. Three highly polymorphic nuclear microsatellites are used to determine the differentiation among provenances in particular between the northern and southern Polish distribution area. Our results demonstrate that the frequency of all types of IADFs is determined by the growing conditions, and only the IADFs in the transition zone are determined by provenance origin, The highest variability between provenances in response to climatic differences was observed for IADF in transition zone, where temperatures and rainfall of spring and early summer differentiated northern and southern provenances. Moreover, the IADF frequency was influenced by drought, thereby differentiating provenances in terms of their sensitivity to severe conditions. Furthermore, environmental differentiation of climate-growth reaction was expressed by a stronger impact of climatic conditions observed in the western than in the northeastern trial site. The results of this study confirm the dependency of climate related changes in wood characteristics on provenance origin. Therefore, density fluctuations seem to be an appropriate adaptive trait for selecting Norway spruce provenances with varying degree of drought sensitivity.

We investigated the occurrence and pathogenicity of Beauveria spp. (Hypocreales: Cordycipitaceae) in forest soils in Poland, in outbreak areas of cockchafers (Coleoptera: Scarabaeidae): Melolontha melolontha L. and M. hippocastani F. We also examined the occurrence of Beauveria in relation to soil pH. Beauveria spp. isolates were characterised at species and genotype levels using ITS and microsatellite markers. Beauveria spp., which were detected at over 80% of sites, were sensitive to pH, preferring neutral or alkaline soils. This suggests that the acidity of forest soils in Poland can affect their efficacy as biological control agents (BCAs). B. brongniartii (Sacc.) Petch as a pathogen of cockchafers occurred at 41% of sites, but often at densities below the threshold values for infection, and it infected only 1.3% of cockchafer grubs. Our results suggest that B. brongniartii genotype isolated from cockchafers in forest soils can potentially expand the pool of BCAs in this environment.

In forestry management, artificially produced planting material is mainly used for renewal the tree population. Seedlings are cultivated in two systems: in the ground (the bare-root seedlings) and in controlled conditions (container seedlings). The aim of the study was to analyse the microsatellite markers of nuclear and chloroplast DNA, in terms of the number and frequency of rare, private, low frequency and common alleles in the planting material of Scots pine. The rare alleles included alleles occurring with less than 1% in analyzed group of seedlings and low frequency alleles occurred with a frequency of less than 25%. The private alleles were detected only in one group of seedlings. Genetic pools of seedlings from traditional (soil) and container production were compared. Planting material came from nurseries in the Olsztynek (N Poland) and the Oleszyce (S Poland) forest district. With the similar number of observed nDNA and cpDNA alleles in both analyzed locations, a higher number of rare, low frequency and private alleles was found within container seedlings. Most private alleles were a rare allele. Rare and private alleles are supposed to be responsible for adaptation to changing climatic conditions and a stressful environment. It seems reasonable to continue research on the meaning of rare and private alleles under conditions of strong selective pressure.

The common cockchafer (Melolontha melolontha) and the forest cockchafer (Melolontha hippocastani) are among the most destructive insect pests in many European countries. Larvae feed on the roots of numerous plant species, thus inflicting severe damage and heavy economic losses. The two species are often discussed together because they are difficult to distinguish during the larval stage. However, they differ slightly in ecology and development. The aim of this study was to develop a quick PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) method for easily identifying the two Melolontha species through tissue samples or larvae, when reliable morphological identification is lacking. The strength of the method was tested on 43M melolontha and 37 M. hippocastani individuals. We demonstrate that the technique is rapid and inexpensive, with strong implications for the effective management of these insect pests.