Evaluation of Antioxidative Mechanisms In Vitro and Triterpenes Composition of Extracts from Silver Birch (Betula pendula Roth) and Black Birch (Betula obscura Kotula) Barks by FT-IR and HPLC-PDA.

Silver birch, Betula pendula Roth, is one of the most common trees in Europe. Due to its content of many biologically active substances, it has long been used in medicine and cosmetics, unlike the rare black birch, Betula obscura Kotula. The aim of the study was therefore to compare the antioxidant properties of extracts from the inner and outer bark layers of both birch trees towards the L929 line treated with acetaldehyde. Based on the lactate dehydrogenase test and the MTT test, 10 and 25% concentrations of extracts were selected for the antioxidant evaluation. All extracts at tested concentrations reduced the production of hydrogen peroxide, superoxide anion radical, and 25% extract decreased malonic aldehyde formation in acetaldehyde-treated cells. The chemical composition of bark extracts was accessed by IR and HPLC-PDA methods and surprisingly, revealed a high content of betulin and lupeol in the inner bark extract of B. obscura. Furthermore, IR analysis revealed differences in the chemical composition of the outer bark between black and silver birch extracts, indicating that black birch may be a valuable source of numerous biologically active substances. Further experiments are required to evaluate their potential against neuroinflammation, cancer, viral infections, as well as their usefulness in cosmetology.

Reproduction strategies of the silver birch (Betula pendula Roth) at post-industrial sites.

The study aimed to evaluate the parameters of reproductive traits, specimens' fertility and reproductive efficiency observed in Betula pendula populations growing at different types of sites (zinc-lead heaps, coal mine heaps and unpolluted site). The leaf biomass and the biometric characteristics of inflorescences and fructifications were identified. Moreover, the biometric parameters of B. pendula seedlings were evaluated for examined sites. Seed-originated trees mostly of age 40 were randomly selected and from each tree, a branches from 1.70 m height and orientation N-S, W-E to the cardinal points of the stem were chosen. In the laboratory, selected soil parameters, the viability of pollen and the seeding value of seeds were analysed. According to the multidimensional statistical analysis the populations of B. pendula growing on post-industrial wastelands represent different morphotypes with lower values of almost all the reproductive traits, compared to the unpolluted birch population. Such traits as the male:female catkin number ratio and the non-embryo seed number were positively correlated with the heavy metal content at the zinc-lead heaps; at the same time these traits were negatively correlated with soil fertility. The fully developed seed number and the mature female catkin number were strongly correlated with the available potassium and phosphorus soil content but also with the leaf number on the generative shoots. The specimens of birch growing in these three habitats did not develop a universal reproductive strategy. Some differences in fecundity, the condition of seeds and the patterns of seed germination were found. The resulting seedling survival is determined by the plasticity of biometric traits, sheltered places for germination, etc. Seedlings that originated from heaps (local gene resources) are more suitable for use in the reclamation of large amounts of waste.

Resolving phylogeny and polyploid parentage using genus-wide genome-wide sequence data from birch trees.

Numerous plant genera have a history including frequent hybridisation and polyploidisation (allopolyploidisation), which means that their phylogeny is a network of reticulate evolution that cannot be accurately depicted as a bifurcating tree with a single tip per species. The genus Betula, which contains many ecologically important tree species, is a case in point. We generated genome-wide sequence reads for 27 diploid and 36 polyploid Betula species or subspecies using restriction site associated DNA (RAD) sequences. These reads were assembled into contigs with a mean length of 675 bp. We reconstructed the evolutionary relationships among diploid Betula species using both supermatrix (concatenation) and species tree methods. We identified the closest diploid relatives of the polyploids according to the relative rates at which reads from polyploids mapped to contigs from different diploid species within a concatenated reference sequence. By mapping reads from allopolyploids to their different putative diploid relatives we assembled contigs from the putative sub-genomes of allopolyploid taxa. We used these to build new phylogenies that included allopolyploid sub-genomes as separate tips. This approach yielded a highly evidenced phylogenetic hypothesis for the genus Betula, including the complex reticulate origins of the majority of its polyploid taxa. Our phylogeny divides the genus into two well supported clades, which, interestingly, differ in their seed-wing morphology. We therefore propose to split Betula into two subgenera.

Genetic and morphometric variability between populations of Betula ×oycoviensis from Poland and Czechia: A revised view of the taxonomic treatment of the Ojców birch.

Birches are generally known for their high genetic and morphological variability, which has resulted in the description of many species. Ojców birch was described in 1809 by Willibald Suibert Joseph Gottlieb Besser in Poland. Since then, several studies assessing its taxonomy were conducted. Today, various authors present Ojców birch at different taxonomic ranks. In Czechia, the Ojców birch is classified a critically endangered taxon and confirmed at one locality consisting of several tens of individuals. However, before a strategy for its conservation can be applied, we consider it necessary to assess the taxonomic position of the endangered Czech population and to evaluate its relationship to the original Polish population. This study aimed to evaluate the morphometric and genetic variability between populations of B. ×oycoviensis in Poland and the Czechia and their relationship to regional populations of B. pendula, one of the putative parental species of the Ojców birch. Altogether, 106 individuals were sampled, including the holotype of B. szaferi, the second putative parental species of B. ×oycoviensis, received from the herbarium of W. Szafer, which is deposited at the Institute of Botany in Kraków. Morphological analyses identified differences in leaves between B. ×oycoviensis and B. pendula. However, no significant differences were found in genome size between selected taxa/working units except for B. pendula sampled in Czechia. The identified difference of the Czech population of B. pendula is probably caused by population variability. Genetic variability between all the taxa under comparison, regardless of their origin, was also very low; only the benchmark taxa (B. nana and B. humilis) clearly differed from all samples analyzed. The results indicate minute morphological and negligible genetic variability between the Czech and Polish populations of B. ×oycoviensis. In light of our results, the classification of B. ×oycoviensis as B. pendula var. oycoviensis seems more accurate than all hitherto presented alternatives (e.g. B. ×oycoviensis as a separate species).

Characterization of chloroplast genomes of Alnus rubra and Betula cordifolia, and their use in phylogenetic analyses in Betulaceae.

OBJECTIVE: Betulaceae is a relatively small birch family that comprises about 160 deciduous trees and shrubs. Chloroplast (cp) genome sequencing of Alnus rubra and Betula cordifolia was carried out to elucidate their molecular features and phylogenetic relationship among species in Betulaceae family. METHODS: Chloroplast genome sequencing was carried out using next generation sequencing method. Molecular and genomic features of the two cp genomes were characterized with other cp genomes in Betulaceae. Also, molecular phylogenetic analysis was performed using the whole cp genome sequences. RESULTS: The average cp genome length was 160,136 bp among the Betulaceae species. Base compositions of the cp genomes were skewed toward a high AT ratio, with an average of 63.4%. We identified 117 different genes 83 with protein coding, 4 with ribosomal RNA, and 30 with tRNA. Eighteen genes contained introns which were conserved among the cp genomes of all Betulaceae. We mined 82 SSRs from the cp genomes of A. rubra, A. cordifolia, and A. nana. The SSRs were variable in motif repeat numbers and presence/absence among the cp genomes. CONCLUSION: Chloroplast genome-wide sequence comparison from 11 Betulaceae species and one cp genome of evergreen oak revealed that the patterns of sequence variations were congruent with two subfamily classification Betuloideae (Alnus and Betula) and Corylaceae (Corylus, Ostrya, and Carpinus). Subsequent phylogenetic analysis also supports the sub-classifications of these species.

Complete chloroplast genome sequence of Betula platyphylla: gene organization, RNA editing, and comparative and phylogenetic analyses.

BACKGROUND: Betula platyphylla is a common tree species in northern China that has high economic and medicinal value. Our laboratory has been devoted to genome research on B. platyphylla for approximately 10 years. As primary organelle genomes, the complete genome sequences of chloroplasts are important to study the divergence of species, RNA editing and phylogeny. In this study, we sequenced and analyzed the complete chloroplast (cp) genome sequence of B. platyphylla. RESULTS: The complete cp genome of B. platyphylla was 160,518 bp in length, which included a pair of inverted repeats (IRs) of 26,056 bp that separated a large single copy (LSC) region of 89,397 bp and a small single copy (SSC) region of 19,009 bp. The annotation contained a total of 129 genes, including 84 protein-coding genes, 37 tRNA genes and 8 rRNA genes. There were 3 genes using alternative initiation codons. Comparative genomics showed that the sequence of the Fagales species cp genome was relatively conserved, but there were still some high variation regions that could be used as molecular markers. The IR expansion event of B. platyphylla resulted in larger cp genomes and rps19 pseudogene formation. The simple sequence repeat (SSR) analysis showed that there were 105 SSRs in the cp genome of B. platyphylla. RNA editing sites recognition indicated that at least 80 RNA editing events occurred in the cp genome. Most of the substitutions were C to U, while a small proportion of them were not. In particular, three editing loci on the rRNA were converted to more than two other bases that had never been reported. For synonymous conversion, most of them increased the relative synonymous codon usage (RSCU) value of the codons. The phylogenetic analysis suggested that B. platyphylla had a closer evolutionary relationship with B. pendula than B. nana. CONCLUSIONS: In this study, we not only obtained and annotated the complete cp genome sequence of B. platyphylla, but we also identified new RNA editing sites and predicted the phylogenetic relationships among Fagales species. These findings will facilitate genomic, genetic engineering and phylogenetic studies of this important species.

Foliar fungi of Betula pendula: impact of tree species mixtures and assessment methods.

Foliar fungi of silver birch (Betula pendula) in an experimental Finnish forest were investigated across a gradient of tree species richness using molecular high-throughput sequencing and visual macroscopic assessment. We hypothesized that the molecular approach detects more fungal taxa than visual assessment, and that there is a relationship among the most common fungal taxa detected by both techniques. Furthermore, we hypothesized that the fungal community composition, diversity, and distribution patterns are affected by changes in tree diversity. Sequencing revealed greater diversity of fungi on birch leaves than the visual assessment method. One species showed a linear relationship between the methods. Species-specific variation in fungal community composition could be partially explained by tree diversity, though overall fungal diversity was not affected by tree diversity. Analysis of specific fungal taxa indicated tree diversity effects at the local neighbourhood scale, where the proportion of birch among neighbouring trees varied, but not at the plot scale. In conclusion, both methods may be used to determine tree diversity effects on the foliar fungal community. However, high-throughput sequencing provided higher resolution of the fungal community, while the visual macroscopic assessment detected functionally active fungal species.

Multispecies genetic structure and hybridization in the Betula genus across Eurasia.

Boreal and cool temperate forests are the major land cover of northern Eurasia, and information about continental-scale genetic structure and past demographic history of forest species is important from an evolutionary perspective and has conservation implications. However, although many population genetic studies of forest tree species have been conducted in Europe or Eastern Asia, continental-scale genetic structure and past demographic history remain poorly known. Here, we focus on the birch genus Betula, which is commonly distributed in boreal and cool temperate forests, and examine 129 populations of two tetraploid and four diploid species collected from Iceland to Japan. All individuals were genotyped at seven to 18 nuclear simple sequence repeats (nSSRs). Pairwise FST' among the six species ranged from 0.285 to 0.903, and genetic differentiation among them was clear. structure analysis suggested that Betula pubescens is an allotetraploid and one of the parental species was Betula pendula. In both species pairs of B. pendula and B. plathyphylla, and B. pubescens and B. ermanii, genetic diversity was highest in central Siberia. A hybrid zone was detected around Lake Baikal for eastern and western species pairs regardless of ploidy level. Approximate Bayesian computation suggested that the divergence of B. pendula and B. platyphylla occurred around the beginning of the last ice age (36 300 years BP, 95% CI: 15 330-92 700) and hybridization between them was inferred to have occurred after the last glacial maximum (1614 years BP, 95% CI: 561-4710), with B. pendula providing a higher contribution to hybrids.

Two Birch Species Demonstrate Opposite Latitudinal Patterns in Infestation by Gall-Making Mites in Northern Europe.

Latitudinal patterns in herbivory, i.e. variations in plant losses to animals with latitude, are generally explained by temperature gradients. However, earlier studies suggest that geographical variation in abundance and diversity of gall-makers may be driven by precipitation rather than by temperature. To test the above hypothesis, we examined communities of eriophyoid mites (Acari: Eriophyoidea) on leaves of Betula pendula and B. pubescens in boreal forests in Northern Europe. We sampled ten sites for each of five latitudinal gradients from 2008-2011, counted galls of six morphological types and identified mites extracted from these galls. DNA analysis revealed cryptic species within two of six morphologically defined mite species, and these cryptic species induced different types of galls. When data from all types of galls and from two birch species were pooled, the percentage of galled leaves did not change with latitude. However, we discovered pronounced variation in latitudinal changes between birch species. Infestation by eriophyoid mites increased towards the north in B. pendula and decreased in B. pubescens, while diversity of galls decreased towards the north in B. pendula and did not change in B. pubescens. The percentage of galled leaves did not differ among geographical gradients and study years, but was 20% lower in late summer relative to early summer, indicating premature abscission of infested leaves. Our data suggest that precipitation has little effect on abundance and diversity of eriophyoid mites, and that climate warming may impose opposite effects on infestation of two birch species by galling mites, favouring B. pendula near the northern tree limit.

Drivers of Change in a 7300-Year Holocene Diatom Record from the Hemi-Boreal Region of Ontario, Canada.

A Holocene lake sediment record spanning the past 7300 years from Wishart Lake in the Turkey Lakes Watershed in the Hemi-Boreal of central Ontario, Canada, was used to evaluate the potential drivers of long-term change in diatom assemblages at this site. An analysis of diatom assemblages found that benthic and epiphytic taxa dominated the mid-Holocene (7300-4000 cal yr BP), indicating shallow, oligotrophic, circum-neutral conditions, with macrophytes present. A significant shift in diatom assemblages towards more planktonic species (mainly Cyclotella sensu lato, but also several species of Aulacoseira, and Tabellaria flocculosa) occurred ~4000 cal yr BP. This change likely reflects an increase in lake level, coincident with the onset of a more strongly positive moisture balance following the drier climates of the middle Holocene, established by numerous regional paleoclimate records. Pollen-inferred regional changes in vegetation around 4000 yrs BP, including an increase in Betula and other mesic taxa, may have also promoted changes in diatom assemblages through watershed processes mediated by the chemistry of runoff. A more recent significant change in limnological conditions is marked by further increases in Cyclotella sensu lato beginning in the late 19th century, synchronous with the Ambrosia pollen rise and increases in sediment bulk density, signaling regional and local land clearance at the time of Euro-Canadian settlement (1880 AD). In contrast to the mid-Holocene increase in planktonic diatoms, the modern increase in Cyclotella sensu lato likely indicates a response to land use and vegetation change, and erosion from the watershed, rather than a further increase in water level. The results from Wishart Lake illustrate the close connection between paleoclimate change, regional vegetation, watershed processes, and diatom assemblages and also provides insight into the controls on abundance of Cyclotella sensu lato, a diatom taxonomic group which has shown significant increases and complex dynamics in the post-industrial era in lakes spanning temperate to Arctic regions.

Comparative analyses of plastid and AFLP data suggest different colonization history and asymmetric hybridization between Betula pubescens and B. nana.

Birches (Betula spp.) hybridize readily, confounding genetic signatures of refugial isolation and postglacial migration. We aimed to distinguish hybridization from range-shift processes in the two widespread and cold-adapted species B. nana and B. pubescens, previously shown to share a similarly east-west-structured variation in plastid DNA (pDNA). We sampled the two species throughout their ranges and included reference samples of five other Betula species and putative hybrids. We analysed 901 individual plants using mainly nuclear high-resolution markers (amplified fragment length polymorphisms; AFLPs); a subset of 64 plants was also sequenced for two pDNA regions. Whereas the pDNA variation as expected was largely shared between B. nana and B. pubescens, the two species were distinctly differentiated at AFLP loci. In B. nana, both the AFLP and pDNA results corroborated the former pDNA-based hypothesis that it expanded from at least two major refugia in Eurasia, one south of and one east of the North European ice sheets. In contrast, B. pubescens showed a striking lack of geographic structuring of its AFLP variation. We identified a weak but significant increase in nuclear (AFLP) gene flow from B. nana into B. pubescens with increasing latitude, suggesting hybridization has been most frequent at the postglacial expansion front of B. pubescens and that hybrids mainly backcrossed to B. pubescens. Incongruence between pDNA and AFLP variation in B. pubescens can be explained by efficient expansion from a single large refugium combined with leading-edge hybridization and plastid capture from B. nana during colonization of new territory already occupied by this more cold-tolerant species.

Molecular footprints of the Holocene retreat of dwarf birch in Britain.

Past reproductive interactions among incompletely isolated species may leave behind a trail of introgressed alleles, shedding light on historical range movements. Betula pubescens is a widespread native tetraploid tree species in Britain, occupying habitats intermediate to those of its native diploid relatives, B. pendula and B. nana. Genotyping 1134 trees from the three species at 12 microsatellite loci, we found evidence of introgression from both diploid species into B. pubescens, despite the ploidy difference. Surprisingly, introgression from B. nana, a dwarf species whose present range is highly restricted in northern, high-altitude peat bogs, was greater than introgression from B. pendula, which is morphologically similar to B. pubescens and has a substantially overlapping range. A cline of introgression from B. nana was found extending into B. pubescens populations far to the south of the current B. nana range. We suggest that this genetic pattern is a footprint of a historical decline and/or northwards shift in the range of B. nana populations due to climate warming in the Holocene. This is consistent with pollen records that show a broader, more southerly distribution of B. nana in the past. Ecological niche modelling predicts that B. nana is adapted to a larger range than it currently occupies, suggesting additional factors such as grazing and hybridization may have exacerbated its decline. We found very little introgression between B. nana and B. pendula, despite both being diploid, perhaps because their distributions in the past have rarely overlapped. Future conservation of B. nana may partly depend on minimization of hybridization with B. pubescens, and avoidance of planting B. pendula near B. nana populations.

Genome sequence of dwarf birch (Betula nana) and cross-species RAD markers.

New sequencing technologies allow development of genome-wide markers for any genus of ecological interest, including plant genera such as Betula (birch) that have previously proved difficult to study due to widespread polyploidy and hybridization. We present a de novo reference genome sequence assembly, from 66× short read coverage, of Betula nana (dwarf birch) - a diploid that is the keystone woody species of subarctic scrub communities but of conservation concern in Britain. We also present 100 bp PstI RAD markers for B. nana and closely related Betula tree species. Assembly of RAD markers in 15 individuals by alignment to the reference B. nana genome yielded 44-86k RAD loci per individual, whereas de novo RAD assembly yielded 64-121k loci per individual. Of the loci assembled by the de novo method, 3k homologous loci were found in all 15 individuals studied, and 35k in 10 or more individuals. Matching of RAD loci to RAD locus catalogues from the B. nana individual used for the reference genome showed similar numbers of matches from both methods of RAD locus assembly but indicated that the de novo RAD assembly method may overassemble some paralogous loci. In 12 individuals hetero-specific to B. nana 37-47k RAD loci matched a catalogue of RAD loci from the B. nana individual used for the reference genome, whereas 44-60k RAD loci aligned to the B. nana reference genome itself. We present a preliminary study of allele sharing among species, demonstrating the utility of the data for introgression studies and for the identification of species-specific alleles.

[Spatial patterns of dominant tree species in sub-alpine Betula-Abies forest in West Sichuan of China].

Based on the investigation in a 4 hm2 Betula-Abies forest plot in sub-alpine area in West Sichuan of China, and by using point pattern analysis method in terms of O-ring statistics, the spatial patterns of dominant species Betula albo-sinensis and Abies faxoniana in different age classes in study area were analyzed, and the intra- and inter-species associations between these age classes were studied. B. albo-sinensis had a unimodal distribution of its DBH frequency, indicating a declining population, while A. faxoniana had a reverse J-shaped pattern, showing an increasing population. All the big trees of B. albo-sinensis and A. faxoniana were spatially in random at all scales, while the medium age and small trees were spatially clumped at small scales and tended to be randomly or evenly distributed with increasing spatial scale. The maximum aggregation degree decreased with increasing age class. Spatial association mainly occurred at small scales. A. faxoniana generally showed positive intra-specific association, while B. albo-sinensis generally showed negative intra-specific association. For the two populations, big and small trees had no significant spatial association, but middle age trees had negative spatial association. Negative inter-specific associations of the two populations were commonly found in different age classes. The larger the difference of age class, the stronger the negative inter-specific association.

[Genetic linkage map of Betula pendula Roth and Betula platyphylla Suk based on random amplified polymorphisms DNA markers].

Based on the genetic inheritance and segregation of random amplified polymorphism DNA (RAPDs) markers, the first mid-density linkage map for silver birch was constructed by using a pseudo-testcross mapping strategy. A segregating population including 80 progenies from the cross between Betula pendula Roth and B. platyphylla Suk was obtained. A set of 1,200 random oligonucleotide primers were screened, and 208 primers were selected to generate RAPD markers within a sample of 80 F1 progenies. A total of 364 segregating sites were identified. Among them, 307 belonged to 1 : 1 segregating site, and 36 belonged to 3 : 1 segregating site, others were found distorted from the normal 1 : 1 ratio. Altogether 307 sites segregating 1 : 1 (testcross configuration) were used to construct parent-specific linkage maps, 145 for B. pendula and 162 for B. platyphylla. The resulting linkage maps consisted of 145 marker sites in 14 groups (four or more sites per group), 6 triples and 6 pairs for B. pendula, which covered the map distance about 955.6 cM (Kosambi units). The average map distance between adjacent markers was 14.9 cM, and 162 linked marker site for B. platyphylla were mapped onto 15 groups (four or more sites per group), 4 triples and 6 pairs, which covered the map distance about 1,545.8 cM, and the average map distance between adjacent markers was 15.2 cM. Further study is warranted to integrate the two maps to one density map and to locate important genes on the maps.

HPLC analysis of leaf surface flavonoids for the preliminary classification of birch species.

Flavonoid aglycones found on the surfaces of birch (Betula spp.) leaves may constitute up to 10% of the dry weight of the leaf. A facile extraction and HPLC procedure has been developed that can be used for the preliminary classification of birch species according to the patterns of their leaf surface flavonoids. The procedure involves no complex sample preparation steps, and is able to provide HPLC chromatograms from fresh leaves in less than 30 min. If necessary, leaves do not even need to be removed from the tree. Since the genus Betula is taxonomically complex and separation of different birch species can be problematic, the developed method was applied to 15 Betula species and four sub-species of Betula pendula Seven of the studied species were classified as B. pubescens and eight as B. pendula-type birches. The remaining four species did not belong to either of these two classes on account of their unique pattern of external flavonoids. The difference between the leaf surface flavonoid composition of B. pubescens and B. pendula type birch species was unambiguously clear, and the developed method could reliably distinguish between the two species. Whilst leaf surface flavonoids can be valuable chemotaxonomic markers, they classify birch species differently from morphological markers. Birch species with diploid chromosome sets did not contain any of the flavanones that were present in the leaves of other species. The close relationship between the occurrence of some flavonoid aglycones and the ploidy level of Betula species suggests that these chemotaxonomic markers may be useful both in taxonomic and phylogenetic analyses.

Differential responses of silver birch (Betula pendula) ecotypes to short-day photoperiod and low temperature.

We investigated interrelations of dormancy and freezing tolerance and the role of endogenous abscisic acid (ABA) in the development of silver birch (Betula pendula Roth) ecotypes in controlled environments. Short-day treatment induced growth cessation, bud set and dormancy development, as well as initiation of cold acclimation and an increase in freezing tolerance. Subsequent low temperature and short days (12-h photoperiod) resulted in a significant increase in freezing tolerance, whereas bud dormancy was gradually released. The concentration of ABA increased in response to short days and then remained high, but ABA concentrations fluctuated irregularly when the dormant plants were subsequently exposed to low temperature during short days. Although there was a parallel development of freezing tolerance and bud dormancy in response to short days, subsequent exposure to low temperature had opposite effects on these processes, enhancing freezing tolerance and releasing dormancy. Compared with the southern ecotype, the northern ecotype was more responsive to short days and low temperature, exhibiting earlier initiation of cold acclimation, growth cessation and an increase in ABA concentrations in short days, and higher freezing tolerance, faster dormancy release and greater alteration in ABA concentrations when subsequently exposed to low temperature during short days. The rates and extent of the increases in ABA concentration may be related to increases in freezing tolerance and dormancy development during short days, whereas the extent of the fluctuations in ABA concentration may play an important role in enhancing freezing tolerance and releasing dormancy during a subsequent exposure to low temperature during short days.

Changes in leaf trichomes and epicuticular flavonoids during leaf development in three birch taxa.

BACKGROUND AND AIMS: Changes in number of trichomes and in composition and concentrations of their exudates throughout leaf development may have important consequences for plant adaptation to abiotic and biotic factors. In the present study, seasonal changes in leaf trichomes and epicuticular flavonoid aglycones in three Finnish birch taxa (Betula pendula, B. pubescens ssp. pubescens, and B. pubescens ssp. czerepanovii) were followed. METHODS: Trichome number and ultrastructure were studied by means of light, scanning and transmission electron microscopy, while flavonoid aglycones in ethanolic leaf surface extracts were analysed by high-pressure liquid chromatography. KEY RESULTS: Density of both glandular and non-glandular trichomes decreased drastically with leaf expansion while the total number of trichomes per leaf remained constant, indicating that the final number of trichomes is established early in leaf development. Cells of glandular trichomes differentiate before those of the epidermis and produce secreted material only during the relatively short period (around 1-2 weeks) of leaf unfolding and expansion. In fully expanded leaves, glandular trichomes appeared to be at the post-secretory phase and function mainly as storage organs; they contained lipid droplets and osmiophilic material (probably phenolics). Concentrations (mg g(-1) d. wt) of surface flavonoids decreased with leaf age in all taxa. However, the changes in total amount ( microg per leaf) of flavonoids during leaf development were taxon-specific: no changes in B. pubescens ssp. czerepanovii, increase in B. pendula and in B. pubescens ssp. pubescens followed by the decline in the latter taxon. Concentrations of most of the individual leaf surface flavonoids correlated positively with the density of glandular trichomes within species, suggesting the participation of glandular trichomes in production of surface flavonoids. CONCLUSIONS: Rapid decline in the density of leaf trichomes and in the concentrations of flavonoid aglycones with leaf age suggests that the functional role of trichomes is likely to be most important at the early stages of birch leaf development.

Postglacial recolonization and cpDNA variation of silver birch, Betula pendula.

Chloroplast PCR-RFLP markers were used to reconstruct the history of the silver birch, Betula pendula Roth, in Europe since the last glacial maximum (LGM). In birch, fossil pollen maps do not reveal a clear chronological sequence of postglacial spread. If anything, the pollen record suggests that most of Europe was recolonized by birches as early as 10000 bp, probably from populations that remained close to the ice sheets during the LGM. The geographical distribution of haplotypes supports a scenario of early colonization. Two of the 13 haplotypes that were observed are common, representing 35% and 49% of the total sample, respectively. Although one of the common haplotypes is predominant in the NW and the other in the SE, both are present throughout most of the investigated geographical area. Rare haplotypes are geographically restricted. The distribution of the haplotypes reveals five genetic boundaries between groups of haplotypes and allows us to infer patterns of postglacial recolonization. Europe was re-occupied by two main waves of recolonization: one eastern and one western, with origins at intermediate latitudes. Populations in the Iberian Peninsula and in Italy did not take part in the postglacial recolonization of Europe.