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Three European representatives of the genus Fraxinus were studied for the first time for their rDNA and heterochromatin patterns. The physical mapping of two rRNA gene families 5S and 18S-5.8S-26S (45S) and the distributional pattern of GC-rich regions in the chromosomes have been established by means of fluorescence in situ hybridization (FISH) and fluorochrome banding with chromomycin A. The genome size was assessed by flow cytometry. Heterochromatin and rDNA organization was conserved and almost identical for two species from Fraxinus section ( F. angustifolia and F. excelsior). The number and position of rDNA loci in F. ornus (section Ornus) were similar; however, the organization of genes was quite different. In this species the 5S and 45S rRNA genes were colocalized at the level of satellites of two chromosome pairs bearing nucleolar organizing regions (NORs). One 5S locus was also observed under the 45S one of one chromosome pair. In F. angustifolia and F. excelsior, only 45S loci were situated at the level of satellites and secondary constrictions, while 5S was located just under 45S in the distal part of the short arm of one out of two marked pairs. The number and position of GC-rich DNA correspond to those of 45S loci. The genome size (2C value) was of 1.54 and 1.68 pg for F. angustifolia and F. excelsior, respectively . Fraxinus ornus possessed the highest 2 C DNA value (1.98 pg). In the light of these cytogenetic features the clear differentiation between two sections ( Fraxinus and Ornus) was observed both at the rDNA and genome size levels. [ABSTRACT FROM AUTHOR]

The cosmopolitan genus Fraxinus, which comprises about 40 species of temperate trees and shrubs occupying various habitats in the Northern Hemisphere, represents a useful model to study speciation in long-lived angiosperms. We used nuclear external transcribed spacers (nETS), phantastica gene sequences, and two chloroplast loci (trnH-psbA and rpl32-trnL) in combination with previously published and newly obtained nITS sequences to produce a time-calibrated multi-locus phylogeny of the genus. We then inferred the biogeographic history and evolution of floral morphology. An early dispersal event could be inferred from North America to Asia during the Oligocene, leading to the diversification of the section Melioides sensus lato. Another intercontinental dispersal originating from the Eurasian section of Fraxinus could be dated from the Miocene and resulted in the speciation of F. nigra in North America. In addition, vicariance was inferred to account for the distribution of the other Old World species (sections Sciadanthus, Fraxinus and Ornus). Geographic speciation likely involving dispersal and vicariance could also be inferred from the phylogenetic grouping of geographically close taxa. Molecular dating suggested that the initial divergence of the taxonomical sections occurred during the middle and late Eocene and Oligocene periods, whereas diversification within sections occurred mostly during the late Oligocene and Miocene, which is consistent with the climate warming and accompanying large distributional changes observed during these periods. These various results underline the importance of dispersal and vicariance in promoting geographic speciation and diversification in Fraxinus. Similarities in life history, reproductive and demographic attributes as well as geographical distribution patterns suggest that many other temperate trees should exhibit similar speciation patterns. On the other hand, the observed parallel evolution and reversions in floral morphology would imply a major influence of environmental pressure. The phylogeny obtained and its biogeographical implications should facilitate future studies on the evolution of complex adaptive characters, such as habitat preference, and their possible roles in promoting divergent evolution in trees. [ABSTRACT FROM AUTHOR]

Tree species with wide distributions often exhibit different levels of genetic structuring correlated to their environment. However, understanding how environmental heterogeneity influences genetic variation is difficult because the effects of gene flow, drift and selection are confounded. We investigated the genetic variation and its ecological correlates in a wind-pollinated Mediterranean tree species, Fraxinus angustifolia Vahl, within a recognised glacial refugium in Croatia. We sampled 11 populations from environmentally divergent habitats within the Continental and Mediterranean biogeographical regions. We combined genetic data analyses based on nuclear microsatellite loci, multivariate statistics on environmental data and ecological niche modelling (ENM). We identified a geographic structure with a high genetic diversity and low differentiation in the Continental region, which contrasted with the significantly lower genetic diversity and higher population divergence in the Mediterranean region. The positive and significant correlation between environmental and genetic distances after controlling for geographic distance suggests an important influence of ecological divergence of the sites in shaping genetic variation. The ENM provided support for niche differentiation between the populations from the Continental and Mediterranean regions, suggesting that contemporary populations may represent two divergent ecotypes. Ecotype differentiation was also supported by multivariate environmental and genetic distance analyses. Our results suggest that despite extensive gene flow in continental areas, long-term stability of heterogeneous environments have likely promoted genetic divergence of ashes in this region and can explain the present- day genetic variation patterns of these ancient populations. [ABSTRACT FROM AUTHOR]

The utility of DNA barcoding for identifying representative specimens of the circumpolar tree genus Fraxinus (56 species) was investigated. We examined the genetic variability of several loci suggested in chloroplast DNA barcode protocols such as matK, rpoB, rpoC1 and trnH-psbA in a large worldwide sample of Fraxinus species. The chloroplast intergenic spacer rpl32- trnL was further assessed in search for a potentially variable and useful locus. The results of the study suggest that the proposed cpDNA loci, alone or in combination, cannot fully discriminate among species because of the generally low rates of substitution in the chloroplast genome of Fraxinus. The intergenic spacer trnH-psbA was the best performing locus, but genetic distance-based discrimination was moderately successful and only resulted in the separation of the samples at the subgenus level. Use of the BLAST approach was better than the neighbor-joining tree reconstruction method with pairwise Kimura's two-parameter rates of substitution, but allowed for the correct identification of only less than half of the species sampled. Such rates are substantially lower than the success rate required for a standardised barcoding approach. Consequently, the current cpDNA barcodes are inadequate to fully discriminate Fraxinus species. Given that a low rate of substitution is common among the plastid genomes of trees, the use of the plant cpDNA "universal" barcode may not be suitable for the safe identification of tree species below a generic or sectional level. Supplementary barcoding loci of the nuclear genome and alternative solutions are proposed and discussed. [ABSTRACT FROM AUTHOR]

Highlights: [•] We studied the phylogeography of the Eurasian species in the genus Fraxinus. [•] The nITS retrieved the classical view of the evolution of the section. [•] The nETS showed a separation of F. angustifolia in two paraphyletic groups. [•] This incongruence indicated an old reticulation between European and Asian ashes. [•] We provide a documented evidence of multiple reticulations within these temperate trees. [Copyright &y& Elsevier]