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International audience; Water, being the universal solvent, acts as a competing agent in fundamental processes, such as folding, aggregation or biomolecular recognition. A molecular understanding of hydrophobic hydration is of central importance to understanding the subtle free energy differences, which dictate function. Ab initio and classical molecular dynamics simulations yield two distinct hydration water populations in the hydration shell of solvated tert-butanol noted as “HB-wrap” and “HB-hydration2bulk”. The experimentally observed hydration water spectrum can be dissected into two modes, centered at 164 and 195 cm–1. By comparison to the simulations, these two bands are attributed to the “HB-wrap” and “HB-hydration2bulk” populations, respectively. We derive a quantitative correlation between the population in each of these two local water coordination motifs and the temperature dependence of the solvation entropy. The crossover from entropy to enthalpy dominated solvation at elevated temperatures, as predicted by theory and observed experimentally, can be rationalized in terms of the distinct temperature stability and thermodynamic signatures of “HB-wrap” and “HB-hydration2bulk”.

Significance For centuries the double layer at the solid/electrolyte interface has been a central concept in electrochemistry. Today, it is still crucial for virtually all renewable energy storage and conversion technologies. Here, the double-layer formation is probed by THz spectroscopy with ultrabright synchrotron light as a source. Our results capture the molecular details of double-layer formation at positively/negatively charged Au electrodes for an NaCl electrolyte. We reveal a contrasting response applying positive versus negative bias, which is dictated by the interfacial water network and rationalized by accompanying molecular dynamics simulations and electronic-structure calculations. While Na+ is directly attracted toward the negatively charged electrode, stripping of the Cl− hydration shell is observed only at larger potential values., The double layer at the solid/electrolyte interface is a key concept in electrochemistry. Here, we present an experimental study combined with simulations, which provides a molecular picture of the double-layer formation under applied voltage. By THz spectroscopy we are able to follow the stripping away of the cation/anion hydration shells for an NaCl electrolyte at the Au surface when decreasing/increasing the bias potential. While Na+ is attracted toward the electrode at the smallest applied negative potentials, stripping of the Cl− hydration shell is observed only at higher potential values. These phenomena are directly measured by THz spectroscopy with ultrabright synchrotron light as a source and rationalized by accompanying molecular dynamics simulations and electronic-structure calculations.

The human telomeric G-quadruplex structural motif of DNA has come to be known as a new and stimulating target for anticancer drug discovery. Small molecules that interact with G-quadruplex structures in a selective way have gained impressive interest in recent years as they may serve as potential therapeutic agents. Here, we show how circular dichroism, UV resonance Raman and small angle X-ray scattering spectroscopies can be effectively combined to provide insights into structural and molecular aspects of the interaction between human telomeric quadruplexes and ligands. This study focuses on the ability of berberine and palmatine to bind with human telomeric quadruplexes and provides analysis of the conformational landscape visited by the relevant complexes upon thermal unfolding. With increasing temperature, both free and bound G-quadruplexes undergo melting through a multi-state process, populating different intermediate states. Despite the structural similarity of the two ligands, valuable distinctive features characterising their interaction with the G-quadruplex emerged from our multi-technique approach.

Ion hydration is of pivotal importance for many fundamental processes. Various spectroscopic methods are used to study the retardation of the hydration bond dynamics in the vicinity of anions and cations. Here we introduce THz-FTIR spectroscopy as a powerful method to answer the open questions. We show through dissection of THz spectra that we can pinpoint characteristic absorption features that can be attributed to the rattling modes of strongly hydrating ions within their hydration cages as well as vibrationally induced charge fluctuations in the case of weakly hydrating ions. Further analysis yields information on anion-cation cooperativity, the size of the dynamic hydration shell, as well as the lifetimes of these collective ion-hydration water modes and their connecting thermal bath states. Our study provides evidence for a non-additive behavior, thus questioning the simplified Hofmeister model. THz spectroscopy enables ion pairing to be observed and quantified at a high salt concentration.

Anthocyanins have long been suggested as having great potential in offering photoprotection to plants facing high light irradiance. Nonetheless, their effective ability in protecting the photosynthetic apparatus from supernumerary photons has been questioned by some authors, based upon the inexact belief that anthocyanins almost exclusively absorb green photons, which are poorly absorbed by chlorophylls. Here we focus on the blue light absorbing features of anthocyanins, a neglected issue in anthocyanin research. Anthocyanins effectively absorb blue photons: the absorbance of blue relative to green photons increases from tri- to mono-hydroxy B-ring substituted structures, reaching up to 50% of green photons absorption. We offer a comprehensive picture of the molecular events activated by low blue-light availability, extending our previous analysis in purple and green basil, which we suggest to be responsible for the "shade syndrome" displayed by cyanic leaves. While purple leaves display overexpression of genes promoting chlorophyll biosynthesis and light harvesting, in green leaves it is the genes involved in the stability/repair of photosystems that are largely overexpressed. As a corollary, this adds further support to the view of an effective photoprotective role of anthocyanins. We discuss the profound morpho-anatomical adjustments imposed by the epidermal anthocyanin shield, which reflect adjustments in light harvesting capacity under imposed shade and make complex the analysis of the photosynthetic performance of cyanic versus acyanic leaves.

Based upon precise terahertz (THz) measurements of the solvated amino acid glycine and accompanying ab‐initio molecular‐dynamics simulations, we show that the N‐C‐C‐O open/close mode at 315 cm−1 serves as a sensitive, label‐free probe for the local protonation of the amide group. Experimentally, we can show that this holds not only for glycine but also for diglycine and valine. The approach is more general, since the changes due to protonation result in intensity changes which can be probed by THz time domain (0–50 cm−1) as well as by precise THz‐FT spectroscopy (50–400 cm−1). A detailed analysis allows us to directly correlate the titration spectra with pK a values. This demonstrates the potential of THz spectroscopy to probe the charge state of a natural amino acid in water in a label‐free manner., Based upon precise terahertz (THz) measurements of the solvated amino acid glycine and accompanying ab‐initio molecular‐dynamics simulations, it is shown that the N‐C‐C‐O open/close mode at 315 cm−1 serves as a sensitive, label‐free probe for the local protonation of the amide group.

Significance Using a supramolecular assembly that catalyzes substrates in water, we show that the nanocage encapsulates a water cluster that is structurally and dynamically distinct from any known phase of water. It plays an important role in the driving force for guest encapsulation: The release of the highly unusual encapsulated water droplet creates a strong thermodynamic drive for the high-affinity binding of guests in aqueous solution for the supramolecular construct., Water under nanoconfinement at ambient conditions has exhibited low-dimensional ice formation and liquid–solid phase transitions, but with structural and dynamical signatures that map onto known regions of water’s phase diagram. Using terahertz (THz) absorption spectroscopy and ab initio molecular dynamics, we have investigated the ambient water confined in a supramolecular tetrahedral assembly, and determined that a dynamically distinct network of 9 ± 1 water molecules is present within the nanocavity of the host. The low-frequency absorption spectrum and theoretical analysis of the water in the Ga4L612− host demonstrate that the structure and dynamics of the encapsulated droplet is distinct from any known phase of water. A further inference is that the release of the highly unusual encapsulated water droplet creates a strong thermodynamic driver for the high-affinity binding of guests in aqueous solution for the Ga4L612− supramolecular construct.

Tracking the excitation of water molecules in the homogeneous liquid is challenging due to the ultrafast dissipation of rotational excitation energy through the hydrogen-bonded network. Here we demonstrate strong transient anisotropy of liquid water through librational excitation using single-color pump-probe experiments at 12.3 THz. We deduce a third order response of chi^3 exceeding previously reported values in the optical range by three orders of magnitude. Using a theory that replaces the nonlinear response with a material response property amenable to molecular dynamics simulation, we show that the rotationally damped motion of water molecules in the librational band is resonantly driven at this frequency, which could explain the enhancement of the anisotropy in the liquid by the external Terahertz field. By addition of salt (MgSO4), the hydration water is instead dominated by the local electric field of the ions, resulting in reduction of water molecules that can be dynamically perturbed by THz pulses.

A multi-technique approach, combining circular dichroism spectroscopy, ultraviolet resonance Raman spectroscopy and small angle scattering techniques, has been deployed to elucidate how the structural features of the human telomeric G-quadruplex d[A(GGGTTA)3GGG] (Tel22) change upon thermal unfolding. The system is studied both in the free form and when it is bound to Actinomycin D (ActD), an anticancer ligand with remarkable conformational flexibility. We find that at room temperature binding of Tel22 with ActD involves end-stacking upon the terminal G-tetrad. Structural evidence for drug-driven dimerization of a significant fraction of the G-quadruplexes is provided. When the temperature is raised, both free and bound Tel22 undergo melting through a multi-state process. We show that in the intermediate states of Tel22 the conformational equilibrium is shifted toward the (3+1) hybrid-type, while a parallel structure is promoted in the complex. The unfolded state of the free Tel22 is consistent with a self-avoiding random-coil conformation, whereas the high-temperature state of the complex is observed to assume a quite compact form. Such an unprecedented high-temperature arrangement is caused by the persistent interaction between Tel22 and ActD, which stabilizes compact conformations even in the presence of large thermal structural fluctuations.

Isolated populations are usually subject to low fitness and reduced genetic diversity, both of which may negatively affect their survival and adaptive potential. Hence, these issues cannot be neglected when planning conservation actions for isolated populations. The Italian population of Cistus laurifolius subsp. laurifolius is extremely isolated. Furthermore, it is affected by fragmentation, being constituted by a single larger subpopulation, surrounded by three much smaller subpopulations, a few hundred metres to a few kilometres apart. In order to fill gaps in demographic and genetic knowledge concerning the Italian population, its area of occupancy, size, age-stage structure and phenology were investigated and its reproductive fitness, pollination strategies and genetic variability were assessed. The population was inferred as fully xenogamous and showed good reproductive performance. Despite this, its genetic variability was low and it showed relatively high levels of inbreeding depression (Fis), seemingly not affected by sub-population size. These results suggest that the Italian population recently suffered fragmentation and reduction in size. The low genetic diversity observed could be explained by the high percentage of mature individuals found in the population, possibly established before fragmentation. For these reasons, the Italian population of C. laurifolius subsp. laurifolius should be monitored and concrete actions aimed at its conservation planned.

Viele Naturphänomene hängen mit der Nukleation von CaCO3 zusammen, wobei die Rolle des Wassers bis jetzt weitgehend unklar war. Änderungen in der Absorption im THz-Frequenzbereich während der frühen Stadien der CaCO3-Nukleation belegen Veränderungen in den gekoppelten Bewegungen der hydratisierten Calcium- und Carbonat-Ionen. Der direkte Zusammenhang zwischen diesen Effekten und der kontinuierlichen Entwicklung des Ionenaktivitätsproduktes zeigt die Lage einer flüssig-flüssig-binodalen Grenze. Die Daten lassen stark darauf schließen, dass sich proto-strukturiertes, amorphes CaCO3 über die Verfestigung von anfänglich flüssigen Vorstufen bildet. Außerdem erhöhen Polycarboxylate, die flüssige Vorstufen von CaCO3 stabilisieren, die kinetische Stabilität des metastabilen flüssig-flüssigen Zustands signifikant, aber sie beeinflussen nicht die Lage der binodalen Grenze. Die mechanistische Bedeutung der Dynamik von Wassernetzwerken bei der Phasentrennung kann als alternativer Weg der Nukleation über Pränukleationscluster rationalisiert werden und ist wahrscheinlich allgemein für wässrige Systeme gültig. published

Many phenomena depend on CaCO3 nucleation where the role of water remains enigmatic. Changes in THz absorption during the early stages of CaCO3 nucleation evidence altered coupled motions of hydrated calcium and carbonate ions. The direct link between these changes and the continuous development of the ion activity product reveals the locus of a liquid–liquid binodal limit. The data strongly suggest that proto-structured amorphous CaCO3 forms through solidification of initially liquid precursors. Furthermore, polycarboxylates, which stabilize liquid precursors of CaCO3, significantly enhance the kinetic stability of the metastable liquid–liquid state, but they do not affect the locus of the binodal limit. The importance of water network dynamics in phase separation mechanisms can be understood based on the notions of the pre-nucleation cluster pathway, and is likely to be more general for aqueous systems. published

Whether flavonoids play significant antioxidant roles in plants challenged by photooxidative stress of different origin has been largely debated over the last few decades. A critical review of the pertinent literature and our experimentation as well, based on a free-of-scale approach, support an important antioxidant function served by flavonoids in plants exposed to a wide range of environmental stressors, the significance of which increases with the severity of stress. On the other side, some questions need conclusive answers when the putative antioxidant functions of plant flavonoids are examined at the level of both the whole-cell and cellular organelles. This partly depends upon a conclusive, robust, and unbiased definition of “a plant antioxidant”, which is still missing, and the need of considering the subcellular re-organization that occurs in plant cells in response to severe stress conditions. This likely makes our deterministic-based approach unsuitable to unveil the relevance of flavonoids as antioxidants in extremely complex biological systems, such as a plant cell exposed to an ever-changing stressful environment. This still poses open questions about how to measure the occurred antioxidant action of flavonoids. Our reasoning also evidences the need of contemporarily evaluating the changes in key primary and secondary components of the antioxidant defense network imposed by stress events of increasing severity to properly estimate the relevance of the antioxidant functions of flavonoids in an in planta situation. In turn, this calls for an in-depth analysis of the sub-cellular distribution of primary and secondary antioxidants to solve this still intricate matter.

Local and propagating vibrations occurring in DNA in the terahertz domain are essential for processes at the basis of cellular metabolism. Here we report the results of a study on the B-DNA terahertz dynamics where we combined high-resolution inelastic x-ray experiments and incoherent inelastic neutron scattering. By using two different high-hydration conditions we could study the effect of packing interactions between double helices, while by selecting Na and Cs counterions we could inspect how the mass loading affects the DNA low-frequency modes. The pattern of coherent excitation energies is well represented in terms of two branches, an acoustic-like one, with an associated propagation velocity of 3000 ± 100 m/s, and the other almost dispersionless at ~2 meV. This picture is also supported by the vibrational density of states projected on the hydrogen atoms. The acoustic-like mode is assigned to DNA excitations, despite its intriguing similarity with the analogous acoustic mode of bulk water at low wave-vector transfers. We also provide evidence for the intrahelical optic-like character of the low-energy mode, that we ascribe to large scale relative motions of DNA sub-domains.

Heterobasidion irregulare is a causal agent of root and butt-rot disease in conifers, and is native to North America. In 1944 it was introduced in central Italy in a Pinus pinea stand, where it shares the same niche with the native species Heterobasidion annosum. The introduction of a non-native pathogen may have significant negative effects on a naive host tree and the ecosystem in which it resides, requiring a better understanding of the system. We compared the spatio-temporal phenotypic, transcriptional and metabolic host responses to inoculation with the two Heterobasidion species in a large experiment with P. pinea seedlings. Differences in length of lesions at the inoculation site (IS), expression of host genes involved in lignin pathway and in cell rescue and defence, and analysis of terpenes at both IS and 12 cm above the IS (distal site, DS), were assessed at 3, 14 and 35 days post inoculation (dpi). Results clearly showed that both species elicit similar physiological and biochemical responses in P. pinea seedlings. The analysis of host transcripts and total terpenes showed differences between inoculation sites and between pathogen and mock inoculated plants. Both pathogen and mock inoculations induced antimicrobial peptide and phenylalanine ammonia-lyase overexpression at IS beginning at 3 dpi; while at DS all the analysed genes, except for peroxidase, were overexpressed at 14 dpi. A significantly higher accumulation of terpenoids was observed at 14 dpi at IS, and at 35 dpi at DS. The terpene blend at IS showed significant variation among treatments and sampling times, while no significant differences were ever observed in DS tissues. Based on our results, H. irregulare does not seem to have competitive advantages over the native species H. annosum in terms of pathogenicity towards P. pinea trees; this may explain why the non-native species has not widely spread over the 73 years since its putative year of introduction into central Italy.

Molecular mechanisms that are the base of the strategies adopted by Mediterranean plants to cope with the challenges imposed by limited or excessive solar radiation during the summer season have received limited attention. In our study, conducted on C. incanus plants growing in the shade or in full sunlight, we performed measurements of relevant physiological traits, such as leaf water potential, gas exchange and PSII photochemistry, RNA-Seq with de-novo assembly, and the analysis of differentially expressed genes. We also identified and quantified photosynthetic pigments, abscisic acid, and flavonoids. Here, we show major mechanisms regulating light perception and signaling which, in turn, sustain the shade avoidance syndrome displayed by the &lsquo, sun loving&rsquo, C. incanus. We offer clear evidence of the detrimental effects of excessive light on both the assembly and the stability of PSII, and the activation of a suite of both repair and effective antioxidant mechanisms in sun-adapted leaves. For instance, our study supports the view of major antioxidant functions of zeaxanthin in sunny plants concomitantly challenged by severe drought stress. Finally, our study confirms the multiple functions served by flavonoids, both flavonols and flavanols, in the adaptive mechanisms of plants to the environmental pressures associated to Mediterranean climate.

There is evidence that the ABA signaling pathway has greatly contributed to increase the complexity of land plants, thereby sustaining their ability to adapt in an ever-changing environment. The regulatory functions of the ABA signaling pathway go well beyond the movements of stomata and the dormancy of seeds. For instance, the ABA signaling regulates the flavonoid biosynthesis, consistent with the high integration of ABA and light signaling pathways, which occurs at the level of key signaling components, such as the bZIP transcription factors HY5 and ABI5. Here we focus on the regulation of 'colorless' (UV-absorbing) flavonol biosynthesis by the ABA signaling and, about how flavonols may regulate, in turn, the ABA signaling network. We discuss very recent findings that quercetin regulates the ABA signaling pathway, and hypothesize this might occur at the level of second messenger and perhaps of primary signaling components as well. We critically review old and recent suggestions of the primary roles played by flavonols, the ancient class of flavonoids already present in bryophytes, in the evolution of terrestrial plants. Our reasoning strongly supports the view that the ABA-flavonol relationship may represent a robust trait of land plants, and might have contributed to their adaptation on land.

The distribution of plant species around the Mediterranean basin is a product of the influence of both geographical barriers and of climatic changes experienced during the Tertiary, with the transition from a warm to cool periods. Several species, once largely distributed across the Northern Hemisphere, retracted to refugial areas in southern Europe where they are described as Tertiary relicts. Platanus orientalis is a typical representative of Tertiary flora in southwest Eurasia; its distribution spreads from the Caucasus to the Mediterranean, with its western border in Southern Italy and Sicily. We analysed genetic diversity and differentiation in the centre and western part of its distribution range using nuclear microsatellites and compared genetic parameters between core and western populations. We found an overall decrease in genetic diversity estimates (heterozygosity, private allelic richness) from central towards western populations, with those from Southern Italy and Sicily showing the lowest values. The low level of genetic diversity probably results from historic range fragmentation experienced by P. orientalis in its westernmost distribution as confirmed by high level genetic isolation of these populations. Ornamental hybrids were genetically distinguished from P. orientalis as contained private alleles, indicating that gene flow with natural populations is rare. Population assignment and neighbour-joining (NJ) analysis of populations identified four groups belonging to two main phyletic groups (the Southern Italian-Sicilian-Balkan and Cretan-Bulgarian-Turkish lineages) that seem to have different biogeographic origin and also excluded an artificial origin for southern Italian and Sicilian populations as previously suggested. These results show that quantifying the genetic variation of a Tertiary relict in a geographical context, and the potential effect of hybridization with introduced ornamental hybrids, can provide useful insights on factors influencing population genetic structure. Such information is crucial to predict how such taxa will respond to increasing anthropogenic influence on the environment., Quantifying the genetic diversity of species is important for understanding their evolutionary history. We investigated the genetic diversity of a Tertiary relict, the tree Platanus orientalis, which has an unusual distribution in the Mediterranean with its largest populations in the east, and it is increasingly rare in the west. We found an overall decrease in genetic diversity from eastern to western populations, with populations in southern Italy containing the lowest levels of genetic diversity. As P. orientalis requires habitats with high moisture levels, these results provide crucial genetic information which may influence its potential to respond to environmental change.

Skin affections after sulfur mustard (SM) exposure include erythema, blister formation and severe inflammation. An antidote or specific therapy does not exist. Anti-inflammatory compounds as well as substances counteracting SM-induced cell death are under investigation. In this study, we investigated the benzylisoquinoline alkaloide berberine (BER), a metabolite in plants like berberis vulgaris, which is used as herbal pharmaceutical in Asian countries, against SM toxicity using a well-established in vitro approach. Keratinocyte (HaCaT) mono-cultures (MoC) or HaCaT/THP-1 co-cultures (CoC) were challenged with 100, 200 or 300 mM SM for 1 h. Post-exposure, both MoC and CoC were treated with 10, 30 or 50 mu M BER for 24 h. At that time, supernatants were collected and analyzed both for interleukine (IL) 6 and 8 levels and for content of adenylate-kinase (AK) as surrogate marker for cell necrosis. Cells were lysed and nucleosome formation as marker for late apoptosis was assessed. In parallel, AK in cells was determined for normalization purposes. BER treatment did not influence necrosis, but significantly decreased apoptosis. Anti-inflammatory effects were moderate, but also significant, primarily in CoC. Overall, BER has protective effects against SM toxicity in vitro. Whether this holds true should be evaluated in future in vivo studies.

The old observation that plants preferentially synthesize flavonoids with respect to the wide range of phenylpropanoid structures when exposed to high doses of UV-B radiation has supported the view that flavonoids are primarily involved in absorbing the shortest solar wavelengths in photoprotection. However, there is compelling evidence that the biosynthesis of flavonoids is similarly upregulated in response to high photosynthetically active radiation in the presence or in the absence of UV-radiation, as well as in response to excess metal ions and photosynthetic redox unbalance. This supports the hypothesis that flavonoids may play prominent roles as scavengers of reactive oxygen species (ROS) generated by light excess. These ‘antioxidant’ functions of flavonoids appears robust, as maintained between different life kingdoms, e.g., plants and animals. The ability of flavonoids to buffer stress-induced large alterations in ROS homeostasis and, hence, to modulate the ROS-signaling cascade, is at the base of well-known functions of flavonoids as developmental regulators in both plants and animals. There is both long and very recent evidence indeed that, in plants, flavonoids may strongly affect phytohormone signaling, e.g., auxin and abscisic acid signaling. This function is served by flavonoids in a very low (nM) concentration range and involves the ability of flavonoids to inhibit the activity of a wide range of protein kinases, including but not limited to mitogen-activated protein kinases, that operate downstream of ROS in the regulation of cell growth and differentiation. For example, flavonoids inhibit the transport of auxin acting on serine–threonine PINOID (PID) kinases that regulate the localization of auxin efflux facilitators PIN-formed (PIN) proteins. Flavonoids may also determine auxin gradients at cellular and tissue levels, and the consequential developmental processes, by reducing auxin catabolism. Recent observations lead to the hypothesis that regulation/modulation of auxin transport/signaling is likely an ancestral function of flavonoids. The antagonistic functions of flavonoids on ABA-induced stomatal closure also offer novel hypotheses on the functional role of flavonoids in plant–environment interactions, in early as well as in modern terrestrial plants. Here, we surmise that the regulation of phytohormone signaling might have represented a primary function served by flavonols for the conquest of land by plants and it is still of major significance for the successful acclimation of modern terrestrial plants to a severe excess of radiant energy.

Spectroscopy in the terahertz frequency regime is a sensitive tool to probe solvation-induced effects in aqueous solutions. Yet, a systematic understanding of spectral lineshapes as a result of distinct solvation contributions remains terra incognita. We demonstrate that modularization of amino acids in terms of functional groups allows us to compute their distinct contributions to the total terahertz response. Introducing the molecular cross-correlation analysis method provides unique access to these site-specific contributions. Equivalent groups in different amino acids lead to look-alike spectral contributions, whereas side chains cause characteristic but additive complexities. Specifically, hydrophilic solvation of the zwitterionic groups in valine and glycine leads to similar terahertz responses which are fully decoupled from the side chain. The terahertz response due to H-bonding within the large hydrophobic solvation shell of valine turns out to be nearly indistinguishable from that in bulk water in direct comparison to the changes imposed by the charged functional groups that form strong H-bonds with their hydration shells. Thus, the hydrophilic groups and their solvation shells dominate the terahertz absorption difference, while on the same intensity scale, the influence of hydrophobic water can be neglected.

In CaCO 3 nucleation, the role of water remains enigmatic. Changes in THz absorption during the early stages of CaCO 3 nucleation evidence altered coupled motions of hydrated calcium and carbonate ions. By high precision THz absorption measurements we were able to follow the changes in the hydration bond dynamics during nucleation. Our THz data strongly suggest that amorphous CaCO 3 forms through solidification of initially liquid precursors. Polycarboxylates, which stabilize CaCO 3 liquid precursors, significantly enhance the kinetic stability of the metastable liquid-liquid state. The importance of water network dynamics in phase separation mechanisms as tested by THz absorption measurements using a p-Ge spectrometer is likely to be more general for aqueous systems.

Solid phases of water, such as ice (Ih) and clathrate hydrates, form characteristic hydrogen bond network motifs, such as hexagonal ice, pentagons, and dodecahedrons. The same motifs might be present in supercooled water and in the hydration structure around hydrophobes. Here, we present the characteristic low frequency fingerprints of ice (Ih), tetrahydrofuran (THF) clathrate hydrates, and tetrabutyl-ammonium bromide (TBAB) semiclathrate close to their melting point, as well as supercooled water at 266.6 K and aqueous alcohol solutions. Interestingly, we find in all these cases two characteristic resonances in the THz frequency range: at least, one intensive band in the frequency range between 190 cm−1 and 220 cm−1 which is a characteristic of a tetrahedral hydrogen bond network configuration and a second band in the frequency range between 140 cm−1 and 170 cm−1, indicating a component with weaker hydrogen bonds. For solvated alcohols, we find spectroscopic fingerprints of a clathratelike structure at 164 cm−1 as well as a tetrahedral network structure at 194 cm−1, which is close to one of ice (Ih) at 192 cm−1. We propose that in the hydration shell of hydrophobes, both structural motifs are present. In the case of supercooled water—unlike ice—only one peak was found in the frequency range between 190 cm−1 and 220 cm−1. Interestingly, the latter peak center-frequency (204 cm−1) corresponds to the average of those of the two peaks observed for ice Ih (191 cm−1 and 215 cm−1). This indicates a homogeneous intermediate hydrogen bonding, providing no evidence for any heterogeneity in two high-density and low-density phases.Solid phases of water, such as ice (Ih) and clathrate hydrates, form characteristic hydrogen bond network motifs, such as hexagonal ice, pentagons, and dodecahedrons. The same motifs might be present in supercooled water and in the hydration structure around hydrophobes. Here, we present the characteristic low frequency fingerprints of ice (Ih), tetrahydrofuran (THF) clathrate hydrates, and tetrabutyl-ammonium bromide (TBAB) semiclathrate close to their melting point, as well as supercooled water at 266.6 K and aqueous alcohol solutions. Interestingly, we find in all these cases two characteristic resonances in the THz frequency range: at least, one intensive band in the frequency range between 190 cm−1 and 220 cm−1 which is a characteristic of a tetrahedral hydrogen bond network configuration and a second band in the frequency range between 140 cm−1 and 170 cm−1, indicating a component with weaker hydrogen bonds. For solvated alcohols, we find spectroscopic fingerprints of a clathratelike structure at 16...

Photosynthetic performance and the expression of genes involved in light signaling and the biosynthesis of isoprenoids and phenylpropanoids were analysed in green ('Tigullio', TIG) and red ('Red Rubin', RR) basil. The aim was to detect the physiological and molecular response mechanisms to high sunlight. The attenuation of blue-green light by epidermal anthocyanins was shown to evoke shade-avoidance responses with consequential effects on leaf morpho-anatomical traits and gas exchange performance. Red basil had a lower mesophyll conductance, partially compensated by the less effective control of stomatal movements, in comparison with TIG. Photosynthesis decreased more in TIG than in RR in high sunlight, because of larger stomatal limitations and the transient impairment of PSII photochemistry. The methylerythritol 4-phosphate pathway promoted above all the synthesis and de-epoxidation of violaxanthin-cycle pigments in TIG and of neoxanthin and lutein in RR. This enabled the green leaves to process the excess radiant energy effectively, and the red leaves to optimize light harvesting and photoprotection. The greater stomatal closure observed in TIG than in RR was due to enhanced abscisic acid (ABA) glucose ester deglucosylation and reduced ABA oxidation, rather than to superior de novo ABA synthesis. This study shows a strong competition between anthocyanin and flavonol biosynthesis, which occurs at the level of genes regulating the oxidation of the C2-C3 bond in the dihydro-flavonoid skeleton.

International audience; Genomic resources have recently been developed for a number of species of Fagaceae, with the purpose of identifying the genetic factors underlying the adaptation of these long-lived, biologically predominant, commercially and ecologically important species to their environment. The sequencing of genomes of the size of the oak genome (740 Mb/C) is now becoming both possible and affordable due to breakthroughs in sequencing technology. However, an understanding of the composition and structure of the oak genome is required before launching a sequencing initiative. We constructed random (Rd) and hypomethylated (Hp) genomic libraries for pedunculate oak (Quercus robur) and carried out a sample sequencing of 2.33 and 2.36 Mb of shotgun DNA from the Rd and Hp libraries, respectively, to provide a first insight into the repetitive element and gene content of the oak genome. We found striking similarities between Rd sequences and previously analyzed BAC end sequences of pedunculate oak, with a similar percentage of known repeat elements (5.56%), an almost identical simple sequence repeat density (i.e., 29 SSRs per 100 kb), an identical profile of SSR motifs (in descending order of frequency—dinucleotide, pentanucleotide, trinucleotide, tetranucleotide, and hexanucleotide motifs). Conversely, the Hp fraction was, as expected, enriched in nuclear genes (2.44-fold enrichment). This enrichment was associated with a lower frequency of retrotransposons than for Rd sequences. We also identified twice as many SSR motifs in the Rd library as in the Hp library. This work provides useful information before opening a new chapter in oak genome sequencing.

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 genetics 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 2 tetraploid and 4 diploid species collected from Iceland to Japan. All individuals were genotyped at 7 to 18 nuclear simple sequence repeats (nSSRs). Pairwise F’ST 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 B. 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 (ABC) 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 (1,614 years BP, 95%CI: 561 – 4,710), with B. pendula providing a higher contribution to hybrids. This article is protected by copyright. All rights reserved.

We have developed a set of eight polymorphic nuclear microsatellite markers for the Mediterranean shrub Pistacia lentiscus by means of an enriched library method. Characterization for the eight loci was carried out on 42 individuals from two populations sampled in southern Spain. The overall number of alleles detected was 59, ranging from three to 13 per locus. Expected heterozygosity per locus and population ranged from 0.139 to 0.895. Two loci albeit only in one population (Seville) departed significantly from Hardy–Weinberg equilibrium expectations and no linkage disequilibrium between pairs of loci was detected. These markers will be used in studies of gene flow across a fragmented landscape.

• Premise of the study Pinus pinea is one of the few widespread plant species that are also genetically depauperate. It is also an important commercial species with high market value seeds. A deeper knowledge of the existing population genetic variation was needed. • Methods and Results Twelve nuclear microsatellites were isolated from genomic and cDNA sequences and screened for variability in 729 individuals from 33 natural populations. Low level of genetic variability was confirmed with average expected heterozygosity of 0.11. Hardy-Weinberg equilibrium expectations were not met in only ~10% of the possible locus/population combinations. All loci were in linkage equilibrium, and the frequency of null alleles was very low (≤ 1% in 332 out of 396 locus/ population combinations). Nine out of the 12 microsatellites were successfully transferred to P. halepensis. • Conclusions Despite low polymorphism, these new markers will be useful to resolve population structure and hold potential for seed origin identification and traceability. © 2012 Botanical Society of America.

Scots pine (Pinus sylvestris L.) is one of the most widespread forest trees in the world, ranging from southern Mediterranean mountains to eastern Siberia. Ten polymorphic microsatellite loci were isolated from Scots pine cDNA sequences and were screened for variability in three natural populations. High levels of genetic variability were observed with effective number of alleles per locus ranging from 1.0 to 4.6 and average expected heterozygosity per population of 0.79. With only two exceptions, Hardy-Weinberg expectations were confirmed. All loci were in linkage equilibrium and there was little evidence for confounding null alleles. These new markers will be used to resolve population structure and gene flow patterns in this major Eurasian forest tree. © 2011 Springer Science+Business Media B.V.

Premise of the study To characterize the level of genetic diversity and gene flow, as well as to identify unambiguously two African tropical tree species, Erythrophleum ivorense and E. suaveolens, we have developed a set of nuclear SSR (Simple Sequence Repeats) markers. Methods and Results Nine SSRs that display polymorphism in both species were identified. The nine newly developed SSR markers can be amplified in only two multiplexed reactions. Levels of polymorphism were assessed in two populations per species, yielding two to fifteen alleles per locus in E. ivorense and three to sixteen alleles per locus in E. suaveolens. Conclusions The SSR markers developed here are promising to study the spatial distribution of genetic diversity and the genetic delimitation of two Erythrophleum species from central Africa. © 2011 Botanical Society of America.

International audience; This study combines neutrality tests and environmental correlations to identify non-neutral patterns of evolution in candidate genes related to drought stress in two closely-related Mediterranean conifers, Pinus pinaster Ait. and Pinus halepensis Mill. Based on previous studies, we selected 12 amplicons covering six candidate genes that were sequenced in a large sample spanning the full range of these two species. Neutrality tests relatively robust to demography (DHEW compound test and ML-HKA test) were used to detect selection events at different temporal scales. Environmental associations between variation at candidate genes and climatic variables were also examined. These combined approaches detected distinct genes that may be targeted by selection, most of them specific to only one of the two conifers, despite their recent divergence (< 10 Ma). An exception was 4-coumarate: CoA ligase (4cl), a gene involved in the production of various important secondary products that appeared to play a role in local adaptation processes of both pines. Another remarkable result was that all significant environmental correlations involved temperature indices, highlighting the importance of this climatic factor as a selective driver on Mediterranean pines. The ability to detect natural selection at the DNA sequence level depends on the nature and the strength of the selection events, on the timescale at which they occurred and on the sensitivity of the methods to other evolutionary forces that can mimic selection (e.g. demography, population structure). Using complementary approaches can help to capture different aspects of the evolutionary processes that govern molecular variation at both intra- and interspecific levels.

5 pages; International audience; This article documents the addition of 411 microsatellite marker loci and 15 pairs of Single Nucleotide Polymorphism (SNP) sequencing primers to the Molecular Ecology Resources Database. Loci were developed for the following species: Acanthopagrus schlegeli, Anopheles lesteri, Aspergillus clavatus, Aspergillus flavus, Aspergillus fumigatus, Aspergillus oryzae, Aspergillus terreus, Branchiostoma japonicum, Branchiostoma belcheri, Colias behrii, Coryphopterus personatus, Cynogolssus semilaevis, Cynoglossus semilaevis, Dendrobium officinale, Dendrobium officinale, Dysoxylum malabaricum, Metrioptera roeselii, Myrmeciza exsul, Ochotona thibetana, Neosartorya fischeri, Nothofagus pumilio, Onychodactylus fischeri, Phoenicopterus roseus, Salvia officinalis L., Scylla paramamosain, Silene latifo, Sula sula, and Vulpes vulpes. These loci were cross-tested on the following species: Aspergillus giganteus, Colias pelidne, Colias interior, Colias meadii, Colias eurytheme, Coryphopterus lipernes, Coryphopterus glaucofrenum, Coryphopterus eidolon, Gnatholepis thompsoni, Elacatinus evelynae, Dendrobium loddigesii Dendrobium devonianum, Dysoxylum binectariferum, Nothofagus antarctica, Nothofagus dombeyii, Nothofagus nervosa, Nothofagus obliqua, Sula nebouxii, and Sula variegata. This article also documents the addition of 39 sequencing primer pairs and 15 allele specific primers or probes for Paralithodes camtschaticus.

For the isolation of the microsatellite markers, we directly sequenced a large number of clones from a non-enriched random genomic library. Briefl y, we extracted ca. 2 μ g of total genomic DNA from a single myrtle individual with the Invisorb Spin Plant Mini Kit (Invitek, Berlin, Germany) following the manufacturer ’ s protocol. Genomic DNA was sheared with a Nebulizer Kit (Invitrogen Corporation, Carlsbad, CA) applying high-pressure nitrogen to obtain DNA fragments ranging between 500 and 1000bp. Sheared DNA was repaired with the End-It DNA End-Repair Kit (Epicentre Biotechnologies, Madison, WI), and then the fragments were routinely cloned using the Zero Background Cloning Kit (Invitrogen Corporation, Carlsbad, CA). Four hundred twenty-fi ve randomly chosen clones were sequenced in the forward direction with the M13 universal primer and using the Big Dye Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) and run on an ABI 3730 DNA Analyzer (Applied Biosystems, Foster City, CA) automatic sequencer. Sequences were checked for the occurrence of di-, tri, and tetranucleotide repeats with the on-line software Sputnik (available at http://www.cbib.ubordeaux2.fr/pise/sputnik.html). Microsatellite motifs were detected in 56 (13%) of the sequences, among which half were discarded because microsatellite motifs were too short, nucleotide repeats were too close to the vector for primer design, or because the clones showed high sequence homology. We sequenced the reverse direction of the selected clones and designed 28 primer 1

International audience; Tree species are thought to be relatively resistant to habitat fragmentation because of their longevity and their aptitude for extensive gene flow, although recent empirical studies have reported negative genetic consequences, in particular after long-term habitat fragmentation in European temperate regions. Yet the response of each species to habitat loss may differ greatly depending on their biological attributes, in particular seed dispersal ability. In this study, we used demographic and molecular data to investigate the genetic consequences of chronic habitat fragmentation in remnant populations of Taxus baccata in the Montseny Mountains, northeast Spain. The age structure of populations revealed demographic bottlenecks and recruitment events associated with exploitation and management practices. We found a strong genetic structure, both at the landscape and within-population levels. We also detected high levels of inbreeding for a strictly outcrossing species. Chronic forest fragmentation resulting from long-term exploitation in the Montseny Mountains seems the most plausible explanation for the strong genetic structure observed. Our results support the view that, contrary to some predictions, tree species are not buffered from the adverse effects of habitat fragmentation, even in the case of species with a high dispersal potential.

Background Expressed Sequence Tags (ESTs) are a source of simple sequence repeats (SSRs) that can be used to develop molecular markers for genetic studies. The availability of ESTs for Quercus robur and Quercus petraea provided a unique opportunity to develop microsatellite markers to accelerate research aimed at studying adaptation of these long-lived species to their environment. As a first step toward the construction of a SSR-based linkage map of oak for quantitative trait locus (QTL) mapping, we describe the mining and survey of EST-SSRs as well as a fast and cost-effective approach (bin mapping) to assign these markers to an approximate map position. We also compared the level of polymorphism between genomic and EST-derived SSRs and address the transferability of EST-SSRs in Castanea sativa (chestnut). Results A catalogue of 103,000 Sanger ESTs was assembled into 28,024 unigenes from which 18.6% presented one or more SSR motifs. More than 42% of these SSRs corresponded to trinucleotides. Primer pairs were designed for 748 putative unigenes. Overall 37.7% (283) were found to amplify a single polymorphic locus in a reference full-sib pedigree of Quercus robur. The usefulness of these loci for establishing a genetic map was assessed using a bin mapping approach. Bin maps were constructed for the male and female parental tree for which framework linkage maps based on AFLP markers were available. The bin set consisting of 14 highly informative offspring selected based on the number and position of crossover sites. The female and male maps comprised 44 and 37 bins, with an average bin length of 16.5 cM and 20.99 cM, respectively. A total of 256 EST-SSRs were assigned to bins and their map position was further validated by linkage mapping. EST-SSRs were found to be less polymorphic than genomic SSRs, but their transferability rate to chestnut, a phylogenetically related species to oak, was higher. Conclusion We have generated a bin map for oak comprising 256 EST-SSRs. This resource constitutes a first step toward the establishment of a gene-based map for this genus that will facilitate the dissection of QTLs affecting complex traits of ecological importance.

This study represents the first large-scale analysis using nuclear molecular markers to assess genetic diversity and structure of Cupressus sempervirens L. Genetic and fossil data were combined to infer the possible role of human activity and evolutionary history in shaping the diversity of cypress populations. We analysed 30 populations with six polymorphic nuclear microsatellite markers. Dramatic reductions in heterozygosity and allelic richness were observed from east to west across the species range. Structure analysis assigned individuals to two main groups separating central Mediterranean and eastern populations. The two main groups could be further divided into five subgroups which showed the following geographical distributions Turkey with the Greek islands Rhodes and Samos, Greece (Crete), Southern Italy, Northern Italy, Tunisia with Central Italy. This pattern of genetic structure is also supported by samova and Barrier analyses. Palaeobotanical data indicated that Cupressus was present in Italy in the Pliocene, Pleistocene and Holocene. Furthermore, our molecular survey showed that Italian cypress populations experienced bottlenecks that resulted in reduced genetic diversity and allelic richness and greater genetic differentiation. Recent colonization or introduction may also have influenced levels of diversity detected in the Italian populations, as most individuals found in this range today have multilocus genotypes that are also present in the eastern range of the species. The data reveal a new interpretation of the history of cypress distribution characterized by ancient eastern populations (Turkey and Greek islands) and a mosaic of recently introduced trees and remnants of ancient, depauperate populations in the central Mediterranean range. © 2009 Blackwell Publishing Ltd.

English yew (Taxus baccata L.;Taxaceae), a Tertiary relict, provides a seminal example of a widespread albeit locally endangered (often close to extinction) tree species. In order to gain detailed insights into the evolutionary dynamics of the species on a broad geographical scale, over 1000 trees from 91 populations of English yew in the western Mediterranean were analyzed using seven nuclear microsatellite markers. Our results revealed contrasting patterns of genetic structure at different spatial scales genetic variation was highly structured at the local scale, while only a low proportion of the observed variation was attributed to regional differences. We also found a geographic gradient of decreasing diversity and increasing population divergence from northwest (central Europe and northern Iberian Peninsula) to southeast (Mediterranean Iberia and North Africa). The patterns revealed in this study probably reflect the combined effects of Quaternary climatic changes and recent impact of human activities, and potentially also more ancient events dating back to the Tertiary. Both climatic and anthropogenic factors seem to have conducted to a long history of population isolation, which may have contributed significantly to enhance population divergence through restricted gene flow and genetic drift. © 2010 Elsevier Inc. All rights reserved.

International audience; *The evolutionary outcomes of range expansion/contraction depend on the biological system considered and the interactions among the evolutionary forces in place. In this study, we examined the demographic history and the local polymorphism patterns of candidate genes linked to drought tolerance of a widespread Mediterranean conifer (Pinus halepensis). *To that end, we used cpSSRs and coalescence modelling of nuclear genes to infer the demographic history of natural populations covering the species range. Ten drought-response candidate genes were then examined for their patterns of polymorphism and tested for selection considering plausible demographic scenarios. *Our results revealed a marked loss of genetic diversity from the relictual Greek population towards the western range of the species, as well as molecular signatures of intense bottlenecks. Moreover, we found an excess of derived polymorphisms in several genes sampled in the western part of the range - a potential result of the action of natural selection on populations confronted with new environments following long-range colonization. *Wide-range expansions-contractions of forest trees are accompanied by strong selective pressures, resulting in distinct evolutionary units. This knowledge is of crucial importance for the conservation and management of forests in the face of climate change.

Seven polymorphic nuclear microsatellite markers for Taxus baccata L. (English yew) were developed using an enriched-library method. An additional polymorphic SSR was obtained by testing eight primer pairs from the congeneric species Taxus sumatrana. Mendelian inheritance for the seven Taxus baccata SSRs was proved by genotyping 17 individuals and 124 megagametophytes (conifer seed haploid tissue). A total of 96 individuals from 5 different populations (10-26 samples per population) were used to estimate genetic diversity parameters. High levels of genetic diversity, with values ranging from 0.533 to 0.929 (6-28 alleles per SSR) were found. No linkage disequilibrium between pairs of loci was detected. All loci but one showed significant departures from Hardy-Weinberg equilibrium. Excess of homozygosity was probably due to high inbreeding in English yew populations, an outcome of low effective population size and/or fragmented distribution. Highly polymorphic SSRs will be used to conduct population genetic studies at different geographical scales and to monitor gene flow. © 2008 Springer Science+Business Media B.V.

Genetic variation is generally considered a prerequisite for adaptation to new environmental conditions. Thus the discovery of genetically depauperate but geographically widespread species is unexpected. We used 12 paternally inherited chloroplast microsatellites to estimate population genetic variation across the full range of an emblematic circum-Mediterranean conifer, stone pine (Pinus pinea L.). The same chloroplast DNA haplotype is fixed in nearly all of the 34 investigated populations. Such a low level of variation is consistent with a previous report of very low levels of diversity at nuclear loci in this species. Stone pine appears to have passed through a severe and prolonged demographic bottleneck, followed by subsequent natural- and human-mediated dispersal across the Mediterranean Basin. No other abundant and widespread plant species has as little genetic diversity as P. pinea at both chloroplast and nuclear markers. However, the species harbors a nonnegligible amount of variation at adaptive traits. Thus a causal relationship between genetic diversity, as measured by marker loci, and the evolutionary precariousness of a species, cannot be taken for granted. © 2007 The Author(s).

The within-population spatial structure of genetic diversity is shaped by demographic processes, including historical accidents such as forest perturbations. Information drawn from the analysis of the spatial distribution of genetic diversity is therefore inherently linked to demographic-historical processes that ultimately determine the fate of populations. All adult trees and saplings in a 1.4-ha plot within a mixed Norway spruce (Picea abies [L.] Karst) stand were characterised by means of chloroplast (paternally inherited) markers, and a large sub-sample of these were genotyped at mitochondrial (maternally inherited) molecular markers. These data were used to analyse the spatial distribution of genetic variation and to compare the patterns corresponding to the two marker types. The plot presented non-homogeneous local stem density in the younger cohorts, and the indirect effect of this source of variation on the spatial genetic structure was investigated. Results suggest that (i) spatially limited seed dispersal induced patchiness in genotype distribution, while pollen flow had a homogenising effect; (ii) deviations from random spatial structure were stronger in the demographically most stable portions of the stand, while they were weaker where sudden bursts of regeneration occurred; (iii) spatially overlapping adult and sapling cohorts displayed the same spatial genetic structure (stronger on stable areas, weaker in portions of the stand undergoing events of intense regeneration), which was substantiated by the influence of local demographic processes. Regeneration dynamics as modulated by demography thus influences the distribution of genetic diversity within the stand both in the younger life stages and in the adult population.

Some 1339 trees from 48 Pinus pinaster stands were characterized by five chloroplast microsatellites, detecting a total of 103 distinct haplotypes. Frequencies for the 16 most abundant haplotypes (pk > 0.01) were spatially interpolated over a lattice made by 430 grid points. Fitting of spatially interpolated values on raw haplotype frequencies at the same geographical location was tested by regression analysis. A range-wide 'diversity map' based on interpolated haplotype frequencies allowed the identification of one 'hotspot' of diversity in central and southeastern Spain, and two areas of low haplotypic diversity located in the western Iberian peninsula and Morocco. Principal component analysis (PCA) carried out on haplotypes frequency surfaces allowed the construction of a colour-based 'synthetic' map of the first three PC components, enabling the detection of the main range-scale genetic trends and the identification of three main 'gene pools' for the species (i) a 'southeastern' gene pool, including southeastern France, Italy, Corsica, Sardinia, Pantelleria and northern Africa; (ii) an 'Atlantic' gene pool, including all the western areas of the Iberian peninsula; and (iii) a 'central' gene pool, located in southeastern Spain. Multivariate and amova analyses carried out on interpolated grid point frequency values revealed the existence of eight major clusters ('gene zones'), whose genetic relationships were related with the history of the species. In addition, demographic models showed more ancient expansions in the eastern and southern ranges of maritime pine probably associated to early postglacial recolonization. The delineation of the gene zones provides a baseline for designing conservation areas in this key Mediterranean pine. © 2007 The Authors.

International audience; We investigated range-wide phylogeographic variation in three European ash species (Fraxinus sp., Oleaceae). Chloroplast DNA (cpDNA) microsatellites were typed in the thermophilous Fraxinus angustifolia and Fraxinus ornus and the observed haplotypes and the geographic distribution of diversity were compared to cpDNA data previously obtained in the more cold-tolerant Fraxinus excelsior. We found wide-ranging haplotype sharing between the phylogenetically close F. angustifolia and F. excelsior, suggesting hybridization (i) in common glacial refuges in the Iberian Peninsula, northern Italy, the eastern and/or Dinaric Alps and the Balkan Peninsula, and/or (ii) during postglacial recolonization. The data allowed us to propose additional glacial refuges for F. angustifolia in southern Italy and in Turkey, and populations from the latter region were particularly polymorphic. There was evidence for refuge areas in Italy, the Balkan Peninsula and Turkey for F. ornus, which did not share any single chloroplast haplotype with the other species. In both F. angustifolia and F. ornus, cpDNA diversity (h(S) = 0.027 and h(S) = 0.009, respectively) was lower and fixation levels (G(ST) = 0.964 and G(ST) = 0.983, respectively) higher than in sympatric F. excelsior (h(S) = 0.096, G(ST) = 0.870). These diversity patterns could be due to temperature tolerance or the demographic history.

We developed 20 polymorphic nuclear microsatellites from Pinus pinaster using genomic libraries enriched with repeated motifs. A total of 25 individuals from five different populations were used to estimate genetic diversity parameters. © 2005 Blackwell Publishing Ltd.

Nothofagus (southern beech), with an 80-million-year-old fossil record, has become iconic as a plant genus whose ancient Gondwanan relationships reach back into the Cretaceous era. Closely associated with Wegener's theory of “Kontinentaldrift”, Nothofagus has been regarded as the “key genus in plant biogeography”. This paradigm has the New Zealand species as passengers on a Moa's Ark that rafted away from other landmasses following the breakup of Gondwana. An alternative explanation for the current transoceanic distribution of species seems almost inconceivable given that Nothofagus seeds are generally thought to be poorly suited for dispersal across large distances or oceans. Here we test the Moa's Ark hypothesis using relaxed molecular clock methods in the analysis of a 7.2-kb fragment of the chloroplast genome. Our analyses provide the first unequivocal molecular clock evidence that, whilst some Nothofagus transoceanic distributions are consistent with vicariance, trans-Tasman Sea distributions can only be explained by long-distance dispersal. Thus, our analyses support the interpretation of an absence of Lophozonia and Fuscospora pollen types in the New Zealand Cretaceous fossil record as evidence for Tertiary dispersals of Nothofagus to New Zealand. Our findings contradict those from recent cladistic analyses of biogeographic data that have concluded transoceanic Nothofagus distributions can only be explained by vicariance events and subsequent extinction. They indicate that the biogeographic history of Nothofagus is more complex than envisaged under opposing polarised views expressed in the ongoing controversy over the relevance of dispersal and vicariance for explaining plant biodiversity. They provide motivation and justification for developing more complex hypotheses that seek to explain the origins of Southern Hemisphere biota., A phylogenetic analysis of Nothofagus species provides evidence for their transoceanic dispersal during the Tertiary, and helps resolve the debate about the origins of plant biodiversity in the Southern Hemisphere