Integrated biological and chemical characterisation of a pair of leonardesque canal lock gates.

The Museo Nazionale della Scienza e della Tecnologia "Leonardo da Vinci" in Milan is exposing two pairs of canal lock gates, used to control the water flow in Milan canal system, whose design appears in the Leonardo's Codex Atlanticus. The wood present in the gates has been deeply characterised by mean of a multidisciplinary investigation involving i) DNA barcoding of wood fragments; ii) microbial community characterisation, and iii) chemical analyses. DNA barcoding revealed that two fragments of the gates belonged to wood species widely used in the middle age: Fagus sylvatica and Picea abies. The chemical characterisations were based on the use of ionic liquid as dissolving medium in order to analyse the entire cell wall material by means of Gel Permeation Chromatography (GPC) and 2D-NMR-HSQC techniques. This multidisciplinary analytical approach was able to highlight the complex nature of the degradation occurred during the gate operation (XVI-XVIII centuries): an intricate interplay between microbial populations (i.e. Shewanella), inorganic factors (i.e. iron from nails), physical factors and the lignocellulosic material.

Effects of root and leaf litter identity and diversity on oribatid mite abundance, species richness and community composition.

Habitat heterogeneity is an important driver of aboveground species diversity but few studies have investigated effects on soil communities. Trees shape their surrounding by both leaf litter and roots generating small scale heterogeneity and potentially governing community patterns of soil organisms. To assess the role of vegetation for the soil fauna, we studied whether tree species (Fagus sylvatica L., Acer pseudoplatanus L., Fraxinus excelsior L., Tilia cordata Mill.), markedly differing in leaf litter quality and root associated mycorrhizal symbionts, affect oribatid mite communities by shaping below- and aboveground resources and habitat complexity and availability. Oribatid mite abundance, species richness, community structure and the proportion of litter living and parthenogenetic individuals were analyzed and related to microbial biomass and the amount of remaining litter mass. Although leaf litter species with higher nutritional values decomposed considerably faster, microbial biomass only slightly differed between leaf litter species. Neither root species nor leaf litter species affected abundance, species richness or community structure of oribatid mites. However, root species had an effect on the proportion of parthenogenetic individuals with increased proportions in the presence of beech roots. Overall, the results suggest that identity and diversity of vegetation via leaf litter or roots are of minor importance for structuring oribatid mite communities of a temperate forest ecosystem.

[Beech (Fagus sylvatica) germination and seedling growth under climatic and allelopathic constraints]. / Germination et croissance des plantules de hêtre (Fagus sylvatica) sous contraintes climatiques et allélopathiques.

Recruitment is a key process for forest sustainability, especially in warm margin of distribution area. The influence of climate (temperate or warm), of soil water availability, and of allelopathic interactions from different forest species have been tested on the germination of Fagus sylvatica in controlled climatic conditions. Germination rates of non-dormant Fagus seeds were improved by relatively warm temperatures (20°C), but reversibly stopped under heat constraint (27°C). The relative growth rate of Fagus seedlings was better under temperate climatic conditions. Foliar extracts of Hedera helix showed the highest allelopathic effect on Fagus recruitment, especially in temperate conditions. Our results suggest a limitation of Fagus recruitment in warm margin of its distribution area, and a modulation of recruitment success according to the identity of plant neighbourhood.

Microsatellite primers for the endangered beech tree, Fagus hayatae (Fagaceae).

PREMISE OF THE STUDY: Microsatellite primers were developed for the endangered species Fagus hayatae (Fagaceae) to investigate the genetic diversity of the population and to investigate species delimitation within Fagus. METHODS AND RESULTS: Ten polymorphic simple sequence repeat markers were developed for F. hayatae using a magnetic bead enrichment method. The primers amplified trinucleotides, hexanucleotides, and complex repeats with six to 16 alleles per locus. The observed and expected heterozygosities across loci varied, with a range of 0.05-0.71 and 0.63-0.91, respectively. Most of the primers also amplified DNA from F. crenata, F. grandifolia, F. japonica, F. longipetiolata, F. lucida, F. orientalis, and F. sylvatica. CONCLUSIONS: These results indicate that these polymorphic loci of F. hayatae will be potentially useful for future studies of the population genetic diversity within the species. In addition, the interspecific amplification indicates that these transferable microsatellite markers will also be useful for future phylogeographic and speciation studies among close Fagus species.

Wide variation in spatial genetic structure between natural populations of the European beech (Fagus sylvatica) and its implications for SGS comparability.

Identification and quantification of spatial genetic structure (SGS) within populations remains a central element of understanding population structure at the local scale. Understanding such structure can inform on aspects of the species' biology, such as establishment patterns and gene dispersal distance, in addition to sampling design for genetic resource management and conservation. However, recent work has identified that variation in factors such as sampling methodology, population characteristics and marker system can all lead to significant variation in SGS estimates. Consequently, the extent to which estimates of SGS can be relied on to inform on the biology of a species or differentiate between experimental treatments is open to doubt. Following on from a recent report of unusually extensive SGS when assessed using amplified fragment length polymorphisms in the tree Fagus sylvatica, we explored whether this marker system led to similarly high estimates of SGS extent in other apparently similar populations of this species. In the three populations assessed, SGS extent was even stronger than this previously reported maximum, extending up to 360 m, an increase in up to 800% in comparison with the generally accepted maximum of 30-40 m based on the literature. Within this species, wide variation in SGS estimates exists, whether quantified as SGS intensity, extent or the Sp parameter. Consequently, we argue that greater standardization should be applied in sample design and SGS estimation and highlight five steps that can be taken to maximize the comparability between SGS estimates.

Multiplexed microsatellite markers for genetic studies of beech.

European beech (Fagus sylvatica L.) is one of the economically most important broadleaved tree species in Europe and has become a model for studying climate change effects on forests. Multiplex PCR of microsatellites is a fast and cost-effective technique allowing high-throughput genotyping. Here we present the procedure used to develop two multiplex kits (8-plexes) for European beech. We paid particular attention to quality control throughout all steps of the multiplex kits development (null allele detection, error rate measurements, linkage disequilibrium). Preliminary assays suggest that the 16 amplified loci are largely devoid of null alleles and allow rapid and cost-effective genotyping of beech with low error rates. The two kits, which differ in their levels of polymorphism, most likely due to marker origin, were also informative in seven other beech species tested.

Testing the impact of calibration on molecular divergence times using a fossil-rich group: the case of Nothofagus (Fagales).

Although temporal calibration is widely recognized as critical for obtaining accurate divergence-time estimates using molecular dating methods, few studies have evaluated the variation resulting from different calibration strategies. Depending on the information available, researchers have often used primary calibrations from the fossil record or secondary calibrations from previous molecular dating studies. In analyses of flowering plants, primary calibration data can be obtained from macro- and mesofossils (e.g., leaves, flowers, and fruits) or microfossils (e.g., pollen). Fossil data can vary substantially in accuracy and precision, presenting a difficult choice when selecting appropriate calibrations. Here, we test the impact of eight plausible calibration scenarios for Nothofagus (Nothofagaceae, Fagales), a plant genus with a particularly rich and well-studied fossil record. To do so, we reviewed the phylogenetic placement and geochronology of 38 fossil taxa of Nothofagus and other Fagales, and we identified minimum age constraints for up to 18 nodes of the phylogeny of Fagales. Molecular dating analyses were conducted for each scenario using maximum likelihood (RAxML + r8s) and Bayesian (BEAST) approaches on sequence data from six regions of the chloroplast and nuclear genomes. Using either ingroup or outgroup constraints, or both, led to similar age estimates, except near strongly influential calibration nodes. Using "early but risky" fossil constraints in addition to "safe but late" constraints, or using assumptions of vicariance instead of fossil constraints, led to older age estimates. In contrast, using secondary calibration points yielded drastically younger age estimates. This empirical study highlights the critical influence of calibration on molecular dating analyses. Even in a best-case situation, with many thoroughly vetted fossils available, substantial uncertainties can remain in the estimates of divergence times. For example, our estimates for the crown group age of Nothofagus varied from 13 to 113 Ma across our full range of calibration scenarios. We suggest that increased background research should be made at all stages of the calibration process to reduce errors wherever possible, from verifying the geochronological data on the fossils to critical reassessment of their phylogenetic position.

Unexpected presence of Fagus orientalis complex in Italy as inferred from 45,000-year-old DNA pollen samples from Venice lagoon.

BACKGROUND: Phylogeographic analyses on the Western Euroasiatic Fagus taxa (F. orientalis, F. sylvatica, F. taurica and F. moesiaca) is available, however, the subdivision of Fagus spp. is unresolved and there is no consensus on the phylogeny and on the identification (both with morphological than molecular markers) of Fagus Eurasiatic taxa. For the first time molecular analyses of ancient pollen, dated at least 45,000 years ago, were used in combination with the phylogeny analysis on current species, to identify the Fagus spp. present during the Last Interglacial period in Italy. In this work we aim at testing if the trnL-trnF chloroplast DNA (cpDNA) region, that has been previously proved efficient in discriminating different Quercus taxa, can be employed in distinguishing the Fagus species and in identifying the ancient pollen. RESULTS: 86 populations from 4 Western Euroasistic taxa were sampled, and sequenced for the trnL-trnF region to verify the efficiency of this cpDNA region in identifying the Fagus spp.. Furthermore, Fagus crenata (2 populations), Fagus grandifolia (2 populations), Fagus japonica, Fagus hayatae, Quercus species and Castanea species were analysed to better resolve the phylogenetic inference. Our results show that this cpDNA region harbour some informative sites that allow to infer relationships among the species within the Fagaceae family. In particular, few specific and fixed mutations were able to discriminate and identify all the different Fagus species. Considering a short fragment of 176 base pairs within the trnL intron, 2 transversions were found able in distinguishing the F. orientalis complex taxa (F. orientalis, F. taurica and F. moesiaca) from the remaining Fagus spp. (F. sylvatica, F. japonica, F. hayataea, F. crenata and F. grandifolia). This permits to analyse this fragment also in ancient samples, where DNA is usually highly degraded. The sequences data indicate that the DNA recovered from ancient pollen belongs to the F. orientalis complex since it displays the informative sites characteristic of this complex. CONCLUSION: The ancient DNA sequences demonstrate for the first time that, in contrast to current knowledge based on palynological and macrofossil data, the F. orientalis complex was already present during the Tyrrhenian period in what is now the Venice lagoon (Italy). This is a new and important insight considering that nowadays West Europe is not the natural area of Fagus orientalis complex, and up to now nobody has hypothesized the presence during the Last Interglacial period of F. orientalis complex in Italy.

[Use of ISSR-markers for determination of convergent evolutionary relations in Fagus genus]. / Vykorystannia ISSR-markeriv dlia vstanovlennia konverhentnykh evoliutsiinykh zv'iazkiv rodu Fagus.

Genetic relationships between members of Fagus genus were assessed using ISSR markers and amplification. The taxonomic status of Fagus sylvatica L. and Fagus orientalis LYPSKY. species in Ukraine has been ascertained more precisely. Intraspecies mean genetic distances were compared according to Nei & Li and respective dendrogram was constructed with the complete joining method.