A novel comparative research platform designed to determine the functional significance of tree species diversity in European forests

One of the current advances in functional biodiversity research is the move away from short-lived test systems towards the exploration of diversity-ecosystem functioning relationships in structurally more complex ecosystems. In forests, assumptions about the functional significance of tree species diversity have only recently produced a new generation of research on ecosystem processes and services. Novel experimental designs have now replaced traditional forestry trials, but these comparatively young experimental plots suffer from specific difficulties that are mainly related to the tree size and longevity. Tree species diversity experiments therefore need to be complemented with comparative observational studies in existing forests. Here we present the design and implementation of a new network of forest plots along tree species diversity gradients in six major European forest types: the FunDivEUROPE Exploratory Platform. Based on a review of the deficiencies of existing observational approaches and of unresolved research questions and hypotheses, we discuss the fundamental criteria that shaped the design of our platform. Key features include the extent of the species diversity gradient with mixtures up to five species, strict avoidance of a dilution gradient, special attention to community evenness and minimal covariation with other environmental factors. The new European research platform permits the most comprehensive assessment of tree species diversity effects on forest ecosystem functioning to date since it offers a common set of research plots to groups of researchers from very different disciplines and uses the same methodological approach in contrasting forest types along an extensive environmental gradient.     

Clear‐felling effects on colonization rates of shade‐tolerant forest herbs into a post‐agricultural forest adjacent to ancient forest

Question: Does clear‐felling influence forest herb colonization into post‐agricultural forest? Location: A stand of poplar cultivars with a dense understorey of Acer pseudoplatanus in Muizen forest (northern Belgium), planted in 1952 on farmland adjacent to ancient forest and clear‐felled in 1997. Methods: Shade‐tolerant forest herbs were surveyed in 112 grid‐based sample plots: just before clear‐felling, and 5 and 10 yr afterwards. Shade‐tolerant herbs were subdivided into ancient forest species (AFS) and other shade‐tolerant species (OSS). Effects of clear‐felling on species number per plot, total cover per plot and colonization rate of species groups were compared using non‐parametrical tests. Species number per plot was modelled by means of generalized linear mixed models (GLMMs), with inventory time, distance to the nearest parcel edge, and cover of light‐loving species (LS) as explanatory variables. The C‐S‐R signature (competitive, stress‐tolerant and ruderal strategies, respectively) shift of sample plots was calculated on the selected shade‐tolerant species. Results: Frequency of most species increased during the 10‐yr period. Number of OSS increased more and faster than that of AFS. OSS increased to the level of the adjacent forest, but was lower where LS cover remained high. There was a positive correlation between the change of the colonization rate and the competitive plant strategy. Conclusions: We assume that clear‐felling stimulated generative reproduction of shade‐tolerant herbs, whereas quickly emerging woody species controlled competitive exclusion by LS. Succession of dark and light phases, such as provided by an understorey managed as a coppice, could promote colonization of shade‐tolerant herbs into post‐agricultural forest.     

Discovery and characterization of artifactual mutations in deep coverage targeted capture sequencing data due to oxidative DNA damage during sample preparation

As researchers begin probing deep coverage sequencing data for increasingly rare mutations and subclonal events, the fidelity of next generation sequencing (NGS) laboratory methods will become increasingly critical. Although error rates for sequencing and polymerase chain reaction (PCR) are well documented, the effects that DNA extraction and other library preparation steps could have on downstream sequence integrity have not been thoroughly evaluated. Here, we describe the discovery of novel C > A/G > T transversion artifacts found at low allelic fractions in targeted capture data. Characteristics such as sequencer read orientation and presence in both tumor and normal samples strongly indicated a non-biological mechanism. We identified the source as oxidation of DNA during acoustic shearing in samples containing reactive contaminants from the extraction process. We show generation of 8-oxoguanine (8-oxoG) lesions during DNA shearing, present analysis tools to detect oxidation in sequencing data and suggest methods to reduce DNA oxidation through the introduction of antioxidants. Further, informatics methods are presented to confidently filter these artifacts from sequencing data sets. Though only seen in a low percentage of reads in affected samples, such artifacts could have profoundly deleterious effects on the ability to confidently call rare mutations, and eliminating other possible sources of artifacts should become a priority for the research community.     

Macromolecular mass spectrometry and electron microscopy as complementary tools for investigation of the heterogeneity of bacteriophage portal assemblies

The success of electron-cryo microscopy (cryo-EM) and image reconstruction of cyclic oligomers, such as the viral and bacteriophage portals, depends on the accurate knowledge of their order of symmetry. A number of statistical methods of image analysis address this problem, but often do not provide unambiguous results. Direct measurement of the oligomeric state of multisubunit protein assemblies is difficult when the number of subunits is large and one subunit renders only a small increment to the full size of the oligomer. Moreover, when mixtures of different stochiometries are present techniques such as analytical centrifugation or size-exclusion chromatography are also less helpful. Here, we use electrospray ionization mass spectrometry to directly determine the oligomeric states of the in vitro assembled portal oligomers of the phages P22, Phi-29 and SPP1, which range in mass from 430 kDa to about 1 million Da. Our data unambiguously reveal that the oligomeric states of Phi-29 and SPP1 portals were 12 and 13, respectively, in good agreement with crystallographic and electron microscopy data. However, in vitro assembled P22 portals were a mixture of 11- and 12-mer species in an approximate ratio of 2:1, respectively. A subsequent reference-free alignment of electron microscopy images of the P22 portal confirmed this mixture of oligomeric states. We conclude that macromolecular mass spectrometry is a valuable tool in structural biology that can aide in the determination of oligomeric states and symmetry of assemblies, providing a good starting point for improved image analysis of cryo-EM data.