Aging at Evolutionary Crossroads: Longitudinal Gene Co-expression Network Analyses of Proximal and Ultimate Causes of Aging in Bats.

How, when, and why do organisms, their tissues, and their cells age remain challenging issues, although researchers have identified multiple mechanistic causes of aging, and three major evolutionary theories have been developed to unravel the ultimate causes of organismal aging. A central hypothesis of these theories is that the strength of natural selection decreases with age. However, empirical evidence on when, why, and how organisms age is phylogenetically limited, especially in natural populations. Here, we developed generic comparisons of gene co-expression networks that quantify and dissect the heterogeneity of gene co-expression in conspecific individuals from different age-classes to provide topological evidence about some mechanical and fundamental causes of organismal aging. We applied this approach to investigate the complexity of some proximal and ultimate causes of aging phenotypes in a natural population of the greater mouse-eared bat Myotis myotis, a remarkably long-lived species given its body size and metabolic rate, with available longitudinal blood transcriptomes. M. myotis gene co-expression networks become increasingly fragmented with age, suggesting an erosion of the strength of natural selection and a general dysregulation of gene co-expression in aging bats. However, selective pressures remain sufficiently strong to allow successive emergence of homogeneous age-specific gene co-expression patterns, for at least 7 years. Thus, older individuals from long-lived species appear to sit at an evolutionary crossroad: as they age, they experience both a decrease in the strength of natural selection and a targeted selection for very specific biological processes, further inviting to refine a central hypothesis in evolutionary aging theories.

Consecutive action of two BAHD acyltransferases promotes tetracoumaroyl spermine accumulation in chicory.

Fully substituted phenolamide accumulation in the pollen coat of Eudicotyledons is a conserved evolutionary chemical trait. Interestingly, spermidine derivatives are replaced by spermine derivatives as the main phenolamide accumulated in the Asteraceae family. Here, we show that the full substitution of spermine in chicory (Cichorium intybus) requires the successive action of two enzymes, that is spermidine hydroxycinnamoyl transferase-like proteins 1 and 2 (CiSHT1 and CiSHT2), two members of the BAHD enzyme family. Deletion of these genes in chicory using CRISPR/Cas9 gene editing technology evidenced that CiSHT2 catalyzes the first N-acylation steps, whereas CiSHT1 fulfills the substitution to give rise to tetracoumaroyl spermine. Additional experiments using Nicotiana benthamiana confirmed these findings. Expression of CiSHT2 alone promoted partially substituted spermine accumulation, and coexpression of CiSHT2 and CiSHT1 promoted synthesis and accumulation of the fully substituted spermine. Structural characterization of the main product of CiSHT2 using nuclear magnetic resonance revealed that CiSHT2 preferentially catalyzed N-acylation of secondary amines to form N5,N10-dicoumaroyl spermine, whereas CiSHT1 used this substrate to synthesize tetracoumaroyl spermine. We showed that spermine availability may be a key determinant toward preferential accumulation of spermine derivatives over spermidine derivatives in chicory. Our results reveal a subfunctionalization among the spermidine hydroxycinnamoyl transferase that was accompanied by a modification of free polyamine metabolism that has resulted in the accumulation of this new phenolamide in chicory and most probably in all Asteraceae. Finally, genetically engineered yeast (Saccharomyces cerevisiae) was shown to be a promising host platform to produce these compounds.

Alignment-free inference of hierarchical and reticulate phylogenomic relationships.

We are amidst an ongoing flood of sequence data arising from the application of high-throughput technologies, and a concomitant fundamental revision in our understanding of how genomes evolve individually and within the biosphere. Workflows for phylogenomic inference must accommodate data that are not only much larger than before, but often more error prone and perhaps misassembled, or not assembled in the first place. Moreover, genomes of microbes, viruses and plasmids evolve not only by tree-like descent with modification but also by incorporating stretches of exogenous DNA. Thus, next-generation phylogenomics must address computational scalability while rethinking the nature of orthogroups, the alignment of multiple sequences and the inference and comparison of trees. New phylogenomic workflows have begun to take shape based on so-called alignment-free (AF) approaches. Here, we review the conceptual foundations of AF phylogenetics for the hierarchical (vertical) and reticulate (lateral) components of genome evolution, focusing on methods based on k-mers. We reflect on what seems to be successful, and on where further development is needed.

Efficient Genome Editing Using CRISPR/Cas9 Technology in Chicory.

CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated with protein CAS9) is a genome-editing tool that has been extensively used in the last five years because of its novelty, affordability, and feasibility. This technology has been developed in many plant species for gene function analysis and crop improvement but has never been used in chicory (Cichorium intybus L.). In this study, we successfully applied CRISPR/Cas9-mediated targeted mutagenesis to chicory using Agrobacterium rhizogenes-mediated transformation and protoplast transfection methods. A U6 promoter (CiU6-1p) among eight predicted U6 promoters in chicory was selected to drive sgRNA expression. A binary vector designed to induce targeted mutations in the fifth exon of the chicory phytoene desaturase gene (CiPDS) was then constructed and used to transform chicory. The mutation frequency was 4.5% with the protoplast transient expression system and 31.25% with A. rhizogenes-mediated stable transformation. Biallelic mutations were detected in all the mutant plants. The use of A. rhizogenes-mediated transformation seems preferable as the regeneration of plants is faster and the mutation frequency was shown to be higher. With both transformation methods, foreign DNA was integrated in the plant genome. Hence, selection of vector (transgene)-free segregants is required. Our results showed that genome editing with CRISPR/Cas9 system can be efficiently used with chicory, which should facilitate and accelerate genetic improvement and functional biology.

A BAHD neofunctionalization promotes tetrahydroxycinnamoyl spermine accumulation in the pollen coat of the Asteraceae family.

In eudicotyledons, accumulation of trihydroxycinnamoyl spermidine that is restricted to the pollen wall constitutes an evolutionary conserved trait. However, the role of this compound, which is synthetized by the BAHD enzyme spermidine hydroxycinnamoyl transferase (SHT), is still a matter of debate. Here, we show that this particular phenolamide is replaced by tetrahydroxycinnamoyl spermine in the pollen coat of the Asteraceae. Phylogenetic analyses combined with quantitative RT-PCR experiments allowed the identification of two homologous genes from Cichorium intybus (chicory) putatively involved in its metabolism. In vitro biochemical characterization of the two enzymes, named CiSHT1 and CiSHT2, confirmed the capability of recombinant proteins to synthesize spermine as well as spermidine derivatives. The wild-type metabolic phenotype was partially restored in an Arabidopsis sht mutant expressing CiSHT2. Strikingly, the transgenic plants also accumulated spermine derivatives that were absent in the wild-type. Overexpression of CiSHT2 in chicory hairy roots led to the accumulation of spermine derivatives, confirming its in vivo function. Complementary sequence analyses revealed the presence of an amino acid motif typical of the SHTs among the BAHD enzyme family. Our results highlight a recent neofunctionalization among the SHTs that has promoted the emergence of new phenolamides in the Asteraceae, which could potentially have contributed to the evolutionary success of this family.

Molecular phylogenetics before sequences: oligonucleotide catalogs as k-mer spectra.

From 1971 to 1985, Carl Woese and colleagues generated oligonucleotide catalogs of 16S/18S rRNAs from more than 400 organisms. Using these incomplete and imperfect data, Carl and his colleagues developed unprecedented insights into the structure, function, and evolution of the large RNA components of the translational apparatus. They recognized a third domain of life, revealed the phylogenetic backbone of bacteria (and its limitations), delineated taxa, and explored the tempo and mode of microbial evolution. For these discoveries to have stood the test of time, oligonucleotide catalogs must carry significant phylogenetic signal; they thus bear re-examination in view of the current interest in alignment-free phylogenetics based on k-mers. Here we consider the aims, successes, and limitations of this early phase of molecular phylogenetics. We computationally generate oligonucleotide sets (e-catalogs) from 16S/18S rRNA sequences, calculate pairwise distances between them based on D 2 statistics, compute distance trees, and compare their performance against alignment-based and k-mer trees. Although the catalogs themselves were superseded by full-length sequences, this stage in the development of computational molecular biology remains instructive for us today.

Marker-based C-arm self-calibration with unknown calibration pattern.

BACKGROUND: Accurate tomographic reconstructions require the knowledge of the actual acquisition geometry. Many mobile C-arm CT scanners have poorly reproducible acquisition geometries and thus need acquisition-specific calibration procedures. Most of geometric self-calibration methods based on projection data either need prior information or are limited to the estimation of a low number of geometric calibration parameters. Other self-calibration methods generally use a calibration pattern with known geometry and are hardly implementable in practice for clinical applications. PURPOSE: We present a three-step marker based self-calibration method which does not require the prior knowledge of the calibration pattern and thus enables the use of calibration patterns with arbitrary markers positions. METHODS: The first step of the method aims at detecting the set of markers of the calibration pattern in each projection of the CT scan and is performed using the YOLO (You Only Look Once) Convolutional Neural Network. The projected marker trajectories are then estimated by a sequential projection-wise marker association scheme based on the Linear Assignment Problem which uses Kalman filters to predict the markers 2D positions in the projections. The acquisition geometry is finally estimated from the marker trajectories using the Bundle-adjustment algorithm. RESULTS: The calibration method has been tested on realistic simulated images of the ICRP (International Commission on Radiological Protection) phantom, using calibration patterns with 10 and 20 markers. The backprojection error was used to evaluate the self-calibration method and exhibited sub-millimeter errors. Real images of two human knees with 10 and 30 markers calibration patterns were then used to perform a qualitative evaluation of the method, which showed a remarkable artifacts reduction and bone structures visibility improvement. CONCLUSIONS: The proposed calibration method gave promising results that pave the way to patient-specific geometric self-calibrations in clinics.

Leveraging historical data to optimize the number of covariates and their explained variance in the analysis of randomized clinical trials.

The amount of data collected from patients involved in clinical trials is continuously growing. All baseline patient characteristics are potential covariates that could be used to improve clinical trial analysis and power. However, the limited number of patients in phases I and II studies restricts the possible number of covariates included in the analyses. In this paper, we investigate the cost/benefit ratio of including covariates in the analysis of clinical trials with a continuous outcome. Within this context, we address the long-running question "What is the optimum number of covariates to include in a clinical trial?" To further improve the benefit/cost ratio of covariates, historical data can be leveraged to pre-specify the covariate weights, which can be viewed as the definition of a new composite covariate. Here we analyze the use of a composite covariate to improve the estimated treatment effect in small clinical trials. A composite covariate limits the loss of degrees of freedom and the risk of overfitting.

Inferring Phylogenomic Relationship of Microbes Using Scalable Alignment-Free Methods.

Inferring phylogenetic relationships among hundreds or thousands of microbial genomes is an increasingly common task. The conventional phylogenetic approach adopts multiple sequence alignment to compare gene-by-gene, concatenated multigene or whole-genome sequences, from which a phylogenetic tree would be inferred. These alignments follow the implicit assumption of full-length contiguity among homologous sequences. However, common events in microbial genome evolution (e.g., structural rearrangements and genetic recombination) violate this assumption. Moreover, aligning hundreds or thousands of sequences is computationally intensive and not scalable to the rate at which genome data are generated. Therefore, alignment-free methods present an attractive alternative strategy. Here we describe a scalable alignment-free strategy to infer phylogenetic relationships using complete genome sequences of bacteria and archaea, based on short, subsequences of length k (k-mers). We describe how this strategy can be extended to infer evolutionary relationships beyond a tree-like structure, to better capture both vertical and lateral signals of microbial evolution.

Effect of a microemulsion system on hapten-peptide reactivity studies: examples of hydroxycitronellal and citral, fragrance skin sensitizers, with glutathione.

In chemico methods, based on the assessment of hapten reactivity toward peptides, have been proposed as alternative methods for the assessment of the skin sensitizing potential of chemicals. However, even if these approaches seem very promising, a major drawback inherent to most in vitro methods is the poor water solubility of many organic molecules in aqueous media. Thus, semiorganic media based on buffer solutions and organic cosolvents such as ethanol or acetonitrile have been proposed, but a narrow equilibrium should be found between the peptide and chemical solubilities. Microemulsions have been shown to be very valuable when reacting a lipophilic organic compound soluble in hydrophobic media with a very hydrophilic organic substance insoluble in most organic solvents. However, the reaction rate between polar and apolar reactants can be influenced, in some cases, by the use of microemulsions. On the basis of NMR experiments, we have compared the reactivity of hydroxycitronellal 1 and citral 2, two weak fragrance sensitizers of major clinical relevance, toward glutathione used as a model nucleophile in a water/acetonitrile 2:1 mixture and in a microemulsion based on chloroform/water/tert-butanol/sodium dodecylsulphate. Hydroxycitronellal and citral were found to react with the thiol group of glutathione to form, in both media, identical adducts, but the observed reaction rates were found to be different. In the case of hydroxycitronellal, the observed reaction rate of glutathione addition on the aldehyde was found to be about three times higher in the microemulsion compared to the classical semiorganic mixture. In the case of citral, the situation was more complex as the Michael addition of glutathione on the conjugated double bond was found to be significantly faster in the classical semiorganic mixture, while the subsequent reaction of a second glutathione molecule on the aldehyde was found to be faster in the microemulsion. This chloroform/water/tert-butanol/sodium dodecylsulphate microemulsion, apparently of the bicontinuous type according to DOSY data, could be of potential interest for the in chemico evaluation of lipophilic chemicals toward peptides to solve solubility problems even if the impact on the chemical rate needs to be further investigated.

The Distribution of Genes Associated With Regulated Cell Death Is Decoupled From the Mitochondrial Phenotypes Within Unicellular Eukaryotic Hosts.

Genetically regulated cell death (RCD) occurs in all domains of life. In eukaryotes, the evolutionary origin of the mitochondrion and of certain forms of RCD, in particular apoptosis, are thought to coincide, suggesting a central general role for mitochondria in cellular suicide. We tested this mitochondrial centrality hypothesis across a dataset of 67 species of protists, presenting 5 classes of mitochondrial phenotypes, including functional mitochondria, metabolically diversified mitochondria, functionally reduced mitochondria (Mitochondrion Related Organelle or MRO) and even complete absence of mitochondria. We investigated the distribution of genes associated with various forms of RCD. No homologs for described mammalian regulators of regulated necrosis could be identified in our set of 67 unicellular taxa. Protists with MRO and the secondarily a mitochondriate Monocercomonoides exilis display heterogeneous reductions of apoptosis gene sets with respect to typical mitochondriate protists. Remarkably, despite the total lack of mitochondria in M. exilis, apoptosis-associated genes could still be identified. These same species of protists with MRO and M. exilis harbored non-reduced autophagic cell death gene sets. Moreover, transiently multicellular protist taxa appeared enriched in apoptotic and autophagy associated genes compared to free-living protists. This analysis suggests that genes associated with apoptosis in animals and the presence of the mitochondria are significant yet non-essential biological components for RCD in protists. More generally, our results support the hypothesis of a selection for RCD, including both apoptosis and autophagy, as a developmental mechanism linked to multicellularity.

Online mobile C-arm calibration using inertial sensors: a preliminary study in order to achieve CBCT.

PURPOSE: Cone beam computed tomography (CBCT) became increasingly popular over the last years. It allows more accurate diagnosis and treatment planning with a lower effective radiation dose. However, volume reconstruction algorithms require a very precise knowledge of the imaging geometry. Due to mechanical instabilities, mobile C-arms are incompatible with existing tomography algorithms. Therefore, C-arm online calibration is essential in order to achieve an accurate volume reconstruction. METHODS: We present an online calibration method for mobile C-arms. It is based on tracking the detector and the X-ray source of the C-arm using three-axis gyroscopes and accelerometers. It aims to be precise and noninvasive. The performance of the calibration algorithm is evaluated in regard to the precision of the sensors and to whether or not dynamic models are considered. In addition, we present an algorithm which propagate the errors from the positions and orientations estimates to the 2D projections on the detector plane. Thus, we can evaluate the impact of the estimation errors on the acquired images. RESULTS: The experiments are conducted on an experimental C-arm. The reached accuracy is [Formula: see text] for orientation and [Formula: see text] for position. These errors propagate as an error of [Formula: see text] for the 2D projections on the detector plane. CONCLUSIONS: The proposed calibration algorithm achieves an accuracy comparable to the precision of existing calibration methods. The required angle accuracy by CBCT algorithms is reached. However, improvements are needed to achieve the required position precision. The in-plane translations of the X-ray source and the detector are the most crucial parameters to estimate in order to conduct CBCT on mobile C-arms.

MeJA Elicitation of Chicory Hairy Roots Promotes Efficient Increase of 3,5-diCQA Accumulation, a Potent Antioxidant and Antibacterial Molecule.

Cichorium intybus L. (Asteraceae) is an important industrial crop, as well as a medicinal plant which produces some bioactive compounds implicated in various biological effects with potential applications in human health. Particularly, roots produce hydroxycinnamic acids like 5-caffeoyquinic acid and 3,5-dicaffeoylquinic acid (di-CQA). The present investigation relates to the use of methyl jasmonate for enhancing phenolic compounds accumulation and production in hairy root cultures of C. intybus. Elicitated hairy root growth rate increased 13.3 times compared with the initial inoculum in a period of 14 days and di-CQA production represented about 12% of DW. The elicitation has also promoted the production of tricaffeoylquinic acid never described in the chicory roots and identified as 3,4,5-tricaffeoyquinic acid by means of nuclear magnetic resonance. Our study confirmed the strong anti-oxidant effect of di-CQA. Our results also confirmed globally a selectivity of action of di-CQA against Gram-positive bacteria, in particular against some strains of Staphylococcus and Streptococcus. However, a non-negligible antibacterial activity of di-CQA against Pseudomonas aeruginosa was also underlined (MIC = 0.156 mg.mL-1 against some P. aeruginosa strains). The influence of di-CQA has been explored to evaluate its impact on the physiology of P. aeruginosa. Di-CQA showed no effect on the biofilm formation and the production of extracellular pyocyanin. However, it demonstrated an effect on virulence through the production of pyoverdine with a dose-dependent manner by more than 7-fold when treated at a concentration of 128 µg·mL-1, thus suggesting a link between di-CQA and iron sequestration. This study shows that elicitated hairy root cultures of chicory can be developed for the production of di-CQA, a secondary metabolite with high antibacterial potential.

Is a low content in atranol/chloroatranol safe in oak moss-sensitized individuals?

BACKGROUND: Chloroatranol and atranol are degradation products of chloroatranorin and atranorin, respectively, and have been identified as important contact allergens in oak moss absolute (Evernia prunastri). OBJECTIVES: To investigate whether chemically modified extracts of oak moss produce positive patch test reactions in previously sensitized subjects. METHODS: A sample of oak moss was treated by a polymer-based method to reduce the content of the two main allergens, namely atranol and chloroatranol, from 3.4% to less than 75 p.p.m. and from 1.8% to less than 25 p.p.m., respectively. Fourteen subjects with positive reactions to oak moss from Trolab and/or Chemotechnique were patch tested to this sample, diluted 1% in petrolatum. RESULTS: The chemically modified sample reacted negatively in six but gave still positive reactions in eight subjects, with the same intensity as the commercially available oak moss patch test materials. CONCLUSIONS: Polymer-based treatment of oak moss extract reduces the allergenic elicitation potential in previously sensitized individuals only to a minor extent. The residual concentrations of atranol and chloroatranol being less than 75 p.p.m. and 25 p.p.m., respectively, are unsafe for the consumer.

The diversity of benthic diatoms affects ecosystem productivity in heterogeneous coastal environments.

The current decrease in biodiversity affects all ecosystems, and the impacts of diversity on ecosystem functioning need to be resolved. So far, marine studies about diversity-ecosystem productivity-relationships have concentrated on small-scale, controlled experiments, with often limited relevance to natural ecosystems. Here, we provide a real-world study on the effects of microorganismal diversity (measured as the diversity of benthic diatom communities) on ecosystem productivity (using chlorophyll a concentration as a surrogate) in a heterogeneous marine coastal archipelago. We collected 78 sediment cores at 17 sites in the northern Baltic Sea and found exceptionally high diatom diversity (328 observed species). We used structural equation models and quantile regression to explore relationships between diatom diversity and productivity. Previous studies have found contradictory results in the relationship between microorganismal diversity and ecosystem productivity, but we showed a linear and positive basal relationship between diatom diversity and productivity, which indicates that diatom diversity most likely forms the lowest boundary for productivity. Thus, although productivity can be high even when diatom diversity is low, high diatom diversity supports high productivity. The trait composition was more effective than taxonomical composition in showing such a relationship, which could be due to niche complementarity. Our results also indicated that environmental heterogeneity leads to substantial patchiness in the diversity of benthic diatom communities, mainly induced by the variation in sediment organic matter content. Therefore, future changes in precipitation and river runoff and associated changes in the quality and quantity of organic matter in the sea, will also affect diatom communities and, hence, ecosystem productivity. Our study suggests that benthic microorganisms are vital for ecosystem productivity, and together with the substantial heterogeneity of coastal ecosystems, they should be considered when evaluating the potential productivity of coastal areas.

Declining maerl vitality and habitat complexity across a dredging gradient: Insights from in situ sediment profile imagery (SPI).

Maerl beds form complex biogenic benthic habitats, characterized by high productivity as well as diverse biological communities. Disturbances associated with extraction and/or fishing activities using mobile bottom-contacting gears such as clam-dredges induce the most severe and long-term effects on these fragile habitats. We here investigated the effects of dredge-fishing on maerl in the bay of Brest (France). We quantified maerl beds structure and vitality across a fine scale quantified dredging intensity gradient through the acquisition of in-situ images of beds cross-section using Sediment Profile Imaging system (SPI). Declines in the proxies of maerl vitality and habitat complexity were measured across the gradient, and were associated with significant changes in the vertical distribution of live and dead maerl as well as of interstitial space. Fishing with dredges caused maerl mortality, substratum compaction, and decreasing habitat complexity. SPI imaging techniques also allowed for an assessment of changes in spatial heterogeneity that dredging created on several aspects of the structure and vitality of maerl beds. It suggests that direct and indirect disturbances induced by dredging are not acting at the same spatial scale, and can thereby differentially affect the ecosystem functions linked to vitality and habitat complexity.

Benchmarking of alignment-free sequence comparison methods.

BACKGROUND: Alignment-free (AF) sequence comparison is attracting persistent interest driven by data-intensive applications. Hence, many AF procedures have been proposed in recent years, but a lack of a clearly defined benchmarking consensus hampers their performance assessment. RESULTS: Here, we present a community resource (http://afproject.org) to establish standards for comparing alignment-free approaches across different areas of sequence-based research. We characterize 74 AF methods available in 24 software tools for five research applications, namely, protein sequence classification, gene tree inference, regulatory element detection, genome-based phylogenetic inference, and reconstruction of species trees under horizontal gene transfer and recombination events. CONCLUSION: The interactive web service allows researchers to explore the performance of alignment-free tools relevant to their data types and analytical goals. It also allows method developers to assess their own algorithms and compare them with current state-of-the-art tools, accelerating the development of new, more accurate AF solutions.

Characterisation and in vivo ecotoxicity evaluation of double-wall carbon nanotubes in larvae of the amphibian Xenopus laevis.

Because of their outstanding properties, carbon nanotubes (CNTs) are being assessed for inclusion in many manufactured products. Due to their massive production and growing number of potential applications, the impact of CNTs on the environment must be taken into consideration. The present investigation evaluates the ecotoxicological potential of double-walled carbon nanotubes (DWNTs) in the amphibian larvae Xenopus laevis at a large range of concentrations in water (from 10 to 500 mgL(-1)). Acute toxicity and genotoxicity were analysed after 12 days of static exposure in laboratory conditions. Acute toxicity was evaluated according to the mortality and the growth of larvae. The genotoxic effects were analysed by scoring the micronucleated erythrocytes of the circulating blood of larvae according to the International Standard micronucleus assay. Moreover, histological preparations of larval intestine were prepared after 12 days of exposure for observation using optical and transmission electron microscopy (TEM). Finally, the intestine of an exposed larva was prepared on a slide for analyse by Raman imaging. The results showed no genotoxicity in erythrocytes of larvae exposed to DWNTs in water, but acute toxicity at every concentration of DWNTs studied which was related to physical blockage of the gills and/or digestive tract. Indeed, black masses suggesting the presence of CNTs were observed inside the intestine using optical microscopy and TEM, and confirmed by Raman spectroscopy analysis. Assessing the risks of CNTs requires better understanding, especially including mechanistic and environmental investigations.

Spatial assessment of fishing effort around European marine reserves: implications for successful fisheries management.

We examined the spatial dynamic of artisanal fishing fleets around five European marine protected areas (MPAs) to derive general implications for the evaluation of MPAs as fisheries management tools. The coastal MPAs studied were located off France, Malta and Spain and presented a variety of spatial designs and processes of establishment. We developed a standardized methodology to define factors influencing effort allocation and to produce fishing effort maps by merging GIS with geostatistical modelling techniques. Results revealed that in most cases the factors "distance to the no-take", "water depth", and "distance to the port" had a significant influence on effort allocation by the fishing fleets. Overall, we found local concentration of fishing effort around the MPA borders. Thus, neglecting the pattern of fishing effort distribution in evaluating MPA benefits, such as spillover of biomass, could hamper sound interpretation of MPAs as fisheries management tools.

k-mer Similarity, Networks of Microbial Genomes, and Taxonomic Rank.

Microbial genomes have been shaped by parent-to-offspring (vertical) descent and lateral genetic transfer. These processes can be distinguished by alignment-based inference and comparison of phylogenetic trees for individual gene families, but this approach is not scalable to whole-genome sequences, and a tree-like structure does not adequately capture how these processes impact microbial physiology. Here we adopted alignment-free approaches based on k-mer statistics to infer phylogenomic networks involving 2,783 completely sequenced bacterial and archaeal genomes and compared the contributions of rRNA, protein-coding, and plasmid sequences to these networks. Our results show that the phylogenomic signal arising from ribosomal RNAs is strong and extends broadly across all taxa, whereas that from plasmids is strong but restricted to closely related groups, particularly Proteobacteria. However, the signal from the other chromosomal regions is restricted in breadth. We show that mean k-mer similarity can correlate with taxonomic rank. We also link the implicated k-mers to genome annotation (thus, functions) and define core k-mers (thus, core functions) in specific phyletic groups. Highly conserved functions in most phyla include amino acid metabolism and transport as well as energy production and conversion. Intracellular trafficking and secretion are the most prominent core functions among Spirochaetes, whereas energy production and conversion are not highly conserved among the largely parasitic or commensal Tenericutes. These observations suggest that differential conservation of functions relates to niche specialization and evolutionary diversification of microbes. Our results demonstrate that k-mer approaches can be used to efficiently identify phylogenomic signals and conserved core functions at the multigenome scale. IMPORTANCE Genome evolution of microbes involves parent-to-offspring descent, and lateral genetic transfer that convolutes the phylogenomic signal. This study investigated phylogenomic signals among thousands of microbial genomes based on short subsequences without using multiple-sequence alignment. The signal from ribosomal RNAs is strong across all taxa, and the signal of plasmids is strong only in closely related groups, particularly Proteobacteria. However, the signal from other chromosomal regions (∼99% of the genomes) is remarkably restricted in breadth. The similarity of subsequences is found to correlate with taxonomic rank and informs on conserved and differential core functions relative to niche specialization and evolutionary diversification of microbes. These results provide a comprehensive, alignment-free view of microbial genome evolution as a network, beyond a tree-like structure.