Chromosome-level genome assembly of the European green woodpecker Picus viridis.

The European green woodpecker, Picus viridis, is a widely distributed species found in the Western Palearctic region. Here, we assembled a highly contiguous genome assembly for this species using a combination of short- and long-read sequencing and scaffolded with chromatin conformation capture (Hi-C). The final genome assembly was 1.28 Gb and features a scaffold N50 of 37 Mb and a scaffold L50 of 39.165 Mb. The assembly incorporates 89.4% of the genes identified in birds in OrthoDB. Gene and repetitive content annotation on the assembly detected 15,805 genes and a ∼30.1% occurrence of repetitive elements, respectively. Analysis of synteny demonstrates the fragmented nature of the P. viridis genome when compared to the chicken (Gallus gallus). The assembly and annotations produced in this study will certainly help for further research into the genomics of P. viridis and the comparative evolution of woodpeckers. Five historical and seven contemporary samples have been resequenced and may give insights on the population history of this species.

CNCA aligns small annotated genomes.

BACKGROUND: To explore the evolutionary history of sequences, a sequence alignment is a first and necessary step, and its quality is crucial. In the context of the study of the proximal origins of SARS-CoV-2 coronavirus, we wanted to construct an alignment of genomes closely related to SARS-CoV-2 using both coding and non-coding sequences. To our knowledge, there is no tool that can be used to construct this type of alignment, which motivated the creation of CNCA. RESULTS: CNCA is a web tool that aligns annotated genomes from GenBank files. It generates a nucleotide alignment that is then updated based on the protein sequence alignment. The output final nucleotide alignment matches the protein alignment and guarantees no frameshift. CNCA was designed to align closely related small genome sequences up to 50 kb (typically viruses) for which the gene order is conserved. CONCLUSIONS: CNCA constructs multiple alignments of small genomes by integrating both coding and non-coding sequences. This preserves regions traditionally ignored in conventional back-translation methods, such as non-coding regions.

Chance Favors the Prepared Genomes: Horizontal Transfer Shapes the Emergence of Antibiotic Resistance Mutations in Core Genes.

Bacterial lineages acquire novel traits at diverse rates in part because the genetic background impacts the successful acquisition of novel genes by horizontal transfer. Yet, how horizontal transfer affects the subsequent evolution of core genes remains poorly understood. Here, we studied the evolution of resistance to quinolones in Escherichia coli accounting for population structure. We found 60 groups of genes whose gain or loss induced an increase in the probability of subsequently becoming resistant to quinolones by point mutations in the gyrase and topoisomerase genes. These groups include functions known to be associated with direct mitigation of the effect of quinolones, with metal uptake, cell growth inhibition, biofilm formation, and sugar metabolism. Many of them are encoded in phages or plasmids. Although some of the chronologies may reflect epidemiological trends, many of these groups encoded functions providing latent phenotypes of antibiotic low-level resistance, tolerance, or persistence under quinolone treatment. The mutations providing resistance were frequent and accumulated very quickly. Their emergence was found to increase the rate of acquisition of other antibiotic resistances setting the path for multidrug resistance. Hence, our findings show that horizontal gene transfer shapes the subsequent emergence of adaptive mutations in core genes. In turn, these mutations further affect the subsequent evolution of resistance by horizontal gene transfer. Given the substantial gene flow within bacterial genomes, interactions between horizontal transfer and point mutations in core genes may be a key to the success of adaptation processes.

A General Framework for Neutrality Tests Based on the Site Frequency Spectrum.

One of the main necessities for population geneticists is the availability of sensitive statistical tools that enable to accept or reject the standard Wright-Fisher model of neutral evolution. A number of statistical tests have been developed to detect specific deviations from the null frequency spectrum in different directions (e.g., Tajima's D, Fu and Li's F and D tests, Fay and Wu's H). A general framework exists to generate all neutrality tests that are linear functions of the frequency spectrum. In this framework, it is possible to develop a family of optimal tests with almost maximum power against a specific alternative evolutionary scenario. In this paper we provide a thorough discussion of the structure and properties of linear and nonlinear neutrality tests. First, we present the general framework for linear tests and emphasise the importance of the property of scalability with the sample size (that is, the interpretation of the tests should not depend on the sample size), which, if missing, can lead to errors in interpreting the data. After summarising the motivation and structure of linear optimal tests, we present a more general framework for the optimisation of linear tests, leading to a new family of tunable neutrality tests. In a further generalisation, we extend the framework to nonlinear neutrality tests and we derive nonlinear optimal tests for polynomials of any degree in the frequency spectrum.

The Crown Pearl V2: an improved genome assembly of the European freshwater pearl mussel Margaritifera margaritifera (Linnaeus, 1758).

Contiguous assemblies are fundamental to deciphering the composition of extant genomes. In molluscs, this is considerably challenging owing to the large size of their genomes, heterozygosity, and widespread repetitive content. Consequently, long-read sequencing technologies are fundamental for high contiguity and quality. The first genome assembly of Margaritifera margaritifera (Linnaeus, 1758) (Mollusca: Bivalvia: Unionida), a culturally relevant, widespread, and highly threatened species of freshwater mussels, was recently generated. However, the resulting genome is highly fragmented since the assembly relied on short-read approaches. Here, an improved reference genome assembly was generated using a combination of PacBio CLR long reads and Illumina paired-end short reads. This genome assembly is 2.4 Gb long, organized into 1,700 scaffolds with a contig N50 length of 3.4 Mbp. The ab initio gene prediction resulted in 48,314 protein-coding genes. Our new assembly is a substantial improvement and an essential resource for studying this species' unique biological and evolutionary features, helping promote its conservation.

Interpreting the pervasive observation of U-shaped Site Frequency Spectra.

The standard neutral model of molecular evolution has traditionally been used as the null model for population genomics. We gathered a collection of 45 genome-wide site frequency spectra from a diverse set of species, most of which display an excess of low and high frequency variants compared to the expectation of the standard neutral model, resulting in U-shaped spectra. We show that multiple merger coalescent models often provide a better fit to these observations than the standard Kingman coalescent. Hence, in many circumstances these under-utilized models may serve as the more appropriate reference for genomic analyses. We further discuss the underlying evolutionary processes that may result in the widespread U-shape of frequency spectra.

Cultural transmission of reproductive success impacts genomic diversity, coalescent tree topologies, and demographic inferences.

Cultural transmission of reproductive success has been observed in many human populations as well as other animals. Cultural transmission of reproductive success consists of a positive correlation of nongenetic origin between the progeny size of parents and children. This correlation can result from various factors, such as the social influence of parents on their children, the increase of children's survival through allocare from uncles and aunts, or the transmission of resources. Here, we study the evolution of genomic diversity over time under cultural transmission of reproductive success. Cultural transmission of reproductive success has a threefold impact on population genetics: (1) the effective population size decreases when cultural transmission of reproductive success starts, mimicking a population contraction, and increases back to its original value when cultural transmission of reproductive success stops; (2) coalescent tree topologies are distorted under cultural transmission of reproductive success, with higher imbalance and a higher number of polytomies; and (3) branch lengths are reduced nonhomogenously, with a higher impact on older branches. Under long-lasting cultural transmission of reproductive success, the effective population size stabilizes but the distortion of tree topology and the nonhomogenous branch length reduction remain, yielding U-shaped site frequency spectra under a constant population size. We show that this yields a bias in site frequency spectra-based demographic inference. Considering that cultural transmission of reproductive success was detected in numerous human and animal populations worldwide, one should be cautious because inferring population past histories from genomic data can be biased by this cultural process.

mold, a novel software to compile accurate and reliable DNA diagnoses for taxonomic descriptions.

DNA data are increasingly being used for phylogenetic inference, and taxon delimitation and identification, but scarcely for the formal description of taxa, despite their undisputable merits in taxonomy. The uncertainty regarding the robustness of DNA diagnoses, however, remains a major impediment to their use. We have developed a new program, mold, that identifies diagnostic nucleotide combinations (DNCs) in DNA sequence alignments for selected taxa, which can be used to provide formal diagnoses of these taxa. To test the robustness of DNA diagnoses, we carry out iterated haplotype subsampling for selected query species in published DNA data sets of varying complexity. We quantify the reliability of diagnosis by diagnosing each query subsample and then checking if this diagnosis remains valid against the entire data set. We demonstrate that widely used types of diagnostic DNA characters are often absent for a query taxon or are not sufficiently reliable. We thus propose a new type of DNA diagnosis, termed "redundant DNC" (or rDNC), which takes into account unsampled genetic diversity, and constitutes a much more reliable descriptor of a taxon. mold successfully retrieves rDNCs for all but two species in the analysed data sets, even in those comprising hundreds of species. mold shows unparalleled efficiency in large DNA data sets and is the only available software capable of compiling DNA diagnoses that suit predefined criteria of reliability.

A Minimal yet Flexible Likelihood Framework to Assess Correlated Evolution.

An evolutionary process is reflected in the sequence of changes of any trait (e.g., morphological or molecular) through time. Yet, a better understanding of evolution would be procured by characterizing correlated evolution, or when two or more evolutionary processes interact. Previously developed parametric methods often require significant computing time as they rely on the estimation of many parameters. Here, we propose a minimal likelihood framework modeling the joint evolution of two traits on a known phylogenetic tree. The type and strength of correlated evolution are characterized by a few parameters tuning mutation rates of each trait and interdependencies between these rates. The framework can be applied to study any discrete trait or character ranging from nucleotide substitution to gain or loss of a biological function. More specifically, it can be used to 1) test for independence between two evolutionary processes, 2) identify the type of interaction between them, and 3) estimate parameter values of the most likely model of interaction. In the current implementation, the method takes as input a phylogenetic tree with discrete evolutionary events mapped on its branches. The method then maximizes the likelihood for one or several chosen scenarios. The strengths and limits of the method, as well as its relative power compared to a few other methods, are assessed using both simulations and data from 16S rRNA sequences in a sample of 54 $\gamma$-enterobacteria. We show that, even with data sets of fewer than 100 species, the method performs well in parameter estimation and in evolutionary model selection. [Correlated evolution; maximum likelihood; model.].

Involvement of ADGRV1 Gene in Familial Forms of Genetic Generalized Epilepsy.

Background: Genetic generalized epilepsies (GGE) including childhood absence epilepsy (CAE), juvenile absence epilepsy (JAE), juvenile myoclonic epilepsy (JME), and GGE with tonic-clonic seizures alone (GGE-TCS), are common types of epilepsy mostly determined by a polygenic mode of inheritance. Recent studies showed that susceptibility genes for GGE are numerous, and their variants rare, challenging their identification. In this study, we aimed to assess GGE genetic etiology in a Sudanese population. Methods: We performed whole-exome sequencing (WES) on DNA of 40 patients from 20 Sudanese families with GGE searching for candidate susceptibility variants, which were prioritized by CADD software and functional features of the corresponding gene. We assessed their segregation in 138 individuals and performed genotype-phenotype correlations. Results: In a family including three sibs with GGE-TCS, we identified a rare missense variant in ADGRV1 encoding an adhesion G protein-coupled receptor V1, which was already involved in the autosomal recessive Usher type C syndrome. In addition, five other ADGRV1 rare missense variants were identified in four additional families and absent from 119 Sudanese controls. In one of these families, an ADGRV1 variant was found at a homozygous state, in a female more severely affected than her heterozygous brother, suggesting a gene dosage effect. In the five families, GGE phenotype was statistically associated with ADGRV1 variants (0R = 0.9 103). Conclusion: This study highly supports, for the first time, the involvement of ADGRV1 missense variants in familial GGE and that ADGRV1 is a susceptibility gene for CAE/JAE and GGE-TCS phenotypes.

ASAP: assemble species by automatic partitioning.

Here, we describe Assemble Species by Automatic Partitioning (ASAP), a new method to build species partitions from single locus sequence alignments (i.e., barcode data sets). ASAP is efficient enough to split data sets as large 104 sequences into putative species in several minutes. Although grounded in evolutionary theory, ASAP is the implementation of a hierarchical clustering algorithm that only uses pairwise genetic distances, avoiding the computational burden of phylogenetic reconstruction. Importantly, ASAP proposes species partitions ranked by a new scoring system that uses no biological prior insight of intraspecific diversity. ASAP is a stand-alone program that can be used either through a graphical web-interface or that can be downloaded and compiled for local usage. We have assessed its power along with three others programs (ABGD, PTP and GMYC) on 10 real COI barcode data sets representing various degrees of challenge (from small and easy cases to large and complicated data sets). We also used Monte-Carlo simulations of a multispecies coalescent framework to assess the strengths and weaknesses of ASAP and the other programs. Through these analyses, we demonstrate that ASAP has the potential to become a major tool for taxonomists as it proposes rapidly in a full graphical exploratory interface relevant species hypothesis as a first step of the integrative taxonomy process.

No HIV-1 molecular evolution on long-term antiretroviral therapy initiated during primary HIV-1 infection.

OBJECTIVE: Most studies about HIV-1 molecular evolution have shown the lack of viral evolution on effective antiretroviral therapy (ART), although controversial results have been documented. We therefore aimed to look for evidence of HIV-1 evolution in patients who initiated ART at the time of primary HIV-1 infection (PHI). DESIGN: We included retrospectively 20 patients diagnosed at PHI, treated at the time of acute infection and with subsequent effective long-term suppressive ART (HIV viral load <20 copies/ml during at least 5 years without any blips). METHODS: Longitudinal blood samples were deep sequenced using Illumina Miseq. Drug-resistance-associated mutations were retained at 2% cutoff and interpreted using the latest Agence Nationale de Recherches sur le Sida et les Hépatites Virales resistance algorithm. Viral evolution was established when temporal structure on maximum-likelihood phylogenetic tree and significant change over time of HIV-1 genetic diversity measured as the average pairwise distance was observed. RESULTS: Emergences or disappearances of drug-resistance-associated mutations were detected in the blood cells during follow-up despite sustained virological control. In all patients, tree topologies showed an absence of segregation between sequences and blood viral populations from all time-points were intermingled. Comparison of the average pairwise distance showed the absence of significant viral diversity at the time of primary infection and afterwards during 5 years of full virological control under ART. CONCLUSION: Despite a slight variation of minority resistance-associated mutation variants, there was no clear evidence of viral evolution during a prolonged period of time in this population of highly controlled adult patients treated at time of PHI.

The genomic view of diversification.

The process of species diversification is traditionally summarized by a single tree, the species tree, whose reconstruction from molecular data is hindered by frequent conflicts between gene genealogies. Here, we argue that instead of seeing these conflicts as nuisances, we can exploit them to inform the diversification process itself. We adopt a gene-based view of diversification to model the ubiquitous presence of gene flow between diverging lineages, one of the most important processes explaining disagreements among gene trees. We propose a new framework for modelling the joint evolution of gene and species lineages relaxing the hierarchy between the species tree and gene trees inherent to the standard view, as embodied in a popular model known as the multispecies coalescent (MSC). We implement this framework in two alternative models called the gene-based diversification models (GBD): (a) GBD-forward following all evolving genomes through time and (b) GBD-backward based on coalescent theory. They feature four parameters tuning colonization, gene flow, genetic drift and genetic differentiation. We propose an inference method based on differences between gene trees. Applied to two empirical data sets prone to gene flow, we find better support for the GBD-backward model than for the MSC model. Along with the increasing awareness of the extent of gene flow, this work shows the importance of considering the richer signal contained in genomic histories, rather than in the mere species tree, to better apprehend the complex evolutionary history of species.

Testing for population decline using maximal linkage disequilibrium blocks.

Only 6% of known species have a conservation status. Methods that assess conservation statuses are often based on individual counts and are thus too laborious to be generalized to all species. Population genomics methods that infer past variations in population size are easy to use but limited to the relatively distant past. Here we propose a population genomics approach that tests for recent population decline and may be used to assess species conservation statuses. More specifically, we study Maximal Recombination Free (MRF) blocks, that are segments of a sequence alignment inherited from a common ancestor without recombination. MRF blocks are relatively longer in small than in large populations. We use the distribution of MRF block lengths rescaled by their mean to test for recent population decline. However, because MRF blocks are difficult to detect, we also consider Maximal Linkage Disequilibrium (MLD) blocks, which are runs of single nucleotide polymorphisms compatible with a single tree. We develop a new method capable of inferring a very recent decline (e.g. with a detection power of 50% for populations whose size was halved to N, 0.05 ×N generations ago) from rescaled MLD block lengths. Our framework could serve as a basis for quantitative tools to assess conservation status in a wide range of species.

Is reproductive strategy a key factor in understanding the evolutionary history of Southern Ocean Asteroidea (Echinodermata)?

Life traits such as reproductive strategy can be determining factors of species evolutionary history and explain the resulting diversity patterns. This can be investigated using phylogeographic analyses of genetic units. In this work, the genetic structure of five asteroid genera with contrasting reproductive strategies (brooding: Diplasterias, Notasterias and Lysasterias versus broadcasting: Psilaster and Bathybiaster) was investigated in the Southern Ocean. Over 1,400 mtDNA cytochrome C oxidase subunit I (COI) sequences were analysed using five species delineation methods (ABGD, ASAP, mPTP, sGMYC and mGMYC), two phylogenetic reconstructions (ML and BA), and molecular clock calibrations, in order to examine the weight of reproductive strategy in the observed differences among phylogeographic patterns. We hypothesised that brooding species would show higher levels of genetic diversity and species richness along with a clearer geographic structuring than broadcasting species. In contrast, genetic diversity and species richness were not found to be significantly different between brooders and broadcasters, but broadcasters are less spatially structured than brooders supporting our initial hypothesis and suggesting more complex evolutionary histories associated to this reproductive strategy. Broadcasters' phylogeography can be explained by different scenarios including deep-sea colonisation routes, bipolarity or cosmopolitanism, and sub-Antarctic emergence for the genus Bathybiaster; Antarctic- New Zealand faunal exchanges across the Polar Front for the genus Psilaster. Brooders' phylogeography could support the previously formulated hypothesis of a past trans-Antarctic seaway established between the Ross and the Weddell seas during the Plio-Pleistocene. Our results also show, for the first time, that the Weddell Sea is populated by a mixed asteroid fauna originating from both the East and West Antarctic.

Evolutionary constraints in fitness landscapes.

In the last years, several genotypic fitness landscapes-combinations of a small number of mutations-have been experimentally resolved. To learn about the general properties of "real" fitness landscapes, it is key to characterize these experimental landscapes via simple measures of their structure, related to evolutionary features. Some of the most relevant measures are based on the selectively acessible paths and their properties. In this paper, we present some measures of evolutionary constraints based on (i) the similarity between accessible paths and (ii) the abundance and characteristics of "chains" of obligatory mutations, that are paths going through genotypes with a single fitter neighbor. These measures have a clear evolutionary interpretation. Furthermore, we show that chains are only weakly correlated to classical measures of epistasis. In fact, some of these measures of constraint are non-monotonic in the amount of epistatic interactions, but have instead a maximum for intermediate values. Finally, we show how these measures shed light on evolutionary constraints and predictability in experimentally resolved landscapes.

The neutral frequency spectrum of linked sites.

We introduce the conditional Site Frequency Spectrum (SFS) for a genomic region linked to a focal mutation of known frequency. An exact expression for its expected value is provided for the neutral model without recombination. Its relation with the expected SFS for two sites, 2-SFS, is discussed. These spectra derive from the coalescent approach of Fu (1995) for finite samples, which is reviewed. Remarkably simple expressions are obtained for the linked SFS of a large population, which are also solutions of the multi-allelic Kolmogorov equations. These formulae are the immediate extensions of the well known single site θ∕f neutral SFS. Besides the general interest in these spectra, they relate to relevant biological cases, such as structural variants and introgressions. As an application, a recipe to adapt Tajima's D and other SFS-based neutrality tests to a non-recombining region containing a neutral marker is presented.

Clinical and genetic study of Tunisian families with genetic generalized epilepsy: contribution of CACNA1H and MAST4 genes.

Genetic generalized epilepsies (GGE) (childhood absence epilepsy (CAE), juvenile myoclonic epilepsy (JME) and epilepsy with generalized tonic-clonic seizures (GTCS)) are mainly determined by genetic factors. Since few mutations were identified in rare families with autosomal dominant GGE, a polygenic inheritance was suspected in most patients. Recent studies on large American or European cohorts of sporadic cases showed that susceptibility genes were numerous although their variants were rare, making their identification difficult. Here, we reported clinical and genetic characteristics of 30 Tunisian GGE families, including 71 GGE patients. The phenotype was close to that in sporadic cases. Nineteen pedigrees had a homogeneous type of GGE (JME-CAE-CGTS), and 11 combined these epileptic syndromes. Rare non-synonymous variants were selected in probands using a targeted panel of 30 candidate genes and their segregation was determined in families. Molecular studies incriminated different genes, mainly CACNA1H and MAST4. The segregation of at least two variants in different genes in some pedigrees was compatible with the hypothesis of an oligogenic inheritance, which was in accordance with the relatively low frequency of consanguineous probands. Since at least 2 susceptibility genes were likely shared by different populations, genetic factors involved in the majority of Tunisian GGE families remain to be discovered. Their identification should be easier in families with a homogeneous type of GGE, in which an intra-familial genetic homogeneity could be suspected.

Comparative Genomics of Tenacibaculum dicentrarchi and "Tenacibaculum finnmarkense" Highlights Intricate Evolution of Fish-Pathogenic Species.

The genus Tenacibaculum encompasses several species pathogenic for marine fish. Tenacibaculum dicentrarchi and "Tenacibaculum finnmarkense" (Quotation marks denote species that have not been validly named.) were retrieved from skin lesions of farmed fish such as European sea bass or Atlantic salmon. They cause a condition referred to as tenacibaculosis and severe outbreaks and important fish losses have been reported in Spanish, Norwegian, and Chilean marine farms. We report here the draft genomes of the T. dicentrarchi and "T. finnmarkense" type strains. These genomes were compared with draft genomes from field isolates retrieved from Chile and Norway and with previously published Tenacibaculum genomes. We used Average Nucleotide Identity and core genome-based phylogeny as a proxy index for species boundary delineation. This work highlights evolution of closely related fish-pathogenic species and suggests that homologous recombination likely contributes to genome evolution. It also corrects the species affiliation of strain AYD7486TD claimed by Grothusen et al. (2016).