Spruce giga-genomes: structurally similar yet distinctive with differentially expanding gene families and rapidly evolving genes.

Spruces (Picea spp.) are coniferous trees widespread in boreal and mountainous forests of the northern hemisphere, with large economic significance and enormous contributions to global carbon sequestration. Spruces harbor very large genomes with high repetitiveness, hampering their comparative analysis. Here, we present and compare the genomes of four different North American spruces: the genome assemblies for Engelmann spruce (Picea engelmannii) and Sitka spruce (Picea sitchensis) together with improved and more contiguous genome assemblies for white spruce (Picea glauca) and for a naturally occurring introgress of these three species known as interior spruce (P. engelmannii × glauca × sitchensis). The genomes were structurally similar, and a large part of scaffolds could be anchored to a genetic map. The composition of the interior spruce genome indicated asymmetric contributions from the three ancestral genomes. Phylogenetic analysis of the nuclear and organelle genomes revealed a topology indicative of ancient reticulation. Different patterns of expansion of gene families among genomes were observed and related with presumed diversifying ecological adaptations. We identified rapidly evolving genes that harbored high rates of non-synonymous polymorphisms relative to synonymous ones, indicative of positive selection and its hitchhiking effects. These gene sets were mostly distinct between the genomes of ecologically contrasted species, and signatures of convergent balancing selection were detected. Stress and stimulus response was identified as the most frequent function assigned to expanding gene families and rapidly evolving genes. These two aspects of genomic evolution were complementary in their contribution to divergent evolution of presumed adaptive nature. These more contiguous spruce giga-genome sequences should strengthen our understanding of conifer genome structure and evolution, as their comparison offers clues into the genetic basis of adaptation and ecology of conifers at the genomic level. They will also provide tools to better monitor natural genetic diversity and improve the management of conifer forests. The genomes of four closely related North American spruces indicate that their high similarity at the morphological level is paralleled by the high conservation of their physical genome structure. Yet, the evidence of divergent evolution is apparent in their rapidly evolving genomes, supported by differential expansion of key gene families and large sets of genes under positive selection, largely in relation to stimulus and environmental stress response.

Soybean (Glycine max) Haplotype Map (GmHapMap): a universal resource for soybean translational and functional genomics.

Here, we describe a worldwide haplotype map for soybean (GmHapMap) constructed using whole-genome sequence data for 1007 Glycine max accessions and yielding 14.9 million variants as well as 4.3 M tag single-nucleotide polymorphisms (SNPs). When sampling random subsets of these accessions, the number of variants and tag SNPs plateaued beyond approximately 800 and 600 accessions, respectively. This suggests extensive coverage of diversity within the cultivated soybean. GmHapMap variants were imputed onto 21 618 previously genotyped accessions with up to 96% success for common alleles. A local association analysis was performed with the imputed data using markers located in a 1-Mb region known to contribute to seed oil content and enabled us to identify a candidate causal SNP residing in the NPC1 gene. We determined gene-centric haplotypes (407 867 GCHs) for the 55 589 genes and showed that such haplotypes can help to identify alleles that differ in the resulting phenotype. Finally, we predicted 18 031 putative loss-of-function (LOF) mutations in 10 662 genes and illustrated how such a resource can be used to explore gene function. The GmHapMap provides a unique worldwide resource for applied soybean genomics and breeding.

DepthFinder: a tool to determine the optimal read depth for reduced-representation sequencing.

MOTIVATION: Identification of DNA sequence variations such as single nucleotide polymorphisms (SNPs) is a fundamental step toward genetic studies. Reduced-representation sequencing methods have been developed as alternatives to whole genome sequencing to reduce costs and enable the analysis of many more individual. Amongst these methods, restriction site associated sequencing (RSAS) methodologies have been widely used for rapid and cost-effective discovery of SNPs and for high-throughput genotyping in a wide range of species. Despite the extensive improvements of the RSAS methods in the last decade, the estimation of the number of reads (i.e. read depth) required per sample for an efficient and effective genotyping remains mostly based on trial and error. RESULTS: Herein we describe a bioinformatics tool, DepthFinder, designed to estimate the required read counts for RSAS methods. To illustrate its performance, we estimated required read counts in six different species (human, cattle, spruce budworm, salmon, barley and soybean) that cover a range of different biological (genome size, level of genome complexity, level of DNA methylation and ploidy) and technical (library preparation protocol and sequencing platform) factors. To assess the prediction accuracy of DepthFinder, we compared DepthFinder-derived results with independent datasets obtained from an RSAS experiment. This analysis yielded estimated accuracies of nearly 94%. Moreover, we present DepthFinder as a powerful tool to predict the most effective size selection interval in RSAS work. We conclude that DepthFinder constitutes an efficient, reliable and useful tool for a broad array of users in different research communities. AVAILABILITY AND IMPLEMENTATION: https://bitbucket.org/jerlar73/DepthFinder. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

A genomic perspective on an old question: Salmo trouts or Salmo trutta (Teleostei: Salmonidae)?

There are particular challenges in defining the taxonomic status of recently radiated groups due to the low level of phylogenetic signal. Members of the Salmo trutta species-complex, which mostly evolved during and following the Pleistocene, show high morphological and ecological diversity that, along with their very wide geographic distribution, have led to morphological description of 47 extant nominal species. However, many of these species have not been supported by previous phylogenetic studies, which could be partly due to lack of significant genetic differences among them, the limited resolution offered by molecular methods previously used, as well as the often local scale of these studies. The development of next-generation sequencing (NGS) and related analytical tools have enhanced our ability to address such challenging questions. In this study, Genotyping-by-Sequencing (GBS) of 15,169 filtered SNPs and mitochondrial DNA (mtDNA) D-loop sequences were combined to assess the phylogenetic relationships among 166 brown trouts representing 21 described species and three undescribed groups collected from 84 localities throughout their natural distribution in Europe, west Asia, and North Africa. The data were analysed using different clustering algorithms (admixture analysis and discriminant analysis of principal components-DAPC), a Bayes Factor Delimitation (BFD) test, species tree reconstruction, gene flow tests (three- and four-population tests), and Rogue taxa identification tests. Genomic contributions of the Atlantic lineage brown trout were found in all major sea basins excluding the North African and Aral Sea basins, suggesting introgressive hybridization of native brown trouts driven by stocking using strains of the Atlantic lineage. After removing the phylogenetic noise caused by the Atlantic brown trout, admixture clusters and DAPC clustering based on GBS data, respectively, resolved 11 and 13 clusters among the previously described brown trout species, which were also supported by BFD test results. Our results suggest that natural hybridization between different brown trout lineages has probably played an important role in the origin of several of the putative species, including S. marmoratus, S. carpio, S. farioides, S. pellegrini, S. caspius (in the Kura River drainage) and Salmo sp. in the Danube River basin. Overall, our results support a multi-species taxonomy for brown trouts. They also resolve some species in the Adriatic-Mediterranean and Black Sea drainages as members of very closely related genomic clusters that may need taxonomic revision. However, any final conclusions pertaining to the taxonomy of the brown trout complex should be based on an integrative approach combining genomic, morphological, and ecological data. To avoid challenges in taxonomy and conservation of species complexes like brown trouts, it is suggested to describe species based on genomic clusters of populations instead of describing species based only on morphologically differentiated single type populations.

SRG extractor: a skinny reference genome approach for reduced-representation sequencing.

MOTIVATION: Reduced-representation sequencing is a genome-wide scanning method for simultaneous discovery and genotyping of thousands to millions of single nucleotide polymorphisms that is used across a wide range of species. However, in this method a reproducible but very small fraction of the genome is captured for sequencing, while the resulting reads are typically aligned against the entire reference genome. RESULTS: Here we present a skinny reference genome approach in which a simplified reference genome is used to decrease computing time for data processing and to increase single nucleotide polymorphism counts and accuracy. A skinny reference genome can be integrated into any reduced-representation sequencing analytical pipeline. AVAILABILITY AND IMPLEMENTATION: https://bitbucket.org/jerlar73/SRG-Extractor. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Fast-GBS v2.0: an analysis toolkit for genotyping-by-sequencing data.

Genotyping-by-sequencing (GBS) is a rapid, flexible, low-cost, and robust genotyping method that simultaneously discovers variants and calls genotypes within a broad range of samples. These characteristics make GBS an excellent tool for many applications and research questions from conservation biology to functional genomics in both model and non-model species. Continued improvement of GBS relies on a more comprehensive understanding of data analysis, development of fast and efficient bioinformatics pipelines, accurate missing data imputation, and active post-release support. Here, we present the second generation of Fast-GBS (v2.0) that offers several new options (e.g., processing paired-end reads and imputation of missing data) and features (e.g., summary statistics of genotypes) to improve the GBS data analysis process. The performance assessment analysis showed that Fast-GBS v2.0 outperformed other available analytical pipelines, such as GBS-SNP-CROP and Gb-eaSy. Fast-GBS v2.0 provides an analysis platform that can be run with different types of sequencing data, modest computational resources, and allows for missing-data imputation for various species in different contexts.

Identification of candidate domestication-related genes with a systematic survey of loss-of-function mutations.

Domestication is an important key co-evolutionary process through which humans have extensively altered the genomic make-up and appearance of both plants and animals. The identification of domestication-related genes remains very arduous. In this study, we present a systematic analytical approach that harnesses two recent advances in genomics, whole-genome sequencing (WGS) and prediction of loss-of-function (LOF) mutations, to greatly facilitate the assembly of an enriched catalogue of domestication-related candidate genes. Using WGS data for 296 cultivated (Glycine max) and 64 wild soybean accessions, we identified 8699 LOF variants, and 116 genes that are uniquely fixed for one or more LOF allele(s) in domesticated soybeans. Existing soybean transcriptomic data led us to overcome analytical challenges associated with whole-genome duplications and to identify neo- or subfunctionalized genes. This systematic approach allowed us to identify 110 candidate domestication-related genes in an efficient and rapid way. This catalogue contains previously well characterized domestication genes in soybean, as well as some orthologs from other domesticated crop species. In addition, it comprises many promising candidate domestication genes. Overall, this collection of candidate domestication-related genes in soybean is almost twice as large as the sum of all previously reported candidate genes in all other crops. We believe this systematic approach could readily be used in wide range of species.

A catalog of annotated high-confidence SNPs from exome capture and sequencing reveals highly polymorphic genes in Norway spruce (Picea abies).

BACKGROUND: Norway spruce [Picea abies (L.) Karst.] is ecologically and economically one of the most important conifer worldwide. Our main goal was to develop a large catalog of annotated high confidence gene SNPs that should sustain the development of genomic tools for the conservation of natural and domesticated genetic diversity resources, and hasten tree breeding efforts in this species. RESULTS: Targeted sequencing was achieved by capturing P. abies exome with probes previously designed from the sequenced transcriptome of white spruce (Picea glauca (Moench) Voss). Capture efficiency was high (74.5%) given a high level of exome conservation between the two species. Using stringent criteria, we delimited a set of 61,771 high-confidence SNPs across 13,543 genes. To validate SNPs, a high-throughput genotyping array was developed for a subset of 5571 predicted SNPs representing as many different gene loci, and was used to genotype over 1000 trees. The estimated true positive rate of the resource was 84.2%, which was comparable with the genotyping success rate obtained for P. abies control SNPs recycled from previous genotyping efforts. We also analyzed SNP abundance across various gene functional categories. Several GO terms and gene families involved in stress response were found over-represented in highly polymorphic genes. CONCLUSION: The annotated high-confidence SNP catalog developed herein represents a valuable genomic resource, being representative of over 13 K genes distributed across the P. abies genome. This resource should serve a variety of population genomics and breeding applications in Norway spruce.

Comprehensive description of genomewide nucleotide and structural variation in short-season soya bean.

Next-generation sequencing (NGS) and bioinformatics tools have greatly facilitated the characterization of nucleotide variation; nonetheless, an exhaustive description of both SNP haplotype diversity and of structural variation remains elusive in most species. In this study, we sequenced a representative set of 102 short-season soya beans and achieved an extensive coverage of both nucleotide diversity and structural variation (SV). We called close to 5M sequence variants (SNPs, MNPs and indels) and noticed that the number of unique haplotypes had plateaued within this set of germplasm (1.7M tag SNPs). This data set proved highly accurate (98.6%) based on a comparison of called genotypes at loci shared with a SNP array. We used this catalogue of SNPs as a reference panel to impute missing genotypes at untyped loci in data sets derived from lower density genotyping tools (150 K GBS-derived SNPs/530 samples). After imputation, 96.4% of the missing genotypes imputed in this fashion proved to be accurate. Using a combination of three bioinformatics pipelines, we uncovered ~92 K SVs (deletions, insertions, inversions, duplications, CNVs and translocations) and estimated that over 90% of these were accurate. Finally, we noticed that the duplication of certain genomic regions explained much of the residual heterozygosity at SNP loci in otherwise highly inbred soya bean accessions. This is the first time that a comprehensive description of both SNP haplotype diversity and SV has been achieved within a regionally relevant subset of a major crop.

Convergent herbivory on conifers by Choristoneura moths after boreal forest formation.

Mitogenomes are useful markers for phylogenetic studies across a range of taxonomic levels. Here, we focus on mitogenome variation across the tortricid moth genus Choristoneura and particularly the spruce budworm (Choristoneura fumiferana) species complex, a notorious pest group of North American conifer forests. Phylogenetic relationships of Tortricidae, representing two subfamilies, four tribes and nine genera, were analyzed using 21 mitogenomes. These included six newly-sequenced mitogenomes for species in the spruce budworm complex plus three additional Choristoneura species and 12 previously published mitogenomes from other tortricids and one from the Cossidae. We evaluated the phylogenetic informativeness of the mitogenomes and reconstructed a time-calibrated tree with fossil and secondary calibrations. We found that tortricid mitogenomes had conserved protein and ribosomal regions, and analysis of all protein-coding plus ribosomal genes together provided an efficient marker at any taxonomic rank. The time-calibrated phylogeny showed evolutionary convergence of conifer feeding within Choristoneura, with two independent lineages, the Nearctic spruce budworm complex and the Palearctic species Choristoneura murinana, both shifting onto conifers about 11 million years ago from angiosperms. These two host-plant shifts both occurred after the formation of boreal forest in the late Miocene. Haplotype diversification within the spruce budworm complex occurred in the last 4 million years, and is probably linked to the initial cooling cycles of the Northern Hemisphere in the Pliocene.

Fast-GBS: a new pipeline for the efficient and highly accurate calling of SNPs from genotyping-by-sequencing data.

BACKGROUND: Next-generation sequencing (NGS) technologies have accelerated considerably the investigation into the composition of genomes and their functions. Genotyping-by-sequencing (GBS) is a genotyping approach that makes use of NGS to rapidly and economically scan a genome. It has been shown to allow the simultaneous discovery and genotyping of thousands to millions of SNPs across a wide range of species. For most users, the main challenge in GBS is the bioinformatics analysis of the large amount of sequence information derived from sequencing GBS libraries in view of calling alleles at SNP loci. Herein we describe a new GBS bioinformatics pipeline, Fast-GBS, designed to provide highly accurate genotyping, to require modest computing resources and to offer ease of use. RESULTS: Fast-GBS is built upon standard bioinformatics language and file formats, is capable of handling data from different sequencing platforms, is capable of detecting different kinds of variants (SNPs, MNPs, and Indels). To illustrate its performance, we called variants in three collections of samples (soybean, barley, and potato) that cover a range of different genome sizes, levels of genome complexity, and ploidy. Within these small sets of samples, we called 35 k, 32 k and 38 k SNPs for soybean, barley and potato, respectively. To assess genotype accuracy, we compared these GBS-derived SNP genotypes with independent data sets obtained from whole-genome sequencing or SNP arrays. This analysis yielded estimated accuracies of 98.7, 95.2, and 94% for soybean, barley, and potato, respectively. CONCLUSIONS: We conclude that Fast-GBS provides a highly efficient and reliable tool for calling SNPs from GBS data.

Genes Required for Free Phage Production are Essential for Pseudomonas aeruginosa Chronic Lung Infections.

The opportunistic pathogen Pseudomonas aeruginosa causes chronic lung infection in patients with cystic fibrosis. The Liverpool Epidemic Strain LESB58 is highly resistant to antibiotics, transmissible, and associated with increased morbidity and mortality. Its genome contains 6 prophages and 5 genomic islands. We constructed a polymerase chain reaction (PCR)-based signature-tagged mutagenesis library of 9216 LESB58 mutants and screened the mutants in a rat model of chronic lung infection. A total of 162 mutants were identified as defective for in vivo maintenance, with 11 signature-tagged mutagenesis mutants having insertions in prophage and genomic island genes. Many of these mutants showed both diminished virulence and reduced phage production. Transcription profiling by quantitative PCR and RNA-Seq suggested that disruption of these prophages had a widespread trans-acting effect on the transcriptome. This study demonstrates that temperate phages play a pivotal role in the establishment of infection through modulation of bacterial host gene expression.

High-density genetic linkage mapping in Sitka spruce advances the integration of genomic resources in conifers.

In species with large and complex genomes such as conifers, dense linkage maps are a useful resource for supporting genome assembly and laying the genomic groundwork at the structural, populational, and functional levels. However, most of the 600+ extant conifer species still lack extensive genotyping resources, which hampers the development of high-density linkage maps. In this study, we developed a linkage map relying on 21,570 single nucleotide polymorphism (SNP) markers in Sitka spruce (Picea sitchensis [Bong.] Carr.), a long-lived conifer from western North America that is widely planted for productive forestry in the British Isles. We used a single-step mapping approach to efficiently combine RAD-seq and genotyping array SNP data for 528 individuals from 2 full-sib families. As expected for spruce taxa, the saturated map contained 12 linkages groups with a total length of 2,142 cM. The positioning of 5,414 unique gene coding sequences allowed us to compare our map with that of other Pinaceae species, which provided evidence for high levels of synteny and gene order conservation in this family. We then developed an integrated map for P. sitchensis and Picea glauca based on 27,052 markers and 11,609 gene sequences. Altogether, these 2 linkage maps, the accompanying catalog of 286,159 SNPs and the genotyping chip developed, herein, open new perspectives for a variety of fundamental and more applied research objectives, such as for the improvement of spruce genome assemblies, or for marker-assisted sustainable management of genetic resources in Sitka spruce and related species.

A spruce gene map infers ancient plant genome reshuffling and subsequent slow evolution in the gymnosperm lineage leading to extant conifers.

BACKGROUND: Seed plants are composed of angiosperms and gymnosperms, which diverged from each other around 300 million years ago. While much light has been shed on the mechanisms and rate of genome evolution in flowering plants, such knowledge remains conspicuously meagre for the gymnosperms. Conifers are key representatives of gymnosperms and the sheer size of their genomes represents a significant challenge for characterization, sequencing and assembling. RESULTS: To gain insight into the macro-organisation and long-term evolution of the conifer genome, we developed a genetic map involving 1,801 spruce genes. We designed a statistical approach based on kernel density estimation to analyse gene density and identified seven gene-rich isochors. Groups of co-localizing genes were also found that were transcriptionally co-regulated, indicative of functional clusters. Phylogenetic analyses of 157 gene families for which at least two duplicates were mapped on the spruce genome indicated that ancient gene duplicates shared by angiosperms and gymnosperms outnumbered conifer-specific duplicates by a ratio of eight to one. Ancient duplicates were much more translocated within and among spruce chromosomes than conifer-specific duplicates, which were mostly organised in tandem arrays. Both high synteny and collinearity were also observed between the genomes of spruce and pine, two conifers that diverged more than 100 million years ago. CONCLUSIONS: Taken together, these results indicate that much genomic evolution has occurred in the seed plant lineage before the split between gymnosperms and angiosperms, and that the pace of evolution of the genome macro-structure has been much slower in the gymnosperm lineage leading to extent conifers than that seen for the same period of time in flowering plants. This trend is largely congruent with the contrasted rates of diversification and morphological evolution observed between these two groups of seed plants.

Complete genome sequences of three Pseudomonas aeruginosa isolates with phenotypes of polymyxin B adaptation and inducible resistance.

Clinical "superbug" isolates of Pseudomonas aeruginosa were previously observed to be resistant to several antibiotics, including polymyxin B, and/or to have a distinct, reproducible adaptive polymyxin resistance phenotype, identified by observing "skipped" wells (appearance of extra turbid wells) during broth microdilution testing. Here we report the complete assembled draft genome sequences of three such polymyxin resistant P. aeruginosa strains (9BR, 19BR, and 213BR).

Parallel and lineage-specific molecular adaptation to climate in boreal black spruce.

In response to selective pressure, adaptation may follow different genetic pathways throughout the natural range of a species due to historical differentiation in standing genetic variation. Using 41 populations of black spruce (Picea mariana), the objectives of this study were to identify adaptive genetic polymorphisms related to temperature and precipitation variation across the transcontinental range of the species, and to evaluate the potential influence of historical events on their geographic distribution. Population structure was first inferred using 50 control nuclear markers. Then, 47 candidate gene SNPs identified in previous genome scans were tested for relationship with climatic factors using an F(ST) -based outlier method and regressions between allele frequencies and climatic variations. Two main intraspecific lineages related to glacial vicariance were detected at the transcontinental scale. Within-lineage analyses of allele frequencies allowed the identification of 23 candidate SNPs significantly related to precipitation and/or temperature variation, among which seven were common to both lineages, eight were specific to the eastern lineage and eight were specific to the western lineage. The implication of these candidate SNPs in adaptive processes was further supported by gene functional annotations. Multiple evidences indicated that the occurrence of lineage-specific adaptive SNPs was better explained by selection acting on historically differentiated gene pools rather than differential selection due to heterogeneity of interacting environmental factors and pleiotropic effects. Taken together, these findings suggest that standing genetic variation of potentially adaptive nature has been modified by historical events, hence affecting the outcome of recent selection and leading to different adaptive routes between intraspecific lineages.

The Spruce Budworm Genome: Reconstructing the Evolutionary History of Antifreeze Proteins.

Insects have developed various adaptations to survive harsh winter conditions. Among freeze-intolerant species, some produce "antifreeze proteins" (AFPs) that bind to nascent ice crystals and inhibit further ice growth. Such is the case of the spruce budworm, Choristoneura fumiferana (Lepidoptera: Tortricidae), a destructive North American conifer pest that can withstand temperatures below -30°C. Despite the potential importance of AFPs in the adaptive diversification of Choristoneura, genomic tools to explore their origins have until now been limited. Here we present a chromosome-scale genome assembly for C. fumiferana, which we used to conduct comparative genomic analyses aimed at reconstructing the evolutionary history of tortricid AFPs. The budworm genome features 16 genes homologous to previously reported C. fumiferana AFPs (CfAFPs), 15 of which map to a single region on chromosome 18. Fourteen of these were also detected in five congeneric species, indicating Choristoneura AFP diversification occurred before the speciation event that led to C. fumiferana. Although budworm AFPs were previously considered unique to the genus Choristoneura, a search for homologs targeting recently sequenced tortricid genomes identified seven CfAFP-like genes in the distantly related Notocelia uddmanniana. High structural similarity between Notocelia and Choristoneura AFPs suggests a common origin, despite the absence of homologs in three related tortricids. Interestingly, one Notocelia AFP formed the C-terminus of a "zonadhesin-like" protein, possibly representing the ancestral condition from which tortricid AFPs evolved. Future work should clarify the evolutionary path of AFPs between Notocelia and Choristoneura and assess the role of the "zonadhesin-like" protein as precursor of tortricid AFPs.

Scanning the genome for gene SNPs related to climate adaptation and estimating selection at the molecular level in boreal black spruce.

Outlier detection methods were used to scan the genome of the boreal conifer black spruce (Picea mariana [Mill.] B.S.P.) for gene single-nucleotide polymorphisms (SNPs) potentially involved in adaptations to temperature and precipitation variations. The scan involved 583 SNPs from 313 genes potentially playing adaptive roles. Differentiation estimates among population groups defined following variation in temperature and precipitation were moderately high for adaptive quantitative characters such as the timing of budset or tree height (Q(ST) = 0.189-0.314). Average differentiation estimates for gene SNPs were null, with F(ST) values of 0.005 and 0.006, respectively, among temperature and precipitation population groups. Using two detection approaches, a total of 26 SNPs from 25 genes distributed among 11 of the 12 linkage groups of black spruce were detected as outliers with F(ST) as high as 0.078. Nearly half of the outlier SNPs were located in exons and half of those were nonsynonymous. The functional annotations of genes carrying outlier SNPs and regression analyses between the frequencies of these SNPs and climatic variables supported their implication in adaptive processes. Several genes carrying outlier SNPs belonged to gene families previously found to harbour outlier SNPs in a reproductively isolated but largely sympatric congeneric species, suggesting differential subfunctionalization of gene duplicates. Selection coefficient estimates (S) were moderate but well above the magnitude of drift (>>1/N(e)), indicating that the signature of natural selection could be detected at the nucleotide level despite the recent establishment of these populations during the Holocene.

The parasite Schistocephalus solidus secretes proteins with putative host manipulation functions.

BACKGROUND: Manipulative parasites are thought to liberate molecules in their external environment, acting as manipulation factors with biological functions implicated in their host's physiological and behavioural alterations. These manipulation factors are part of a complex mixture called the secretome. While the secretomes of various parasites have been described, there is very little data for a putative manipulative parasite. It is necessary to study the molecular interaction between a manipulative parasite and its host to better understand how such alterations evolve. METHODS: Here, we used proteomics to characterize the secretome of a model cestode with a complex life cycle based on trophic transmission. We studied Schistocephalus solidus during the life stage in which behavioural changes take place in its obligatory intermediate fish host, the threespine stickleback (Gasterosteus aculeatus). We produced a novel genome sequence and assembly of S. solidus to improve protein coding gene prediction and annotation for this parasite. We then described the whole worm's proteome and its secretome during fish host infection using LC-MS/MS. RESULTS: A total of 2290 proteins were detected in the proteome of S. solidus, and 30 additional proteins were detected specifically in the secretome. We found that the secretome contains proteases, proteins with neural and immune functions, as well as proteins involved in cell communication. We detected receptor-type tyrosine-protein phosphatases, which were reported in other parasitic systems to be manipulation factors. We also detected 12 S. solidus-specific proteins in the secretome that may play important roles in host-parasite interactions. CONCLUSIONS: Our results suggest that S. solidus liberates molecules with putative host manipulation functions in the host and that many of them are species-specific.