Mahesh Panchal

A global genome database of all of Earth's species diversity could be a treasure trove of scientific discoveries. However, regardless of the major advances in genome sequencing technologies, only a tiny fraction of species have genomic information available. To contribute to a more complete planetary genomic database, scientists and institutions across the world have united under the Earth BioGenome Project (EBP), which plans to sequence and assemble high-quality reference genomes for all ~1.5 million recognized eukaryotic species through a stepwise phased approach. As the initiative transitions into Phase II, where 150,000 species are to be sequenced in just four years, worldwide participation in the project will be fundamental to success. As the European node of the EBP, the European Reference Genome Atlas (ERGA) seeks to implement a new decentralised, accessible, equitable and inclusive model for producing high-quality reference genomes, which will inform EBP as it scales. To embark on this mission, ERGA launched a Pilot Project to establish a network across Europe to develop and test the first infrastructure of its kind for the coordinated and distributed reference genome production on 98 European eukaryotic species from sample providers across 34 European countries. Here we outline the process and challenges faced during the development of a pilot infrastructure for the production of reference genome resources, and explore the effectiveness of this approach in terms of high-quality reference genome production, considering also equity and inclusion. The outcomes and lessons learned during this pilot provide a solid foundation for ERGA while offering key learnings to other transnational and national genomic resource projects.

Endoparasitoid wasps are important natural enemies of many insect species and are major selective forces on the host immune system. Despite increased interest in insect antiparasitoid immunity, there is sparse information on the evolutionary dynamics of biological pathways and gene regulation involved in host immune defense outside Drosophila species. We de novo assembled transcriptomes from two beetle species and used time-course differential expression analysis to investigate gene expression differences in closely related species Galerucella pusilla and G. calmariensis that are, respectively, resistant and susceptible against parasitoid infection by Asecodes parviclava parasitoids. Approximately 271 million and 224 million paired-ended reads were assembled and filtered to form 52,563 and 59,781 transcripts for G. pusilla and G. calmariensis, respectively. In the whole-transcriptome level, an enrichment of functional categories related to energy production, biosynthetic process, and metabolic process was exhibited in both species. The main difference between species appears to be immune response and wound healing process mounted by G. pusilla larvae. Using reciprocal BLAST against the Drosophila melanogaster proteome, 120 and 121 immune-related genes were identified in G. pusilla and G. calmariensis, respectively. More immune genes were differentially expressed in G. pusilla than in G. calmariensis, in particular genes involved in signaling, hematopoiesis, and melanization. In contrast, only one gene was differentially expressed in G. calmariensis. Our study characterizes important genes and pathways involved in different immune functions after parasitoid infection and supports the role of signaling and hematopoiesis genes as key players in host immunity in Galerucella against parasitoid wasps.

As a part of the ELIXIR-EXCELERATE efforts in capacity building, we present here 10 steps to facilitate researchers getting started in genome assembly and genome annotation. The guidelines given are broadly applicable, intended to be stable over time, and cover all aspects from start to finish of a general assembly and annotation project. Intrinsic properties of genomes are discussed, as is the importance of using high quality DNA. Different sequencing technologies and generally applicable workflows for genome assembly are also detailed. We cover structural and functional annotation and encourage readers to also annotate transposable elements, something that is often omitted from annotation workflows. The importance of data management is stressed, and we give advice on where to submit data and how to make your results Findable, Accessible, Interoperable, and Reusable (FAIR).

The observation of habitat‐specific phenotypes suggests the action of natural selection. The three‐spined stickleback (Gasterosteus aculeatus) has repeatedly colonized and adapted to diverse freshwater habitats across the northern hemisphere since the last glaciation, while giving rise to recurring phenotypes associated with specific habitats. Parapatric lake and river populations of sticklebacks harbour distinct parasite communities, a factor proposed to contribute to adaptive differentiation between these ecotypes. However, little is known about the transcriptional response to the distinct parasite pressure of those fish in a natural setting. Here, we sampled wild‐caught sticklebacks across four geographical locations from lake and river habitats differing in their parasite load. We compared gene expression profiles between lake and river populations using 77 whole‐transcriptome libraries from two immune‐relevant tissues, the head kidney and the spleen. Differential expression analyses revealed 139 genes with habitat‐specific expression patterns across the sampled population pairs. Among the 139 differentially expressed genes, eight are annotated with an immune function and 42 have been identified as differentially expressed in previous experimental studies in which fish have been immune challenged. Together, these findings reinforce the hypothesis that parasites contribute to adaptation of sticklebacks in lake and river habitats.

Endoparasites with complex life cycles are faced with several biological challenges, as they need to occupy various ecological niches throughout their development. Host phenotypes that increase the parasite’s transmission rate to the next host have been extensively described, but few mechanistic explanations have been proposed to describe their proximate causes. In this study we explore the possibility that host phenotypic changes are triggered by the production of mimicry proteins from the parasite by using an ecological model system consisting of the infection of the threespine stickleback (Gasterosteus aculeatus) by the cestode Schistocephalus solidus. Using RNA-seq data, we assembled 9,093 protein-coding genes from which ORFs were predicted to generate a reference proteome. Based on a previously published method, we built two complementary analysis pipelines to i) establish a general classification of protein similarity among various species (pipeline A) and ii) identify candidate mimicry proteins showing specific host-parasite similarities (pipeline B), a key feature underlying the possibility of molecular mimicry. Ninety-four tapeworm proteins showed high local sequence homology with stickleback proteins. Four of these candidates correspond to secreted or membrane proteins that could be produced by the parasite and eventually be released in or be in contact with the host to modulate physiological pathways involved in various phenotypes (e.g. behaviors). One of these candidates belongs to the Wnt family, a large group of signaling molecules involved in cell-to-cell interactions and various developmental pathways. The three other candidates are involved in ion transport and post-translational protein modifications. We further confirmed that these four candidates are expressed in three different developmental stages of the cestode by RT-PCR, including the stages found in the host. In this study, we identified mimicry candidate peptides from a behavior-altering cestode showing specific sequence similarity with host proteins. Despite their potential role in modulating host pathways that could lead to parasite-induced phenotypic changes and despite our confirmation that they are expressed in the developmental stage corresponding to the altered host behavior, further investigations will be needed to confirm their mechanistic role in the molecular cross-talk taking place between S. solidus and the threespine stickleback.

The patterns of genomic divergence during ecological speciation are shaped by a combination of evolutionary forces. Processes such as genetic drift, local reduction of gene flow around genes causing reproductive isolation, hitchhiking around selected variants, variation in recombination and mutation rates are all factors that can contribute to the heterogeneity of genomic divergence. On the basis of 60 fully sequenced three-spined stickleback genomes, we explore these different mechanisms explaining the heterogeneity of genomic divergence across five parapatric lake and river population pairs varying in their degree of genetic differentiation. We find that divergent regions of the genome are mostly specific for each population pair, while their size and abundance are not correlated with the extent of genome-wide population differentiation. In each pair-wise comparison, an analysis of allele frequency spectra reveals that 25–55% of the divergent regions are consistent with a local restriction of gene flow. Another large proportion of divergent regions (38–75%) appears to be mainly shaped by hitchhiking effects around positively selected variants. We provide empirical evidence that alternative mechanisms determining the evolution of genomic patterns of divergence are not mutually exclusive, but rather act in concert to shape the genome during population differentiation, a first necessary step towards ecological speciation. A variety of evolutionary forces influence the genomic landscape of divergence during ecological speciation. Here we characterize the evolution of genomic divergence patterns based on 60 fully sequenced three-spined stickleback genomes, contrasting lake and river populations that differ in parasite abundance. Our comparison of the size and abundance of divergent regions in the genomes across a continuum of population differentiation suggests that selection and the hitchhiking effect on neutral sites mainly contributes to the observed heterogeneous patterns of genomic divergence. Additional divergent regions of the genome can be explained by a local reduction of gene flow. Our description of genomic divergence patterns across a continuum of population differentiation combined with an analysis of molecular signatures of evolution highlights how adaptation shapes the differentiation of sticklebacks in freshwater habitats.

Duplicate genes emerge as copy-number variations (CNVs) at the population level, and remain copy-number polymorphic until they are fixed or lost. The successful establishment of such structural polymorphisms in the genome plays an important role in evolution by promoting genetic diversity, complexity and innovation. To characterize the early evolutionary stages of duplicate genes and their potential adaptive benefits, we combine comparative genomics with population genomics analyses to evaluate the distribution and impact of CNVs across natural populations of an eco-genomic model, the three-spined stickleback. With whole genome sequences of 66 individuals from populations inhabiting three distinct habitats, we find that CNVs generally occur at low frequencies and are often only found in one of the 11 populations surveyed. A subset of CNVs, however, displays copy-number differentiation between populations, showing elevated within-population frequencies consistent with local adaptation. By comparing teleost genomes to identify lineage-specific genes and duplications in sticklebacks, we highlight rampant gene content differences among individuals in which over 30% of young duplicate genes are CNVs. These CNV genes are evolving rapidly at the molecular level and are enriched with functional categories associated with environmental interactions, depicting the dynamic early copy-number polymorphic stage of genes during population differentiation. After a locus is duplicated in a genome, individuals from a population instantaneously differ in the number of copies of this locus producing a copy-number variation (CNV). Over time, the joint effects of selection and other evolutionary forces will act to either eliminate the extra genetic copy or retain it. Depending on this evolutionary interplay, young duplications, including newly duplicated genes, can persist for millions of years as CNVs. CNVs may especially be prevalent between populations that have colonized and adapted to disparate environments in which selective pressures differ. Using whole genome sequences from several populations of three-spined sticklebacks that inhabit different environments, we find that a third of young duplicated genes are CNVs. These young CNV genes are enriched with environmental response functions and evolving rapidly at the molecular level, making them promising candidates for a role in the rapid ecological adaptation to novel environments.

Since the end of the Pleistocene, the three‐spined stickleback (Gasterosteus aculeatus) has repeatedly colonized and adapted to various freshwater habitats probably originating from ancestral marine populations. Standing genetic variation and the underlying genomic architecture both have been speculated to contribute to recent adaptive radiations of sticklebacks. Here, we expand on the current genomic resources of this fish by providing extensive genome‐wide variation data from six individuals from a marine (North Sea) stickleback population. Using next‐generation sequencing and a combination of paired‐end and mate‐pair libraries, we detected a wide size range of genetic variation. Among the six individuals, we found more than 7% of the genome is polymorphic, consisting of 2 599 111 SNPs, 233 464 indels and structural variation (SV) (>50 bp) such as 1054 copy‐number variable regions (deletions and duplications) and 48 inversions. Many of these polymorphisms affect gene and coding sequences. Based on SNP diversity, we determined outlier regions concordant with signatures expected under adaptive evolution. As some of these outliers overlap with pronounced regions of copy‐number variation, we propose the consideration of such SV when analysing SNP data from re‐sequencing approaches. We further discuss the value of this resource on genome‐wide variation for further investigation upon the relative contribution of standing variation on the parallel evolution of sticklebacks and the importance of the genomic architecture in adaptive radiation.

Nested clade phylogeographic analysis (NCPA) is a widely used method that aims to identify past demographic events that have shaped the history of a population. In an earlier study, NCPA has been fully automated, allowing it to be tested with simulated data sets generated under a null model in which samples simulated from a panmictic population are geographically distributed. It was noted that NCPA was prone to inferring false positives, corroborating earlier findings. The present study aims to evaluate both single-locus and multilocus NCPA under the scenario of restricted gene flow among spatially distributed populations. We have developed a new program, ANeCA-ML, which implements multilocus NCPA. Data were simulated under 3 models of gene flow: a stepping stone model, an island model, and a stepping stone model with some long-distance dispersal. Results indicate that single-locus NCPA tends to give a high frequency of false positives, but, unlike the random-mating scenario presented previously, inferences are not limited to restricted gene flow with isolation by distance or contiguous range expansion. The proportion of single-locus data sets that contained false inferences was 76% for the panmictic case, 87% for the stepping stone model, 79% for the stepping stone model with long-distance dispersal, and more than 99% for the island model. The frequency of inferences is inversely related to the amount of gene flow between demes. We performed multilocus NCPA by grouping the simulated loci into data sets of 5 loci. The false-positive rate was reduced in multilocus NCPA for some inferences but remained high for others. The proportion of multilocus data sets that contained false inferences was 17% for the panmictic case, 30% for the stepping stone model, 4% for the stepping stone model with long-distance dispersal, and 54% for the island model. Multilocus NCPA reduces the false-positive rate by restricting the sensitivity of the method but does not appear to increase the accuracy of the approach. Three classical tests—the analysis of molecular variance method, Fu's Fs, and the Mantel test—show that there is information in the data that gives rise to explicable results using these standard approaches. In conclusion, for the scenarios that we have examined, our simulation study suggests that the NCPA method is unreliable and its inferences may be misleading. We suggest that the NCPA method should not be used without objective simulation-based testing by independent researchers.