Cellular development and evolution of the mammalian cerebellum.

The expansion of the neocortex, a hallmark of mammalian evolution1,2, was accompanied by an increase in cerebellar neuron numbers3. However, little is known about the evolution of the cellular programmes underlying the development of the cerebellum in mammals. In this study we generated single-nucleus RNA-sequencing data for around 400,000 cells to trace the development of the cerebellum from early neurogenesis to adulthood in human, mouse and the marsupial opossum. We established a consensus classification of the cellular diversity in the developing mammalian cerebellum and validated it by spatial mapping in the fetal human cerebellum. Our cross-species analyses revealed largely conserved developmental dynamics of cell-type generation, except for Purkinje cells, for which we observed an expansion of early-born subtypes in the human lineage. Global transcriptome profiles, conserved cell-state markers and gene-expression trajectories across neuronal differentiation show that cerebellar cell-type-defining programmes have been overall preserved for at least 160 million years. However, we also identified many orthologous genes that gained or lost expression in cerebellar neural cell types in one of the species or evolved new expression trajectories during neuronal differentiation, indicating widespread gene repurposing at the cell-type level. In sum, our study unveils shared and lineage-specific gene-expression programmes governing the development of cerebellar cells and expands our understanding of mammalian brain evolution.

The molecular evolution of spermatogenesis across mammals.

The testis produces gametes through spermatogenesis and evolves rapidly at both the morphological and molecular level in mammals1-6, probably owing to the evolutionary pressure on males to be reproductively successful7. However, the molecular evolution of individual spermatogenic cell types across mammals remains largely uncharacterized. Here we report evolutionary analyses of single-nucleus transcriptome data for testes from 11 species that cover the three main mammalian lineages (eutherians, marsupials and monotremes) and birds (the evolutionary outgroup), and include seven primates. We find that the rapid evolution of the testis was driven by accelerated fixation rates of gene expression changes, amino acid substitutions and new genes in late spermatogenic stages, probably facilitated by reduced pleiotropic constraints, haploid selection and transcriptionally permissive chromatin. We identify temporal expression changes of individual genes across species and conserved expression programs controlling ancestral spermatogenic processes. Genes predominantly expressed in spermatogonia (germ cells fuelling spermatogenesis) and Sertoli (somatic support) cells accumulated on X chromosomes during evolution, presumably owing to male-beneficial selective forces. Further work identified transcriptomal differences between X- and Y-bearing spermatids and uncovered that meiotic sex-chromosome inactivation (MSCI) also occurs in monotremes and hence is common to mammalian sex-chromosome systems. Thus, the mechanism of meiotic silencing of unsynapsed chromatin, which underlies MSCI, is an ancestral mammalian feature. Our study illuminates the molecular evolution of spermatogenesis and associated selective forces, and provides a resource for investigating the biology of the testis across mammals.

Transcriptome and translatome co-evolution in mammals.

Gene-expression programs define shared and species-specific phenotypes, but their evolution remains largely uncharacterized beyond the transcriptome layer1. Here we report an analysis of the co-evolution of translatomes and transcriptomes using ribosome-profiling and matched RNA-sequencing data for three organs (brain, liver and testis) in five mammals (human, macaque, mouse, opossum and platypus) and a bird (chicken). Our within-species analyses reveal that translational regulation is widespread in the different organs, in particular across the spermatogenic cell types of the testis. The between-species divergence in gene expression is around 20% lower at the translatome layer than at the transcriptome layer owing to extensive buffering between the expression layers, which especially preserved old, essential and housekeeping genes. Translational upregulation specifically counterbalanced global dosage reductions during the evolution of sex chromosomes and the effects of meiotic sex-chromosome inactivation during spermatogenesis. Despite the overall prevalence of buffering, some genes evolved faster at the translatome layer-potentially indicating adaptive changes in expression; testis tissue shows the highest fraction of such genes. Further analyses incorporating mass spectrometry proteomics data establish that the co-evolution of transcriptomes and translatomes is reflected at the proteome layer. Together, our work uncovers co-evolutionary patterns and associated selective forces across the expression layers, and provides a resource for understanding their interplay in mammalian organs.

Convergent origination of a Drosophila-like dosage compensation mechanism in a reptile lineage.

Sex chromosomes differentiated from different ancestral autosomes in various vertebrate lineages. Here, we trace the functional evolution of the XY Chromosomes of the green anole lizard (Anolis carolinensis), on the basis of extensive high-throughput genome, transcriptome and histone modification sequencing data and revisit dosage compensation evolution in representative mammals and birds with substantial new expression data. Our analyses show that Anolis sex chromosomes represent an ancient XY system that originated at least ≈160 million years ago in the ancestor of Iguania lizards, shortly after the separation from the snake lineage. The age of this system approximately coincides with the ages of the avian and two mammalian sex chromosomes systems. To compensate for the almost complete Y Chromosome degeneration, X-linked genes have become twofold up-regulated, restoring ancestral expression levels. The highly efficient dosage compensation mechanism of Anolis represents the only vertebrate case identified so far to fully support Ohno's original dosage compensation hypothesis. Further analyses reveal that X up-regulation occurs only in males and is mediated by a male-specific chromatin machinery that leads to global hyperacetylation of histone H4 at lysine 16 specifically on the X Chromosome. The green anole dosage compensation mechanism is highly reminiscent of that of the fruit fly, Drosophila melanogaster Altogether, our work unveils the convergent emergence of a Drosophila-like dosage compensation mechanism in an ancient reptilian sex chromosome system and highlights that the evolutionary pressures imposed by sex chromosome dosage reductions in different amniotes were resolved in fundamentally different ways.

Comparative genome analysis of Pseudogymnoascus spp. reveals primarily clonal evolution with small genome fragments exchanged between lineages.

BACKGROUND: Pseudogymnoascus spp. is a wide group of fungi lineages in the family Pseudorotiaceae including an aggressive pathogen of bats P. destructans. Although several lineages of P. spp. were shown to produce ascospores in culture, the vast majority of P. spp. demonstrates no evidence of sexual reproduction. P. spp. can tolerate a wide range of different temperatures and salinities and can survive even in permafrost layer. Adaptability of P. spp. to different environments is accompanied by extremely variable morphology and physiology. RESULTS: We sequenced genotypes of 14 strains of P. spp., 5 of which were extracted from permafrost, 1 from a cryopeg, a layer of unfrozen ground in permafrost, and 8 from temperate surface environments. All sequenced genotypes are haploid. Nucleotide diversity among these genomes is very high, with a typical evolutionary distance at synonymous sites dS ≈ 0.5, suggesting that the last common ancestor of these strains lived >50 Mya. The strains extracted from permafrost do not form a separate clade. Instead, each permafrost strain has close relatives from temperate environments. We observed a strictly clonal population structure with no conflicting topologies for ~99% of genome sequences. However, there is a number of short (~100-10,000 nt) genomic segments with the total length of 67.6 Kb which possess phylogenetic patterns strikingly different from the rest of the genome. The most remarkable case is a MAT-locus, which has 2 distinct alleles interspersed along the whole-genome phylogenetic tree. CONCLUSIONS: Predominantly clonal structure of genome sequences is consistent with the observations that sexual reproduction is rare in P. spp. Small number of regions with noncanonical phylogenies seem to arise due to some recombination events between derived lineages of P. spp., with MAT-locus being transferred on multiple occasions. All sequenced strains have heterothallic configuration of MAT-locus.

Crossing-over in a hypervariable species preferentially occurs in regions of high local similarity.

Recombination between double-stranded DNA molecules is a key genetic process which occurs in a wide variety of organisms. Usually, crossing-over (CO) occurs during meiosis between genotypes with 98.0-99.9% sequence identity, because within-population nucleotide diversity only rarely exceeds 2%. However, some species are hypervariable and it is unclear how CO can occur between genotypes with less than 90% sequence identity. Here, we study CO in Schizophyllum commune, a hypervariable cosmopolitan basidiomycete mushroom, a frequently encountered decayer of woody substrates. We crossed two haploid individuals, from the United States and from Russia, and obtained genome sequences for their 17 offspring. The average genetic distance between the parents was 14%, making it possible to study CO at very high resolution. We found reduced levels of linkage disequilibrium between loci flanking the CO sites indicating that they are mostly confined to hotspots of recombination. Furthermore, CO events preferentially occurred in regions under stronger negative selection, in particular within exons that showed reduced levels of nucleotide diversity. Apparently, in hypervariable species CO must avoid regions of higher divergence between the recombining genomes due to limitations imposed by the mismatch repair system, with regions under strong negative selection providing the opportunity for recombination. These patterns are opposite to those observed in a number of less variable species indicating that population genomics of hypervariable species may reveal novel biological phenomena.

The miniature genome of a carnivorous plant Genlisea aurea contains a low number of genes and short non-coding sequences.

BACKGROUND: Genlisea aurea (Lentibulariaceae) is a carnivorous plant with unusually small genome size - 63.6 Mb - one of the smallest known among higher plants. Data on the genome sizes and the phylogeny of Genlisea suggest that this is a derived state within the genus. Thus, G. aurea is an excellent model organism for studying evolutionary mechanisms of genome contraction. RESULTS: Here we report sequencing and de novo draft assembly of G. aurea genome. The assembly consists of 10,687 contigs of the total length of 43.4 Mb and includes 17,755 complete and partial protein-coding genes. Its comparison with the genome of Mimulus guttatus, another representative of higher core Lamiales clade, reveals striking differences in gene content and length of non-coding regions. CONCLUSIONS: Genome contraction was a complex process, which involved gene loss and reduction of lengths of introns and intergenic regions, but not intron loss. The gene loss is more frequent for the genes that belong to multigenic families indicating that genetic redundancy is an important prerequisite for genome size reduction.

Insertions and deletions trigger adaptive walks in Drosophila proteins.

Maps that relate all possible genotypes or phenotypes to fitness--fitness landscapes--are central to the evolution of life, but remain poorly known. An insertion or a deletion (indel) of one or several amino acids constitutes a substantial leap of a protein within the space of amino acid sequences, and it is unlikely that after such a leap the new sequence corresponds precisely to a fitness peak. Thus, one can expect an indel in the protein-coding sequence that gets fixed in a population to be followed by some number of adaptive amino acid substitutions, which move the new sequence towards a nearby fitness peak. Here, we study substitutions that occur after a frame-preserving indel in evolving proteins of Drosophila. An insertion triggers 1.03 ± 0.75 amino acid substitutions within the protein region centred at the site of insertion, and a deletion triggers 4.77 ± 1.03 substitutions within such a region. The difference between these values is probably owing to a higher fraction of effectively neutral insertions. Almost all of the triggered amino acid substitutions can be attributed to positive selection, and most of them occur relatively soon after the triggering indel and take place upstream of its site. A high fraction of substitutions that follow an indel occur at previously conserved sites, suggesting that an indel substantially changes selection that shapes the protein region around it. Thus, an indel is often followed by an adaptive walk of length that is in agreement with the theory of molecular adaptation.

Developmental and evolutionary dynamics of cis-regulatory elements in mouse cerebellar cells.

Organ development is orchestrated by cell- and time-specific gene regulatory networks. In this study, we investigated the regulatory basis of mouse cerebellum development from early neurogenesis to adulthood. By acquiring snATAC-seq (single-nucleus assay for transposase accessible chromatin using sequencing) profiles for ~90,000 cells spanning 11 stages, we mapped cerebellar cell types and identified candidate cis-regulatory elements (CREs). We detected extensive spatiotemporal heterogeneity among progenitor cells and a gradual divergence in the regulatory programs of cerebellar neurons during differentiation. Comparisons to vertebrate genomes and snATAC-seq profiles for ∼20,000 cerebellar cells from the marsupial opossum revealed a shared decrease in CRE conservation during development and differentiation as well as differences in constraint between cell types. Our work delineates the developmental and evolutionary dynamics of gene regulation in cerebellar cells and provides insights into mammalian organ development.

Short indels are subject to insertion-biased gene conversion.

Recombination between homologous loci is accompanied by formation of heteroduplexes. Repairing mismatches in heteroduplexes often leads to single nucleotide substitutions in a process known as gene conversion. Gene conversion was shown to be GC-biased in different organisms; that is, a W(A or T)→S(G or C) substitution is more likely in this process than a S→W substitution. Here, we show that the insertion/deletion ratio for short noncoding indels that reach fixation between species is positively correlated with the recombination rate in Drosophila melanogaster, Homo sapiens, and Saccharomyces cerevisiae. This correlation is both due to an increase of the fixation rate of insertions and decrease of the fixation rate of deletions in regions of high recombination. Whole-genome data on indel polymorphism and divergence in D. melanogaster rule out mutation biases and selection as the cause of this trend, pointing to insertion-biased gene conversion as the most likely explanation. The bias toward insertions is the strongest for single-nucleotide indels, and decreases with indel length. In regions of high recombination rate this bias leads to an up to ∼5-fold excess of fixed short insertions over deletions, and substantially affects the evolution of DNA segments.

Strong mutational bias toward deletions in the Drosophila melanogaster genome is compensated by selection.

Insertions and deletions (collectively indels) obviously have a major impact on genome evolution. However, before large-scale data on indel polymorphism became available, it was difficult to estimate the strength of selection acting on indel mutations. Here, we analyze indel polymorphism and divergence in different compartments of the Drosophila melanogaster genome: exons, introns of different lengths, and intergenic regions. Data on low-frequency polymorphisms indicate that 0.036-0.039 short (1-30 nt) insertion mutations and 0.085-0.092 short deletion mutations, with mean lengths 3.23 and 4.78, respectively, occur per single-nucleotide substitution. The excess of short deletion over short insertion mutations implies that indel mutations of these lengths should lead to a loss of approximately 0.30 nt per single-nucleotide replacement. However, polymorphism and divergence data show that this deletion bias is almost completely compensated by selection: Negative selection is stronger against deletions, whereas insertions are more likely to be favored by positive selection. Among the inframe low-frequency polymorphic mutations in exons, long introns, and intergenic regions, selection prevents a larger fraction of deletions (80-87%, depending on the type of the compartment) than of insertions (70-82%) or single-nucleotide substitutions (49-73%), from reaching high frequencies. The corresponding fractions were the lowest in short introns: 66%, 47%, and 15%, respectively, consistent with the weakest selective constraint in them. The McDonald-Kreitman test shows that 32-46% of the deletions and 60-73% of the insertions that were fixed in the recent evolution of D. melanogaster are adaptive, whereas this fraction is only 0-29% for single-nucleotide substitutions.