Vocal learning-associated convergent evolution in mammalian proteins and regulatory elements.

Vocal production learning ("vocal learning") is a convergently evolved trait in vertebrates. To identify brain genomic elements associated with mammalian vocal learning, we integrated genomic, anatomical, and neurophysiological data from the Egyptian fruit bat (Rousettus aegyptiacus) with analyses of the genomes of 215 placental mammals. First, we identified a set of proteins evolving more slowly in vocal learners. Then, we discovered a vocal motor cortical region in the Egyptian fruit bat, an emergent vocal learner, and leveraged that knowledge to identify active cis-regulatory elements in the motor cortex of vocal learners. Machine learning methods applied to motor cortex open chromatin revealed 50 enhancers robustly associated with vocal learning whose activity tended to be lower in vocal learners. Our research implicates convergent losses of motor cortex regulatory elements in mammalian vocal learning evolution.

syntenyPlotteR: a user-friendly R package to visualize genome synteny, ideal for both experienced and novice bioinformaticians.

Motivation: The rapid increase in the number of chromosome-scale genome assemblies has renewed interest in chromosome evolution studies. The visualization of syntenic relationships between genomes is a crucial initial step in the study of chromosome rearrangements and evolution. There are few tools available that serve this purpose, and they can be difficult to learn. Moreover, these tools are limited in the number of species comparisons that can be visualized and the size of chromosome rearrangements identified. Thus, the development of novel visualization tools is in strong need. Results: Here, we present syntenyPlotteR, an R package developed to visualize homologous synteny blocks in a pairwise or multispecies manner. This package contains three functions that allow users to generate publication-quality representations of syntenic relationships easily and quickly between genomes of interest. Availability and implementation: SyntenyPlotteR can be installed from CRAN with the documentation found in https://farre-lab.github.io/syntenyPlotteR/.

An atlas of regulatory elements in chicken: A resource for chicken genetics and genomics.

A comprehensive characterization of regulatory elements in the chicken genome across tissues will have substantial impacts on both fundamental and applied research. Here, we systematically identified and characterized regulatory elements in the chicken genome by integrating 377 genome-wide sequencing datasets from 23 adult tissues. In total, we annotated 1.57 million regulatory elements, representing 15 distinct chromatin states, and predicted about 1.2 million enhancer-gene pairs and 7662 super-enhancers. This functional annotation of the chicken genome should have wide utility on identifying regulatory elements accounting for gene regulation underlying domestication, selection, and complex trait regulation, which we explored. In short, this comprehensive atlas of regulatory elements provides the scientific community with a valuable resource for chicken genetics and genomics.

A genomic timescale for placental mammal evolution.

The precise pattern and timing of speciation events that gave rise to all living placental mammals remain controversial. We provide a comprehensive phylogenetic analysis of genetic variation across an alignment of 241 placental mammal genome assemblies, addressing prior concerns regarding limited genomic sampling across species. We compared neutral genome-wide phylogenomic signals using concatenation and coalescent-based approaches, interrogated phylogenetic variation across chromosomes, and analyzed extensive catalogs of structural variants. Interordinal relationships exhibit relatively low rates of phylogenomic conflict across diverse datasets and analytical methods. Conversely, X-chromosome versus autosome conflicts characterize multiple independent clades that radiated during the Cenozoic. Genomic time trees reveal an accumulation of cladogenic events before and immediately after the Cretaceous-Paleogene (K-Pg) boundary, implying important roles for Cretaceous continental vicariance and the K-Pg extinction in the placental radiation.

Evolutionary constraint and innovation across hundreds of placental mammals.

Zoonomia is the largest comparative genomics resource for mammals produced to date. By aligning genomes for 240 species, we identify bases that, when mutated, are likely to affect fitness and alter disease risk. At least 332 million bases (~10.7%) in the human genome are unusually conserved across species (evolutionarily constrained) relative to neutrally evolving repeats, and 4552 ultraconserved elements are nearly perfectly conserved. Of 101 million significantly constrained single bases, 80% are outside protein-coding exons and half have no functional annotations in the Encyclopedia of DNA Elements (ENCODE) resource. Changes in genes and regulatory elements are associated with exceptional mammalian traits, such as hibernation, that could inform therapeutic development. Earth's vast and imperiled biodiversity offers distinctive power for identifying genetic variants that affect genome function and organismal phenotypes.

Evolution of the ancestral mammalian karyotype and syntenic regions.

Decrypting the rearrangements that drive mammalian chromosome evolution is critical to understanding the molecular bases of speciation, adaptation, and disease susceptibility. Using 8 scaffolded and 26 chromosome-scale genome assemblies representing 23/26 mammal orders, we computationally reconstructed ancestral karyotypes and syntenic relationships at 16 nodes along the mammalian phylogeny. Three different reference genomes (human, sloth, and cattle) representing phylogenetically distinct mammalian superorders were used to assess reference bias in the reconstructed ancestral karyotypes and to expand the number of clades with reconstructed genomes. The mammalian ancestor likely had 19 pairs of autosomes, with nine of the smallest chromosomes shared with the common ancestor of all amniotes (three still conserved in extant mammals), demonstrating a striking conservation of synteny for ∼320 My of vertebrate evolution. The numbers and types of chromosome rearrangements were classified for transitions between the ancestral mammalian karyotype, descendent ancestors, and extant species. For example, 94 inversions, 16 fissions, and 14 fusions that occurred over 53 My differentiated the therian from the descendent eutherian ancestor. The highest breakpoint rate was observed between the mammalian and therian ancestors (3.9 breakpoints/My). Reconstructed mammalian ancestor chromosomes were found to have distinct evolutionary histories reflected in their rates and types of rearrangements. The distributions of genes, repetitive elements, topologically associating domains, and actively transcribed regions in multispecies homologous synteny blocks and evolutionary breakpoint regions indicate that purifying selection acted over millions of years of vertebrate evolution to maintain syntenic relationships of developmentally important genes and regulatory landscapes of gene-dense chromosomes.

Augmin-dependent microtubule self-organization drives kinetochore fiber maturation in mammals.

Chromosome segregation in mammals relies on the maturation of a thick bundle of kinetochore-attached microtubules known as k-fiber. How k-fibers mature from initial kinetochore microtubule attachments remains a fundamental question. By combining molecular perturbations and phenotypic analyses in Indian muntjac fibroblasts containing the lowest known diploid chromosome number in mammals (2N = 6) and distinctively large kinetochores, with fixed/live-cell super-resolution coherent-hybrid stimulated emission depletion (CH-STED) nanoscopy and laser microsurgery, we demonstrate a key role for augmin in kinetochore microtubule self-organization and maturation, regardless of pioneer centrosomal microtubules. In doing so, augmin promotes kinetochore and interpolar microtubule turnover and poleward flux. Tracking of microtubule growth events within individual k-fibers reveals a wide angular dispersion, consistent with augmin-mediated branched microtubule nucleation. Augmin depletion reduces the frequency of kinetochore microtubule growth events and hampers efficient repair after acute k-fiber injury by laser microsurgery. Together, these findings underscore the contribution of augmin-mediated microtubule amplification for k-fiber self-organization and maturation in mammals.

Conservation of chromatin conformation in carnivores.

High throughput chromatin conformation capture (Hi-C) of leukocyte DNA was used to investigate the evolutionary stability of chromatin conformation at the chromosomal level in 11 species from three carnivore families: Felidae, Canidae, and Ursidae. Chromosome-scale scaffolds (C-scaffolds) of each species were initially used for whole-genome alignment to a reference genome within each family. This approach established putative orthologous relationships between C-scaffolds among the different species. Hi-C contact maps for all C-scaffolds were then visually compared and found to be distinct for a given reference chromosome or C-scaffold within a species and indistinguishable for orthologous C-scaffolds having a 1:1 relationship within a family. The visual patterns within families were strongly supported by eigenvectors from the Hi-C contact maps. Analysis of Hi-C contact maps and eigenvectors across the three carnivore families revealed that most cross-family orthologous subchromosomal fragments have a conserved three-dimensional (3D) chromatin structure and thus have been under strong evolutionary constraint for ∼54 My of carnivore evolution. The most pronounced differences in chromatin conformation were observed for the X chromosome and the red fox genome, whose chromosomes have undergone extensive rearrangements relative to other canids. We also demonstrate that Hi-C contact map pattern analysis can be used to accurately identify orthologous relationships between C-scaffolds and chromosomes, a method we termed "3D comparative scaffotyping." This method provides a powerful means for estimating karyotypes in de novo sequenced species that have unknown karyotype and no physical mapping information.

Towards complete and error-free genome assemblies of all vertebrate species.

High-quality and complete reference genome assemblies are fundamental for the application of genomics to biology, disease, and biodiversity conservation. However, such assemblies are available for only a few non-microbial species1-4. To address this issue, the international Genome 10K (G10K) consortium5,6 has worked over a five-year period to evaluate and develop cost-effective methods for assembling highly accurate and nearly complete reference genomes. Here we present lessons learned from generating assemblies for 16 species that represent six major vertebrate lineages. We confirm that long-read sequencing technologies are essential for maximizing genome quality, and that unresolved complex repeats and haplotype heterozygosity are major sources of assembly error when not handled correctly. Our assemblies correct substantial errors, add missing sequence in some of the best historical reference genomes, and reveal biological discoveries. These include the identification of many false gene duplications, increases in gene sizes, chromosome rearrangements that are specific to lineages, a repeated independent chromosome breakpoint in bat genomes, and a canonical GC-rich pattern in protein-coding genes and their regulatory regions. Adopting these lessons, we have embarked on the Vertebrate Genomes Project (VGP), an international effort to generate high-quality, complete reference genomes for all of the roughly 70,000 extant vertebrate species and to help to enable a new era of discovery across the life sciences.

Platypus and echidna genomes reveal mammalian biology and evolution.

Egg-laying mammals (monotremes) are the only extant mammalian outgroup to therians (marsupial and eutherian animals) and provide key insights into mammalian evolution1,2. Here we generate and analyse reference genomes of the platypus (Ornithorhynchus anatinus) and echidna (Tachyglossus aculeatus), which represent the only two extant monotreme lineages. The nearly complete platypus genome assembly has anchored almost the entire genome onto chromosomes, markedly improving the genome continuity and gene annotation. Together with our echidna sequence, the genomes of the two species allow us to detect the ancestral and lineage-specific genomic changes that shape both monotreme and mammalian evolution. We provide evidence that the monotreme sex chromosome complex originated from an ancestral chromosome ring configuration. The formation of such a unique chromosome complex may have been facilitated by the unusually extensive interactions between the multi-X and multi-Y chromosomes that are shared by the autosomal homologues in humans. Further comparative genomic analyses unravel marked differences between monotremes and therians in haptoglobin genes, lactation genes and chemosensory receptor genes for smell and taste that underlie the ecological adaptation of monotremes.

Vertebrate Chromosome Evolution.

The study of chromosome evolution is undergoing a resurgence of interest owing to advances in DNA sequencing technology that facilitate the production of chromosome-scale whole-genome assemblies de novo. This review focuses on the history, methods, discoveries, and current challenges facing the field, with an emphasis on vertebrate genomes. A detailed examination of the literature on the biology of chromosome rearrangements is presented, specifically the relationship between chromosome rearrangements and phenotypic evolution, adaptation, and speciation. A critical review of the methods for identifying, characterizing, and visualizing chromosome rearrangements and computationally reconstructing ancestral karyotypes is presented. We conclude by looking to the future, identifying the enormous technical and scientific challenges presented by the accumulation of hundreds and eventually thousands of chromosome-scale assemblies.

Broad host range of SARS-CoV-2 predicted by comparative and structural analysis of ACE2 in vertebrates.

The novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of COVID-19. The main receptor of SARS-CoV-2, angiotensin I converting enzyme 2 (ACE2), is now undergoing extensive scrutiny to understand the routes of transmission and sensitivity in different species. Here, we utilized a unique dataset of ACE2 sequences from 410 vertebrate species, including 252 mammals, to study the conservation of ACE2 and its potential to be used as a receptor by SARS-CoV-2. We designed a five-category binding score based on the conservation properties of 25 amino acids important for the binding between ACE2 and the SARS-CoV-2 spike protein. Only mammals fell into the medium to very high categories and only catarrhine primates into the very high category, suggesting that they are at high risk for SARS-CoV-2 infection. We employed a protein structural analysis to qualitatively assess whether amino acid changes at variable residues would be likely to disrupt ACE2/SARS-CoV-2 spike protein binding and found the number of predicted unfavorable changes significantly correlated with the binding score. Extending this analysis to human population data, we found only rare (frequency <0.001) variants in 10/25 binding sites. In addition, we found significant signals of selection and accelerated evolution in the ACE2 coding sequence across all mammals, and specific to the bat lineage. Our results, if confirmed by additional experimental data, may lead to the identification of intermediate host species for SARS-CoV-2, guide the selection of animal models of COVID-19, and assist the conservation of animals both in native habitats and in human care.

Broad Host Range of SARS-CoV-2 Predicted by Comparative and Structural Analysis of ACE2 in Vertebrates.

The novel coronavirus SARS-CoV-2 is the cause of Coronavirus Disease-2019 (COVID-19). The main receptor of SARS-CoV-2, angiotensin I converting enzyme 2 (ACE2), is now undergoing extensive scrutiny to understand the routes of transmission and sensitivity in different species. Here, we utilized a unique dataset of 410 vertebrates, including 252 mammals, to study cross-species conservation of ACE2 and its likelihood to function as a SARS-CoV-2 receptor. We designed a five-category ranking score based on the conservation properties of 25 amino acids important for the binding between receptor and virus, classifying all species from very high to very low. Only mammals fell into the medium to very high categories, and only catarrhine primates in the very high category, suggesting that they are at high risk for SARS-CoV-2 infection. We employed a protein structural analysis to qualitatively assess whether amino acid changes at variable residues would be likely to disrupt ACE2/SARS-CoV-2 binding, and found the number of predicted unfavorable changes significantly correlated with the binding score. Extending this analysis to human population data, we found only rare (<0.1%) variants in 10/25 binding sites. In addition, we observed evidence of positive selection in ACE2 in multiple species, including bats. Utilized appropriately, our results may lead to the identification of intermediate host species for SARS-CoV-2, justify the selection of animal models of COVID-19, and assist the conservation of animals both in native habitats and in human care.

An integrated chromosome-scale genome assembly of the Masai giraffe (Giraffa camelopardalis tippelskirchi).

BACKGROUND: The Masai giraffe (Giraffa camelopardalis tippelskirchi) is the largest-bodied giraffe and the world's tallest terrestrial animal. With its extreme size and height, the giraffe's unique anatomical and physiological adaptations have long been of interest to diverse research fields. Giraffes are also critical to ecosystems of sub-Saharan Africa, with their long neck serving as a conduit to food sources not shared by other herbivores. Although the genome of a Masai giraffe has been sequenced, the assembly was highly fragmented and suboptimal for genome analysis. Herein we report an improved giraffe genome assembly to facilitate evolutionary analysis of the giraffe and other ruminant genomes. FINDINGS: Using SOAPdenovo2 and 170 Gbp of Illumina paired-end and mate-pair reads, we generated a 2.6-Gbp male Masai giraffe genome assembly, with a scaffold N50 of 3 Mbp. The incorporation of 114.6 Gbp of Chicago library sequencing data resulted in a HiRise SOAPdenovo + Chicago assembly with an N50 of 48 Mbp and containing 95% of expected genes according to BUSCO analysis. Using the Reference-Assisted Chromosome Assembly tool, we were able to order and orient scaffolds into 42 predicted chromosome fragments (PCFs). Using fluorescence in situ hybridization, we placed 153 cattle bacterial artificial chromosomes onto giraffe metaphase spreads to assess and assign the PCFs on 14 giraffe autosomes and the X chromosome resulting in the final assembly with an N50 of 177.94 Mbp. In this assembly, 21,621 protein-coding genes were identified using both de novo and homology-based predictions. CONCLUSIONS: We have produced the first chromosome-scale genome assembly for a Giraffidae species. This assembly provides a valuable resource for the study of artiodactyl evolution and for understanding the molecular basis of the unique adaptive traits of giraffes. In addition, the assembly will provide a powerful resource to assist conservation efforts of Masai giraffe, whose population size has declined by 52% in recent years.

Splice-Break: exploiting an RNA-seq splice junction algorithm to discover mitochondrial DNA deletion breakpoints and analyses of psychiatric disorders.

Deletions in the 16.6 kb mitochondrial genome have been implicated in numerous disorders that often display muscular and/or neurological symptoms due to the high-energy demands of these tissues. We describe a catalogue of 4489 putative mitochondrial DNA (mtDNA) deletions, including their frequency and relative read rate, using a combinatorial approach of mitochondria-targeted PCR, next-generation sequencing, bioinformatics, post-hoc filtering, annotation, and validation steps. Our bioinformatics pipeline uses MapSplice, an RNA-seq splice junction detection algorithm, to detect and quantify mtDNA deletion breakpoints rather than mRNA splices. Analyses of 93 samples from postmortem brain and blood found (i) the 4977 bp 'common deletion' was neither the most frequent deletion nor the most abundant; (ii) brain contained significantly more deletions than blood; (iii) many high frequency deletions were previously reported in MitoBreak, suggesting they are present at low levels in metabolically active tissues and are not exclusive to individuals with diagnosed mitochondrial pathologies; (iv) many individual deletions (and cumulative metrics) had significant and positive correlations with age and (v) the highest deletion burdens were observed in major depressive disorder brain, at levels greater than Kearns-Sayre Syndrome muscle. Collectively, these data suggest the Splice-Break pipeline can detect and quantify mtDNA deletions at a high level of resolution.

A near-chromosome-scale genome assembly of the gemsbok (Oryx gazella): an iconic antelope of the Kalahari desert.

Background: The gemsbok (Oryx gazella) is one of the largest antelopes in Africa. Gemsbok are heterothermic and thus highly adapted to live in the desert, changing their feeding behavior when faced with extreme drought and heat. A high-quality genome sequence of this species will assist efforts to elucidate these and other important traits of gemsbok and facilitate research on conservation efforts. Findings: Using 180 Gbp of Illumina paired-end and mate-pair reads, a 2.9 Gbp assembly with scaffold N50 of 1.48 Mbp was generated using SOAPdenovo. Scaffolds were extended using Chicago library sequencing, which yielded an additional 114.7 Gbp of DNA sequence. The HiRise assembly using SOAPdenovo + Chicago library sequencing produced a scaffold N50 of 47 Mbp and a final genome size of 2.9 Gbp, representing 90.6% of the estimated genome size and including 93.2% of expected genes according to Benchmarking Universal Single-Copy Orthologs analysis. The Reference-Assisted Chromosome Assembly tool was used to generate a final set of 47 predicted chromosome fragments with N50 of 86.25 Mbp and containing 93.8% of expected genes. A total of 23,125 protein-coding genes and 1.14 Gbp of repetitive sequences were annotated using de novo and homology-based predictions. Conclusions: Our results provide the first high-quality, chromosome-scale genome sequence assembly for gemsbok, which will be a valuable resource for studying adaptive evolution of this species and other ruminants.

Reconstruction of avian ancestral karyotypes reveals differences in the evolutionary history of macro- and microchromosomes.

BACKGROUND: Reconstruction of ancestral karyotypes is critical for our understanding of genome evolution, allowing for the identification of the gross changes that shaped extant genomes. The identification of such changes and their time of occurrence can shed light on the biology of each species, clade and their evolutionary history. However, this is impeded by both the fragmented nature of the majority of genome assemblies and the limitations of the available software to work with them. These limitations are particularly apparent in birds, with only 10 chromosome-level assemblies reported thus far. Algorithmic approaches applied to fragmented genome assemblies can nonetheless help define patterns of chromosomal change in defined taxonomic groups. RESULTS: Here, we make use of the DESCHRAMBLER algorithm to perform the first large-scale study of ancestral chromosome structure and evolution in birds. This algorithm allows us to reconstruct the overall genome structure of 14 key nodes of avian evolution from the Avian ancestor to the ancestor of the Estrildidae, Thraupidae and Fringillidae families. CONCLUSIONS: Analysis of these reconstructions provides important insights into the variability of rearrangement rates during avian evolution and allows the detection of patterns related to the chromosome distribution of evolutionary breakpoint regions. Moreover, the inclusion of microchromosomes in our reconstructions allows us to provide novel insights into the evolution of these avian chromosomes, specifically.

Chromosome-level assembly reveals extensive rearrangement in saker falcon and budgerigar, but not ostrich, genomes.

BACKGROUND: The number of de novo genome sequence assemblies is increasing exponentially; however, relatively few contain one scaffold/contig per chromosome. Such assemblies are essential for studies of genotype-to-phenotype association, gross genomic evolution, and speciation. Inter-species differences can arise from chromosomal changes fixed during evolution, and we previously hypothesized that a higher fraction of elements under negative selection contributed to avian-specific phenotypes and avian genome organization stability. The objective of this study is to generate chromosome-level assemblies of three avian species (saker falcon, budgerigar, and ostrich) previously reported as karyotypically rearranged compared to most birds. We also test the hypothesis that the density of conserved non-coding elements is associated with the positions of evolutionary breakpoint regions. RESULTS: We used reference-assisted chromosome assembly, PCR, and lab-based molecular approaches, to generate chromosome-level assemblies of the three species. We mapped inter- and intrachromosomal changes from the avian ancestor, finding no interchromosomal rearrangements in the ostrich genome, despite it being previously described as chromosomally rearranged. We found that the average density of conserved non-coding elements in evolutionary breakpoint regions is significantly reduced. Fission evolutionary breakpoint regions have the lowest conserved non-coding element density, and intrachromomosomal evolutionary breakpoint regions have the highest. CONCLUSIONS: The tools used here can generate inexpensive, efficient chromosome-level assemblies, with > 80% assigned to chromosomes, which is comparable to genomes assembled using high-density physical or genetic mapping. Moreover, conserved non-coding elements are important factors in defining where rearrangements, especially interchromosomal, are fixed during evolution without deleterious effects.

Chromosome Segregation Is Biased by Kinetochore Size.

Chromosome missegregation during mitosis or meiosis is a hallmark of cancer and the main cause of prenatal death in humans. The gain or loss of specific chromosomes is thought to be random, with cell viability being essentially determined by selection. Several established pathways including centrosome amplification, sister-chromatid cohesion defects, or a compromised spindle assembly checkpoint can lead to chromosome missegregation. However, how specific intrinsic features of the kinetochore-the critical chromosomal interface with spindle microtubules-impact chromosome segregation remains poorly understood. Here we used the unique cytological attributes of female Indian muntjac, the mammal with the lowest known chromosome number (2n = 6), to characterize and track individual chromosomes with distinct kinetochore size throughout mitosis. We show that centromere and kinetochore functional layers scale proportionally with centromere size. Measurement of intra-kinetochore distances, serial-section electron microscopy, and RNAi against key kinetochore proteins confirmed a standard structural and functional organization of the Indian muntjac kinetochores and revealed that microtubule binding capacity scales with kinetochore size. Surprisingly, we found that chromosome segregation in this species is not random. Chromosomes with larger kinetochores bi-oriented more efficiently and showed a 2-fold bias to congress to the equator in a motor-independent manner. Despite robust correction mechanisms during unperturbed mitosis, chromosomes with larger kinetochores were also strongly biased to establish erroneous merotelic attachments and missegregate during anaphase. This bias was impervious to the experimental attenuation of polar ejection forces on chromosome arms by RNAi against the chromokinesin Kif4a. Thus, kinetochore size is an important determinant of chromosome segregation fidelity.

Reconstruction of the diapsid ancestral genome permits chromosome evolution tracing in avian and non-avian dinosaurs.

Genomic organisation of extinct lineages can be inferred from extant chromosome-level genome assemblies. Here, we apply bioinformatic and molecular cytogenetic approaches to determine the genomic structure of the diapsid common ancestor. We then infer the events that likely occurred along this lineage from theropod dinosaurs through to modern birds. Our results suggest that most elements of a typical 'avian-like' karyotype (40 chromosome pairs, including 30 microchromosomes) were in place before the divergence of turtles from birds ~255 mya. This genome organisation therefore predates the emergence of early dinosaurs and pterosaurs and the evolution of flight. Remaining largely unchanged interchromosomally through the dinosaur-theropod route that led to modern birds, intrachromosomal changes nonetheless reveal evolutionary breakpoint regions enriched for genes with ontology terms related to chromatin organisation and transcription. This genomic structure therefore appears highly stable yet contributes to a large degree of phenotypic diversity, as well as underpinning adaptive responses to major environmental disruptions via intrachromosomal repatterning.