A region of suppressed recombination misleads neoavian phylogenomics.

Genomes are typically mosaics of regions with different evolutionary histories. When speciation events are closely spaced in time, recombination makes the regions sharing the same history small, and the evolutionary history changes rapidly as we move along the genome. When examining rapid radiations such as the early diversification of Neoaves 66 Mya, typically no consistent history is observed across segments exceeding kilobases of the genome. Here, we report an exception. We found that a 21-Mb region in avian genomes, mapped to chicken chromosome 4, shows an extremely strong and discordance-free signal for a history different from that of the inferred species tree. Such a strong discordance-free signal, indicative of suppressed recombination across many millions of base pairs, is not observed elsewhere in the genome for any deep avian relationships. Although long regions with suppressed recombination have been documented in recently diverged species, our results pertain to relationships dating circa 65 Mya. We provide evidence that this strong signal may be due to an ancient rearrangement that blocked recombination and remained polymorphic for several million years prior to fixation. We show that the presence of this region has misled previous phylogenomic efforts with lower taxon sampling, showing the interplay between taxon and locus sampling. We predict that similar ancient rearrangements may confound phylogenetic analyses in other clades, pointing to a need for new analytical models that incorporate the possibility of such events.

Complexity of avian evolution revealed by family-level genomes.

Despite tremendous efforts in the past decades, relationships among main avian lineages remain heavily debated without a clear resolution. Discrepancies have been attributed to diversity of species sampled, phylogenetic method and the choice of genomic regions1-3. Here we address these issues by analysing the genomes of 363 bird species4 (218 taxonomic families, 92% of total). Using intergenic regions and coalescent methods, we present a well-supported tree but also a marked degree of discordance. The tree confirms that Neoaves experienced rapid radiation at or near the Cretaceous-Palaeogene boundary. Sufficient loci rather than extensive taxon sampling were more effective in resolving difficult nodes. Remaining recalcitrant nodes involve species that are a challenge to model due to either extreme DNA composition, variable substitution rates, incomplete lineage sorting or complex evolutionary events such as ancient hybridization. Assessment of the effects of different genomic partitions showed high heterogeneity across the genome. We discovered sharp increases in effective population size, substitution rates and relative brain size following the Cretaceous-Palaeogene extinction event, supporting the hypothesis that emerging ecological opportunities catalysed the diversification of modern birds. The resulting phylogenetic estimate offers fresh insights into the rapid radiation of modern birds and provides a taxon-rich backbone tree for future comparative studies.

TRAILS: Tree reconstruction of ancestry using incomplete lineage sorting.

Genome-wide genealogies of multiple species carry detailed information about demographic and selection processes on individual branches of the phylogeny. Here, we introduce TRAILS, a hidden Markov model that accurately infers time-resolved population genetics parameters, such as ancestral effective population sizes and speciation times, for ancestral branches using a multi-species alignment of three species and an outgroup. TRAILS leverages the information contained in incomplete lineage sorting fragments by modelling genealogies along the genome as rooted three-leaved trees, each with a topology and two coalescent events happening in discretized time intervals within the phylogeny. Posterior decoding of the hidden Markov model can be used to infer the ancestral recombination graph for the alignment and details on demographic changes within a branch. Since TRAILS performs posterior decoding at the base-pair level, genome-wide scans based on the posterior probabilities can be devised to detect deviations from neutrality. Using TRAILS on a human-chimp-gorilla-orangutan alignment, we recover speciation parameters and extract information about the topology and coalescent times at high resolution.

Worldwide Late Pleistocene and Early Holocene population declines in extant megafauna are associated with Homo sapiens expansion rather than climate change.

The worldwide extinction of megafauna during the Late Pleistocene and Early Holocene is evident from the fossil record, with dominant theories suggesting a climate, human or combined impact cause. Consequently, two disparate scenarios are possible for the surviving megafauna during this time period - they could have declined due to similar pressures, or increased in population size due to reductions in competition or other biotic pressures. We therefore infer population histories of 139 extant megafauna species using genomic data which reveal population declines in 91% of species throughout the Quaternary period, with larger species experiencing the strongest decreases. Declines become ubiquitous 32-76 kya across all landmasses, a pattern better explained by worldwide Homo sapiens expansion than by changes in climate. We estimate that, in consequence, total megafauna abundance, biomass, and energy turnover decreased by 92-95% over the past 50,000 years, implying major human-driven ecosystem restructuring at a global scale.

Single-cell analysis identifies genes facilitating rhizobium infection in Lotus japonicus.

Legume-rhizobium signaling during establishment of symbiotic nitrogen fixation restricts rhizobium colonization to specific cells. A limited number of root hair cells allow infection threads to form, and only a fraction of the epidermal infection threads progress to cortical layers to establish functional nodules. Here we use single-cell analysis to define the epidermal and cortical cell populations that respond to and facilitate rhizobium infection. We then identify high-confidence nodulation gene candidates based on their specific expression in these populations, pinpointing genes stably associated with infection across genotypes and time points. We show that one of these, which we name SYMRKL1, encodes a protein with an ectodomain predicted to be nearly identical to that of SYMRK and is required for normal infection thread formation. Our work disentangles cellular processes and transcriptional modules that were previously confounded due to lack of cellular resolution, providing a more detailed understanding of symbiotic interactions.

Characterizing the evolution and phenotypic impact of ampliconic Y chromosome regions.

A major part of the human Y chromosome consists of palindromes with multiple copies of genes primarily expressed in testis, many of which have been claimed to affect male fertility. Here we examine copy number variation in these palindromes based on whole genome sequence data from 11,527 Icelandic men. Using a subset of 7947 men grouped into 1449 patrilineal genealogies, we infer 57 large scale de novo copy number mutations affecting palindrome 1. This corresponds to a mutation rate of 2.34 × 10-3 mutations per meiosis, which is 4.1 times larger than our phylogenetic estimate of the mutation rate (5.72 × 10-4), suggesting that de novo mutations on the Y are lost faster than expected under neutral evolution. Although simulations indicate a selection coefficient of 1.8% against non-reference copy number carriers, we do not observe differences in fertility among sequenced men associated with their copy number genotype, but we lack statistical power to detect differences resulting from weak negative selection. We also perform association testing of a diverse set of 341 traits to palindromic copy number without any significant associations. We conclude that large-scale palindrome copy number variation on the Y chromosome has little impact on human phenotype diversity.

Genome-wide coancestry reveals details of ancient and recent male-driven reticulation in baboons.

Baboons (genus Papio) are a morphologically and behaviorally diverse clade of catarrhine monkeys that have experienced hybridization between phenotypically and genetically distinct phylogenetic species. We used high-coverage whole-genome sequences from 225 wild baboons representing 19 geographic localities to investigate population genomics and interspecies gene flow. Our analyses provide an expanded picture of evolutionary reticulation among species and reveal patterns of population structure within and among species, including differential admixture among conspecific populations. We describe the first example of a baboon population with a genetic composition that is derived from three distinct lineages. The results reveal processes, both ancient and recent, that produced the observed mismatch between phylogenetic relationships based on matrilineal, patrilineal, and biparental inheritance. We also identified several candidate genes that may contribute to species-specific phenotypes.

Pervasive incomplete lineage sorting illuminates speciation and selection in primates.

Incomplete lineage sorting (ILS) causes the phylogeny of some parts of the genome to differ from the species tree. In this work, we investigate the frequencies and determinants of ILS in 29 major ancestral nodes across the entire primate phylogeny. We find up to 64% of the genome affected by ILS at individual nodes. We exploit ILS to reconstruct speciation times and ancestral population sizes. Estimated speciation times are much more recent than genomic divergence times and are in good agreement with the fossil record. We show extensive variation of ILS along the genome, mainly driven by recombination but also by the distance to genes, highlighting a major impact of selection on variation along the genome. In many nodes, ILS is reduced more on the X chromosome compared with autosomes than expected under neutrality, which suggests higher impacts of natural selection on the X chromosome. Finally, we show an excess of ILS in genes with immune functions and a deficit of ILS in housekeeping genes. The extensive ILS in primates discovered in this study provides insights into the speciation times, ancestral population sizes, and patterns of natural selection that shape primate evolution.

Genome-wide coancestry reveals details of ancient and recent male-driven reticulation in baboons.

Baboons (genus Papio ) are a morphologically and behaviorally diverse clade of catarrhine monkeys that have experienced hybridization between phenotypically and genetically distinct phylogenetic species. We used high coverage whole genome sequences from 225 wild baboons representing 19 geographic localities to investigate population genomics and inter-species gene flow. Our analyses provide an expanded picture of evolutionary reticulation among species and reveal novel patterns of population structure within and among species, including differential admixture among conspecific populations. We describe the first example of a baboon population with a genetic composition that is derived from three distinct lineages. The results reveal processes, both ancient and recent, that produced the observed mismatch between phylogenetic relationships based on matrilineal, patrilineal, and biparental inheritance. We also identified several candidate genes that may contribute to species-specific phenotypes. One-Sentence Summary: Genomic data for 225 baboons reveal novel sites of inter-species gene flow and local effects due to differences in admixture.

Evolution of the germline mutation rate across vertebrates.

The germline mutation rate determines the pace of genome evolution and is an evolving parameter itself1. However, little is known about what determines its evolution, as most studies of mutation rates have focused on single species with different methodologies2. Here we quantify germline mutation rates across vertebrates by sequencing and comparing the high-coverage genomes of 151 parent-offspring trios from 68 species of mammals, fishes, birds and reptiles. We show that the per-generation mutation rate varies among species by a factor of 40, with mutation rates being higher for males than for females in mammals and birds, but not in reptiles and fishes. The generation time, age at maturity and species-level fecundity are the key life-history traits affecting this variation among species. Furthermore, species with higher long-term effective population sizes tend to have lower mutation rates per generation, providing support for the drift barrier hypothesis3. The exceptionally high yearly mutation rates of domesticated animals, which have been continually selected on fecundity traits including shorter generation times, further support the importance of generation time in the evolution of mutation rates. Overall, our comparative analysis of pedigree-based mutation rates provides ecological insights on the mutation rate evolution in vertebrates.

PRDM9 losses in vertebrates are coupled to those of paralogs ZCWPW1 and ZCWPW2.

In most mammals and likely throughout vertebrates, the gene PRDM9 specifies the locations of meiotic double strand breaks; in mice and humans at least, it also aids in their repair. For both roles, many of the molecular partners remain unknown. Here, we take a phylogenetic approach to identify genes that may be interacting with PRDM9 by leveraging the fact that PRDM9 arose before the origin of vertebrates but was lost many times, either partially or entirely-and with it, its role in recombination. As a first step, we characterize PRDM9 domain composition across 446 vertebrate species, inferring at least 13 independent losses. We then use the interdigitation of PRDM9 orthologs across vertebrates to test whether it coevolved with any of 241 candidate genes coexpressed with PRDM9 in mice or associated with recombination phenotypes in mammals. Accounting for the phylogenetic relationship among a subsample of 189 species, we find two genes whose presence and absence is unexpectedly coincident with that of PRDM9: ZCWPW1, which was recently shown to facilitate double strand break repair, and its paralog ZCWPW2, as well as, more tentatively, TEX15 and FBXO47ZCWPW2 is expected to be recruited to sites of PRDM9 binding; its tight coevolution with PRDM9 across vertebrates suggests that it is a key interactor within mammals and beyond, with a role either in recruiting the recombination machinery or in double strand break repair.

The Mutationathon highlights the importance of reaching standardization in estimates of pedigree-based germline mutation rates.

In the past decade, several studies have estimated the human per-generation germline mutation rate using large pedigrees. More recently, estimates for various nonhuman species have been published. However, methodological differences among studies in detecting germline mutations and estimating mutation rates make direct comparisons difficult. Here, we describe the many different steps involved in estimating pedigree-based mutation rates, including sampling, sequencing, mapping, variant calling, filtering, and appropriately accounting for false-positive and false-negative rates. For each step, we review the different methods and parameter choices that have been used in the recent literature. Additionally, we present the results from a 'Mutationathon,' a competition organized among five research labs to compare germline mutation rate estimates for a single pedigree of rhesus macaques. We report almost a twofold variation in the final estimated rate among groups using different post-alignment processing, calling, and filtering criteria, and provide details into the sources of variation across studies. Though the difference among estimates is not statistically significant, this discrepancy emphasizes the need for standardized methods in mutation rate estimations and the difficulty in comparing rates from different studies. Finally, this work aims to provide guidelines for computational and statistical benchmarks for future studies interested in identifying germline mutations from pedigrees.

Studying mutation rate evolution in primates-a need for systematic comparison of computational pipelines.

The lack of consensus methods to estimate germline mutation rates from pedigrees has led to substantial differences in computational pipelines in the published literature. Here, we answer Susanne Pfeifer's opinion piece discussing the pipeline choices of our recent article estimating the germline mutation rate of rhesus macaques (Macaca mulatta). We acknowledge the differences between the method that we applied and the one preferred by Pfeifer. Yet, we advocate for full transparency and justification of choices as long as rigorous comparison of pipelines remains absent because it is the only way to conclude on best practices for the field.

Small RNAs in Seminal Plasma as Novel Biomarkers for Germ Cell Tumors.

Circulating miRNAs secreted by testicular germ cell tumors (TGCT) show great potential as novel non-invasive biomarkers for diagnosis of TGCT. Seminal plasma (SP) represents a biofluid closer to the primary site. Here, we investigate whether small RNAs in SP can be used to diagnose men with TGCTs or the precursor lesions, germ cell neoplasia in situ (GCNIS). Small RNAs isolated from SP from men with TGCTs (n = 18), GCNIS-only (n = 5), and controls (n = 25) were sequenced. SP from men with TGCT/GCNIS (n = 37) and controls (n = 22) were used for validation by RT-qPCR. In general, piRNAs were found at lower levels in SP from men with TGCTs. Ten small RNAs were found at significantly (q-value < 0.05) different levels in SP from men with TGCT/GCNIS than controls. Random forests classification identified sets of small RNAs that could detect either TGCT/GCNIS or GCNIS-only with an area under the curve of 0.98 and 1 in ROC analyses, respectively. RT-qPCR validated hsa-miR-6782-5p to be present at 2.3-fold lower levels (p = 0.02) in the SP from men with TGCTs compared with controls. Small RNAs in SP show potential as novel biomarkers for diagnosing men with TGCT/GCNIS but validation in larger cohorts is needed.

The germline mutational process in rhesus macaque and its implications for phylogenetic dating.

BACKGROUND: Understanding the rate and pattern of germline mutations is of fundamental importance for understanding evolutionary processes. RESULTS: Here we analyzed 19 parent-offspring trios of rhesus macaques (Macaca mulatta) at high sequencing coverage of ∼76× per individual and estimated a mean rate of 0.77 × 10-8de novo mutations per site per generation (95% CI: 0.69 × 10-8 to 0.85 × 10-8). By phasing 50% of the mutations to parental origins, we found that the mutation rate is positively correlated with the paternal age. The paternal lineage contributed a mean of 81% of the de novo mutations, with a trend of an increasing male contribution for older fathers. Approximately 3.5% of de novo mutations were shared between siblings, with no parental bias, suggesting that they arose from early development (postzygotic) stages. Finally, the divergence times between closely related primates calculated on the basis of the yearly mutation rate of rhesus macaque generally reconcile with divergence estimated with molecular clock methods, except for the Cercopithecoidea/Hominoidea molecular divergence dated at 58 Mya using our new estimate of the yearly mutation rate. CONCLUSIONS: When compared to the traditional molecular clock methods, new estimated rates from pedigree samples can provide insights into the evolution of well-studied groups such as primates.

Evaluating genetic causes of azoospermia: What can we learn from a complex cellular structure and single-cell transcriptomics of the human testis?

Azoospermia is a condition defined as the absence of spermatozoa in the ejaculate, but the testicular phenotype of men with azoospermia may be very variable, ranging from full spermatogenesis, through arrested maturation of germ cells at different stages, to completely degenerated tissue with ghost tubules. Hence, information regarding the cell-type-specific expression patterns is needed to prioritise potential pathogenic variants that contribute to the pathogenesis of azoospermia. Thanks to technological advances within next-generation sequencing, it is now possible to obtain detailed cell-type-specific expression patterns in the testis by single-cell RNA sequencing. However, to interpret single-cell RNA sequencing data properly, substantial knowledge of the highly sophisticated data processing and visualisation methods is needed. Here we review the complex cellular structure of the human testis in different types of azoospermia and outline how known genetic alterations affect the pathology of the testis. We combined the currently available single-cell RNA sequencing datasets originating from the human testis into one dataset covering 62,751 testicular cells, each with a median of 2637 transcripts quantified. We show what effects the most common data-processing steps have, and how different visualisation methods can be used. Furthermore, we calculated expression patterns in pseudotime, and show how splicing rates can be used to determine the velocity of differentiation during spermatogenesis. With the combined dataset we show expression patterns and network analysis of genes known to be involved in the pathogenesis of azoospermia. Finally, we provide the combined dataset as an interactive online resource where expression of genes and different visualisation methods can be explored ( https://testis.cells.ucsc.edu/ ).

Integration and reanalysis of transcriptomics and methylomics data derived from blood and testis tissue of men with 47,XXY Klinefelter syndrome indicates the primary involvement of Sertoli cells in the testicular pathogenesis.

Klinefelter syndrome (KS; 47,XXY) is the most common sex chromosomal anomaly and causes a multitude of symptoms. Often the most noticeable symptom is infertility caused by azoospermia with testicular histology showing hyalinization of tubules, germ cells loss, and Leydig cell hyperplasia. The germ cell loss begins early in life leading to partial hyalinization of the testis at puberty, but the mechanistic drivers behind this remain poorly understood. In this systematic review, we summarize the current knowledge on developmental changes in the cellularity of KS gonads supplemented by a comparative analysis of the fetal and adult gonadal transcriptome, and blood transcriptome and methylome of men with KS. We identified a high fraction of upregulated genes that escape X-chromosome inactivation, thus supporting previous hypotheses that these are the main drivers of the testicular phenotype in KS. Enrichment analysis showed overrepresentation of genes from the X- and Y-chromosome and testicular transcription factors. Furthermore, by re-evaluation of recent single cell RNA-sequencing data originating from adult KS testis, we found novel evidence that the Sertoli cell is the most affected cell type. Our results are consistent with disturbed cross-talk between somatic and germ cells in the KS testis, and with X-escapee genes acting as mediators.

Association Mapping and Disease: Evolutionary Perspectives.

In this chapter, we give a short introduction to the genetics of complex diseases emphasizing evolutionary models for disease genes and the effect of different models on the genetic architecture, and we give a survey of the state-of-the-art of genome-wide association studies (GWASs).

Breaking Free: The Genomics of Allopolyploidy-Facilitated Niche Expansion in White Clover.

The merging of distinct genomes, allopolyploidization, is a widespread phenomenon in plants. It generates adaptive potential through increased genetic diversity, but examples demonstrating its exploitation remain scarce. White clover (Trifolium repens) is a ubiquitous temperate allotetraploid forage crop derived from two European diploid progenitors confined to extreme coastal or alpine habitats. We sequenced and assembled the genomes and transcriptomes of this species complex to gain insight into the genesis of white clover and the consequences of allopolyploidization. Based on these data, we estimate that white clover originated ∼15,000 to 28,000 years ago during the last glaciation when alpine and coastal progenitors were likely colocated in glacial refugia. We found evidence of progenitor diversity carryover through multiple hybridization events and show that the progenitor subgenomes have retained integrity and gene expression activity as they traveled within white clover from their original confined habitats to a global presence. At the transcriptional level, we observed remarkably stable subgenome expression ratios across tissues. Among the few genes that show tissue-specific switching between homeologous gene copies, we found flavonoid biosynthesis genes strongly overrepresented, suggesting an adaptive role of some allopolyploidy-associated transcriptional changes. Our results highlight white clover as an example of allopolyploidy-facilitated niche expansion, where two progenitor genomes, adapted and confined to disparate and highly specialized habitats, expanded to a ubiquitous global presence after glaciation-associated allopolyploidization.

Dynamic Copy Number Evolution of X- and Y-Linked Ampliconic Genes in Human Populations.

Ampliconic genes are multicopy, with the majority found on sex chromosomes and enriched for testis-expressed genes. While ampliconic genes have been associated with the emergence of hybrid incompatibilities, we know little about their copy number distribution and their turnover in human populations. Here, we explore the evolution of human X- and Y-linked ampliconic genes by investigating copy number variation (CNV) and coding variation between populations using the Simons Genome Diversity Project. We develop a method to assess CNVs using the read depth on modified X and Y chromosome targets containing only one repetition of each ampliconic gene. Our results reveal extensive standing variation in copy number both within and between human populations for several ampliconic genes. For the Y chromosome, we can infer multiple independent amplifications and losses of these gene copies even within closely related Y haplogroups, that diversified < 50,000 years ago. Moreover, X- and Y-linked ampliconic genes seem to have a faster amplification dynamic than autosomal multicopy genes. Looking at expression data from another study, we also find that X- and Y-linked ampliconic genes with extensive CNV are significantly more expressed than genes with no CNV during meiotic sex chromosome inactivation (for both X and Y) and postmeiotic sex chromosome repression (for the Y chromosome only). While we cannot rule out that the XY-linked ampliconic genes are evolving neutrally, this study gives insights into the distribution of copy number within human populations and demonstrates an extremely fast turnover in copy number of these regions.