Prevalence and characteristics of genetic disease in adult kidney stone formers.

BACKGROUND: Molecular mechanisms of kidney stone formation remain unknown in most patients. Previous studies showed high a heritability of nephrolithiasis, but data on prevalence and characteristics of genetic disease in unselected adults with nephrolithiasis are lacking. This study was conducted to fill this important knowledge gap. METHODS: We performed whole exome sequencing in 787 participants of the Bern Kidney Stone Registry, an unselected cohort of adults with ≥ 1 past kidney stone episode (KSF), and 114 non-stone-forming individuals (NKSF). An exome-based panel of 34 established nephrolithiasis genes was analyzed and variants assessed according to ACMG criteria. Pathogenic (P) or likely pathogenic (LP) variants were considered diagnostic. RESULTS: Mean age of KSF was 47±15 years, and 18% were first time KSF. A Mendelian kidney stone disease was present in 2.9% (23 of 787) of KSF. The most common genetic diagnoses were cystinuria (SLC3A1, SLC7A9; n=13), Vitamin D-24 hydroxylase deficiency (CYP24A1; n=5) and primary hyperoxaluria (AGXT, GRHPR, HOGA1; n=3). 8.1% (64 of 787) of KSF were monoallelic for LP/P variants predisposing to nephrolithiasis, most frequently in SLC34A1/A3 or SLC9A3R1 (n=37), CLDN16 (n=8) and CYP24A1 (n=8). KSF with Mendelian disease had a lower age at the first stone event (30±14 years vs. 36±14 years, p=0.003), were more likely to have cystine stones (23.4% vs. 1.4%) and less likely to have calcium oxalate monohydrates stones (31.9% vs. 52.5%) compared to KSF without genetic diagnosis. The phenotype of KSF with variants predisposing to nephrolithiasis was subtle and showed significant overlap with KSF without diagnostic variants. In NKSF, no Mendelian disease was detected, and LP/P variants were significantly less prevalent compared to KSF (1.8% vs. 8.1%). CONCLUSION: Mendelian disease is uncommon in unselected adult KSF, yet variants predisposing to nephrolithiasis are significantly enriched in adult KSF.

Molecular parallelism in the evolution of a master sex-determining role for the anti-Mullerian hormone receptor 2 gene (amhr2) in Midas cichlids.

The evolution of sex chromosomes and their differentiation from autosomes is a major event during genome evolution that happened many times in several lineages. The repeated evolution and lability of sex-determination mechanisms in fishes makes this a well-suited system to test for general patterns in evolution. According to current theory, differentiation is triggered by the suppression of recombination following the evolution of a new master sex-determining gene. However, the molecular mechanisms that establish recombination suppression are known from few examples, owing to the intrinsic difficulties of assembling sex-determining regions (SDRs). The development of forward-genetics and long-read sequencing have generated a wealth of data questioning central aspects of the current theory. Here, we demonstrate that sex in Midas cichlids is determined by an XY system, and identify and assemble the SDR by combining forward-genetics, long-read sequencing and optical mapping. We show how long-reads aid in the detection of artefacts in genotype-phenotype mapping that arise from incomplete genome assemblies. The male-specific region is restricted to a 100-kb segment on chromosome 4 that harbours transposable elements and a Y-specific duplicate of the anti-Mullerian receptor 2 gene, which has evolved master sex-determining functions repeatedly. Our data suggest that amhr2Y originated by an interchromosomal translocation from chromosome 20 to 4 pre-dating the split of Midas and Flier cichlids. In the latter, it is pseudogenized and translocated to another chromosome. Duplication of anti-Mullerian genes is a common route to establishing new sex determiners, highlighting the role of molecular parallelism in the evolution of sex determination.

Early stages of sympatric homoploid hybrid speciation in crater lake cichlid fishes.

Homoploid hybrid speciation (i.e., hybrid speciation without a change in ploidy) has traditionally been considered to be rare in animals. Only few accepted empirical examples of homoploid hybrid speciation in nature exist, and in only one previous case (insects) was it convincingly shown that this process occurred in complete sympatry. Here, we report an instance of sympatric homoploid hybrid speciation in Midas cichlid fishes in Crater Lake Xiloá, Nicaragua. The hybrid lineage, albeit at an early stage of speciation, has genomically and phenotypically diverged from both of its two parental species. Together with a distinct stable isotope signature this suggests that this hybrid lineages occupies a different trophic niche compared to the other sympatric Midas cichlid species in Crater Lake Xiloá.

An intronic transposon insertion associates with a trans-species color polymorphism in Midas cichlid fishes.

Polymorphisms have fascinated biologists for a long time, but their genetic underpinnings often remain elusive. Here, we aim to uncover the genetic basis of the gold/dark polymorphism that is eponymous of Midas cichlid fish (Amphilophus spp.) adaptive radiations in Nicaraguan crater lakes. While most Midas cichlids are of the melanic "dark morph", about 10% of individuals lose their melanic pigmentation during their ontogeny and transition into a conspicuous "gold morph". Using a new haplotype-resolved long-read assembly we discover an 8.2 kb, transposon-derived inverted repeat in an intron of an undescribed gene, which we term goldentouch in reference to the Greek myth of King Midas. The gene goldentouch is differentially expressed between morphs, presumably due to structural implications of inverted repeats in both DNA and/or RNA (cruciform and hairpin formation). The near-perfect association of the insertion with the phenotype across independent populations suggests that it likely underlies this trans-specific, stable polymorphism.

Genome sequences reveal global dispersal routes and suggest convergent genetic adaptations in seahorse evolution.

Seahorses have a circum-global distribution in tropical to temperate coastal waters. Yet, seahorses show many adaptations for a sedentary, cryptic lifestyle: they require specific habitats, such as seagrass, kelp or coral reefs, lack pelvic and caudal fins, and give birth to directly developed offspring without pronounced pelagic larval stage, rendering long-range dispersal by conventional means inefficient. Here we investigate seahorses' worldwide dispersal and biogeographic patterns based on a de novo genome assembly of Hippocampus erectus as well as 358 re-sequenced genomes from 21 species. Seahorses evolved in the late Oligocene and subsequent circum-global colonization routes are identified and linked to changing dynamics in ocean currents and paleo-temporal seaway openings. Furthermore, the genetic basis of the recurring "bony spines" adaptive phenotype is linked to independent substitutions in a key developmental gene. Analyses thus suggest that rafting via ocean currents compensates for poor dispersal and rapid adaptation facilitates colonizing new habitats.

Different Sources of Allelic Variation Drove Repeated Color Pattern Divergence in Cichlid Fishes.

The adaptive radiations of East African cichlid fish in the Great Lakes Victoria, Malawi, and Tanganyika are well known for their diversity and repeatedly evolved phenotypes. Convergent evolution of melanic horizontal stripes has been linked to a single locus harboring the gene agouti-related peptide 2 (agrp2). However, where and when the causal variants underlying this trait evolved and how they drove phenotypic divergence remained unknown. To test the alternative hypotheses of standing genetic variation versus de novo mutations (independently originating in each radiation), we searched for shared signals of genomic divergence at the agrp2 locus. Although we discovered similar signatures of differentiation at the locus level, the haplotypes associated with stripe patterns are surprisingly different. In Lake Malawi, the highest associated alleles are located within and close to the 5' untranslated region of agrp2 and likely evolved through recent de novo mutations. In the younger Lake Victoria radiation, stripes are associated with two intronic regions overlapping with a previously reported cis-regulatory interval. The origin of these segregating haplotypes predates the Lake Victoria radiation because they are also found in more basal riverine and Lake Kivu species. This suggests that both segregating haplotypes were present as standing genetic variation at the onset of the Lake Victoria adaptive radiation with its more than 500 species and drove phenotypic divergence within the species flock. Therefore, both new (Lake Malawi) and ancient (Lake Victoria) allelic variation at the same locus fueled rapid and convergent phenotypic evolution.

The comparative genomic landscape of adaptive radiation in crater lake cichlid fishes.

Factors ranging from ecological opportunity to genome composition might explain why only some lineages form adaptive radiations. While being rare, particular systems can provide natural experiments within an identical ecological setting where species numbers and phenotypic divergence in two closely related lineages are notably different. We investigated one such natural experiment using two de novo assembled and 40 resequenced genomes and asked why two closely related Neotropical cichlid fish lineages, the Amphilophus citrinellus species complex (Midas cichlids; radiating) and Archocentrus centrarchus (Flyer cichlid; nonradiating), have resulted in such disparate evolutionary outcomes. Although both lineages inhabit many of the same Nicaraguan lakes, whole-genome inferred demography suggests that priority effects are not likely to be the cause of the dissimilarities. Also, genome-wide levels of selection, transposable element dynamics, gene family expansion, major chromosomal rearrangements and the number of genes under positive selection were not markedly different between the two lineages. To more finely investigate particular subsets of the genome that have undergone adaptive divergence in Midas cichlids, we also examined if there was evidence for 'molecular pre-adaptation' in regions identified by QTL mapping of repeatedly diverging adaptive traits. Although most of our analyses failed to pinpoint substantial genomic differences, we did identify functional categories containing many genes under positive selection that provide candidates for future studies on the propensity of Midas cichlids to radiate. Our results point to a disproportionate role of local, rather than genome-wide factors underlying the propensity for these cichlid fishes to adaptively radiate.

Contrasting signatures of genomic divergence during sympatric speciation.

The transition from 'well-marked varieties' of a single species into 'well-defined species'-especially in the absence of geographic barriers to gene flow (sympatric speciation)-has puzzled evolutionary biologists ever since Darwin1,2. Gene flow counteracts the buildup of genome-wide differentiation, which is a hallmark of speciation and increases the likelihood of the evolution of irreversible reproductive barriers (incompatibilities) that complete the speciation process3. Theory predicts that the genetic architecture of divergently selected traits can influence whether sympatric speciation occurs4, but empirical tests of this theory are scant because comprehensive data are difficult to collect and synthesize across species, owing to their unique biologies and evolutionary histories5. Here, within a young species complex of neotropical cichlid fishes (Amphilophus spp.), we analysed genomic divergence among populations and species. By generating a new genome assembly and re-sequencing 453 genomes, we uncovered the genetic architecture of traits that have been suggested to be important for divergence. Species that differ in monogenic or oligogenic traits that affect ecological performance and/or mate choice show remarkably localized genomic differentiation. By contrast, differentiation among species that have diverged in polygenic traits is genomically widespread and much higher overall, consistent with the evolution of effective and stable genome-wide barriers to gene flow. Thus, we conclude that simple trait architectures are not always as conducive to speciation with gene flow as previously suggested, whereas polygenic architectures can promote rapid and stable speciation in sympatry.

Reconstructing the Evolutionary History of Chromosomal Races on Islands: A Genome-Wide Analysis of Natural House Mouse Populations.

Chromosomal evolution is widely considered to be an important driver of speciation, as karyotypic reorganization can bring about the establishment of reproductive barriers between incipient species. One textbook example for genetic mechanisms of speciation are large-scale chromosomal rearrangements such as Robertsonian (Rb) fusions, a common class of structural variants that can drastically change the recombination landscape by suppressing crossing-over and influence gene expression by altering regulatory networks. Here, we explore the population structure and demographic patterns of a well-known house mouse Rb system in the Aeolian archipelago in Southern Italy using genome-wide data. By analyzing chromosomal regions characterized by different levels of recombination, we trace the evolutionary history of a set of Rb chromosomes occurring in different geographical locations and test whether chromosomal fusions have a single shared origin or occurred multiple times. Using a combination of phylogenetic and population genetic approaches, we find support for multiple, independent origins of three focal Rb chromosomes. The elucidation of the demographic patterns of the mouse populations within the Aeolian archipelago shows that an interplay between fixation of newly formed Rb chromosomes and hybridization events has contributed to shaping their current karyotypic distribution. Overall, our results illustrate that chromosome structure is much more dynamic than anticipated and emphasize the importance of large-scale chromosomal translocations in speciation.

How Linked Selection Shapes the Diversity Landscape in Ficedula Flycatchers.

There is an increasing awareness that selection affecting linked neutral sites strongly influences on how diversity is distributed across the genome. In particular, linked selection is likely involved in the formation of heterogenous landscapes of genetic diversity, including genomic regions with locally reduced effective population sizes that manifest as dips in diversity, and "islands" of differentiation between closely related populations or species. Linked selection can be in the form of background selection or selective sweeps, and a long-standing quest in population genetics has been to unveil the relative importance of these processes. Here, we analyzed the theoretically expected reduction of diversity caused by linked selection in the collared flycatcher (Ficedula albicollis) genome and compared this with population genomic data on the distribution of diversity across the flycatcher genome. By incorporating data on recombination rate variation and the density of target sites for selection (including both protein-coding genes and conserved noncoding elements), we found that background selection can explain most of the observed baseline variation in genetic diversity. However, positive selection was necessary to explain the pronounced local diversity dips in the collared flycatcher genome. We confirmed our analytical findings by comprehensive simulations. Therefore, our study demonstrates that even though both background selection and selective sweeps contribute to the heterogeneous diversity landscape seen in this avian system, they play different roles in shaping it.

Genomes reveal marked differences in the adaptive evolution between orangutan species.

BACKGROUND: Integrating demography and adaptive evolution is pivotal to understanding the evolutionary history and conservation of great apes. However, little is known about the adaptive evolution of our closest relatives, in particular if and to what extent adaptions to environmental differences have occurred. Here, we used whole-genome sequencing data from critically endangered orangutans from North Sumatra (Pongo abelii) and Borneo (P. pygmaeus) to investigate adaptive responses of each species to environmental differences during the Pleistocene. RESULTS: Taking into account the markedly disparate demographic histories of each species after their split ~ 1 Ma ago, we show that persistent environmental differences on each island had a strong impact on the adaptive evolution of the genus Pongo. Across a range of tests for positive selection, we find a consistent pattern of between-island and species differences. In the more productive Sumatran environment, the most notable signals of positive selection involve genes linked to brain and neuronal development, learning, and glucose metabolism. On Borneo, however, positive selection comprised genes involved in lipid metabolism, as well as cardiac and muscle activities. CONCLUSIONS: We find strikingly different sets of genes appearing to have evolved under strong positive selection in each species. In Sumatran orangutans, selection patterns were congruent with well-documented cognitive and behavioral differences between the species, such as a larger and more complex cultural repertoire and higher degrees of sociality. However, in Bornean orangutans, selective responses to fluctuating environmental conditions appear to have produced physiological adaptations to generally lower and temporally more unpredictable food supplies.

Biased Inference of Selection Due to GC-Biased Gene Conversion and the Rate of Protein Evolution in Flycatchers When Accounting for It.

The rate of recombination impacts on rates of protein evolution for at least two reasons: it affects the efficacy of selection due to linkage and influences sequence evolution through the process of GC-biased gene conversion (gBGC). We studied how recombination, via gBGC, affects inferences of selection in gene sequences using comparative genomic and population genomic data from the collared flycatcher (Ficedula albicollis). We separately analyzed different mutation categories ("strong"-to-"weak," "weak-to-strong," and GC-conservative changes) and found that gBGC impacts on the distribution of fitness effects of new mutations, and leads to that the rate of adaptive evolution and the proportion of adaptive mutations among nonsynonymous substitutions are underestimated by 22-33%. It also biases inferences of demographic history based on the site frequency spectrum. In light of this impact, we suggest that inferences of selection (and demography) in lineages with pronounced gBGC should be based on GC-conservative changes only. Doing so, we estimate that 10% of nonsynonymous mutations are effectively neutral and that 27% of nonsynonymous substitutions have been fixed by positive selection in the flycatcher lineage. We also find that gene expression level, sex-bias in expression, and the number of protein-protein interactions, but not Hill-Robertson interference (HRI), are strong determinants of selective constraint and rate of adaptation of collared flycatcher genes. This study therefore illustrates the importance of disentangling the effects of different evolutionary forces and genetic factors in interpretation of sequence data, and from that infer the role of natural selection in DNA sequence evolution.

Morphometric, Behavioral, and Genomic Evidence for a New Orangutan Species.

Six extant species of non-human great apes are currently recognized: Sumatran and Bornean orangutans, eastern and western gorillas, and chimpanzees and bonobos [1]. However, large gaps remain in our knowledge of fine-scale variation in hominoid morphology, behavior, and genetics, and aspects of great ape taxonomy remain in flux. This is particularly true for orangutans (genus: Pongo), the only Asian great apes and phylogenetically our most distant relatives among extant hominids [1]. Designation of Bornean and Sumatran orangutans, P. pygmaeus (Linnaeus 1760) and P. abelii (Lesson 1827), as distinct species occurred in 2001 [1, 2]. Here, we show that an isolated population from Batang Toru, at the southernmost range limit of extant Sumatran orangutans south of Lake Toba, is distinct from other northern Sumatran and Bornean populations. By comparing cranio-mandibular and dental characters of an orangutan killed in a human-animal conflict to those of 33 adult male orangutans of a similar developmental stage, we found consistent differences between the Batang Toru individual and other extant Ponginae. Our analyses of 37 orangutan genomes provided a second line of evidence. Model-based approaches revealed that the deepest split in the evolutionary history of extant orangutans occurred ∼3.38 mya between the Batang Toru population and those to the north of Lake Toba, whereas both currently recognized species separated much later, about 674 kya. Our combined analyses support a new classification of orangutans into three extant species. The new species, Pongo tapanuliensis, encompasses the Batang Toru population, of which fewer than 800 individuals survive. VIDEO ABSTRACT.

Whole-genome patterns of linkage disequilibrium across flycatcher populations clarify the causes and consequences of fine-scale recombination rate variation in birds.

Recombination rate is heterogeneous across the genome of various species and so are genetic diversity and differentiation as a consequence of linked selection. However, we still lack a clear picture of the underlying mechanisms for regulating recombination. Here we estimated fine-scale population recombination rate based on the patterns of linkage disequilibrium across the genomes of multiple populations of two closely related flycatcher species (Ficedula albicollis and F. hypoleuca). This revealed an overall conservation of the recombination landscape between these species at the scale of 200 kb, but we also identified differences in the local rate of recombination despite their recent divergence (<1 million years). Genetic diversity and differentiation were associated with recombination rate in a lineage-specific manner, indicating differences in the extent of linked selection between species. We detected 400-3,085 recombination hotspots per population. Location of hotspots was conserved between species, but the intensity of hotspot activity varied between species. Recombination hotspots were primarily associated with CpG islands (CGIs), regardless of whether CGIs were at promoter regions or away from genes. Recombination hotspots were also associated with specific transposable elements (TEs), but this association appears indirect due to shared preferences of the transposition machinery and the recombination machinery for accessible open chromatin regions. Our results suggest that CGIs are a major determinant of the localization of recombination hotspots, and we propose that both the distribution of TEs and fine-scale variation in recombination rate may be associated with the evolution of the epigenetic landscape.

Evidence that the rate of strong selective sweeps increases with population size in the great apes.

Quantifying the number of selective sweeps and their combined effects on genomic diversity in humans and other great apes is notoriously difficult. Here we address the question using a comparative approach to contrast diversity patterns according to the distance from genes in all great ape taxa. The extent of diversity reduction near genes compared with the rest of intergenic sequences is greater in a species with larger effective population size. Also, the maximum distance from genes at which the diversity reduction is observed is larger in species with large effective population size. In Sumatran orangutans, the overall genomic diversity is ∼30% smaller than diversity levels far from genes, whereas this reduction is only 9% in humans. We show by simulation that selection against deleterious mutations in the form of background selection is not expected to cause these differences in diversity among species. Instead, selective sweeps caused by positive selection can reduce diversity level more severely in a large population if there is a higher number of selective sweeps per unit time. We discuss what can cause such a correlation, including the possibility that more frequent sweeps in larger populations are due to a shorter waiting time for the right mutations to arise.

Genomic distribution and estimation of nucleotide diversity in natural populations: perspectives from the collared flycatcher (Ficedula albicollis) genome.

Properly estimating genetic diversity in populations of nonmodel species requires a basic understanding of how diversity is distributed across the genome and among individuals. To this end, we analysed whole-genome resequencing data from 20 collared flycatchers (genome size ≈1.1 Gb; 10.13 million single nucleotide polymorphisms detected). Genomewide nucleotide diversity was almost identical among individuals (mean = 0.00394, range = 0.00384-0.00401), but diversity levels varied extensively across the genome (95% confidence interval for 200-kb windows = 0.0013-0.0053). Diversity was related to selective constraint such that in comparison with intergenic DNA, diversity at fourfold degenerate sites was reduced to 85%, 3' UTRs to 82%, 5' UTRs to 70% and nondegenerate sites to 12%. There was a strong positive correlation between diversity and chromosome size, probably driven by a higher density of targets for selection on smaller chromosomes increasing the diversity-reducing effect of linked selection. Simulations exploring the ability of sequence data from a small number of genetic markers to capture the observed diversity clearly demonstrated that diversity estimation from finite sampling of such data is bound to be associated with large confidence intervals. Nevertheless, we show that precision in diversity estimation in large outbred population benefits from increasing the number of loci rather than the number of individuals. Simulations mimicking RAD sequencing showed that this approach gives accurate estimates of genomewide diversity. Based on the patterns of observed diversity and the performed simulations, we provide broad recommendations for how genetic diversity should be estimated in natural populations.

Evolution and demography of the great apes.

The great apes are the closest living relatives of humans. Chimpanzees and bonobos group together with humans, while gorillas and orangutans are more divergent from humans. Here, we review insights into their evolution pertaining to the topology of species and subspecies and the reconstruction of their demography based on genome-wide variation. These advances have only become possible recently through next-generation sequencing technologies. Given the close relationship to humans, they provide an important evolutionary context for human genetics.

Divergence in gene expression within and between two closely related flycatcher species.

Relatively little is known about the character of gene expression evolution as species diverge. It is for instance unclear if gene expression generally evolves in a clock-like manner (by stabilizing selection or neutral evolution) or if there are frequent episodes of directional selection. To gain insights into the evolutionary divergence of gene expression, we sequenced and compared the transcriptomes of multiple organs from population samples of collared (Ficedula albicollis) and pied flycatchers (F. hypoleuca), two species which diverged less than one million years ago. Ordination analysis separated samples by organ rather than by species. Organs differed in their degrees of expression variance within species and expression divergence between species. Variance was negatively correlated with expression breadth and protein interactivity, suggesting that pleiotropic constraints reduce gene expression variance within species. Variance was correlated with between-species divergence, consistent with a pattern expected from stabilizing selection and neutral evolution. Using an expression PST approach, we identified genes differentially expressed between species and found 16 genes uniquely expressed in one of the species. For one of these, DPP7, uniquely expressed in collared flycatcher, the absence of expression in pied flycatcher could be associated with a ≈20-kb deletion including 11 of 13 exons. This study of a young vertebrate speciation model system expands our knowledge of how gene expression evolves as natural populations become reproductively isolated.

Recombination Rate Variation Modulates Gene Sequence Evolution Mainly via GC-Biased Gene Conversion, Not Hill-Robertson Interference, in an Avian System.

The ratio of nonsynonymous to synonymous substitution rates (ω) is often used to measure the strength of natural selection. However, ω may be influenced by linkage among different targets of selection, that is, Hill-Robertson interference (HRI), which reduces the efficacy of selection. Recombination modulates the extent of HRI but may also affect ω by means of GC-biased gene conversion (gBGC), a process leading to a preferential fixation of G:C ("strong," S) over A:T ("weak," W) alleles. As HRI and gBGC can have opposing effects on ω, it is essential to understand their relative impact to make proper inferences of ω. We used a model that separately estimated S-to-S, S-to-W, W-to-S, and W-to-W substitution rates in 8,423 avian genes in the Ficedula flycatcher lineage. We found that the W-to-S substitution rate was positively, and the S-to-W rate negatively, correlated with recombination rate, in accordance with gBGC but not predicted by HRI. The W-to-S rate further showed the strongest impact on both dN and dS. However, since the effects were stronger at 4-fold than at 0-fold degenerated sites, likely because the GC content of these sites is farther away from its equilibrium, ω slightly decreases with increasing recombination rate, which could falsely be interpreted as a consequence of HRI. We corroborated this hypothesis analytically and demonstrate that under particular conditions, ω can decrease with increasing recombination rate. Analyses of the site-frequency spectrum showed that W-to-S mutations were skewed toward high, and S-to-W mutations toward low, frequencies, consistent with a prevalent gBGC-driven fixation bias.