Chromosome-Level Assemblies of the Pieris mannii Butterfly Genome Suggest Z-Origin and Rapid Evolution of the W Chromosome.

The insect order Lepidoptera (butterflies and moths) represents the largest group of organisms with ZW/ZZ sex determination. While the origin of the Z chromosome predates the evolution of the Lepidoptera, the W chromosomes are considered younger, but their origin is debated. To shed light on the origin of the lepidopteran W, we here produce chromosome-level genome assemblies for the butterfly Pieris mannii and compare the sex chromosomes within and between P. mannii and its sister species Pieris rapae. Our analyses clearly indicate a common origin of the W chromosomes of the two Pieris species and reveal similarity between the Z and W in chromosome sequence and structure. This supports the view that the W in these species originates from Z-autosome fusion rather than from a redundant B chromosome. We further demonstrate the extremely rapid evolution of the W relative to the other chromosomes and argue that this may preclude reliable conclusions about the origins of W chromosomes based on comparisons among distantly related Lepidoptera. Finally, we find that sequence similarity between the Z and W chromosomes is greatest toward the chromosome ends, perhaps reflecting selection for the maintenance of recognition sites essential to chromosome segregation. Our study highlights the utility of long-read sequencing technology for illuminating chromosome evolution.

Analysis of genetically determined gene expression suggests role of inflammatory processes in exfoliation syndrome.

BACKGROUND: Exfoliation syndrome (XFS) is an age-related systemic disorder characterized by excessive production and progressive accumulation of abnormal extracellular material, with pathognomonic ocular manifestations. It is the most common cause of secondary glaucoma, resulting in widespread global blindness. The largest global meta-analysis of XFS in 123,457 multi-ethnic individuals from 24 countries identified seven loci with the strongest association signal in chr15q22-25 region near LOXL1. Expression analysis have so far correlated coding and a few non-coding variants in the region with LOXL1 expression levels, but functional effects of these variants is unclear. We hypothesize that analysis of the contribution of the genetically determined component of gene expression to XFS risk can provide a powerful method to elucidate potential roles of additional genes and clarify biology that underlie XFS. RESULTS: Transcriptomic Wide Association Studies (TWAS) using PrediXcan models trained in 48 GTEx tissues leveraging on results from the multi-ethnic and European ancestry GWAS were performed. To eliminate the possibility of false-positive results due to Linkage Disequilibrium (LD) contamination, we i) performed PrediXcan analysis in reduced models removing variants in LD with LOXL1 missense variants associated with XFS, and variants in LOXL1 models in both multiethnic and European ancestry individuals, ii) conducted conditional analysis of the significant signals in European ancestry individuals, and iii) filtered signals based on correlated gene expression, LD and shared eQTLs, iv) conducted expression validation analysis in human iris tissues. We observed twenty-eight genes in chr15q22-25 region that showed statistically significant associations, which were whittled down to ten genes after statistical validations. In experimental analysis, mRNA transcript levels for ARID3B, CD276, LOXL1, NEO1, SCAMP2, and UBL7 were significantly decreased in iris tissues from XFS patients compared to control samples. TWAS genes for XFS were significantly enriched for genes associated with inflammatory conditions. We also observed a higher incidence of XFS comorbidity with inflammatory and connective tissue diseases. CONCLUSION: Our results implicate a role for connective tissues and inflammation pathways in the etiology of XFS. Targeting the inflammatory pathway may be a potential therapeutic option to reduce progression in XFS.

Quality assurance within the context of genome diagnostics (a german perspective).

The rapid and dynamic implementation of Next-Generation Sequencing (NGS)-based assays has revolutionized genetic testing, and in the near future, nearly all molecular alterations of the human genome will be diagnosable via massive parallel sequencing. While this progress will further corroborate the central role of human genetics in the multidisciplinary management of patients with genetic disorders, it must be accompanied by quality assurance measures in order to allow the safe and optimal use of knowledge ascertained from genome diagnostics. To achieve this, several valuable tools and guidelines have been developed to support the quality of genome diagnostics. In this paper, authors with experience in diverse aspects of genomic analysis summarize the current status of quality assurance in genome diagnostics, with the aim of facilitating further standardization and quality improvement in one of the core competencies of the field.

Dysregulated Retinoic Acid Signaling in the Pathogenesis of Pseudoexfoliation Syndrome.

Pseudoexfoliation (PEX) syndrome, a stress-induced fibrotic matrix process, is the most common recognizable cause of open-angle glaucoma worldwide. The recent identification of PEX-associated gene variants uncovered the vitamin A metabolic pathway as a factor influencing the risk of disease. In this study, we analyzed the role of the retinoic acid (RA) signaling pathway in the PEX-associated matrix metabolism and evaluated its targeting as a potential candidate for an anti-fibrotic intervention. We provided evidence that decreased expression levels of RA pathway components and diminished RA signaling activity occur in an antagonistic crosstalk with TGF-ß1/Smad signaling in ocular tissues and cells from PEX patients when compared with age-matched controls. Genetic and pharmacologic modes of RA pathway inhibition induced the expression and production of PEX-associated matrix components by disease-relevant cell culture models in vitro. Conversely, RA signaling pathway activation by natural and synthetic retinoids was able to suppress PEX-associated matrix production and formation of microfibrillar networks via antagonization of Smad-dependent TGF-ß1 signaling. The findings indicate that deficient RA signaling in conjunction with hyperactivated TGF-ß1/Smad signaling is a driver of PEX-associated fibrosis, and that restoration of RA signaling may be a promising strategy for anti-fibrotic intervention in patients with PEX syndrome and glaucoma.

Why and how we should join the shift from significance testing to estimation.

A paradigm shift away from null hypothesis significance testing seems in progress. Based on simulations, we illustrate some of the underlying motivations. First, p-values vary strongly from study to study, hence dichotomous inference using significance thresholds is usually unjustified. Second, 'statistically significant' results have overestimated effect sizes, a bias declining with increasing statistical power. Third, 'statistically non-significant' results have underestimated effect sizes, and this bias gets stronger with higher statistical power. Fourth, the tested statistical hypotheses usually lack biological justification and are often uninformative. Despite these problems, a screen of 48 papers from the 2020 volume of the Journal of Evolutionary Biology exemplifies that significance testing is still used almost universally in evolutionary biology. All screened studies tested default null hypotheses of zero effect with the default significance threshold of p = 0.05, none presented a pre-specified alternative hypothesis, pre-study power calculation and the probability of 'false negatives' (beta error rate). The results sections of the papers presented 49 significance tests on average (median 23, range 0-390). Of 41 studies that contained verbal descriptions of a 'statistically non-significant' result, 26 (63%) falsely claimed the absence of an effect. We conclude that studies in ecology and evolutionary biology are mostly exploratory and descriptive. We should thus shift from claiming to 'test' specific hypotheses statistically to describing and discussing many hypotheses (possible true effect sizes) that are most compatible with our data, given our statistical model. We already have the means for doing so, because we routinely present compatibility ('confidence') intervals covering these hypotheses.

The ectodysplasin-A receptor is a candidate gene for lateral plate number variation in stickleback fish.

Variation in lateral plating in stickleback fish represents a classical example of rapid and parallel adaptation in morphology. The underlying genetic architecture involves polymorphism at the ectodysplasin-A gene (EDA). However, lateral plate number is influenced by additional loci that remain poorly characterized. Here, we search for such loci by performing genome-wide differentiation mapping based on pooled whole-genome sequence data from a European stickleback population variable in the extent of lateral plating, while tightly controlling for the phenotypic effect of EDA. This suggests a new candidate locus, the EDA receptor gene (EDAR), for which additional support is obtained by individual-level targeted Sanger sequencing and by comparing allele frequencies among natural populations. Overall, our study illustrates the power of pooled whole-genome sequencing for searching phenotypically relevant loci and opens opportunities for exploring the population genetics and ecological significance of a new candidate locus for stickleback armor evolution.

The maintenance of standing genetic variation: Gene flow vs. selective neutrality in Atlantic stickleback fish.

Adaptation to derived habitats often occurs from standing genetic variation. The maintenance within ancestral populations of genetic variants favourable in derived habitats is commonly ascribed to long-term antagonism between purifying selection and gene flow resulting from hybridization across habitats. A largely unexplored alternative idea based on quantitative genetic models of polygenic adaptation is that variants favoured in derived habitats are neutral in ancestral populations when their frequency is relatively low. To explore the latter, we first identify genetic variants important to the adaptation of threespine stickleback fish (Gasterosteus aculeatus) to a rare derived habitat-nutrient-depleted acidic lakes-based on whole-genome sequence data. Sequencing marine stickleback from six locations across the Atlantic Ocean then allows us to infer that the frequency of these derived variants in the ancestral habitat is unrelated to the likely opportunity for gene flow of these variants from acidic-adapted populations. This result is consistent with the selective neutrality of derived variants within the ancestor. Our study thus supports an underappreciated explanation for the maintenance of standing genetic variation, and calls for a better understanding of the fitness consequences of adaptive variation across habitats and genomic backgrounds.

Clinal genomic analysis reveals strong reproductive isolation across a steep habitat transition in stickleback fish.

How ecological divergence causes strong reproductive isolation between populations in close geographic contact remains poorly understood at the genomic level. We here study this question in a stickleback fish population pair adapted to contiguous, ecologically different lake and stream habitats. Clinal whole-genome sequence data reveal numerous genome regions (nearly) fixed for alternative alleles over a distance of just a few hundred meters. This strong polygenic adaptive divergence must constitute a genome-wide barrier to gene flow because a steep cline in allele frequencies is observed across the entire genome, and because the cline center closely matches the habitat transition. Simulations confirm that such strong divergence can be maintained by polygenic selection despite high dispersal and small per-locus selection coefficients. Finally, comparing samples from near the habitat transition before and after an unusual ecological perturbation demonstrates the fragility of the balance between gene flow and selection. Overall, our study highlights the efficacy of divergent selection in maintaining reproductive isolation without physical isolation, and the analytical power of studying speciation at a fine eco-geographic and genomic scale.

Genomic release-recapture experiment in the wild reveals within-generation polygenic selection in stickleback fish.

How rapidly natural selection sorts genome-wide standing genetic variation during adaptation remains largely unstudied experimentally. Here, we present a genomic release-recapture experiment using paired threespine stickleback fish populations adapted to selectively different lake and stream habitats. First, we use pooled whole-genome sequence data from the original populations to identify hundreds of candidate genome regions likely under divergent selection between these habitats. Next, we generate F2 hybrids from the same lake-stream population pair in the laboratory and release thousands of juveniles into a natural stream habitat. Comparing the individuals surviving one year of stream selection to a reference sample of F2 hybrids allows us to detect frequency shifts across the candidate regions toward the genetic variants typical of the stream population-an experimental outcome consistent with polygenic directional selection. Our study reveals that adaptation in nature can be detected as a genome-wide signal over just a single generation.

Widespread intersex differentiation across the stickleback genome - The signature of sexually antagonistic selection?

Females and males within a species commonly have distinct reproductive roles, and the associated traits may be under perpetual divergent natural selection between the sexes if their sex-specific control has not yet evolved. Here, we explore whether such sexually antagonistic selection can be detected based on the magnitude of differentiation between the sexes across genome-wide genetic polymorphisms by whole-genome sequencing of large pools of female and male threespine stickleback fish. We find numerous autosomal genome regions exhibiting intersex allele frequency differences beyond the range plausible under pure sampling stochasticity. Alternative sequence alignment strategies rule out that these high-differentiation regions represent sex chromosome segments misassembled into the autosomes. Instead, comparing allele frequencies and sequence read depth between the sexes reveals that regions of high intersex differentiation arise because autosomal chromosome segments got copied into the male-specific sex chromosome (Y), where they acquired new mutations. Because the Y chromosome is missing in the stickleback reference genome, sequence reads derived from DNA copies on the Y chromosome still align to the original homologous regions on the autosomes. We argue that this phenomenon hampers the identification of sexually antagonistic selection within a genome, and can lead to spurious conclusions from population genomic analyses when the underlying samples differ in sex ratios. Because the hemizygous sex chromosome sequence (Y or W) is not represented in most reference genomes, these problems may apply broadly.

Correction: Berner, D. Allele Frequency Difference AFD-An Intuitive Alternative to FST for Quantifying Genetic Population Differentiation. Genes 2019, 10, 308.

This note is to correct an error in my paper, concerning the Shannon differentiation metric (DShannon) (Reference [43] in the paper). The paper states that DShannon is undefined mathematically whenever one or both populations are monomorphic, that is, fixed for a single allele. Accordingly, the DShannon curve in Figure 1a, showing population differentiation in relation to allele counts for the case in which the pooled minor allele frequency (MAF) is maximal, did not extend across the full range of allele counts; the rightmost data point reflecting complete population differentiation was missing. Moreover, DShannon was completely missing in Figure 1b visualizing the continuum of allele frequency differentiation when the MAF is minimal (one population monomorphic across the entire allele count range).

De Novo Sequencing, Assembly, and Annotation of Four Threespine Stickleback Genomes Based on Microfluidic Partitioned DNA Libraries.

: The threespine stickleback is a geographically widespread and ecologically highly diverse fish that has emerged as a powerful model system for evolutionary genomics and developmental biology. Investigations in this species currently rely on a single high-quality reference genome, but would benefit from the availability of additional, independently sequenced and assembled genomes. We present here the assembly of four new stickleback genomes, based on the sequencing of microfluidic partitioned DNA libraries. The base pair lengths of the four genomes reach 92-101% of the standard reference genome length. Together with their de novo gene annotation, these assemblies offer a resource enhancing genomic investigations in stickleback. The genomes and their annotations are available from the Dryad Digital Repository (https://doi.org/10.5061/dryad.113j3h7).

The protective variant rs7173049 at LOXL1 locus impacts on retinoic acid signaling pathway in pseudoexfoliation syndrome.

LOXL1 (lysyl oxidase-like 1) has been identified as the major effect locus in pseudoexfoliation (PEX) syndrome, a fibrotic disorder of the extracellular matrix and frequent cause of chronic open-angle glaucoma. However, all known PEX-associated common variants show allele effect reversal in populations of different ancestry, casting doubt on their biological significance. Based on extensive LOXL1 deep sequencing, we report here the identification of a common non-coding sequence variant, rs7173049A>G, located downstream of LOXL1, consistently associated with a decrease in PEX risk (odds ratio, OR = 0.63; P = 6.33 × 10-31) in nine different ethnic populations. We provide experimental evidence for a functional enhancer-like regulatory activity of the genomic region surrounding rs7173049 influencing expression levels of ISLR2 (immunoglobulin superfamily containing leucine-rich repeat protein 2) and STRA6 [stimulated by retinoic acid (RA) receptor 6], apparently mediated by allele-specific binding of the transcription factor thyroid hormone receptor beta. We further show that the protective rs7173049-G allele correlates with increased tissue expression levels of ISLR2 and STRA6 and that both genes are significantly downregulated in tissues of PEX patients together with other key components of the STRA6 receptor-driven RA signaling pathway. siRNA-mediated downregulation of RA signaling induces upregulation of LOXL1 and PEX-associated matrix genes in PEX-relevant cell types. These data indicate that dysregulation of STRA6 and impaired retinoid metabolism are involved in the pathophysiology of PEX syndrome and that the variant rs7173049-G, which represents the first common variant at the broad LOXL1 locus without allele effect reversal, mediates a protective effect through upregulation of STRA6 in ocular tissues.

Allele Frequency Difference AFD⁻An Intuitive Alternative to FST for Quantifying Genetic Population Differentiation.

Measuring the magnitude of differentiation between populations based on genetic markers is commonplace in ecology, evolution, and conservation biology. The predominant differentiation metric used for this purpose is FST. Based on a qualitative survey, numerical analyses, simulations, and empirical data, I here argue that FST does not express the relationship to allele frequency differentiation between populations generally considered interpretable and desirable by researchers. In particular, FST (1) has low sensitivity when population differentiation is weak, (2) is contingent on the minor allele frequency across the populations, (3) can be strongly affected by asymmetry in sample sizes, and (4) can differ greatly among the available estimators. Together, these features can complicate pattern recognition and interpretation in population genetic and genomic analysis, as illustrated by empirical examples, and overall compromise the comparability of population differentiation among markers and study systems. I argue that a simple differentiation metric displaying intuitive properties, the absolute allele frequency difference AFD, provides a valuable alternative to FST. I provide a general definition of AFD applicable to both bi- and multi-allelic markers and conclude by making recommendations on the sample sizes needed to achieve robust differentiation estimates using AFD.

How clonal are clones? A quest for loss of heterozygosity during asexual reproduction in Daphnia magna.

Due to the lack of recombination, asexual organisms are predicted to accumulate mutations and show high levels of within-individual allelic divergence (heterozygosity); however, empirical evidence for this prediction is largely missing. Instead, evidence of genome homogenization during asexual reproduction is accumulating. Ameiotic crossover recombination is a mechanism that could lead to long genomic stretches of loss of heterozygosity (LOH) and unmasking of mutations that have little or no effect in heterozygous state. Therefore, LOH might be an important force for inducing variation among asexual offspring and may contribute to the limited longevity of asexual lineages. To investigate the genetic consequences of asexuality, here we used high-throughput sequencing of Daphnia magna for assessing the rate of LOH over a single generation of asexual reproduction. Comparing parthenogenetic daughters with their mothers at several thousand genetic markers generated by restriction site-associated DNA (RAD) sequencing resulted in high LOH rate estimation that largely overlapped with our estimates for the error rate. To distinguish these two, we Sanger re-sequenced the top 17 candidate RAD-loci for LOH, and all of them proved to be false positives. Hence, even though we cannot exclude the possibility that short stretches of LOH occur in genomic regions not covered by our markers, we conclude that LOH does not occur frequently during asexual reproduction in D. magna and ameiotic crossovers are very rare or absent. This finding suggests that clonal lineages of D. magna will remain genetically homogeneous at least over time periods typically relevant for experimental work.

Predictable genome-wide sorting of standing genetic variation during parallel adaptation to basic versus acidic environments in stickleback fish.

Genomic studies of parallel (or convergent) evolution often compare multiple populations diverged into two ecologically different habitats to search for loci repeatedly involved in adaptation. Because the shared ancestor of these populations is generally unavailable, the source of the alleles at adaptation loci, and the direction in which their frequencies were shifted during evolution, remain elusive. To shed light on these issues, we here use multiple populations of threespine stickleback fish adapted to two different types of derived freshwater habitats-basic and acidic lakes on the island of North Uist, Outer Hebrides, Scotland-and the present-day proxy of their marine ancestor. In a first step, we combine genome-wide pooled sequencing and targeted individual-level sequencing to demonstrate that ecological and phenotypic parallelism in basic-acidic divergence is reflected by genomic parallelism in dozens of genome regions. Exploiting data from the ancestor, we next show that the acidic populations, residing in ecologically more extreme derived habitats, have adapted by accumulating alleles rare in the ancestor, whereas the basic populations have retained alleles common in the ancestor. Genomic responses to selection are thus predictable from the ecological difference of each derived habitat type from the ancestral one. This asymmetric sorting of standing genetic variation at loci important to basic-acidic divergence has further resulted in more numerous selective sweeps in the acidic populations. Finally, our data suggest that the maintenance in marine fish of standing variation important to adaptive basic-acidic differentiation does not require extensive hybridization between the marine and freshwater populations. Overall, our study reveals striking genome-wide determinism in both the loci involved in parallel divergence, and in the direction in which alleles at these loci have been selected.

Meta-analysis of chromosome-scale crossover rate variation in eukaryotes and its significance to evolutionary genomics.

Understanding the distribution of crossovers along chromosomes is crucial to evolutionary genomics because the crossover rate determines how strongly a genome region is influenced by natural selection on linked sites. Nevertheless, generalities in the chromosome-scale distribution of crossovers have not been investigated formally. We fill this gap by synthesizing joint information on genetic and physical maps across 62 animal, plant and fungal species. Our quantitative analysis reveals a strong and taxonomically widespread reduction of the crossover rate in the centre of chromosomes relative to their peripheries. We demonstrate that this pattern is poorly explained by the position of the centromere, but find that the magnitude of the relative reduction in the crossover rate in chromosome centres increases with chromosome length. That is, long chromosomes often display a dramatically low crossover rate in their centre, whereas short chromosomes exhibit a relatively homogeneous crossover rate. This observation is compatible with a model in which crossover is initiated from the chromosome tips, an idea with preliminary support from mechanistic investigations of meiotic recombination. Consequently, we show that organisms achieve a higher genome-wide crossover rate by evolving smaller chromosomes. Summarizing theory and providing empirical examples, we finally highlight that taxonomically widespread and systematic heterogeneity in crossover rate along chromosomes generates predictable broad-scale trends in genetic diversity and population differentiation by modifying the impact of natural selection among regions within a genome. We conclude by emphasizing that chromosome-scale heterogeneity in crossover rate should urgently be incorporated into analytical tools in evolutionary genomics, and in the interpretation of resulting patterns.

Posttranscriptional Regulation of LOXL1 Expression Via Alternative Splicing and Nonsense-Mediated mRNA Decay as an Adaptive Stress Response.

Purpose: Alternative mRNA splicing coupled to nonsense-mediated decay (NMD) is a common mRNA surveillance pathway also known to dynamically modulate gene expression in response to cellular stress. Here, we investigated the involvement of this pathway in the regulation of lysyl oxidase-like 1 (LOXL1) expression in response to pseudoexfoliation (PEX)-associated pathophysiologic factors. Methods: Transcript levels of LOXL1 isoforms were determined in ocular tissues obtained from donor eyes without and with PEX syndrome. Pseudoexfoliation-relevant cell types, including human Tenon's capsule fibroblasts (hTCF) and trabecular meshwork cells (hTMC), were exposed to puromycin, caffeine, TGF-ß1, homocysteine, IL-6, retinoic acid, UV-B radiation, oxidative stress, and mechanical stress for up to 48 hours. Western blot analysis was carried out using antibodies against LOXL1, (phosphorylated-) eukaryotic initiation factor 2-α (eIF2-α), and regulator of nonsense transcripts 2 (UPF2). RNA interference was used to knockdown UPF1-3 and Serine/threonine-protein kinase (SMG1). Results: Constitutive expression of wild-type LOXL1 and alternatively spliced LOXL1-a transcripts was detected in all ocular tissues showing highest levels in trabecular meshwork and differential expression between PEX and control specimens. LOXL1-a transcripts were upregulated in hTCF and hTMC by NMD inhibitors puromycin and caffeine (≥6-fold; P < 0.01) or after knockdown of NMD core factors (≥2-fold; P < 0.05), whereas mRNA and protein levels of LOXL1 were reduced (≤0.8 fold; P < 0.05). Exposure of cells to various PEX-associated (stress) factors, including TGF-ß1, UV-B light, oxidative stress, mechanical stress, and retinoic acid enhanced LOXL1-a transcript levels (≥1.5-fold; P < 0.05), while partially downregulating LOXL1 levels (≤0.7-fold; P < 0.05). Stress-induced inhibition of NMD was dependent on phosphorylation of eIF2α. Conclusions: These findings provide evidence for a functional role of alternative splicing coupled to NMD in the posttranscriptional regulation of LOXL1 gene expression and suggest this mechanism to represent a dynamic mode of adapting LOXL1 expression to PEX-associated environmental and nutritional cues.