A recombineering pipeline to clone large and complex genes in Chlamydomonas.

The ability to clone genes has greatly advanced cell and molecular biology research, enabling researchers to generate fluorescent protein fusions for localization and confirm genetic causation by mutant complementation. Most gene cloning is polymerase chain reaction (PCR)�or DNA synthesis-dependent, which can become costly and technically challenging as genes increase in size, particularly if they contain complex regions. This has been a long-standing challenge for the Chlamydomonas reinhardtii research community, as this alga has a high percentage of genes containing complex sequence structures. Here we overcame these challenges by developing a recombineering pipeline for the rapid parallel cloning of genes from a Chlamydomonas bacterial artificial chromosome collection. To generate fluorescent protein fusions for localization, we applied the pipeline at both batch and high-throughput scales to 203 genes related to the Chlamydomonas CO2 concentrating mechanism (CCM), with an overall cloning success rate of 77%. Cloning success was independent of gene size and complexity, with cloned genes as large as 23 kb. Localization of a subset of CCM targets confirmed previous mass spectrometry data, identified new pyrenoid components, and enabled complementation of mutants. We provide vectors and detailed protocols to facilitate easy adoption of this technology, which we envision will open up new possibilities in algal and plant research.

Superoxide is promoted by sucrose and affects amplitude of circadian rhythms in the evening.

Plants must coordinate photosynthetic metabolism with the daily environment and adapt rhythmic physiology and development to match carbon availability. Circadian clocks drive biological rhythms which adjust to environmental cues. Products of photosynthetic metabolism, including sugars and reactive oxygen species (ROS), are closely associated with the plant circadian clock, and sugars have been shown to provide metabolic feedback to the circadian oscillator. Here, we report a comprehensive sugar-regulated transcriptome of Arabidopsis and identify genes associated with redox and ROS processes as a prominent feature of the transcriptional response. We show that sucrose increases levels of superoxide (O2-), which is required for transcriptional and growth responses to sugar. We identify circadian rhythms of O2--regulated transcripts which are phased around dusk and find that O2- is required for sucrose to promote expression of TIMING OF CAB1 (TOC1) in the evening. Our data reveal a role for O2- as a metabolic signal affecting transcriptional control of the circadian oscillator in Arabidopsis.

Chromosomal assembly of the nuclear genome of the endosymbiont-bearing trypanosomatid Angomonas deanei.

Angomonas deanei is an endosymbiont-bearing trypanosomatid with several highly fragmented genome assemblies and unknown chromosome number. We present an assembly of the A. deanei nuclear genome based on Oxford Nanopore sequence that resolves into 29 complete or close-to-complete chromosomes. The assembly has several previously unknown special features; it has a supernumerary chromosome, a chromosome with a 340-kb inversion, and there is a translocation between two chromosomes. We also present an updated annotation of the chromosomal genome with 10,365 protein-coding genes, 59 transfer RNAs, 26 ribosomal RNAs, and 62 noncoding RNAs.

A novel terpene synthase controls differences in anti-aphrodisiac pheromone production between closely related Heliconius butterflies.

Plants and insects often use the same compounds for chemical communication, but not much is known about the genetics of convergent evolution of chemical signals. The terpene (E)-ß-ocimene is a common component of floral scent and is also used by the butterfly Heliconius melpomene as an anti-aphrodisiac pheromone. While the biosynthesis of terpenes has been described in plants and microorganisms, few terpene synthases (TPSs) have been identified in insects. Here, we study the recent divergence of 2 species, H. melpomene and Heliconius cydno, which differ in the presence of (E)-ß-ocimene; combining linkage mapping, gene expression, and functional analyses, we identify 2 novel TPSs. Furthermore, we demonstrate that one, HmelOS, is able to synthesise (E)-ß-ocimene in vitro. We find no evidence for TPS activity in HcydOS (HmelOS ortholog of H. cydno), suggesting that the loss of (E)-ß-ocimene in this species is the result of coding, not regulatory, differences. The TPS enzymes we discovered are unrelated to previously described plant and insect TPSs, demonstrating that chemical convergence has independent evolutionary origins.

How do predators generalize warning signals in simple and complex prey communities? Insights from a videogame.

The persistence of distinct warning signals within and between sympatric mimetic communities is a puzzling evolutionary question because selection favours convergence of colour patterns among toxic species. Such convergence is partly shaped by predators' reaction to similar but not identical stimulus (i.e. generalization behaviour), and generalization by predators is likely to be shaped by the diversity of local prey. However, studying generalization behaviour is generally limited to simple variations of prey colour patterns. Here, we used a computer game played by humans as surrogate predators to investigate generalization behaviours in simple (4 morphs) and complex (10 morphs) communities of unprofitable (associated with a penalty) and profitable butterflies. Colour patterns used in the game are observed in the natural populations of unprofitable butterfly species such as Heliconius numata. Analyses of 449 game participants' behaviours show that players avoided unprofitable prey more readily in simple than in complex communities. However, generalization was observed only in players that faced complex communities, enhancing the protection of profitable prey that looked similar to at least one unprofitable morph. Additionally, similarity among unprofitable prey also reduced attack rates only in complex communities. These results are consistent with previous studies using avian predators but artificial colour patterns and suggest that mimicry is more likely to evolve in complex communities where increases in similarity are more likely to be advantageous.

Genomic architecture and introgression shape a butterfly radiation.

We used 20 de novo genome assemblies to probe the speciation history and architecture of gene flow in rapidly radiating Heliconius butterflies. Our tests to distinguish incomplete lineage sorting from introgression indicate that gene flow has obscured several ancient phylogenetic relationships in this group over large swathes of the genome. Introgressed loci are underrepresented in low-recombination and gene-rich regions, consistent with the purging of foreign alleles more tightly linked to incompatibility loci. Here, we identify a hitherto unknown inversion that traps a color pattern switch locus. We infer that this inversion was transferred between lineages by introgression and is convergent with a similar rearrangement in another part of the genus. These multiple de novo genome sequences enable improved understanding of the importance of introgression and selective processes in adaptive radiation.

Ancestral Admixture Is the Main Determinant of Global Biodiversity in Fission Yeast.

Mutation and recombination are key evolutionary processes governing phenotypic variation and reproductive isolation. We here demonstrate that biodiversity within all globally known strains of Schizosaccharomyces pombe arose through admixture between two divergent ancestral lineages. Initial hybridization was inferred to have occurred ∼20-60 sexual outcrossing generations ago consistent with recent, human-induced migration at the onset of intensified transcontinental trade. Species-wide heritable phenotypic variation was explained near-exclusively by strain-specific arrangements of alternating ancestry components with evidence for transgressive segregation. Reproductive compatibility between strains was likewise predicted by the degree of shared ancestry. To assess the genetic determinants of ancestry block distribution across the genome, we characterized the type, frequency, and position of structural genomic variation using nanopore and single-molecule real-time sequencing. Despite being associated with double-strand break initiation points, over 800 segregating structural variants exerted overall little influence on the introgression landscape or on reproductive compatibility between strains. In contrast, we found strong ancestry disequilibrium consistent with negative epistatic selection shaping genomic ancestry combinations during the course of hybridization. This study provides a detailed, experimentally tractable example that genomes of natural populations are mosaics reflecting different evolutionary histories. Exploiting genome-wide heterogeneity in the history of ancestral recombination and lineage-specific mutations sheds new light on the population history of S. pombe and highlights the importance of hybridization as a creative force in generating biodiversity.

Genetic dissection of assortative mating behavior.

The evolution of new species is made easier when traits under divergent ecological selection are also mating cues. Such ecological mating cues are now considered more common than previously thought, but we still know little about the genetic changes underlying their evolution or more generally about the genetic basis for assortative mating behaviors. Both tight physical linkage and the existence of large-effect preference loci will strengthen genetic associations between behavioral and ecological barriers, promoting the evolution of assortative mating. The warning patterns of Heliconius melpomene and H. cydno are under disruptive selection due to increased predation of nonmimetic hybrids and are used during mate recognition. We carried out a genome-wide quantitative trait locus (QTL) analysis of preference behaviors between these species and showed that divergent male preference has a simple genetic basis. We identify three QTLs that together explain a large proportion (approximately 60%) of the difference in preference behavior observed between the parental species. One of these QTLs is just 1.2 (0-4.8) centiMorgans (cM) from the major color pattern gene optix, and, individually, all three have a large effect on the preference phenotype. Genomic divergence between H. cydno and H. melpomene is high but broadly heterogenous, and admixture is reduced at the preference-optix color pattern locus but not the other preference QTLs. The simple genetic architecture we reveal will facilitate the evolution and maintenance of new species despite ongoing gene flow by coupling behavioral and ecological aspects of reproductive isolation.

Recombination rate variation shapes barriers to introgression across butterfly genomes.

Hybridisation and introgression can dramatically alter the relationships among groups of species, leading to phylogenetic discordance across the genome and between populations. Introgression can also erode species differences over time, but selection against introgression at certain loci acts to maintain postmating species barriers. Theory predicts that species barriers made up of many loci throughout the genome should lead to a broad correlation between introgression and recombination rate, which determines the extent to which selection on deleterious foreign alleles will affect neutral alleles at physically linked loci. Here, we describe the variation in genealogical relationships across the genome among three species of Heliconius butterflies: H. melpomene (mel), H. cydno (cyd), and H. timareta (tim), using whole genomes of 92 individuals, and ask whether this variation can be explained by heterogeneous barriers to introgression. We find that species relationships vary predictably at the chromosomal scale. By quantifying recombination rate and admixture proportions, we then show that rates of introgression are predicted by variation in recombination rate. This implies that species barriers are highly polygenic, with selection acting against introgressed alleles across most of the genome. In addition, long chromosomes, which have lower recombination rates, produce stronger barriers on average than short chromosomes. Finally, we find a consistent difference between two species pairs on either side of the Andes, which suggests differences in the architecture of the species barriers. Our findings illustrate how the combined effects of hybridisation, recombination, and natural selection, acting at multitudes of loci over long periods, can dramatically sculpt the phylogenetic relationships among species.

Sexually dimorphic gene expression and transcriptome evolution provide mixed evidence for a fast-Z effect in Heliconius.

Sex chromosomes have different evolutionary properties compared to autosomes due to their hemizygous nature. In particular, recessive mutations are more readily exposed to selection, which can lead to faster rates of molecular evolution. Here, we report patterns of gene expression and molecular evolution for a group of butterflies. First, we improve the completeness of the Heliconius melpomene reference annotation, a neotropical butterfly with a ZW sex determination system. Then, we analyse RNA from male and female whole abdomens and sequence female ovary and gut tissue to identify sex- and tissue-specific gene expression profiles in H. melpomene. Using these expression profiles, we compare (a) sequence divergence and polymorphism; (b) the strength of positive and negative selection; and (c) rates of adaptive evolution, for Z and autosomal genes between two species of Heliconius butterflies, H. melpomene and H. erato. We show that the rate of adaptive substitutions is higher for Z than autosomal genes, but contrary to expectation, it is also higher for male-biased than female-biased genes. Additionally, we find no significant increase in the rate of adaptive evolution or purifying selection on genes expressed in ovary tissue, a heterogametic-specific tissue. Our results contribute to a growing body of literature from other ZW systems that also provide mixed evidence for a fast-Z effect where hemizygosity influences the rate of adaptive substitutions.

Reducing the rate of post-surgical urinary tract infections in orthopedic patients.

Urinary tract infection (UTI) is the fourth leading cause of healthcare-associated infections, with approximately 70%-80% being attributed to the inappropriate use of indwelling catheters. In many cases, indwelling catheters are used inappropriately without any valid indication, creating potentially avoidable and significant patient distress, discomfort, pain and activity restrictions, together with substantial care burden, cost and hospitalisation. In the Division of Orthopedic Surgery at Toronto Western Hospital (TWH), we identified UTI rate reduction as a quality improvement priority. Patients who underwent total hip and knee joint replacements and hip fracture repairs at TWH were monitored for the incidence of UTI and the usage of catheters. The data collected as part of the American College of Surgeons National Surgical Quality Improvement Program (ACS NSQIP) revealed UTI rate of 2.1% among 666 patients who were treated between January and June 2016. Data collected through a custom field in the ACS NSQIP workstation further revealed that indwelling catheters were overused, with 55.2% of patients receiving indwelling catheters in the same time period. These data were presented to the orthopaedic leadership group and surgeons at TWH in July 2016 to set the quality improvement target and create the working group. Nursing staff was provided education to strictly follow the institutional catheter-associated UTI prevention guidelines and change ideas based on the guidelines were implemented in July 2016. As a result, the rate of UTI decreased to 1.1% and the use of indwelling catheter decreased to 19.8% among 883 patients who were treated between July 2016 and March 2017. The study indicated that a systematic approach, engaging all front-line staff including nurse educators and nurse practitioners, helps to facilitate implementation of practice changes. We expect that ongoing reminders and education ensure that the changes are sustainable.

No evidence for maintenance of a sympatric Heliconius species barrier by chromosomal inversions.

Mechanisms that suppress recombination are known to help maintain species barriers by preventing the breakup of coadapted gene combinations. The sympatric butterfly species Heliconius melpomene and Heliconius cydno are separated by many strong barriers, but the species still hybridize infrequently in the wild, and around 40% of the genome is influenced by introgression. We tested the hypothesis that genetic barriers between the species are maintained by inversions or other mechanisms that reduce between-species recombination rate. We constructed fine-scale recombination maps for Panamanian populations of both species and their hybrids to directly measure recombination rate within and between species, and generated long sequence reads to detect inversions. We find no evidence for a systematic reduction in recombination rates in F1 hybrids, and also no evidence for inversions longer than 50 kb that might be involved in generating or maintaining species barriers. This suggests that mechanisms leading to global or local reduction in recombination do not play a significant role in the maintenance of species barriers between H. melpomene and H. cydno.

Formin Is Associated with Left-Right Asymmetry in the Pond Snail and the Frog.

While components of the pathway that establishes left-right asymmetry have been identified in diverse animals, from vertebrates to flies, it is striking that the genes involved in the first symmetry-breaking step remain wholly unknown in the most obviously chiral animals, the gastropod snails. Previously, research on snails was used to show that left-right signaling of Nodal, downstream of symmetry breaking, may be an ancestral feature of the Bilateria [1 and 2]. Here, we report that a disabling mutation in one copy of a tandemly duplicated, diaphanous-related formin is perfectly associated with symmetry breaking in the pond snail. This is supported by the observation that an anti-formin drug treatment converts dextral snail embryos to a sinistral phenocopy, and in frogs, drug inhibition or overexpression by microinjection of formin has a chirality-randomizing effect in early (pre-cilia) embryos. Contrary to expectations based on existing models [3, 4 and 5], we discovered asymmetric gene expression in 2- and 4-cell snail embryos, preceding morphological asymmetry. As the formin-actin filament has been shown to be part of an asymmetry-breaking switch in vitro [6 and 7], together these results are consistent with the view that animals with diverse body plans may derive their asymmetries from the same intracellular chiral elements [8].

Major Improvements to the Heliconius melpomene Genome Assembly Used to Confirm 10 Chromosome Fusion Events in 6 Million Years of Butterfly Evolution.

The Heliconius butterflies are a widely studied adaptive radiation of 46 species spread across Central and South America, several of which are known to hybridize in the wild. Here, we present a substantially improved assembly of the Heliconius melpomene genome, developed using novel methods that should be applicable to improving other genome assemblies produced using short read sequencing. First, we whole-genome-sequenced a pedigree to produce a linkage map incorporating 99% of the genome. Second, we incorporated haplotype scaffolds extensively to produce a more complete haploid version of the draft genome. Third, we incorporated ∼20x coverage of Pacific Biosciences sequencing, and scaffolded the haploid genome using an assembly of this long-read sequence. These improvements result in a genome of 795 scaffolds, 275 Mb in length, with an N50 length of 2.1 Mb, an N50 number of 34, and with 99% of the genome placed, and 84% anchored on chromosomes. We use the new genome assembly to confirm that the Heliconius genome underwent 10 chromosome fusions since the split with its sister genus Eueides, over a period of about 6 million yr.

Evaluating the use of ABBA-BABA statistics to locate introgressed loci.

Several methods have been proposed to test for introgression across genomes. One method tests for a genome-wide excess of shared derived alleles between taxa using Patterson's D statistic, but does not establish which loci show such an excess or whether the excess is due to introgression or ancestral population structure. Several recent studies have extended the use of D by applying the statistic to small genomic regions, rather than genome-wide. Here, we use simulations and whole-genome data from Heliconius butterflies to investigate the behavior of D in small genomic regions. We find that D is unreliable in this situation as it gives inflated values when effective population size is low, causing D outliers to cluster in genomic regions of reduced diversity. As an alternative, we propose a related statistic ƒ(d), a modified version of a statistic originally developed to estimate the genome-wide fraction of admixture. ƒ(d) is not subject to the same biases as D, and is better at identifying introgressed loci. Finally, we show that both D and ƒ(d) outliers tend to cluster in regions of low absolute divergence (d(XY)), which can confound a recently proposed test for differentiating introgression from shared ancestral variation at individual loci.

Estimation of the spontaneous mutation rate in Heliconius melpomene.

We estimated the spontaneous mutation rate in Heliconius melpomene by genome sequencing of a pair of parents and 30 of their offspring, based on the ratio of number of de novo heterozygotes to the number of callable site-individuals. We detected nine new mutations, each one affecting a single site in a single offspring. This yields an estimated mutation rate of 2.9 × 10(-9) (95% confidence interval, 1.3 × 10(-9)-5.5 × 10(-9)), which is similar to recent estimates in Drosophila melanogaster, the only other insect species in which the mutation rate has been directly estimated. We infer that recent effective population size of H. melpomene is about 2 million, a substantially lower value than its census size, suggesting a role for natural selection reducing diversity. We estimate that H. melpomene diverged from its Müllerian comimic H. erato about 6 Ma, a somewhat later date than estimates based on a local molecular clock.

A conserved set of maternal genes? Insights from a molluscan transcriptome.

The early animal embryo is entirely reliant on maternal gene products for a 'jump-start' that transforms a transcriptionally inactive embryo into a fully functioning zygote. Despite extensive work on model species, it has not been possible to perform a comprehensive comparison of maternally-provisioned transcripts across the Bilateria because of the absence of a suitable dataset from the Lophotrochozoa. As part of an ongoing effort to identify the maternal gene that determines left-right asymmetry in snails, we have generated transcriptome data from 1 to 2-cell and ~32-cell pond snail (Lymnaea stagnalis) embryos. Here, we compare these data to maternal transcript datasets from other bilaterian metazoan groups, including representatives of the Ecydysozoa and Deuterostomia. We found that between 5 and 10% of all L. stagnalis maternal transcripts (~300-400 genes) are also present in the equivalent arthropod (Drosophila melanogaster), nematode (Caenorhabditis elegans), urochordate (Ciona intestinalis) and chordate (Homo sapiens, Mus musculus, Danio rerio) datasets. While the majority of these conserved maternal transcripts ("COMATs") have housekeeping gene functions, they are a non-random subset of all housekeeping genes, with an overrepresentation of functions associated with nucleotide binding, protein degradation and activities associated with the cell cycle. We conclude that a conserved set of maternal transcripts and their associated functions may be a necessary starting point of early development in the Bilateria. For the wider community interested in discovering conservation of gene expression in early bilaterian development, the list of putative COMATs may be useful resource.

Fine mapping of the pond snail left-right asymmetry (chirality) locus using RAD-Seq and fibre-FISH.

The left-right asymmetry of snails, including the direction of shell coiling, is determined by the delayed effect of a maternal gene on the chiral twist that takes place during early embryonic cell divisions. Yet, despite being a well-established classical problem, the identity of the gene and the means by which left-right asymmetry is established in snails remain unknown. We here demonstrate the power of new genomic approaches for identification of the chirality gene, "D". First, heterozygous (Dd) pond snails Lymnaea stagnalis were self-fertilised or backcrossed, and the genotype of more than six thousand offspring inferred, either dextral (DD/Dd) or sinistral (dd). Then, twenty of the offspring were used for Restriction-site-Associated DNA Sequencing (RAD-Seq) to identify anonymous molecular markers that are linked to the chirality locus. A local genetic map was constructed by genotyping three flanking markers in over three thousand snails. The three markers lie either side of the chirality locus, with one very tightly linked (<0.1 cM). Finally, bacterial artificial chromosomes (BACs) were isolated that contained the three loci. Fluorescent in situ hybridization (FISH) of pachytene cells showed that the three BACs tightly cluster on the same bivalent chromosome. Fibre-FISH identified a region of greater that ∼0.4 Mb between two BAC clone markers that must contain D. This work therefore establishes the resources for molecular identification of the chirality gene and the variation that underpins sinistral and dextral coiling. More generally, the results also show that combining genomic technologies, such as RAD-Seq and high resolution FISH, is a robust approach for mapping key loci in non-model systems.

A physiologically required G protein-coupled receptor (GPCR)-regulator of G protein signaling (RGS) interaction that compartmentalizes RGS activity.

G protein-coupled receptors (GPCRs) can interact with regulator of G protein signaling (RGS) proteins. However, the effects of such interactions on signal transduction and their physiological relevance have been largely undetermined. Ligand-bound GPCRs initiate by promoting exchange of GDP for GTP on the Gα subunit of heterotrimeric G proteins. Signaling is terminated by hydrolysis of GTP to GDP through intrinsic GTPase activity of the Gα subunit, a reaction catalyzed by RGS proteins. Using yeast as a tool to study GPCR signaling in isolation, we define an interaction between the cognate GPCR (Mam2) and RGS (Rgs1), mapping the interaction domains. This reaction tethers Rgs1 at the plasma membrane and is essential for physiological signaling response. In vivo quantitative data inform the development of a kinetic model of the GTPase cycle, which extends previous attempts by including GPCR-RGS interactions. In vivo and in silico data confirm that GPCR-RGS interactions can impose an additional layer of regulation through mediating RGS subcellular localization to compartmentalize RGS activity within a cell, thus highlighting their importance as potential targets to modulate GPCR signaling pathways.

The role of the RACK1 ortholog Cpc2p in modulating pheromone-induced cell cycle arrest in fission yeast.

The detection and amplification of extracellular signals requires the involvement of multiple protein components. In mammalian cells the receptor of activated C kinase (RACK1) is an important scaffolding protein for signal transduction networks. Further, it also performs a critical function in regulating the cell cycle by modulating the G1/S transition. Many eukaryotic cells express RACK1 orthologs, with one example being Cpc2p in the fission yeast Schizosaccharomyces pombe. In contrast to RACK1, Cpc2p has been described to positively regulate, at the ribosomal level, cells entry into M phase. In addition, Cpc2p controls the stress response pathways through an interaction with Msa2p, and sexual development by modulating Ran1p/Pat1p. Here we describe investigations into the role, which Cpc2p performs in controlling the G protein-mediated mating response pathway. Despite structural similarity to Gß-like subunits, Cpc2p appears not to function at the G protein level. However, upon pheromone stimulation, cells overexpressing Cpc2p display substantial cell morphology defects, disorientation of septum formation and a significantly protracted G1 arrest. Cpc2p has the potential to function at multiple positions within the pheromone response pathway. We provide a mechanistic interpretation of this novel data by linking Cpc2p function, during the mating response, with its previous described interactions with Ran1p/Pat1p. We suggest that overexpressing Cpc2p prolongs the stimulated state of pheromone-induced cells by increasing ste11 gene expression. These data indicate that Cpc2p regulates the pheromone-induced cell cycle arrest in fission yeast by delaying cells entry into S phase.