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Repeated Duplication of Argonaute2 Is Associated with Strong Selection and Testis Specialization in Drosophila
Samuel H. Lewis, Claire L. Webster, Heli Salmela and Darren J. Obbard
Genetics October 1, 2016 vol. 204 no. 2 757-769; https://doi.org/10.1534/genetics.116.192336
Samuel H. Lewis
Institute of Evolutionary Biology, University of Edinburgh, Ashworth Laboratories, EH9 3FL, United Kingdom
For correspondence: sam.lewis@gen.cam.ac.uk
Claire L. Webster
Heli Salmela
Department of Biosciences, Centre of Excellence in Biological Interactions, University of Helsinki, Finland
Darren J. Obbard
Institute of Evolutionary Biology, University of Edinburgh, Ashworth Laboratories, EH9 3FL, United KingdomCentre for Immunity, Infection and Evolution, University of Edinburgh, Ashworth Laboratories, EH9 3FL, United Kingdom
Argonaute2 (Ago2) is a rapidly evolving nuclease in the Drosophila melanogaster RNA interference (RNAi) pathway that targets viruses and transposable elements in somatic tissues. Here we reconstruct the history of Ago2 duplications across the D. obscura group and use patterns of gene expression to infer new functional specialization. We show that some duplications are old, shared by the entire species group, and that losses may be common, including previously undetected losses in the lineage leading to D. pseudoobscura. We find that while the original (syntenic) gene copy has generally retained the ancestral ubiquitous expression pattern, most of the novel Ago2 paralogs have independently specialized to testis-specific expression. Using population genetic analyses, we show that most testis-specific paralogs have significantly lower genetic diversity than the genome-wide average. This suggests recent positive selection in three different species, and model-based analyses provide strong evidence of recent hard selective sweeps in or near four of the six D. pseudoobscura Ago2 paralogs. We speculate that the repeated evolution of testis specificity in obscura group Ago2 genes, combined with their dynamic turnover and strong signatures of adaptive evolution, may be associated with highly derived roles in the suppression of transposable elements or meiotic drive. Our study highlights the lability of RNAi pathways, even within well-studied groups such as Drosophila, and suggests that strong selection may act quickly after duplication in RNAi pathways, potentially giving rise to new and unknown RNAi functions in nonmodel species.
Argonaute
ARGONAUTE genes are found in almost all eukaryotes, where they play a key role in antiviral immune defense, gene regulation, and genome stability. They perform this diverse range of functions through their role in RNA interference (RNAi) mechanisms, an ancient system of nucleic acid manipulation in which small RNA (sRNA) molecules guide Argonaute proteins to nucleic acid targets through base complementarity (reviewed in Meister 2013). Gene duplication has occurred throughout the evolution of the Argonaute gene family, with ancient duplication events characteristic of some lineages—such as three duplications early in plant evolution (Singh et al. 2015) and multiple expansions and losses throughout the evolution of nematodes (reviewed in Buck and Blaxter 2013) and the Diptera (Lewis et al. 2016). After duplication, Argonautes have often undergone functional divergence, involving changes in expression patterns and altered sRNA binding partners (Lu et al. 2011; Leebonoi et al. 2015; Miesen et al. 2015). Duplication early in eukaryotic evolution produced two distinct Argonaute subfamilies, Ago and Piwi, which have since been retained in the vast majority of Metazoa (Cerutti and Casas-Mollano 2006). Members of the Ago subfamily are expressed in both somatic and germline tissue, and variously bind sRNAs derived from host transcripts (miRNAs, endo-siRNAs) or transposable elements (TE endo-siRNAs) and viruses (viRNAs). In contrast, in most vertebrates and arthropods, the Piwi subfamily members are expressed primarily in association with the germline (reviewed in Ross et al. 2014), and bind sRNAs from TEs and host loci (piRNAs), suggesting that the Piwi subfamily specialized to a germline-specific role on the lineages leading to vertebrates and arthropods.
After the early divergence of the Ago and Piwi subfamilies, subsequent duplications gave rise to three Piwi subfamily members [Ago3, Aubergine (Aub), and Piwi] and two Ago subfamily members (Ago1 and Ago2) in Drosophila melanogaster. All three Piwi subfamily genes are associated with the germline and bind piRNAs derived from TEs and other repetitive genomic elements: Ago3 and Aub amplify the piRNA signal through the "Ping-Pong" cycle (reviewed in Luteijn and Ketting 2013), and Piwi suppresses transposition by directing heterochromatin formation (Sienski et al. 2012). These functional differences are associated with contrasting selective regimes, with Aub evolving under positive selection (Kolaczkowski et al. 2011) and more rapidly than Ago3 and Piwi (Obbard et al. 2009a). In contrast, Ago1 binds miRNAs and regulates gene expression by inhibiting translation and marking transcripts for degradation (reviewed in Eulalio et al. 2008). This function imposes strong selective constraint on Ago1, resulting in slow evolution and very few adaptive substitutions (Obbard et al. 2006, 2009a; Kolaczkowski et al. 2011). Finally, Ago2 binds siRNAs from viruses (viRNAs) and TEs (endo-siRNAs), and functions in gene regulation (Wen et al. 2015), dosage compensation (Menon and Meller 2012), and the ubiquitous suppression of viruses (Li et al. 2002; van Rij et al. 2006) and TEs (Chung et al. 2008; Czech et al. 2008). Ago2 also evolves under strong positive selection, with frequent selective sweeps (Obbard et al. 2006, 2009a,b, 2011; Kolaczkowski et al. 2011), possibly driven by an arms race with virus-encoded suppressors of RNAi (VSRs) (Obbard et al. 2006; Marques and Carthew 2007; van Mierlo et al. 2014).
In contrast to D. melanogaster, from which most functional knowledge of Ago2 in arthropods is derived, an expansion of Ago2 has been reported in D. pseudoobscura (Hain et al. 2010), providing an opportunity to study how the RNAi pathway evolves after duplication. Given the roles of D. melanogaster Ago2 in antiviral defense (Li et al. 2002; van Rij et al. 2006), TE suppression (Chung et al. 2008; Czech et al. 2008), dosage compensation (Menon and Meller 2012), and gene regulation (Wen et al. 2015), we hypothesized that these D. pseudoobscura Ago2 paralogs may have diverged in function. To elucidate the evolution and function of Ago2 paralogs in D. pseudoobscura and its relatives, we identified and dated Ago2 duplication events across available Drosophila genomes and transcriptomes, tested for divergence in expression patterns between the Ago2 paralogs in D. subobscura, D. obscura, and D. pseudoobscura, and quantified the evolutionary rate and positive selection acting on each of these paralogs. We find that testis specificity of Ago2 paralogs has evolved repeatedly in the obscura group, and that the majority of paralogs show evidence of recent positive selection.
Identification of Ago2 homologs in the Drosophilidae
We used tBLASTx to identify Ago2 homologs in transcriptomes and genomes of 39 species of the Drosophilidae, using previously characterized Ago2 from the closest possible relative to provide the query for each species. If blast returned partial hits, we aligned all hits from the target species to all Argonautes from the query species and assigned hits to the appropriate Ago lineage based on a neighbor-joining tree. For each query sequence, we then manually curated partial blast hits into complete genes using Geneious v5.6.2 (http://www.geneious.com, Kearse et al. 2012) (see Supplemental Material, File S3 for sequence accessions).
Additionally, we used degenerate PCR to identify Ago2 paralogs in D. azteca and D. affinis, and paralog-specific PCR with a touchdown amplification cycle to validate the Ago2 paralogs identified in D. subobscura, D. obscura, and D. pseudoobscura. For each reaction, unincorporated primers were removed with ExonucleaseI (New England Biolabs) and 5′ phosphates were removed with Antarctic Phosphatase (NEB). The PCR products were sequenced by Edinburgh Genomics using BigDye V3 reagents on a capillary sequencer (Applied Biosystems, Foster City, CA), and Sanger sequence reads were trimmed and assembled using Geneious v.5.6.2 (http://www.geneious.com, Kearse et al. 2012). We also used a combination of PCR and blast searches to locate D. pseudoobscura Ago2a1 and Ago2a3, which lie on the unplaced "Unknown_contig_265" in release 3.03 of the D. pseudoobscura genome (all PCR primers are detailed in Table S4).
Phylogenetic analysis of drosophilid Ago2 paralogs
To characterize the evolutionary relationships between Ago2 homologs in the Drosophilidae, we aligned sequences using translational MAFFT (Katoh et al. 2002) with default parameters (File S1). We noted that there is a high degree of codon usage bias (CUB) in D. pseudoobscura Ago2e [effective number of codons (ENC) = 34.24] and D. obscura Ago2e (ENC = 40.36), and a lesser degree in D. subobscura Ago2f (ENC = 45.63) and D. obscura Ago2f (ENC = 48.39), and comparison with genome-wide patterns of codon usage bias placed these genes in the lower half of the distribution of ENC (Figure S5). To reduce the impact of CUB, which disproportionately affects synonymous sites, we stripped all third position sites in this analysis (Behura and Severson 2013). We then inferred a gene tree using the Bayesian approach implemented in BEAST v1.8.1 (Drummond et al. 2012) under a nucleotide model, assuming a general time reversible (GTR) substitution model, variation between sites modeled by a γ-distribution with four categories, and base frequencies estimated from the data. We used the default priors for all parameters, except tree shape (for which we specified a birth–death speciation model) and the date of the Drosophila–Sophophora split. To estimate a time scale for the tree, we specified a normal distribution for the date of this node using values based on mutation rate estimates in Obbard et al. 2012, with a mean value of 32 MYA, standard deviation of 7 MYA, and lower and upper bounds of 15 MYA and 50 MYA, respectively. We ran the analysis for 50 million steps, recording samples from the posterior every 1000 steps, and inferred a maximum clade credibility tree with TreeAnnotator v1.8.1 (Drummond et al. 2012). Note that precise date estimates are not a primary focus of this study, but that other calibrations (Russo et al. 1995; Tamura 2004) would lead to more ancient estimates of divergence, and thus stronger evidence for selective maintenance.
Domain architecture and structural modeling of Ago2 paralogs in the obscura group
To infer the location of each domain in each paralog identified in D. subobscura, D. obscura, and D. pseudoobscura, we searched the Pfam database (Finn et al. 2009). To test for structural differences between the D. pseudoobscura paralogs, we built structural models of each paralog, based on the published X-ray crystallographic structure of human Ago2 (Schirle and Macrae 2012). We used the MODELER software in the Discovery Studio 4.0 Modeling Environment [Accelrys Software, San Diego (2013)] to calculate 10 models, selected the most energetically favorable for each protein, and assessed model quality with the 3D-profile option in the software. To assess variation in selective pressure across the structure of each paralog, we mapped variable residues onto each structure (Figure S7) using PyMol v.1.7.4.1 (Schrödinger).
Quantification of virus-induced expression of Ago2 paralogs
We exposed 48- to 96-hr posteclosion virgin males and females of D. melanogaster, D. subobscura, D. obscura, and D. pseudoobscura to Drosophila C virus (DCV), by puncturing the thorax with a pin contaminated with DCV at a dose of ∼4 × 107 tissue culture infective dose 50 (TCID50) per milliliter. Infection with DCV using this method has previously been shown to lead to a rapid and ultimately fatal increase in DCV titer in D. melanogaster and obscura group species (Longdon et al. 2015). All flies were incubated at 18C under a 12L:12D light cycle, with D. melanogaster on Lewis medium and D. subobscura, D. obscura, and D. pseudoobscura on banana medium. We sampled four to seven individuals per species at 0, 8, 16, 24, 48, and 72 hr postinfection. At each time point we extracted RNA using TRIzol reagent (Ambion) and a chloroform/isopropanol extraction, treated twice with TURBO DNase (Ambion), and reverse transcribed using M-MLV reverse transcriptase (Promega, Madison, WI) primed with random hexamers. We then quantified the expression of Ago2 paralogs in these samples by quantitative PCR (qPCR), using Fast Sybr Green (Applied Biosystems) and custom-designed paralog-specific qPCR primer pairs (see Table S5 for primer sequences). Due to their high level of sequence similarity (99.9% identity), no primer pair could distinguish between D. pseudoobscura Ago2a1 and Ago2a3, so combined expression of these two genes is presented as "Ago2a." All qPCR reactions for each sample were run in duplicate and scaled to the internal reference gene Ribosomal Protein L32 (RpL32). To capture the widest possible biological variation, the three biological replicates for each species each used a different wild-type genetic background (see Table S3 for backgrounds used).
Quantification of Ago2 paralog expression in different tissues and life stages
For D. subobscura, D. obscura, and D. pseudoobscura, we extracted RNA from the head, testis/ovaries, and carcass of 48- to 96-hr posteclosion virgin adults, with males and females extracted separately. Each sample consisted of 8–15 individuals in D. subobscura, 10 individuals in D. obscura, and 15 individuals in D. pseudoobscura. We then used qPCR to quantify the expression of each Ago2 paralog in each tissue, with two technical replicates per sample (reagents, primers, and cycling conditions as above). We carried out five replicates per species, each using a different wild-type background (see Table S3 for details of backgrounds used). To provide an informal comparison with the expression pattern of Ago2 before duplication (an "ancestral" expression pattern), we used the bases per kilobase of gene model per million mapped bases (BPKM) values for Ago2 calculated from RNA-sequencing (RNA-seq) data from the body (carcass and digestive system), head, ovary, and testis of 4-day-old D. melanogaster adults by Brown et al. 2014, scaling each BPKM value to the value for RpL32 in each tissue. Due to the design of that experiment, the body data are derived from pooled samples of males and females (Brown et al. 2014).
To quantify expression of Ago2 paralogs in D. pseudoobscura embryos, we collected eggs within 30 min of laying and used qPCR to measure the expression of each Ago2 paralog (reagents and primers as above) in two separate wild-type genetic backgrounds (MV8 and MV10). As above, we estimated an ancestral expression pattern of Ago2 before duplication from the BPKM values for Ago2 in 0- to 2-hr-old D. melanogaster embryos according to Brown et al. 2014, scaled to the BPKM value for RpL32 in embryos. To determine any changes in the expression of other D. pseudoobscura Argonautes (Ago1, Ago3, Aub, and Piwi) that are associated with Ago2 duplication, we measured their expression in adult tissues and embryos, as detailed above, and compared this with the expression of the Argonautes in D. melanogaster as measured by Brown et al. 2014.
Testing for evolutionary rate changes associated with tissue specificity of Ago2
We used codeml (PAML v4.4, Yang 1997) to fit variants of the M0 model (a single dN/dS ratio, ω) to the 65 drosophilid Ago2 homologs shown in Figure 1. All analyses of sequence evolution excluded the highly repetitive N-terminal glutamine-rich repeat regions, as these regions are effectively unalignable and are unlikely to conform to simple models of sequence evolution (Palmer and Obbard 2016). In contrast to the tree topology, which was based on first and second positions only, the alignment for the codeml analysis included all positions (File S2). To compare the evolutionary rates of ubiquitously expressed and testis-specific Ago2 paralogs, we fitted a model specifying one ω for the Ago2 paralogs that were shown to be testis-specific by qPCR (7 homologs), and another ω for the rest of the tree (58 homologs). We also fitted two models to account for rate variation between the obscura group Ago2 subclades. The first model specified a separate ω for the Ago2a subclade (17 homologs), the Ago2e subclade (8 homologs), the Ago2f subclade (5 homologs), and the rest of the tree (35 homologs). The second model additionally incorporated an extra ω specified for the D. pseudoobscura–D. persimilis Ago2a–Ago2b subclade (3 homologs, all of which are testis-specific, in contrast with the rest of the obscura group Ago2a subclade). We used Akaike weights to assess which model provided the best fit to the data, given the number of parameters. As mentioned above, the high CUB seen in some Ago2 paralogues may affect PAML analyses by decreasing synonymous site divergence (dS) in those lineages, thereby inflating the dN/dS ratio (ω). However, we find no link between levels of CUB and the value of ω, suggesting that CUB is not impacting our PAML analyses.
An approximately time-scaled Bayesian gene tree of Ago2 in the Drosophilidae. Duplication events are marked by yellow diamonds, Bayesian posterior support is shown for nodes for which it is <100% and the genes and species that are the focus of the present study are marked in boldface type. Ago2 has duplicated at least 12 times in the Drosophilidae: 7 times in the obscura group, twice early in the melanogaster group, and once each in the lineages leading to D. willistoni, S. deflexa and D. kikkawai. There has also been a potentially recent duplication of Ago2a on the D. affinis/D. azteca lineage (∼5 MYA), although the low support for this node may suggest that these paralogs could also nest within the D. pseudoobscura/D. persimilis expansion, with one paralog sister to the Ago2a–Ago2b subclade and the other sister to the Ago2c–Ago2d subclade. After duplication, Ago2 paralogs in the obscura group have specialized to the testis 3 times independently (marked with ♂) and have been retained for an extended period of time (>10 MY in the case of Ago2e), suggesting an adaptive basis for testis specificity. The labeling a–e of paralogous clades corresponds to Hain et al. (2010) and is retained for consistency with subsequent publications, which also use these labels, while clade f is newly reported here. All genes were identified by BLAST, apart from the following, which were found by PCR: D. teissieri Ago2; D. santomea Ago2; D. azteca Ago2a, Ago2b, and Ago2e; and D. pseudoobscura Ago2a1 and Ago2a3.
Sequencing of Ago2 paralog haplotypes from D. subobscura, D. obscura, and D. pseudoobscura
To obtain genotype data for the Ago2 paralogs in D. subobscura, D. obscura, and D. pseudoobscura, we sequenced the Ago2 paralogs from six males and six females of each species, each from a different wild-collected line (detailed in Table S3, sequence polymorphism data in File S4). We extracted genomic DNA from each individual using the DNeasy Blood and Tissue kit (QIAGEN, Valencia, CA) and amplified and Sanger sequenced each Ago2 paralog from each individual (reagents and PCR primers as above, sequencing primers detailed in Table S5). We trimmed and assembled Sanger sequence reads using Geneious v.5.6.2 (http://www.geneious.com, Kearse et al. 2012), and identified polymorphic sites by eye. After sequencing Ago2a (annotated as a single gene in the D. pseudoobscura genome), we discovered two very recent Ago2a paralogs (which we denote Ago2a1 and Ago2a3), which had been cross-amplified. For each D. pseudoobscura individual, we therefore resequenced Ago2a3 using one primer targeted to its neighboring locus GA22965, and used this sequence to resolve polymorphic sites in the Ago2a1/Ago2a3 composite sequence, thereby gaining both genotypes for each individual. For each Ago2 paralog, we inferred haplotypes from these sequence data using PHASE (Stephens et al. 2001), apart from the X-linked paralogs (Ago2a1, Ago2a3, and Ago2d) in D. pseudoobscura males, for which phase was obtained directly from the sequence data. The hemizygous haploid X-linked sequences were used in phase inference and should substantially improve the inferred phasing of female genotypes.
To quantify differences between paralogs in their population genetic characteristics, we aligned haplotypes using translational MAFFT (Katoh et al. 2002), and used DnaSP v.5.10.01 (Librado and Rozas 2009) to calculate the following summary statistics for each Ago2 paralogue: π (pairwise diversity, with Jukes–Cantor correction as described in Lynch and Crease 1990) at nonsynonymous (πa) and synonymous (πs) sites, Tajima's D (Tajima 1989) and ENC (Wright 1990). To compare the ENC for each gene with the genome as a whole, we used codonW v1.4.2 (Peden 1995) to calculate the ENC for the longest ORF from each gene or transcript in the genomes or transcriptomes of D. subobscura, D. obscura, and D. pseudoobscura (ORF sets detailed below). In each species, we then compared the ENC values of each Ago2 paralog with this genome-wide ENC distribution.
Testing for positive selection on Ago2 paralogs in the obscura group
We used McDonald–Kreitman (MK) tests (McDonald and Kreitman 1991) to test for positive selection on each Ago2 paralog. For each paralog, we chose an outgroup with divergence at synonymous sites (KS) in the range 0.1–0.2 where possible. However, the prevalence of duplications and losses of Ago2 paralogs in the obscura group meant that for some tests no suitably divergent extant outgroup existed. In these cases, we reconstructed hypothetical ancestral sequences using the M0 model provided by codeml from PAML (Yang 1997). To assess the effect of these outgroup choices on our results, we repeated each test with another outgroup and found no effect of outgroup choice on the significance of any tests, and only marginal differences in estimates of α and ωα (results of tests using primary and alternative outgroups are detailed in Table S1 and Table S2).
A complementary approach to identifying positive selection is to test for reduced diversity at a locus compared with the genome as a whole. To compare the diversity of each D. pseudoobscura Ago2 paralog with the genome-wide distribution of synonymous site diversity, we used genomic data for 12 lines generated by McGaugh et al. 2012. We mapped short reads to the longest ORF for each gene in the R3.2 gene set using Bowtie2 v2.1.0 (Langmead et al. 2009) and estimated synonymous site diversity (θW based on fourfold synonymous sites) at each ORF using PoPoolation (Kofler et al. 2011). We then plotted the distribution of synonymous site diversity, limited to genes in the size range of 0.75–3 kb for comparability with the Ago2 paralogs, and compared the fourfold synonymous site diversity levels of each D. pseudoobscura Ago2 paralog with this distribution. Some D. pseudoobscura paralogs are located on autosomes (Ago2b, Ago2c, and Ago2e) and some on the X chromosome (Ago2a1, Ago2a3, and Ago2d). Therefore, because of the different population genetic expectations for autosomal and X-linked genes (Vicoso and Charlesworth 2006), we examined separate distributions for autosomal and X-linked genes. To provide an additional test for reduced diversity at D. pseudoobscura Ago2 paralogs, we performed maximum-likelihood Hudson–Kreitman–Aguadé (HKA) tests (Wright and Charlesworth 2004), using divergence from D. affinis and intraspecific polymorphism data for 84 D. pseudoobscura loci generated by Haddrill et al. (2010). We performed 63 tests to encompass all one-, two-, three-, four-, five-, and six-way combinations of the paralogs and calculated Akaike weights from the resulting likelihood estimates to provide an estimate of the level of support for each combination.
To infer a genome-wide distribution of synonymous site diversity for D. obscura and D. subobscura, for which genomic data are unavailable, we used pooled transcriptome data from wild-collected adult male flies that had previously been generated for surveys of RNA viruses (van Mierlo et al. 2014; Webster et al. 2016). To generate a de novo transcriptome for each species, we assembled short reads with Trinity r20140717 (Grabherr et al. 2011). For each species, we mapped short reads from the pooled sample to the longest ORF for each transcript, estimated synonymous site diversity at each locus using PoPoolation (Kofler et al. 2011), and plotted the distribution of diversity (as described above for D. pseudoobscura). The presence of heterozygous sites in males (identified by Sanger sequencing) confirmed that all Ago2 paralogs in D. subobscura and D. obscura are autosomal: we therefore compared the synonymous site diversity for these paralogs with the autosomal distribution and do not show the distributions for putatively X-linked genes. Our use of transcriptome data for D. obscura and D. subobscura will bias the resulting diversity distributions in three ways. First, variation in expression level will cause individuals displaying high levels of expression to be overrepresented among reads, downwardly biasing diversity. Second, highly expressed genes are easier to assemble, and highly expressed genes tend to display lower genetic diversity (Pal et al. 2001; Lemos et al. 2005). Third, high-diversity genes are harder to assemble, per se. However, as all three biases will tend to artifactually reduce diversity in the genome-wide data set relative to Ago2, this makes our finding that Ago2 paralogs display unusually low diversity conservative.
Identifying selective sweeps in Ago2 paralogs of D. pseudoobscura
To test whether the unusually low diversity seen in the D. pseudoobscura Ago2 paralogs is due to recent selection or generally reduced diversity in that region of the genome, we compared diversity at each paralog to diversity in their neighboring regions. We obtained sequence data for the 50 kb either side of each of these paralogs from the 11 whole genomes detailed in McGaugh et al. 2012 (SRA044960.1, SRA044955.2, and SRA044956.1). Note that the very high similarity of these Ago2 paralogues means that they cannot be accurately assembled from short read data, and are not present in the data from McGaugh et al. 2012. For each genome, we therefore replaced the poorly assembled region corresponding to the paralog with one of our own Sanger-sequenced haplotypes, making a set of 11 ∼102-kb sequences for each paralog. We aligned these sequences using PRANK (Löytynoja and Goldman 2005) with default settings and calculated Watterson's θ at all sites in a sliding window across each alignment, with a window size of 5 kb and a step of 1 kb. For Ago2a1 and Ago2a3, which are located in tandem, we analyzed the same genomic region. Since our Ago2 haplotypes were sampled from a different North American population of D. pseudoobscura than those of McGaugh et al. 2012, an apparent reduction in local diversity might result from differences in diversity between the two populations. We therefore also repeated these analyses on a data set in which our Sanger sequenced haplotypes were removed, leaving missing data.
To test explicitly for selective sweeps at each region, we used Sweepfinder (Nielsen et al. 2005b) to calculate the likelihood and location of a sweep in or near each Ago2 paralog. We specified a grid size of 20,000, a folded frequency spectrum for all sites, and included invariant sites. To infer the significance of any observed peaks in the composite likelihood ratio, we used ms (Hudson 2002) to generate 1000 samples of 11 sequences under a neutral coalescent model. We generated separate samples for each region surrounding an Ago2 paralog, conditioning on the number of polymorphic sites observed in that region, the sequence length equal to the alignment length, and an effective population size of 106 (based on a previous estimate for D. melanogaster by Li and Stephan 2006). We specified the recombination rate at 5 cM/Mb, a conservative value based on previous estimates for D. pseudoobscura (McGaugh et al. 2012), which will lead to larger segregating linkage groups and therefore a more stringent significance threshold.
All new sequences produced in this study have been submitted to GenBank as KX016642–KX016771.
Ago2 has undergone numerous ancient and recent duplications in the obscura group
Ago2 duplications had previously been noted in D. pseudoobscura (Hain et al. 2010), but their age and distribution in other species was unknown. We used BLAST (Altschul et al. 1997) and PCR to identify 65 Ago2 homologs in 39 species sampled across the Drosophilidae, including 30 homologs in 9 obscura group species. Using PCR and Sanger sequencing, we verified that the paralogs in D. subobscura, D. obscura, and D. pseudoobscura are genuine distinct loci, and not artifacts of erroneous assembly. Additionally, we verified that all paralogs possess introns and so are most likely to be the product of segmental duplication rather than retrotransposition. This is perhaps unsurprising, given that segmental duplicates are generally retained at a higher rate than retrotransposed duplicates, despite the rate of retrotransposition being higher than segmental duplication (Hahn 2009).
To characterize the relationships between Ago2 homologs in the obscura group and the other Drosophilidae, and estimate the date of the duplication events that produced them, we carried out a strict clock Bayesian phylogenetic analysis (Figure 1). This showed that there are early diverging Ago2 clades in the obscura group: the Ago2e subclade that diverged from other Ago2 paralogs ∼21 MYA (±10 MY) and the Ago2a and Ago2f subclades that were produced by a gene duplication event ∼16 MYA (±7 MY). Subsequently there have been a series of more recent duplications in the D. pseudoobscura subgroup Ago2a–d lineage. Using published genomes, transcriptomes, and PCR, we were unable to identify Ago2e in D. subobscura, Ago2e or Ago2f in D. lowei, or Ago2f in D. pseudoobscura, D. persimilis, and D. azteca. While apparent losses may reflect a lack of genomic data (D. subobscura, D. lowei, and D. azteca), incomplete genome assemblies (D. pseudoobscura and D. persimilis), or unexpressed genes in transcriptome surveys, we attempted to validate the losses observable in D. pseudoobscura and D. subobscura by extensive PCR and were again unable to recover these genes from those two species.
In release 3.03 of the D. pseudoobscura genome, the paralogs Ago2b–Ago2e have confirmed locations, but Ago2a1 and Ago2a3 (the very recent paralogs newly identified here) lie in tandem on an unplaced contig with a third incomplete copy (Ago2a2) between them. We used PCR to confirm the existence, orientation, and relative positioning of these genes and to identify the location of this contig, which lies in reverse orientation on chromosome XL-group1a (predicted coordinates 3,463,701–3,489,689). We then combined this information with our phylogenetic analysis to reconstruct the positional evolution of D. pseudoobscura Ago2 paralogs (Figure S1). We found that D. pseudoobscura Ago2d is syntenic with D. melanogaster Ago2, indicating that Ago2d is the ancestral paralog in this species. We also found that Ago2 paralogs have translocated throughout the D. pseudoobscura genome (Figure S1) and are situated on autosomes (Ago2b, Ago2c, and Ago2e) and both arms of the X chromosome (Ago2a1, Ago2a3, and Ago2d). It should be noted that a lack of genomic data precludes similar synteny analysis for any other obscura group species; our naming of the Ago2 paralogs in these species as Ago2a (or Ago2a and Ago2b in the case of D. affinis and D. azteca) reflects their position within the Ago2a subclade, rather than a syntenic relationship or otherwise with D. pseudoobscura Ago2a1 and Ago2a3.
Ago2 paralogs in D. subobscura, D. obscura, and D. pseudoobscura are probably functional
Our phylogenetic analysis (Figure 1) revealed that the Ago2 paralogs in the obscura group have retained coding sequences for millions of generations, showing that they have remained functional for this period. They have also retained PAZ and PIWI domains and a bilobal structure (characteristic of Argonaute proteins), suggesting that they are part of a functional RNAi pathway. In D. melanogaster Ago2 plays a key role in antiviral immunity, but is ubiquitously and highly expressed in both males and females and is not strongly induced by viral challenge (Figure 2a, Aliyari et al. 2008). To test whether this expression pattern has been conserved after Ago2 duplication, or whether any Ago2 paralogs have become inducible by viral challenge, we measured the expression of each Ago2 paralog in female and male D. subobscura, D. obscura, and D. pseudoobscura after infection with DCV. These species are separated by ∼10 MY of evolution and represent the three major clades within the obscura group. Members of the obscura group are highly susceptible to DCV, supporting high viral titres and displaying rapid mortality (Longdon et al. 2015). We found that only one paralog is expressed in both sexes at a high level in D. subobscura (Ago2a), D. obscura (Ago2a), and D. pseudoobscura (Ago2c). These paralogs show a similar pattern of expression to D. melanogaster Ago2, being expressed constitutively throughout the time course rather than induced by viral infection (Figure 2). Unexpectedly, and with only one exception, the other Ago2 paralogs in all species were expressed exclusively in males (Figure 2, b–d), raising the possibility that these duplicates have specialized to a sex-specific role. The one exception was D. pseudoobscura Ago2d, which is the ancestral paralog in this species (inferred by synteny), and for which we could not detect any expression.
Expression patterns of Ago2 paralogs under challenge with DCV. In each obscura group species, only one Ago2 paralog has retained the ancestral pattern of ubiquitous stable expression in each sex (illustrated by D. melanogaster). In contrast, all other paralogs are expressed in males only (in D. pseudoobscura females, Ago2a, Ago2b, Ago2d, and Ago2e are all unexpressed throughout the time course). The only exception to this is D. pseudoobscura Ago2d, which is unexpressed in either sex. The high degree of sequence similarity between Ago2a1 and Ago2a3 prevented us from amplifying these genes separately in qPCR, and here they are combined as "Ago2a." Error bars indicate one standard error estimated from two technical replicates in each of three different genetic backgrounds. Apparent differences in expression between sexes and species should be interpreted with caution, as these may be driven by differences in expression levels of the reference gene (RpL32).
Ago2 paralogs have repeatedly specialized to the testis
To determine whether the strongly male-biased expression pattern is associated with a testis-specific role, we quantified the tissue-specific expression patterns of Ago2 paralogs in D. subobscura, D. obscura, and D. pseudoobscura. In D. melanogaster, the single copy of Ago2 was expressed in all adult tissues (Figure 3D), and transcripts were present in the embryo (Figure S2). In D. subobscura, D. obscura, and D. pseudoobscura, we found that the Ago2 paralogs exhibited striking differences in their tissue-specific patterns of expression (Figure 3, A–C). In each species, one paralog has retained the ancestral ubiquitous expression pattern in adult tissues. In contrast, every other paralog was expressed only in the testis, except for the nonexpressed D. pseudoobscura Ago2d. None of the testis-specific paralogs in D. pseudoobscura was detectable in embryos (Figure S2).
Tissue-specific expression patterns of Ago2 paralogs. In each of the three obscura group species tested, one paralog has retained the ancestral ubiquitous expression pattern, while the others have specialized to the testis (with the exception of D. pseudoobscura Ago2d). The high degree of sequence similarity between Ago2a1 and Ago2a3 prevented us from amplifying these genes separately in qPCR, and here they are combined as "Ago2a." Error bars indicate one standard error estimated from two technical replicates in each of five different genetic backgrounds. D. melanogaster expression levels were taken from a single RNA-seq experiment (Brown et al. 2014).
Interestingly, the ubiquitously expressed paralog in D. subobscura and D. obscura is the ancestral gene (Ago2a in both cases, as inferred by synteny with D. melanogaster), but in D. pseudoobscura another paralog (Ago2c) has evolved the ubiquitous expression pattern, and the ancestral gene (Ago2d) was not expressed at a detectable level in any tissue. When interpreted in the context of the phylogenetic relationships between these paralogs, the most parsimonious explanation is that testis specificity evolved at least three times: first at the base of the Ago2e clade, second at the base of the Ago2f clade, and third at the base of the D. pseudoobscura–D. persimilis Ago2a–Ago2b subclade (Figure 1).
Testis specificity is associated with faster protein evolution
To test for differences in evolutionary rate between testis-specific and ubiquitously expressed Ago2 paralogs, we fitted sequence evolution models to the set of drosophilid Ago2 sequences depicted in Figure 1 using codeml (PAML, Yang 1997). These tests estimate separate dN/dS ratios (ω) for different subclades in the gene tree, providing a test for differential rates of protein evolution. We found that most support (Akaike weight = 0.99) falls behind a model specifying a different ω for each obscura group Ago2 subclade, and another separate ω for the D. pseudoobscura–D. persimilis Ago2a–Ago2b subclade. Under this model, the testis-specific D. pseudoobscura–D. persimilis Ago2a–Ago2b subclade has the highest rate of protein evolution (ω = 0.32 ± 0.047 SE), followed by the testis-specific Ago2f subclade (ω = 0.21 ± 0.014), the ubiquitous Ago2a subclade (ω = 0.19 ± 0.012), the testis-specific Ago2e subclade (ω = 0.16 ± 0.010), and finally the other Drosophilid Ago2 sequences (ω = 0.12 ± 0.002). This shows that the evolution of testis specificity was accompanied by an increase in the rate of protein evolution following two of the three duplications. We also used the Bayes empirical Bayes sites test in codeml to identify codons evolving under positive selection across the entire gene tree, and the branch-sites test to identify codons under positive selection in the obscura group Ago2 subclade. While we found no positively selected codons with the sites test, we identified three codons under positive selection (297, 338, and 360) in the obscura group Ago2 subclade with the branch-sites test (likelihood ratio test M8 vs. M8a, P < 0.005).
MK tests identify strong positive selection on D. pseudoobscura Ago2e
Changes in evolutionary rate after the evolution of testis specificity may occur as a result of changes in positive selection or changes in selective constraint. However, unless there are multiple substitutions within single codons, this will be hard to detect using methods such as codeml. Therefore, as a second test for positive selection on Ago2 paralogs in D. subobscura, D. obscura, and D. pseudoobscura, we gathered intraspecies polymorphism data for each Ago2 paralog in these species (File S4) and performed MK tests (Table S1). The MK test uses a comparison of the numbers of fixed differences between species at nonsynonymous (Dn) and synonymous (Ds) sites, and polymorphisms within a species at nonsynonymous (Pn) and synonymous (Ps) sites to infer the action of positive selection. If all mutations are either neutral or strongly deleterious, the Dn/Ds ratio should be approximately equal to the Pn/Ps ratio; however, if there is positive selection, an excess of nonsynonymous differences is expected (McDonald and Kreitman 1991). The majority of MK tests were nonsignificant (Fisher's exact test, P > 0.1), despite often displaying relatively high KA/KS ratios e.g., D. pseudoobscura Ago2a1 (KA/KS = 0.34), Ago2b (KA/KS = 0.43), and Ago2d (KA/KS = 0.36). However, the low diversity at these loci (<10 polymorphic sites in most cases; see below) means that the MK approach has little power, and that estimates of the proportion of substitutions that are adaptive (α) are likely to be poor. In contrast to the other loci, our MK analysis identified strong positive selection acting on D. pseudoobscura Ago2e—which has relatively high genetic diversity—with α at 100% (α = 1.00; Fisher's exact test, P = 0.0004). This result is driven by the extreme dearth of nonsynonymous-to-synonymous polymorphisms (0 Pn to 17 Ps), despite substantial numbers of fixed differences (77 Dn to 120 Ds), and its statistical significance is robust to the choice of outgroup (Table S2).
The majority of Ago2 paralogs have extremely low levels of sequence diversity
When strong selection acts to reduce genetic diversity at a locus, it can also reduce diversity at linked loci before recombination can break up linkage (Maynard Smith and Haigh 1974). Recent positive selection can therefore be inferred from a reduction in synonymous site diversity compared with other genes. Because MK tests can detect only multiple long-term substitutions, and are hampered by low diversity, diversity-based approaches offer a complementary way to detect very recent strong selection. We therefore compared the synonymous site diversity at each Ago2 paralog in D. pseudoobscura with the distribution of genome-wide synonymous site diversity. We found that all D. pseudoobscura paralogs have unusually low diversity, relative to other loci: Ago2a1, Ago2b, and Ago2c fall into the lowest percentile, Ago2a3 and Ago2d into the second lowest percentile, and Ago2e into the eighth lowest percentile (Figure S4). A multilocus extension of the HKA test (ML-HKA, Wright and Charlesworth 2004) confirmed that the diversity of Ago2a1–Ago2e is significantly lower than the D. pseudoobscura genome as a whole (Akaike weight = 0.98).
Unfortunately, population-genomic data are not available for D. subobscura and D. obscura, preventing a similar analysis. However, we found similar results for Ago2a and Ago2e when comparing the diversity of D. subobscura and D. obscura Ago2 paralogs to levels of diversity inferred from transcriptome data (data from Webster et al. 2016), suggesting that this effect is not limited to D. pseudoobscura, and these genes may therefore have been recent targets of selection in multiple species. In D. obscura, Ago2a and Ago2e fall into the 2nd and 4th lowest diversity percentile, respectively, whereas Ago2f falls into the 19th percentile (Figure S4). In D. subobscura, Ago2a falls into the 7th percentile, whereas Ago2f falls into the 16th percentile (Figure S4). The prevalence of low intraspecific diversity for testis-specific paralogs is consistent with recent selective sweeps, suggesting that positive selection, not merely relaxation of constraint, has contributed to the increased evolutionary rate seen after specialization to the testis.
Four of six D. pseudoobscura Ago2 duplicates show a strong signature of recent hard selective sweeps
The impact of selection on linked diversity (a selective sweep) is expected to leave a characteristic footprint in local genetic diversity around the site of selection, and this forms the basis of explicit model-based approaches to detect the recent action of positive selection (Nielsen et al. 2005a). For D. pseudoobscura, population genomic data for 11 haplotypes is available from McGaugh et al. (2012), permitting an explicit model-based test for recent hard selective sweeps near to Ago2 paralogs. We therefore combined our Ago2 data with 111-kb-long haplotypes from McGaugh et al. (2012) to analyze the neighboring region around each paralog. Ago2a1 and Ago2a3 form a tandem repeat and were therefore analyzed together as a single potential sweep. We found strong evidence for recent selective sweeps at or very close to Ago2a1/3, Ago2b, and Ago2c, which display sharp troughs in their diversity levels and large peaks in the composite likelihood of a sweep, which far exceed a significance threshold derived from coalescent simulation (P < 0.01; Figure 4). These localized reductions in diversity remain when our own Ago2 haplotype data are removed, showing the results are robust to the fact that our Ago2 sequence data are derived from a different population to the genome-wide data of McGaugh et al. (2012) [Figure S6; note that sequence data for Ago2 paralogs cannot be derived from the data of McGaugh et al. (2012), because of their extreme similarity]. In addition, there is ambiguous evidence for a sweep at Ago2d, in the form of one significant (P < 0.01) likelihood peak just upstream of the paralog, but two other peaks ∼1 kb and ∼3 kb further upstream. There is no evidence for a hard sweep at Ago2e, which has no diversity trough or likelihood peak.
Selective sweeps at D. pseudoobscura Ago2 paralogs. For each paralog, diversity at all sites (Watterson's θ) is displayed in red, and the likelihood of a sweep centered at that site (composite likelihood ratio, CLR) is displayed in blue. The gene region containing the paralog is represented by the shaded vertical bar, and the significance threshold for the CLR is displayed by the horizontal dotted line (P < 0.01, derived from the 10th-highest CLR out of 1000 coalescent simulations, assuming constant recombination rate and Ne). There is strong evidence for sweeps at Ago2a, Ago2b, and Ago2c, indicated by troughs in their diversity levels and peaks in the likelihood of a sweep.
Testis specificity may indicate a loss of antiviral function
We have found that Ago2 paralogs in the obscura group have repeatedly evolved divergent expression patterns after duplication, with the majority of paralogs specializing to the testis. This is the first report of testis specificity for any arthropod Ago2, which is ubiquitously expressed in D. melanogaster (Celniker et al. 2009), and provides a strong indication that these paralogs have diverged in function. This testis specificity (Figure 3) suggests that these Argonautes are likely to have lost their ancestral ubiquitous antiviral role. Additionally, the constant level of expression of testis-specific paralogs under DCV infection (Figure 2) suggests that they have not evolved an inducible response to viral infection, either restricted to the testis or in other tissues. In contrast, one paralog in each species has retained the ubiquitous expression pattern seen in D. melanogaster (D. subobscura Ago2a, D. obscura Ago2a, and D. pseudoobscura Ago2c; Figure 3), suggesting that these paralogs have retained roles in antiviral defense (Li et al. 2002; van Rij et al. 2006), dosage compensation (Menon and Meller 2012), and/or somatic TE suppression (Chung et al. 2008; Czech et al. 2008).
Both ubiquitous and testis-specific Ago2 paralogs show evidence of recent positive selection
We identified selective sweeps at the ubiquitously expressed Ago2 paralog in D. pseudoobscura Ago2c, and very low diversity in the ubiquitously expressed Ago2 paralogs of D. subobscura and D. obscura (Ago2a), suggesting that all of these genes may have recently experienced strong positive selection. Four randomly chosen genes that are testis-specific in D. melanogaster (Mikhaylova et al. 2008) do not fall into the low-diversity tails of the genome-wide diversity distributions of D. obscura [β-tubulin (85D), Hsp60C, sungrazer and roughex] and D. subobscura [β-tubulin (85D), Hsp60C, sungrazer, and Calcutta cup], suggesting that the low diversity of testis-specific Ago2 paralogs in these species is not a general consequence of testis-specific expression. This is consistent with previous findings of strong selection and rapid evolution of Ago2 in D. melanogaster (Obbard et al. 2006, 2009b, 2011), which has also experienced recent sweeps in D. melanogaster, D. simulans, and D. yakuba (Obbard et al. 2011), and across Drosophila more broadly (Kolaczkowski et al. 2011). It has previously been suggested that this is driven by arms-race coevolution with viruses (Obbard et al. 2009a; Kolaczkowski et al. 2011), some of which encode viral suppressors of RNAi (VSRs) that block Ago2 function (Bronkhorst and van Rij 2014). The presence of VSR-encoding viruses, such as Nora virus, in natural obscura group populations (Webster et al. 2016), combined with the host specificity that can be displayed by VSRs (van Mierlo et al. 2014), suggest that arms-race dynamics may also be driving the rapid evolution of ubiquitously expressed Ago2 paralogs in the obscura group.
Potential testis-specific functions
In contrast to their ancestral ubiquitous expression pattern, the dominant fate for Ago2 paralogs in the obscura group appears to have been specialization to the testis. Paralogs often undergo a brief period of testis specificity soon after duplication (Assis and Bachtrog 2013, 2015), and this has given rise to the "out-of-the-testis" hypothesis, in which new paralogs are initially testis-specific before evolving functions in other tissues (Kaessmann 2010). However, two lines of evidence suggest an adaptive basis for the testis specificity observed for the obscura group Ago2 paralogs. First, testis specificity has been retained for >10 MY in Ago2e and Ago2f, in contrast to the broadening of expression over time expected under the out-of-the-testis hypothesis (Kaessmann 2010; Assis and Bachtrog 2013). Second, all testis-specific Ago2 paralogs in D. pseudoobscura show evidence either of long-term positive selection (MK test for the high-diversity Ago2e) or of recent selective sweeps (in low-diversity Ago2a1/3 and Ago2b), and the testis-specific D. obscura Ago2e displays a reduction in diversity, potentially driven by selection.
Under a subfunctionalization model for Ago2 testis specialization, five candidate selective pressures seem likely: testis-specific dosage compensation, antiviral defense, gene regulation, TE suppression, and/or the suppression of meiotic drive. Of these, testis-specific dosage compensation seems the least likely to drive testis specificity because the MSL complex, which Ago2 directs to X-linked genes to carry out dosage compensation in the soma of D. melanogaster, is absent from testis (Conrad and Akhtar 2012). Testis-specific antiviral defense seems similarly unlikely, as the only known paternally transmitted Drosophila viruses (Sigmaviruses; Rhabdoviridae) pass through both the male and female gametes (Longdon and Jiggins 2012), and so the potential benefits of testis specificity seem unclear. Alternatively, testis-specific Ago2 duplicates could be coevolving with other testis-specific genes through the hairpin RNA pathway, in which siRNAs generated from endogenous hairpin-forming RNAs (hpRNAs) bind Ago2 and regulate the expression of host genes (Okamura et al. 2008). In D. melanogaster, hpRNA-derived siRNAs target testis-specific genes involved in male fertility and coevolve with these targets to maintain base complementarity (Wen et al. 2015). If a similar pathway operates in the obscura group, Ago2 paralogs could have specialized to the hpRNA pathway in order to regulate testis-specific genes more effectively.
Finally, the suppression of TEs or meiotic drive seem promising candidate selective forces. First, numerous TEs transpose preferentially in the testis, such as Penelope in D. virilis (Rozhkov et al. 2010) and copia in D. melanogaster (Pasyukova et al. 1997; Morozova et al. 2009), which could impose a selection pressure on Ago2 paralogs to provide a testis-specific TE suppression mechanism. It should be noted that all members of the canonical anti-TE Piwi subfamily (Ago3, Aub, and Piwi) are also expressed in obscura group testis (Figure S3), suggesting that if Ago2 paralogs have specialized to suppress TEs, they are doing so alongside the existing TE suppression mechanism. Second, testis specificity could have evolved to suppress meiotic drive, which is prevalent (in the form of sex-ratio distortion) in the obscura group (Gershenson 1928; Sturtevant and Dobzhansky 1936; Wu and Beckenbach 1983; Jaenike 2001; Unckless et al. 2015), and which is suppressed by RNAi-based mechanisms in other species (Tao et al. 2007; Kotelnikov et al. 2009; Gell and Reenan 2013). A high level of meiotic drive in the obscura group could therefore impose selection for the evolution of novel suppression mechanisms, leading to the repeated specialization of Ago2 paralogs to the testis.
Prospects for novel functions during the evolution of RNAi
The functional specialization that we observe for obscura group Ago2 paralogs raises the prospect of undiscovered derived functions following Argonaute expansions in other lineages. Ago2 has duplicated frequently across the arthropods, with expansions present in insects (D. willistoni (Figure 1) and Musca domestica, Scott et al. 2014), crustaceans (Penaeus monodon, Leebonoi et al. 2015), and chelicerates (Tetranychus urticae, Ixodes scapularis, Mesobuthus martensii, and Parasteatoda tepidariorum, Palmer and Jiggins 2015). The prevalence of testis specificity in obscura group Ago2 paralogs raises the possibility that specialization to the germline may be more widespread following Argonaute duplication. The expression of Ago2 paralogs has previously been characterized in P. monodon and shows that one paralog has indeed specialized to the germline of both males and females, but not the testis alone (Leebonoi et al. 2015). Publicly available RNA-seq data from the head, gonad, and carcass of male and female M. domestica (GSE67065, Meisel et al. 2015) suggest that neither M. domestica Ago2 paralog has specialized to the testis (Figure S8). However, public data from the head, thorax, and abdomen of male and female D. willistoni (GSE31723, Meisel et al. 2012) show that one D. willistoni Ago2 paralog (FBgn0212615) is expressed ubiquitously, while the other (FBgn0226485) is expressed only in the male abdomen (Figure S8), consistent with the evolution of testis specificity after duplication. This raises the possibility that a testis-specific selection pressure may be driving the retention and specialization of Ago2 paralogs across the arthropods.
In conclusion, we have identified rapid and repeated evolution of testis specificity after the duplication of Ago2 in the obscura group, associated with low genetic diversity and signatures of strong selection. Ago2 and other RNAi genes have undergone frequent expansions in different eukaryotic lineages (Mukherjee et al. 2013; Lewis et al. 2016) and have been shown to switch between ubiquitous and germline- or ovary-specific functions in isolated species. This study provides evidence for the evolution of a new testis-specific RNAi function and suggests that positive selection may act on young paralogs to drive the rapid evolution of novel RNAi mechanisms across the eukaryotes.
We thank Ben Longdon and Brian Charlesworth for providing us with strains of D. obscura and D. pseudoobscura, respectively, and Francis Jiggins for providing us with DCV. This work was supported by a Natural Environment Research Council doctoral training grant (NERC DG NE/J500021/1 to S.H.L.), the Academy of Finland (265971 to H.S.), a University of Edinburgh chancellor's fellowship, a Wellcome Trust research career development fellowship (WT085064 to D.J.O.), and a Wellcome Trust strategic award to the Centre for Immunity, Infection and Evolution (WT095831).
Communicating editor: D. A. Barbash
Supplemental material is available online at www.genetics.org/lookup/suppl/doi:10.1534/genetics.116.192336/-/DC1.
Received June 6, 2016.
Accepted August 12, 2016.
Copyright © 2016 by the Genetics Society of America
Available freely online through the author-supported open access option.
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Team Page | Past Results | Remaining Schedule | Player Stats | Player Roster
Remaining Schedule
Date Time vs Last Matchup Result
1/19/20 2:00 PM ET Binghamton 3/2/19 L 60-83
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redpajama_set_name: RedPajamaCommonCrawl


Braun, Carl, German bass; b. Meisenheim, Prussia, June 2, 1885; d. Hamburg, April 19, 1960. He studied with Hermann Gausche in Kreuznach and later with Eugen Robert Weiss. He sang at the Wiesbaden Opera (1906–11) and at the Vienna Court Opera (1911–12); then was engaged at the Berlin City Opera (1912–14). He also appeared at the Bayreuth Festivals (1906–31). On Feb. 8, 1913, he made his American debut at the Metropolitan Opera in N.Y. as King Marke, but was dismissed as an enemy alien in the spring of 1917 when the U.S. entered the war against Germany. In 1922–23 he made a South American tour; also sang in the U.S. in 1928 and 1931. In 1933 he was engaged as a stage director at the German Opera in Berlin, and in 1935–36 held similar posts at the Berlin Volksoper and at the Danzig Municipal Theater. In 1937 he retired from the stage and was thereafter active mainly as a concert agent in Hamburg. He was particularly esteemed for his Wagnerian roles.
"Braun, Carl." Baker's Biographical Dictionary of Musicians. . Encyclopedia.com. 22 Apr. 2019 <https://www.encyclopedia.com>.


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Universal Thee At Beresford Lounge 30th May 2014 – Pat McGuire
Posted on July 28, 2014 by my voice
I'm sitting in the Variety Bar with my friend Tom Perrie (Brother of Andrew Perrie, bassist with Universal Thee) waiting for another friend "Half-day McVey" to arrive. We are ruminating if the goldfish are real. There is a faux fireplace on a screen in the corner so it's entirely plausible that the fish in the tank above the stairs are also just clever fakes, perhaps a few LCD screens in a box configuration? They could even be in a real tank but just fake robotic fish? Anything is possible these days. Maybe if I took one out and put it in my pint we would find out the truth? Luckily the band come in just as I'm about to test my theory. I'll find out next time but for now the goldfish are safe.
Universal Thee have just finished sound check for the gig tonight and require some liquid refreshment. So we huddle up under the fake fireplace and get some drinks in. As a band they have the right dynamic even sans instruments. James Russell (singer/guitarist) is a slight chap with an air of inquisitiveness, always looking around as if he is soaking in information all the time. His wife and fellow singer Lisa Russell is charming and chatty and very down to earth. Robin Spivey (guitarist) is quiet and seems shy (but when he has his guitar strapped on it's a different story, more about this later). Andrew Perrie (Bassist) is affable and intelligent and only has an iPhone for "work reasons". He also plays bass with his eyes closed a lot (this becomes apparent as I work through the photographs I took on the night). Drummer Kevin Haddow is tall and slightly scary looking-as all good drummers should be. It has been said that the best bands are like a gang, if so then Universal Thee are a gang that could beat you in a pub quiz, and perhaps also in a fight in the car park afterwards if you cheated.
Back in March I reviewed their Album-Back to Earth and missed seeing them live. So I'm really looking forward to getting some shots and seeing them tonight. As an added bonus I get to spend some time with them and their Manager James Scott pre gig too. As we leave The Variety Bar the band are wondering if they can take some food into the venue. I suggest getting a "pizza pass" to go with their band passes, I'm not sure if it worked.
We cross the road and sit outside The Beresford. Sipping drinks in the sunshine that pours down Sauchiehall street and chat about comedy TV shows. Synthesizer Patel and Wilco being a key topic at one point. Their Manager James and I have a few surprising things in common too, involving criminal gangs in Wishaw, and schools in Viewpark and him having a hairy time in Bellshill once. It's a small world. I hasten to add none of us were in criminal gangs in Wishaw…
This is not the bands first gig in Glasgow, they have played here a few times before. But it's my first time seeing them live so I'm champing at the bit for the venue to open so I can experience some Universal Thee up close, and personal. I've never been in the Beresford Lounge in its present incarnation before. But it would have been nicer for the acts on the bill if there was a sign outside advising that a show was on, and perhaps one directing people downstairs to the gig too. This is no fault of the promoter(Laura Scott of the Scottish Tour Collective). The fact that a few bands have gigs/album launches on at nearby venues doesn't help either, but that is just bad luck. (Holy Ghosts were doing an album launch in Nice N Sleazies. Eastcoastdefector were playing at Broadcast too.)
Before Universal Thee come on however we have a nice acoustic set from Madeline Orr, followed by Scott Cowie doing comedy and guitar. Both enjoyable sets that were only really hampered by there being a small crowd. And a key thing here too, the bar downstairs was closed, causing people to drift upstairs if they wanted a drink.
All of that aside though I was there for Universal Thee live. So I stocked up on booze and nicotine prior to their set thus enabling me to take my pictures and enjoy the show. And what a show it was. On the LP the band might sound slightly reserved, live however they rip it up, rock out and get tore in. Their songs have a certain oomph factor. Structured noise and melody whilst still being faithful to the recorded versions on the LP.
They open with Bear in the Hospital which was my favourite track on the album when I reviewed it back in March. To quote myself from that article – "A wonderful yet minimalistic song that covers all the bases for good indie rock." Played live it is heavier and of course louder. That is why we go and see bands in the first place is it not? To hear a song organically and happening in front of us. Out front the sound is good and the band could and should be playing to a packed house. As it is though some of their friends from fellow Edinburgh band Ded Rabbit who were playing the Attic at The Garage next door are there. Wesley and Fraser from Kilmarnock's own One Last Secret too. Along with a few punters who hung around from earlier. And of course Tom, "Half-day McVey" and myself.
The band crack open the bottle of fuzzy pop that is Bone Collector next. This is one of their songs that openly shows their Pixies influence. I'm almost back in my early 20's when I saw The Pixies in London, if I closed my eyes I could be there. Robin Spivey's guitar licks and the song's dynamic really make me think is Joey Santiago here tonight? Tiger Tiger has an short but effective intro before the song springs to life. Wonderful stuff. James and Lisa's vocals intertwine and move around the song like a spider walking up your spine. Bendy guitars take us to the end, but I still want it to go on, and on and on. Universal Thee then do two songs that I hadn't heard before and which are not on the LP. Why do you have to be so unkind and Xang. Causing me to malfunction in operating my camera so I retire to a table to have a sip or two of the drinks that Tom and "Half-day McVey" have bought me. Crisis resolved I go back to shooting pictures. Andrew Perrie is still playing his bass with his eyes closed. Kevin Haddow is hammering the drums(despite having to use a spare hi hat stand as a cymbal stand) and Make a little Money begins with more bendy guitar and segues into lovely vocals and quirky walking bass lines.
As the set draws to the last third. The band take it to Down which while the song's lyrical content might be about something unpleasant, strangely lifts my spirits with it's lovely noisy passages and heavy drums. Robin Spivey is not a shy guitarist, he is evoking guitar gods as he pulls something ethereal from his Telecaster. I'm hearing echoes of Sonic Youth as my ears get blasted from the side monitors and the amps. Wolves of the Netherworld then rips us all a new one! I'm genuinely scared at one point as the lyrics seem to be about what their manager James and I were talking about before the gig (criminal gangs…) but I'm sure it is not.
Always end on a high note and leave them asking for more. So the last song does this indeed. Aranis Natas is rocking us out on a high. I don't know if the sound guy set his faders to malky, or if it's just my ears, but it sounds loud even in the quiet parts. Menacing and almost industrial. Indie rock has never sounded so good, really!
I'm sad that the gig had to end, but it left me wanting more. I'm glad I finally got to see them live, meet them in person and do some pictures and review. It vindicates me, makes me feel whole. It might have changed my life too. And to quote "Half-day McVey" (not his real name, he is a musician and producer in his own right, and hopefully I will be doing a feature on his projects soon.)-"Reminds me of Uresei Yatsura crossed with Pavement. In a good way!"
After the gig I wandered over to Sleazies with "Half-day McVey" and the band went back to Edinburgh. It was a cracking night all in all. When Universal Thee come back to Glasgow I'll be there to see them and I urge you to give them a listen too. They are one band who are definitely going places. I foresee bigger venues and maybe even some festivals on the horizon. Catch them while you can, up close and personal.
Pat McGuire.
Universal Thee gigs coming up :
27th of June in the Green Room in Perth.
5th July at River in Glasgow.
11th July Sneaky Pete's in Edinburgh.
18th July Audio Soup Festival in Dunbar.
26th July The Corrina in Perth.
https://www.facebook.com/universalthee
http://www.abadgeoffriendship.com/artists/universal-thee
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Leveling the backyard, part 1
As we have excavated the central back yard, we piled up the dirt on part of the Bottoms that has already been excavated.
But as we got more and more of the central back yard dug up, and particularly after lowering the main irrigation water line, it seemed we could move the dirt back to where it will eventually be. So we took some time to dig all that dirt up out of the Bottoms
and move it to the ditches that were the result of our digging, levelling off the back yard, more or less.
This "level" is still several inches below where the lawn level will eventually be, still leaves some substantial ditches, adjacent to where we will be continuing to dig.
But it gets things to the point that we will begin to cover up things we may eventually need to find, specifically with the underground irrigation system.
The control value for Zone 5, which controls the sprinklers for the Iris bed and the Herb Garden beds, is just off the sidewalk. It's about 7 inches from the sidewalk and 11 inches down from the level of the sidewalk.
The main irrigation water line runs way down at bedrock, until it gets to the River of Rocks, where the River of Rocks terminates at the back sidewalk. Then it comes up to a more "normal" depth and continues around the house. It rises up to 8 inches below the level of the River of Rocks, and the sidewalk, and is 13 inches away from the sidewalk. (It basically runs parallel to the sidewalk, 12 to 13 inches away from the sidewalk, for the entire length of the house.)
The valves for Zones 6 and 7, which will be used to water the central backyard and the Bottoms (respectively), and buried in the Bottoms, just off of the Rock Retaining wall. One of the rocks in the Retaining wall is more or less rectangular and sticks up an inch or two above the top of the rest of the wall. From the top of that rock, it's 21 inches down to the valve covers and 14 inches away from the Retaining wall itself.


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Little Miss Sunshine and American Optimism
Is the incredibly wicked satire of last year's independent film hit Little Miss Sunshine accessible to non-Americans? I wonder.
Do other cultures breed a hyper-competitive drive for "number one" rankings in everything from piloting USAF jets to talent contests to wealth and recognition on the motivational circuit to academic studies of Proust? Do other cultures quickly label everyone who competes but doesn't win "a loser"? Do other cultures heap such lopsided praise on "winners" and offer so little to the "rest of us" that suicide (at least in this film) seems like a better alternative than slipping in the rankings? I honestly don't know, but I suspect not.
In the event other cultures may be mystified by the dark humor and rapier-like parody in this film, I offer up the following as context for understanding this justifiably popular movie.
I am not immune to competitive juices, so I say this not as a smarmy putdown of competition but to draw the distinction between the honor implicit in competing and the pathological emphasis now placed on "winning" "the top spot" in American society. I played on basketball teams for five years in junior and high school and a year of football in 10th grade (hard-working talentless benchwarmer, but hey, I had fun being part of the teams), and I was a contractor/carpenter in the deep recession of the early 80s.
You want a "Darwinian fight to the death?" That was the "interest rate is 16%, nobody's building anything" reality then. And if you want a competition where winning is literally one shot in a million--try writing. 30 million blogs (or is it 300 million?), 5,000 novels published each year, 100,000 books in print, blah blah blah. The point is: has our society veered into a pathology of winning, in which parents are punching Little League coaches and screaming at little kids for not scoring more than their opponents?
This film says yes, definitely, categorically, yes.
Critics generally describe this as a comedy about a dysfunctional family. It isn't that at all; it's a biting critique of a dysfunctional society.
The family is actually the only source of support and solace available to the beleagered individuals depicted in the film as they each strive--and painfully fail to reach--the pinnacle of the American Dream: success through hard work and meritocracy.
The banality of this goal is ruthlessly satirized as the movie progresses. Every conceivable form of American Success with a Capital S is savagely revealed as distorting and empty.
The wholesome Dad swears under his breath as his dream of a motivational-speaker-writer empire crumbles before him; his family is disgusted by his hollow cheerleader motivational patter--the soundtrack, if you will, of the American Dream.
The acerbic grandfather shoots heroin to control his various aches and pains, and rudely defines success as having sex with as many women as possible (hey, it worked for JFK, Bill Clinton and innumerable "sports heroes")--a crass, unwholesome portrayal of the "real American Dream"-- or at least the male one.
The harried, chain-smoking mother is frayed by the insuperable demands of holding the family together while she puts fast-food on the table and rescues her mentally unstable brother.
Her brother, an openly gay aspiring academic superstar, is suicidal because another academic has replaced him as the number one Proust scholar--and snagged his gay lover to boot.
The son, who has taken a vow of silence to express his open hatred of his family, single-mindedly works out in order to achieve his goal of being accepted to the Air Force Academy and becoming a pilot. His dreams are dashed by a weakness beyond his control, and he is inconsolable.
The slightly chubby daughter pursues her dream of winning a sickeningly saccharine talent contest in California, "Little Miss Sunshine." Despite its wholesome veneer, the entire contest is deeply subversive of childhood, a bizarre distortion in which girls are made up to look like small adult women and perform "Miss America" type song-and-dance routines.
The daughter's painfully bawdy routine--a searing exaggeration of the contest's subtext of pushing adulthood onto little girls--draws predictable howls of outrage from the contest authorities, and rallies the family to her side.
When Granddad unexpectedly passes away, the American bureaucracy takes a mirthful beating as the family is forced to sneak the body out of the hospital and load it in their decrepit VW van.
Though other bits of American life get similarly skewered, the key satire is of The American Dream: wealth and recognition achieved via hard work and "pursuing your dreams." As the wheels fall off the U.S. and global economies, I have to wonder how Americans will adapt to the narrowing of opportunities and the ever-tightening strictures of debt and job losses.
I wonder if they will begin to understand the myths they bought into and clung to so tenaciously as their plight deepened, ("the Ownership Society funded by leveraged debt", for instance) and realise that if they bothered to vote (recall that U.S. voter turnout is a miserable 40% compared to 80% in France and other well-established democracies), they might regain the political power which they have so passively ceded in their individual obsession with competitive triumph and public recognition.


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Q: Casting Syntax to Loop Through Array of Arrays in Swift I have an NSArray consisting of NSArrays of strings created in Objective-C.
I now want to loop through the items in the array in a swift class and am having trouble with the syntax.
The original Objective-C Array of arrays looks like the following:
NSArray* shapes =@[@[@"square",@"square.png"],@[@"circle",@"circle.png"],@[@"square",@"square.png"]];

I am able to get and print the Array from the Objective-C class using:
let shapes:Array = Utilities.sharedInstance().getShapes

The following to loop through the array, however, is not compiling:
var term : String = ""
var pic : String = ""
for shape in shapes  {
term  = shape[1] //ERROR HERE
pic = shape[2] //SAME ERROR HERE
            }

It gives the error:  Type 'Any' has no subscript members
What is the proper syntax to loop through the elements?

A: You can try
 let shapes = Utilities.sharedInstance().getShapes as! [[String]]

Your Array elements are of type Any so you can't use [] with them until you cast , it's always the case when you use bridged code from objective-c , so you have to be specific about the actual type you use , also i encourage
struct Item {
   let term,pic:String
}

Then
let res:[Item] = shapes.map { Item(term:$0[0],pic:$0[1]) }

an irrelevant note but important you can do
NSArray* shapes = @[@"square",@"circle",@"square"];

then the matter of appending .png is simple instead of having an [[String]] directly it's [String]

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text:
LEsporte Clube XV de Novembro, noto anche semplicemente come XV de Piracicaba, è una società calcistica brasiliana con sede nella città di Piracicaba, nello stato di San Paolo.

Storia
Il 15 novembre 1913 venne fondato il club, che dalla data prese il proprio nome. Per distinguerlo da altre compagini con la medesima denominazione, viene solitamente chiamato "XV de Piracicaba". Debuttò nella prima divisione del Campionato Paulista nel 1949, in seguito alla vittoria nella Série A2 del 1948. Nel club militava Nílton de Sordi, futuro membro della Nazionale di calcio del Brasile. Vi giocò agli albori della sua carriera (1954-1956) anche José Altafini, futuro protagonista del calcio italiano, nativo proprio di Piracicaba. Il XV de Piracicaba continuò a militare nella massima serie statale sino al 1965, anno in cui fu retrocesso: tornò poi nel 1967, dopo aver vinto la seconda divisione. Nel 1976 il club raggiunse il secondo posto del torneo, venendo preceduto solo dal Palmeiras; l'anno seguente fece il suo esordio nel Brasileirão, giocando in casa contro il Centro Sportivo Alagoano. La compagine bianco-nera disputò la III Copa Brasil, terminando all'ottavo posto in classifica finale. Nel successivo torneo giocò sedici incontri, raccogliendo tre vittorie, cinque pareggi e otto sconfitte, mentre il 1979 vide l'ultima partecipazione del XV de Piracicaba: l'ultimo incontro in massima serie fu quello del 9 dicembre contro il Coritiba allo stadio Major Antônio Couto Pereira. Nel 1995, invece, ci fu l'ultima gara del XV de Piracicaba nella Serie A1 del campionato statale: retrocesso al termine della stagione, la formazione dopo qualche tempo scese ulteriormente di categoria, finendo in terza serie. Ciononostante, continuava a partecipare a competizioni a livello nazionale, e stabilì il record di vittorie consecutive ottenute nelle prime partite del Campeonato Brasileiro Série B: nel 1998, con sette partite vinte nelle prime sette gare.

Palmarès

Competizioni nazionali
 Campeonato Brasileiro Série C: 1
1995

Competizioni regionali
 Copa Brasil Central: 1
1969

Competizioni statali

1931, 1947, 1948, 1967, 1983, 2011

2016, 2022

Altri piazzamenti

Secondo posto: 1976

Note

Collegamenti esterni

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Chaskis Theatre will take over Upstairs at The Arts Theatre for a 3 week festival to present the world premieres of: Human Emotional Process by José Rivera, Desert Rats by Nate Rufus Edelman and the European premieres of: Mothergun and Slow Falling Bird by Christine Evans.
Desert Rats: Estranged brothers Frank and Jesse reunite to plan a kidnapping in a squalid motel room on a hellish day in Barstow. When day turns into night and their victim is brought out of the trunk, the siblings find their troubles have just begun.
Mothergun: A group of three war survivors with no common language struggle to survive in the mud of a refugee camp "somewhere between Europe and Hell".
Slow Falling Bird: Set in an Immigration Detention Centre in the dead centre of the Australian desert, Slow Falling Bird moves from gritty realism to hallucinatory sequences as warping social and mental conditions on both sides of the wire lead to an explosive show-down.
Human Emotional Process by José Rivera; Desert Rats by Nate Rufus Edelman; Mothergun and Slow Falling Bird by Christine Evans.
Aaron Anthony, Emma Drinkwater, Julian Protheroe, Josie Dunn, Huw Parmenter, Rowan Polonski, Tuyen Do, Tom Higgins, Andrew Jardine, Bradley Taylor, Rosie Akerman, Aaron Anthony, Josie Dunn, Rob Falconer, Hannah Harvey, Tim McFarland, Lorin Merhart, Huw Parmenter and Zoe Thorne.
Las Americas Above closed on 15 August 2016.


Muthaiga Golf Club's Greg Snow held his nerve on the final day of the second leg of the Safari Tour Golf Series, that was held at the Vet Lab Sports Club, to fend off a mounting challenge from a resurgent Dimas Indiza to claim the tournament's top prize. Snow finished 1-over par 73 for the round to take his total for the tournament to 6-under par 282; while Indiza shot an impressive 4-under par 68 for the final round to return a similar tally, and to tie for the lead.
A three-hole playoff, on the Par-4, first, the uphill dogleg left Par-4, 17th and on the Par-4, 18th ensued to decide the winner of the tournament. Both made par on the first and 17th holes in the playoff, but a bogey on the 18th for Indiza and a par for Snow secured the latter's lead.
Sigona Golf Club's Sujan Shah returned level par for the round to take his overall tally for the tournament to 3-Under Par 285 and finish third overall. For their efforts, Greg Snow took home KES 150,000; Indiza KES 100,000 and Sujan claimed KES 80,000.
Indiza's impressive outing on the second leg of the Safari Tour takes him top of the rankings after two rounds with 38 points overall. Greg Snow comes in second with 35.5 points while Nyali Golf Club's champion., David Wakhu (Golf Park) comes in third with 34 points. Sigona's Sujan Shah who made this Safari Tour debut at Vet Lab is ranked eighth with a commendable 18 points.
Action now moves to the Limuru Country Club for the third leg of the Safari Tour Golf Series from the 27th to the 31st of October this year.


text:
SN 2010ko – supernowa typu Ia odkryta 5 grudnia 2010 roku w galaktyce NGC 1954. W momencie odkrycia miała maksymalną jasność 16,80.

Przypisy

Supernowe zaobserwowane w 2010 roku