The Conserved miR-51 microRNA Family Is Redundantly Required for Embryonic Development and Pharynx Attachment in Caenorhabditis elegans

A major question about cytokinesis concerns the role of the septin proteins, which localize to the division site in all animal and fungal cells but are essential for cytokinesis only in some cell types. For example, in Schizosaccharomyces pombe, four septins localize to the division site, but deletion of the four genes produces only a modest delay in cell separation. To ask if the S. pombe septins function redundantly in cytokinesis, we conducted a synthetic-lethal screen in a septin-deficient strain and identified seven mutations. One mutation affects Cdc4, a myosin light chain that is an essential component of the cytokinetic actomyosin ring. Five others cause frequent cell lysis during cell separation and map to two loci. These mutations and their dosage suppressors define a signaling pathway (including Rho1 and a novel arrestin) for repairing cell-wall damage. The seventh mutation affects the poorly understood RNA-binding protein Scw1 and severely delays cell separation when combined either with a septin mutation or with a mutation affecting the septin-interacting, anillin-like protein Mid2, suggesting that Scw1 functions in a pathway parallel to that of the septins. Taken together, our results suggest that the S. pombe septins participate redundantly in one or more pathways that cooperate with the actomyosin ring during cytokinesis and that a septin defect causes septum defects that can be repaired effectively only when the cell-integrity pathway is intact.THE fission yeast Schizosaccharomyces pombe provides an outstanding model system for studies of cytokinesis (McCollum and Gould 2001; Balasubramanian et al. 2004; Pollard and Wu 2010). As in most animal cells, successful cytokinesis in S. pombe requires an actomyosin ring (AMR). The AMR begins to assemble at the G₂/M transition and involves the type II myosin heavy chains Myo2 and Myp2 and the light chains Cdc4 and Rlc1 (Wu et al. 2003). Myo2 and Cdc4 are essential for cytokinesis under all known conditions, Rlc1 is important at all temperatures but essential only at low temperatures, and Myp2 is essential only under stress conditions. As the AMR constricts, a septum of cell wall is formed between the daughter cells. The primary septum is sandwiched by secondary septa and subsequently digested to allow cell separation (Humbel et al. 2001; Sipiczki 2007). Because of the internal turgor pressure of the cells, the proper assembly and structural integrity of the septal layers are essential for cell survival.Septum formation involves the β-glucan synthases Bgs1/Cps1/Drc1, Bgs3, and Bgs4 (Ishiguro et al. 1997; Le Goff et al. 1999; Liu et al. 1999, 2002; Martín et al. 2003; Cortés et al. 2005) and the α-glucan synthase Ags1/Mok1 (Hochstenbach et al. 1998; Katayama et al. 1999). These synthases are regulated by the Rho GTPases Rho1 and Rho2 and the protein kinase C isoforms Pck1 and Pck2 (Arellano et al. 1996, 1997, 1999; Nakano et al. 1997; Hirata et al. 1998; Calonge et al. 2000; Sayers et al. 2000; Ma et al. 2006; Barba et al. 2008; García et al. 2009b). The Rho GTPases themselves appear to be regulated by both GTPase-activating proteins (GAPs) and guanine-nucleotide-exchange factors (GEFs) (Nakano et al. 2001; Calonge et al. 2003; Iwaki et al. 2003; Tajadura et al. 2004; Morrell-Falvey et al. 2005; Mutoh et al. 2005; García et al. 2006, 2009a,b). In addition, septum formation and AMR function appear to be interdependent. In the absence of a normal AMR, cells form aberrant septa and/or deposit septal materials at random locations, whereas a mutant defective in septum formation (bgs1) is also defective in AMR constriction (Gould and Simanis 1997; Le Goff et al. 1999; Liu et al. 1999, 2000). Both AMR constriction and septum formation also depend on the septation initiation network involving the small GTPase Spg1 (McCollum and Gould 2001; Krapp and Simanis 2008). Despite this considerable progress, many questions remain about the mechanisms and regulation of septum formation and its relationships to the function of the AMR.One major question concerns the role(s) of the septins. Proteins of this family are ubiquitous in fungal and animal cells and typically localize to the cell cortex, where they appear to serve as scaffolds and diffusion barriers for other proteins that participate in a wide variety of cellular processes (Longtine et al. 1996; Gladfelter et al. 2001; Hall et al. 2008; Caudron and Barral 2009). Despite the recent progress in elucidating the mechanisms of septin assembly (John et al. 2007; Sirajuddin et al. 2007; Bertin et al. 2008; McMurray and Thorner 2008), the details of septin function remain obscure. However, one prominent role of the septins and associated proteins is in cytokinesis. Septins concentrate at the division site in every cell type that has been examined, and in Saccharomyces cerevisiae (Hartwell 1971; Longtine et al. 1996; Lippincott et al. 2001; Dobbelaere and Barral 2004) and at least some Drosophila (Neufeld and Rubin 1994; Adam et al. 2000) and mammalian (Kinoshita et al. 1997; Surka et al. 2002) cell types, the septins are essential for cytokinesis. In S. cerevisiae, the septins are required for formation of the AMR (Bi et al. 1998; Lippincott and Li 1998). However, this cannot be their only role, because the AMR itself is not essential for cytokinesis in this organism (Bi et al. 1998; Korinek et al. 2000; Schmidt et al. 2002). Moreover, there is no evidence that the septins are necessary for AMR formation or function in any other organism. A further complication is that in some cell types, including most Caenorhabditis elegans cells (Nguyen et al. 2000; Maddox et al. 2007) and some Drosophila cells (Adam et al. 2000; Field et al. 2008), the septins do not appear to be essential for cytokinesis even though they localize to the division site.S. pombe has seven septins, four of which (Spn1, Spn2, Spn3, and Spn4) are expressed in vegetative cells and localize to the division site shortly before AMR constriction and septum formation (Longtine et al. 1996; Berlin et al. 2003; Tasto et al. 2003; Wu et al. 2003; An et al. 2004; Petit et al. 2005; Pan et al. 2007; Onishi et al. 2010). Spn1 and Spn4 appear to be the core members of the septin complex (An et al. 2004; McMurray and Thorner 2008), and mutants lacking either of these proteins do not assemble the others at the division site. Assembly of a normal septin ring also depends on the anillin-like protein Mid2, which colocalizes with the septins (Berlin et al. 2003; Tasto et al. 2003). Surprisingly, mutants lacking the septins are viable and form seemingly complete septa with approximately normal timing. These mutants do, however, display a variable delay in separation of the daughter cells, suggesting that the septins play some role(s) in the proper completion of the septum or in subsequent processes necessary for cell separation (Longtine et al. 1996; An et al. 2004; Martín-Cuadrado et al. 2005).It is possible that the septins localize to the division site and yet are nonessential for division in some cell types because their role is redundant with that of some other protein(s) or pathway(s). To explore this possibility in S. pombe, we screened for mutations that were lethal in combination with a lack of septins. The results suggest that the septins cooperate with the AMR during cytokinesis and that, in the absence of septin function, the septum is not formed properly, so that an intact system for recognizing and repairing cell-wall damage becomes critical for cell survival.     

Inferring Bacterial Genome Flux While Considering Truncated Genes

Bacterial gene content variation during the course of evolution has been widely acknowledged and its pattern has been actively modeled in recent years. Gene truncation or gene pseudogenization also plays an important role in shaping bacterial genome content. Truncated genes could also arise from small-scale lateral gene transfer events. Unfortunately, the information of truncated genes has not been considered in any existing mathematical models on gene content variation. In this study, we developed a model to incorporate truncated genes. Maximum-likelihood estimates (MLEs) of the new model reveal fast rates of gene insertions/deletions on recent branches, suggesting a fast turnover of many recently transferred genes. The estimates also suggest that many truncated genes are in the process of being eliminated from the genome. Furthermore, we demonstrate that the ignorance of truncated genes in the estimation does not lead to a systematic bias but rather has a more complicated effect. Analysis using the new model not only provides more accurate estimates on gene gains/losses (or insertions/deletions), but also reduces any concern of a systematic bias from applying simplified models to bacterial genome evolution. Although not a primary purpose, the model incorporating truncated genes could be potentially used for phylogeny reconstruction using gene family content.GENE content variation as a key feature of bacterial genome evolution has been well recognized (Garcia-Vallvé et al. 2000; Ochman and Jones 2000; Snel et al. 2002; Welch et al. 2002; Kunin and Ouzounis 2003; Fraser-Liggett 2005; Tettelin et al. 2005) and gained increasing attention in recent years. Various methods have been employed to study the variation of gene content in the form of gene insertions/deletions (or gene gains/losses); there are studies of population dynamics (Nielsen and Townsend 2004), birth-and-death evolutionary models (Berg and Kurland 2002; Novozhilov et al. 2005), phylogeny-dependent studies including parsimony methods (Mirkin et al. 2003; Daubin et al. 2003a,b; Hao and Golding 2004), and maximum-likelihood methods (Hao and Golding 2006, 2008b; Cohen et al. 2008; Cohen and Pupko 2010; Spencer and Sangaralingam 2009). The pattern of gene presence/absence also contains phylogenetic signals (Fitz-Gibbon and House 1999; Snel et al. 1999; Tekaia et al. 1999) and has been used for phylogenetic reconstruction (Dutilh et al. 2004; Gu and Zhang 2004; Huson and Steel 2004; Zhang and Gu 2004; Spencer et al. 2007a,b). All these studies make use of the binary information of gene presence or absence and neglect the existence of gene segments or truncated genes.Bacterial genomes are known to harbor pseudogenes. An intracellular species Mycobacterium leprae is an extreme case for both the proportion and the number of pseudogenes: estimated as 40% of the 3.2-Mb genome and 1116 genes (Cole et al. 2001). In free-living bacteria, pseudogenes can make up to 8% of the annotated genes in the genome (Lerat and Ochman 2004). Many pseudogenes result from the degradation of native functional genes (Cole et al. 2001; Mira et al. 2001). Pseudogenes could also result from the degradation of transferred genes and might even be acquired directly via lateral gene transfer. For instance, in plant mitochondrial genomes, which have an α-proteobacterial ancestry, most, if not all, of the laterally transferred genes are pseudogenes (Richardson and Palmer 2007). Furthermore, evidence has been documented that gene transfer could take place at the subgenic level in a wide range of organisms, e.g., among bacteria (Miller et al. 2005; Choi and Kim 2007; Chan et al. 2009), between ancient duplicates in archaea (Archibald and Roger 2002), between different organelles (Hao and Palmer 2009; Hao 2010), and between eukaryotes (Keeling and Palmer 2001). A large fraction of pseudogenes have been shown to arise from failed lateral transfer events (Liu et al. 2004) and most of them are transient in bacterial genomes (Lerat and Ochman 2005). Zhaxybayeva et al. (2007) reported that genomes with truncated homologs might erroneously lead to false inferences of “gene gain” rather than multiple instances of “gene loss.” This raises the question of how a false diagnosis of gene absence affects the estimation of insertion/deletion rates. Recently, we showed that the effect of a false diagnosis of gene absence on estimation of insertion/deletion rates is not systematic, but rather more complicated (Hao and Golding 2008a). To further address the problem, a study incorporating the information of truncated genes is highly desirable. This will not only yield more accurate estimates of the rates of gene insertions/deletions, but also provide a quantitative view of the effect of truncated genes on rate estimation, which has been understudied in bacterial genome evolution.In this study, we developed a model that considers the information of truncated genes and makes use of a parameter-rich time-reversible rate matrix. Rate variation among genes is allowed in the model by incorporating a discrete Γ-distribution. We also allow rates to vary on different parts of the phylogeny (external branches vs. internal branches). Consistent with previous studies, the rates of gene insertions/deletions are comparable to or larger than the rates of nucleotide substitution and the rates of gene insertions/deletions are further inflated in closely related groups and on external branches, suggesting high rates of gene turnover of recently transferred genes. The results from the new model also suggest that many recently truncated genes are in the process of being rapidly deleted from the genome. Some other interesting estimates in the model are also presented and discussed. One implication of the study, though not primary, is that the state of truncated genes could serve as an additional phylogenetic character for phylogenetic reconstruction using gene family content.     

Profiling Sex-Specific piRNAs in Zebrafish

Piwi proteins and their partner small RNAs play an essential role in fertility, germ-line stem cell development, and the basic control and evolution of animal genomes. However, little knowledge exists regarding piRNA biogenesis. Utilizing microfluidic chip analysis, we present a quantitative profile of zebrafish piRNAs expressed differentially between testis and ovary. The sex-specific piRNAs are derived from separate loci of repeat elements in the genome. Ovarian piRNAs can be categorized into groups that reach up to 92 members, indicating a sex-specific arrangement of piRNA genes in the genome. Furthermore, precursor piRNAs preferentially form a hairpin structure at the 3′end, which seem to favor the generation of mature sex-specific piRNAs. In addition, the mature piRNAs from both the testis and the ovary are 2′-O-methylated at their 3′ ends.SMALL RNAs, ranging from 19 to 30 nucleotides (nt) in length, constitute a large family of regulatory molecules with diverse functions in invertebrates, vertebrates, plants, and fungi (Bartel 2004; Nakayashiki 2005). Two major classes of small RNAs are microRNAs (miRNAs) and small interfering RNAs (siRNAs). The functions of small RNAs have been conserved through evolution; they have been shown to inhibit gene expression at the levels of mRNA degradation, translational repression, chromatin modification, heterochromatin formation, and DNA elimination (Mochizuki et al. 2002; Bartel 2004; Kim et al. 2005; Brodersen and Voinnet 2006; Lee and Collins 2006; Vaucheret 2006).Over the past few years, focus on the genetics of small RNAs has helped clarify the mechanisms behind the regulation of these molecules. While hundreds of small RNAs have been identified from mammalian somatic tissues, relatively little is known about small RNAs in germ cells. A recent breakthrough has been the identification of small RNAs that associate with Piwi proteins (piRNAs) from Drosophila and mammalian gonads (Aravin et al. 2001, 2006; Girard et al. 2006; Grivna et al. 2006; Vagin et al. 2006; Watanabe et al. 2006). piRNAs and their interacting proteins Ziwi/Zili have also been identified in zebrafish (Houwing et al. 2007, 2008). Increasing evidence indicates that piRNAs play roles mainly in germ cell differentiation and genomic stability (Carthew 2006; Lau et al. 2006; Vagin et al. 2006; Brennecke et al. 2007; Chambeyron et al. 2008; Klattenhoff and Theurkauf 2008; Kuramochi-Miyagawa et al. 2008; Kim et al. 2009; Lim et al. 2009; Unhavaithaya et al. 2009). Moreover, although piRNAs are mostly expressed in germ line cells, recent studies showed piRNA expression in nongerm cells, for example, T-cell lines (Jurkat cells and MT4) (Azuma-Mukai et al. 2008; Yeung et al. 2009), indicating other functions such as in the immune system. piRNAs do not appear to be derived from double-stranded RNA precursors, and their biogenesis mechanisms, although unclear, may be distinct from those of siRNA and miRNA. Recently, two distinct piRNA production pathways were further proposed: the “ping-pong” model (Brennecke et al. 2007; Gunawardane et al. 2007) and the Ago3-independent piRNA pathway centered on Piwi in somatic cells (Li et al. 2009; Malone et al. 2009). However, the mechanistic pathways of piRNA activity and their biogenesis are still largely unknown.Teleost fishes comprise >24,000 species, accounting for more than half of extant vertebrate species, displaying remarkable variation in morphological and physiological adaptations (see review in Zhou et al. 2001). Recently, Houwing et al. (2007, 2008) reported findings on Ziwi/Zili and associated piRNAs, implicating roles in germ cell differentiation, meiosis, and transposon silencing in the germline of the zebrafish. However, some of the identified zebrafish piRNAs are nonrepetitive and nontransposon-related piRNAs, suggesting that piRNAs may have additional unknown roles. In this study, we show that for males and females, piRNAs are specifically derived from separate loci of the repeat elements, and that ovarian piRNAs are far more often associated in groups. Genomic analysis of piRNAs indicates a tendency to folding at the 3′ end of the piRNA precursor, which may favor cleavage of the piRNA precursor to generate mature sex-specific piRNAs. Furthermore, methylation modification occurs at the 2′-O-hydroxyl group on the ribose of the final 3′ nucleotide in both the testis and the ovary.     

Patterns and Processes of Genome-Wide Divergence Between North American and African Drosophila melanogaster

Genomic tools and analyses are now being widely used to understand genome-wide patterns and processes associated with speciation and adaptation. In this article, we apply a genomics approach to the model organism Drosophila melanogaster. This species originated in Africa and subsequently spread and adapted to temperate environments of Eurasia and the New World, leading some populations to evolve reproductive isolation, especially between cosmopolitan and Zimbabwean populations. We used tiling arrays to identify highly differentiated regions within and between North America (the United States and Caribbean) and Africa (Cameroon and Zimbabwe) across 63% of the D. melanogaster genome and then sequenced representative fragments to study their genetic divergence. Consistent with previous findings, our results showed that most differentiation was between populations living in Africa vs. outside of Africa (i.e., “out-of-Africa” divergence), with all other geographic differences being less substantial (e.g., between cosmopolitan and Zimbabwean races). The X chromosome was much more strongly differentiated than the autosomes between North American and African populations (i.e., greater X divergence). Overall differentiation was positively associated with recombination rates across chromosomes, with a sharp reduction in regions near centromeres. Fragments surrounding these high F_ST sites showed reduced haplotype diversity and increased frequency of rare and derived alleles in North American populations compared to African populations. Nevertheless, despite sharp deviation from neutrality in North American strains, a small set of bottleneck/expansion demographic models was consistent with patterns of variation at the majority of our high F_ST fragments. Although North American populations were more genetically variable compared to Europe, our simulation results were generally consistent with those previously based on European samples. These findings support the hypothesis that most differentiation between North America and Africa was likely driven by the sorting of African standing genetic variation into the New World via Europe. Finally, a few exceptional loci were identified, highlighting the need to use an appropriate demographic null model to identify possible cases of selective sweeps in species with complex demographic histories.THE study of genetic differentiation between populations and species has recently been empowered by the use of genomic techniques and analysis (e.g., Noor and Feder 2006; Stinchcombe and Hoekstra 2008). In the past decade, genetic studies of adaptation and speciation have taken advantage of emerging molecular techniques to scan the genomes of diverging populations for highly differentiated genetic regions (e.g., Wilding et al. 2001; Emelianov et al. 2003; Beaumont and Balding 2004; Campbell and Bernatchez 2004; Scotti-Saintagne et al. 2004; Achere et al. 2005; Turner et al. 2005; Vasemagi et al. 2005; Bonin et al. 2006, 2007; Murray and Hare 2006; Savolainen et al. 2006; Yatabe et al. 2007; Nosil et al. 2008, 2009; Turner et al. 2008a,b; Kulathinal et al. 2009). As a result, genome scans can identify candidate regions that may be associated with adaptive evolution between diverging populations and, more broadly, are able to describe genome-wide patterns and processes of population differentiation (Begun et al. 2007; Stinchcombe and Hoekstra 2008).Genome scans in well-studied genetic model species such as Drosophila melanogaster gain particular power because differentiated loci are mapped to a well-annotated genome. Moreover, the evolutionary history of D. melanogaster is rich with adaptive and demographic events with many parallels to human evolution. Most notable is the historical out-of-Africa migration and subsequent adaptation to temperate ecological environments of Europe, Asia, North America, and Australia. This has resulted in widespread genetic and phenotypic divergence between African and non-African populations (e.g., David and Capy 1988; Begun and Aquadro 1993; Capy et al. 1994; Colegrave et al. 2000; Rouault et al. 2001; Takahashi et al. 2001; Caracristi and Schlötterer 2003; Baudry et al. 2004; Pool and Aquadro 2006; Schmidt et al. 2008; Yukilevich and True 2008a,b). Further, certain populations in Africa and in the Caribbean vary in their degree of reproductive isolation from populations in more temperate regions (Wu et al. 1995; Hollocher et al. 1997; Yukilevich and True 2008a,b). In particular, the Zimbabwe and nearby populations of southern Africa are strongly sexually isolated from all other populations, designating them as a distinct behavioral race (Wu et al. 1995).D. melanogaster has received a great deal of attention from the population geneticists in studying patterns of sequence variation across African and non-African populations. Many snapshots have been taken of random microsatellite and SNP variants spread across X and autosomes, and these have generated several important conclusions. Polymorphism patterns in European populations are characterized by reduced levels of nucleotide and haplotype diversity, an excess of high frequency-derived polymorphisms, and elevated levels of linkage disequilibrium relative to African populations (e.g., Begun and Aquadro 1993; Andolfatto 2001; Glinka et al. 2003; Haddrill et al. 2005; Ometto et al. 2005; Thornton and Andolfatto 2006; Hutter et al. 2007; Singh et al. 2007). These results have been generally interpreted as compatible with population size reduction/bottlenecks followed by recent population expansions. On the other hand, African populations are generally assumed either to have been relatively constant in size over time or to have experienced population size expansions. They generally show higher levels of nucleotide and haplotype diversity, an excess of rare variants, and a deficit of high frequency-derived alleles (Glinka et al. 2003; Ometto et al. 2005; Pool and Aquadro 2006; Hutter et al. 2007; but see Haddrill et al. 2005 for evidence of bottlenecks in Africa).Previous work also shows that the ratio of X-linked to autosomal polymorphism deviates from neutral expectations in opposite directions in African and European populations with more variation on the X than expected in Africa and less variation on the X than expected in Europe (Andolfatto 2001; Kauer et al. 2002; Hutter et al. 2007; Singh et al. 2007). The deviation from neutrality in the ratio of X-autosome polymorphism may be explained by positive selection being more prevalent on the X in Europe and/or by a combination of bottlenecks and male-biased sex ratios in Europe and female-biased sex ratios in Africa (Charlesworth 2001; Hutter et al. 2007; Singh et al. 2007). The selective explanation stems from the argument that, under the hitchhiking selection model, X-linked loci are likely to be more affected by selective sweeps than autosomal loci (Maynard Smith and Haigh 1974; Charlesworth et al. 1987; Vicoso and Charlesworth 2006, 2009).The relative contribution of selective and demographic processes in shaping patterns of genomic variation and differentiation is highly debated (Wall et al. 2002; Glinka et al. 2003; Haddrill et al. 2005; Ometto et al. 2005; Schöfl and Schlötterer 2004; Thornton and Andolfatto 2006; Hutter et al. 2007; Singh et al. 2007; Shapiro et al. 2007; Stephan and Li 2007; Hahn 2008; Macpherson et al. 2008; Noor and Bennett 2009; Sella et al. 2009). This is especially the case in D. melanogaster because derived non-African populations have likely experienced a complex set of demographic events during their migration out of Africa (e.g., Thornton and Andolfatto 2006; Singh et al. 2007; Stephan and Li 2007), making population genetics signatures of demography and selection difficult to tease apart (e.g., Macpherson et al. 2008). Thus it is still unclear what role selection has played in shaping overall patterns of genomic variation and differentiation relative to demographic processes in this species.While there is a long tradition in studying arbitrarily or opportunistically chosen sequences in D. melanogaster, genomic scans that focus particularly on highly differentiated sites across the genome have received much less attention. Such sites are arguably the best candidates to resolve the debate on which processes have shaped genomic differentiation within species (e.g., Przeworski 2002). Recently, a genome-wide scan of cosmopolitan populations in the United States and in Australia was performed to investigate clinal genomic differentiation on the two continents (Turner et al. 2008a). Many single feature polymorphisms differentiating Northern and Southern Hemisphere populations were identified. Among the most differentiated loci in common between continents, 80% were differentiated in the same orientation relative to the Equator, implicating selection as the likely explanation (Turner et al. 2008a). Larger regions of genomic differentiation within and between African and non-African populations have also been discovered, some of them possibly being driven by divergent selection (e.g., Dopman and Hartl 2007; Emerson et al. 2008; Turner et al. 2008a, Aguade 2009). Despite this recent progress, we still know relatively little about large-scale patterns of genomic differentiation in this species, especially between African and non-African populations, and whether most of this differentiation is consistent with demographic processes alone or if it requires selective explanations.In this work, we explicitly focus on identifying differentiated sites across the genome between U.S., Caribbean, West African, and Zimbabwean populations. This allows us to address several fundamental questions related to genomic evolution in D. melanogaster, such as the following: (1) Do genome-wide patterns of differentiation reflect patterns of reproductive isolation? (2) Is genomic differentiation random across and within chromosomes or are some regions overrepresented? (3) What are the population genetics properties of differentiated sites and their surrounding sequences? (4) Can demographic historical processes alone explain most of the observed differentiation on a genome-wide level or is it necessary to involve selection in their explanation?In general, our findings revealed that most genomic differentiation within D. melanogaster shows an out-of-Africa genetic signature. These results are inconsistent with the notion that most genomic differentiation occurs between cosmopolitan and Zimbabwean reproductively isolated races. Further, we found that the X is more differentiated between North American and African populations and more strongly deviates from pure neutrality in North American populations relative to autosomes. Nevertheless, our article shows that much of this deviation from neutrality is broadly consistent with several demographic null models, with a few notable exceptions. Athough this does not exclude selection as a possible alternative mechanism for the observed patterns, it supports the idea that most differentiation in D. melanogaster was likely driven by the sorting of African standing genetic variation into the New World.