New genes are added to most genomes on a steady basis. A new gene can either begin as a copy of an existing gene from elsewhere in the genome, or is created entirely ‘from scratch’ from a DNA sequence that had not previously encoded for a protein. New genes that are not found in other related species are called orphan genes—and these genes can account for up to 30% of all the genes in the well-studied genomes. However, for reasons that are not fully understood, the total number of genes in most genomes remains fairly constant despite these regular additions. Now, Palmieri et al. have investigated this paradox by following the evolutionary fate of orphan genes in a small group of related species of fruit fly.

Palmieri et al. discovered that most orphan genes are very short-lived, even though they showed clear signals of carrying out important functions. Most orphan genes died out quickly due to mutations that made them unable to be expressed as functional proteins, and a small number were deleted entirely from the genome. Unexpectedly, new orphan genes were more likely to die out than those that had been around for a while.

Palmieri et al. also found that the expression levels of orphan genes determined their probability of dying with those genes that were expressed to the highest levels being most likely to persist longer. Furthermore, genes that were expressed more in males than in females were also less likely to die. The next challenge will be to identify the mechanisms that determine which orphan genes survive and which do not.

Orphans are genes with limited phylogenetic distribution and represent a considerable fraction (up to 30%) of the gene catalog in all sequenced genomes (Khalturin et al., 2009). Studies conducted in different eukaryotes showed that orphans emerge at high rates (Domazet-Loso et al., 2007; Wissler et al., 2013). While gene duplication and exaptation from transposable elements often result in orphan genes (Toll-Riera et al., 2009), they also originate frequently de novo from non-coding DNA (Cai et al., 2008; Heinen et al., 2009; Knowles and McLysaght, 2009; Wu et al., 2011; Yang and Huang, 2011; Xie et al., 2012; Neme and Tautz, 2013; Wu and Zhang, 2013), probably through intermediate proto-genes (Carvunis et al., 2012). Compared to evolutionary conserved genes, orphans are overall shorter (Lipman et al., 2002), fast evolving (Domazet-Loso and Tautz, 2003), have lower and more tissue-restricted expression (Lemos et al., 2005). Moreover, they often show testis-biased expression (Levine et al., 2006; Begun et al., 2007), probably due to frequent origination in testis (Kaessmann, 2010).

In Drosophila the rate of orphan emergence is particulary high (Domazet-Loso et al., 2007) and many orphans become quickly essential (Chen et al., 2010). Although the function of only a few orphan genes has been studied, it has been proposed that orphans might serve an important role in speciation and adaptation to different environments (Khalturin et al., 2008; Khalturin et al., 2009; Colbourne et al., 2011). The high rate of orphan origination would predict an increase in gene content over time. However, gene content in eukaryotes is remarkably stable compared to genome size, as highlighted by Tautz and Domazet-Loso (2011). To solve this paradox Tautz and Domazet-Loso proposed that orphans have only a short lifetime (‘rapid-turnover’ hypothesis) (Tautz and Domazet-Loso, 2011). Thus, although orphans are continuously created, most of them might be lost in a relatively short evolutionary time. Relaxed selective costraints in orphans (Cai and Petrov, 2010) might also contribute to the high rate of orphan loss.

Moreover, since orphans are typically identifed by the comparison of distantly related species, their evolutionary stability has been so far neglected. This contrasts the comprehensive analysis of evolutionary patterns of gains and losses of non-orphan genes (Hahn et al., 2007). In this study, several partially interrelated factors affect gene loss, including gene expression levels, number of protein–protein interactions, gene dispensability, and rate of sequence evolution (Krylov et al., 2003; Cai and Petrov, 2010).

This study focuses for the first time on the evolutionary stability of orphan genes. We investigate the factors contributing to orphan loss and find that orphan age, male-biased gene expression, and microsatellite content are correlated with orphan stability. Surprisingly, differences in evolutionary rates cannot explain orphan loss and we propose that orphan loss is driven by lineage-specific evolutionary constraints. Overall, orphan genes are lost at a significantly higher rate than non-orphan genes, supporting the ‘rapid-turnover’ hypothesis.

Orphans are commonly detected by BLASTing the genes of a given organism against a set of outgroup species (Domazet-Loso and Tautz, 2003; Toll-Riera et al., 2009). A BLASTP cutoff of 10⁻³–10⁻⁴ was found to be optimal to maximize sensitivity and specificity in Drosophila (Domazet-Loso and Tautz, 2003). To identify orphans we used a BLASTP cutoff of 10⁻⁴ combined with a TBLASTN cutoff of 10⁻⁴, to exclude genes with unannotated orthologs in other species. Following these criteria, we searched in Drosophila pseudoobscura for genes with no sequence conservation in 10 Drosophila species outside the Drosophila obscura group (Figure 6—figure supplement 1). In total, we identified 1152 orphans, corresponding to 7% of all the D. pseudoobscura genes. Our estimate is slightly lower than a previous one (Zhang et al., 2010), due to our different filtering procedure, but still consistent with a high rate of orphan gain in Drosophila (Domazet-Loso and Tautz, 2003; Domazet-Loso et al., 2007; Zhou et al., 2008; Wissler et al., 2013). Our data clearly indicate that orphan genes are subject to purifying selection, as they show several hallmarks of functional protein-coding sequences (Figures 1, 2). A comparison of orphan genes preserved between D. pseudoobscura and D. affinis resulted in a distribution of dN/dS significantly lower than 1 with a median of 0.44 (Figure 1—figure supplement 1, one-sided Wilcoxon signed-rank test, p<1.0 × 10⁻¹⁵), as expected for protein-coding sequences. Moreover, dN/dS for orphans is significantly lower (Mann–Whitney test, p=2.7 × 10⁻¹⁴) than dN/dS calculated on a random set of intergenic regions with the same length distribution of orphans (see ‘Materials and methods’, section ‘Evolutionary rates’) (Figure 1A). Consistent with this, we also found orphans to be more conserved than intergenic regions (Figure 1B, Figure 1—figure supplement 2). The codon usage bias of orphans is intermediate to that of old genes and intergenic regions (Figure 1C).

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To further test for purifying selection acting on orphans, we used a polymorphism dataset of 45 strains being re-sequenced for the third chromosome of D. pseudoobscura (‘Materials and methods’). We calculated the ratio of synonymous to non-synonymous polymorphism (pN/pS), since it provides an indication of purifying selection. We found that pN/pS for orphans is significantly lower compared to intergenic regions (Mann–Whitney test, p=0.02182) (Figure 2), and significantly greater for old genes (Mann–Whitney test, p<1.0 × 10⁻¹⁵), consistent with purifying selection operating on orphans.

In agreement with studies in other species (Domazet-Loso and Tautz, 2003; Toll-Riera et al., 2009; Wolf et al., 2009; Cai and Petrov, 2010; Capra et al., 2010; Carvunis et al., 2012), we also find that orphan genes are shorter (median length for orphans = 344 bp, median length for old genes = 1470 bp), have a lower GC content (median GC content for orphans = 0.54, median GC content for old genes = 0.55), are expressed at lower levels (expression in D. pseudoobscura males: mean expression for orphans = 29 FPKM, mean expression for old genes = 41 FPKM) than old genes (Figure 3). Using CD-Hit (Li and Godzik, 2006), we found the fraction of genes with a paralog (>90% protein similarity) to be similar for orphans (6.9%) and old genes (6.4%). Orphans are more enriched in microsatellites, also consistent with previous findings in vertebrates (Toll-Riera et al., 2012) and rice (Guo et al., 2007). Furthermore, unlike mammals (Toll-Riera et al., 2009), none of the D. pseudoobscura orphans was found to be associated with transposable elements (see ‘Transposons detection’).

Figure 3

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The distribution of orphans is heterogeneous across chromosomes (χ²-test, p<1.0 × 10⁻¹⁵), with the X chromosome having the highest fraction of orphans. In the obscura group, the two X-chromosome arms have a different evolutionary history. XL corresponds to Muller’s element A and is homologous to the X chromosome in D. melanogaster. XR, however, has been recently derived from an autosome (Muller’s element D, 3L in D. melanogaster). Analyzing the old-X and neo-X chromosomes separately, we observed a striking difference in the number of orphans despite similar chromosome sizes, with the old-X responsible for the excess of X-linked orphan genes, and the neo-X showing a similar number of orphans as the autosomes (Figure 4). For each chromosomal arm, we computed genomic features in 100 kb windows to correlate them with the difference in orphan content between old-X and neo-X. We found that average GC content, microsatellite density, transposon density, and length of intergenic regions differ between the two chromosomal arms (Figure 5).

Figure 4

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Figure 5

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We hypothesized that this pronounced difference between the two chromosome arms might reflect a different history of X-linkage. If orphan genes emerge at a higher rate on the X-chromosome (Levine et al., 2006), the shorter history of X-linkage on the neo-X could explain the paucity of orphans on the neo-X compared to old-X. In this case, the difference in orphan number between old-X and neo-X chromosomes should date back to the time before the origin of the neo-X, with a similar number of orphans originating after the creation of the neo-X. We therefore used the genomic sequences of five members of the D. obscura group (D. pseudoobscura [Richards et al., 2005], D. miranda [Zhou and Bachtrog, 2012], and the de novo assembled D. persimilis, D. lowei, and D. affinis) to date the origin of the orphan genes to different ancestral nodes in the phylogenetic tree of these species (Beckenbach et al., 1993). We distinguished five groups of genes: old genes (non orphans) and four different orphan age classes (Figure 6). Surprisingly, we observed a consistent paucity of orphans on XR relative to XL across all age classes (Figure 7). This persistent difference in orphan number between XL and XR in all age classes suggests that X-linkage is not sufficient to explain the enrichment of orphans on XL. We conclude that the former autosome differs from the ancestral X chromosomal arm by a yet unidentified feature that affects the emergence of new orphans.

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Figure 7

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The analysis of orphans that have putatively lost their function via the acquisition of a stop codon or a frame shift causing insertion/deletion (pseudogenized/lost orphans) reveals another interesting feature of the XL–XR fusion. The oldest orphans in our dataset (age class 4) show a pronounced excess of pseudogenized orphans on XR in D. affinis and D. miranda (Figure 8A). This trend was not observed for orphans that emerged on XR after the XL–XR fusion (Figure 8B,C), nor for old genes (Figure 8D) and is not due to an increased rate of orphan gain on XR (Figure 9). Since the oldest orphans (age class 4) on XR are a mixture of autosomal (i.e., before the fusion) and sex-chromosomal (i.e., after the fusion) orphans, we speculate that the high rate of pseudogenization of orphans on the XR may reflect the new X-linkage of previously autosomal orphans. A previous study (Meisel et al., 2009) found that the XR chromosome has experienced a burst of gene duplications to autosomes after its creation. It is plausible that after the conversion of the XR from autosome to sex-chromosome, orphans might have been duplicated to autosomes, whereas the XR ancestral copy would have become pseudogenized. To test this hypothesis, we looked for evidence of gene duplications for the orphans lost on the XR at node 4 (Figure 6). We aligned the sequences of these genes in D. lowei and D. miranda to the respective genomes using BLASTN (cutoff 10⁻⁵). Upon manual inspection of the alignments, we found that only 1 out of 21 genes in D. miranda (gene ID: GA23486) and 1 out of 14 genes in D. lowei (gene ID: GA23807) had a second hit on an autosome covering at least 50% of the length of the query gene. Other genes either produced a single best hit on the XR chromosome or spurious short hits on other chromosomes (data not shown). Thus, we conclude that duplication of orphans cannot explain the excess of pseudogenized orphans on XR. Nevertheless, our analysis clearly indicates that the emergence of the neo-X chromosome influenced the orphan dynamics on XR, affecting rates of both gain and loss, thus we excluded this chromosome arm for our analyses of the rate of orphan turnover.

Figure 8

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Figure 9

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For each age class, we determined the number of pseudogenized orphans (Tautz and Domazet-Loso, 2011). In the D. persimilis lineage, orphan pseudogenization can be studied for three different age classes. If orphans of all age classes were functionally equivalent, no difference in the rate of orphan pseudogenization would be expected. We observe, however, that the fraction of orphan pseudogenes decreases with age (Figure 10). The D. miranda lineage also shows a higher loss of young orphan genes. The relatively small number of observations, however, precludes statistical testing of this trend. Overall, orphan genes are lost significantly more often than old genes (Fisher’s exact test, p=3.3 × 10⁻⁸), consistent with the rapid turnover hypothesis. The unequal conservation of orphans of different age classes is also apparent after normalizing by coding sequence length (Figure 11), to account for the fact that longer coding sequences (CDS) have a greater chance of acquiring ORF-disrupting mutations. When looking at the distribution of premature termination codons (PTC) along the open reading frame (ORF) of all genes, we observed that PTCs are enriched at the beginning and at the end of the ORF (Figure 12), consistent with previous results in D. melanogaster (Lee and Reinhardt, 2012) and D. pseudoobscura (Hoehn et al., 2012). Since ORF-disrupting mutations occuring at the end of the ORF might have little impact on gene function, we redefined pseudogenes by considering only ORF-disrupting mutations localized in the first half of the ORF and confirmed that orphans of age class 3 are lost more often than those of age class 4 (Figure 13). Age class 2 was intermediate, most likely not reflecting a biological phenomenon, but due to a high sampling variance associated with the small number of observations (9 orphans). Finally, the pattern is also robust to a more conservative criterion for ortholog assignment (see ‘Annotation of the obscura species’, Figure 14).

Figure 10

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Figure 11

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Figure 12

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Figure 13

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Figure 14

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To determine features associated with the differences in disabling mutations among orphans from different age classes, we contrasted orphans lost in D. lowei and/or D. persimilis (lost orphans) vs orphans conserved in all the obscura species (conserved orphans). Genes in both classes evolve at the same rate, are of similar length, and have similar codon usage bias (Figure 15A–E). Conserved orphans have a higher GC content, contain fewer microsatellites, are expressed at a higher level and are more male-biased (Figure 15B,D–F,G,H) compared to lost orphans. Conserved orphans tend to increase their expression level as they become older (Figure 16A), whereas the opposite pattern is true for lost orphans (Figure 16B).

Figure 15

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Figure 16

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Orphan genes are frequently expressed in the testis (Levine et al., 2006; Begun et al., 2007) and have a male-biased gene expression pattern (Metta and Schlötterer, 2008). This pattern could be generated by pervasive gene expression in testis, which facilitates the functional recruitment of non-specific expression (Kaessmann, 2010). Another explanation is that expression in testis does not require a complex architecture of regulatory modules (Sassone-Corsi, 2002; Kleene, 2005; Kaessmann, 2010), so that fewer substitutions are required to obtain a functional regulatory module for expressing a novel gene in testis compared to other tissues. We scrutinized these explanations by comparing the fraction of male-biased genes among orphan genes from different age classes. Unexpectedly, the fraction of male-biased genes increases with the age of the orphan genes (Figure 17). This increase of male-biased orphans among the older age classes is the result of a preferential loss of orphans with an unbiased gene expression (Figure 18). To confirm that male-biased gene expression is associated with orphan retention rather than emergence, we analyzed the sex-bias in D. miranda for orphans with and without an open reading frame. Consistent with the gene expression pattern in D. pseudoobscura, we found that lost orphans have a significantly lower male-bias in D. miranda (Figure 19). We conclude that the previously reported male-biased gene expression of orphan genes is not the result of a preferential recruitment of male-biased transcripts, nor do orphans gradually acquire male-biased gene expression. Rather, male-biased orphans are more likely to be retained.

Figure 17

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Figure 18

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Figure 19

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Our study provides the missing link to understand orphan dynamics. Until now, orphan evolution was primarily studied on long phylogenetic branches. Although this approach is well suited to discover new orphans, it does not allow tracing the evolution of orphans. Previous studies showed a high rate of orphan gain, which is not reflected in an increase in gene number. To resolve this apparent paradox, it has been postulated that orphans must be lost at a high rate as well (Tautz and Domazet-Loso, 2011). In this study, we used the framework of closely related species in the obscura group to study the patterns of orphan gain and losses. We show that orphans not only emerge at high rates, but that they are also rapidly lost (Figure 10). Interestingly, most losses (∼76%) were due to disabling mutations rather than deletions of the orphan gene. Although under equilibrium conditions the number of losses balances the number of orphan gains, here, we observed a surplus of orphan gains (Figure 6). We caution that this discrepancy probably does not imply an increase of gene number, but rather reflects the limited evolutionary time to acquire mutations. Using a rather conservative criterion for disabling mutations, either premature stop codons or frameshift indels, we have probably not identified all orphans that have lost their function. Furthermore, we do not account for the possibility of loss of function due to changes in gene regulation.

Importantly, codon usage bias, dN/dS values and sequence conservation clearly suggest that orphan genes are functionally constrained and these constraints do not differ among orphans that are conserved in the obscura group and those that lost function in at least one species of the group. Hence, it may be possible that orphan loss is stochastic and reflects weak purifying selection. Nevertheless, lost orphans differ in some aspects from conserved ones. Orphans that are lost contain more microsatellite stretches and have a lower, less sex-biased gene expression than retained ones. Furthermore, we also found that the rate of orphan loss decreases with orphan age, a result consistent with orphans serving a functional role only temporarily. Previous work suggested that orphans are important for adaptation to novel environments (Khalturin et al., 2009; Colbourne et al., 2011), but it is also possible that orphans contribute to stabilize new connections in gene networks (Capra et al., 2010; Warnefors and Eyre-Walker, 2011) and become obsolete once such new connections have been optimized. Our data suggest that orphans become quickly functional, which is reflected in their codon usage bias, dN/dS ratio and sequence conservation.

The chromosomal translocation resulting in the neo-X chromosome provides another interesting perspective on the evolution of orphan genes. Despite the fact that the neo-X is now fully dosage compensated (Abraham and Lucchesi, 1974), and has obtained a similar base composition as the XL (Gallach et al., 2007), we noted that the translocation resulted in a preferential loss of orphan genes on the neo-X. Since this pattern is restricted to orphans that most likely originated before the chromosomal fusion, we argue that the change in chromosomal environment has affected the function of orphan genes, most likely via expression differences. We speculate that the selective advantage conferred by these orphans has diminished, which resulted in a higher loss rate. Interestingly, the elevated rate of orphan loss after the neo-X formation seems to be still ongoing. This differential loss of orphan genes point in a similar direction as the observation that the gene composition of the neo-X has been altered by gene duplication (Meisel et al., 2009). Hence, both (orphan) gene loss and duplication contribute to fast gene content remodeling on a newly formed sex chromosome in Drosophila.

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Author details

1. Nicola Palmieri

   Institut für Populationsgenetik, Vetmeduni Vienna, Vienna, Austria

   Contribution

   NP, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

2. Carolin Kosiol

   Institut für Populationsgenetik, Vetmeduni Vienna, Vienna, Austria

   Contribution

   CK, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

3. Christian Schlötterer

   Institut für Populationsgenetik, Vetmeduni Vienna, Vienna, Austria

   Contribution

   CS, Conception and design, Acquisition of data, Drafting or revising the article

   For correspondence

   christian.schloetterer@vetmeduni.ac.at

   Competing interests

   The authors declare that no competing interests exist.

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