Author response: Expansion and loss of sperm nuclear basic protein genes in Drosophila correspond with genetic conflicts between sex chromosomes

Abstract

Article Figures and data Abstract Editor's evaluation eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Many animal species employ sperm nuclear basic proteins (SNBPs) or protamines to package sperm genomes tightly. SNBPs vary across animal lineages and evolve rapidly in mammals. We used a phylogenomic approach to investigate SNBP diversification in Drosophila species. We found that most SNBP genes in Drosophila melanogaster evolve under positive selection except for genes essential for male fertility. Unexpectedly, evolutionarily young SNBP genes are more likely to be critical for fertility than ancient, conserved SNBP genes. For example, CG30056 is dispensable for male fertility despite being one of three SNBP genes universally retained in Drosophila species. We found 19 independent SNBP gene amplification events that occurred preferentially on sex chromosomes. Conversely, the montium group of Drosophila species lost otherwise-conserved SNBP genes, coincident with an X-Y chromosomal fusion. Furthermore, SNBP genes that became linked to sex chromosomes via chromosomal fusions were more likely to degenerate or relocate back to autosomes. We hypothesize that autosomal SNBP genes suppress meiotic drive, whereas sex-chromosomal SNBP expansions lead to meiotic drive. X-Y fusions in the montium group render autosomal SNBPs dispensable by making X-versus-Y meiotic drive obsolete or costly. Thus, genetic conflicts between sex chromosomes may drive SNBP rapid evolution during spermatogenesis in Drosophila species. Editor's evaluation Chang et al. used a previously published set of highly contiguous genomes to infer the drivers of the evolution of sperm nuclear basic proteins and find several instances of gene duplication mainly occurring in sex chromosomes. Moreover, they provide a genetic characterization of one such protein (CG30056). The paper was initially reviewed by experts in the field through Review Commons. The three reviewers were enthusiastic about the potential of the paper and made a set of suggestions to make the paper stronger. The authors incorporated the suggestions when relevant, added a new (and relevant) experiment, edited the manuscript as requested, and clarified some instances that could be further developed. https://doi.org/10.7554/eLife.85249.sa0 Decision letter eLife's review process eLife digest In sperm, DNA is packaged more tightly than in other cells thanks to small proteins called ‘sperm nuclear basic proteins’ (SNBPs), also called protamines in mammals. SNBPs are important for sperm to develop properly and correctly perform their role during fertilization. Although the evolution of SNBPs has been studied in mammals, these proteins have not been as thoroughly examined in invertebrates. Chang et al. took advantage of the availability of high-quality sequences for the genomes of 78 species of Drosophila flies to investigate the evolution of the genes that code for SNBPs in these flies. The results showed that, just like in mammals, in Drosophila the protein sequences of SNBPs evolve rapidly. However, unlike mammals, Chang et al. also found that Drosophila species frequently gained and lost genes coding for SNBPs. Interestingly, the ‘older’ genes (genes that appeared earlier in evolution) that code for SNBPs are not essential for reproduction in the fruit fly Drosophila melanogaster. This is an unexpected finding because older genes usually have essential roles for survival and reproduction, which require them to be passed on to the next generation and remain in the genome. In contrast, younger SNBP genes that had appeared more recently and were not shared between different species of Drosophila were often essential for fertility. These results, combined with other observations about where SNBP genes are located in the genome, led Chang et al. to hypothesize that SNBPs present in sex chromosomes act as ‘meiotic drivers’ while those on other chromosomes (known as autosomes) suppress meiotic drive. In other words, SNBP genes present in the sex chromosomes may be responsible for killing sister sperm cells that do not carry those genes, while SNBP genes that are not located on sex chromosomes may suppress this activity. This is of particular interest because it indicates that SNBPs are involved in genetic conflicts between the two sex chromosomes: sperm that carry SNBPs on the X chromosome may kill sperm with a Y chromosome, and vice versa. The results of Chang et al. shed light on the mysterious evolution of SNBPs in Drosophila flies. Although previous hypotheses regarding the rapid evolution of SNBPs evolution have focused on their role in genome packaging, this new analysis suggests that much of the evolutionary change is likely driven by genetic conflicts between sex chromosomes. Introduction Chromatin plays a critical role in organizing genomes and regulating gene expression. Histones are the primary protein components of chromatin in most eukaryotes. Due to their conserved, essential functions, most histone proteins are ancient and subject to strong evolutionary constraints, although there are distinct exceptions among histone variants (Raman et al., 2022; Molaro et al., 2020; Talbert and Henikoff, 2021). Many animal species replace most histones with sperm nuclear basic proteins (SNBPs) to package their genomes more tightly into tiny sperm heads during spermatogenesis (Sassone-Corsi, 2002; Ward and Coffey, 1991). Like histones, most SNBPs are small (<200 amino acids) and positively charged. Many SNBPs also contain a high proportion of lysine, arginine, and cysteine residues, which form disulfide bridges to further condense DNA within sperm heads (Török et al., 2016; Eirín-López and Ausió, 2009). As a result of their tighter DNA packaging, SNBPs can reduce the size of the sperm nuclei by 20–200-fold compared to histone-enriched nuclei (Brewer et al., 1999). Based on their role in condensing sperm nuclei, the prevailing hypothesis is that sexual selection for competitive sperm shapes led to the evolutionary origins of SNBP genes in most animal taxa (Lüke et al., 2014; Lüpold et al., 2016). SNBPs have been most well-studied in mammals (Balhorn, 2007). Mammalian SNBPs in mature sperm include protamine 1 (PRM1) and protamine 2 (PRM2), which are encoded in an autosomal gene cluster that includes Transition Protein 2 (TNP2) and protamine 3 (PRM3) (Martin-Coello et al., 2011). Although these four genes share moderate homology, TPN2 is only expressed during the histone-to-protamine transition (Nayernia et al., 1996), whereas PRM3 only localizes to the cytoplasm of elongated spermatids (Martin-Coello et al., 2011). Both PRM1 and PRM2 are essential for fertility in humans and mice; their expression levels directly affect sperm quality (Balhorn, 2007). Loss of PRM1 or PRM2 leads to defects in sperm head morphology and fertility in mice and humans (Cho et al., 2003; Cho et al., 2001). Yet, PRM2 has undergone pseudogenization in bulls (Balhorn, 2007). Thus, even SNBPs essential for male fertility can be subject to evolutionary turnover in some species. Although SNBPs play a similar genome-packaging role to histones, they differ dramatically from histones in their evolutionary origins and trajectories. Whereas histones have ancient origins, SNBPs were independently derived from different ancestral proteins across taxa (Eirín-López and Ausió, 2009; Reynolds and Wolfe, 1984; Török and Gornik, 2018). For example, SNBPs arose from linker histone H1 gene variants in liverworts and tunicates (Lewis et al., 2004; D’Ippolito et al., 2019), whereas they arose from histone H2B gene variants in cnidarians and echinoderms (Török et al., 2016; Green and Poccia, 1988; Eirín-López et al., 2006). SNBPs in other animals lack apparent homology to other existing proteins, obscuring their evolutionary origins (Eirín-López et al., 2006). In addition to their convergent evolution and turnover, SNBPs differ dramatically from histones in their evolutionary rates of amino acids. For example, PRM1 and PRM2 are among the most rapidly evolving protein-coding genes encoded in mammalian genomes (Saperas and Ausió, 2013) and evolve under positive selection in many lineages (Lüke et al., 2014; Wyckoff and Wang, 2000). The positive selection of SNBPs results in changes to their amino acid composition. For example, the arginine content of PRM1 is partially correlated across species with sperm head length, which may reflect the selective pressures of sperm competition (Lüke et al., 2016b). The rapid evolution of PRM1 and PRM2 is consistent with sexual selection on sperm heads driving SNBP origins and rapid evolution in mammals (Wyckoff and Wang, 2000; Lüke et al., 2016b; Lüke et al., 2016a), although this hypothesis has yet to be experimentally tested. Moreover, the evolutionary trajectories of SNBP genes and their underlying causes have not been deeply investigated outside mammals. Drosophila species provide an excellent model to study SNBP function and evolution due to the ease of genetic manipulations and sperm biology characterization, and the availability of high-quality genome sequences from many closely related species. Previous studies have shown that Drosophila SNBPs independently arose from proteins encoding high mobility group box (HMG-box) DNA-binding proteins (Gärtner et al., 2015); thus, they have distinct origins and likely functions from mammalian protamines. Five HMG-box SNBP genes have been previously identified in Drosophila melanogaster: ProtA, ProtB, ddbt, Mst77F, and Prtl99C (Tirmarche et al., 2014; Kimura and Loppin, 2016; Jayaramaiah Raja and Renkawitz-Pohl, 2005; Eren-Ghiani et al., 2015; Yamaki et al., 2016). Each of these five SNBPs is incorporated into nuclei independently of each other, suggesting that they play distinct roles in sperm formation (Kimura and Loppin, 2016; Eren-Ghiani et al., 2015; Rathke et al., 2010). Based on the common HMG-box motifs found in these five SNBPs, 10 other male-specific proteins with the same motif were later identified in D. melanogaster (Gärtner et al., 2015; Eren-Ghiani et al., 2015), and 4 of them were later shown to be enriched in sperm nuclei (Gärtner et al., 2015). Other proteins without any HMG-box are also demonstrated to locate in sperm nuclei, but it is unclear whether they bind to DNA (Rivard et al., 2021; Hempel et al., 2006; Harhangi et al., 1999). Recent studies in Drosophila have suggested an alternate hypothesis other than sperm competition—meiotic drive and its suppression—to explain the rapid diversification and innovation of SNBP-like proteins (Vedanayagam et al., 2021; Muirhead and Presgraves, 2021). Meiotic drivers are selfish elements that can bias their transmission via hijacking meiosis or post-meiosis processes, e.g., killing sperm that do not carry the driver. These genetic drivers exist in widespread lineages, including plants, animals, and fungi (Courret et al., 2019). One of the first identified drivers is Segregation Distorter in D. melanogaster, whose drive strength can be further enhanced by the knockdown of ProtA/ProtB genes (Gingell and McLean, 2020). Thus, ProtA/ProtB serve as suppressors of meiotic drive through an unknown mechanism. The second piece of evidence emerged from studies of Distorter on the X (Dox), an X-chromosomal driver in Drosophila simulans (Tao et al., 2007a). Dox emerged via the stepwise acquisition of multiple gene segments, mostly from ProtA/ProtB. Dox produces chromosome condensation defects in Y chromosome-containing sperm during spermatogenesis, ultimately leading to X-chromosomal bias among functional sperm and sex-ratio bias in resulting progeny (Tao et al., 2007a; Faulhaber, 1967; Tao et al., 2007b). In D. simulans and sister species, Dox-like genes have amplified and diversified on the X chromosome in an escalating battle between X and Y chromosomes for transmission through the male germline (Vedanayagam et al., 2021; Muirhead and Presgraves, 2021). Thus, genetic conflicts between sex chromosomes and their suppression of those conflicts could provide an alternate explanation for the recurrent diversification of SNBP genes in Drosophila species. Here, we systemically explored the evolution of SNBP genes via a detailed phylogenomic analysis across Drosophila species. We found that SNBP genes are rapidly evolving, and most of them are under positive selection in Drosophila, like in mammals. Thus, the rapid sequence changes of SNBP genes are common to many animal taxa. Interestingly, we found an inverse relationship between age and essentiality; young SNBPs are essential for male fertility in D. melanogaster, whereas ancient, conserved SNBPs are not. Moreover, SNBP genes essential for male fertility in D. melanogaster are frequently lost in other Drosophila species. Unexpectedly, we found 19 independent amplification events from eight different SNBP genes on either X or Y chromosomes in Drosophila species. Conversely, species with reduced conflicts between sex chromosomes due to chromosomal fusions do not undergo SNBP amplification, but instead lose SNBP genes. Thus, we conclude that rapid diversification of SNBP genes might be largely driven by genetic conflicts between sex chromosomes in Drosophila. Results SNBP genes in Drosophila species To study SNBP evolution in Drosophila species, we performed a detailed survey of all testis-specific genes encoding HMG boxes in D. melanogaster. Our survey did not reveal any additional genes beyond the 15 previously identified autosomal SNBP genes, which function at different stages of spermatogenesis (Table 1). For example, CG14835, ProtA, ProtB, Mst77F, Prtl99C, and ddbt all encode SNBP proteins present in the mature sperm head (Jayaramaiah Raja and Renkawitz-Pohl, 2005; Eren-Ghiani et al., 2015; Yamaki et al., 2016). In contrast, Tpl94D, tHMG-1, tHMG-2, and CG30356 encode transition SNBP proteins during the transition between histone and protamines but are not retained in mature sperm (Gärtner et al., 2015; Rathke et al., 2007). The five remaining SNBP genes (Mst33A, CG30056, CG31010, CG34269, and CG42355) remain cytologically uncharacterized (Eren-Ghiani et al., 2015). Using single-cell transcriptomic data (Witt et al., 2021), we confirmed that all candidate SNBP genes are transcribed in male germline cells, with the highest level of expression of most SNBP genes occurring in late spermatocytes. The only exceptions are CG34269, which is transcribed earlier in late spermatogonia, and CG30056, which is transcribed later in late spermatids (Figure 1—figure supplement 1). SNBP proteins in D. melanogaster tend to be short (<200 a.a.) and mostly have high isoelectric points (>10), consistent with their basic charge and potential function in tight packaging of DNA (Table 1). A closer examination revealed that 11 SNBP genes encode a single HMG box, whereas four genes (Tpl94D, Prtl99C, Mst33A, and CG42355) encode two HMG boxes (Figure 1—figure supplement 2). Table 1 McDonald–Kreitman tests for positive selection on sperm nuclear basic protein (SNBP) genes in two Drosophila species. NameLocation (Mb)LengthpI*# of HMGD. melanogasterD. serrataAlphaχ2 p-valueAlphaχ2 p-valueExpression stage†PhenotypeCitationsCG300562R:12.613711.05 (10.70)1-50.090.750.208UndefinedUndefinedaCG303562R:8.714910.65 (10.89)10.7850.0110.440.226‡Pre-individualizationUndefinedb,cCG310103R:30.72544.77 (8.10)10.5350.0340.610.001UndefinedUndefinedaCG342693L:0.519110.7 (10.34)1–0.2630.6130.7360.001UndefinedUndefinedaCG423553L:2.016111.29 (10.89)20.6820.0120.6820.036UndefinedUndefinedb,cMst33A2L:11.635910.61 (10.14)20.2080.391NANAUndefinedUndefinedcddbt3L:0.311712.3 (11.76)1–0.3160.642–0.3330.647Mature spermSteriledMst77F3L:20.821510.34 (9.95)1–0.3080.628NANAMature spermSterileePrtl99C2R:29.820111.25 (10.57)20.2420.483NANAMature spermSterilefTpl94D2R:23.016411.3 (10.11)20.5710.0230.520.074‡Pre-individualizationFertilegCG148353L:7.415210.43 (4.81)10.3320.416NANAMature spermFertileaProtA§2R:14.914611.12 (11.52)10.0270.9410.6670.012Mature spermLow fertilityeProtB§2R:14.914410.8 (11.60)1–0.0290.9450.9520Mature spermLow fertilityetHMG-13R:22.51267.67 (6.11)10.6590.02NANAPre-individualizationFertilebtHMG-23R:22.51338.94 (7.19)10.4430.199NANAPre-individualizationFertileb We only show results from unpolarized MK tests using all (including rare) SNPs. Other variations of these results (e.g., polarized, excluding rare SNPs) are shown in Supplementary file 5. Citations: aYamaki, 2018, bGärtner et al., 2015, cDoyen et al., 2015, dYamaki et al., 2016, eJayaramaiah Raja and Renkawitz-Pohl, 2005, fEren-Ghiani et al., 2015, gRathke et al., 2007. Genes with any evidence of positive selection have p-values in bold. * Isoelectric point of either the whole protein or just HMG domains only (in parentheses). † Post-meiotic protein expression. ‡ A significant signature of positive selection is obtained after removing low-frequency SNPs (<5%) and/or after polarizing changes (see Supplementary file 5). § Independent duplications in two species. To investigate the retention of SNBP genes across Drosophila species, we expanded our analysis to homologs of D. melanogaster SNBP genes found in published genome assemblies from 15 Drosophila species with NCBI gene annotation. We also included Scaptodrosophila lebanonensis as an outgroup species. Our phylogenomic analyses revealed that two SNBP genes (tHMG and Prot) underwent recent gene duplications in D. melanogaster. Both are present as closely related paralogs (tHMG-1 and tHMG-2, ProtA and ProtB) in D. melanogaster but only in one copy in closely related species (Figure 1; Tirmarche et al., 2014; Jayaramaiah Raja and Renkawitz-Pohl, 2005). Five SNBP genes are found only in the Sophophora subgenus: CG42355, Mst33A, Mst77F, Prtl99C, and Tpl94D (Figure 1), and are, therefore, less than 40 million years old. At the other extreme, we found orthologs of eight D. melanogaster SNBP genes (CG14835, CG30056, CG30356, CG31010, CG34529, ddbt, tHMG, and Prot) in the outgroup species, S. lebanonensis. Thus, these eight SNBP genes are at least 50 million years old (Suvorov et al., 2022). Figure 1 with 4 supplements see all Download asset Open asset Origins and evolution of Drosophila sperm nuclear basic protein (SNBP) genes. (A) Phylogenomic analysis of 13–15 SNBP genes from D. melanogaster organized into three groups (dotted lines): required for male fertility, not required for male fertility, or untested in previous analyses. We identified homologs of these genes in 14 other Drosophila species and an outgroup species, S. lebanonensis, whose phylogenetic relationships and divergence times are indicated on the left (Kumar et al., 2017). Genes retained in autosomal syntenic locations are indicated by black squares, whereas paralogs located in non-syntenic autosomal locations, or X-chromosomes, or Y-chromosomes are indicated in gray, blue and red squares, respectively. Numbers within the squares show the copy number, if >1, of different genes, e.g., D. melanogaster has two paralogs each of both Prot and tHMG genes. An empty square with a line across it indicates that only a pseudogene can be found in the shared syntenic location, whereas an ‘X’ indicates that no ortholog is found, even though one is expected based on the phylogenomic inference of SNBP age. Based on this analysis, we infer that eight SNBP genes are at least 50 million years old, but only three genes are strictly retained in all 16 species (CG30056, CG31010, and Prot). Indeed, none of the SNBP genes required for male fertility in D. melanogaster are strictly conserved in other Drosophila species, either arising more recently (Mst77F, Prtl99C) or having been lost in at least one species after birth (ddbt). We also marked the montium group species, D. kikkawai, in red, because it has unusually lost six SNBP genes. (B, C) We compared dN/dS (B) or dN (C) values for all orthologous SNBP genes (red dots) in D. melanogaster compared to a histogram of the same values for the genome-wide distribution (gray bars) obtained from an analysis using six species by the 12 Drosophila genomes project (Clark et al., 2007). Our analyses reveal that most SNBP genes are at or beyond the 95th or 99th percentile for dN/dS or dN values (blue dashed lines). The values of CG34269 are calculated using only five species because it is lost in one of the surveyed species, D. ananassae; therefore; we do not show its dN, as it is not comparable to other genes. Our inability to detect homologs beyond the reported species does not appear to result from their rapid sequence evolution. Indeed, abSENSE analyses (Weisman et al., 2020) support the finding that Prtl99C, Mst77F, Mst33A, Tpl94, and CG42355 were recently acquired in Sophophora within 40 MYA. For example, the probability of a true homolog being undetected for Prtl99C and Mst77F is 0.07 and 0.18 (using E-value = 1), respectively (Supplementary file 1, 'Materials and methods'). We also examined the syntenic regions of SNBP genes (conserved genomic neighboring genes) to confirm the loss of SNBP genes in some representative species, e.g., D. kikkawai, D. ananassae, D. pseudoobscura, D. willistoni, D. albomicans, D. virilis, and S. lebanonensis. Although abSENSE and synteny analyses rule out the absence of true homologs, they cannot rule out the less parsimonious possibility that SNBP genes are older but were lost multiple times in non-Sophophora species. Similarly, our analysis focuses on SNBP genes present in D. melanogaster, but other Drosophila species may have additional, unrelated SNBP genes. We confirmed that Drosophila SNBP gene expression is primarily male-limited across species using publicly available RNA-seq data; their expression is particularly enriched in testes (Figure 1—figure supplement 3A; Supplementary file 2). The only exception is a CG42355 paralog in D. takahashii that also has weak expression in females (~9 TPM; Figure 1—figure supplement 3A; Supplementary file 2). We observed a moderate to high correlation (Spearman’s rho = 0.142–0.753; Figure 1—figure supplement 4) for the expression of SNBP genes between species. Like in D. melanogaster, most Drosophila SNBP proteins are small, possess at least one HMG box domain, and have high isoelectric points, suggesting that these features are crucial for their function (Supplementary file 3). In addition to orthologs of these SNBP genes found in shared syntenic locations on autosomes, we also found sex chromosome-linked paralogs of SNBP genes in several species. The most dramatic example is the presence of 34 copies of tHMG paralogs in the poorly assembled X chromosomal region of D. simulans (Figure 1). These are discussed in more detail later in this study. Rapid evolution and positive selection of Drosophila SNBP genes Based on the precedent of rapidly evolving protamines in mammals, we next investigated whether Drosophila SNBP genes also evolve rapidly. We calculated protein evolution rates (non-synonymous substitution rates over synonymous substitution rates, dN/dS) for 13 of 15 D. melanogaster SNBP genes for six species in the melanogaster group (Supplementary file 4). We excluded two SNBP genes, ProtB and tHMG-2, since these duplicates are not found outside D. melanogaster. We found that 11 of 13 SNBP genes (except CG30056 and ProtA) evolve faster (higher dN/dS) than 95% of protein-coding genes across the genome (Figure 1B). These high protein evolution rates are due to high dN instead of low dS (Figure 1C), suggesting that SNBPs evolve under either extensive positive selection or reduced functional constraints. We used McDonald–Kreitman tests to test the possibility of recent positive selection in the D. melanogaster lineage, taking advantage of many sequenced strains from this species (Rathke et al., 2007; Witt et al., 2021; Yang, 2007; Yang et al., 2000; Kasinathan et al., 2020; Bayes and Malik, 2009; Kim et al., 2021; Altschul et al., 1990; Manni et al., 2021; Vedanayagam et al., 2022). The McDonald–Kreitman test compares the ratio of non-synonymous to synonymous substitutions fixed during inter-species divergence to the ratio of these polymorphisms segregating within species. If there is an excess of non-synonymous changes fixed during inter-species divergence, this results from positive selection. Indeed, our tests reveal that five SNBP genes in D. melanogaster have evolved under positive selection during its divergence from D. simulans (Table 1). By polarizing the test using the inferred ancestral sequences of tHMG-1 and tHMG-2, we find that tHMG-1, but not its paralog, tHMG-2, evolved under positive selection in D. melanogaster (Supplementary file 5). We also took advantage of genome sequences from 110 D. serrata strains to carry out McDonald–Kreitman tests of SNBP genes from D. serrata compared to its sister species, D. bunnanda in the montium group (Reddiex et al., 2018; Bronski et al., 2020). Among the SNBP genes shared between D. melanogaster and D. serrata, we found that four genes (CG30356, CG31010, CG42355, Tpl94D) evolved under positive selection in both D. melanogaster and D. serrata, whereas two genes (CG30056, ddbt) do not show a signature of positive selection in either species (Supplementary file 5). Three additional genes (CG34269 and two ProtA/B duplicates) evolved under positive selection only in D. serrata (ProtA/B underwent independent gene duplications in D. melanogaster and D. serrata). Finally, we used maximum-likelihood analyses using the site model of the codeml program in the PAML package (Yang, 2007; Yang et al., 2000) to investigate whether any residues in the SNBP genes had evolved under recurrent positive selection. We limited our analyses to 17 species of the melanogaster group to avoid saturation of synonymous substitutions. Although we recapitulated a previously published positive selection result using ddbt genes from just five Drosophila species (Yamaki et al., 2016), analyses using 17 melanogaster group species did not find a significant signature of site-specific positive selection in any SNBP gene (Supplementary file 6). The discrepancy between the McDonald–Kreitman tests and the PAML results indicates that although many SNBP genes evolve under positive selection, either SNBP genes or the exact residues evolving under recurrent positive selection vary throughout Drosophila evolution. What determines SNBP essentiality for male fertility Of 15 SNBP genes, nine have been previously characterized for their roles in male fertility based on gene knockout or knockdown experiments in D. melanogaster (Table 1). These genes show differences in their importance for male fertility in D. melanogaster. Three genes (Mst77F, Prtl99C, and ddbt) are essential for male fertility (Tirmarche et al., 2014; Kimura and Loppin, 2016; Jayaramaiah Raja and Renkawitz-Pohl, 2005; Eren-Ghiani et al., 2015; Rathke et al., 2010), but separate knockouts of six individual genes (CG14835, ProtA, ProtB Tpl94D, tHMG-1, and tHMG-2) do not appear to impair male fertility under standard laboratory conditions. Further experimentation has revealed a fertility cost for double knockouts of ProtA/ProtB but only in conditions of sperm exhaustion via mating with excess numbers of females (Tirmarche et al., 2014). No information is currently available for the remaining six SNBP genes. We also found nearly strict retention of all SNBP genes in all sequenced strains of D. melanogaster, no matter whether they are essential for male fertility in laboratory experiments or not (Supplementary file 7). There are a few distinguishing characteristics common to SNBP genes required for male fertility. Neither the number of HMG domains nor expression levels of SNBP are associated with essentiality. Instead, proteins essential for male fertility (Mst77F, Prtl99C, and ddbt) are more likely to be present in the mature sperm head, whereas transition SNBPs (Tpl94D, tHMG-1, and tHMG-2) are more likely to be dispensable, potentially due to functional redundancy with each other. Moreover, SNBP genes important for male fertility in D. melanogaster show no signature of positive selection according to McDonald–Kreitman tests. In contrast, two of the three identified transition SNBP genes evolve under positive selection (Tpl94D and tHMG-1). This suggests that genes with redundant function or less critical for male fertility are more likely to evolve under positive selection, although we note that several SNBP genes remain functionally uncharacterized or have not been tested exclusively (Table 1). How does SNBP essentiality in D. melanogaster correlate with age and retention across Drosophila species evolution? We find that two essential SNBP genes (Prtl99C and Mst77F) are evolutionarily young, i.e., they arose relatively recently in Drosophila evolution. Moreover, both genes have been lost at least once in the montium group species since their birth. The third essential SNBP gene, ddbt, arose before the separation of Drosophila and S. lebanonensis, but it has also been lost at least once (in D. willistoni) among the 15 species analyzed (Yamaki et al., 2016 Figure 1A). Based on these findings, we infer that not only are these three essential SNBP genes subject to evolutionary turnover, but they also gain or lose essential function across Drosophila evolution. Our findings are reminiscent of recent studies that show the high evolutionary lability of many genes involved in essential heterochromatin or centromere function in Drosophila (Kasinathan et al., 2020; Bayes and Malik, 2009). CG30056 is dispensable for male fertility in D. melanogaster despite being universally retained in Drosophila species Given th