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

  1. Ching-Ho Chang  Is a corresponding author
  2. Isabel Mejia Natividad
  3. Harmit S Malik
  1. Division of Basic Sciences, Fred Hutchinson Cancer Center, United States
  2. Howard Hughes Medical Institute, Fred Hutchinson Cancer Center, United States


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.


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.


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.


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. serrata
Alphaχ2 p-valueAlphaχ2 p-valueExpression stagePhenotypeCitations
CG300562R:12.613711.05 (10.70)1-50.090.750.208UndefinedUndefineda
CG303562R:8.714910.65 (10.89)10.7850.0110.440.226Pre-individualizationUndefinedb,c
CG310103R:30.72544.77 (8.10)10.5350.0340.610.001UndefinedUndefineda
CG342693L:0.519110.7 (10.34)1–0.2630.6130.7360.001UndefinedUndefineda
CG423553L:2.016111.29 (10.89)20.6820.0120.6820.036UndefinedUndefinedb,c
Mst33A2L:11.635910.61 (10.14)20.2080.391NANAUndefinedUndefinedc
ddbt3L:0.311712.3 (11.76)1–0.3160.642–0.3330.647Mature spermSteriled
Mst77F3L:20.821510.34 (9.95)1–0.3080.628NANAMature spermSterilee
Prtl99C2R:29.820111.25 (10.57)20.2420.483NANAMature spermSterilef
Tpl94D2R:23.016411.3 (10.11)20.5710.0230.520.074Pre-individualizationFertileg
CG148353L:7.415210.43 (4.81)10.3320.416NANAMature spermFertilea
ProtA§2R:14.914611.12 (11.52)10.0270.9410.6670.012Mature spermLow fertilitye
ProtB§2R:14.914410.8 (11.60)1–0.0290.9450.9520Mature spermLow fertilitye
tHMG-13R:22.51267.67 (6.11)10.6590.02NANAPre-individualizationFertileb
tHMG-23R:22.51338.94 (7.19)10.4430.199NANAPre-individualizationFertileb
  1. 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.

  2. Genes with any evidence of positive selection have p-values in bold.

  3. *

    Isoelectric point of either the whole protein or just HMG domains only (in parentheses).

  4. Post-meiotic protein expression.

  5. A significant signature of positive selection is obtained after removing low-frequency SNPs (<5%) and/or after polarizing changes (see Supplementary file 5).

  6. §

    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
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 the high evolutionary turnover of SNBP genes in our sampling of 15 Drosophila species, we investigated whether any SNBP genes are universally retained across all Drosophila species. For this purpose, we expanded our phylogenomic analysis of SNBP evolution to a recently published dataset of 78 highly contiguous and complete Drosophila genomes (Kim et al., 2021), using tblastn and reciprocal blastx searches (Altschul et al., 1990). Based on this analysis, we find only two SNBP genes have been strictly retained across all Drosophila species — Prot and CG30056 (Figure 1A), while the CG31010 gene has only been lost in one species. Prot and CG30056 also have the lowest dN/dS among SNBP genes (Figure 1B), suggesting that they evolve under a higher degree of selective constraint than other SNBP genes.

Previous studies have shown that a dual loss of ProtA/ProtB reduces male fertility in conditions of sperm exhaustion in D. melanogaster (Tirmarche et al., 2014). However, CG30056 remains functionally uncharacterized for its role in male fertility despite being one of only two well-retained and highly conserved SNBP genes in Drosophila species (Figure 1B). To study its contribution to male fertility, we generated a complete deletion knockout of CG30056 gene using CRISPR/Cas9 (Figure 2A). CG30056 is located in an intron of frazzled, a gene essential for development and morphogenesis (Figure 2A). We co-injected a construct encoding two gRNAs designed to target sequences immediately flanking CG30056 together with a repair construct containing 3xP3-DsRed (a visible eye marker). We successfully obtained transgenic flies encoding DsRed and validated the deletion of CG30056 using PCR (Figure 2A). Homozygous knockout flies were viable, confirming that the removal of CG30056 did not disrupt the essential frazzled gene.

The strictly retained, highly conserved sperm nuclear basic protein (SNBP) gene, CG30056, is dispensable for male fertility in D. melanogaster.

(A) The SNBP gene, CG30056, is encoded co-directionally in an intron of the essential frazzled gene. Using guide RNAs designed to match sites flanking CG30056, and a healing construct encoding eye-specific DsRed, we created a knockout allele replacing CG30056 with DsRed. The knockout was verified using PCR and primers flanking the CG30056 locus (right). Note that balancer lines encode a wildtype copy of CG30056. (B) We performed fertility assays comparing CG30056 homozygous knockout flies with heterozygous controls, either KO/Balancer or KO/wt (gray ovals). Each dot represents a single replicate, and the average and 95% confidence interval based on standard errors are shown in the figures. Fertility assays were performed either for a few days or to sperm exhaustion (gray ovals). We also assayed fertility of knockout strains for the fertility-essential Mst77F gene, and the fertility-nonessential Tpl94D gene. We also documented the sex ratios of the resulting progeny in (C). Consistent with previous findings, we found that Mst77F knockout males are essentially sterile and Tpl94D knockout males were indistinguishable from their heterozygous controls. We found either no or weak evidence of fertility impairments in two different crosses with homozygous CG30056 knockout males compared to KO/Balancer controls. However, we found no evidence of CG30056 requirement for male fertility in more stringent ‘sperm exhaustion’ fertility experiments compared to KO/wildtype controls (gray ovals). (C) We observed no significant evidence of sex-ratio distortion that would suggest an X-versus-Y meiotic drive in progeny resulting from either CG30056, Mst77F, or Tpl94D knockout males. Although there is suggestive evidence of sex-ratio distortion in progeny of one of the Mst77F genotypes, this is inconsistent between the two crosses and most likely due to stochastic effects of having very few resulting progeny. The raw data of (B) and (C) are shown in Supplementary file 8.

Next, we tested how CG30056 contributes to male fertility. We first generated CG30056-KO homozygous males by crossing two different CG30056-KO founder lines either to each other or to a D. melanogaster strain with a large deletion spanning CG30056 (Df(2R)BSC880). This ‘trans-heterozygote’ strategy prevents our phenotypes from being affected by CRISPR/Cas9-generated off-target mutations generated in the same founder chromosome. As controls, we used CG30056/SM6a or CG30056/CyO heterozygous males (SM6a and CyO are balancer chromosomes with an intact copy of CG30056). We compared the fertility of these males by mating them with two wildtype females at room temperature and counting their adult offspring. We found only modest differences in offspring number between heterozygous controls and homozygous knockout males (Figure 2B; Supplementary file 8), implying that loss of CG30056 does not significantly lower male fertility. We also detected no evidence of sex-ratio distortion in our crosses (Figure 2C; Supplementary file 8). Parallel fertility experiments conducted with mutants of Mst77F or Tpl94D recapitulated previous findings, confirming the essentiality of Mst77F and the dispensability of Tpl94D (Figure 2B; Supplementary file 8; Gärtner et al., 2015; Kimura and Loppin, 2016).

Comparisons to balancer chromosome-containing males could mask more subtle fertility effects of CG30056-KO. Therefore, we increased the stringency of our fertility assay in two ways. First, we compared the fertility of CG30056-KO ‘trans-heterozygote’ males with heterozygous males carrying one CG30056-KO allele and one wildtype chromosome, which was the parental chromosome used to create the deletion. Second, we carried out fertility experiments by crossing individual males with 10 wildtype females to exhaust male sperm; similar assays were used to reveal fertility consequences of ProtA/ProtB double deletions (Tirmarche et al., 2014). In spite of this increased stringency, we did not find a significant fertility difference between CG30056-KO and the control sibling males (Figure 2B). Thus, despite its strict retention for more than 50 million years of Drosophila evolution, standard laboratory conditions reveal no male fertility requirement of CG30056. However, CG30056 might play another function that we did not assay for, such as in sperm storage or sperm precedence that would explain its strict retention.

Thus, two of three SNBP genes (Mst77F and Prtl99C) essential for male fertility are evolutionarily young and poorly retained. In contrast, one of the most well-conserved and well-retained SNBP genes (CG30056) is not essential. Our work suggests that newly arisen Drosophila SNBP genes are more likely to encode essential, non-redundant male fertility functions than ancient, well-retained ones.

Recurrent amplification of a subset of SNBP genes on sex chromosomes

Our analysis of SNBP genes across a limited set of Drosophila species had already revealed significant evidence of evolutionary turnover (Figure 1). We further analyzed evolutionary turnover of SNBP genes in 78 Drosophila species and two other Drosophilidae species, most of which lack detailed gene annotation (Kim et al., 2021). We inferred gains and losses of SNBP genes in these species, which we represent on a circular phylogram of all species. To assign the chromosomal location of SNBP genes, we estimated coverage of publicly available Illumina and Nanopore reads (represented in Figure 3 and Supplementary file 9) of either male or mixed-sex flies from various Drosophila species. We also assigned location to specific Muller elements based on 3285 BUSCO (Benchmarking Universal Single-Copy Orthologs) genes on the contigs (Manni et al., 2021). We could readily assign male-specific regions to the Y chromosomes for species where sequencing reads were available from male and female flies separately. However, we could not ascribe a sex-chromosomal linked location of a contig to either the X or Y chromosome in cases where there was no linkage information from BUSCO genes and no read data available from females, only from males and mixed-sex flies.

Figure 3 with 2 supplements see all
Recurrent amplifications of Drosophila sperm nuclear basic protein (SNBP) genes are biased for sex-chromosomal linkage.

(A) Using reciprocal BLAST (see 'Materials and methods'), we searched for homologs of each D. melanogaster SNBP gene in 78 distinct Drosophila species and two outgroup species (shown in dot lines). We depict our findings using the circular phylogram representation for SNBP gene CG31010. The innermost circle is a circular phylogeny of the species (Kim et al., 2021). The next circle ring indicates autosomal copies, with colors to indicate copy number (scale bar, top left; note that scales are different for each gene). Thus, CG31010 is present in one autosomal copy in all but one Drosophila species (gray bar). The third circle indicates sex-chromosomal copies. Red and blue frames in the middle ring indicate X- or Y-linkage if that can be reliably assigned. Dotted frames indicate copies that might not be real orthologs based on phylogeny, whereas solid frames indicate five or more copies. For example, CG31010 is present in five copies on the X-chromosome of D. obscura. The outermost circle shows copies with ambiguous chromosomal location: there are no such copies for CG31010. (B) Using the same representation scheme, we indicate gene retention and amplification for seven other SNBP genes for which we find robust evidence of amplification, from a copy number of five (CG14835) to nearly 50 (tHMG). We also marked the montium group species that lost many SNBP genes with yellow lines. We note that assemblies of Lordiphosa species have lower quality, and the data need to be interpreted carefully. (C) SNBP gene amplifications (five or more copies) are heavily biased for sex chromosomal linkage. Given the relative size of sex chromosomes and autosomes, this pattern is highly non-random (test of proportions, p=2.3e-5).

All ancestrally-retained SNBP genes are located on autosomes in D. melanogaster. This is also the case in most Drosophila species. On the circular phylogram, we represented losses or duplications of the ancestral autosomal gene in the innermost circle, gains on the sex chromosomes are represented in the next (middle) concentric circle, and gains with ambiguous chromosomal location are represented in the outermost concentric circle (Figure 3, Figure 3—figure supplement 1). We use the CG31010 gene to illustrate this representation. CG31010 has been retained in all Drosophila species except one, which is shown by a gray bar in the innermost concentric circle. In addition, CG31010 amplified to a total of five X-linked copies in D. obscura, represented by a dark red bar in the middle concentric circle (Figure 3A).

Our expanded survey reinforced our initial findings (Figure 1A) that multiple SNBP genes are subject to lineage-specific amplifications (more than five copies in one species). We found that 8 of 13 D. melanogaster SNBP genes investigated (CG14835, ddbt, Mst33A, Mst77F, Prtl99C, Prot, tHMG, and CG31010) underwent amplification in at least one species (Figure 3 and Table 2). In total, we found that SNBP genes have experienced 20 independent amplification events, including one event in the outgroup species, Leucophenga varia (Figure 3A and B). Most SNBP amplifications are evolutionarily young (<10 million years old; Figure 3—figure supplement 2), and 15 of them are specific to a single surveyed species (Figure 3A and B). Like their parental genes, most amplified copies encode positively charged HMG domain-containing proteins (Supplementary file 3) and have male-specific, mostly testis-specific, expression (Figure 1—figure supplement 3B). Thus, amplified copies are also likely to function as SNBP genes. Many amplified SNBP genes are arranged in tandem arrays, making their sequences hard to assemble. Moreover, some amplified SNBP genes, e.g., Dox-related genes derived from ProtA/B in D. simulans (Vedanayagam et al., 2021; Muirhead and Presgraves, 2021), are too diverged from the parental genes for unambiguous assignment and are missing in our survey. Therefore, our reported numbers are thus likely underestimates.

Table 2
Summary of evolution events of Drosophila sperm nuclear basic protein (SNBP) genes.
NamePhenotypeAge (My)Expression level (TPM)*Evolutionary rate (dN/dS)Positive selection(MK test)Amplification event(s)§Number of loss events in 80 speciesNumber of loss events in the montium group(X-Y fusion)
tHMGFertile>65935,11940.39+2X; 1U51
  1. *

    Gene expression level in D. melanogaster testes (Supplementary file 2).

  2. dN/dS in D. melanogaster subgroup species (Supplementary file 4).

  3. Positive selection based on McDonald–Kreitman tests in D. melanogaster and/or D. serrata.

  4. §

    Any specific location with five or more copies of any one SNBP gene. A represents all autosomes combined, X represents the X chromosome, Y represents the Y chromosome, X/Y represents either the X or Y chromosome, and U represents regions with unknown chromosome locations.

  5. Number of potential loss events inferred by the phylogeny (Figure 3 and Figure 3—figure supplement 1). Some of these may represent false negatives due to incomplete genome assemblies.

We found that eight amplifications are X-linked and four are Y-linked, whereas, for four amplifications, we can infer sex chromosomal linkage but cannot distinguish between X- or Y-linkage (Figure 3; Supplementary file 10). Thus, we conclude that 80% (16/20, Figure 3B) of amplifications occurred on sex chromosomes. This high fraction is significantly higher than the null expectation (Test of Proportions, p=2.3e-5) if SNBP amplifications were randomly distributed between sex chromosomes or autosomes. Under the null expectation, ~33% should be on sex chromosomes, given each chromosome arm, except the dot chromosome, has a similar size.

To better understand the evolutionary origins and potential function of SNBP gene amplifications, we investigated the amplification of tHMG in D. simulans and sister species (Figure 4). We traced its evolutionary history using a combination of phylogenetic and shared synteny analyses. From these analyses, we inferred that tHMG copies on the heterochromatic X chromosome (tHMG-hetX) experienced two duplications before the amplification. The first of these occurred onto the euchromatic X region, proximal to CG12691. Subsequently, a second duplication spanning X-linked tHMG and part of CG12691 occurred onto pericentromeric X (Figure 4A). We analyzed their sequences to assess the evolutionary pressures shaping the amplified tHMG-hetX relative to the parental tHMG copies (tHMG-Anc). Within an 81 amino acid residue stretch, we found 19 non-synonymous changes that occurred within tHMG-hetX compared to just 4 non-synonymous changes that occurred within tHMG-Anc (Figure 4B). Thus, tHMG-hetX evolves considerably faster than tHMG-Anc. Branch analyses using PAML revealed that both tHMG-hetX and tHMG-Anc branches have significantly higher protein evolution rates than branches in other species (Figure 4C; ω = 1.6, LRT test, p=0.007; Supplementary file 11). However, the branch-site test did not reveal clear evidence of positive selection on any residues (LRT test, p=0.23; Supplementary file 11). Our analyses on the tHMG expansion thus reveal a rapid, complex series of chromosome rearrangements leading to sex chromosome SNBP amplifications, which bear hallmarks of positive selection.

Tracing the duplication and amplification of tHMG genes in D.simulans and close relatives.

(A) Using a combination of genome assemblies and phylogenetic analyses, we traced the evolutionary origins and steps that led to the massive amplification of tHMG genes on the D. simulans X chromosome. The first step in this process was the duplication of the ancestral tHMG gene (flanked by CCT1 and Octb1R) on the 3R chromosomal arm to a new location on 3R (tHMG-3R#2 now flanked by CG31468 and Gba1a) and to a location on the X chromosome euchromatin, where tHMG-euX is flanked by CG12691 and CG15572. We infer that this CG12691-tHMG-euX locus then duplicated to another locus in X-heterochromatin, between Atbp and the flamenco locus, and further amplified. These resulting copies experienced different fates in D. simulans and its sibling species. For example, in D. sechellia, tHMG-3R#2, tHMG-euX, and tHMG-hetX were all lost but a degenerated copy of tHMG-3R#2 and flanking genes can be found on its Y chromosome. In contrast, in D. mauritiana, tHMG-3R#2 pseudogenized on 3R, tHMG-euX was retained while tHMG-hetX underwent an amplification to a copy number of 15 tandemly arrayed genes in the X heterochromatin. Finally, in D. simulans, tHMG-3R#2 was completely lost, tHMG-euX was pseudogenized, and tHMG-hetX amplified to a copy number of 15 on the X heterochromatin. We note that the amplification unit sizes are different between D. simulans and D. mauritiana, suggesting that these were independent amplifications. Moreover, we detected different copy numbers (all more than 30) of tHMG-hetX across three sequenced strains of D. simulans we surveyed. This difference is likely due to both incomplete assemblies of this region and strain-specific differences. In addition to this X chromosomal expansion, we also found a few degenerated copies of tHMG on the 3R heterochromatic region and the Y chromosome. (B) The alignment shows the divergence between different tHMG copies in the D. simulans clade and D. melanogaster. Surprisingly, we X-linked tHMG duplicates diverged more from parental genes on autosomes, indicating that they experienced different evolutionary forces than the parental copies. Among 243 aligned nucleotide sites, we found 19 non-synonymous changes and only 3 synonymous changes shared in all X-linked copies after they diverged from the parental copy. Similarly, four non-synonymous changes and no synonymous change occurred on the parental copy in the ancestral species of the simulans clade. Most non-synonymous changes are in the DNA-binding HMG box. As a result, parental copies and new X-linked copies in D. simulans and D. mauritiana only share ~70% protein identity, which is very low given the <3 MY divergence. Our branch test using PAML further shows that both branches have significantly higher protein evolution rates (ω = 1.6, LRT test, p=0.007; Supplementary file 11). However, we did not find evidence of positive selection using a branch-site test (LRT test, p=0.23; Supplementary file 11). (C) Phylogenetic analyses of the various tHMG genes confirm the chronology of events outlined in (A) and find strong evidence of concerted evolution among the amplified tHMG-hetX copies on D. mauritiana and D. simulans, in which copies from the X-linked heterochromatic region are highly homogeneous within species, but diverged between species. For comparison, we showed the species tree on the left, and the phylogeny of three D. simulans clade species is not solved due to lineage sorting and gene flow. To simplify the analysis, we only used sequences that are annotated in NCBI databases.

Short life of sex chromosome-linked SNBP genes

Previous studies showed that the Dox meiotic driver arose from an SNBP partial gene amplification (ProtA/B) in D. simulans (Vedanayagam et al., 2021; Muirhead and Presgraves, 2021; Vedanayagam et al., 2022). We hypothesize that the Drosophila sex-chromosomal SNBP amplifications we have found might similarly be involved in genetic conflicts between sex chromosomes across the Drosophila genus, via X-versus-Y meiotic drive. In contrast, all ancestral single-copy SNBP genes that gave rise to these amplifications are encoded on autosomal loci and thus are more likely to encode suppressors of meiotic drive, as is the case for the ProtA/B genes against Segregation Distorter (Gingell and McLean, 2020).

If our hypothesis for this duality of SNBP gene functions is correct, we would further predict that ancestrally autosomal SNBP genes that became linked to sex chromosomes would preferentially amplify to become meiotic drivers or be lost due to the loss of ancestral functional requirements. To test this hypothesis, we surveyed SNBP genes that became linked to sex chromosomes via chromosome fusions. We found three SNBP genes, CG14835, CG34269, and ddbt, which are widely retained in Drosophila species on the ancestrally autosomal Muller D element. Both CG14835 and ddbt genes independently became X-linked in the D. willistoni, D. pseudoobscura, and D. repletoides clades, while CG34269 became X-linked in the two former clades. Consistent with our hypothesis, some newly X-linked SNBP genes either degenerated (two instances out of eight) or translocated back to autosomes (one instance out of eight). In five cases, SNBP genes were still retained on sex chromosomes (Figure 5; Supplementary file 12). Among these five cases, we observed one amplification event –ddbt amplified to six copies in D. repletoides (Figure 3B), consistent with the idea that it may act as a meiotic driver. In contrast, we found no instances of pseudogenization or subsequent translocation to the X chromosome of SNBP genes that are still preserved on their original autosomal locations or involved in chromosome fusions between autosomes (0/16). This difference is highly significant (Figure 5; Supplementary file 12; 3:5 versus 0:16, Fisher’s exact test, p=0.03). However, this pattern may have alternate explanations, including previous findings that male-biased genes preferentially avoid the X chromosome (Sturgill et al., 2007).

Evolutionary retention, degeneration, or translocation of sperm nuclear basic protein (SNBP) genes following chromosomal fusions.

SNBP genes are ancestrally encoded on autosomes. Following chromosome fusion over Drosophila evolution, we found eight cases in which three SNBP genes (CG14835, CG34269, and ddbt) became linked to sex chromosomes. In 1/8 cases, SNBP genes translocated back to an autosome. In 2/8 cases, the sex chromosome-linked SNBP genes degenerated despite being otherwise widely conserved in non-montium Drosophila species. In 5/8 cases, SNBP genes were retained on neo-sex chromosomes in 5/8 cases. Among these, we observed one amplification event; ddbt amplified to six copies in D. repletoides. In contrast to sex chromosomal linkage, SNBP genes that remained linked to autosomes despite chromosomal fusions were strictly retained in 16/16 cases. These retention patterns differ significantly between sex chromosomes and autosomes (Fisher’s exact test, p=0.03).

Loss of SNBP genes in the montium group coincides with X-Y chromosomal fusion

Our phylogenomic analyses also highlighted one Drosophila clade—the montium group of species (including D. kikkawai)—which suffered a precipitous loss of at least five SNBP genes (Figure 3). In contrast to D. kikkawai, three of these five SNBP genes are retained in all other D. melanogaster group species. For the other two genes, Mst77F was lost twice and tHMG was lost three times (D. fuyamai has lost both Mst77F and tHMG). A closer examination allowed us to infer that six different SNBP genes underwent 11 independent degeneration events in the montium group (Figure 6A). Intriguingly, five of six SNBP genes lost in the montium clade (Mst77F, Prtl99C, Mst33A, tHMG, CG14835) are also among the eight SNBP genes subject to sex chromosome-specific amplifications in other Drosophila species (Figure 3 and Table 2).

Figure 6 with 1 supplement see all
A dramatic loss of sperm nuclear basic protein (SNBP) genes coincided with a fusion of X and Y chromosomes in the montium group species.

(A) Using a phylogeny of species from the montium group, we traced the retention or loss of SNBP genes that are otherwise primarily conserved across other Drosophila species. Genes retained in autosomal syntenic locations are indicated in black squares, whereas pseudogenes are indicated by an empty square with a diagonal line. We traced a total of 11 independent pseudogenization events. Three of these pseudogenization events occurred early such that all species from this group have lost CG14835, Mst33A, and tHMG. Three other SNBP genes were lost later (in some cases on multiple occasions) and are, therefore, missing only in a subset of species. For example, we infer that CG34629 was lost on at least five independent occasions (and also in outgroup species D. ananassae). We correlated this dramatic loss of otherwise-conserved SNBP genes with the X-chromosome linkage of genes that are ancestrally Y-linked in other Drosophila species, shown on the right. For example, of 12 Y-chromosomal genes in most related species, including D. melanogaster and D. ananassae, most are now X-linked in montium group species (e.g., 11/12 in D. triauraria, 9/11 in D. jambulina, and 7/10 in D. bocqueti and D. kikkawai). We note these species still harbor a Y chromosome; however, this Y-chromosome lacks most ancestrally Y-linked genes. (B) We traced the chromosomal arrangement and linkage of ancestrally Y-linked genes in D. triauraria using new genome assembly (NCBI accession: GCA_014170315.2) and genetic crosses in (C). We were able to show that the D. triauraria X chromosome represents a fusion of the X chromosome (e.g., from D. melanogaster) and chromosomal segments containing 11 protein-coding genes that are typically found on the Y chromosome (e.g., from D. melanogaster). Genetic crosses confirmed the X-linkage of 9 of these previously Y-linked genes. The lack of allelic differences in D. triauraria prevented us from confirming this for the other two genes: CCY and WDY. (C) An example of the genetic cross used to verify X-linkage. Using genetic crosses between different D. triauraria strains with allelic variation in ancestral Y-linked genes, we evaluated whether male flies inherit these genes maternally, paternally, or from both parents. We observed only maternal inheritance, confirming the X-chromosomal linkage of these genes.

Notably, we did not find SNBP amplification events in any species of the montium clade. Given our hypothesis that autosomal SNBP genes might be linked to the suppression of meiotic drive (above), we speculated that the loss of these genes in the montium group of Drosophila species may have coincided with reduced genetic conflicts between sex chromosomes in this clade.

How could such reduction in sex chromosomal genetic conflicts arise? An important clue came from a previous study, which showed that many ancestrally Y-linked genes are present in females because of possible relocation to other chromosomes in the montium group (Dupim et al., 2018). We revisited this question to pinpoint which Y chromosomal gene translocations coincided with SNBP degeneration in this lineage. Using the available assemblies with Illumina-based chromosome assignment, we surprisingly found that most ancestrally Y-linked genes are actually linked to the X chromosome (Figure 6A). For example, we were able to unambiguously infer X-chromosomal linkage for most ancestrally Y-linked genes in D. kikkawai (7/10), D. jambulina (9/11), D. bocqueti (7/10), D. aff. chauvacae (7/8), and D. triauraria (11/12) (Figure 6A), although some ancestrally Y-linked genes are not present in the assemblies. Moreover, in D. triauraria, we found that 11 of 12 ancestrally Y-linked genes, i.e., all except JYalpha, are located on the same region of the X chromosome (Figure 6B). The most parsimonious explanation for these findings is a single translocation fused most of the Y chromosome to the X chromosome in the ancestor of the montium group of species. Although montium group species still harbor a Y chromosome, their Y chromosome is missing most ancestrally Y-linked genes. After this ancient X-Y chromosomal fusion, some Y-linked genes, e.g., PRY, relocated back to the Y chromosome in some species (Figure 6—figure supplement 1).

We carried out genetic mapping studies to confirm our unexpected inference of a Y-to-X translocation in the montium group. For this, we performed genetic crosses between different D. triauraria strains harboring polymorphisms on ancestrally Y-linked genes (Conner et al., 2021). To unambiguously infer the chromosomal location of these genes, we focused on whether SNPs in these genes were paternally or maternally inherited. If the SNPs were Y-linked, we would expect them to be strictly paternally inherited, whereas we would expect both paternal and maternal inheritance in the case of autosomal linkage. In contrast to these expectations, all F1 males inherit alleles of ancestral Y-linked genes from their mother, unambiguously indicating their X-chromosomal linkage (Figure 6C). Our finding of an X-Y fusion via both computational and genetic linkage analyses is remarkable because X-Y fusion is an extremely rare evolutionary event that has only previously been documented in one vole species (Couger et al., 2021).

The translocation of a large segment of the Y-chromosome to the X-chromosome in the montium group would render any X-versus-Y meiotic drive encoded in this chromosomal region obsolete or costly. As a result, there would be active selection to jettison such meiotic drive systems on both the X and Y chromosomes. Indeed, no meiotic drive has been documented in the montium species (Courret et al., 2019), although this absence could simply reflect the inadequate number of studies for meiotic drive. Following an X-Y fusion, any autosomal SNBP genes required to suppress meiotic drive would become dispensable, leading to their degeneration. Thus, our hypothesis of genetic conflicts between sex chromosomes can explain the loss or degeneration of SNBP genes following alleviation of the sex-chromosomal conflict as well (Figure 7).

Genetic conflict between sex chromosomes may explain the rapid turnover of sperm nuclear basic protein (SNBP) genes in Drosophila species.

SNBP genes are ancestrally encoded on autosomes where we hypothesize that some of them act to suppress meiotic drive between sex chromosomes (e.g., ProtA/B). However, in some cases, paralogs of these SNBP genes duplicate onto sex chromosomes where they undergo dramatic amplification. We propose that this amplification creates an opportunity for them to act as meiotic drive elements themselves (e.g., Dox), imbuing sex chromosomes that inherit them with transmission advantages. A fusion of the sex chromosomes (e.g., D. montium species group) leads to a loss of meiotic competition between sex chromosomes, which will subsequently lead to the loss or degeneration of the suppressing SNBP genes on autosomes since their drive suppression functions are rendered superfluous.


Our analyses of Drosophila SNBP genes reveal many similar patterns to rapid protamine evolution observed in mammals, including a pervasive trend of positive selection. However, there are also some dramatic differences, which may stem from distinct biological functions and selective pressures. One of the most dramatic differences is the evolutionary turnover of SNBP genes in Drosophila versus mammals. Most mammals share four SNBP genes: TNP1, TNP2, PRM1, and PRM2. In contrast, SNBP repertoires vary extensively between Drosophila species, with both dramatic gains and losses. We discovered several independent, species-specific amplifications of SNBP genes from 5 to >50 copies that preferentially occurred on sex chromosomes (Figure 3 and Table 2). Conversely, we also found that several SNBP genes were lost in the montium group, coinciding with an X-Y chromosome fusion event (Figure 6 and Table 2). In other lineages, three SNBP genes that became linked to sex chromosomes via chromosomal fusions either degenerated or translocated back to autosomes (Figure 5).

Based on all these observations, we hypothesize that many SNBP genes, such as the ProtA/B genes in D. melanogaster, arise and are retained on autosomes to act as suppressors of meiotic drive (Gingell and McLean, 2020). In contrast, sex chromosomal amplifications of SNBP genes, like the Dox amplification, act as meiotic drivers, potentially by disrupting the histone-to-protamine transition directly via their protein products or indirectly by affecting the expression or function of parental autosomal SNBP genes (Vedanayagam et al., 2021; Muirhead and Presgraves, 2021; Tao et al., 2007a). X-Y chromosome fusions eliminate the circumstances that facilitate meiotic drive and may lead to the degeneration of otherwise conserved SNBP genes, whose functions as drive suppressors are no longer required. Thus, unlike in mammals, sex chromosome-associated meiotic drive might be one of the primary causes of SNBP evolution in Drosophila species.

Why would meiotic drive only influence Drosophila, but not mammalian, SNBP evolution? One important distinction may arise from the timing of SNBP transcription. In D. melanogaster, most SNBP genes are transcribed before meiosis but translated after meiosis (Jayaramaiah Raja and Renkawitz-Pohl, 2005; Rathke et al., 2007; Barckmann et al., 2013). Thus, SNBP transcripts from a single allele, e.g., an X-linked allele, can be inherited and translated by all sperm, regardless of which chromosomes they carry. Consequently, they can act as meiotic drivers by causing chromatin dysfunction in sperm without the allele, e.g., Y-bearing sperm. In contrast, mammalian SNBPs are only expressed post-meiosis (Hecht et al., 1986; Peschon et al., 1987) and, therefore, individual SNBP alleles can only affect the chromatin states of sperm in which they reside. Therefore, mammalian SNBP genes may be less likely to evolve as meiotic drivers.

Our findings do not rule out the possibility that forces other than meiotic drive might also be important for driving the rapid evolution and turnover of SNBP genes in Drosophila species. Indeed, we note that only some SNBP genes undergo both amplifications and losses across Drosophila species (Figure 3 and Table 2). Other SNBP genes that do not experience such dramatic turnover still evolve rapidly (Table 2), including in D. serrata, a montium group species with a fused X-Y chromosome (Table 1). Signatures of positive selection that do not correspond to sex chromosomal meiotic drive could be instead explained by some form of haploid selection in spermatogenesis (Raices et al., 2019), such as sperm competition, and influence the evolution of sperm morphologies, as has been proposed in mammals.

Another possible selective pressure on SNBP genes may come from endosymbiotic Wolbachia bacteria. One means by which Wolbachia manipulate Drosophila hosts to ensure their preferential propagation is by mediating cytoplasmic incompatibility. Embryos produced between infected males and uninfected females fail to de-compact the paternal pronucleus and arrest in development (Perreault, 1992; Loppin et al., 2000). Recent studies have revealed that Wolbachia produces toxins that prevent the deposition of SNBP during spermatogenesis (Kaur et al., 2022) and delay SNBP removal in embryos post-fertilization to accomplish this cytoplasmic incompatibility (Beckmann et al., 2019; Landmann et al., 2009). Indeed, deletions of ProtA/ProtB exacerbate the intensity of cytoplasmic incompatibility imposed by Wolbachia in D. melanogaster (Kaur et al., 2022). Thus, selective pressure from Wolbachia toxins could also provide selective pressures for SNBP innovation.

Our analyses have focused on SNBP genes that can be readily identified because they possess HMG DNA-binding domains. However, this does not represent a comprehensive list of SNBP genes. Indeed, recent findings have shown that the atlas SNBP gene from D. melanogaster does not encode an HMG domain entirely and has arisen de novo (Rivard et al., 2021). Such genes might use an alternate means to bind DNA or may indirectly affect the localization of other SNBP proteins and therefore sperm chromatin. However, even focusing on the HMG-domain containing SNBP genes reveals an unexpected relationship between their essential function and evolution. SNBP genes encoding essential fertility functions arose recently and have been frequently lost in Drosophila species. In contrast, SNBP genes less essential for male fertility are more prone to evolve under positive selection. This suggests that SNBP genes with redundant roles in male fertility are more likely to acquire new function.

One clue for this unexpected lability emerges from discovering SNBP genes that are essential for D. melanogaster fertility but were lost in other species. For example, ddbt is a conserved SNBP gene with essential sperm telomere-capping function in D. melanogaster; loss of ddbt leads to defects in telomere capping and induction of telomeric fusions in embryos (Yamaki et al., 2016). We find that the two lineages that lost ddbt—all D. willistoni subgroup species, and D. albomicans in the D. immigrans species group—have a pair of neo-sex chromosomes due to the fusion of Muller element D and sex chromosomes (Sturtevant and Novitski, 1941; Chang et al., 2010; Zhou et al., 2012; Ellison and Bachtrog, 2019). We hypothesize that the loss of ddbt might have led to these chromosome fusions that cause the independently evolved neo-sex chromosomes. Alternatively, since telomeric sequences rapidly evolve in an ‘arms-race’-like dynamic with telomere-binding proteins across Drosophila species (Saint-Leandre and Levine, 2020), particular chromatin rearrangements may have obviated the essential function of ddbt in some species, as has been hypothesized for other paternal-effect lethal chromatin genes (Levine et al., 2015). The binding specificity and function of ddbt also hint that other SNBPs might also bind to specific genomic regions and have other important biological roles, e.g., gene regulation and protecting against DNA damage (Rathke et al., 2010). This suggests that the nature of essential SNBP fertility functions can itself be idiosyncratic and species-specific, arising from the underlying rapid evolution of chromatin, as has been hypothesized for evolutionarily young but essential genes involved in centromere and heterochromatin function (Kasinathan et al., 2020; Ross et al., 2013).

Materials and methods

Molecular evolutionary analyses of SNBP genes

Request a detailed protocol

We use PAML 4.9 (Yang, 2007; RRID:SCR_014932) to calculate protein evolution rates (dN/dS) of SNBP genes and conduct site-model, branch-model, and branch-site model analyses. We compare the protein evolution rates of SNBP genes to the genome-wide rates (8521 genes) from the 12 Drosophila genomes project (Clark et al., 2007). Many SNBP genes analyzed in our study were missed in previous analyses because they are rapidly evolving and hard to align. Therefore, we aligned the coding sequences of orthologous SNBP genes from the same six Drosophila species used to calculate dN/dS previously (Clark et al., 2007), taking care to only use orthologous sequences. For all phylogenic analyses, we first constructed maximum-likelihood trees using iqtree 2.1.3 (Minh et al., 2020) using parameters '-m MFP -nt AUTO -alrt 1000 -bb 1000 -bnni'. We then calculated protein evolution rates of SNBP genes using the same parameters with the generated gene trees (model = 0 and CodonFreq = 2).

For the site-model codeml analyses, we analyzed 9–17 unambiguous orthologs from species in the melanogaster group to increase the power and accuracy. We compared NS sites models M1a to M2a, and M7 or M8a, to M8 using likelihood ratio tests to ask whether models allowing a class of sites where dN/dS exceeds 1 (e.g., M8) provide a better fit to the data than models that disallow positive selection (e.g., M7, M8a). We also used different codon parameter values (CodonFreq = 0, 2, 3) in our analyses to check whether our results were robust.

For the branch-model codeml analyses, we first simplified the tree by reconstructing the ancestral sequences of ampliconic genes in each species using MEGAX (10.1.8) (Stecher et al., 2020). We then compared models by assigning different protein evolution rates to different branches using PAML (CodonFreq = 2) and likelihood ratio tests. We tested several models, starting with the null model, which models the same protein evolutionary rate across branches (model = 0). We compared this null model to alternative models in which all X-linked branches share a common protein evolutionary rate that is different from other branches (model = 2). Finally, we compared a model in which the early duplication branches on both the X-linked copies and the parental copy share different protein evolutionary rates than in other branches (model = 2). We found that this latter model, with a higher protein evolution rate at early duplication branches, fits best across all models, so we applied this setting to conduct the branch-site test. We compared two models with all sites sharing the same protein evolution rate (fix_omega = 1) and various evolution rates (fix_omega = 0).

To look for positive selection in two individual lineages, D. melanogaster and D. serrata, we applied McDonald–Kreitman tests to compare within-species polymorphism and between-species divergence (McDonald and Kreitman, 1991). We used D. simulans as the closely related outgroup species for the D. melanogaster analysis, and D. bunnanda for the D. serrata analysis. To polarize the changes in each lineage, we inferred ancestral sequences of D. melanogaster and D. simulans using seven species in the D. melanogaster group (D. melanogaster, D. simulans, D. mauritiana, D. sechellia, D. yakuba, D. erecta, and other well-aligned outgroup species: D. biarmipes, D. elegans, D. eugracilis, D. ficusphila, or D. rhopaloa) using MEGAX (10.1.8; RRID:SCR_000667; Stecher et al., 2020). Similarly, we used five species in the montium group (D. serrata, D. bunnanda, D. birchii, D. truncata, and D. mayri) to polarize the D. serrata-specific changes. We extracted population data from public datasets of >1000 D. melanogaster strains (Hervas et al., 2017; Lack et al., 2016) and 111 D. serrata strains (Reddiex et al., 2018). We conducted both polarized and unpolarized McDonald–Kreitman tests using R scripts (https://github.com/jayoung/MKtests_JY) and confirmed our findings using an online server (Egea et al., 2008; http://mkt.uab.es/mkt/MKT.asp).

Searching for homologs of SNBP genes in Drosophila and outgroup species

Request a detailed protocol

We used tblastn and reciprocal blastx to search homologs of all SNBP genes across all genome assemblies (RRID:SCR_004870; Altschul et al., 1990) using D. melanogaster protein sequences as queries. Since SNBP genes are rapidly evolving, we used the following parameters: e-value < 1e-2, amino acid identity > 20%, and blast score > 10. We further required that the best reciprocal blastx hit when searching D. melanogaster genes was the original query gene to ensure that we were recovering true orthologs. To further confirm questionable orthologs, e.g., only one species with the homolog in the lineage, we examined its synteny, anticipating that orthologs should also have shared syntenic contexts. We also examined the syntenic regions of SNBP genes and conducted blastx using a lower threshold (e-value < 1) to confirm the loss of SNBP genes in some lineages, especially in the montium group species.

We used the abSENSE package (RRID:SCR_023223; Weisman et al., 2020) to calculate the probability of not detecting homologs in more diverged species (using E-value = 1), enabling us to distinguish whether our inability to detect homologs was due to rapid divergence or true absence.

Transcriptomic analyses of SNBP genes

Request a detailed protocol

We combined the public gene annotations with our own annotated SNBP gene annotations and mapped publicly available transcriptome datasets (Supplementary file 13) to the genome assemblies using HiSAT2 (v2.2.1 with parameters –exon and –ss to specify the exon positions and splice sites; RRID:SCR_015530; Kim et al., 2019). We then estimated the expression levels using the gene annotations as input for Stringtie (v2.1.4 with parameters -dta -G to specify annotation files; RRID:SCR_016323; Kovaka et al., 2019). For single-cell transcriptomic data, we downloaded and used the scripts and data from (Witt et al., 2021). We modified the published scripts to extract and plot the expression level of SNBP genes using the NormalizeData function of Seurat (RRID:SCR_007322; Hao et al., 2021).

Assigning sex-linkage of contigs in genome assemblies

Request a detailed protocol

We mapped Nanopore and Illumina reads from male samples (sometimes from mixed-sex or female samples) to genome assemblies using minimap2 (RRID:SCR_018550; Li, 2021) and bwa-mem (RRID:SCR_022192; Vasimuddin et al., 2019) using the default parameters. We calculated coverage of each site using samtools depth and estimated the median coverage of each 10 kb window and each contig. We then examined the genome-wide distribution of 10 kb window coverage using the density function in R. We called two peaks of coverage using turnpoints function in R. As we expected, the prominent peak with higher coverage mostly represents autosomal regions, whereas the lower coverage peak mostly represents sex-linked regions. We used the average of coverage from the two peaks as the threshold to assign autosomal and sex-chromosomal contigs.

We also confirmed our assignments using the sequence-homology method. We identified orthologs of 3285 highly conserved genes in all species using BUSCO v5.0.0 with default setting and diptera_odb10 database (RRID:SCR_015008; Manni et al., 2021). Since the X chromosome is conserved across Drosophila species, we identified orthologs of X-linked genes to assign X-linked contigs. Lastly, for species with Illumina data from both males and females, we distinguished X from Y chromosome-linked contigs using a previously described method (Chang and Larracuente, 2019). We could not reliably distinguish their sex chromosomes from autosomes in Lordiphosa species (probably due to high heterozygosity or lower assembly quality) and therefore could not confidently assign the chromosomal location of their SNBP amplifications.

We used genetic mapping to examine the X-linkage of ancestral Y-linked genes in D. triauraria. We first identified polymorphic SNPs in D. triauraria SNBP genes using publicly available Illumina data (Conner et al., 2021) and called SNPs (bcftools call -m -Oz; RRID:SCR_005227) in each strain (Li et al., 2009; Li, 2011). We took advantage of the fact that F1 males will only inherit maternal X alleles. Four D. triauraria strains (14028-0691.01 [National Drosophila Species Stock Center], KMJ1 [Ehime-Fly: E-15304], OKNG12-6 [Ehime-Fly: E-15309] and YKS-MTK [Ehime-Fly: E-15303]) were kindly provided by Dr. Brandon Cooper. We crossed two strains with different alleles of ancestrally Y-linked genes (14028-0691.01 with KMJ1 and OKNG12-6 with YKS-MTK) and genotyped these genes in F1 males using PCR (Supplementary file 14) and Sanger sequencing to examine whether their allele-specific inheritance patterns were more consistent with Y-linkage (paternal inheritance), X-linkage (maternal inheritance), or autosomal linkage (both paternal and maternal inheritance).

CRISPR/Cas9 knockout and fertility assays

Request a detailed protocol

To generate the CG30056 knockout strain, we first cloned two guide RNAs, targeting either the 5′ or 3′ end of CG30056, into pCFD4-U6:1_U6:3tandemgRNAs (RRID:Addgene_49411) using NEBuilder HiFi DNA Assembly Master Mix (NEB catalog E2621). For the repairing construct, we used independent PCR reactions to amplify 3xP3 DsRed, the backbone of pDsRed-attP (RRID:Addgene_51019), and ~1 kb homologous sequences of upstream and downstream of CG30056 using PCR independently. These four fragments were annealed using NEBuilder HiFi DNA Assembly Master Mix. We then used the Q5 Site-Directed Mutagenesis Kit (NEB catalog E0554S) to mutate the gRNA target PAM sites on the repairing construct so that they would not also be targeted by the guide RNAs we used. All primers are listed in Supplementary file 14. These two constructs were injected into y[1] M{GFP[E.3xP3]=vas-Cas9.RFP-}ZH-2A w[1118] (RRID:BDSC_55821) embryos by BestGene. The resulting transgenic flies were backcrossed to yw, selected using the DsRed marker, and balanced by CyO. The transgenic flies were further confirmed by PCR using independent primer sets (Figure 3A and Supplementary file 14).

For comparisons, we also tested male fertility effects of two SNBP genes previously shown to be essential (Mst77F) or not (Tpl94D) for male fertility. We obtained Mst77F (Δ1) knockout flies (kindly provided by Dr. Benjamin Loppin Kimura and Loppin, 2016) and two fly strains carrying large deletions spanning Mst77F (RRID:BDSC_24956 and RRID:BDSC_27369). For Tpl94D, we used a mutant from the Drosophila Gene Disruption Project (RRID:BDSC_26333; Bellen et al., 2004) and a fly carrying a large deletion spanning this gene (BDSC_7672; Parks et al., 2004).

We obtained males for fertility assays by crossing female virgin transgenic flies with other transgenic flies or flies carrying a large deletion (RRID:BDSC_30585). To measure their fertility, each resulting 2–5-day-old F1 male was crossed to two 2–5-day-old virgin females from the wildtype Oregon R D. melanogaster strain (kindly provided by Dr. Courtney Schroeder; RRID:BDSC_5 and Wolbachia cured) at room temperature. We transferred the mating pairs to new vials every three days and counted all resulting offspring from the first 12 days.

To detect subtle fertility defects in CG30056 knockout flies, we replaced the balancer chromosome with the wildtype chromosome from the strain used for the CRISPR experiment (RRID:BDSC_55821). We also performed a sperm exhaustion assay in which we crossed each 2–5-day-old F1 male to ten 2–5-day-old virgin females from the wildtype Oregon R D. melanogaster strain at 25°C. We transferred the mating pairs to new vials every 3–4 days and counted all resulting offspring from the first 14 days. We excluded crosses that produced less than 20 offspring at the end, except those from Mst77F mutants. Most of these crosses did not have progeny because they did not mate well. Since Mst77F mutants are sterile (Kimura and Loppin, 2016), we cannot exclude such data.

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting file; Source Data files have been provided for all Figures. No sequence data have been generated for this manuscript.


    1. Chang TP
    2. Tsai TH
    3. Chang HY
    Fusions of muller’s elements during chromosome evolution of Drosophila albomicans
    Zoological Studies 49:152.
    1. Clark AG
    2. Eisen MB
    3. Smith DR
    4. Bergman CM
    5. Oliver B
    6. Markow TA
    7. Kaufman TC
    8. Kellis M
    9. Gelbart W
    10. Iyer VN
    11. Pollard DA
    12. Sackton TB
    13. Larracuente AM
    14. Singh ND
    15. Abad JP
    16. Abt DN
    17. Adryan B
    18. Aguade M
    19. Akashi H
    20. Anderson WW
    21. Aquadro CF
    22. Ardell DH
    23. Arguello R
    24. Artieri CG
    25. Barbash DA
    26. Barker D
    27. Barsanti P
    28. Batterham P
    29. Batzoglou S
    30. Begun D
    31. Bhutkar A
    32. Blanco E
    33. Bosak SA
    34. Bradley RK
    35. Brand AD
    36. Brent MR
    37. Brooks AN
    38. Brown RH
    39. Butlin RK
    40. Caggese C
    41. Calvi BR
    42. Bernardo de Carvalho A
    43. Caspi A
    44. Castrezana S
    45. Celniker SE
    46. Chang JL
    47. Chapple C
    48. Chatterji S
    49. Chinwalla A
    50. Civetta A
    51. Clifton SW
    52. Comeron JM
    53. Costello JC
    54. Coyne JA
    55. Daub J
    56. David RG
    57. Delcher AL
    58. Delehaunty K
    59. Do CB
    60. Ebling H
    61. Edwards K
    62. Eickbush T
    63. Evans JD
    64. Filipski A
    65. Findeiss S
    66. Freyhult E
    67. Fulton L
    68. Fulton R
    69. Garcia ACL
    70. Gardiner A
    71. Garfield DA
    72. Garvin BE
    73. Gibson G
    74. Gilbert D
    75. Gnerre S
    76. Godfrey J
    77. Good R
    78. Gotea V
    79. Gravely B
    80. Greenberg AJ
    81. Griffiths-Jones S
    82. Gross S
    83. Guigo R
    84. Gustafson EA
    85. Haerty W
    86. Hahn MW
    87. Halligan DL
    88. Halpern AL
    89. Halter GM
    90. Han MV
    91. Heger A
    92. Hillier L
    93. Hinrichs AS
    94. Holmes I
    95. Hoskins RA
    96. Hubisz MJ
    97. Hultmark D
    98. Huntley MA
    99. Jaffe DB
    100. Jagadeeshan S
    101. Jeck WR
    102. Johnson J
    103. Jones CD
    104. Jordan WC
    105. Karpen GH
    106. Kataoka E
    107. Keightley PD
    108. Kheradpour P
    109. Kirkness EF
    110. Koerich LB
    111. Kristiansen K
    112. Kudrna D
    113. Kulathinal RJ
    114. Kumar S
    115. Kwok R
    116. Lander E
    117. Langley CH
    118. Lapoint R
    119. Lazzaro BP
    120. Lee S-J
    121. Levesque L
    122. Li R
    123. Lin C-F
    124. Lin MF
    125. Lindblad-Toh K
    126. Llopart A
    127. Long M
    128. Low L
    129. Lozovsky E
    130. Lu J
    131. Luo M
    132. Machado CA
    133. Makalowski W
    134. Marzo M
    135. Matsuda M
    136. Matzkin L
    137. McAllister B
    138. McBride CS
    139. McKernan B
    140. McKernan K
    141. Mendez-Lago M
    142. Minx P
    143. Mollenhauer MU
    144. Montooth K
    145. Mount SM
    146. Mu X
    147. Myers E
    148. Negre B
    149. Newfeld S
    150. Nielsen R
    151. Noor MAF
    152. O’Grady P
    153. Pachter L
    154. Papaceit M
    155. Parisi MJ
    156. Parisi M
    157. Parts L
    158. Pedersen JS
    159. Pesole G
    160. Phillippy AM
    161. Ponting CP
    162. Pop M
    163. Porcelli D
    164. Powell JR
    165. Prohaska S
    166. Pruitt K
    167. Puig M
    168. Quesneville H
    169. Ram KR
    170. Rand D
    171. Rasmussen MD
    172. Reed LK
    173. Reenan R
    174. Reily A
    175. Remington KA
    176. Rieger TT
    177. Ritchie MG
    178. Robin C
    179. Rogers Y-H
    180. Rohde C
    181. Rozas J
    182. Rubenfield MJ
    183. Ruiz A
    184. Russo S
    185. Salzberg SL
    186. Sanchez-Gracia A
    187. Saranga DJ
    188. Sato H
    189. Schaeffer SW
    190. Schatz MC
    191. Schlenke T
    192. Schwartz R
    193. Segarra C
    194. Singh RS
    195. Sirot L
    196. Sirota M
    197. Sisneros NB
    198. Smith CD
    199. Smith TF
    200. Spieth J
    201. Stage DE
    202. Stark A
    203. Stephan W
    204. Strausberg RL
    205. Strempel S
    206. Sturgill D
    207. Sutton G
    208. Sutton GG
    209. Tao W
    210. Teichmann S
    211. Tobari YN
    212. Tomimura Y
    213. Tsolas JM
    214. Valente VLS
    215. Venter E
    216. Venter JC
    217. Vicario S
    218. Vieira FG
    219. Vilella AJ
    220. Villasante A
    221. Walenz B
    222. Wang J
    223. Wasserman M
    224. Watts T
    225. Wilson D
    226. Wilson RK
    227. Wing RA
    228. Wolfner MF
    229. Wong A
    230. Wong GK-S
    231. Wu C-I
    232. Wu G
    233. Yamamoto D
    234. Yang H-P
    235. Yang S-P
    236. Yorke JA
    237. Yoshida K
    238. Zdobnov E
    239. Zhang P
    240. Zhang Y
    241. Zimin AV
    242. Baldwin J
    243. Abdouelleil A
    244. Abdulkadir J
    245. Abebe A
    246. Abera B
    247. Abreu J
    248. Acer SC
    249. Aftuck L
    250. Alexander A
    251. An P
    252. Anderson E
    253. Anderson S
    254. Arachi H
    255. Azer M
    256. Bachantsang P
    257. Barry A
    258. Bayul T
    259. Berlin A
    260. Bessette D
    261. Bloom T
    262. Blye J
    263. Boguslavskiy L
    264. Bonnet C
    265. Boukhgalter B
    266. Bourzgui I
    267. Brown A
    268. Cahill P
    269. Channer S
    270. Cheshatsang Y
    271. Chuda L
    272. Citroen M
    273. Collymore A
    274. Cooke P
    275. Costello M
    276. D’Aco K
    277. Daza R
    278. De Haan G
    279. DeGray S
    280. DeMaso C
    281. Dhargay N
    282. Dooley K
    283. Dooley E
    284. Doricent M
    285. Dorje P
    286. Dorjee K
    287. Dupes A
    288. Elong R
    289. Falk J
    290. Farina A
    291. Faro S
    292. Ferguson D
    293. Fisher S
    294. Foley CD
    295. Franke A
    296. Friedrich D
    297. Gadbois L
    298. Gearin G
    299. Gearin CR
    300. Giannoukos G
    301. Goode T
    302. Graham J
    303. Grandbois E
    304. Grewal S
    305. Gyaltsen K
    306. Hafez N
    307. Hagos B
    308. Hall J
    309. Henson C
    310. Hollinger A
    311. Honan T
    312. Huard MD
    313. Hughes L
    314. Hurhula B
    315. Husby ME
    316. Kamat A
    317. Kanga B
    318. Kashin S
    319. Khazanovich D
    320. Kisner P
    321. Lance K
    322. Lara M
    323. Lee W
    324. Lennon N
    325. Letendre F
    326. LeVine R
    327. Lipovsky A
    328. Liu X
    329. Liu J
    330. Liu S
    331. Lokyitsang T
    332. Lokyitsang Y
    333. Lubonja R
    334. Lui A
    335. MacDonald P
    336. Magnisalis V
    337. Maru K
    338. Matthews C
    339. McCusker W
    340. McDonough S
    341. Mehta T
    342. Meldrim J
    343. Meneus L
    344. Mihai O
    345. Mihalev A
    346. Mihova T
    347. Mittelman R
    348. Mlenga V
    349. Montmayeur A
    350. Mulrain L
    351. Navidi A
    352. Naylor J
    353. Negash T
    354. Nguyen T
    355. Nguyen N
    356. Nicol R
    357. Norbu C
    358. Norbu N
    359. Novod N
    360. O’Neill B
    361. Osman S
    362. Markiewicz E
    363. Oyono OL
    364. Patti C
    365. Phunkhang P
    366. Pierre F
    367. Priest M
    368. Raghuraman S
    369. Rege F
    370. Reyes R
    371. Rise C
    372. Rogov P
    373. Ross K
    374. Ryan E
    375. Settipalli S
    376. Shea T
    377. Sherpa N
    378. Shi L
    379. Shih D
    380. Sparrow T
    381. Spaulding J
    382. Stalker J
    383. Stange-Thomann N
    384. Stavropoulos S
    385. Stone C
    386. Strader C
    387. Tesfaye S
    388. Thomson T
    389. Thoulutsang Y
    390. Thoulutsang D
    391. Topham K
    392. Topping I
    393. Tsamla T
    394. Vassiliev H
    395. Vo A
    396. Wangchuk T
    397. Wangdi T
    398. Weiand M
    399. Wilkinson J
    400. Wilson A
    401. Yadav S
    402. Young G
    403. Yu Q
    404. Zembek L
    405. Zhong D
    406. Zimmer A
    407. Zwirko Z
    408. Jaffe DB
    409. Alvarez P
    410. Brockman W
    411. Butler J
    412. Chin C
    413. Gnerre S
    414. Grabherr M
    415. Kleber M
    416. Mauceli E
    417. MacCallum I
    418. Drosophila 12 Genomes Consortium
    (2007) Evolution of genes and genomes on the Drosophila phylogeny
    Nature 450:203–218.
    1. Nayernia K
    2. Adham I
    3. Kremling H
    4. Reim K
    5. Schlicker M
    6. Schluter G
    Stage and developmental specific gene expression during mammalian spermatogenesis
    The International Journal of Developmental Biology 40:379–383.

Decision letter

  1. Daniel R Matute
    Reviewing Editor; University of North Carolina, Chapel Hill, United States
  2. Molly Przeworski
    Senior Editor; Columbia University, United States

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

[Editors' note: this paper was reviewed by Review Commons.]


Author response

Overall comments

We are pleased by the reviewers’ comments and appreciate their suggestions for improvements. In addition to correcting small typos throughout the manuscript, the major changes we did in response to their comments are as follows:

  • Changed the title of our paper to reflect the strong evolutionary correlation more accurately between sex chromosomal meiotic drive and gains/losses of SNBP genes in Drosophila.

  • New experiments to test the role of the well-conserved, universally retained SNBP, CG30056, in male fertility in D. melanogaster. Although reviewers had suggested we could eliminate this section, we felt that this would add a lot of weight to the unexpectedly inverse relationship between age/retention and fertility functions of SNBP genes. Thus, over the past few months, we have carried out new experiments with increased sample sizes, better controls, and sperm exhaustion. These new results strengthen our earlier analyses.

  • Better clarification of the X-Y chromosome fusion, which is a new observation, in the montium group via careful rewriting as partly suggested by Reviewer #2.

  • Highlighting that the genetic conflicts hypothesis does not rule out a role for sperm competition or other conflicts in shaping SNBP evolution in a revised Discussion.

All changes in response to the reviewer’s comments have been detailed in our point-by-point response (below). You will see that we have addressed almost all the suggestions made, including with new experiments. The only reviewer suggestions (all optional from Reviewer 3), which we did not directly address in our revision are:

  • Branch specific protamine evolution analyses for sex chromosome amplified SNBP genes: given the state of SNBP gene annotation and the difficulties of assembling these genes in large tandem arrays, this will require considerable work and is beyond the scope of the paper.

  • Covariation between SNBP evolution and sperm morphology: We cannot perform these experiments as there is a paucity of sperm morphology data currently. Obtaining this data reliably is a significant undertaking.

  • Are SNBP genes more prone to be lost than average in the montium group: We have not comprehensively examined all loss events in the montium group or any other Drosophila group. This is also a non-trivial analysis, albeit it would be very interesting. However, we believe the more relevant comparison is whether these lost SNBP genes are more likely to be retained in non-montium species, which they are, as we now highlight.

We hope you will favorably judge our good faith efforts to address all other reviewers’ comments, and their laudatory comments during the previous round of reviews.

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

Chang and Malik present a comprehensive evolutionary analysis of sperm nuclear basic proteins (SNBPs) in Drosophila. In addition, they provide a preliminary functional characterization of one such protein (CG30056) and describe a newly discovered X-Y chromosomal fusion in the Drosophila montium species group. All of these findings are interesting and important, but the headline from this study is the well-supported possibility that SNBPs, or at least a large fraction of them, function in suppressing X vs. Y chromosome meiotic drive. While this hypothesis is challenging to test experimentally, the authors provide strong correlational evidence that SNBPs are associated with drive by documenting these proteins' rapid evolution. This rapid evolution takes the form of sequence changes (as predicted by coevolution between drivers and suppressors of drive), gene amplification in cases when SNBPs move to sex chromosomes (consistent with the SNBP becoming a potential agent of drive for its new "home chromosome"), and gene loss in species with X-Y chromosome fusions (in which drive is not predicted to occur).

Overall, this is an excellent, comprehensive study. The phylogenetic and genomic analyses are first- rate (and one of the first to make use of the new 101 Drosophila genomes); the logic is very well explained; conclusions are supported by multiple lines of evidence; the writing and figures are clear and accessible; and, the findings are fascinating. It's a good sign that it is easy to imagine several experiments one could do to follow up on this study, but I do not feel any are required in revision, as the manuscript is comprehensive as is. Thus, I have just a few minor points the authors may wish to consider in making revisions and a few suggestions for clarity/typos.

We thank the reviewer for their positive comments on our work.

1) I would be interested in whether the authors think that all SNBPs in a given Drosophila species function(ed) in meiotic drive, or whether some fraction may play other roles, such as sexual selection or chromatin compaction, which have been the traditional hypotheses for SNBP function. Relatedly, given the high turnover of SNBPs the authors observe and the fact that some melanogaster-essential SNBPs are younger genes, would they like to comment on whether the subsets of SNBPs involved in drive/suppression vs. chromatin packaging/sperm traits/Wolbachia defense are likely to differ from across fly species?

The reviewer raises an excellent point. In our revised discussion, we now speculate that different SNBPs might have distinct functions. For example, the same subset of SNBPs is subject to gene amplification and loss whereas other SNBPs are subject to less turnover. Moreover, even this stable set of SNBPs evolves rapidly, including in the montium group of species that have undergone dramatic SNBP loss. As the reviewer suggests, sperm competition or pressures from Wolbachia toxins might be is a driving force for sperm evolution. We discuss these possibilities and conclude in our discussion: “Our findings do not rule out the possibility that forces other than meiotic drive are also important for driving the rapid evolution and turnover of SNBP genes in Drosophila species.”

2) What do the authors make of the lower isoelectric points for a few of the SNBPs (e.g., CG31010 with π = 4.77 in Table 1)? These proteins have identifiable HMG box domains, so is the π driven lower by other parts of the protein sequence?

We thank the reviewer for raising this point. We found that the π of HMG domains can range from 6 to 12. Thus, the π is driven by both HMG domains and other parts of proteins. We now include the π of the whole SNBP protein and the HMG domain alone in Table 1. We do not have enough biochemical information to speculate on how these differences could alter SNBP function.

3) For readers less familiar with the field, it may help to spell out (e.g., on p. 6) why the authors consider ProtA/B to be important for fertility. Some of the previous papers on these genes describe them as dispensable – though the present authors are correct that these previous studies do detect fertility defects of various magnitudes under some conditions.

We agree with the reviewer. Previous studies are in disagreement about the importance of ProtA/ProtB for male fertility- while no significant effects were seen under standard fertility assays, sperm exhaustion conditions (mating with excess females) did reveal fertility effects. We have now added these references and discussed ProtA/ProtB more fully in our revision.

4) On p. 9, paragraph 2, the data showing that "six different SNBP genes underwent 11 independent degeneration events in the montium group" are shown in Figure 6A, not 5A.

Thank you. This has been fixed in our revision.

5) The summary Table 2 is useful, but I wonder whether including relative levels of expression and dN/dS in addition to ordinal rankings might help clarify. For instance, if there were a drop off in mean expression level between the 5th and 6th most highly expressed SNBP, this wouldn't be evident from the table.

We agree with this suggestion and have now added this information.

6) In Figure 3, I like the use of the clean CG31010 figure in panel A to illustrate the circular representations. In addition, though, it might be useful to show Prot's graph at this same, larger size, since it's the most complicated and will likely be most closely examined.

We agree with this suggestion and have now amended this figure in line with the reviewer’s suggestion.

7) In Figure 4, the end of the legend says that the species tree is shown "on the right," but it's on the left in the figure.

Thank you. This has been fixed in our revision.

Cross-Consultation Comments

I agree with both Reviewers 2 and 3 that the title could be changed to be a bit more tentative. I'd had this thought as well.

We agree with this suggestion. We have now amended this title to “Expansion and loss of sperm nuclear basic protein genes in Drosophila correspond with genetic conflicts between sex chromosomes.”

I agree with Reviewer 2 that the fertility assay could be conducted with a larger sample size and a better control in order to be better compared with how the authors described other published fertility phenotypes for SNBPs. For the control, crossing the deletion line to y w (or w1118) and using the resulting heterozygotes (KO/+) would be better than using the mutation over the balancer chromosome (KO/CyO).

We agree with both suggestions. We now compare fertility between KO/KO and KO/+ males in sperm exhaustion assays. Our more stringent fertility assays find no evidence of CG30056 role in male fertility, strengthening our previous findings. We have now added the motivation for the new assays and the new results to our Revision.

I agree with Reviewer 3's third bullet point about spending a bit more time on the different possible roles that SNBPs could play in spermatids. (This is a more eloquent version of my review point #1.)

We have now expanded our discussion of other possibilities in our revision.

I agree in principle with Reviewer 3's first bullet point about examining whether SNBP evolution correlates with changes in sperm morphology, but this feels like it could be a whole, fascinating study on its own, while this manuscript is already packed with data. I'd welcome the authors' thoughts about this in discussion, but wouldn't personally require a formal analysis of this to be added prior to publication.

We also agree that this would be an interesting test. However, we are not able to do the test due to the scarcity of sperm phenotype data in Drosophila. We also think that our original version unintentionally downplayed this possibility. Our revised discussion makes clear that the rapid evolution of some Drosophila SNBP genes may be driven by sperm competition, just as in mammals, and influence the evolution of sperm morphologies.

Reviewer #1 (Significance (Required)):

This study describes an important conceptual advancing in our understanding of the evolution and potential functions of sperm nuclear basic proteins (SNBPs) in Drosophila, which stands in interesting contrast to the functional roles of equivalent proteins in primates. It should be of broad interest to biologists studying spermatogenesis, meiotic drive, and genome evolution, both in and out of Drosophila.

We thank the reviewer for their positive appraisal.

To contextualize the work, paternal DNA is typically compacted during spermatogenesis. This process involves the replacement of histones with other small, positively charged proteins in a sequential order, ending with protamines that bind DNA in mature sperm. In Drosophila, work over the last two decades (largely from the labs of R. Renkawitz-Pohl, B. Loppin and B. Wakimoto) has identified more than a dozen sperm nuclear basic proteins that localize to condensing/condensed spermatid nuclei.

Two interesting observations have been that many of these proteins are dispensable for male fertility, and the proteins vary in their degree of evolutionary conservation. Recent work from Eric Lai's lab (J Vedanayagam et al. 2021, Nat Ecol Evol) showed that in D. simulans and sister species, at least one of these SNBP genes (Prot) underwent gene amplification and now acts in those species as a meiotic driver. This finding suggested the hypothesis, tested thoroughly in the present study, that the rapidly evolving SNBP gene family could be involved in causing or suppressing meiotic drive. Consistent with this idea, the authors here find that SNBP genes expand in copy number more frequently when they move from autosomes to sex chromosomes (consistent with the idea that they may cause or contribute to drive), and that otherwise well-conserved SNBP genes are lost in a group of species in which sex chromosome meiotic drive is not expected to occur. These findings are based on a thorough and well conducted phylogenomic and molecular evolutionary analysis of SNBPs across dozens of Drosophila species. Overall, this work generates exciting new hypotheses about the function of SNBPs and should be widely read both within and outside of the field.

We are grateful for the reviewer’s accurate summary of our work and its significance. We share the reviewer’s excitement and expect that more studies will explore the new function of SNBPs in multiple taxa soon.

Keyword describing my field of expertise: Drosophila, molecular evolution, reproduction, genetics, genome evolution.

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

The paper describes interesting patterns on the evolution of Drosophila SNBP genes, and proposes a very interesting explanation, namely, that meiotic drive is the main evolutionary force behind these patterns. Some of these observations have recently been made by other authors in a single case (the Dox genes in D. simulans), but not in the scale and breadth of the present manuscript. The manuscript combines an extensive investigation of available genomes with expert analysis, and new experimental data. In particular, the finding that the ancestral Y became incorporated into de X in montium species is very exciting, and may provide a smoking gun for the explanation proposed by the authors. Overall, I think it is a very good paper. I do have several criticisms and suggestions that may help to improve it.

We are grateful for the positive comments of the reviewer and for their constructive criticism and suggestions, which we have incorporated into our revision.

The paper has a speculative side that it almost unavoidable given its novelty and breadth. I do not see this as a problem per se, but I think the uncertain/unsupported/problematic points should be more openly presented to the readers. The main cases I noted are:

1) The title of the manuscript states that "Genetic conflicts between sex chromosomes drive expansion and loss of sperm nuclear basic protein genes in Drosophila", but the evidence is somewhat circumstantial, and the patterns may be explained also by other known phenomena (e.g., demasculinization of the sex chromosomes; below). I think the tone of the end of the Introduction reflects more faithfully the strength of the evidence ("Thus, we conclude that rapid diversification of SNBP genes might be largely driven by genetic conflicts between sex chromosomes in Drosophila."). I understand the temptation of writing a bold title, but I think it is a bit misleading in the present case. I.e., it would be desirable that the title conveys the uncertainties of the data and their interpretation.

We agree with this suggestion. We have now amended this title to “Expansion and loss of sperm nuclear basic protein genes in Drosophila correspond with genetic conflicts between sex chromosomes.” However, we also want to highlight that de-masculinization of the X chromosome cannot explain the observed amplification and loss patterns of SNBP genes, except in cases of sex chromosome fusions. We now highlight the de-masculinization hypothesis for the latter case, but still strongly favor the genetic conflicts hypothesis.

2) "In contrast, we found no instances of pseudogenization or subsequent translocation to the X chromosome of SNBP genes that are still preserved on their original autosomal locations or involved in chromosome fusions between autosomes (0/16). This difference is highly significant (Figure 5 and Table S11; 3:5 versus 0:16, Fisher's exact test, P=0.03). " Readers should be warned that this pattern can also be explained by the well-known demasculinization of X chromosomes (e.g., Sturgill et al. Nature 2007, 450, 238-241)

We agree with this point and thank the reviewer for pointing this out. We now expressly raise the ‘de- masculinization of X chromosomes’ as one potential explanation of the pattern we observe here.

3) "Indeed, no meiotic drive has been documented in the montium species even though it is rampant in many other Drosophila lineages [38]." Two remarks here:

a) the authors should make clear that they are referring to sex-chromosome meiotic drive.

b) I think the evidence is much weaker than the sentence implies. Sex-chromosome meiotic drive is known in less than 20 Drosophila species, scattered throughout the phylogeny. As far as I know all cases were discovered by accident, so the sampling is biased towards model species (e.g., the obscura group, which was very popular around 1930-1960). So we do not know the true frequency of sex-ratio meiotic drive among Drosophila species, nor, say, if it is more common in the Drosophila or Sophophora species, if it is suspiciously absent in the montium group (as suggested by the authors), etc. I think these uncertainties should be acknowledged or, perhaps, given the weakness of the argument, the sentence should be deleted or attenuated.

We agree with this comment and have now removed this argument in our revision.

4) "X-Y chromosome fusions eliminate the extent of meiotic drive and may lead to the degeneration of otherwise conserved SNBP genes, whose functions as drive suppressors are no longer required. Thus, unlike in mammals, sex chromosome-associated meiotic drive appears to be the primary cause of SNBP evolutionary turnover in Drosophila species." The authors found that in the montium species the ancestral Y became incorporated into de X chromosome, and that montium species seem to have an inordinate amount of SNBP gene losses. They combine these two observations by suggesting that these SNBP became dispensable or deleterious because they originally were involved in XY meiotic drive. I think many readers will think that males in montium species are X/0, whereas in fact in all of them carry a Y chromosome (just, in most cases, more gene poor than "normal" Y-chromosomes). I do not think this is a fatal flaw for the explanation proposed by the authors, but certainly is a difficulty that should be acknowledged.

We agree with this point. It was not our intention to suggest that montium group males are X/O, but this could be misinterpreted as we originally stated. We now add a clarification that montium group males still harbor a Y chromosome, which is missing most ancestrally Y-linked genes.

Problems/suggestions with experiments and data analysis

5) There is a section titled "CG30056 is universally retained in Drosophila but dispensable for male fertility in D. melanogaster". In this section and in the figures, it is stated, "Although CG30056 is the most conserved SNBP we surveyed, we found no clear difference in offspring number between heterozygous controls and homozygous knockout males (Figure 2B). (…) We found either no or weak evidence of fertility impairments in two different crosses with homozygous CG30056 knockout males.".

I think the fertility data are weak for the purpose of the authors, and I strongly suspect that this conclusion is wrong. Let me explain why. At other passages of the manuscript, the authors classify the SNBP genes in three groups. (i) essential/important for male fertility: "Three genes (Mst77F, Prtl99C and ddbt) are essential for male fertility while knockdown or knockout of two other SNBP genes (ProtA, and ProtB) leads to significant reduction in male fertility [27-30, 32]." (ii) genes that do not appear to impair male fertility at all. (iii) untested. CG30056 was in the last group, and hence the authors produced knockouts, tested their effect in male fertility, and concluded that it belongs to the second group.

Now, look at Figure 3B. The numbers of tested males are too small (it seems to range from 3 to 10), and male fertility is known to be a very noisy phenotype (as shown by the huge scatter in the authors' data). Furthermore, two different knockouts were tested, and both were nominally less fertile than the controls, and in one of them the difference is statistically significant. Taken at the face value, the knockouts seem to be perhaps ~25% less fertile than the controls. Another potentially big problem is that the "control males" actually carry visible dominant mutants (the balancers CyO or SM6) which certainly reduce their fitness, whereas the experimental males are wild-type for these mutants. Without the detrimental effect of these visible mutants in the controls, the difference to the CG30056 knockouts will probably be even larger. Note that the fertility effects of the genes ProtA, and ProtB (a.k.a. "Mst35B") , which the authors put in group "essential/important for male fertility" would not had been detected if assayed as the CG30056 gene: Tirmarche et al. (2014; the reference cited by the authors) stated that: "In fact, the impact of Mst35B on male fertility was only revealed when mutant males were allowed to mate with a large excess of virgin females (1 for 10; Figure 3F) but not with a 1:1 sex ratio (not shown). " The authors' fertility test did not used this type of challenge.

My general impression is that the fertility effects of CG30056 may actually be similar to ProtA and ProtB. I think the authors should do a proper fertility test of CG30056, or remove this section. Another possibly useful approach would be to classify the SNBP genes in those essential for male fertility and those that are not essential, because "experimentally speaking" this is a safer distinction (e.g., the fertility testes reported by other authors may also had been quick tests). Since these genes only function in sperm and are under purifying selection (otherwise they would have been lost; also, all have dN/dS < 1 ), they all most likely affect male fertility to some extent. In case the section on male fertility stays, it will be necessary to provide more details. How many males were crossed for each genotype? In some cases in Figure 2B, it seems that as low as 3, but it may be data superposition in the graph. Please provide the raw data in the supplementary material.

We are very appreciative of the many important points raised by the reviewer. Rather than removing this conclusion, which is not central to our paper, we have now performed additional, well-controlled experiments to address the reviewer’s concerns, which we summarize below:

We hope these changes will satisfy the reviewer's concerns about this section of our paper.

6) "Our phylogenomic analyses also highlighted one Drosophila clade- the montium group of species (including D. kikkawai)- which suffered a precipitous loss of at least five SNBP genes that are otherwise conserved in sister and outgroup species (Figure 3). (…) Given our hypothesis that autosomal SNBP genes might be linked to the suppression of meiotic drive (above), we speculated that the loss of these genes in the montium group of Drosophila species may have coincided with reduced genetic conflicts between sex chromosomes in this clade." The montium data is an important part of the paper. I think the authors should test the statistical significance of this pattern.

We appreciate the reviewer’s suggestion. However, we are unable to perform the statistical tests suggested for technical reasons. We note that three loss events occurred in the ancestor of D. montium species, while two happened in the ancestors of most D. montium species. Since it’s hard to estimate the evolutionary rates using these internal branches, we can’t directly compare them to other branches using statistics. However, in response to the reviewer’s comments, we now more clearly contrast the fate of SNBPs between D. montium species and other melanogaster group species, noting that three of five genes lost in the montium group are retained in all other melanogaster group species.

Other points:

7) "The five remaining SNBP genes (Mst33A, CG30056, CG31010, CG34269, and CG42355) remain cytologically uncharacterized [30]." I think it will be interesting if the authors look at other potentially useful resources: Vibranovski et al. papers which looked at gene expression in mitotic, meiotic and post-meiotic cells (https://mnlab.uchicago.edu/sppress/index.php), and the papers by several labs on testis single-cell transcriptomic data (Witt et al. 2021 PLOS Genetics. 17(8):e1009728 ; Nat Commun. 2021;12: 892). These may provide additional clues on the function of SNBP genes. There is also a recent report on sperm proteome (doi: https://doi.org/10.1101/2022.02.14.480191)

We are grateful to the reviewer for this suggestion. We now add the data from single-cell expression analyses from Witt et al. in Table 1—figure supplement 1. We found most SNBPs are expressed at late spermatocytes and early spermatids, although CG30056 is primarily expressed in late spermatids, whereas CG34269 is expressed earlier in late spermagonia. The data from Vibrranovski et al. also show similar patterns but don’t have four of these genes, including CG34269. The data from Mahadevaraju et al. are from larva testes, and lack some critical stages during spermatogenesis. Thus, we only report the data from Witt et al.

We also surveyed the proteome data as the reviewer suggested, but we only found 3 SNBPs (ProtA, ProtB, and Prtl99C) in the data. This did not include, Mst77F, which is the most highly expressed (see Table 2) and well-studied SNBP, so we suspect the proteomic study might be biased toward proteins from sperm tails.

Therefore, we decide not to include this analysis.

8) "Our inability to detect homologs beyond the reported species does not appear to result from their rapid sequence evolution. Indeed, abSENSE analyses [45] 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 (Table S1, Methods)." This should be complemented by synteny analysis.

It may not have been clear from our original version that we did perform synteny analyses for all SNBP genes. We have now restated this more clearly in our revision.

9) I found the following sentence unclear: "However, we could only ascribe a sex chromosomal linked location for species if no data was available from either BUSCO genes or females (only males and mixed-sex flies)."

We modified the sentence to make it clearer: “However, we could not ascribe a sex-chromosomal linked location of a contig to either the X or Y chromosome in cases where there was no linkage information from BUSCO genes and no read data available from females, only from males and mixed-sex flies.”

10) "Using the available assemblies with Illumina-based chromosome assignment, we surprisingly found that most ancestrally Y-linked genes are not linked to autosomes as was previously suggested [by Dupim et al. 2018] (Figure 6A)."

The new result of X-linkage is exciting, but the sentence is not exact: Dupim et al. 2018 made clear that they could only separate X/A from Y-linkage. E.g., the legend of their Figure 3: "Phylogeny and gene content of the Y chromosome in the montium subgroup. "M" means amplification only in males (i.e., Y- linkage), whereas "MF" means amplification in both sexes (autosomal or X-linkage)."

We are grateful to the reviewer for this correction. We now modified the sentence to make clear that Dupim et al. had “showed that many ancestrally Y-linked genes are present in females because of possible relocation to other chromosomes in the montium group.”

11) "The most parsimonious explanation for these findings is a single translocation of most of the Y chromosome to the X chromosome via a chromosome fusion in the ancestor of the montium group of species. Afterward, some of these genes relocated back to the Y chromosome in some species (Figure S6; Supplementary text)." Explanations for this pattern of "return to the Y" have been extensively discussed and tested in Dupim et al. 2008 (see their section "Why genes seem to return to the Y chromosome after Y incorporations?" ) The available evidence strongly suggests that it is not a case of relocation to the Y.

We thank the reviewer for raising this point. However, our conclusions disagree slightly with those from Dupim et al. 2018, in part because of additional sequencing in this clade. Dupim et al. suggested the possibility that most Y chromosomal loci duplicated to other chromosomes in the ancestor of the D. montium clade, following which each species degenerated either Y-linked or autosomal copies of genes. If this was the case, Y-linked copies should have diverged from X-linked copies since the ancestor of the D. montium clade. In contrast to this expectation, our phylogenetic analyses found that D. kikkawai Y-linked PRY is more closely related to X- linked PRY in all other related species (Figure 6—figure supplement 1). This result is much less parsimoniously explained by the ancient duplication event proposed by Dupim et al. and is more consistent with a ‘return-to-Y’ that we propose. We also make clear that, unlike PRY, we can’t differentiate the two hypotheses in the case of kl-2.

12) Figure 6B suggests that the authors assembled the "translocated Y" in D. triauraria. However, no direct data or account for this assembly is provided. Please clarify.

This was not our assembly. We searched all publicly available assemblies in the montium group and found one assembly (NCBI accession GCA_014170315.2) that assembled all ancestral Y-linked regions. We now clarify this in our revision.

13) "Why would meiotic drive only influence Drosophila, but not mammalian, SNBP evolution? One important distinction may arise from the timing of SNBP transcription. In D. melanogaster, SNBP genes are transcribed before meiosis but translated after meiosis [29, 43, 57]. Thus, SNBP transcripts from a single allele, e.g., Xlinked allele, are inherited and translated by all sperm, regardless of which chromosomes they carry. Consequently, they can act as meiotic drivers by causing chromatin dysfunction in sperm without the allele, e.g., Y-bearing sperm." During spermatogenesis Drosophila haploid cells actually are syncytial, which has interesting consequences for the evolution of male genes (Raices et al., Genome Res. 1115-1122, 2019). This may be relevant for the present paper.

We thank the reviewer for this suggestion. We now gratefully include this citation in our revision.

Reviewer #2 (Significance (Required)):

See above

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

This manuscript by Chang and Malik consider the evolution of HMG-box-containing sperm nuclear basic proteins (SNBPs) across Drosophila species in phylogenetic context. Previous work in mammals had highlighted fast evolution of proteins involved in chromatin remodeling during spermatogenesis. Here, the authors provide evidence for widespread positive selection and likely involvement in genetic conflict in a set of proteins with analogous functions in Drosophila. Amongst other findings, the authors highlight biased amplification of SNBP paralogs on sex chromosomes along several Drosophila lineages, a tendency towards loss/pseudogenization following translocation onto a sex chromosome, and an intriguing concerted SNBP loss event in the montium group where parts of the Y chromosome have become fused to the X, thus nullifying the chance that genetic conflicts can play out via distorted segregation of sex chromosomes. The authors suggest that, taken together, their findings support widespread of SNBPs involvement (as instigators and repressors) in meiotic drive. Overall, I found the manuscript to be well written and thorough in its exploration of the evolutionary dynamics of SNBPs in this clade.

We thank the reviewer for the accurate summary and the kind comments.

Below, I have highlighted some aspects that I think would benefit from further attention, none of them major.

Following their exploration of patterns of SNBP evolution in Drosophila, the authors highlight support of their data for genetic conflict between sex chromosomes. They also rightly acknowledge that other evolutionary drivers such as sperm competition might also play a role in, for example, fast evolution of certain SNBPs. Yet those (not mutually exclusive) alternatives are never pitted directly against each other. The focus is firmly on exploring the support for the sex chromosome genetic conflict model. Given that the authors highlight Drosophila as a great model in part because of its well characterized sperm biology (including comparative morphology), I wondered why the authors had not made an explicit attempt to see if SNBP evolution covaries with aspects of sperm morphology across Drosophila.

We do agree with the reviewer that it will be very interesting to test whether SNBP evolution covaries with sperm morphology in Drosophila. However, data on sperm morphology is scant in most Drosophila species. Indeed, this trait has only been well studied in clades with heteromorphic (different-sized) sperm but we agree this will be an exciting topic to consider in the future.

We also clarify better in our revised discussion that our analyses do not rule out a role for sperm competition or sperm morphology in driving the evolution of at least some SNBP genes. We note that a subset of SNBP genes undergo gene amplifications and loss, but most SNBP genes evolve rapidly including in species with gene loss. Thus, the meiotic drive hypothesis is not to the exclusion of other hypotheses.

The most intriguing part of the manuscript for me was the exploration of SNBP fate in the montium group, where the authors find evidence for an ancestral fusion event between the X and parts of the Y chromosome. The loss of SNBPs is certainly consistent with the conflict model but I was wondering to what extent this lineage is characterized more broadly by unusual evolution at the chromosomal level. Is there simply a lot of upheaval in montium, with more frequent gain/loss across the board? How specific is SNBP loss in the context of other orthologous groups? This could be investigated by looking at retention of other genes in other orthologous groups (in montium and some other control group) or perhaps by looking at synteny conservation.

This is a good suggestion. Using the same methodology as used in this paper, we found that very few D. melanogaster essential genes (<3% of >2000) are lost in any single species we surveyed here (unpublished data). However, we have not carried out similar analyses for all genes; given vastly different rates of evolution, this would be a significant undertaking. Thus, we are not able to make a direct comparison between SNBP genes and a control group, that would include other testes-specific or fertility-essential genes. Instead, we highlight the fact that since we identify SNBPs using syntenic analyses, we have known that the neighboring genes of SNBPs are much better conserved than the SNBP genes themselves in the montium group species.

In introducing SNBPs, the authors focus on their role as packaging agents. Clearly, SNBPs do package the genome in the sense that they bind to DNA and lead to reduced chromosome volume. But is this all packaging for packaging's sake (as portrayed by the sperm shape hypothesis)? Or is the situation a bit more nuanced, where condensation leads to a reduction of volume but also to a shutdown of transcription, protection from DNA damage, etc.? I think the focus on packaging alone is somewhat limiting when it comes to imagining how these proteins might act in the context of genomic conflicts. The authors may want to broaden their description of SNBPs in the Introduction accordingly.

We completely agree with the reviewer and are currently exploring these possibilities in follow-up studies on SNBP function. However, it is fair to add that this hypothesis has not been well-recognized, and we, therefore, prefer to include it in our revised Discussion rather than Introduction. However, we also think that SNBP packaging function might be targeted by Wolbachia-encoded toxins, speeding up their evolution (revised Discussion). We think there are many molecular possibilities for SNBPs.

The authors highlight that some SNBPs are expressed in mature sperm whereas others are transition proteins. The evidence for positive selection chiefly comes from the latter group (and "undefined" proteins that could also be transition proteins). Can the authors comment on whether this is expected/unexpected? Along the same lines, the authors highlight differences between Drosophila and mammals when it comes to the timing of transcription/translation during meiosis, suggesting that meiotic drive can happen in Drosophila because alleles are expressed early and can exert an effect after meiosis regardless of whether the associated locus is present in the gamete. I wonder how this relates, if at all, to the author's finding that transition SNBPs are more likely to be part of conflicts (as indicated by positive selection signals) compared to SNBPs in mature sperm.

We thank the reviewer for this comment. We expect that many genes expressed explicitly in spermatogenesis, including SNBP genes, would be under position selection, regardless of whether they are associated with X-Y conflicts. The positive selection signals could come from either X-Y conflicts, sperm competition, or conflicts with Wolbachia; we now discuss all of these in the Discussion.

In contrast, the amplification and loss of a subset of Drosophila SNBPs are more likely associated with X-Y conflicts. We note that known SNBPs retained in mature sperm are more likely to be subject to amplification than known transition proteins.

Regarding the timing of expression, it is true that transition SNBPs act earlier in spermatogenesis than SNBPs retained in mature sperm. However, for the meiotic drive hypothesis to apply, all it requires is for SNBP expression to precede sperm individualization, which it does for most SNBPs, including transition proteins.

It is not entirely clear from the text (and also e.g. Table S4) how dN and dS (and subsequently dN/dS) where calculated. I presume as a single estimate across the whole phylogeny? If so, how heterogeneous is dN/dS across the phylogeny and can the authors identify specific branches on which selective regimes are different? A branch-level analysis should be better powered than the site-level analysis the authors present, which requires repeated selection on the same set of sites to get a strong enough signal. A branch-specific assessment of evolution would be particularly valuable in combination when combined with the assessment of amplifications/losses.

We thank the reviewer for this question. The reviewer is correct. We estimated dN and dS in Supplementary file 4 across the whole phylogeny. We conducted branch tests for the amplification of tHMG only in the Dsim clade (Supplementary file 11).

We are interested in how SNBP amplification happened across species, but we need better gene annotation for their structure in many of these 19 independent cases. Moreover, we hope to combine these with transcriptomic analyses with detailed sequence analyses to reveal how the event happened and how gene conversion, gene duplication, and mutations affect their evolution. Each of these analyses requires extensive additional resources and analyses, and we feel are beyond the scope of this current paper.

The authors suggest that young SNBPs are more likely to encode essential, non-redundant male fertility functions (p7, third paragraph). I'm not sure whether this generalization is appropriate given the small sample. Tpl94D is as young as Mst77F/Prtl99C, tHMG and CG14835 homologs have been lost along different lineages and most of the events are in a single lineage leading up to D. kikkawai. Do the authors really feel that this generalization is warranted?

We agree with the reviewer. However, it is striking that the known fertility essential genes are either young or not universally conserved. We have therefore reworded our conclusion to make this contrast more accurate.

How do the sex-chromosomal amplifications differ in sequence from the ancestral autosomal copies? The authors suggest that the sex chromosomal copies might be involved in meiotic drive? Does the sequence offer a function as to how? (e.g. loss of charged residues/DNA-binding capacity)?

These are good questions. We do not know mechanistically how the sex-chromosome amplifications may cause meiotic drive. We did not observe the loss of positive charge or HMG domain in most sex-chromosomal amplified copies (Supplementary file 3). Our current working hypothesis is that they compete for the DNA binding with autosomal SNBP, and might interact with other proteins, e.g., heterochromatin proteins, to disturb sperm function. How they might function to cause meiotic drive is an active area of investigation in our and other labs.

I think it would be nice to have a final table/figure to summarizing the different lines of evidence for all the genes in Table 1 (i.e. positive selection yes/no, amplification in some lineages yes/no, sex chromosome translocations yes/no), for different lineages, including whether any of the HMG-box genes are unlikely to act as SNBPs.

We agree with this suggestion. We have now significantly revised and added to Table 2 to include this added information.

The evidence the authors present is often consistent with genetic conflicts between sex chromosomes. Is it cogent? Arguably not since direct tests of the mechanism are provided. I would therefore suggest a more cautious title than one stating that conflicts drive expansion and loss of SNBPs.

We agree with all three reviewers and have amended our title to highlight the correlation. We also discuss other possibilities that can drive SNBP evolution in our revised Discussion.

Typographical errors etc.:

– P3. First paragraph: "One of the driving forces.… " I found this sentence a bit odd in terms of causality (changes in composition being portrayed as a force that leads to selection)

We thank the reviewer for pointing out the confusing construction. We modified the sentence to “The positive selection of SNBPs results in changes to their amino acid composition.”

– P3. Second paragraph: should be "HMG-box" rather than "HMB-box"


– P3. Fourth paragraph "…, consistent with the observation in mammals". I think "consistent" should be reserved for two observations that speak to the same phenomenon. SNBPs could evolve with no evidence for positive selection in Drosophila and that wouldn't exactly be "inconsistent" with mammals. It would just be different.

Fixed. We changed “consistent with” to “similar to”.

– P5. Fifth paragraph: should be "in the PAML package" rather than "in PAML package"


– P9. Second paragraph: "… montium group (Figure 5A…)" should be Figure 6A.


Cross-Consultation Comments

I have not much to add. The other reviews seem fair and well-informed from my somewhat-outside perspective. I don't know how tricky/time-consuming the suggested additional fly mating experiments are but want to note that, in general, I'm loath to "punish" authors of principally bioinformatic work for including some experiments. If experimental shortcomings can be addressed with appropriate caveats, that should be an option, as should removal of experimental data that – by the experts – would be considered too preliminary.

We thank the reviewer for their support. However, we felt that improved experiments on CG30056 role in fertility could broaden the scope of this paper, despite the additional time and labor commitment. We have now finished these experiments and they do reinforce our original conclusions with much greater support.

Reviewer #3 (Significance (Required)):

I'm not enough of an expert in the field of SNBPs to assess the level of advance provided by this study.


Article and author information

Author details

  1. Ching-Ho Chang

    Division of Basic Sciences, Fred Hutchinson Cancer Center, Seattle, United States
    Conceptualization, Data curation, Formal analysis, Funding acquisition, Validation, Investigation, Visualization, Writing – original draft, Project administration, Writing – review and editing
    For correspondence
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9361-1190
  2. Isabel Mejia Natividad

    1. Division of Basic Sciences, Fred Hutchinson Cancer Center, Seattle, United States
    2. Howard Hughes Medical Institute, Fred Hutchinson Cancer Center, Seattle, United States
    Investigation, Writing – review and editing
    Competing interests
    No competing interests declared
  3. Harmit S Malik

    1. Division of Basic Sciences, Fred Hutchinson Cancer Center, Seattle, United States
    2. Howard Hughes Medical Institute, Fred Hutchinson Cancer Center, Seattle, United States
    Conceptualization, Supervision, Funding acquisition, Visualization, Writing – original draft, Project administration, Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6005-0016


Damon Runyon Cancer Research Foundation (DRG 2438-21)

  • Ching-Ho Chang

National Institute of General Medical Sciences (R01-GM74108)

  • Harmit S Malik

Howard Hughes Medical Institute

  • Harmit S Malik

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.


We thank Tobias Warnecke and two anonymous reviewers for their comments and suggestions to improve the manuscript. We are grateful to Cécile Courret, Janet Young, Phoebe Hsieh, Pravrutha Raman, and Rupinder Kaur for their comments on the manuscript. Finally, we thank Barbara Wakimoto, Amanda Larracuente, John Sproul, and members of the Malik, Ahmad, and Henikoff labs for fruitful discussions. We also thank Benjamin Loppin, Brandon Cooper and the Bloomington Drosophila Stock Center (supported by NIH P40OD018537) for the Drosophila strains used, and acknowledge Flybase (supported by NHGRI Award U41HG000739) for helping build bioinformatic tools across various Drosophila species’ genomes. We are supported by a Damon-Runyon Cancer Research Foundation postdoctoral fellowship DRG 2438-21 (to C-HC) and National Institutes of Health grant R01-GM74108 (to HSM). The funders played no role in the study design, data collection and interpretation, or the decision to publish this study. HSM is an Investigator of the Howard Hughes Medical Institute. This article is subject to HHMI’s Open Access to Publications policy. HHMI lab heads have previously granted a nonexclusive CC BY 4.0 license to the public and a sublicensable license to HHMI in their research articles. Pursuant to those licenses, the author-accepted manuscript of this article can be made freely available under a CC BY 4.0 license immediately upon publication.

Senior Editor

  1. Molly Przeworski, Columbia University, United States

Reviewing Editor

  1. Daniel R Matute, University of North Carolina, Chapel Hill, United States

Version history

  1. Preprint posted: June 9, 2022 (view preprint)
  2. Received: November 30, 2022
  3. Accepted: January 4, 2023
  4. Version of Record published: February 10, 2023 (version 1)


© 2023, Chang et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.


  • 1,271
    Page views
  • 145
  • 4

Article citation count generated by polling the highest count across the following sources: PubMed Central, Crossref, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Ching-Ho Chang
  2. Isabel Mejia Natividad
  3. Harmit S Malik
Expansion and loss of sperm nuclear basic protein genes in Drosophila correspond with genetic conflicts between sex chromosomes
eLife 12:e85249.

Further reading

    1. Evolutionary Biology
    Hironori Funabiki, Isabel E Wassing ... Thomas Carroll
    Research Article

    5-Methylcytosine (5mC) and DNA methyltransferases (DNMTs) are broadly conserved in eukaryotes but are also frequently lost during evolution. The mammalian SNF2 family ATPase HELLS and its plant ortholog DDM1 are critical for maintaining 5mC. Mutations in HELLS, its activator CDCA7, and the de novo DNA methyltransferase DNMT3B, cause immunodeficiency-centromeric instability-facial anomalies (ICF) syndrome, a genetic disorder associated with the loss of DNA methylation. We here examine the coevolution of CDCA7, HELLS and DNMTs. While DNMT3, the maintenance DNA methyltransferase DNMT1, HELLS, and CDCA7 are all highly conserved in vertebrates and green plants, they are frequently co-lost in other evolutionary clades. The presence-absence patterns of these genes are not random; almost all CDCA7 harboring eukaryote species also have HELLS and DNMT1 (or another maintenance methyltransferase, DNMT5). Coevolution of presence-absence patterns (CoPAP) analysis in Ecdysozoa further indicates coevolutionary linkages among CDCA7, HELLS, DNMT1 and its activator UHRF1. We hypothesize that CDCA7 becomes dispensable in species that lost HELLS or DNA methylation, and/or the loss of CDCA7 triggers the replacement of DNA methylation by other chromatin regulation mechanisms. Our study suggests that a unique specialized role of CDCA7 in HELLS-dependent DNA methylation maintenance is broadly inherited from the last eukaryotic common ancestor.

    1. Developmental Biology
    2. Evolutionary Biology
    Nico Posnien, Vera S Hunnekuhl, Gregor Bucher
    Review Article

    Gene expression has been employed for homologizing body regions across bilateria. The molecular comparison of vertebrate and fly brains has led to a number of disputed homology hypotheses. Data from the fly Drosophila melanogaster have recently been complemented by extensive data from the red flour beetle Tribolium castaneum with its more insect-typical development. In this review, we revisit the molecular mapping of the neuroectoderm of insects and vertebrates to reconsider homology hypotheses. We claim that the protocerebrum is non-segmental and homologous to the vertebrate fore- and midbrain. The boundary between antennal and ocular regions correspond to the vertebrate mid-hindbrain boundary while the deutocerebrum represents the anterior-most ganglion with serial homology to the trunk. The insect head placode is shares common embryonic origin with the vertebrate adenohypophyseal placode. Intriguingly, vertebrate eyes develop from a different region compared to the insect compound eyes calling organ homology into question. Finally, we suggest a molecular re-definition of the classic concepts of archi- and prosocerebrum.