Parallel Evolution of Sperm Hyper-Activation Ca2+ Channels.

Sperm hyper-activation is a dramatic change in sperm behavior where mature sperm burst into a final sprint in the race to the egg. The mechanism of sperm hyper-activation in many metazoans, including humans, consists of a jolt of Ca2+ into the sperm flagellum via CatSper ion channels. Surprisingly, all nine CatSper genes have been independently lost in several animal lineages. In Drosophila, sperm hyper-activation is performed through the cooption of the polycystic kidney disease 2 (pkd2) Ca2+ channel. The parallels between CatSpers in primates and pkd2 in Drosophila provide a unique opportunity to examine the molecular evolution of the sperm hyper-activation machinery in two independent, nonhomologous calcium channels separated by > 500 million years of divergence. Here, we use a comprehensive phylogenomic approach to investigate the selective pressures on these sperm hyper-activation channels. First, we find that the entire CatSper complex evolves rapidly under recurrent positive selection in primates. Second, we find that pkd2 has parallel patterns of adaptive evolution in Drosophila. Third, we show that this adaptive evolution of pkd2 is driven by its role in sperm hyper-activation. These patterns of selection suggest that the evolution of the sperm hyper-activation machinery is driven by sexual conflict with antagonistic ligands that modulate channel activity. Together, our results add sperm hyper-activation channels to the class of fast evolving reproductive proteins and provide insights into the mechanisms used by the sexes to manipulate sperm behavior.

Introduction

Sexual conflict shapes sperm development and sperm dynamics (Swanson and Vacquier 2002; Clark et al. 2006; Turner et al. 2008; Wilburn and Swanson 2016). Both male–female interactions and intermale competition drive rapid changes in male reproductive proteins, whose constant innovation has been likened to a molecular arms race. These rapid changes in reproductive proteins have the potential to establish barriers to fertilization between populations and lead to the evolution of new species (Parker and Partridge 1998; Gavrilets 2000; Howard et al. 2009; Moyle et al. 2014). The best-known examples of this phenomenon include the rapid evolution of reproductive proteins in abalone, mammals, and Drosophila (Lee et al. 1995; Swanson and Vacquier 1997; Kresge et al. 2001; Swanson et al. 2003; Gomendio et al. 2006; Clark et al. 2007; Hamm et al. 2007; Findlay et al. 2014; Vicens et al. 2015). Molecular evolutionary studies on how sexual conflicts shape reproductive proteins have focused on several aspects of sperm biology such as direct sperm-egg interactions, seminal fluid proteins and sperm behavior (Swanson and Vacquier 2002; Panhuis et al. 2006; Fisher et al. 2016). Here, we uncover the patterns of molecular evolution of the sperm hyper-activation machinery in animals, which remains a fundamental but largely unexplored aspect of sperm biology.

When spermatogenesis is complete, the resulting mature sperm are motile but quiescent. After copulation, however, sperm cease normal swimming and burst into a sprint. This dramatic postmating acceleration of sperm is known as sperm hyper-activation (Suarez et al. 1991). Sperm hyper-activation was first observed in mammals through studies on the golden hamster (Yanagimachi 1969, 1970), and has since been since described in many other taxa (Cosson et al. 2008; Suarez and Ho 2003). When sperm hyper-activate, they go through a cellular change that alters the motion of the sperm flagellum from a slow, low amplitude, symmetric beat to a whip-like, high amplitude, asymmetric beat (Ooi et al. 2014). This transition is not subtle; hyper-activated sperm swimming at top speed are propelled with a force several times that of normal swimming (Ishijima 2011). This hyper-activated acceleration of sperm is necessary for successful fertilization, and is an integral part of sperm capacitation.

The proximate molecular mechanism of sperm hyper-activation consists of a jolt of Ca2+ ions to the sperm flagellum, which triggers a complex intracellular chain of events to drive accelerated swimming (Ho et al. 2002; Carlson et al. 2009). The use of Ca2+ influx as a sperm hyper-activation trigger is an ancient and widely conserved mechanism across metazoans (Cai et al. 2014). In most metazoans, the Ca2+ influx arrives via the cation channels of sperm (CatSper) complex, which forms ion channels on the sperm flagellum (Cai et al. 2014). The CatSper complex consists of four core proteins that form the Ca2+ pore and five auxiliary proteins (Chung et al. 2017). Sperm that do not hyper-activate fail to fertilize eggs, and males with mutations that disable sperm hyper-activation are often sterile despite producing morphologically normal sperm (Ren et al. 2001).

All CatSper proteins—core and auxiliary—are necessary for sperm hyper-activation and male fertility (Loux et al. 2013). Despite the conserved role of the CatSper complex in sperm hyper-activation across metazoan taxa, the sequences of CatSper proteins provide hints of being evolutionarily labile. Previous analyses of CatSper evolution have been focused on the first exon of CATSPER1, which shows signs of adaptive evolution in the form of an accelerated accumulation of indels and nonsynonymous changes in mammals (Cai and Clapham 2008; Podlaha and Zhang 2003; Podlaha et al. 2005). More surprisingly, the entire suite of CatSper genes has been lost in several animal taxa, including those leading to arthropods, nematodes, mollusks, jawless fishes, bony fishes, birds, frogs, etc. (Cai and Clapham 2008). Some taxa that have lost the CatSper complex, such as frogs, no longer hyper-activate their sperm during fertilization (Dziminski et al. 2009). In contrast, many other taxa, such as flies and birds, are known to hyper-activate sperm despite lacking a functional CatSper complex (Köttgen et al. 2011; Yang and Lu 2011; O’Brien et al. 2011; Nguyen et al. 2014; Zhou et al. 2015). These patterns suggest that some taxa that are missing the CatSper complex may have compensated for the loss through the cooption of other mechanisms to perform sperm hyper-activation. The evolutionary forces that drive the repeated turnover of the sperm hyper-activation machinery remain unaddressed.

The mechanism of CatSper-independent sperm hyper-activation remains unknown in many taxa, but is best understood in Drosophila melanogaster. None of the CatSper genes are present in the D. melanogaster genome. Yet, Drosophila males hyper-activate their sperm postcopulation as a necessary step for successful fertilization. CatSper-independent sperm hyper-activation in Drosophila is performed by the protein polycystic kidney disease 2 (pkd2) (Yang and Lu 2011; Köttgen et al. 2011). Similar to the CatSper proteins in mammals, Drosophila pkd2 is a Ca2+ ion channel protein on the fly sperm flagellum. Drosophila pkd2 null sperm are morphologically normal, but do not hyper-activate after transfer to the female reproductive tract. As a result, Drosophila pkd2-deficient sperm fail to reach the storage organs and are not retained in the female (Gao et al. 2003; Watnick et al. 2003). Drosophila, therefore, appear to have compensated for the loss of CatSper ion channels by using the pkd2 channel to trigger sperm hyper-activation.

Sperm hyper-activation is a powerful and tightly controlled behavioral switch for the final sprint in the race to the egg (Montoto et al. 2011). The sperm hyper-activation machinery may, therefore, be vulnerable intermale sperm competition, female choice based selection, or both. An evolutionary arms race over the modulation of sperm hyper-activation can manifest as the rapid evolution of sperm hyper-activation channels. The parallels between CatSpers in primates and pkd2 in Drosophila provide a unique opportunity to examine the how selection has shaped sperm hyper-activation machinery in two independent, nonhomologous calcium channels. Here, we use a comprehensive phylogenomic approach with CatSper in primates and pkd2 in Drosophila to investigate the selective pressures on the Ca2+ channels required for sperm hyper-activation. First, we find that all core and auxiliary proteins of the CatSper complex evolve rapidly under recurrent positive selection in primates. Second, we find that pkd2 has similar patterns of positive selection in Drosophila, including increased amino-acid substitution and an accumulation of indels. Third, we show that the selective pressures of Drosophila pkd2 and primate PKD2 are radically different; primate PKD2, which has no role in sperm hyper-activation, is highly conserved rather than rapidly evolving. This provides a unique example where an otherwise slow-evolving “housekeeping” gene is dragged into an evolutionary conflict and experiences adaptive evolution. Together, our study provides the first comprehensive analysis of the molecular evolutionary patterns of the sperm hyper-activation Ca2+ channels in primates and flies.

Materials and Methods

Collection of Primate and Drosophila Gene Sequences

We acquired sequences from up to 17 species of primates and flies each for our phylogenetic analyses. Previous methods for finding homologs for phylogenetic analyses have relied on direct sequencing or manual curating of annotated genomes. Because a reliance on gene annotation models is prone to errors due to mis-annotation, and also limits the number of species that may be analyzed (Markova-Raina and Petrov 2011), we used a different approach to scan sequenced genomes and identify high quality sequences. This method is similar to the one used recently to study positive selection in the synaptonemal complex in Drosophila (Hemmer and Blumenstiel 2016), and utilizes the broad range of available sequenced genomes to maximize the power of our analyses. First, we gathered the annotated H. sapiens CDS sequence for each CatSper gene to use as bait for the searches. For each CatSper gene, we gathered homologous sequences from up to 17 primate species that provide a well distributed phylogenetic sampling (table 1). Next, we identified the genomic region containing each gene using tBlastn (NCBI) (Madden 2003). Using Exonerate (Slater and Birney 2005) on the narrowed genomic region, we identified the homologous coding regions for each gene, and predicted intron–exon splice sites. We used this two-step method because while exonerate is highly accurate at identifying homologs, it is slow at searching through full genomes. Focusing on smaller contigs that contained our gene of interest accelerated our analyses.

Table 1

Species Used in This Study

A		B
Primates	Group	Drosophila	Subgroup
Pan troglodyte	Hominid	D. melanogaster	melanogaster
Pan paniscus	Hominid	D. simulans	melanogaster
Homo sapiens	Hominid	D. sechellia	melanogaster
Gorilla gorilla	Hominid	D. mauritiana	melanogaster
Pongo abeli	Hominid	D. yakuba	melanogaster
Nomascus leucogenys	Hominid	D. tessieri	melanogaster
Colobus angolensis	Old World Monkeys	D. santomea	melanogaster
Chorocebus sabaeus	Old World Monkeys	D. erecta	melanogaster
Cerocebus atys	Old World Monkeys	D. orena	melanogaster
Mandrillis leucophaeus	Old World Monkeys	D. eugracillis	eugracillis
Macaca fascicularis	Old World Monkeys	D. biarmipes	suzukii
Macaca mulatta	Old World Monkeys	D. takahashii	takahashii
Macaca nemestrina	Old World Monkeys	D. ficusphila	ficusphila
Saimiri boliviensis	New World Monkeys	D. rhopaloa	montium
Aotus nancymae	New World Monkeys	D. elegans	elegans
Callithirx jacchus	New World Monkeys	D. ananassae	ananassae
		D. bipectinata	ananassae

For analyses with Drosophila species, we restricted our survey of Drosophila pkd2 sequences to the melanogaster group of species because including more divergent sequences saturated dS and raised the rate of false positives. For species with annotated genomes, we acquired homologous sequences from Flybase, whereas for species with unannotated genomes, we used our two-step method homology search (table 1). We also PCR amplified, subcloned and directly Sanger sequenced Drosophila pkd2 from D. tessieri, D. santomea, D. mauritiana, and D. orena. These sequences have been deposited in Genbank under accession numbers KY990034-KY990049.

Tests of Recurrent Positive Selection

We developed a pipeline to obtain high quality sequences from available genomic resources, and to modify our analysis to ignore low quality sequences (supplementary fig. 6, Supplementary Material online). Codon based methods of phylogenetic analysis require the accurate alignment of homologous gene sequences. We, therefore, only included sequences from a species when the homolog accounted for at least 90% of the total length of the reference coding region. This method ensured that we did not lose power in our tests because of incomplete gene sequences. To detect patterns of recurrent positive selection in these genes, we prepared and analyzed sequences with the following pipeline. First, we translated all coding sequences to amino acid sequences and aligned the amino acid sequences using CLUSTAL Omega (Sievers et al. 2011). Next, we back-translated the amino acid sequences to their corresponding CDS using Pal2Nal (Suyama et al. 2006), using settings to remove all gaps and stop codons. We then manually reviewed each alignment to confirm that there were no gaps or stop codons. We constructed the phylogenies for our alignment based on modEncode data (Perelman et al. 2011; Chen et al. 2014).

We used the same alignments for two separate analyses: Phylogenetic Analysis by Maximum Likelihood (PAML) (Yang 2007) and Baysean Unrestricted Test For Episodic Diversification (BUSTED) (Murrell et al. 2015). To test for recurrent selection using PAML, we compared NSsites models M7 and M8, using the branch model 0. We calculated a P value using a log-ratio test between the log-likelihood scores for each model (Yang 2007). PAML is highly sensitive to mis-alignments; even slight mis-alignments can easily create false positive signals (Markova-Raina and Petrov 2011). When we observed a P value < 0.05, we repeated our analyses by starting at the beginning of the pipeline, but this time using the T-coffee (Notredame et al. 2000) and Muscle (Edgar 2004) aligners, ensuring that our result was not an artifact of alignment error. We only considered true rejections of the null where a P value < 0.05 was observed with all three aligners, and reported the least significant P value. We ran M8a using the same alignment that generated the least significant P value. Like PAML, BUSTED compares models of selection for homologous sequences over a phylogenetic distribution. Unlike PAML, BUSTED takes a Bayesian approach to build these models. This framework makes BUSTED an independent test from PAML to analyze the molecular evolution of a gene. We ran BUSTED using the Data Monkey server (http://www.datamonkey.org/). When the program did not correctly compute the base tree, we reoriented nodes to correctly reconstruct the phylogeny.

To compare dN/dS between the CatSper genes (fig. 1), we removed the percent alignment threshold for each species so that we could analyze dN/dS over a common phylogeny of 16 species. To calculate dN/dS along branch lengths, we used the branch model 1 with NSsites model 0 (Yang 2007).

. 1.—

The entire CatSper complex evolves adaptively in primates. (A) The CatSper complex evolves under positive selection in almost every lineage in the primate phylogeny. For each CatSper gene, branches where dN/dS >1 are highlighted with a color corresponding to each gene. In cases where multiple genes have a dN/dS >1 for a single branch, the colors are stacked. (B) We detect specific sites under selection in almost every CatSper gene. Extracellular domains are marked as dark segments and intracellular domains as light segments. Orange arrows indicate the sites under selection by the Bayes-Empirical-Bayes test in PAML, with a posterior-probability >0.90 (Yang et al. 2005). Selected sites grouped close together are labeled with a bar specifying the number of sites under selection. The number of extracellular and intracellular sites under selection for each gene are tabulated.

We also used the McDonald–Kreitman (MK) test for positive selection on the narrower timescale of D. melanogaster and D. simulans divergence. For this test, we subcloned and resequenced Drosophila pkd2 from eight lines of D. melanogaster and D. simulans using the method described above. We ran MK tests for Drosophila pkd2 using DnaSP (Librado and Rozas 2009). To identify protein domains, we submitted amino acid sequences to the SMART server (Letunic et al. 2015) using all available databases.

Insertion/Deletion Polymorphism Analysis

To quantitatively assess the extent of indel differences between Drosophila pkd2 in the D. melanogaster group, we developed a null prediction for indel variation per amino-acid site. To do this, we first measured the differences in length for Drosophila pkd2 for the full gene, and determined the length variance per base pair. We measured the difference in length of each region of the gene, using the N-terminus, first trans-membrane domain, PKD channel, and C-terminus as alignment anchors. We then calculated the standard deviation of each of these regions, and divided by the length of the region to calculate a deviation per amino-acid value. For each region, we present the ratio of one deviation per amino-acid of the gene region divided by the full length of the gene.

Computational Modeling of the Drosophila pkd2 Three Dimensional Structure

To model the structure of Drosophila pkd2 we accessed i-Tasser via the web server at http://zhanglab.ccmb.med.umich.edu/I-TASSER/(Roy et al. 2010). We provided the program with the amino acid sequence of Drosophila pkd2 from positions 233-810 to correspond with the published high resolution cryo-EM structure for human PKD2 (Shen et al. 2016). We provided the human PKD2 structure as a scaffold for i-Tasser, and used the option to align the two sequences before structural prediction. Due to the limits of i-Tasser, we modeled a single monomer of Drosophila pkd2. To arrange these monomers in a tetrameric complex, we aligned the Drosophila pkd2 monomer to the four positions of the human PKD2 monomers in the solved structure using PyMOL. We highlighted all nonsynonymous positions between D. melanogaster and D. simulans in the predicted structure.

Results

Accelerated Amino Acid Evolution of the Entire CatSper Complex in Primates

Despite the critical role of the CatSper complex in sperm hyper-activation across a wide variety of metazoa, little is known about its molecular evolution. At the core of the CatSper complex lies a Ca2+ pore composed of a hetero-tetramer of CATSPER1-4. Each core CatSper protein contains a six-pass transmembrane domain with polycystic kidney disease (PKD) domains (Jin et al. 2005; Lobley et al. 2003; Qi et al. 2007, 20; Quill et al. 2001; Ren et al. 2001) (fig. 1). In contrast, four of the five auxiliary proteins, CATSPERβ, CATSPERΔ, CATSPERγ, and CATSPERε, have a large extracellular region with one or two transmembrane domains (Liu et al. 2007; Wang et al. 2009; Chung et al. 2011, 2017). The fifth auxiliary protein, CATSPERζ, is a small intracellular scaffold that helps assemble the complex (Chung et al. 2017). It is found only on the sperm flagellum, with a substantial portion exposed to the external environment of the sperm, and its only known function is in fertilization. The CatSper channel is, in theory, well positioned to be involved in sperm competition. We were, therefore, interested in a comprehensive understanding of the molecular evolutionary patterns of the CatSper complex and in uncovering the evolutionary forces that drive the changes in these genes.

To examine the evolutionary forces that shape the CatSper complex in primates, we gathered and aligned homologous sequences for each CatSper gene from 16 primate species (table 1). This sampling of primate species represents ∼40 million years of divergence. To test if any of any of the CatSper proteins show signatures of rapid accumulation of amino acid changes, we used Phylogenetic Analysis by Maximum Likelihood (PAML) to calculate the synonymous to nonsynonymous substitution rate ratios (dN/dS) (Yang 2007). For each of the nine genes that constitute the CatSper complex, we asked if any branch in the primate phylogeny had a dN/dS >1. We first analyzed the four core proteins CATSPER1-4 that form the Ca2+ pore. We detected elevated rates of dN/dS in each of these four core proteins (fig. 1, supplementary fig. 1, Supplementary Material online). In addition to the core proteins, all five auxiliary transmembrane proteins CATSPERβ, CATSPERΔ, CATSPERγ, CATSPERε and CATSPERζ also show elevated rates of dN/dS. We found that every branch in the primate lineage, with the exception of gorillas, shows a signature of accelerated evolution for at least one of the CatSper genes. These results show that all nine proteins that form the CatSper complex may have evolved under pervasive and strong positive selection across 40 million years of primate evolution.

Patterns of Positively Selected Sites in CatSper Channel Proteins

The positions of the adaptively evolving amino acid sites within a protein can provide insights into the functional properties that are under selection. To identify the adaptively evolving amino acid sites in each of the CatSper genes, we used the NSsites models of PAML. For each CatSper gene, we tested whether its evolution over the primate phylogeny is consistent with neutral evolution (model M7 and M8a) or with recurrent positive selection (model M8). PAML also uses a Bayesian framework to identify the specific sites in a gene that evolve adaptively under recurrent positive selection. We found that all nine CatSper genes show significant evidence of recurrent positive selection with both of these tests (supplementary table 1, Supplementary Material online). In CATSPER1, the site models of PAML did not detect any specific sites under selection, indicating that the selective signature may be broadly dispersed across the gene. CATSPER2, CATSPER3, and CATSPER4 each have several sites that evolve under positive selection, but these are not clustered in any particular functional domain (fig. 1supplementary table 2, Supplementary Material online). The patterns of selection in the auxiliary genes, however, are remarkably different. CATSPERΔ, CATSPERγ, and CATSPERε all have several sites under selection, and CATSPERβ has a dramatic excess of sites under selection compared to any other CatSper gene (supplementary table 2, Supplementary Material online). The extracellular domain CATSPERβ is full of adaptively changing amino acid sites. Because CATSPERβ is such a clear outlier with the greatest number of sites under positive selection, we were concerned about spurious false positives generated from alignment errors. A manual inspection of the CATSPERβ alignment makes it clear that these are not false positives—we find practically no mis-alignment between the 16 homologous protein sequences (supplementary fig. 2, Supplementary Material online). Little is known about the precise molecular properties of CATSPERβ, other than that it requires CATSPER1 for stable localization to the tail of sperm (Liu et al. 2007). These results show that among all components of the CatSper complex, CATSPERβ is the most frequent target of adaptive evolution.

If the evolution of the sperm hyper-activation machinery is driven by factors in the external environment of sperm, this would manifest as an enrichment of adaptively evolving sites in the extra-cellular regions of the CatSper proteins. We observed different patterns of extra and intracellular changes between the core and auxiliary proteins. Consistent with the patterns observed with CATSPERβ, all of the sites under selection in the auxiliary proteins CATSPERΔ, CATSPERγ, and CATSPERε are in extracellular domains. In contrast, only one third of the sites under selection in the core proteins are extracellular (supplementary table 2, Supplementary Material online). These results suggest that in the auxiliary CatSper proteins, adaptive evolution is driven by extracellular interactions. The CatSper complex, therefore, appears locked in an evolutionary conflict that drives its rapid evolution, with CATSPERβ being most directly placed at the interface of this conflict.

The Sperm Hyper-Activation Channel pkd2 Evolves Adaptively in Drosophila

Although all CatSper genes are missing in Drosophila, sperm hyper-activation remains an essential step in successful fertilization (Köttgen et al. 2011). For successful fertilization, Drosophila sperm have to swim from the uterus to the sperm storage organs in the female (Neubaum and Wolfner 1999). Several lines of evidence have shown strong selection on different aspects of Drosophila reproductive biology, including the length of sperm and ducts (Lüpold et al. 2016), and the peptides that control sperm storage and release (Findlay et al. 2014). In D. melanogaster, the gene pkd2 is required to hyper-activate sperm so that they can navigate the ducts that lead to the sperm storage organs (Gao et al. 2003; Köttgen et al. 2011; Watnick et al. 2003). Drosophila pkd2 is highly enriched in the testis, and the primary mutant phenotype is male sterility, along with a subtle effect on smooth muscle contraction (Gao et al. 2004). Like CatSper, Drosophila pkd2 is a Ca2+ channel with a PKD domain. If the CatSper-independent sperm hyper-activation machinery in Drosophila is also involved in a molecular arms race, we predict to find similar signatures of positive selection in Drosophila pkd2.

First, we investigated whether Drosophila pkd2 evolves under positive selection between D. melanogaster and its sister species D. simulans. Indeed, Drosophila pkd2 is notable for having the most significant P value of all genes in the Drosophila genome in previous McDonald–Kreitman comparisons between D. melanogaster and D. simulans (Begun et al. 2007). To conduct more detailed analyses, we performed direct Sanger sequencing of Drosophila pkd2 from eight strains of each species (supplementary table 3, Supplementary Material online). While Drosophila pkd2 is highly polymorphic within populations, there is also a significant excess of fixed nonsynonymous differences between species (McDonald and Kreitman 1991) (fig. 2). The fixed nonsynonymous changes form discrete clusters. Both the polymorphic and fixed nonsynonymous differences are located mostly outside of the PKD domain, indicating the channel pore function may be well conserved while channel activity modulating sites in Drosophila pkd2 change rapidly.

. 2.—

pkd2 evolves adaptively in Drosophila. (A) Fixed nonsynonymous fixed differences between D. melanogaster and D. simulans are marked with purple arrows above the domain structure. Clusters of fixed nonsynonymous changes are labeled with a bar specifying the number of sites. Polymorphic nonsynonymous changes within each species are marked below the domain structure. The gene span of Drosophila pkd2 is annotated as a grey bar with blue rectangles marking the transmembrane domains and a green box marking the coiled-coil domain. Extracellular and intracellular domains are in different shades of grey. (B) McDonald–Kreitman tests show that Drosophila pkd2 evolves under positive selection between D. melanogaster and D. simulans, and this signal is generated by changes in the extracellular domain. The MK table details nonsynonymous fixed (Nf), synonymous fixed (Sf), nonsynonymous polymorphic (Np), and synonymous polymorphic (Sp) sites. We report the Fisher‘s exact (FE) P value, and the alpha-value for each segment of the gene. (C) A polarized McDonald–Kreitman test for the extracellular domain of Drosophila pkd2 demonstrates that this region evolves under positive selection along both lineages. Fixed changes were polarized to the D. melanogaster or D. simulans lineages using D. yakuba as an outgroup species. (D) NSsites model tests using PAML show that Drosophila pkd2 evolves adaptively across many species of Drosophila.

Many of the fixed differences in Drosophila pkd2 are clustered in the first extracellular loop. A recently solved cryo-electron microscopy (cryo-EM) structure of human PKD2 found that this extracellular loop is critical for gating the homo-tetrameric PKD2 channel (Shen et al. 2016). The orientation of the channel exposes this loop to the external environment of the sperm. To see if Drosophila pkd2 has evolved adaptively in the extracellular loop, we separately analyzed Drosophila pkd2 in four regions. Analyses of these separate regions show that it is this extracellular loop that drives the signature of positive selection in Drosophila pkd2. To further identify the lineages that experienced adaptive evolution, we polarized our MK test using D. yakuba as an outgroup species. Our results show that Drosophila pkd2 underwent adaptive evolution along each of the lineages that lead to D. melanogaster and D. simulans (fig. 2).

To better understand the special positioning of the fixed nonsynonymous sites, we modeled a three-dimensional molecular structure of Drosophila pkd2 with the i-Tasser software (Roy et al. 2010) (fig. 3). We used the cryo-EM structure of human PKD2 (Shen et al. 2016) as a template, which includes the region from the first transmembrane domain to the end of the Ca2+ channel domain. A plot of the nonsynonymous changes between D. melanogaster and D. simulans on this predicted structure shows that these sites are not buried in the pore, but are instead directly accessible to the environment of the sperm (fig. 3).

. 3.—

A predicted structural model of Drosophila pkd2 shows that nonsynonymous changes between D. melanogaster and D. simulans reside on the extracellular faces. (A) The homo-tetramer of our predicted Drosophila pkd2 structure. The 2-D gene diagram is shaded for the region that could be successfully modeled. The monomers are alternated in shades of grey for contrast. The extracellular loop of one monomer is colored blue. The diagram to the right describes the orientation of the channel. (B) Sites that diverge between D. melanogaster and D. simulans are on the extracellular region of the channel. The base of the monomer is grey, and the extracellular region is blue. The nonsynonymous changes between the two species shown in yellow.

Because our results provide strong evidence that Drosophila pkd2 evolves under positive selection between D. melanogaster and D. simulans, we next investigated whether a similar signature of recurrent positive selection is seen across a broader range of Drosophila species. We curated sequences for Drosophila pkd2 from 17 species of Drosophila by identifying homologs from reference genomes and by directly Sanger sequencing Drosophila pkd2 from additional species. We used the branch and NSsites models of PAML, and BUSTED to test for signatures of selection in Drosophila pkd2 (fig. 2, supplementary fig. 3, Supplementary Material online) (Murrell et al. 2015). Our results from NSsites and BUSTED analyses provide evidence for episodic positive selection on pkd2 across Drosophila species.

Indel variation is known to drive the rapid diversification of the first exon of CATSPER1 (Podlaha and Zhang 2003). Because there are several fixed indels between D. melanogaster and D. simulans, we wanted to know if the variation in the length of the extracellular region is greater than that in the other domains of Drosophila pkd2. We compared the variance in length of each segment of Drosophila pkd2 to the variance in length of the whole gene. We find that most of the change in the length of Drosophila pkd2 comes from the extracellular domain (supplementary fig. 4, Supplementary Material online). Therefore, the region of Drosophila pkd2 that contains the most indel variability is the same region that contains the majority of the divergent sites between D. melanogaster and D. simulans. Together, these results show that, similar to CatSper genes, both amino acid substitutions and indel differences in regions exposed to the extracellular environment have played an important role in the rapid evolution of Drosophila pkd2.

Primate PKD2, Which Does Not Have a Role in Sperm Hyper-Activation, Does Not Evolve under Positive Selection

In primates, PKD2 is primarily known for its role in autosomal dominant polycystic kidney disease (Cai et al. 1999). Loss of function in primate PKD2 causes male sterility, but this sterility is due to abnormal formation of cysts within the testes rather than a sperm hyper-activation defect as in Drosophila (Nie and Arend 2014). Because PKD2 is required for both somatic and gamete development, it is unclear if its evolution would also be driven by sexual selection. Therefore, we analyzed primate PKD2 to see if we could find patterns of selection similar to Drosophila pkd2 and CatSpers.

We first confirmed that primate PKD2 and Drosophila pkd2 are true orthologs by constructing a maximum-likelihood phylogeny using sequences from the primate PKD2 family, primate PKD1 family, CatSpers, and Drosophila pkd2 (supplementary fig. 5, Supplementary Material online). Next, we examined the molecular evolution of primate PKD2 by collecting sequences from 11 primate species and using the same PAML based approach as with the CatSper complex and Drosophila pkd2. Consistent with our predictions, neither the branch analysis nor the NSsites models suggest any pattern of positive selection (supplementary table 5 and fig. 5 and , Supplementary Material online). Like CatSper proteins, primate PKD2 is part of a complex that contains several other PKD proteins (Tsiokas et al. 1997). We collected sequences and analyzed PKD1, PKD1L1, PKD1L3, PKD2, PKD2L1, and PKD2L2 by the same tests (we did not obtain enough homologous sequences for PKD1L2 to complete our analysis). We found no evidence for positive selection in primate PKD genes, with the exception of PKD1L3 (supplementary table 5, Supplementary Material online). Interestingly, while the rest of the PKD genes are expressed primarily in the heart and kidney, transcript profiling indicates that PKD1L3 is most strongly expressed in the placenta, suggesting that it may be subject to a different set of selective pressures than the other PKD genes (Li et al. 2003). Nevertheless, our data show that PKD2 is well conserved in primates, and suggests that the adaptive evolution of pkd2 in Drosophila is driven by its role in sperm hyper-activation.

Discussion

Hyper-activation of sperm, triggered by the opening of flagellar Ca2+ channels, is a critical behavioral switch in the race to fertilization. Our analyses show that these Ca2+ channels are not conserved, but rather are shaped by recurrent bouts of positive selection. These findings have several implications for understanding the evolutionary and mechanistic aspects of sperm behavior.

First, while much attention has focused on the evolution of the first exon of CatSper1, this region reflects only a small part of a much larger pattern. Our analyses show that the entire CatSper complex, including all core and auxiliary proteins, show robust signatures of positive selection in primate lineages. By viewing the molecular evolutionary patterns of the CatSper complex as a whole, we find that CATSPERβ is the most prevalent target of selection. Little is know about the molecular function of CATSPERβ, and our results suggest that it plays a key role in the regulation of channel activity. Because we find the majority of positively selected sites in the extracellular region of CATSPERβ and the other auxiliary proteins, it is likely that interactions with proteins in the seminal fluid or the female reproductive tract modulate CatSper activity.

Second, we find that the nonorthologous sperm hyper-activation Ca2+ channels in primates and flies, taxa that are separated by >500 million years, experience remarkably similar selective pressures. We find that the positively selected sites in Drosophila pkd2 are not buried within the pore, but instead interface with the external environment. Similar to the CatSper complex, our results predict that the forces that drive changes in the Drosophila pkd2 complex involve interacting proteins that modulate the activity of the sperm hyper-activation machinery. These parallel patterns suggest that the sperm hyper-activation machinery may be engaged in the same evolutionary conflicts in a broad diversity of taxa. Interestingly, these are not the first polycystic kidney disease domain containing genes that have been shown to evolve rapidly in the context of reproductive functions. PKDREJ is a sperm receptor for egg-coat proteins which controls the timing of the acrosome reaction (Sutton et al. 2008), and it has evolved rapidly under positive selection across primates and murine species (Hamm et al. 2007; Vicens et al. 2015). PKDREJ may act as an ion channel via its PKD domain, though its molecular function is not well characterized. The later stages of sperm behavior appear to involve two rapidly evolving PKD domain meditated switches: first CatSper/pkd2 for sperm hyper-activation, and then PKDREJ for the acrosome reaction.

Both sperm competition and female choice have the potential to engage the sperm hyper-activation machinery in sexual conflict. Males may deploy seminal fluid peptides to inactivate the sperm hyper-activation channels of competing sperm, thus providing a massive advantage for their own sperm (fig. 4) (Deborah M. Neubaum and Wolfner 1999; Qazi and Wolfner 2003). Alternatively, females may secrete peptides to inhibit sperm hyper-activation, thus providing a mechanism to modulate fertilization rates and avoid polyspermy (fig. 4) (Aagaard et al. 2013). Under either scenario, the evolutionary arms race between secreted reproductive proteins and sperm hyper-activation channels drives the patterns of recurrent positive selection that we observe in CatSpers and Drosophila pkd2.

. 4.—

Both intermale sperm competition and female choice can drive the rapid evolution of the sperm hyper-activation channels. (A) Males that secrete seminal peptides that block the hyper-activation channels of sperm from competing males gain a selective advantage. (B) Females may secrete peptides that inhibit sperm hyper-activation channels to control fertilization rates. Males that can prevent the inhibition of sperm hyper-activation gain a selective advantage.

Third, the molecular mechanisms of sperm competition remain unclear despite ample evidence for genetic variation in sperm competitive abilities both within and between species (Price et al. 1999; Matute and Coyne 2010; Sweigart 2010; Castillo and Moyle 2014). The patterns of recurrent positive selection on CatSper and Drosophila pkd2 complexes make them strong candidates for a role in sperm competition. While testing the role of CatSpers in sperm competition in primates has obvious experimental limitations, testing the role of pkd2 in sperm competition in Drosophila is imminently feasible. Our results set the stage for experiments involving transgenic allelic swaps between D. melanogaster pkd2 and divergent Drosophila pkd2 alleles from other species in an otherwise D. melanogaster genetic background. If the sperm of individuals bearing these interspecies allele swaps compete poorly with other sperm, this may reveal an important role for Drosophila pkd2 in the molecular mechanisms of sperm competition.

Together, our study introduces the sperm hyper-activation genes as a new class of male reproductive proteins that evolve rapidly. We find parallel patterns of adaptive evolution in nonorthologous proteins that serve as the sperm hyper-activation calcium channels in primates and flies. Identifying the factors that modulate the sperm hyper-activation machinery promises to provide insights into the molecular mechanisms used by the sexes to manipulate sperm behavior to their own advantage.

Supplementary Material

Supplementary data are available at Genome Biology and Evolution online.

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