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Unravelling : size and rspb.royalsocietypublishing.org ejaculate size mediate selection on morphology in free-swimming

Keyne Monro and Dustin J. Marshall Research Centre for Geometric Biology, School of Biological Sciences, Monash University, Melbourne, Cite this article: Monro K, Marshall DJ. 2016 Victoria 3800, Australia Unravelling anisogamy: egg size and ejaculate dimorphism (anisogamy) defines the in most multicellular size mediate selection on morphology in organisms. Theoretical explanations for its maintenance usually emphasize free-swimming sperm. Proc. R. Soc. B 283: the size-related selection pressures of and survival, 20160671. assuming that fertilization of all precludes selection for phenotypes that http://dx.doi.org/10.1098/rspb.2016.0671 enhance fertility. In external fertilizers, however, fertilization is often incom- plete due to sperm limitation, and the risk of polyspermy weakens the advantage of high sperm numbers that is predicted to limit sperm size, allow- ing alternative selection pressures to target free-swimming sperm. We asked Received: 23 March 2016 whether egg size and ejaculate size mediate selection on the free-swimming Accepted: 20 June 2016 sperm of Galeolaria caespitosa, a marine tubeworm with , by comparing relationships between sperm morphology and male fertility across manipulations of egg size and sperm density. Our results suggest that selection pressures exerted by these factors may aid the maintenance of anisogamy in external fertilizers by limiting the of larger Subject Areas: sperm in the absence of competition. In doing so, our study offers a more com- ecology, plete explanation for the stability of anisogamy across the range of sperm environments typical of this and identifies new potential Keywords: for the sexes to coevolve via mutual selection pressures exerted by at fertilization. anisogamy, egg size, external fertilization, selection analysis, sexual coevolution, sperm morphology 1. Background Author for correspondence: Anisogamy is widely held to be the catalyst for the evolution and coevolution of the sexes [1–3]. Key theory for its origin [4] invokes disruptive selection on Keyne Monro gamete size in an isogamous marine ancestor with external fertilization. e-mail: [email protected] Assuming finite resources for reproduction, and that zygote fitness increases with the combined sizes of fusing gametes, fertilization efficiency is increased when one parent makes many small gametes to maximize zygote production, while the other makes fewer large gametes that provide the cytoplasmic reserves needed for zygote viability. Once evolved, anisogamy is said to be maintained by postcopulatory selection pressures (sperm competition and cryptic choice) that reinforce the specialization of male and female gametes [5,6]. Specifically, Parker [7] argued that anisogamy is unstable with- out sperm competition because eggs are kept large by the burden of zygote provisioning, whereas sperm are kept small by vying for fertilizations. Other- wise, males gain no advantage from making larger ejaculates once sperm are numerous enough to fertilize all available eggs and would be better off making larger sperm that contribute to zygote provisioning. Other theory proposed that sperm are small to yield the high numbers needed to ensure fertilization [8–10]. This was dismissed for internal fertilizers, including vertebrates and insects, on the grounds that ejaculates are often vastly diluted without loss of fertility, and fertilization is only incomplete under Electronic supplementary material is available sperm-limited conditions that rarely encounter [7]. Yet for external fer- at http://dx.doi.org/10.1098/rspb.2016.0671 or tilizers, sperm limitation is common due to rapid dilution of sperm after spawning [11,12], giving selection ample scope to favour both numerous tiny via http://rspb.royalsocietypublishing.org. sperm and large eggs via their enhancement of gamete encounter rates

& 2016 The Author(s) Published by the Royal Society. All rights reserved. [13,14]. Accordingly, recent models that simultaneously con- mating system, fertility is a bell-shaped function of gamete 2 sider the effects of sperm competition and limitation on encounter rates that increase with egg size and sperm density. rspb.royalsocietypublishing.org gamete evolution confirm that the latter can, in principle, At one extreme, insufficient encounters leave many eggs unferti- stabilize anisogamy by acting to increase sperm numbers lized due to sperm limitation; at the other extreme, excessive [15,16]. In practice, however, excess sperm cause a decline encounters make many fertilizations unviable due to polyspermy [33]. Since a certain number of polyspermic fertilizations occur in fertility due to polyspermy, a condition that is almost naturally regardless of encounter rates, fertility is to an extent invariably lethal in external fertilizers [17]. Larger ejaculates incomplete even at its maximum [33–36]. Fertility also varies may therefore have less adaptive value than predicted [18] with other factors such as egg fertilizability, sperm swimming and even destabilize anisogamy by favouring smaller eggs behaviour and male–female compatibility [37,38]. Consequently, with lower risk of polyspermy [19–21]. Overall, this suggests selection can potentially target gametes of either via that numeric effects on sperm size driven by competition their effects on fertility. Individuals used in our study were may have limited power to explain anisogamy in external sampled intertidally near Royal Brighton Yacht Club, Victoria, B Soc. R. Proc. fertilizers and that other selection pressures may contribute Australia (37.918 S, 144.998 E), and held in recirculating aquaria to its evolutionary maintenance. at Monash University. To obtain fresh gametes daily for exper- Little is known of how female choice influences the imentation, adults were extracted from tubes and isolated in phenotypes of free-swimming sperm, despite widespread small volumes of filtered seawater to induce spawning. 283 evidence of its potential to shape the outcomes of sperm com- petition and sperm evolution in mammals, birds and insects (b) Manipulation of fertilization environment 20160671 : [22–24]. For internal fertilizers like these, female choice of To understand whether egg size mediates selection on sperm mor- sperm occurs during sperm transport and storage in the phology, and whether its effects interact with sperm limitation, we female reproductive tract, and targets properties (e.g. size, trialled the fertility of 97 focal males of known sperm phenotype at length, quality) that thereby influence male fertility beyond low and high sperm densities against reference cohorts of small the numerical effects of ejaculate size [25,26]. For external fer- and large eggs (figure 1). Each trial consisted of a single male’s tilizers whose sperm undergo selection in the external sperm exposed to a replicate cohort of eggs, which were pooled across 25–30 females to minimize compatibility effects on male fer- environment without the complexity of the female reproduc- tility. Care was taken to ensure that all females contributed a tive tract, female choice of sperm is limited to gamete-level similar number of eggs. We did not conduct trials with sperm com- interactions [27] controlled by accessory reproductive fluids petition because we were interested in alternative mechanisms of [28] or the phenotypes of eggs themselves. The role of gamete sperm evolution and lacked the capacity to assign zygote paternity recognition proteins located on eggs in determining male ferti- via molecular analyses. Free-spawned eggs in nature are often lity and reproductive isolation is well established [29,30]. Egg faced with sperm limitation [11,12], making our trials ecologically size offers another key opportunity for females to exert selection relevant to this mating system (see also [39,40]) but nonetheless on free-swimming sperm via its role in fertilization kinetics, limited to inferring selection pressures in the absence of compe- yet its capacity to do so, its interplay with ejaculate size and tition. Trials were conducted in 13 replicate blocks with 5–11 the broader consequences for the evolutionary maintenance of males per block. Within blocks, each male was mated with the anisogamy are currently unknown. same combination of females, so that trials were standardized with respect to the genetic background of eggs, and differed Here, we present a novel test of this idea, exploring only with respect to our manipulation of fertilization environment whether egg size mediates selection on the free-swimming and male identity. sperm of Galeolaria caespitosa, a dioecious marine tubeworm that sheds male and female gametes into the sea to fuse exter- nally. Specifically, we used the classic multiple-regression (i) Egg size Based on preliminary work, female Galeolaria eggs up to approach of Lande & Arnold [31], implemented in a mixed- 70 mm in diameter and eggs smaller than 55 mmindiameterare model framework, to compare the relationship between not fertilizable. To manipulate egg size for our trials, we filtered sperm morphological traits and male fertility across an exper- all eggs spawned by the unique set of females used in each replicate imental manipulation of egg size. We did so in combination block to remove unviable eggs and partition the rest into two size with an orthogonal manipulation of ejaculate size to further classes. First, we pooled all eggs per block of females in a small explore whether egg-mediated selection interacts with volume of seawater and washed the suspension through 60 mm sperm limitation, given the joint importance of these two fac- mesh. We re-filtered the portion of eggs caught by this mesh size, tors in alternative explanations for anisogamy [13,14,16] (see retaining those caught by it a second time (diameter . 60 mm) as also [10] for a review of theory not covered here). By asking our large size class. Second, we re-filtered the portion of eggs that whether selection pressures surrounding fertilization in the passed through this mesh size using 55 mm mesh, retaining the por- sea act to oppose or reinforce gamete dimorphism in external tion caught by it (55 mm , diameter , 60 mm) as our small size class and discarding those that passed through as unfertilizable. fertilizers, our study has important implications for sexual This difference in diameter translates into approximately 30% coevolution and the evolutionary maintenance of anisogamy difference in mean volume between size classes. Both portions of in this mating system. retained eggs were re-suspended in filtered seawater at densities of approximately 2 Â 103 ml21 until use in trials. To confirm the effectiveness of our manipulations, we compared the sizes of 15 eggs from each size class per block using t-tests. In no case was it 2. Material and methods necessary to discard a block of trials because the mean size of (a) Study species large eggs was no larger than that of small eggs (all p , 0.02). Galeolaria caespitosa (named by genus hereafter) inhabits interti- dal zones of southwestern Australia in dense aggregations of (ii) Sperm density calcareous tubes [32]. Adults are fertile year-round and continu- Since ejaculate size alters patterns of selection on sperm mor- ally spawn gametes that fuse in the water column. Typical of this phology in another Galeolaria species [40], we included a similar reference cohort of eggs 3 rspb.royalsocietypublishing.org 1–n

small eggs large eggs B Soc. R. Proc.

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Figure 1. Experimental manipulation of fertilization environment for trials of male fertility. Free-spawned eggs were pooled across multiple females, then filtered into small and large size classes. Free-spawned sperm were kept isolated by male and diluted to low and high densities (with an aliquot reserved for phenotyping). This created four fertilization environments in which each male’s fertility was trialled. Trials were conducted in 13 replicate blocks, with sperm from each focal male (n ¼ 5–11 per block) exposed to a reference cohort of eggs from the same pool of females (n ¼ 25–30 per block) to isolate the effects of male identity and experimental conditions on male fertility. (Online version in colour.) manipulation here to evaluate the generality of this effect and its at 1000Â magnification, ensuring that the entire cell lay in the interplay with egg size. We first ran preliminary fertilization plane of focus, and tracing the tail during live capture to account trials across 10-fold dilutions of sperm (107 –102 cells ml21)to for any flexing that may have been difficult to interpret from find two densities that (i) imposed sperm limitation without risk- stored images. In IMAGEJ (http://imagej.nih.gov/ij), we measured ing total fertilization failure for eggs in the small size class, given the length of the sperm tail, plus the length and width of the sperm their smaller target size for sperm, and (ii) maximized fertility body. The latter comprises an oval nucleus, a midpiece of four without risking severe polyspermy for eggs in the large size spherical mitochondria, and a cap-like acrosome that extends class, given their larger target size [19,20]. Based on these trials, into a long, branching process upon contact with eggs (see also we used 104 cells ml21 as our low density and 105 cells ml21 as [42] for a detailed explanation of Galeolaria sperm). We measured our high one. These densities fall within the continuum available only normal cells with intact tails and no acrosomal reaction to to Galeolaria eggs in the field, and fertility at our low density is signal contamination of the sample by eggs. comparable with field estimates [41]. We collected each male’s eja- culate (reserving 10 ml for sperm phenotyping; see below) and recorded its initial sperm density (106 –108 cells ml21) from three (d) Fertilization trials replicate haemocytometer counts. To trial male fertility under Fertilization trials, each consisting of 0.1 ml of egg solution equivalent conditions, we standardized initial sperm densities mixed gently with 0.9 ml of sperm solution, were run in small among males before diluting samples from each male to the vials at a constant temperature of 218C. There were four trials required experimental conditions. for each of the 97 males, exposing sperm at low and high den- sities to both large and small eggs (figure 1). Each block of trials also included a vial of sperm-free eggs of each size class, (c) Measurement of sperm morphology to control for errant fertilizations that may have occurred while Sperm morphology was recorded from the reserved sample of extracting adults from tubes. We ran trials for 2 h, shaking each male’s ejaculate. We captured images of at least 15 cells per them gently every 15 min to re-suspend gametes and minimize male using a digital camera mounted to a compound microscope oxygen limitation within vials. After this time, we captured images of all trials using a digital camera mounted to a compound from the same male by fitting an unstructured covariance 4 microscope at 100Â magnification, and scored fertility as the pro- matrix, with diagonal elements estimating male variance in rela- portion of undergoing normal development. Counts tive fertility by fertilization environment, and with off-diagonal rspb.royalsocietypublishing.org were done blind and repeated by two observers. The initial repeat- elements estimating covariance in fertility across environ- ability of counts was high (yielding an intraclass correlation of 0.86), ments due to shared male identity. This model (model 1) was but trials with disparate counts were further re-checked against the baseline for subsequent analyses. images to exclude errors in scoring embryonic development. Next, to ask whether selection on sperm traits differed in response to egg size and sperm density, we used nested LRTs to compare the fits of models in which partial regression (e) Data analyses coefficients (selection gradients) were allowed to vary across fer- (i) Variation in sperm morphology tilization environments with the fits of reduced models in which To evaluate the relative magnitudes of variation in sperm traits gradients were held constant environmentally [46]. Since the among males and within males (i.e. among gametes from the multifactorial nature of our experiment meant that selection gra- B Soc. R. Proc. same ejaculate), we fitted a multivariate linear mixed model dients could potentially differ in response to interactive effects of using restricted maximum likelihood, with tail length, body egg size and sperm density, such complex three-way interactions length and body width as response variables, and with male and had to be evaluated before exploring the simpler, additive effects gamete (nested in male) as random effects. This model estimated of each factor in isolation. We therefore added to our baseline the among-male variance of each trait, plus the among-male model 1 all linear (directional) selection gradients, plus all their 283 covariance (or correlation) between each trait pair, partitioned two- and three-way interactions with both factors. This model 20160671 : from residual, within-male variation (electronic supplementary (model 2) fitted our data no better than a reduced model material, table S1). We tested the significance of all estimates (model 3) from which three-way interactions were omitted 2 using standard likelihood-ratio tests (LRTs) corrected for multi- (x ¼ 0.95, d.f. ¼ 3, p ¼ 0.81), indicating that egg size and sperm plicity [43]. Because fertility was assayed at the level of males, density had, at most, independent effects on directional selection. not gametes, we used male trait means in our analyses of selection. We then compared model 3 with other, further-reduced models Conceptually, doing so assumes that sperm phenotypes are deter- in which gradients were pooled across either egg sizes or mined more by the diploid genomes of males than by the haploid sperm densities to test whether directional selection was modi- genomes of sperm, as seems to be the case for most species [44]. fied by each factor alone. Once directional selection gradients Statistically, doing so assumes that trait values are fixed variables were estimated on the appropriate scales (i.e. within or across measured without error, which is expected to bias our estimates of fertilization environments) identified by our model-selection pro- selection towards zero, but does not bias the hypothesis tests of cess, we used LRTs to evaluate the significance of each gradient these coefficients that are our focus here [45]. and whether it differed among environments. Last, building upon model 3, we repeated the process for non- linear (quadratic and correlational) selection gradients. Again, in (ii) Selection on sperm morphology the absence of any evidence that egg size and sperm density To estimate selection on sperm traits (tail length, body length and had interactive effects on nonlinear selection (x2 ¼ 9.17, d.f. ¼ 6, body width) owing to their impacts on male fertility, we p ¼ 0.16), we fitted reduced models to explore whether nonlinear implemented Lande & Arnold’s [31] multiple-regression approach selection was altered by either factor alone. in a mixed-model framework using maximum likelihood. In this framework, we also implemented a model-selection approach [46] to compare selection across manipulations of egg size and sperm density, thereby testing whether these factors act as 3. Results agents of selection on sperm morphology [47]. Before analysis, sperm traits were transformed to units of stan- (a) Variation in sperm morphology dard deviation [31], while fertility was transformed to a relative Sperm tail length, body length and body width each varied sig- scale by dividing each absolute value by the mean value across nificantly among males (electronic supplementary material, all fertilization environments (see also [40,48]). By preserving figure S1), with male identity explaining 38%, 37% and 27%, environmental differences in mean fertility, this transformation respectively, of the trait’s total variation among gametes (elec- assumes hard selection on free-swimming sperm: in other tronic supplementary material, table S1). Sperm traits also words, that different environments contribute different numbers of to the next generation, favouring genotypes with high- covaried among males, with a significantly positive correlation ¼ est fertility across environments [49,50]. For external fertilizers like (r 0.44) between tail length and body length, and a signifi- Galeolaria, differential fertility due to fine-scale differences in ferti- cantly negative one (r ¼ 20.29) between tail length and body lization environment [41,51] and the subsequent mixing of width. Hence, males with longer-tailed sperm tended to have dispersive zygotes in the water column make this a likely scenario longer-bodied sperm or narrower-bodied sperm, but dimen- (see also [16] for a similar treatment of fitness in modelling the sions of the sperm body were themselves uncorrelated evolution of anisogamy). The key distinction from soft selection (electronic supplementary material, table S1). (which assumes that each environment contributes equally to the next generation) lies in the evolutionary consequences, since hard selection should drive populations to a single phenotypic (b) Variation in fertility across fertilization environments optimum, whereas soft selection is expected to promote local Illustrating the expected kinetics of external fertilization, the optima [52]. Operationally, our transformation simply rescaled mean fertility of males increased in response to egg size gradients in accordance with [31]; their signs and relative magni- (x2 ¼ 90.11, d.f. ¼ 1, p , 0.01) and sperm density (x2 ¼ 94.79, tudes within environments, their directions of change across d.f. ¼ 1, p , 0.01; electronic supplementary material, figure environments and their hypothesis tests were identical to analyses x2¼ ¼ of untransformed data. S2), but their effects were not interactive ( 2.35, d.f. 1, First, we analysed relative fertility in a linear mixed model p ¼ 0.13). Eggs in both size classes were considerably sperm- with egg size and sperm density as factorial fixed effects, and limited at low sperm density, as were small eggs at high with block and male identity as random effects. When modelling sperm density (albeit to less extent; electronic supplementary male identity, we accounted for the non-independence of trials material, figure S2). Large eggs at high sperm density 0.20 p , 0.01; electronic supplementary material, table S2). Com- 5 small eggs paring individual gradients across densities attributed

** rspb.royalsocietypublishing.org large eggs this result to disruptive selection on body length at high 0.15 density, whereas this trait was selectively neutral at low density

) ± s.e. 2 i (gbody lengthÂsperm density: x ¼ 14.06, d.f. ¼ 1, p , 0.01), and * 0.10 to positive correlational selection on tail and body lengths at low density, whereas this trait combination was neutral 2 0.05 at high density (gtail lengthÂbody lengthÂsperm density: x ¼ 6.82, d.f. ¼ 1, p , 0.01). Sperm density also altered quadratic selec- 2 0 tion on body width (gbody width sperm density: x ¼ 4.14, d.f. ¼ 6, p ¼ 0.04) and correlational selection on body length and 2 width (g : x ¼ 5.03, d.f. ¼ 1, B Soc. R. Proc. –0.05 body length body widthÂsperm density p ¼ 0.02), but no individual gradient was significant. Similarly,

tail length was under stabilizing selection at low sperm

directional selection gradient ( b –0.10 * * density and neutral at high density, but this difference across environments was not significant. 283 –0.15 To resolve these latter ambiguous results and to compare tail length body length body width the overall geometry of nonlinear selection across sperm den- 20160671 : sperm morphology sities, we performed an eigenanalysis of each g-matrix [53]. Figure 2. Egg size mediates directional selection on sperm morphology. Each This post hoc procedure complements the usual estimation of nonlinear selection gradients by visualizing nonlinear gradient (bi) measures the direct force of selection to increase or decrease the trait mean (*p , 0.05; **p , 0.01). For each trait, selection differs selection along composite axes (eigenvectors of g, defined significantly in strength across egg size classes. (Online version in colour.) by linear combinations of sperm traits) where curvature (given by eigenvalues of g) is strongest. As such, it does not uncover any more selection than in g, but simply reallo- cates selection to the various coefficients [54]. We calculated approached the maximum fertility indicated by preliminary standard errors and p-values for eigenvalues according to trials (see Material and methods section). Reynolds et al. [55] and plotted significant axes using thin- As estimated in model 1, relative fertility varied signifi- plate splines [54]. cantly among males in all fertilization environments, At each sperm density, we detected two significant axes of offering consistent opportunity for selection to target sperm nonlinear selection (electronic supplementary material, table phenotypes via this fitness component (electronic sup- S3), allowing selection to be visualized as a single fitness plementary material, figure S2). Male variance in fertility surface and the sperm phenotypes that maximize fertility to depended on interactive effects of egg size and sperm density be found by jointly interpreting trait loadings on those axes. (seen in significant loss of fit when their effects were mod- At low density, the surface was a simple peak (figure 3a). elled as independent: x2 ¼ 19.76, d.f. ¼ 4, p , 0.01). The Here, the contrasting loadings of tail length and body dimen- interaction occurred because variance increased from smaller sions on m3 imply that highly fertile males had sperm with eggs to larger eggs at low sperm density (x2 ¼ 18.73, d.f. ¼ 1, average values of all traits, or that combined longer tails with p , 0.01), but was unaltered by egg size at high sperm den- smaller bodies (with the latter kept small by the high loading sity (x2 ¼ 1.13, d.f. ¼ 1, p ¼ 0.29; electronic supplementary of body width on m2). By contrast, the surface at high density material, figure S2). was a saddle (figure 3b). Here, each body dimension contribu-

ted positively to m1 and tail length dominated m3, implying (c) Selection on sperm morphology across fertilization that highly fertile males had sperm with unusually large bodies (or unusually small ones, though for less gains in environments fertility) and tails of average length. Directional selection gradients (b), measuring the direct force of selection to shift the mean values of sperm traits, differed in response to egg size (x2 ¼ 16.46, d.f. ¼ 3, p , 0.01) but not sperm density (x2 ¼ 5.78, d.f. ¼ 3, p ¼ 0.12). Comparing indi- 4. Discussion and conclusion vidual gradients across egg size classes showed that larger Theoretical explanations for the maintenance of anisogamy eggs exert stronger selection on sperm morphology than smaller have traditionally emphasized the size-related selection press- eggs do (figure 2). Specifically, longer sperm tails enhanced male ures of sperm competition and postzygotic survival [4,7], fertility regardless of egg size, but the benefits became more assuming that fertilization of all available eggs precludes 2 extreme for sperm targeting larger eggs (ßtail lengthÂegg size x ¼ selection for phenotypes that enhance fertility [10]. In external 4.26, d.f. ¼ 1, p ¼ 0.03). Similarly, the length and width of the fertilizers, however, fertilization is often incomplete due to sperm body had no impact on a male’s ability to fertilize smaller sperm limitation [11,12], and the risk of polyspermy weakens eggs, but larger eggs favoured males with shorter-bodied sperm the advantage of high sperm numbers that is predicted 2 (ßbody length egg size: x ¼ 10.24, d.f. ¼ 1, p , 0.01) and narrower- to limit sperm size [18]. These issues give scope for alternative 2 bodied sperm (ßbody widthÂegg size: x ¼ 7.17, d.f. ¼ 1, p , 0.01). selection pressures to shape the evolution of free-swimming Nonlinear selection gradients (g), measuring the direct force sperm. Here, we show that egg size and ejaculate size of selection to shift the variances and covariances of sperm traits, independently mediate selection on the morphology of the were unaltered by egg size (x2 ¼ 8.89, d.f. ¼ 6, p ¼ 0.18) free-swimming sperm of Galeolaria, and in ways that could but differed in response to sperm density (x2¼ 22.94, d.f. ¼ 6, potentially reinforce the size dimorphism of male and female (a)(b) 6 low sperm density high sperm density rspb.royalsocietypublishing.org

0.80 2.00

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relative fitness 0.50 1.25 –2 –2 0.40 –1 1.00 –1 B Soc. R. Proc. –1 0 2 0 2 3 0 1 m 1 1 m m 1 2 m 0 2 3 2 1 –1

Figure 3. Sperm density mediates nonlinear selection on sperm morphology. Each axis (mi) is defined by a linear combination of sperm traits (tail length, body 283

length and body width) and measures the direct force of selection to increase or decrease variation in trait combinations. Axes are derived from eigenanalyses of 20160671 : nonlinear selection gradients (g; see details in text). (Online version in colour.) gametes. In doing so, our study offers new insights into sperm different males when given the opportunity to do so remains evolution and the evolutionary maintenance of anisogamy in an open question. external fertilizers. Female choice of sperm is increasingly recognized as an important driver of sperm evolution and sexual coevolution in internal fertilizers, seen in positive associations between (a) Egg size is an agent of directional selection on sperm length and dimensions of the female reproductive tract [22–24]. Evidence of female sperm choice in external fer- sperm morphology tilizers has been slower to accumulate, despite offering one of Our tests revealed egg size as an agent of directional selection the few opportunities for females to exert in this on sperm morphology, actively promoting directional evol- system [12,61]. A notable exception is the body of work on utionary change in sperm traits via their effects on male gamete recognition proteins, demonstrating their facilitation fertility. Specifically, larger eggs intensified positive selection of mate choice and reproductive isolation by mediating male on tail length: that is, longer-tailed sperm enhanced male fertility within and among species [30,62,63]. Other studies fertility regardless of egg size, but the selective advantage of have also suggested that the ovarian fluid surrounding eggs longer tails grew more extreme when sperm targeted larger at spawning controls female choice of conspecific sperm via eggs. Larger eggs also favoured shorter- and narrower-bodied effects on sperm swimming behaviour [28,64], though this sperm, whereas smaller eggs relaxed selection on sperm body phenomenon is absent in our study species [37]. Here, to our dimensions entirely. Based on physical and biomechanical prin- knowledge, we provide the first evidence that eggs may them- ciples [56,57], these results therefore predict that evolutionary selves target morphological, possibly hydrodynamic, aspects increases in egg size will drive the evolution of a more hydro- of sperm performance, adding a new dimension to the argu- dynamic sperm morphology, with reduced drag from a ment that eggs are more than passive receivers of sperm, and smaller body and greater thrust from a longer tail. Evidence external fertilization more than a race among males [63]. from other external fertilizers that low body-to-tail ratios enhance sperm swimming speed [58], and that faster sperm have higher fertilization success [59], supports our interpret- (b) Ejaculate size is an agent of nonlinear selection on ation. This course assumes that the adaptive evolution of sperm phenotype is unconstrained by genetic architecture, sperm morphology which remains unknown for Galeolaria, but is nonetheless Given the role of gamete encounter rates in fertilization kinetics consistent with current evidence from diverse taxa [60]. [19–21], we expected that selection on Galeolaria sperm would The possibility that egg size exerts selection on the depend on interactive effects of egg and ejaculate sizes, with morphology of free-swimming sperm raises the question of egg size effects being stronger under sperm limitation and whether this constitutes an analogue of cryptic female choice. easing as eggs became sperm-saturated. Instead, ejaculate A necessary component of such choice is that females discrimi- size altered nonlinear selection on sperm morphology, acting nate between, and preferentially use, the sperm of different to change the variance of trait combinations, but did not alter males [61]. Here in Galeolaria, eggs of different sizes altered the directional changes favoured in those traits by eggs. To the probability of fertilization by a given male according to understand how these different aspects of the fertilization the morphology of his sperm. Since eggs were pooled across environment target different moments of the distribution of females to isolate the fitness consequences of sperm traits sperm phenotypes, it is worth realizing that nonlinear selection from genetic compatibilities at fertilization, we argue that this shapes trait variation independently of directional selection on phenomenon may indeed reflect the preferential use of differ- trait means [31,53]. Hence, sperm-limited conditions drive the ent sperm phenotypes by eggs. Since our fertilization trials contraction of variance around the longer-tailed, smaller- were not conducted under competitive conditions, however, bodied phenotype favoured by larger eggs, acting to stabilize the capacity for eggs to discriminate between sperm from that phenotype in doing so. While this loss of variance could potentially limit egg-mediated selection by reducing the range midpiece, which stores the resources that drive energy metab- 7 of sperm phenotypes available, sperm-saturated conditions olism and [65], and produces energy in proportion to rspb.royalsocietypublishing.org should have the opposite effect, giving eggs new selective its size [66]. Thus, if the resources gained from a smaller body opportunities by acting to expand variance around the new outweigh those paid for a longer tail (as is likely if swimming phenotype. Such fluctuating effects of sperm density are facili- is powered by the midpiece), then larger eggs may indeed tated in nature, moreover, by variation in population size, shape a sperm morphology that is effectively smaller in terms spawning behaviour and dilution effects [11,12]. of net cost. This argument, while admittedly speculative, Estimates of selection on sperm phenotype remain rare for suggests that the focus on sperm size in models of anisogamy external fertilizers, but past work has revealed broadly similar and sperm competition potentially offers an over-simplified patternsto ours. Forexample, Fitzpatricket al. [39] detected non- view of sperm evolution (see also [56]), but one that could be linear, but not directional, selection in favour of smaller sperm reconciled with results like ours by a better understanding of bodies under sperm-limited conditions in the mussel, Mytilus the energetic costs of different sperm designs. B Soc. R. Proc. galloprovincialis. Our study of Galeolaria gemineoa [40] found Last, sperm and egg sizes are further assumed to coevolve that longer-tailed, smaller-bodied sperm had a selective advan- via their shared investment in zygotes [4,7], but our study tage at higher sperm densities, but driven more by directional argues that gametes of external fertilizers may do so via than nonlinear selection and across a broader range of densities mutual selection pressures exerted by sperm and eggs at fer- 283 than we explored here. Hence, the limited evidence available tilization. Past work on this mating system has established supports two observations. First, selection on sperm mor- the importance of sperm density as an agent of selection on 20160671 : phology is context-dependent, offering a potential explanation egg size, with smaller ejaculates favouring larger eggs that for the diversity of ejaculate characteristics in external fertilizers are more easily located by sperm, but larger ejaculates favour- [27]. Second, regardless of its exact form and source, selection ing smaller eggs with less risk of polyspermy [19–21]. By consistently targets body size relative to tail length, bearing now showing that egg size reciprocates as an agent of selec- out the apriorirecommendation of this ratio as a key predictor tion on sperm, we infer that the evolutionary fates of the of sperm performance via its links to swimming speed [56]. sexes are coupled by interactions between gametes at both pre- and postzygotic stages of the history. Moreover, (c) Implications for the evolutionary maintenance the evolutionary diversification of egg size spans an astonish- ing several orders of magnitude among extant metazoans, of anisogamy and is usually attributed to environmental heterogeneity in Overall, our work suggests that selection pressures exerted by postzygotic selection [67]. The possibility that sperm evol- egg size and sperm limitation may aid the maintenance of ani- ution has been harnessed to this process implies that a key sogamy in external fertilizers by limiting the adaptive value of next step will be to look for signs of gamete coevolution in larger sperm in the absence of competition. This departs from the deeper evolutionary history of this group. the usual assumption that easing the pressure of sperm compe- tition should favour larger sperm that enhance zygote viability Data accessibility. Data available on Dryad (http://dx.doi.org/10.5061/ [1,4,7]. We by no means argue that these pressures are mutually dryad.2bj8g). exclusive, only that those demonstrated here potentially offer a Authors’ contributions. K.M. and D.J.M. conceived and designed the more complete explanation for the stability of anisogamy across study. K.M. analysed the data and drafted the manuscript. Both the range of sperm environments typical of this mating system. authors contributed substantially to revisions and gave final Our argument that larger eggs favour smaller sperm (despite approval for publication. Competing interests. ample trait variation, only weakly constrained by negative cor- We have no competing interests. Funding. relations, to let them evolve proportionately larger; electronic The study was supported by fellowships and grants awarded under the Australian Research Council’s ’Discovery Projects’ scheme. supplementary material, table S1) assumes that the body is Acknowledgements. We thank Amy Hooper for excellent research assist- costlier than the tail. Sperm energetics, however, offer some ance, Craig White, Evatt Chirgwin and several anonymous reviewers support for this interpretation. For free-swimming sperm, for constructive comments on the work and manuscript, and Royal body size is the sum of the nucleus and mitochondrial Brighton Yacht Club for access to field collection sites.

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