Abstract Conflict between the sexes over reproductive interests can drive rapid evolution of reproductive traits and promote speciation. Here we show that inter-species mating between Caenorhabditis nematodes sterilizes maternal individuals. The principal effectors of male-induced harm are sperm cells, which induce sterility and shorten lifespan by displacing conspecific sperm, invading the ovary, and sometimes breaching the gonad to infiltrate other tissues. This sperm-mediated harm is pervasive across species, but idiosyncrasies in its magnitude implicate both independent histories of sexually antagonistic coevolution within species and differences in reproductive mode (self-fertilizing hermaphrodites versus females) in determining its severity. Consistent with this conclusion, in androdioecious species the hermaphrodites are more vulnerable, the males more benign, or both. Patterns of assortative mating and a low incidence of invasive sperm occurring with conspecific mating are indicative of ongoing intra-specific sexual conflict that results in inter-species reproductive incompatibility.

Author Summary The sexes have divergent reproductive interests, and conflict arising from this disparity can drive the rapid evolution of reproductive traits and promote speciation. Here we describe a unique reproductive barrier in Caenorhabditis nematodes that is induced by sperm. We found that mating between species can sterilize maternal worms and even cause premature death, and we were able to attribute this phenomenon directly to the sperm themselves. Sperm from other species can displace sperm from the same species and, in some cases, can invade inappropriate parts of the maternal reproductive system and even their non-reproductive tissues. We find that mating to males of another species harms females far more than does within-species mating. Overall, our observations are consistent with ongoing sexual conflict between the sexes within species, arising as a byproduct of sperm competition among the gametes of different males. Finally, patterns of assortative mating indicate that mating behaviours that reduce the likelihood of costly inter-species mating have evolved in this group of animals. These findings support an important role of sexual selection and gametic interactions contributing to reproductive boundaries between species, as predicted by evolutionary theory.

Citation: Ting JJ, Woodruff GC, Leung G, Shin N-R, Cutter AD, Haag ES (2014) Intense Sperm-Mediated Sexual Conflict Promotes Reproductive Isolation in Caenorhabditis Nematodes. PLoS Biol 12(7): e1001915. https://doi.org/10.1371/journal.pbio.1001915 Academic Editor: Mariana Federica Wolfner, Cornell University, United States of America Received: December 6, 2013; Accepted: June 19, 2014; Published: July 29, 2014 Copyright: © 2014 Ting et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from NSERC and NIH to ADC, NIH award GM79414 to ESH, a Canada Research Chair to ADC, an NSERC Post-Graduate Scholarship to JJT, and an Anne G. Wylie Dissertation Fellowship to GCW. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Abbreviations: df, degree of freedom; DIC, differential interference contrast; Fog, feminization of germline; XX animals, females and hermaphrodites

Introduction Rarely do reproductive interests of males and females perfectly align. Sexual selection can accelerate the evolution of the traits and molecules mediating reproductive encounters, and this can lead to sexual conflict [1],[2]. Components of the reproductive system that mediate male-female interactions, such as reproductive tract morphology, sperm and egg traits, and molecular components of seminal fluid all diverge rapidly between many species [3],[4]. The particularly forceful process of sexual antagonism drives co-evolutionary arms races between sex-limited traits that exact or counteract harmful, but self-serving, effects on the other sex [2],[4],[5]. Ongoing sexually antagonistic coevolution that operates within a species might generate mismatched interactions between gametes or other reproductive tract components when mating occurs between species. When such mismatches interfere with normal conspecific reproduction [6],[7], they have the potential to instigate or magnify reproductive isolation between species [8],[9]. Selection for traits that prevent the deleterious consequences of inter-species mating for the parents or hybrid offspring may result in further trait evolution [10]. Though pre-mating reinforcement behaviours have received much attention and debate [10]–[12], post-mating mechanisms of gametic isolation, such as conspecific sperm precedence, also can play key roles in pre-zygotic reproductive isolation [13],[14]. The prevailing view of gametic isolation between species is that fertilization precedence of conspecific sperm can provide a potent reproductive barrier, mediated by cryptic female choice, sperm competition, or incompatibilities between female reproductive tracts and heterospecific ejaculates [15]–[18]. Conspecific sperm precedence occurs both in species with internal and external fertilization, governed by a broad variety of proximate mechanisms [14],[19],[20]. Alternatively, Drosophila provide examples of inter-species mating harm, for example, owing to an overly engorged “insemination reaction mass” that exacts a fitness cost on females [6],[7], and female Carabus beetles suffer ruptured reproductive tracts from physical damage upon inter-species matings [21]. Within species, male seminal proteins can manipulate female physiology in a manner sub-optimal for females but beneficial to males [22]. While coevolution between the sexes may obscure the traces of such sexual antagonism (as for other forms of genetic conflict [23]), interactions between divergent populations and species can unmask the underlying conflicts by revealing mismatched male and female traits [8]. Caenorhabditis nematodes provide a powerful system to examine both sexual antagonism and its modulation by reproductive mode. Males, females, and hermaphrodites will mate readily and promiscuously in lab culture, and mechanical harm incurred from multiple mating reduces longevity and survival in C. elegans hermaphrodites [24] and C. remanei females [25]. Male-derived chemical cues also are thought to accelerate female and hermaphrodite aging [26]. Following copulation, C. elegans hermaphrodites can expel male ejaculates [27], and males deposit copulatory plugs that inhibit re-mating [28],[29] and induce larger brood sizes in their partners [30]. In response to experimentally elevated sperm competition, C. elegans evolve larger sperm [31]. Though anatomical evolution in Caenorhabditis is conservative, these intra-specific dynamics suggest there may be substantial inter-species divergence in cryptic reproductive traits. Evolutionary transitions in reproductive mode from highly outbreeding to highly self-fertilizing are expected to reduce intra- and inter-sexual conflict [32],[33]. Three species of Caenorhabditis have independently evolved androdioecy (hermaphrodites and males) from dioecy (females and males), such that hermaphrodites are capable of self-fertilization in addition to being fertilized by males [34]. These androdioecious species manifest a “selfing syndrome” analogous to plants that includes reduced sperm size and low mating vigor [35],[36]. Hermaphrodites from such species with relaxed sexual selection might be particularly susceptible to adverse effects of mating to vigorous males from closely related species that have a recent history of strong sexual selection (the “weak inbreeder, strong outbreeder” or WISO hypothesis [33],[37]). Despite the generally limited understanding of Caenorhabditis ecology and inter-species interactions in their rotting fruit and vegetal habitats, some species are sympatric [34],[38], putting them at risk for inter-species encounters. Species readily mate with one another in the laboratory, and the animals' transparent bodies provide literal windows into postmating-prezygotic, and postzygotic, reproductive interactions and barriers [39]–[41]. Here we describe an unprecedented postmating-prezygotic reproductive barrier in Caenorhabditis nematodes, induced directly by sperm cells, that imposes potent fitness costs to females and hermaphrodites. Theory predicts species with selfing hermaphrodites to be more susceptible to inter-species harm and less capable of inducing harm [33],[37]. Theory also predicts that rapid divergence in sexually selected traits will produce heterogeneity in harmful effects between species pairs and consequently may fail to yield phylogenetic signal in the magnitudes of effect [2],[4]. In this counterpoint to mechanisms of conspecific sperm precedence, we affirm a potent role for sexual conflict as a pre-zygotic isolating barrier between species.

Materials and Methods Maintenance Animals were maintained according to standard C. elegans procedures [77], with the exception of increased agar concentration in NGM plates to 2.2% in order to discourage animals from burrowing underneath the surface of the plate. Cultures were maintained at 20°C and 25°C. See Text S1 for strains of each species used for experiments: C. afra (sp. 7), C. brenneri, C. briggsae, C. elegans, C. latens (sp. 23), C. nigoni (sp. 9), C. portoensis (sp. 6), C. remanei, C. tropicalis (sp. 11), C. wallacei (sp. 16), C. sp. 5 [44]. Quantification of Reduced Reproductive Output Crosses consisted of placing one hermaphrodite at the fourth larval stage (L4, penultimate stage of development) with six heterospecific males overnight (18–24 hours) on a 35 mm diameter Petri dish with a 10 mm diameter bacteria spot (E. coli OP50). Hermaphrodites (Figure 1A–1F) that successfully mated (presence of a copulatory plug) were transferred daily and we measured reproductive output as the yield of viable adult progeny from two days of egg laying following the final mating event (representing >90% of lifetime brood size). Control hermaphrodites were individuals allowed to produce self-progeny. For matings involving females (Figure 2A and 2B), we first mated them to conspecific males overnight and the subsequent day we mated treatment females to heterospecific males. In all cases except one (C. briggsae×C. nigoni), matings are incapable of yielding viable hybrid progeny (few hybrids are produced by C. briggsae×C. nigoni [40],[78]). Therefore, reproductive output measures the number of conspecific progeny of females (or, equivalently, self-progeny of hermaphrodites). The single mating treatment (Figure 1D) consisted of placing ten young adult hermaphrodites (C. briggsae) with 40 young adult males (C. nigoni) on a 35 mm Petri dish with a 10 mm diameter bacteria spot. After an hour, mated hermaphrodites were isolated to individual 35 mm diameter Petri dishes, transferred daily, and allowed to lay eggs in order to measure progeny production. Survival Measurements In situations when both female and hermaphrodites are used, they will be referred to as XX animals as they both have two X chromosomes. Seven L4 XX animals, depending on the species, were placed with ten heterospecific or conspecific males per plate and left overnight. The next day, XX animals were assayed for mortality by being touched on the head with an eyebrow hair glued to a toothpick. If the animal performed a backwards locomotive response to the touch, it was scored as alive. If it did not, it was scored as dead. This was performed every day for at least seven days. Every two days, XX animals and males were transferred to new plates in order to prevent the confusion of progeny with parents. Additionally, in these assays, XX animals were kept under continuous mating conditions: when males died or crawled off the plate, they were replaced with new males. XX animals that crawled off the plate were excluded from the lifespan measurements. DNA Staining and Vital Staining of Sperm The nuclei of animals were visualized using Hoechst 33258 staining. Seven XX animals were mated with ten heterospecific or conspecific males per plate for 1–3 days, and then XX animals were fixed in 100% methanol overnight at 4°C. The animals were then washed three times in M9 buffer and incubated in 1 µg/ml Hoechst in M9 buffer for 5 minutes, followed by mounting for fluorescent microscopy and imaging. Male sperm were fluorescently labeled in vivo with MitoTracker Red CMXRos (Invitrogen) [71]. Males were incubated in 1 mM dye for 2–3 hours, and then left on a plate to recover overnight. Subsequently, these males were mated with virgin young adult XX animals for 1–4 hours (matings with C. elegans males were allowed to run overnight). Virginity was assured by isolating XX L4 animals from males before reaching adulthood. Mated XX animals were then mounted on 10% agarose pads [79] or 2% agarose pads and immobilized with 50 mM sodium azide for differential interference contrast (DIC) and fluorescence imaging. Automated time-lapse photography (1–10 frames per second) was performed with the Open Lab software package and a Zeiss Axioskop 2 equipped with DIC and fluorescence microscopy. C. nigoni-fog-3(RNAi) and Scoring Germ Line Feminization Phenotypes A 929 base pair fragment including coding sequence homologous to fog-3 was PCR amplified from C. nigoni genomic DNA using primers flanked with 5′ T7 promoters. The reaction was gel purified using the QIAquick kit (Qiagen), and the resultant template was then used for in vitro transcription using the MAXIscript kit (Ambion) to make dsRNA. The dsRNA was recovered using phenol-chloroform extraction and isopropanol precipitation, and the dsRNA was then introduced into the animals via maternal microinjection. The male progeny of injected animals were scored for the feminization of germline (Fog) phenotype using DIC microscopy via standard methods [77]. The worms were mounted on 2% agarose pads and immobilized with 50 mM sodium azide. Only males with clearly defined oocytes and no observable sperm were used for sterilization and lethality experiments. Fog males were allowed to recover for 30 minutes on a plate in a drop of M9 buffer. These males were capable of performing the mating behaviour and of depositing copulatory plugs (and presumably other seminal fluids). These males were then assayed for their ability to sterilize and prematurely kill C. briggsae hermaphrodites. These males were then used for experiments as described above. Control wild-type males were mounted, immobilized, and allowed to recover for the same amount of time in order to remove these as confounding factors. Assortative Mating We focused our assortative mating assays on C. nigoni males, as their aggressive sperm results in sterility and increased mortality (Figure 1). We expect males to mate indiscriminately [41],[43]; therefore, XX animal behaviours (preference or avoidance) should account for the majority of mating biases observed. Assortative mating assays consisted of placing ten virgin C. nigoni males with ten virgin conspecific and/or heterospecific mating partners on a 35 mm diameter Petri dish. The three treatments involved presenting males to (i) ten conspecific, (ii) ten heterospecific (C. remanei, C. elegans, or C. briggsae), or (iii) a mixture of five conspecific and five heterospecific mating partners. See Text S1 for results of (i) conspecific and (ii) heterospecific treatment. Control assays consisted of males (C. remanei, C. elegans, or C. briggsae) following the same treatments as above with C. nigoni females as the heterospecific species. We recorded successful mating by the presence of a copulatory plug deposited by a male onto an XX animal's vulva. We also recorded whether any XX animals left the 3 mm diameter (5 µl) bacterial spot mating area, which, we reasoned, was effective in avoiding copulation. We limited the mating period to 10 minutes to ensure males only mated once (male∶female ratio >1 was used to more easily observe successful copulations with inefficient males of androdioecious species). This 10 minute mating period was determined by preliminary experiments with a male placed with multiple conspecific females. In order to visually distinguish the two female/hermaphrodite species from one another, strains with pharyngially expressed GFP (C. briggsae PS9391) or RFP (C. nigoni VX0092) markers were used, which we presume exerts no direct effect on mate choice. See Text S1 for observed mating frequencies. Statistical Analyses All statistical analyses were performed using IBM SPSS Statistics v.20, unless otherwise noted. We conducted non-parametric tests for measures of reproductive output, owing to non-normal distributions and heterogeneous variances. To assess the effect of heterospecific matings on reproductive output (i.e., extent of sterilization), we compared the control (selfing for hermaphrodites and conspecific matings for females) to each treatment (heterospecific mating) using Mann-Whitney U tests with Bonferroni correction for multiple testing. We used Kaplan-Meier survival analysis to test for an effect of mating on survival of females or hermaphrodites. The survival analyses were performed with the OASIS online application [80] and SPSS. In experiments that explored assortative mating with a mixed species treatment (five conspecific and five heterospecific mating partners; Figure 6), an index of mating bias was calculated as the difference between the number of mated C. nigoni females and the number of mated individuals of the other maternal species present in the arena, divided by the number of C. nigoni females present in the arena (five). Positive values indicate a mating bias towards C. nigoni females over the female (or hermaphrodite) species that they were paired with, negative values indicate the reciprocal, and a value of zero indicates no mating bias (a lack of preference or avoidance). Negative values were not observed in our experiments. We then tested for a significant difference from zero with two-tailed one sample t-tests.

Supporting Information Figure S1. C. nigoni females mate multiply. (A) DIC image of a C. nigoni adult hermaphrodite after a two hour mating period with conspecific males labeled with vital dyes (red or green). Diakinesis stage oocytes (do) are seen distal to the uterus, but not beyond the bend in the reflexed gonad (asterisk). (B, C) Sperm (sp) from males stained with red and green are localized in the spermatheca. Also denoted is the vulva (v), and embryos localized to the uterus (emb). (D) A merged image of (A–C). (C, D) Auto fluorescence of the gut is visible. Each panel is a mosaic assembled from multiple overlapping images; all scale bars are 100 microns. See Text S1 for quantification of multiple mating experiments. https://doi.org/10.1371/journal.pbio.1001915.s001 (TIF) Figure S2. Inter-species mating harm is neither prevented nor substantially rescued by conspecific mating. C. briggsae hermaphrodites were left unmated (selfing only), treated to one mating period with one set of males (Con only: conspecific C. briggsae or Het only: heterospecific C. nigoni), or treated to two mating periods with two sets of males (Het-Het, Het-Con, Con-Het, Con-Con). A conspecific mating taking place one day after a heterospecific mating does not strongly rescue C. briggsae progeny production (Het-Het versus Het-Con: Mann-Whitney U = 126.5, p = 0.043: not significant after multiple tests corrections). Additionally, a conspecific mating does not prevent sterilization from a subsequent heterospecific mating (Con-Con versus Con-Het: U = 5.5, p≤0.001). Finally, C. briggsae hermaphrodites mated with heterospecific males, regardless of order, experience the same decrease in reproductive output (Het-Con versus Con-Het: U = 208.0, p = 0.599). Dotted line indicates the median of the selfing control for reference; samples sizes are in parentheses. Boxplot whiskers indicate 1.5× (interquartile range). For all mating treatments reproductive output is quantified by adult progeny produced two days following the second mating period. Multiple Mann-Whitney U tests were conducted and Bonferroni correction for multiple tests were applied (corrected α = 0.0125). Asterisk indicate statistical significance and (ns) indicate non-significance following Bonferroni correction. https://doi.org/10.1371/journal.pbio.1001915.s002 (EPS) Figure S3. No correlation between severity of harm by heterospecific males and phylogenetic distance or disparity in sperm size. We constructed an ordinal scale of severity of harm to females (or hermaphrodites) as: 1 = no sterilization and/or ectopic sperm; 2 = weak progeny reduction and/or no ectopic sperm; 3 = moderate progeny reduction and/or some ectopic sperm detected; 4 = strong sterilization and/or ectopic sperm present; 5 = near complete sterilization and/or extensive ectopic sperm. (A) We plotted the metric of severity of harm as a function of discretized evolutionary distance of hermaphrodites to heterospecific males mates, on the basis of the phylogenetic topology among species (see Figure 1). We observed no significant relation between phylogenetic distance and severity of harm, either for all hermaphrodite species pooled (Spearman's ρ = −0.15, p = 0.52) or considered separately (all distances have identical harm index for C. briggsae; C. elegans ρ = −0.54, p = 0.28, C. tropicalis ρ = −0.17, p = 0.74). Although this analysis is crude and is based on only a modest number of species comparisons, a more sophisticated analysis of phylogenetic contrasts is undermined by the topology of phylogenetic relationships among the species included in our study. (B) On the basis of sperm size values for the subset of species available in [48], we computed the difference in sperm cross-sectional area between conspecific and heterospecific males. In the case of matings to hermaphrodites, size of hermaphrodite sperm was used in calculations. Spearman rank correlation between these phylogenetically uncorrected metrics was not significant for matings to heterospecific males from dioecious species (p = 0.32). In the case of matings to androdioecious males, we detected a significant association (p = 0.012) owing to the weak sensitivity of hermaphrodites and no sensitivity of females to heterospecific androdioecious males. https://doi.org/10.1371/journal.pbio.1001915.s003 (EPS) Figure S4. Patterns of maternal survival in different Caenorhabditis crosses. Maternal survival in conspecific and heterospecific crosses of (A) C. tropicalis (hermaphrodite/male) with C. wallacei (female/male); (B) C. sp. 5 (female/male) with C. nigoni (female/male); and (C). C. remanei (female/male) with C. nigoni (female/male). (A) C. wallacei maternal survival was statistically greater in the presence of C. tropicalis males (Kaplan-Meier log-rank test: χ2 = 49.57, df = 1, p≤0.001). However, C. tropicalis maternal survival was not affected by C. wallacei males (χ2 = 0.45, df = 1, p = 0.503). (B) The presence of heterospecific males had no statistically significant effect on survival of C. sp. 5 females (χ2 = 0.002, df = 1, p = 0.961) or C. nigoni females (χ2 = 0.16, df = 1, p = 0.688). (C) C. remanei maternal lifespan over eight days was significantly reduced in the presence of C. nigoni males (χ2 = 8.81, df = 1, p = 0.003). C. nigoni maternal survival was not decreased by C. remanei males (χ2 = 1.65, df = 1, p = 0.199). Sample sizes are in parentheses, asterisks indicate significant difference in survival (p≤0.05), and ns indicate non-significant difference in survival. In all panels, dashed lines represent conspecific crosses and solid lines represent heterospecific crosses, while colours correspond to the maternal species. https://doi.org/10.1371/journal.pbio.1001915.s004 (EPS) Figure S5. C. nigoni sperm can ectopically localize and fertilize oocytes in C. elegans hermaphrodites. (A–D) Two C. elegans hermaphrodites mated for 2–6 hours with vitally stained C. nigoni males. Panels display images under DIC (A, C) and fluorescence microscopy (B, D). Indicated is the presence of ectopically localized C. nigoni sperm (esp). (E) A different focal plane of the animal in panels (C, D) reveals the presence of an ectopic embryo (ece) distal to the spermatheca. Also denoted are sperm (sp), the vulva (v), and a properly localized embryo (emb). The bend of the gonad is noted by an asterisk. All scale bars are 100 microns. https://doi.org/10.1371/journal.pbio.1001915.s005 (TIF) Figure S6. Mislocalization of sperm in different Caenorhabditis crosses. The percent of mated females/hermaphrodites observed to contain fluorescently labeled male sperm that localize outside of the spermatheca and uterus (ectopic, purple), in the uterus (orange), and/or in the spermathecae (blue). (A, B) Species names in black boxes below the x-axis indicate dioecious species; white boxes indicate androdioecious species. (C) C. elegans fog-2 “females” doubly mated to vitally stained males (red or green) show that the smaller C. elegans male sperm are displaced from the spermatheca by larger C. nigoni sperm. (D) The percent of observed C. briggsae hermaphrodites with ectopic C. nigoni male sperm increases with time since mating. The dotted horizontal line denotes a 50% reference line; sample sizes are in parentheses. https://doi.org/10.1371/journal.pbio.1001915.s006 (EPS) Table S1. Statistical results of multiple Mann-Whitney U tests corresponding to Figure 1A–1C. https://doi.org/10.1371/journal.pbio.1001915.s007 (DOCX) Movie S1. Male sperm localization in mated C. briggsae hermaphrodites. (A) Labeled C. briggsae male sperm localize to the spermatheca and uterus of a C. briggsae hermaphrodite. Time-lapse video of a C. briggsae hermaphrodite that has been mated with a fluorescently labeled C. briggsae male. (B) Labeled C. nigoni male sperm ectopically migrate in a C. briggsae hermaphrodite. Time-lapse video of a C. briggsae hermaphrodite that has been mated with a fluorescently labeled C. nigoni male. Numerous sperm are located outside of the hermaphrodite's gonad. The spermatheca and uterus have been outlined in yellow, whereas the proximal and distal gonad have been labeled in white. Scale bar represents 100 microns. Images were taken every 10 seconds and the video is sped up 10×. https://doi.org/10.1371/journal.pbio.1001915.s008 (AVI) Movie S2. Ectopic migration of an individual C. nigoni sperm in a C. briggsae hermaphrodite. Differential interference contrast time-lapse video of a C. briggsae hermaphrodite that has been mated with a C. nigoni male. Three ectopic sperm are visible (indicated by the white arrowheads in the opening frames). One ectopic sperm is observed crawling transversely around the distal gonad (lone arrowhead in subsequent frames). Each frame step represents one second. https://doi.org/10.1371/journal.pbio.1001915.s009 (MP4) Text S1. Supplementary methods and results. Strains used in each experiment. Results of mating frequencies observed from assortative mating assays. Additional experiments examining sperm competition within species, effects of conspecific matings to heterospecifically mated hermaphrodites, and sperm localization in different species of Caenorhabditis. https://doi.org/10.1371/journal.pbio.1001915.s010 (DOC)

Acknowledgments We thank S. Baird, M.A. Félix, and the Caenorhabditis Genetics Center (funded by the NIH Research Infrastructure award P40 OD010440)) for strains, C. Braendle for sharing unpublished data on C. tropicalis sperm size, and G. Wilkinson, H. Rodd, J. Levine, H. Rundle, and L. Rowe for useful discussions.

Author Contributions The author(s) have made the following declarations about their contributions: Conceived and designed the experiments: JJT GCW ADC ESH. Performed the experiments: JJT GCW GL NRS. Analyzed the data: JJT GCW ADC ESH. Contributed reagents/materials/analysis tools: JJT GCW. Wrote the paper: JJT GCW ADC ESH.