PMC

(A) Diagram of the reproductive tract of a XX nematode animal, magnified in (B) to illustrate the typical movement of gametes near the site of fertilization. Sperm must repeatedly crawl from the uterus to the spermatheca via the proximal spermathecal valve (psv) after being pushed out by fertilized eggs, migrating toward attractants secreted by the most proximal oocytes; red arrows indicate direction of sperm movement. Oocytes in the oviduct enter the spermatheca through the distal spermathecal valve (dsv) to be fertilized, then move into the uterus and exit the animal through the vulva; black arrows indicate direction of oocyte and embryo movement. (C) The normal process of self-fertilization in hermaphrodites (adapted from ) becomes disrupted upon heterospecific mating. (D) Heterospecific male sperm inhibit ovulation, invade the proximal gonad, induce premature oocyte maturation and ectopic fertilization, and in some cases invade somatic tissues.

(A) Wild-type C. nigoni male, with spermatocytes (spc) and sperm (sp) in the seminal vesicle. These are extruded through the vas deferens (vd) during copulation. (B) C. nigoni male treated with maternal Cni-fog-3(RNAi). Oocytes (o) develop instead of sperm, while the somatic testis, including the glandular vas deferens (vd), remain unaffected. (C) The number of viable progeny of C. briggsae hermaphrodites do not differ when unmated (blue) or mated (white) to germline-feminized (fog-3 (RNAi)) C. nigoni males (Mann-Whitney U = 51.5, p = 0.419; ns, not significant), unlike when unmated or mated to wild-type C. nigoni males (U = 26.5, p = 0.019). Boxplot whiskers indicate 1.5× (interquartile range). (D) C. briggsae hermaphrodites mated to Cni-fog-3 (RNAi) males (solid line) have significantly reduced mortality compared to when mated to wildtype C. nigoni males (dashed line; Kaplan-Meier log-rank test: χ² = 23.7, df = 1, p≤0.001). Sample sizes are shown in parentheses.

In assay arenas containing a mixture of females from two species and males from one species, (A) C. nigoni females are more likely than C. briggsae hermaphrodites to mate with C. briggsae males (t = 2.828, df = 24, p = 0.009) and C. nigoni males (t = 8.988, df = 24, p≤0.001). (B) A similar female mating bias was also observed when C. elegans hermaphrodites and C. nigoni females were presented to C. elegans males (t = 2.939, df = 19, p = 0.008) and to C. nigoni males (t = 28.996, df = 19, p≤0.001). (C) Mixtures of C. remanei and C. nigoni females show a mating bias toward C. nigoni females over C. remanei females when they are presented with C. nigoni males (t = 2.683, df = 24, p = 0.013), but not when presented with C. remanei males (t = 1.365, df = 24, p = 0.185). Positive values indicate that C. nigoni females mated more readily than the other female (or hermaphrodite) species that they were paired with; negative values indicate the reciprocal, while zero indicate no mating bias. Error bars represent ±1 standard error (SE); sample sizes are shown in parentheses.

(A–F) Paired images of DIC and vitally dyed sperm inside maternal animals after conspecific and heterospecific crosses. (A) In conspecific crosses with C. briggsae, sperm remain completely in the uterus and spermatheca (bracket). (B) Female from C. nigoni conspecific cross displaying sperm localization to the uterus and spermatheca. (C and D) In rare cases of conspecific crosses for C. nigoni (C; 5%) and C. remanei (D; 8%), we observe isolated sperm (arrows) breach the distal spermathecal valve, but this does not cause sterility. (F) When C. nigoni males are mated with C. briggsae hermaphrodites, ectopic sperm are seen in large numbers, and in some cases breach the gonad completely. Wedge, anterior end of ovary; arrowhead, sperm nuclei in head region. (G) Series of frames from time-lapse movie () depicting movement of an individual ectopic C. nigoni sperm in the distal gonad of a C. briggsae hermaphrodite. The yellow crescent outlines the cell body of a sessile sperm, which serves as a reference point to highlight the motion of a second sperm, whose body is outlined in red.

Females from dioecious species (A: C. nigoni, B: C. remanei) were initially mated overnight to conspecific males, followed by overnight mating of treatment females (white) to heterospecific males from either dioecious (C. nigoni, C. remanei, C. brenneri) or androdioecious (C. briggsae, C. elegans) species; control females (red) did not receive a second mating. Reproductive output was then quantified as the number of progeny produced two days post mating treatment. (A, B) Females often became sterilized upon a subsequent mating with heterospecific males from dioecious species compared to controls: (A) C. nigoni females (C. remanei males U = 385.5, p = 0.001; C. brenneri males U = 206.5, p≤0.001), (B) C. remanei females (C. nigoni males U = 239.5, p≤0.001; C. brenneri males U = 259.5, p≤0.001). However, females were resistant to harm induced by males of androdioecious species: (A) C. nigoni (C. briggsae males U = 409.5, p = 0.020; C. elegans males U = 577.0, p = 0.291), (B) C. remanei (C. briggsae males U = 887.0, p = 0.886; C. elegans males U = 863.0, p = 0.731). (C) C. nigoni females mated to heterospecific C. briggsae males (solid red line) did not incur a statistically significant survival cost relative to conspecific mating (dashed black line; Kaplan-Meier log-rank test: χ² = 0.203, df = 1, p = 0.652; ns not significant). Asterisks in (A and B) indicate significant difference from controls (Bonferroni correction for multiple tests were applied; corrected α = 0.0125). Boxplot whiskers in (A and B) indicate 1.5× (interquartile range) and dotted horizontal lines indicate median of viable progeny produced by the controls (females mated with conspecifics); sample sizes are shown in parentheses.

Hermaphrodites of each selfing species were mated to males of dioecious (A–C) or another androdioecious (D, E) species and reproductive output was quantified as the number of self-progeny produced two days post mating treatment. (A) C. briggsae and (B) C. elegans produced significantly fewer progeny after heterospecific matings to males of all species tested (white) compared to selfing or “no mating” (blue). (C) C. tropicalis also produced fewer progeny after heterospecific matings, but only to a subset of the species tested. (D) A single heterospecific mating event was sufficient to induce progeny loss for C. briggsae hermaphrodites crossed to C. nigoni males (Mann-Whitney U = 37.0, p≤0.001). (E, F) Males from hermaphroditic species also reduced hermaphrodite self-progeny production following heterospecific mating (C. briggsae U = 65.0, p = 0.001; C. elegans U = 178.0, p = 0.008). However, males from hermaphroditic species were less effective at reducing progeny compared to dioecious males (C. briggsae U = 122.0, p≤0.001; C. elegans U = 631.0, p≤0.001). (G) C. briggsae hermaphrodites mated to C. nigoni males (solid blue line) suffer higher mortality than when mated to C. briggsae males (dashed black line; Kaplan-Meier log-rank test: χ² = 5.218, df = 1, p = 0.022). Asterisks in (A–F) indicate significant difference from selfing control (Mann-Whitney U test p<0.05; Bonferroni correction applied in (A–C); (A, C) corrected α = 0.007; (B) corrected α = 0.008; ). In (A–F), boxplot whiskers indicate 1.5× (interquartile range) and dotted horizontal lines indicate median of viable progeny produced by the selfing control for reference. Dagger (†) in (C) indicates that no successful matings occurred between C. portoensis males and C. tropicalis hermaphrodites. Sample sizes are shown in parentheses. Phylogeny in (A–C) adapted from .

(A) Shortly after mating, the smaller C. elegans male sperm (red) are displaced from the spermatheca by larger C. nigoni sperm (green) in C. elegans fog-2 “females” doubly mated to vitally dyed males. sth, spermatheca; u, uterus, v, vulva. This panel is a mosaic assembled from multiple overlapping images. (B) C. briggsae adult hermaphrodite after overnight mating with conspecific males. Abundant embryos (e) are restricted to the uterus and reliably laid through the vulva (v). Diakinesis stage oocytes (do) are seen distal to the uterus, but not beyond the bend in the reflexed gonad (asterisk). (C) When mated overnight with C. nigoni males, few embryos are produced despite abundant sperm (sp). Proximal oocytes are abnormal (ao), and diakinesis oocytes are pushed distal to the gonad bend. p, copulatory plug. (D) In hermaphrodites recently inseminated by heterospecific males, the germ line is generally well organized, but ectopic embryos (ece) can be observed distal to the oviduct (od), which is often distended by sperm that have breached it from the spermatheca (sth). Embryos are also seen in the normal uterine position, explaining how some cross progeny are laid . (E) Hoechst DNA staining of conspecifically mated C. briggsae reveals abundant punctate sperm nuclei (sp) that are always restricted to the uterus and spermatheca and multiple diakinesis-stage meiotic nuclei (do). (F) DNA staining of C. briggsae hermaphrodites mated overnight with C. nigoni (similar to B) reveal a zone of endomitotic oocytes (emo) in the proximal region of the ovary abutting a large mass of sperm (sp). (G) In some cases, embryos localized to the uterus (emb) are viable (as judged by their advanced state of development) but not laid through the vulva (v).