Sexual selection is a major driver of trait variation, and the intensity of male competition for mating opportunities has been linked with sperm size across diverse taxa. Mating competition among females may also shape the evolution of sperm traits, but the interplay between female-female competition and male-male competition on sperm morphology is not well understood. We evaluated variation in sperm morphology in two species with socially polyandrous mating systems, in which females compete to mate with multiple males. Northern jacanas (Jacana spinosa) and wattled jacanas (J. jacana) vary in their degree of polyandry and sexual dimorphism, suggesting species differences in the intensity of sexual selection. We compared mean and variance in sperm head, midpiece, and tail length between species and breeding stages, because these measures have been associated with the intensity of sperm competition. We found that the species with greater polyandry, northern jacana, has sperm with longer midpieces and tails, as well as marginally lower intra-ejaculate variation in tail length. Intra-ejaculate variation was also significantly lower in copulating males than in incubating males, suggesting flexibility in sperm production as males cycle between breeding stages. Our results indicate that stronger female-female competition for mating opportunities may also shape more intense male-male competition by selecting for longer and less variable sperm traits. These findings extend frameworks developed in socially monogamous species to reveal that sperm competition may be an important evolutionary force layered atop female-female competition for mates.

We collected samples from a population of northern jacanas (Jacana spinosa) in La Barqueta, Chiriqui (8.207N, 82.579W) and a population of wattled jacanas (J. jacana) near Chepo (9.166N, 79.122W) from 16 May to 1 July 2018. We sampled 11 northern jacana males (5 copulating, 7 incubating) and 7 wattled jacana males (4 copulating, 3 incubating). Ductus deferens were preserved in 5% formaldehyde solution in PBS in the field. We also measured testes mass and volume, and body mass. 

We prepared sperm samples for visualization with brightfield microscopy. Using a dissection microscope at 10x magnification, we used forceps to make an incision in the seminal glomus and squeezed out the fixed ejaculate onto a microscope slide in 15-20 μL of 5% formaldehyde. We then pipetted the ejaculate-formaldehyde mixture into a microcentrifuge tube, mixed it with a pipette, and pipetted it onto a fresh slide with a slipcover. We photographed samples immediately after slide preparation to avoid crystallization of the buffer. We identified at least 10 intact sperm cells per male and photographed them using an EVOS FL fluorescence microscope at 600x magnification.

To compare sperm morphology between the two species, we focused on the length of three sperm traits: tail, midpiece, and head length. We measured the length of each trait using the segment line tool in ImageJ v1.8. We measured the tail based on the end of the midpiece to terminal end of the flagellum. We distinguished the head from the midpiece by a change in transparency and tapering shape, based on guidelines for Charadriiform sperm morphology (Jamieson 2007).

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