In the broadcast-spawning mussel Mytilus galloprovincialis, egg-derived chemicals (chemoattractants) released into seawater bias competitive fertilization success: sperm are differentially attracted up chemoattractant gradients depending on the male–female combination, fertilization is favoured for males less related at nuclear loci but of the same mitochondrial DNA lineage (Lymbery, Kennington & Evans 2017), and chemotaxis strength previously correlated with offspring survival (Oliver & Evans 2014). The phenomenon is therefore established, but its mechanism and evolutionary meaning are not: the chemical identity of the chemoattractants and their among-female variation are unknown; the genetic basis (cyto-nuclear compatibility? male-type mitochondrial transmission?) and fitness consequences of the mitochondrial-lineage bias are untested; the sperm receptors and signalling pathways that mediate differential responses, and their intraspecific variation, are uncharacterized in any species; and the prevalence of chemoattractant-mediated gamete-level mate choice across taxa, its interaction with other female-reproductive-fluid-mediated selection, and its evolutionary significance remain open.

Background

Broadcast spawning is the ancestral reproductive strategy in which males and females release gametes into the water, so that all sexual selection after mating must act on free-swimming sperm and drifting eggs. Sperm chemotaxis is the directed swimming of sperm along a gradient of egg-derived chemicals; such chemoattractants are known across animals and plants and are well characterized in a few model broadcast spawners, for example sea urchin sperm-activating peptides such as speract, which bind guanylyl cyclase receptors on the sperm surface, trigger calcium influx and alter the flagellar beat. Gamete-level mate choice (also called cryptic female choice at the gamete stage) is the idea that females can bias which male's sperm fertilizes their eggs after mating or spawning; ovarian fluid in fishes, released with eggs, is one such mediator. Mytilus galloprovincialis, the Mediterranean or blue mussel, is a broadcast-spawning bivalve with an unusual mitochondrial inheritance system called doubly uniparental inheritance (DUI): females transmit one mitochondrial genome (F-type) to all offspring, while males additionally carry a divergent male-type (M-type) mitochondrial genome inherited from their father through sperm. Australian populations also contain two phylogeographic mitochondrial lineages (Northern and Southern Hemisphere) that admix at nuclear loci. A series of experiments set up the problem: Evans et al. (2012) showed mussel egg chemoattractants alter sperm swimming behaviour in a male–female-specific way; Oliver & Evans (2014) showed these chemically moderated gamete preferences predict offspring fitness; Kekäläinen & Evans (2017) showed egg-derived chemicals remotely regulate sperm physiology; and Lymbery et al. (2016) developed a fluorescent mitochondrial dye method to track the real-time fertilization success of individual competing ejaculates. Using that dye method in a cross-classified competition design, Lymbery, Kennington & Evans (2017) provided the first direct evidence in any species that differential sperm chemotaxis up egg-chemoattractant gradients moderates intraspecific competitive fertilization success, with biases favouring males less related to the female at neutral nuclear markers but sharing her mitochondrial lineage. The paper closes by identifying what is unresolved: the chemoattractant molecules of M. galloprovincialis are not yet identified (and sea urchin speract shows strong purifying selection with little within-genus variation, so a single conserved peptide is unlikely to encode among-female variation); the fitness benefits and cyto-nuclear genetic basis of the mitochondrial-lineage bias, including complications from sperm-transmitted male-type mitochondria, remain to be resolved; no study in any species has examined intraspecific variation in the sperm signalling pathways that respond to egg chemicals; and the generality of chemoattractant-mediated gamete-level mate choice across taxa, its relationship to other female reproductive fluid-mediated selection such as ovarian fluid in fishes, and its role in the evolutionary cascade of sexual selection are untested.

Problem Statement

Building on the demonstration by Lymbery, Kennington & Evans (2017) that egg chemoattractants moderate intraspecific sperm competition in Mytilus galloprovincialis, determine the mechanism and evolutionary significance of this gamete-level mate choice. The overarching objective joins four linked questions: (1) identify the chemical molecules (single peptides, defined mixtures, or broader suites of egg-derived chemicals) that constitute the chemoattractant signal of M. galloprovincialis, characterize how these chemical profiles vary among females, and establish how that variation maps onto differential sperm attraction; (2) determine the genetic and physiological basis of the observed mitochondrial-lineage bias in fertilization, specifically whether lineage-based fertilization preferences reflect cyto-nuclear compatibility involving particular nuclear genes (for example genes involved in mitochondrial function), how male-type mitochondrial transmission through sperm complicates the pattern, and what fitness benefits, if any, accrue to females and offspring from same-lineage fertilizations; (3) characterize the receptors and downstream signalling pathways on sperm that mediate differential responses to intraspecific chemoattractant variation, including the extent of intraspecific variation in those pathways, which to date have not been examined in any species; and (4) test the generality of chemoattractant-mediated gamete-level mate choice across taxa — its prevalence outside broadcast spawners, its interaction with other female-reproductive-fluid-mediated selection such as ovarian fluid in fishes, and its role in the evolutionary cascade of sexual selection.

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