LAMP-based molecular sexing in a gonochoric marine bivalve (Macoma balthica rubra) with divergent sex-specific mitochondrial genomes

Abstract

Taking advantage of the unique system of doubly uniparental inheritance (DUI) of mitochondria, we developed a reliable molecular method to sex individuals of the marine bivalve Macoma balthica rubra. In species with DUI (~100 known bivalves), both sexes transmit their mitochondria: males bear both a male- and female-type mitogenome, while females bear only the female type. Male and female mitotypes are sufficiently divergent to reliably PCR-amplify them specifically. Loop-mediated isothermal amplification (LAMP) is a precise, economical and portable alternative to PCR for molecular sexing and we demonstrate its application in this context. We used 154 individuals sampled along the Atlantic coast of France and sexed microscopically by gonad examination to test for the congruence among gamete type, PCR sexing and LAMP sexing. We show an exact match among the sexing results from these three methods using the male and female mt-cox1 genes. DUI can be disrupted in inter-specific hybrids, causing unexpected distribution of mitogenomes, such as homoplasmic males or heteroplasmic females. To our knowledge, DUI disruption at the intra-specific scale has never been tested. We applied our sexing protocol to control for unexpected heteroplasmy caused by hybridization between divergent genetic lineages and found no evidence of disruption in the mode of mitochondrial inheritance in M. balthica rubra. We propose LAMP as a useful tool to accelerate eco-evolutionary studies of DUI. It offers the opportunity to investigate the potential role of, previously unaccounted-for, sex-specific patterns such as sexual selection or sex-specific dispersal bias in the evolution of free-spawning benthic species.

Keywords: DUI; Macoma balthica; heteroplasmy; loop‐mediated isothermal amplification; mitochondrial DNA; sexing.

FIGURE 1

A model of doubly uniparental inheritance of mitochondria in bivalves. The ovocyte (1) and sperm (2) contain a single mitotype (homoplasmy). The sperm introduces the male mitotype into the ovocyte (3), and zygotes are therefore heteroplasmic (4). In early development, male mitotypes aggregate in the germ line of males (5), while they disperse (to the point of becoming undetectable) in females (6). At the adult stage, females (7) transmit the F mitotype with ovocytes while males (8) transmit the M mitotype with sperm (figure adapted from Breton et al. (2014)).

FIGURE 2

Sampling area. Study sites are depicted as blue dots. The location of the intra‐specific hybridization zone described in Becquet et al. (2012) and Le Cam et al. (2022) is represented as a thick gray line, and spans, based on current knowledge, from Saint‐Brieuc to Mont‐Saint‐Michel Bay.

FIGURE 3

Loop‐mediated isothermal amplification (LAMP) of male cox1 and 16S (rrnL) genes. Sample types: female (♀) and male (♂) gDNA templates and no‐gDNA negative control (NC). Names of primer sets (two per gene) are presented on the right. Yellow reactions correspond to successful amplifications of male‐type genes.