Role of Doubly Uniparental Inheritance of mitochondria in maintaining reproductive isolation – DRIVE

Full male mt genomes will be sequenced from three lineages spanning two hybrid zones in Europe: one at the entrance of the Baltic sea and one in France. Sampling these lineages will insure that we can capture the range of genetic variation within the lineages present in Europe, in the perspective of designing a set of molecular probes to capture mt genes at the population level.

Characterizing nuclear OXPHO genes will consist in testing the hypothesis that a specific set of nuclear OXPHO isoforms are expressed in sperm, compared to somatic tissues and ovocytes, and measure their relative expression. To do so, sexually-mature males and females will be sampled on both sides of the French hybrid zone. The level of sequence divergence between these populations will facilitate bait set design. A catalogue of OXPHO genes and isoforms will be constructed and their differential expression will be measured.

Testing the implication of DUI in MNIs represent the core of project DRIVE. We will design a custom bait set to capture mt and nuclear OXPHO genes from both sperm and somatic tissues. We will then sample 6 sites across the Finistère/Contentin hybrid zone. We will look if the degree of linkage disequilibrium (LD) among genes of different OXPHO complexes, for sperm and somatic tissues within the same individuals. We will test whether LD correlates with the degree of admixture between the northern and southern lineages (i.e. DUI-caused MNIs are more likely in hybrids). Putative MNI mutations will be mapped on protein 3rd structure to investigate functional effects.

During the period concerned, the objectives were to:

1. prepare the communication tools for the project. This objective is fulfilled.

2.prepare a somatic and gametic tissue library for various populations of the target species L. balthica, by optimizing the protocols (a) for maintaining and reproducing populations in the laboratory, (b) for preparing gamete extracts pure for transcriptome sequencing. This objective is fulfilled.

3. sequence, annotate and compare the mitogenomes of several lineages of L. balthica. We have chosen to focus on the description of the male and female mitogenomes of a single line of L. balthica, and to compare them to another species with DUI (Scrobicularia plana) having the same characteristics. exceptional (size of the male mitogenome, and of the cox2 gene in particular; extreme divergence between male and female mitogenomes). This objective is therefore partially achieved.

4. characterize the nuclear and mitochondrial transcriptomes of male and female gametes. Tissue samples to complete this goal are ready to use for RNA extraction and transcriptome sequencing. This objective will be completed in year 3 of the project.

5.Samples were collected along the French Atlantic coasts, during the L. balthica spawning period, during the winters of 2018 and 2019 (9 sites, 248 male individuals sampled). We completed this sampling in 2019 and 2020 (not initially planned) by adding 140 male individuals from 12 sites in Europe. This objective has been achieved (except for the targeted enrichment part of the OXPHOS genes and their sequencing, scheduled for year 3 of the project).

6. knowledge transfer. Our work was presented in the form of two posters at the ESEB Congress in Finland (2019), an A-rank publication, a book chapter, a seminar. We have trained 2 M2, 1 L3, 2 BTS.

DRIVE touches on several key questions in organismal and evolutionary biology, such as how highly dispersive organisms such as marine bivalves can develop and maintain local adaptations at narrow geographical scales. Theory predicts that genetic incompatibilities causing population divergence can be trapped by environmental gradients, complicating tremendously the distinction between intrinsic and extrinsic barriers to gene flow. DRIVE will add a new tool to comprehend how these different forces can participate in population divergence. These questions have bearing for biodiversity research (the Bivalvia is a highly diverse group of over 9000 described species), as population divergence can lead to speciation, but also for aquaculture, as the adaptation of shellfish species to a changing environment is a strong societal and economic concern.

Capt C, Bouvet K, Guerra D, Robicheau BM, Stewart DT, Pante E*, Breton S*. Unorthodox features in two venerid bivalves with doubly uniparental inheritance of mitochondria. Sci Rep 10, 1087 (2020). doi.org/10.1038/s41598-020-57975-y (* contribution égale) hal.archives-ouvertes.fr/hal-02499562v1

Stewart DT, Breton S, Chase EE, Robicheau BM, Bettinazzi S, Pante E, Youssef N, Garrido-Ramos MA (2020) An unusual evolutionary strategy: the origins, genetic repertoire, andimplications of doubly uniparental inheritance of mitochondrial DNA in bivalves. 24th Evolutionary Biology Meeting at Marseilles. HAL submission hal-02927307 being checked.

17th Congress of the European Society for Evolutionary Biology (ESEB), Turku,Finland (2019) Tassé M, Capt C,Pante E, Breton S. The mtDNA-encoded COX2 protein: bivalves have the longest (poster)

17th Congress of the European Society for Evolutionary Biology (ESEB), Turku,Finland (2019) Bremaud J, Viricel A, Dubillot E,Pante E. Comparative phylogeography of a marine bivalve based on mâles- and femâles-type mitochondrial DNA (poster)

Eric Pante (2019-02-12) Etude des mécanismes qui génèrent et maintiennent la biodiversité en milieu marin : le cas des invertébrés à fort pouvoir dispersif. Habilitation à Diriger les Recherches (HDR). La Rochelle Université.

Cyto-nuclear genetic incompatibilities are a type of intrinsic barrier that has recently gained recognition as an important cause of hybrid breakdown and reproductive isolation. These incompatibilities occur when nuclear and cytoplasmic backgrounds that have not co-evolved are tested in hybrids. Mito-nuclear incompatibilities (MNI) are particularly relevant, as they will affect a fundamental function in animals and plants, the production of ATP. While many advances in the comprehension of these mechanisms have been made based on model species, more work has to be done to understand how MNIs affect natural populations facing changing environments.

Marine bivalves are tremendous biological models to study how MNI contribute to building reproductive isolation. Many of these species are characterized by large population sizes, high fecundity and high dispersal abilities, factors that should impede the establishment of local adaptation. Despite these characteristics, strong genetic structuring can be observed at geographical scales that are much finer than the expected scale of larval dispersal. In addition, coastal bivalves were subjected to complex historical population fluctuations and range shifts in response to glacial cycles, leading to secondary contact zones that are conducive to developing MNIs.

Some bivalve species show a remarkable exception of the maternal inheritance of mitochondria in metazoans: the doubly uniparental mode of inheritance (DUI). In this system, females transmit a “female” mitochondrion that persists in oocytes and in somatic tissues of both sexes, while males pass on “male” mitochondria that persist in the male germ line. The male and female mitochondrial genomes can be highly divergent, begging the question of how DUI evolves and is maintained. This system offers tremendous potential for MNI to develop in inter-populational hybrids, as a network of cyto-nuclear interactions exist. Despite this potential, little information is available on how it might participate to reproductive isolation. Indeed, in DUI species, mito-genetic incompatibilities could be expressed not only between female mitochondrial and nuclear genes (in somatic tissues and oocytes), but also between the male mitochondrial and nuclear genes in sperm of inter-populational hybrids.

The project DRIVE aims at determining whether DUI can play a significant role in maintaining barriers to gene flow among divergent populations by the way of MNIs. To meet this objective, we will use the bivalve Limecola balthica as a model system. We will (1) obtain complete male and female mitogenomes from multiple lineages spanning a secondary contact zone, (2) determine, using RNA-seq, whether nuclear isoforms specific to the male mitogenome background are expressed in sperm, (3) use exon capture to measure levels of population differentiation and linkage disequilibrium across nuclear and mitochondrial genes (both males and females) coding for protein complexes implicated in the production of ATP (i.e. the oxidative phosphorylation chain).

DRIVE touches on several key questions in organismal and evolutionary biology, such as how highly dispersive organisms such as marine bivalves can develop and maintain local adaptations at narrow geographical scales. Theory predicts that genetic incompatibilities causing population divergence can be trapped by environmental gradients, complicating tremendously the distinction between intrinsic and extrinsic barriers to gene flow. DRIVE will add a new tool to comprehend how these different forces can participate in population divergence. These questions have bearing for biodiversity research (the Bivalvia is a highly diverse group of over 9000 described species), as population divergence can lead to speciation, but also for aquaculture, as the adaptation of shellfish species to a changing environment is a strong societal and economic concern.