High heteroplasmy is associated with low mitochondrial copy number and selection against non-synonymous mutations in the snail Cepaea nemoralis.

Molluscan mitochondrial genomes are unusual because they show wide variation in size, radical genome rearrangements and frequently show high variation (> 10%) within species. As progress in understanding this variation has been limited, we used whole genome sequencing of a six-generation matriline of the terrestrial snail Cepaea nemoralis, as well as whole genome sequences from wild-collected C. nemoralis, the sister species C. hortensis, and multiple other snail species to explore the origins of mitochondrial DNA (mtDNA) variation. The main finding is that a high rate of SNP heteroplasmy in somatic tissue was negatively correlated with mtDNA copy number in both Cepaea species. In individuals with under ten mtDNA copies per nuclear genome, more than 10% of all positions were heteroplasmic, with evidence for transmission of this heteroplasmy through the germline. Further analyses showed evidence for purifying selection acting on non-synonymous mutations, even at low frequency of the rare allele, especially in cytochrome oxidase subunit 1 and cytochrome b. The mtDNA of some individuals of Cepaea nemoralis contained a length heteroplasmy, including up to 12 direct repeat copies of tRNA-Val, with 24 copies in another snail, Candidula rugosiuscula, and repeats of tRNA-Thr in C. hortensis. These repeats likely arise due to error prone replication but are not correlated with mitochondrial copy number in C. nemoralis. Overall, the findings provide key insights into mechanisms of replication, mutation and evolution in molluscan mtDNA, and so will inform wider studies on the biology and evolution of mtDNA across animal phyla.

Fine mapping of the Cepaea nemoralis shell colour and mid-banded loci using a high-density linkage map.

Molluscs are a highly speciose phylum that exhibits an astonishing array of colours and patterns, yet relatively little progress has been made in identifying the underlying genes that determine phenotypic variation. One prominent example is the land snail Cepaea nemoralis for which classical genetic studies have shown that around nine loci, several physically linked and inherited together as a 'supergene', control the shell colour and banding polymorphism. As a first step towards identifying the genes involved, we used whole-genome resequencing of individuals from a laboratory cross to construct a high-density linkage map, and then trait mapping to identify 95% confidence intervals for the chromosomal region that contains the supergene, specifically the colour locus (C), and the unlinked mid-banded locus (U). The linkage map is made up of 215,593 markers, ordered into 22 linkage groups, with one large group making up ~27% of the genome. The C locus was mapped to a ~1.3 cM region on linkage group 11, and the U locus was mapped to a ~0.7 cM region on linkage group 15. The linkage map will serve as an important resource for further evolutionary and population genomic studies of C. nemoralis and related species, as well as the identification of candidate genes within the supergene and for the mid-banding phenotype.