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.

Flipping Shells! Unwinding LR Asymmetry in Mirror-Image Molluscs.

In seeking to understand the establishment of left-right (LR) asymmetry, a limiting factor is that most animals are ordinarily invariant in their asymmetry, except when manipulated or mutated. It is therefore surprising that the wider scientific field does not appear to fully appreciate the remarkable fact that normal development in molluscs, especially snails, can flip between two chiral types without pathology. Here, I describe recent progress in understanding the evolution, development, and genetics of chiral variation in snails, and place it in context with other animals. I argue that the natural variation of snails is a crucial resource towards understanding the invariance in other animal groups and, ultimately, will be key in revealing the common factors that define cellular and organismal LR asymmetry.

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.

Deep structure, long-distance migration and admixture in the colour polymorphic land snail Cepaea nemoralis.

Although snails of the genus Cepaea have historically been important in studying colour polymorphism, an ongoing issue is that there is a lack of knowledge of the underlying genetics of the polymorphism, as well as an absence of genomic data to put findings in context. We, therefore, used phylogenomic methods to begin to investigate the post-glacial history of Cepaea nemoralis, with a long-term aim to understand the roles that selection and drift have in determining both European-wide and local patterns of colour polymorphism. By combining prior and new mitochondrial DNA data from over 1500 individuals with ddRAD genomic data from representative individuals across Europe, we show that patterns of differentiation are primarily due to multiple deeply diverged populations of snails. Minimally, there is a widespread Central European population and additional diverged groups in Northern Spain, the Pyrenees, as well as likely Italy and South Eastern Europe. The genomic analysis showed that the present-day snails in Ireland and possibly some other locations are likely descendants of admixture between snails from the Pyrenees and the Central European group, an observation that is consistent with prior inferences from mitochondrial DNA alone. The interpretation is that C. nemoralis may have arrived in Ireland via long-distance migration from the Pyrenean region, subsequently admixing with arrivals from elsewhere. This work, therefore, provides a baseline expectation for future studies on the genetics of the colour polymorphism, as well as providing a comparator for similar species.

Ion channel profiling of the Lymnaea stagnalis ganglia via transcriptome analysis.

BACKGROUND: The pond snail Lymnaea stagnalis (L. stagnalis) has been widely used as a model organism in neurobiology, ecotoxicology, and parasitology due to the relative simplicity of its central nervous system (CNS). However, its usefulness is restricted by a limited availability of transcriptome data. While sequence information for the L. stagnalis CNS transcripts has been obtained from EST libraries and a de novo RNA-seq assembly, the quality of these assemblies is limited by a combination of low coverage of EST libraries, the fragmented nature of de novo assemblies, and lack of reference genome. RESULTS: In this study, taking advantage of the recent availability of a preliminary L. stagnalis genome, we generated an RNA-seq library from the adult L. stagnalis CNS, using a combination of genome-guided and de novo assembly programs to identify 17,832 protein-coding L. stagnalis transcripts. We combined our library with existing resources to produce a transcript set with greater sequence length, completeness, and diversity than previously available ones. Using our assembly and functional domain analysis, we profiled L. stagnalis CNS transcripts encoding ion channels and ionotropic receptors, which are key proteins for CNS function, and compared their sequences to other vertebrate and invertebrate model organisms. Interestingly, L. stagnalis transcripts encoding numerous putative Ca2+ channels showed the most sequence similarity to those of Mus musculus, Danio rerio, Xenopus tropicalis, Drosophila melanogaster, and Caenorhabditis elegans, suggesting that many calcium channel-related signaling pathways may be evolutionarily conserved. CONCLUSIONS: Our study provides the most thorough characterization to date of the L. stagnalis transcriptome and provides insights into differences between vertebrates and invertebrates in CNS transcript diversity, according to function and protein class. Furthermore, this study provides a complete characterization of the ion channels of Lymnaea stagnalis, opening new avenues for future research on fundamental neurobiological processes in this model system.

Internet 'shellebrity' reflects on origin of rare mirror-image snails.

While animal bodies are typically bilaterally symmetric on the outside, the internal organs nearly always show an invariant left-right (LR) asymmetry. In comparison, snails are both internally and externally LR asymmetric, outwardly obvious in the shell coiling direction, or chirality. Although some species of snail are naturally variable for chirality, sinistral individuals occur very rarely in most species. The developmental and genetic basis of these rare mirror-imaged individuals remains mysterious. To resolve this issue, the finding of a 'one in a million' sinistral garden snail called 'Jeremy' was used to recruit citizen scientists to find further sinistral snails. These snails were then bred together to understand whether their occurrence is due an inherited condition. The combined evidence shows that rare sinistral garden snails are not usually produced due to a major effect maternal Mendelian locus. Instead, they are likely mainly produced by a developmental accident. This finding has relevance to understanding the common factors that define cellular and organismal LR asymmetry, and the origin of rare reversed individuals in other animal groups that exhibit nearly invariant LR asymmetry.

Discrete or indiscrete? Redefining the colour polymorphism of the land snail Cepaea nemoralis.

Biologists have long tried to describe and name the different phenotypes that make up the shell polymorphism of the land snail Cepaea nemoralis. Traditionally, the view is that the ground colour of the shell is one of a few major colour classes, either yellow, pink or brown, but in practise it is frequently difficult to distinguish the colours, and define different shades of the same colour. To understand whether colour variation is in reality continuous, and to investigate how the variation may be perceived by an avian predator, we applied psychophysical models of colour vision to shell reflectance measures. We found that both achromatic and chromatic variation are indiscrete in Cepaea nemoralis, being continuously distributed over many perceptual units. Nonetheless, clustering analysis based on the density of the distribution did reveal three groups, roughly corresponding to human-perceived yellow, pink and brown shells. We also found large-scale geographic variation in the frequency of these groups across Europe, and some covariance between shell colour and banding patterns. Although further studies are necessary, the observation of continuous variation in colour is intriguing because the traditional theory is that the underlying supergene that determines colour has evolved to prevent phenotypes from "dissolving" into continuous trait distributions. The findings thus have significance for understanding the Cepaea polymorphism, and the nature of the selection that acts upon it, as well as more generally highlighting the need to measure colour objectively in other systems.

Recombination within the Cepaea nemoralis supergene is confounded by incomplete penetrance and epistasis.

Although the land snail Cepaea nemoralis is one of the most thoroughly investigated colour polymorphic species, there have been few recent studies on the inheritance of the shell traits. Previously, it has been shown that the shell polymorphism is controlled by a series of nine or more loci, of which five make a single 'supergene' containing tightly linked colour and banding loci and more loosely linked pigmentation, spread band and punctate loci. However, one limitation of earlier work was that putative instances of recombination between loci within the supergene were not easily verified. We therefore generated a new set of C. nemoralis crosses that segregate for colour, banding and pigmentation, and several other unlinked shell phenotype loci. The snails were genotyped using a set of RAD-seq-derived loci that flank the supergene, and instances of recombination tested by comparing inferred supergene genotype against RAD-marker genotype. We found no evidence that suspected 'recombinant' individuals are recombinant between loci within the supergene. As point estimates of recombination between both colour/banding, and colour/pigmentation loci are zero, incomplete penetrance and epistasis are a better explanation for the apparent 'recombinant' phenotype of some snail shells. Overall, this work, therefore, shows that the architecture of the supergene may not be as previously supposed. It also provides a resource for fine mapping of the supergene and other major shell phenotype loci.

Reconstructed historical distribution and phylogeography unravels non-steppic origin of Caucasotachea vindobonensis (Gastropoda: Helicidae).

Existing data on the phylogeography of European taxa of steppic provenance suggests that species were widely distributed during glacial periods but underwent range contraction and fragmentation during interglacials into "warm-stage refugia." Among the steppe-related invertebrates that have been examined, the majority has been insects, but data on the phylogeography of snails is wholly missing. To begin to fill this gap, phylogeographic and niche modeling studies on the presumed steppic snail Caucasotachea vindobonensis were conducted. Surprisingly, reconstruction of ancestral areas suggests that extant C. vindobonensis probably originated in the Balkans and survived there during the Late Pleistocene glaciations, with a more recent colonization of the Carpatho-Pannonian and the Ponto-Caspian regions. In the Holocene, C. vindobonensis colonized between the Sudetes and the Carpathians to the north, where its recent and current distribution may have been facilitated by anthropogenic translocations. Together, these data suggest a possible non-steppic origin of C. vindobonensis. Further investigation may reveal the extent to which the steppic snail assemblages consist partly of Holocene newcomers.

RAD-Seq derived markers flank the shell colour and banding loci of the Cepaea nemoralis supergene.

Studies on the classic shell colour and banding polymorphism of the land snail Cepaea played a crucial role in establishing the importance of natural selection in maintaining morphological variation. Cepaea is also a pre-eminent model for ecological genetics because the outward colour and banding phenotype is entirely genetically determined, primarily by a 'supergene' of at least five loci. Unfortunately, progress in understanding the evolution and maintenance of the Cepaea polymorphism stalled, partly because of a lack of genetic markers. With a view to re-establish Cepaea as a prominent model of molecular ecology, we made six laboratory crosses of Cepaea nemoralis, five of which segregated for shell ground colour (C) and the presence or absence of bands (B). First, scoring of colour and banding in 323 individuals found no recombination between the C and B loci of the supergene. Second, using restriction site-associated DNA sequencing (RAD-Seq) of two parents and 22 offspring, we identified 44 anonymous markers putatively linked to the colour (C) and banding (B) loci. The genotype of eleven of the most promising RAD-Seq markers was independently validated in the same 22 offspring, then up to a further 146 offspring were genotyped. The closest RAD-Seq markers scored are within ~0.6 centimorgan (cM) of the C-B supergene linkage group, with the combined loci together forming a 35.8 cM linkage map of markers that flank both sides of the Cepaea C-B supergene.

Qualitative and quantitative methods show stability in patterns of Cepaea nemoralis shell polymorphism in the Pyrenees over five decades.

Over the past century, the study of animal color has been critical in establishing some of the founding principles of biology, especially in genetics and evolution. In this regard, one of the emerging strengths of working with the land snail genus Cepaea is that historical collections can be compared against modern-day samples, for instance, to understand the impact of changing climate and habitat upon shell morph frequencies. However, one potential limitation is that prior studies scored shell ground color by eye into three discrete colours yellow, pink, or brown. This incurs both potential error and bias in comparative surveys. In this study, we therefore aimed to use a quantitative method to score shell color and evaluated it by comparing patterns of C. nemoralis shell color polymorphism in the Pyrenees, using both methods on present-day samples, and against historical data gathered in the 1960s using the traditional method. The main finding was that while quantitative measures of shell color reduced the possibility of error and standardized the procedure, the same altitudinal trends were recovered, irrespective of the method. The results also showed that there was a general stability in the local shell patterns over five decades, including altitudinal clines, with just some exceptions. Therefore, although subject to potential error human scoring of snail color data remains valuable, especially if persons have appropriate training. In comparison, while there are benefits in taking quantitative measures of color in the laboratory, there are also several practical disadvantages, mainly in terms of throughput and accessibility. In the future, we anticipate that genomic methods may be used to understand the potential role of selection in maintaining shell morph clines. In addition, photographs generated by citizen scientists conducting field surveys may be used with deep learning-based methods to survey color patterns.

Mobilizing molluscan models and genomes in biology.

Molluscs are among the most ancient, diverse, and important of all animal taxa. Even so, no individual mollusc species has emerged as a broadly applied model system in biology. We here make the case that both perceptual and methodological barriers have played a role in the relative neglect of molluscs as research organisms. We then summarize the current application and potential of molluscs and their genomes to address important questions in animal biology, and the state of the field when it comes to the availability of resources such as genome assemblies, cell lines, and other key elements necessary to mobilising the development of molluscan model systems. We conclude by contending that a cohesive research community that works together to elevate multiple molluscan systems to 'model' status will create new opportunities in addressing basic and applied biological problems, including general features of animal evolution. This article is part of the Theo Murphy meeting issue 'Molluscan genomics: broad insights and future directions for a neglected phylum'.

Quantitative measures and 3D shell models reveal interactions between bands and their position on growing snail shells.

The nature of shell growth in gastropods is useful because it preserves the ontogeny of shape, colour, and banding patterns, making them an ideal system for understanding how inherited variation develops, is established and maintained within a population. However, qualitative scoring of inherited shell characters means there is a lack of knowledge regarding the mechanisms that control fine variation. Here, we combine empirical measures of quantitative variation and 3D modeling of shells to understand how bands are placed and interact. By comparing five-banded Cepaea individuals to shells lacking individual bands, we show that individual band absence has minor but significant impacts upon the position of remaining bands, implying that the locus controlling band presence/absence mainly acts after position is established. Then, we show that the shell grows at a similar rate, except for the region below the lowermost band. This demonstrates that wider bands of Cepaea are not an artifact of greater shell growth on the lower shell; they begin wider and grow at the same rate as other bands. Finally, we show that 3D models of shell shape and banding pattern, inferred from 2D photos using ShellShaper software, are congruent with empirical measures. This work therefore establishes a method that may be used for comparative studies of quantitative banding variation in snail shells, extraction of growth parameters, and morphometrics. In the future, studies that link the banding phenotype to the network of shell matrix proteins involved in biomineralization and patterning may ultimately aid in understanding the diversity of shell forms found in molluscs.

The draft genome sequence of the grove snail Cepaea nemoralis.

Studies on the shell color and banding polymorphism of the grove snail Cepaea nemoralis and the sister taxon Cepaea hortensis have provided compelling evidence for the fundamental role of natural selection in promoting and maintaining intraspecific variation. More recently, Cepaea has been the focus of citizen science projects on shell color evolution in relation to climate change and urbanization. C. nemoralis is particularly useful for studies on the genetics of shell polymorphism and the evolution of "supergenes," as well as evo-devo studies of shell biomineralization, because it is relatively easily maintained in captivity. However, an absence of genomic resources for C. nemoralis has generally hindered detailed genetic and molecular investigations. We therefore generated ∼23× coverage long-read data for the ∼3.5 Gb genome, and produced a draft assembly composed of 28,537 contigs with the N50 length of 333 kb. Genome completeness, estimated by BUSCO using the metazoa dataset, was 91%. Repetitive regions cover over 77% of the genome. A total of 43,519 protein-coding genes were predicted in the assembled genome, and 97.3% of these were functionally annotated from either sequence homology or protein signature searches. This first assembled and annotated genome sequence for a helicoid snail, a large group that includes edible species, agricultural pests, and parasite hosts, will be a core resource for identifying the loci that determine the shell polymorphism, as well as in a wide range of analyses in evolutionary and developmental biology, and snail biology in general.

Genetics and evidence for balancing selection of a sex-linked colour polymorphism in a songbird.

Colour polymorphisms play a key role in sexual selection and speciation, yet the mechanisms that generate and maintain them are not fully understood. Here, we use genomic and transcriptomic tools to identify the precise genetic architecture and evolutionary history of a sex-linked colour polymorphism in the Gouldian finch Erythrura gouldiae that is also accompanied by remarkable differences in behaviour and physiology. We find that differences in colour are associated with an ~72-kbp region of the Z chromosome in a putative regulatory region for follistatin, an antagonist of the TGF-ß superfamily genes. The region is highly differentiated between morphs, unlike the rest of the genome, yet we find no evidence that an inversion is involved in maintaining the distinct haplotypes. Coalescent simulations confirm that there is elevated nucleotide diversity and an excess of intermediate frequency alleles at this locus. We conclude that this pleiotropic colour polymorphism is most probably maintained by balancing selection.

Speciation and gene flow between snails of opposite chirality.

Left-right asymmetry in snails is intriguing because individuals of opposite chirality are either unable to mate or can only mate with difficulty, so could be reproductively isolated from each other. We have therefore investigated chiral evolution in the Japanese land snail genus Euhadra to understand whether changes in chirality have promoted speciation. In particular, we aimed to understand the effect of the maternal inheritance of chirality on reproductive isolation and gene flow. We found that the mitochondrial DNA phylogeny of Euhadra is consistent with a single, relatively ancient evolution of sinistral species and suggests either recent "single-gene speciation" or gene flow between chiral morphs that are unable to mate. To clarify the conditions under which new chiral morphs might evolve and whether single-gene speciation can occur, we developed a mathematical model that is relevant to any maternal-effect gene. The model shows that reproductive character displacement can promote the evolution of new chiral morphs, tending to counteract the positive frequency-dependent selection that would otherwise drive the more common chiral morph to fixation. This therefore suggests a general mechanism as to how chiral variation arises in snails. In populations that contain both chiral morphs, two different situations are then possible. In the first, gene flow is substantial between morphs even without interchiral mating, because of the maternal inheritance of chirality. In the second, reproductive isolation is possible but unstable, and will also lead to gene flow if intrachiral matings occasionally produce offspring with the opposite chirality. Together, the results imply that speciation by chiral reversal is only meaningful in the context of a complex biogeographical process, and so must usually involve other factors. In order to understand the roles of reproductive character displacement and gene flow in the chiral evolution of Euhadra, it will be necessary to investigate populations in which both chiral morphs coexist.

Single-gene speciation: Mating and gene flow between mirror-image snails.

Variation in the shell coiling, or chirality, of land snails provides an opportunity to investigate the potential for "single-gene" speciation, because mating between individuals of opposite chirality is believed not possible if the snails mate in a face-to-face position. However, the evidence in support of single-gene speciation is sparse, mostly based upon single-gene mitochondrial studies and patterns of chiral variation between species. Previously, we used a theoretical model to show that as the chiral phenotype of offspring is determined by the maternal genotype, occasional chiral reversals may take place and enable gene flow between mirror image morphs, preventing speciation. Here, we show empirically that there is recent or ongoing gene flow between the different chiral types of Japanese Euhadra species. We also report evidence of mating between mirror-image morphs, directly showing the potential for gene flow. Thus, theoretical models are suggestive of gene flow between oppositely coiled snails, and our empirical study shows that they can mate and that there is gene flow in Euhadra. More than a single gene is required before chiral variation in shell coiling can be considered to have created a new species.