Marine transmissible cancer navigates urbanized waters, threatening spillover.

Inter-individual transmission of cancer cells represents a unique form of microparasites increasingly reported in marine bivalves. In this study, we sought to understand the ecology of the propagation of Mytilus trossulus Bivalve Transmissible Neoplasia 2 (MtrBTN2), a transmissible cancer affecting four Mytilus mussel species worldwide. We investigated the prevalence of MtrBTN2 in the mosaic hybrid zone of M. edulis and M. galloprovincialis along the French Atlantic coast, sampling contrasting natural and anthropogenic habitats. We observed a similar prevalence in both species, probably due to the spatial proximity of the two species in this region. Our results showed that ports had higher prevalence of MtrBTN2, with a possible hotspot observed at a shuttle landing dock. No cancer was found in natural beds except for two sites close to the hotspot, suggesting spillover. Ports may provide favourable conditions for the transmission of MtrBTN2, such as high mussel density, stressful conditions, sheltered and confined shores or buffered temperatures. Ships may also spread the disease through biofouling. Our results suggest ports may serve as epidemiological hubs, with maritime routes providing artificial gateways for MtrBTN2 propagation. This highlights the importance of preventing biofouling on docks and ship hulls to limit the spread of marine pathogens hosted by fouling species.

Transcriptomics of mussel transmissible cancer MtrBTN2 suggests accumulation of multiple cancer traits and oncogenic pathways shared among bilaterians.

Transmissible cancer cell lines are rare biological entities giving rise to diseases at the crossroads of cancer and parasitic diseases. These malignant cells have acquired the amazing capacity to spread from host to host. They have been described only in dogs, Tasmanian devils and marine bivalves. The Mytilus trossulus bivalve transmissible neoplasia 2 (MtrBTN2) lineage has even acquired the capacity to spread inter-specifically between marine mussels of the Mytilus edulis complex worldwide. To identify the oncogenic processes underpinning the biology of these atypical cancers we performed transcriptomics of MtrBTN2 cells. Differential expression, enrichment, protein-protein interaction network, and targeted analyses were used. Overall, our results suggest the accumulation of multiple cancerous traits that may be linked to the long-term evolution of MtrBTN2. We also highlight that vertebrate and lophotrochozoan cancers could share a large panel of common drivers, which supports the hypothesis of an ancient origin of oncogenic processes in bilaterians.

Traits of a mussel transmissible cancer are reminiscent of a parasitic life style.

Some cancers have evolved the ability to spread from host to host by transmission of cancerous cells. These rare biological entities can be considered parasites with a host-related genome. Still, we know little about their specific adaptation to a parasitic lifestyle. MtrBTN2 is one of the few lineages of transmissible cancers known in the animal kingdom. Reported worldwide, MtrBTN2 infects marine mussels. We isolated MtrBTN2 cells circulating in the hemolymph of cancerous mussels and investigated their phenotypic traits. We found that MtrBTN2 cells had remarkable survival capacities in seawater, much higher than normal hemocytes. With almost 100% cell survival over three days, they increase significantly their chances to infect neighboring hosts. MtrBTN2 also triggered an aggressive cancerous process: proliferation in mussels was ~ 17 times higher than normal hemocytes (mean doubling time of ~ 3 days), thereby favoring a rapid increase of intra-host population size. MtrBTN2 appears to induce host castration, thereby favoring resources re-allocation to the parasites and increasing the host carrying capacity. Altogether, our results highlight a series of traits of MtrBTN2 consistent with a marine parasitic lifestyle that may have contributed to the success of its persistence and dissemination in different mussel populations across the globe.

Implementation of various approaches to study the prevalence, incidence and progression of disseminated neoplasia in mussel stocks.

Marine mussel production is of substantial economic interest in numerous coastal areas worldwide, making crucial the study of pathologies that affect them. Disseminated neoplasia (DN) has recently been suggested to be linked to blue mussel, Mytilus edulis, mortality outbreaks observed in France since 2014, although the evidence remains indirect. In order to improve DN detection and monitoring, we compared the sensitivity of four diagnostic tools, namely haemocytology, histology, flow cytometry, and genetics. Haemocytological examination gave the best results in sensitivity and had the advantage of being non-invasive, allowing disease progression to be followed in affected mussels. Using this approach, we showed that DN progression is usually slow, and we provide evidence of remission events. We observed a high diversity of forms and mitotic features of neoplastic cells located in the vesicular connective tissue but rarely in the haemolymph. Circulating cells occur as four main types but are homogenous in morphology and DNA content within a single individual. Polyploidy proved very high, from 8 N to 18 N. Genetic analysis of haemolymph DNA showed that a Mytilus trossulus genetic signal was associated with almost all the DN cases here diagnosed by haemocytological examination, regardless of the DN type. This result corroborates DN is a transmissible cancer that first originated in a M. trossulus host and subsequently crossed into M. edulis. No pre-neoplastic conditions were detectable. The prevalence of the disease was quite low, which, together with the low morbidity observed in the lab, suggest DN is unlikely to be the direct cause of mortality outbreaks in France.

Interpreting the genomic landscape of speciation: a road map for finding barriers to gene flow.

Speciation, the evolution of reproductive isolation among populations, is continuous, complex, and involves multiple, interacting barriers. Until it is complete, the effects of this process vary along the genome and can lead to a heterogeneous genomic landscape with peaks and troughs of differentiation and divergence. When gene flow occurs during speciation, barriers restricting gene flow locally in the genome lead to patterns of heterogeneity. However, genomic heterogeneity can also be produced or modified by variation in factors such as background selection and selective sweeps, recombination and mutation rate variation, and heterogeneous gene density. Extracting the effects of gene flow, divergent selection and reproductive isolation from such modifying factors presents a major challenge to speciation genomics. We argue one of the principal aims of the field is to identify the barrier loci involved in limiting gene flow. We first summarize the expected signatures of selection at barrier loci, at the genomic regions linked to them and across the entire genome. We then discuss the modifying factors that complicate the interpretation of the observed genomic landscape. Finally, we end with a road map for future speciation research: a proposal for how to account for these modifying factors and to progress towards understanding the nature of barrier loci. Despite the difficulties of interpreting empirical data, we argue that the availability of promising technical and analytical methods will shed further light on the important roles that gene flow and divergent selection have in shaping the genomic landscape of speciation.

Population genomics of sexual and asexual lineages in fissiparous ribbon worms (Lineus, Nemertea): hybridization, polyploidy and the Meselson effect.

Comparative population genetics in asexual vs. sexual species offers the opportunity to investigate the impact of asexuality on genome evolution. Here, we analyse coding sequence polymorphism and divergence patterns in the fascinating Lineus ribbon worms, a group of marine, carnivorous nemerteans with unusual regeneration abilities, and in which asexual reproduction by fissiparity is documented. The population genomics of the fissiparous L. pseudolacteus is characterized by an extremely high level of heterozygosity and unexpectedly elevated πN /πS ratio, in apparent agreement with theoretical expectations under clonal evolution. Analysis of among-species allele sharing and read-count distribution, however, reveals that L. pseudolacteus is a triploid hybrid between Atlantic populations of L. sanguineus and L. lacteus. We model and quantify the relative impact of hybridity, polyploidy and asexuality on molecular variation patterns in L. pseudolacteus and conclude that (i) the peculiarities of L. pseudolacteus population genomics result in the first place from hybridization and (ii) the accumulation of new mutations through the Meselson effect is more than compensated by processes of heterozygosity erosion, such as gene conversion or gene copy loss. This study illustrates the complexity of the evolutionary processes associated with asexuality and identifies L. pseudolacteus as a promising model to study the first steps of polyploid genome evolution in an asexual context.

Population genomic footprints of fine-scale differentiation between habitats in Mediterranean blue tits.

Linking population genetic variation to the spatial heterogeneity of the environment is of fundamental interest to evolutionary biology and ecology, in particular when phenotypic differences between populations are observed at biologically small spatial scales. Here, we applied restriction-site associated DNA sequencing (RAD-Seq) to test whether phenotypically differentiated populations of wild blue tits (Cyanistes caeruleus) breeding in a highly heterogeneous environment exhibit genetic structure related to habitat type. Using 12 106 SNPs in 197 individuals from deciduous and evergreen oak woodlands, we applied complementary population genomic analyses, which revealed that genetic variation is influenced by both geographical distance and habitat type. A fine-scale genetic differentiation supported by genome- and transcriptome-wide analyses was found within Corsica, between two adjacent habitats where blue tits exhibit marked differences in breeding time while nesting < 6 km apart. Using redundancy analysis (RDA), we show that genomic variation remains associated with habitat type when controlling for spatial and temporal effects. Finally, our results suggest that the observed patterns of genomic differentiation were not driven by a small proportion of highly differentiated loci, but rather emerged through a process such as habitat choice, which reduces gene flow between habitats across the entire genome. The pattern of genomic isolation-by-environment closely matches differentiation observed at the phenotypic level, thereby offering significant potential for future inference of phenotype-genotype associations in a heterogeneous environment.

Comparative population genomics in animals uncovers the determinants of genetic diversity.

Genetic diversity is the amount of variation observed between DNA sequences from distinct individuals of a given species. This pivotal concept of population genetics has implications for species health, domestication, management and conservation. Levels of genetic diversity seem to vary greatly in natural populations and species, but the determinants of this variation, and particularly the relative influences of species biology and ecology versus population history, are still largely mysterious. Here we show that the diversity of a species is predictable, and is determined in the first place by its ecological strategy. We investigated the genome-wide diversity of 76 non-model animal species by sequencing the transcriptome of two to ten individuals in each species. The distribution of genetic diversity between species revealed no detectable influence of geographic range or invasive status but was accurately predicted by key species traits related to parental investment: long-lived or low-fecundity species with brooding ability were genetically less diverse than short-lived or highly fecund ones. Our analysis demonstrates the influence of long-term life-history strategies on species response to short-term environmental perturbations, a result with immediate implications for conservation policies.

Can we continue to neglect genomic variation in introgression rates when inferring the history of speciation? A case study in a Mytilus hybrid zone.

The use of molecular data to reconstruct the history of divergence and gene flow between populations of closely related taxa represents a challenging problem. It has been proposed that the long-standing debate about the geography of speciation can be resolved by comparing the likelihoods of a model of isolation with migration and a model of secondary contact. However, data are commonly only fit to a model of isolation with migration and rarely tested against the secondary contact alternative. Furthermore, most demographic inference methods have neglected variation in introgression rates and assume that the gene flow parameter (Nm) is similar among loci. Here, we show that neglecting this source of variation can give misleading results. We analysed DNA sequences sampled from populations of the marine mussels, Mytilus edulis and M. galloprovincialis, across a well-studied mosaic hybrid zone in Europe and evaluated various scenarios of speciation, with or without variation in introgression rates, using an Approximate Bayesian Computation (ABC) approach. Models with heterogeneous gene flow across loci always outperformed models assuming equal migration rates irrespective of the history of gene flow being considered. By incorporating this heterogeneity, the best-supported scenario was a long period of allopatric isolation during the first three-quarters of the time since divergence followed by secondary contact and introgression during the last quarter. By contrast, constraining migration to be homogeneous failed to discriminate among any of the different models of gene flow tested. Our simulations thus provide statistical support for the secondary contact scenario in the European Mytilus hybrid zone that the standard coalescent approach failed to confirm. Our results demonstrate that genomic variation in introgression rates can have profound impacts on the biological conclusions drawn from inference methods and needs to be incorporated in future studies.

Hybridization and speciation.

Hybridization has many and varied impacts on the process of speciation. Hybridization may slow or reverse differentiation by allowing gene flow and recombination. It may accelerate speciation via adaptive introgression or cause near-instantaneous speciation by allopolyploidization. It may have multiple effects at different stages and in different spatial contexts within a single speciation event. We offer a perspective on the context and evolutionary significance of hybridization during speciation, highlighting issues of current interest and debate. In secondary contact zones, it is uncertain if barriers to gene flow will be strengthened or broken down due to recombination and gene flow. Theory and empirical evidence suggest the latter is more likely, except within and around strongly selected genomic regions. Hybridization may contribute to speciation through the formation of new hybrid taxa, whereas introgression of a few loci may promote adaptive divergence and so facilitate speciation. Gene regulatory networks, epigenetic effects and the evolution of selfish genetic material in the genome suggest that the Dobzhansky-Muller model of hybrid incompatibilities requires a broader interpretation. Finally, although the incidence of reinforcement remains uncertain, this and other interactions in areas of sympatry may have knock-on effects on speciation both within and outside regions of hybridization.

Differential introgression from a sister species explains high F(ST) outlier loci within a mussel species.

Scanning genomes for loci with high levels of population differentiation has become a standard of population genetics. F(ST) outlier loci are most often interpreted as signatures of local selection, but outliers might arise for many other reasons too often left unexplored. Here, we tried to identify further the history and genetic basis underlying strong differentiation at F(ST) outlier loci in a marine mussel. A genome scan of genetic differentiation has been conducted between Atlantic and Mediterranean populations of Mytilus galloprovincialis. The differentiation was low overall (F(ST) = 0.03), but seven loci (2%) were strong F(ST) outliers. We then analysed DNA sequence polymorphism at two outlier loci. The genetic structure proved to be the consequence of differential introgression of alleles from the sister-hybridizing species Mytilus edulis. Surprisingly, the Mediterranean population was the most introgressed at these two loci, although the contact zone between the two species is nowadays localized along the Atlantic coasts of France and the British Isles. A historical contact between M. edulis and Mediterranean M. galloprovincialis should have happened during glacial periods. It proved difficult to disentangle two hypotheses: (i) introgression was adaptive, implying edulis alleles have been favoured in Mediterranean populations, or (ii) the genetic architecture of the barrier to edulis gene flow is different between the two M. galloprovincialis backgrounds. Five of the seven outliers between M. galloprovincialis populations were also outliers between M. edulis and Atlantic M. galloprovincialis, which would support the latter hypothesis. Differential introgression across semi-permeable barriers to gene flow is a neglected scenario to interpret outlying loci that may prove more widespread than anticipated.

Evidence for a slightly deleterious effect of intron polymorphisms at the EF1alpha gene in the deep-sea hydrothermal vent bivalve Bathymodiolus.

A multilocus analysis was initiated in order to infer the general effect of demography and the indirect effect of positive selection on some chromosome segments in Bathymodiolus. Mussels of the genus Bathymodiolus inhabit the very hostile, fragmented and variable environment of deep-sea hydrothermal vents which is thought to cause recurrent population bottlenecks via extinction/colonisation processes and adaptation to new environmental conditions. In the course of this work we discovered that the assumption of neutrality of non-coding polymorphisms usually made in genome scan experiments was likely to be violated at one of the loci we analysed. The direct effect of slight purifying selection on non-coding polymorphisms shares many resemblances with the indirect effect of positive selection through genetic hitchhiking. Combining polymorphism with divergence data for several closely related species allowed us to obtain different expectations for the direct effect of negative selection and the indirect effect of positive selection. We observed a strong excess of rare non-coding polymorphisms at the second intron of the EF1alpha gene in the two species Bathymodiolus azoricus and Bathymodiolus thermophilus, while two other loci, the mitochondrial COI gene and an intron of the Lysozyme gene, did not exhibit such a deviation. In addition, the divergence rate of the EF1alpha intron was estimated to be unexpectedly low when calibrated using the closure of the Panama Isthmus that interrupted gene flow between the two species. The polymorphism to divergence ratio was similar to the one observed for the other two loci, in accordance to the hypothesis of purifying selection. We conclude that slight purifying selection is likely to act on polymorphic intronic mutations of the EF1alpha second intron and discuss the possible relationship with the specific biology of Bathymodiolus mussels.

Single Nucleotide polymorphisms and their relationship to codon usage bias in the Pacific oyster Crassostrea gigas.

DNA sequence polymorphism and codon usage bias were investigated in a set of 41 nuclear loci in the Pacific oyster Crassostrea gigas. Our results revealed a very high level of DNA polymorphism in oysters, in the order of magnitude of the highest levels reported in animals to date. A total of 290 single nucleotide polymorphisms (SNPs) were detected, 76 of which being localised in exons and 214 in non-coding regions. Average density of SNPs was estimated to be one SNP every 60 bp in coding regions and one every 40 bp in non-coding regions. Non-synonymous substitutions contributed substantially to the polymorphism observed in coding regions. The non-synonymous to silent diversity ratio was 0.16 on average, which is fairly higher to the ratio reported in other invertebrate species recognised to display large population sizes. Therefore, purifying selection does not appear to be as strong as it could have been expected for a species with a large effective population size. The level of non-synonymous diversity varied greatly from one gene to another, in accordance with varying selective constraints. We examined codon usage bias and its relationship with DNA polymorphism. The table of optimal codons was deduced from the analysis of an EST dataset, using EST counts as a rough assessment of gene expression. As recently observed in some other taxa, we found a strong and significant negative relationship between codon bias and non-synonymous diversity suggesting correlated selective constraints on synonymous and non-synonymous substitutions. Codon bias as measured by the frequency of optimal codons for expression might therefore provide a useful indicator of the level of constraint upon proteins in the oyster genome.

Variation in synonymous codon use and DNA polymorphism within the Drosophila genome.

A strong negative correlation between the rate of amino-acid substitution and codon usage bias in Drosophila has been attributed to interference between positive selection at nonsynonymous sites and weak selection on codon usage. To further explore this possibility we have investigated polymorphism and divergence at three kinds of sites: synonymous, nonsynonymous and intronic in relation to codon bias in D. melanogaster and D. simulans. We confirmed that protein evolution is one of the main explicative parameters for interlocus codon bias variation (r(2) approximately 40%). However, intron or synonymous diversities, which could have been expected to be good indicators of local interference [here defined as the additional increase of drift due to selection on tightly linked sites, also called 'genetic draft' by Gillespie (2000)] did not covary significantly with codon bias or with protein evolution. Concurrently, levels of polymorphism were reduced in regions of low recombination rates whereas codon bias was not. Finally, while nonsynonymous diversities were very well correlated between species, neither synonymous nor intron diversities observed in D. melanogaster were correlated with those observed in D. simulans. All together, our results suggest that the selective constraint on the protein is a stable component of gene evolution while local interference is not. The pattern of variation in genetic draft along the genome therefore seems to be instable through evolutionary times and should therefore be considered as a minor determinant of codon bias variance. We argue that selective constraints for optimal codon usage are likely to be correlated with selective constraints on the protein, both between codons within a gene, as previously suggested, and also between genes within a genome.

Direct selection on allozymes is not required to explain heterogeneity among marker loci across a Mytilus hybrid zone.

Unequal differentiation between two types of loci (allozyme and DNA markers) across a Mytilus hybrid zone has recently been claimed as evidence for direct selection on some allozyme loci. We provide here a counter-example: a noncoding DNA locus that exhibits as much differentiation as the incriminated allozymes do. The levels of genetic differentiation varied widely among both allozymes and noncoding DNA markers and no clear difference emerged between the two types of markers. This suggests that the strong interlocus variance in genetic differentiation has been confounded with a discrepancy between marker types as a result of an insufficient and unbalanced locus sampling. Heterogeneity in differentiation among neutral loci can be created by stochastic variance during the allopatric divergence preceding a secondary contact. In hybrid zones, a further source of variance is differential introgression among chromosomal regions after the secondary contact owing to the local influence of selected genes on more or less distant markers. However, the degree of differentiation alone gives no way to distinguish indirect pseudo-selection (a regular and ubiquitous feature of hybrid zones) from direct selection. More generally, we suggest that comparative neutrality tests based on discrepancies among marker types have to be applied with caution when the presence of semi-permeable genetic barriers to gene exchange is suspected.

Introgression patterns in the mosaic hybrid zone between Mytilus edulis and M. galloprovincialis.

Hybrid zones are fascinating systems to investigate the structure of genetic barriers. Marine hybrid zones deserve more investigation because of the generally high dispersion potential of planktonic larvae which allows migration on scales unrivalled by terrestrial species. Here we analyse the genetic structure of the mosaic hybrid zone between the marine mussels Mytilus edulis and M. galloprovincialis, using three length-polymorphic PCR loci as neutral and diagnostic markers on 32 samples along the Atlantic coast of Europe. Instead of a single genetic gradient from M. galloprovincialis on the Iberian Peninsula to M. edulis populations in the North Sea, three successive transitions were observed in France. From South to North, the frequency of alleles typical of M. galloprovincialis first decreases in the southern Bay of Biscay, remains low in Charente, then increases in South Brittany, remains high in most of Brittany, and finally decreases again in South Normandy. The two enclosed patches observed in the midst of the mosaic hybrid zone in Charente and Brittany, although predominantly M. edulis-like and M. galloprovincialis-like, respectively, are genetically original in two respects. First, considering only the various alleles typical of one species, the patches show differentiated frequencies compared to the reference external populations. Second, each patch is partly introgressed by alleles of the other species. When introgression is taken into account, linkage disequilibria appear close to their maximum possible values, indicating a strong genetic barrier within all transition zones. Some pre- or postzygotic isolation mechanisms (habitat specialization, spawning asynchrony, assortative fertilization and hybrid depression) have been documented in previous studies, although their relative importance remains to be evaluated. We also provided evidence for a recent migratory 'short-cut' connecting M. edulis-like populations of the Charente patch to an external M. edulis population in Normandy and thought to reflect artificial transfer of spat for aquaculture.

Microsatellite analysis of 6-hour-old embryos reveals no preferential intraspecific fertilization between cupped oysters Crassostrea gigas and Crassostrea angulata.

Experimental examination of reproductive isolation is the first step in understanding hybridization processes. Here, we studied preferential fertilization between 2 cupped oyster taxa, Crassostrea angulata and Crassostrea gigas, as a potential prezygotic reproductive isolation. Early examination of sperm competition is now possible by molecular analysis of oyster embryos. This avoids the confounding effect of differential mortality during the larval stage. Six hundred embryos were sampled from 2 crosses. Three microsatellite loci were enough to determine without ambiguity the taxa of contributing sires of embryos. No evidence of preferential fertilization between gametes from the same taxa was shown. A significantly higher contribution of the C. gigas males was revealed with the C. angulata females, but not with the C. gigas females, which might suggest early heterosis or interaction differences between gametes. In the light of these results, natural hybridization between both taxa can be expected in cases of their geographical coexistence, as in the Southern European populations in which both taxa are in contact as a result of aquaculture development.

An inbreeding model of associative overdominance during a population bottleneck.

Associative overdominance, the fitness difference between heterozygotes and homozygotes at a neutral locus, is classically described using two categories of models: linkage disequilibrium in small populations or identity disequilibrium in infinite, partially selfing populations. In both cases, only equilibrium situations have been considered. In the present study, associative overdominance is related to the distribution of individual inbreeding levels (i.e., genomic autozygosity). Our model integrates the effects of physical linkage and variation in inbreeding history among individual pedigrees. Hence, linkage and identity disequilibrium, traditionally presented as alternatives, are summarized within a single framework. This allows studying nonequilibrium situations in which both occur simultaneously. The model is applied to the case of an infinite population undergoing a sustained population bottleneck. The effects of bottleneck size, mating system, marker gene diversity, deleterious genomic mutation parameters, and physical linkage are evaluated. Bottlenecks transiently generate much larger associative overdominance than observed in equilibrium finite populations and represent a plausible explanation of empirical results obtained, for instance, in marine species. Moreover, the main origin of associative overdominance is random variation in individual inbreeding whereas physical linkage has little effect.