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.     

Patterns of larval dispersal and their effect on the maintenance of a blue mussel hybrid zone in southwestern England

Abstract The blue mussels Mytilus edulis and M. galloprovincialis hybridize in southwestern England. Within this hybrid zone environmentally based directional selection favors individuals with alleles specific to M. galloprovincialis . What forces are countering this directional selection and allowing for the maintenance of a stable hybrid population are unknown. We used both the genetics of recently settled larvae and a fine-scale model of the physical oceanography of the region to determine the patterns of larval dispersal throughout the hybrid zone and the bordering parental populations. Evidence from both the model and the genetics suggests that the hybrid zone lies between two barriers to dispersal. Start Point separates the M. edulis population from the hybrid zone and allows minimal dispersal from the hybrid zone into the M. edulis population, but none in the other direction. Likewise, the M. galloprovincialis populations along the northern coast of Cornwall regularly receive immigrating larvae from the hybrid zone, but larvae from the M. galloprovincialis population do not enter the hybrid zone. However, larvae settling at hybrid zone sites have high frequencies of alleles specific to M. edulis , suggesting that reproductive barriers, selection in the larval stage, or gene flow from an undetermined source is effectively balancing the directional selection observed in the adults.     

Conspecific Sperm Precedence Is a Reproductive Barrier between Free-Spawning Marine Mussels in the Northwest Atlantic Mytilus Hybrid Zone

Reproductive isolation at the gamete stage has become a focus of speciation research because of its potential to evolve rapidly between closely related species. Conspecific sperm precedence (CSP), a type of gametic isolation, has been demonstrated in a number of taxa, both marine and terrestrial, with the potential to play an important role in speciation. Free-spawning marine invertebrates are ideal subjects for the study of CSP because of a likely central role for gametic barriers in reproductive isolation. The western Atlantic Mytilus blue mussel hybrid zone, ranging from the Atlantic Canada to eastern Maine, exhibits characteristics conducive to the study of CSP. Previous studies have shown that gametic incompatibility is incomplete, variable in strength and the genotype distribution is bimodal—dominated by the parental species, with a low frequency of hybrids. We conducted gamete crossing experiments using M. trossulus and M. edulis individuals collected from natural populations during the spring spawning season in order to detect the presence or absence of CSP within this hybrid zone. We detected CSP, defined here as a reduction in heterospecific offspring from competitive fertilizations in vitro compared to that seen in non-competitive fertilizations, in five of the twelve crosses in which conspecific crosses were detectable. This is the first finding of CSP in a naturally hybridizing population of a free-spawning marine invertebrate. Our findings support earlier predictions that CSP can promote assortative fertilization in bimodal hybrid zones, further advancing their hypothesized progression towards full speciation. Despite strong CSP numerous heterospecific fertilizations remain, reinforcing the hypothesis that compatible females are a source of hybrid offspring in mixed natural spawns.     

Geographic variation and positive selection on M7 lysin, an acrosomal sperm protein in mussels (Mytilus spp.)

Successful fertilization in free-spawning marine organisms depends on the interactions between genes expressed on the surfaces of eggs and sperm. Positive selection frequently characterizes the molecular evolution of such genes, raising the possibility that some common deterministic process drives the evolution of gamete recognition genes and may even be important for understanding the evolution of prezygotic isolation and speciation in the marine realm. One hypothesis is that gamete recognition genes are subject to selection for prezygotic isolation, namely, reinforcement. In a previous study, positive selection on the gene coding for the acrosomal sperm protein M7 lysin was demonstrated among allopatric populations of mussels in the Mytilus edulis species group (M. edulis, Mytilus galloprovincialis, and Mytilus trossulus). Here, we expand sampling to include M7 lysin haplotypes from populations where mussel species are sympatric and hybridize to determine whether there is a pattern of reproductive character displacement (RCD), which would be consistent with reinforcement driving selection on this gene. We do not detect a strong pattern of RCD; neither are there unique haplotypes in sympatry nor is there consistently greater population structure in comparisons involving sympatric populations. One distinct group of haplotypes, however, is strongly affected by natural selection, and this group of haplotypes is found within M. galloprovincialis populations throughout the Northern Hemisphere concurrent with haplotypes common to M. galloprovincialis and M. edulis. We suggest that balancing selection, perhaps resulting from sexual conflicts between sperm and eggs, maintains old allelic diversity within M. galloprovincialis.     

Intrinsic reproductive isolating mechanisms in the maintenance of a hybrid zone between ecologically divergent subspecies

Understanding factors involved in maintaining stable hybrid zones is important for predicting the ultimate fate of the interacting taxa, but the relative importance of mechanisms such as ecological selection and intrinsic reproductive isolation remains unclear. Most studies of reproductive isolation in hybrid zones have focused either on zones with strongly bimodal patterns in genotype or phenotype frequencies, with relatively strong isolation, or unimodal zones with relatively weak isolation, whereas less is known about more intermediate classes of hybrid zone. Here, we utilize a hybrid zone of this intermediate type occurring between northern and southern subspecies of Atlantic killifish, Fundulus heteroclitus, to identify isolating mechanisms playing a role in maintaining this type of zone. The two subspecies differ in environmental tolerance, and we found some evidence of microhabitat preference between subspecies within a small tidal creek at the centre of the hybrid zone. There was also an association between sex, mitochondrial genotype and habitat within this creek. Fertilization success did not differ between consubspecific and heterosubspecific crosses, but hatching success was significantly lower for crosses involving southern males and northern females, and crosses between southern females and northern males had altered developmental rates. Southern females and northern males showed patterns consistent with positive assortative mating. Together, these results indicate a role for a combination of factors including assortative mating and/or early hybrid inviability in the maintenance of this hybrid zone and suggest that hybrid zones with intermediate levels of reproductive isolation are likely to be maintained by multiple interacting isolating mechanisms.     

Disruption of doubly uniparental inheritance of mitochondrial DNA in hybrid mussels (Mytilus edulis x M. galloprovincialis)

Blue mussels of the genus Mytilus have an unusual mode of mitochondrial DNA inheritance termed doubly uniparental inheritance (DUI). Females are homoplasmic for the F mitotype which is inherited maternally, whereas males are heteroplasmic for this and the paternally inherited M mitotype. In areas where species distributions overlap a varying degree of hybridization occurs; yet genetic differences between allopatric populations are maintained. Observations from natural populations and previous laboratory experiments suggest that DUI may be disrupted by hybridization, giving rise to heteroplasmic females and homoplasmic males. We carried out controlled laboratory crosses between Mytilus edulis and M. galloprovincialis to produce pure species and hybrid larvae of known parentage. DNA markers were used to follow the fate of the F and M mitotypes through larval development. Disruption of the mechanism which determines whether the M mitotype is retained or eliminated occurred in an estimated 38% of M. edulis x M. galloprovincialis hybrid larvae, a level double that previously observed in adult mussels from a natural M. edulis x M. galloprovincialis hybrid population. Furthermore, reciprocal hybrid crosses exhibited contrasting types of DUI disruption. The results indicate that disruption of DUI in hybrid mussels may be associated with increased mortality and hence could be a factor in the maintenance of genetic integrity for each species.     

Evidence of prezygotic isolation,but not assortative mating,between locally adapted populations of Fundulus heteroclitus across a salinity gradient

Selection along environmental gradients can drive reproductive isolation and speciation. Among fishes, salinity is a major factor limiting species distributions, and despite its importance in generating species diversity, speciation events between marine and freshwater are rare. Here, we tested for mechanisms of reproductive isolation between locally adapted freshwater and brackish water-native populations of killifish, Fundulus heteroclitus, from either side of a hybrid zone along a salinity gradient. There was evidence for pre-zygotic endogenous reproductive isolation with reduced fertilization success between crosses of freshwater-native males and brackish water-native females. Exogenous pre-zygotic isolation was also present where females had highest fertilization in their native salinity. We used a replicated mass spawning design to test for mate choice in both brackish and fresh water. After genotyping 187 parents and 2523 offspring at 2347 SNPs across the genome, 85% of offspring were successfully assign to their parents. However, no reinforcing mate choice was observed. These results therefore demonstrate emerging, yet limited, reproductive isolation and incipient speciation across a marine to freshwater salinity gradient and suggest that both endogenous and exogenous mechanisms, but not assortative mating, contribute to divergence.     

Gamete compatibility and sperm competition affect paternity and hybridization between sympatric Asterias sea stars

Gamete interactions may strongly influence speciation and hybridization in sympatric broadcast-spawning marine invertebrates. We examined the role of gamete compatibility in species integrity using cross-fertilization studies between sympatric Asterias sea stars from a secondary contact zone in the northwest Atlantic. In crosses between single males and single females, gametes of both species were compatible and produced viable, fertile hybrid offspring, but with considerable variation in the receptivity of eggs to heterospecific sperm. Differential compatibility of heterospecific gametes was detected in sperm competition studies in which we used a nuclear DNA marker to assign paternity to larval offspring. Several families showed conspecific sperm precedence in A. forbesi eggs, and one family showed competitive superiority of A. forbesi sperm fertilizing A. rubens eggs. Gametic interactions are an important component of prezygotic reproductive isolation in sympatric Asterias. The interaction between gametes of these closely related sea stars is consistent with the function of gamete recognition systems that are known to mediate fertilization success and speciation in other marine invertebrates.     

Positive selection on an acrosomal sperm protein,M7 lysin,in three species of the mussel genus Mytilus

Marine invertebrate sperm proteins are particularly interesting because they are characterized by positive selection and are likely to be involved in prezyogotic isolation and, thus, speciation. Here, we present the first survey of interspecific and intraspecific variation of a bivalve sperm protein among a group of species that regularly hybridize in nature. M7 lysin is found in sperm acrosomes of mussels and dissolves the egg vitelline coat, permitting fertilization. We sequenced multiple alleles of the mature protein-coding region of M7 lysin from allopatric populations of mussels in the Mytilus edulis species group (M. edulis, M. galloprovincialis, and M. trossulus). A significant McDonald-Kreitman test showed an excess of fixed amino acid replacing substitutions between species, consistent with positive selection. In addition, Kolmogorov-Smirnov tests showed significant heterogeneity in polymorphism to divergence ratios for both synonymous variation and combined synonymous and nonsynonymous variation within M. galloprovincialis. These results indicate that there has been adaptive evolution at M7 lysin and, furthermore, show that positive selection on sperm proteins can occur even when postzygotic reproductive isolation is incomplete.     

Local adaptation and species segregation in two mussel (Mytilus edulis x Mytilus trossulus) hybrid zones

Few marine hybrid zones have been studied extensively, the major exception being the hybrid zone between the mussels Mytilus edulis and Mytilus galloprovincialis in southwestern Europe. Here, we focus on two less studied hybrid zones that also involve Mytilus spp.; Mytilus edulis and Mytilus trossulus are sympatric and hybridize on both western and eastern coasts of the Atlantic Ocean. We review the dynamics of hybridization in these two hybrid zones and evaluate the role of local adaptation for maintaining species boundaries. In Scandinavia, hybridization and gene introgression is so extensive that no individuals with pure M. trossulus genotypes have been found. However, M. trossulus alleles are maintained at high frequencies in the extremely low salinity Baltic Sea for some allozyme genes. A synthesis of reciprocal transplantation experiments between different salinity regimes shows that unlinked Gpi and Pgm alleles change frequency following transplantation, such that post-transplantation allelic composition resembles native populations found in the same salinity. These experiments provide strong evidence for salinity adaptation at Gpi and Pgm (or genes linked to them). In the Canadian Maritimes, pure M. edulis and M. trossulus individuals are abundant, and limited data suggest that M. edulis predominates in low salinity and sheltered conditions, whereas M. trossulus are more abundant on the wave-exposed open coasts. We suggest that these conflicting patterns of species segregation are, in part, caused by local adaptation of Scandinavian M. trossulus to the extremely low salinity Baltic Sea environment.     

INCIPIENT REPRODUCTIVE ISOLATION BETWEEN TWO SYMPATRIC MORPHS OF THE INTERTIDAL SNAIL LITTORINA SAXATILIS

The study of speciation in recent populations is essentially a study of the evolution of reproductive isolation mechanisms between sub-groups of a species. Prezygotic isolation can be of central importance to models of speciation, either being a consequence of reinforcement of assortative mating in hybrid zones, or a pleiotropic effect of morphological or behavioral adaptation to different environments. To suggest speciation by reinforcement between incipient species one must at least know that gene flow occurs, or have recently occurred, and that assortative mating has been established in the hybrid zone. In Galician populations of the marine snail Littorina saxatilis, two main morphs appear on the same shores, one on the upper-shore barnacle belt and the other in the lower-shore mussel belt. The two morphs overlap in distribution in the midshore where hybrids are found together with pure forms. Allozyme variation indicates that the two parental morphs share a common gene pool, although within shores, gene flow between morphs is less than gene flow within morphs. In this study, we observed mating behavior in the field, and we found that mating was not random in midshore sites, with a deficiency of heterotypic pairs. Habitat selection, assortative mating, and possibly sexual selection among females contributed to the partial reproductive isolation between the pure morphs. Sizes of mates were often positively correlated, in particular, in the upper shore, indicating size-assortative mating too. However, this seemed to be a consequence of nonrandom microdistributions of snails of different sizes. Because we also argue that the hybrid zone is of primary rather than secondary origin, this seems to be an example of sympatric reproductive isolation, either established by means of reinforcement or as a by-product to divergent selection acting on other characters.     

Evidence of selective mortality in favour of the Mytilus galloprovincialis Lmk phenotype in British mussel populations

Samples of mussels ( Mytilus ) were collected from 17 localities within hybrid zones of Mytilus edulis and Mytilus galloprovincialis in south-west and north-east England. The study of two polymorphic allozyme loci ( esterase-D and octopine dehydrogenase ), which are partially diagnostic for the two forms of mussel, reveal the existence of widespread length-dependent allele frequency variation. Larger mussels tend to have a higher frequency of alleles characteristically at high frequency in Mytilus galloprovincialis. Also at a given shell length galloprovincialis alleles have a higher frequency higher up the shore. Computer simulation is used to demonstrate that length-dependent variation may be generated not only by differential mortality but also by differential growth and in models including or excluding immigration. Evidence supports the hypothesis that selective mortality acting in favour of the galloprovincialis phenotype within hybrid populations in Britain is balanced by immigration of the more abundant Mytilus edulis.     

Fitness landscapes support the dominance theory of post-zygotic isolation in the mussels Mytilus edulis and M. galloprovincialis

We studied the genetic basis of post-zygotic isolation in the marine mussels Mytilus edulis and Mytilus galloprovincialis. Evidence was obtained for a high number of recessive Dobzhansky-Muller substitutions in the genome of these two mussel taxa. We analysed the segregation of unlinked diagnostic markers in the progeny of two backcrosses and an F2 cross, 36 h and 200 days after fertilization. Directional selection favouring M. galloprovincialis genotypes was observed in both kinds of cross. In the F2, epistatic interactions between each pair of chromosome fragments mapped by the markers were identified in addition. Our results imply that homozygous-homozygous interactions are required for breakdown of coadaptation, in accordance with the dominance theory of post-zygotic isolation. Endogenous post-zygotic selection distributed over many loci throughout the genome provides the missing factor explaining the astonishing persistence and strength of barriers to neutral introgression in such a dispersive taxon as Mytilus.     

Electrophoretic investigation of systematic relationships in the marine mussels Modiolus modiolus L., Mytilus edulis L., and Mytilus galloprovincialis Lmk. (Mytilidae; Mollusca)

Genetic variation was assayed electrophoretically at 13–16 loci in Modiolus modiolus, Mytilus edulis, and Mytilus galloprovincialis. High genetic distance ( D ) values were observed between Modiolus modiolus and Mytilus edulis (1.516 ± 0.523) and between Modiolus modiolus and Mytilus galloprovincialis (1.564 ± 0.539), whereas the distance between Mytilus edulis and M. galloprovincialis (0.167 ± 0.118) was rather low. The systematic status ot Mytilus edulis and M. galloprovincialis is discussed in relation to these lindings and the genetic distance values are used to estimate divergence times which in turn are compared with paleontological estimates. The observations of high average heterozygosity in Modiolus modiolus, and high correlations of locus heterozygosity between taxa are discussed briefly.     

The importance of ecological and behavioural data in studies of hybridisation among marine fishes

Natural hybridisation is a widespread phenomenon, particularly well documented in terrestrial and freshwater ecosystems, where it has been ascribed substantial evolutionary and adaptive relevance. Hybridisation has received comparatively less attention in marine systems, though there has been a recent surge of reported marine hybrids, particularly among corals and fishes. This review summarises the current knowledge of hybridisation in marine fishes, with a focus on ecological and behavioural factors that may play a role in hybridisation processes. Rarity of one or both parental species within the hybrid zone, overlap in habitat use, dietary overlap and the breakdown in assortative mating appear to have a role in facilitating hybridisation. Despite this, most of the recent literature on marine fish hybridisation has a strong genetic focus, with little or no quantitative information about the ecological and behavioural factors that initiate or facilitate hybridisation. Future studies should attempt to gather ecological and behavioural data from hybrid zones, thus teasing out which processes are most relevant to overcoming pre-zygotic barriers to reproductive isolation. Not only will this advance our understanding of the adaptive and evolutionary relevance of hybridisation in marine fishes, but it will also provide unique insights into the maintenance of reproductive isolation and the process of speciation in the marine environment.     

Premating barriers to gene exchange and their implications for the structure of a mosaic hybrid zone between Chorthippus brunneus and C. jacobsi (Orthoptera: Acrididae)

Many hybrid zones contain a deficit of hybrid genotypes relative to expectations from tension zone models. This is often associated with separation of parental genotypes into distinct habitats (mosaicism), but sometimes parentals can be found co-occuring in the same local population (bimodality). In both cases, prezygotic isolation may play an important role in determining the genotypic composition of the zone. Chorthippus brunneus and C. jacobsi (Orthoptera: Acrididae) meet and form a complex hybrid zone in northern Spain. Analysis of stridulatory peg numbers reveals partial spatial and seasonal isolation in a 25 km2 area of the zone: C. jacobsi phenotypes predominate in June and July and are present in both valley and mountain habitats; C. brunneus phenotypes predominate in August and are restricted to valley habitats, always in sympatry with C. jacobsi. Strong assortative mating was observed in laboratory mating experiments. Spatial, seasonal and behavioural isolation combine to produce strong premating isolation in the study area. These results suggest a role for premating isolation in maintaining both the mosaic structure and bimodality of this hybrid zone.     

Faster fertilization rate in conspecific versus heterospecific matings in house mice

Barriers to gene flow can arise at any stage in the reproductive sequence. Most studies of reproductive isolation focus on premating or postzygotic phenotypes, leaving the importance of differences in fertilization rate overlooked. Two closely related species of house mice, Mus domesticus and M. musculus , form a narrow hybrid zone in Europe, suggesting that one or more isolating factors operate in the face of ongoing gene flow. Here, we test for differences in fertilization rate using laboratory matings as well as in vitro sperm competition assays. In noncompetitive matings, we show that fertilization occurs significantly faster in conspecific versus heterospecific matings and that this difference arises after mating and before zygotes form. To further explore the mechanisms underlying this conspecific advantage, we used competitive in vitro assays to isolate gamete interactions. Surprisingly, we discovered that M. musculus sperm consistently outcompeted M. domesticus sperm regardless of which species donated ova. These results suggest that in vivo fertilization rate is mediated by interactions between sperm, the internal female environment, and/or contributions from male seminal fluid. We discuss the implications of faster conspecific fertilization in terms of reproductive isolation among these two naturally hybridizing species.     

Plasticity of fertilization rates under varying temperature in the broadcast spawning mussel,Mytilus galloprovincialis

Oceans are a huge sink for the increased heat associated with anthropogenic climate change, and it is vital to understand the heat tolerance of marine organisms at all life stages to accurately predict species’ responses. In broadcast spawning marine invertebrates, reproduction is a vulnerable process in which sperm and eggs are released directly into the open water. Gametes are then exposed to fluctuating environmental conditions that may impact their fertilizing capacity. Using the broadcast spawning Mediterranean mussel, Mytilus galloprovincialis, as a model species, we performed blocks of factorial mating crosses to assess the variance in fertilization rates among individuals under both ambient and elevated temperatures. Overall, we found a small, but significant decline in fertilization rates with elevated temperatures. However, there was substantial plasticity in responses, with particular mussels having increased fertilization under elevated temperatures, although the majority showed decreased fertilization rates. Our results suggest possible future reproductive costs to ocean warming in M. galloprovincialis, although it is also possible that genetic variation for thermal sensitivity may allow for adaptation to changing environmental conditions.