Pumping ions: rapid parallel evolution of ionic regulation following habitat invasions

Marine to freshwater colonizations constitute among the most dramatic evolutionary transitions in the history of life. This study examined evolution of ionic regulation following saline-to-freshwater transitions in an invasive species. In recent years, the copepod Eurytemora affinis has invaded freshwater habitats multiple times independently. We found parallel evolutionary shifts in ion-motive enzyme activity (V-type H(+) ATPase, Na(+) /K(+) -ATPase) across independent invasions and in replicate laboratory selection experiments. Freshwater populations exhibited increased V-type H(+) ATPase activity in fresh water (0 PSU) and declines at higher salinity (15 PSU) relative to saline populations. This shift represented marked evolutionary increases in plasticity. In contrast, freshwater populations displayed reduced Na(+) /K(+) -ATPase activity across all salinities. Most notably, modifying salinity alone during laboratory selection experiments recapitulated the evolutionary shifts in V-type H(+) ATPase activity observed in nature. Maternal and embryonic acclimation could not account for the observed shifts in enzyme activity. V-type H(+) ATPase function has been hypothesized to be critical for freshwater and terrestrial adaptations, but evolution of this enzyme function had not been previously demonstrated in the context of habitat transitions. Moreover, the speed of these evolutionary shifts was remarkable, within a few generations in the laboratory and a few decades in the wild.     

Response to selection and evolvability of invasive populations

While natural selection might in some cases facilitate invasions into novel habitats, few direct measurements of selection response exist for invasive populations. This study examined selection response to changes in salinity using the copepod Eurytemora affinis. This copepod has invaded fresh water from saline habitats multiple times independently throughout the Northern Hemisphere. Selection response to a constant intermediate salinity (5 PSU) was measured in the laboratory for saline source and freshwater invading populations from the St. Lawrence drainage (North America). These populations were reared under three conditions: (1) native salinities (0 or 15 PSU) for at least two generations, (2) 5 PSU for two generations, and (3) 5 PSU for six generations. Full-sib clutches taken from populations reared under these three conditions were split across four salinities (0, 5, 15, and 25 PSU) to determine reaction norms for survival and development time. Contrasts in survival and development time across the three rearing conditions were treated as the selection response. Selection at 5 PSU resulted in a significant decline in freshwater (0 PSU) tolerance for both the saline and freshwater populations. Yet, evolutionary differences in freshwater tolerance persisted between the saline and freshwater populations. The saline and freshwater populations converged in their high-salinity (25 PSU) tolerance, with an increase in the freshwater population and decline in the saline population. Development time did not shift greatly in response to selection at 5 PSU. For all three rearing conditions, the freshwater population exhibited retarded larval development and accelerated juvenile development relative to the saline population. Results from this study indicate that both the saline and freshwater populations exhibit significant responses to selection for a fitness-related trait critical for invasions into a novel habitat. For the Symposium on “Evolvability and Adaptation of Invasive Species,” Society for the Study of Evolution 2004.     

Testing for beneficial reversal of dominance during salinity shifts in the invasive copepod Eurytemora affinis,and implications for the maintenance of genetic variation

Maintenance of genetic variation at loci under selection has profound implications for adaptation under environmental change. In temporally and spatially varying habitats, non‐neutral polymorphism could be maintained by heterozygote advantage across environments (marginal overdominance), which could be greatly increased by beneficial reversal of dominance across conditions. We tested for reversal of dominance and marginal overdominance in salinity tolerance in the saltwater‐to‐freshwater invading copepod Eurytemora affinis. We compared survival of F1 offspring generated by crossing saline and freshwater inbred lines (between‐salinity F1 crosses) relative to within‐salinity F1 crosses, across three salinities. We found evidence for both beneficial reversal of dominance and marginal overdominance in salinity tolerance. In support of reversal of dominance, survival of between‐salinity F1 crosses was not different from that of freshwater F1 crosses under freshwater conditions and saltwater F1 crosses under saltwater conditions. In support of marginal overdominance, between‐salinity F1 crosses exhibited significantly higher survival across salinities relative to both freshwater and saltwater F1 crosses. Our study provides a rare empirical example of complete beneficial reversal of dominance associated with environmental change. This mechanism might be crucial for maintaining genetic variation in salinity tolerance in E. affinis populations, allowing rapid adaptation to salinity changes during habitat invasions.     

Evolution of physiological tolerance and performance during freshwater invasions

Invasive species that penetrate habitat boundaries are likelyto experience strong selection and rapid evolution. This studydocuments evolutionary shifts in tolerance and performance followingthe invasion of fresh water by the predominantly estuarine andsalt marsh copepod Eurytemora affinis. Common-garden experimentswere performed on freshwater-invading (Lake Michigan) and ancestralsaline (St. Lawrence marsh) populations to measure shifts inadult survival (at 0, 5, and 25 PSU), and survival during developmentand development time (both using full-sib clutches split across0, 5, 15, and 25 PSU). Results showed clear evidence of heritableshifts in tolerance and performance associated with freshwaterinvasions. The freshwater population exhibited a gain in low-salinitytolerance and a reduction in high-salinity tolerance relativeto the saline population, suggesting tradeoffs. These tradeoffswere supported by negative genetic correlations between survivalat fresh (0 PSU) versus higher salinities. Mortality in responseto salinity occurred primarily before metamorphosis, suggestingthat selection in response to salinity had acted primarily onthe early life-history stages. The freshwater population exhibitedcurious patterns of life-history evolution across salinities,relative to the saline population, of retarded development tometamorphosis but accelerated development from metamorphosisto adulthood. This pattern might reflect tradeoffs between developmentrate and survival in fresh water at the early life-history stages,but some other selective force acting on later life-historystages. Significant effects of clutch (genotype) and clutch-by-salinityinteraction (G x E) on survival and development time in bothpopulations indicated ample genetic variation as substrate fornatural selection. Variation for high-salinity tolerance waspresent in the freshwater population despite negative geneticcorrelations between high- and low-salinity tolerance. Resultsimplicate the importance of natural selection and document theevolution of reaction norms during freshwater invasions.     

Developmental dissociation in morphological evolution of the stickleback opercle

Oceanic threespine sticklebacks have repeatedly and independently evolved new morphologies upon invasions of freshwater habitats. A consistent derived feature of the freshwater form across populations and geography is a shape change of the opercle, a large early developing facial bone. We show that the principal multivariate axis describing opercle shape development from the young larva to the full adult stage of oceanic fish matches the principal axis of evolutionary change associated with relocation from the oceanic to freshwater habitat. The opercle phenotype of freshwater adults closely resembles the phenotype of the bone in juveniles. Thus, evolution to the freshwater condition is in large part by truncation of development; the freshwater fish do not achieve the full ancestral adult bone shape. Additionally, the derived state includes dissociated ontogenetic changes. Dissociability may reflect an underlying modular pattern of opercle development, and facilitate flexibility of morphological evolution.     

Habitat transitions alter the adaptive landscape and shape phenotypic evolution in needlefishes (Belonidae)

Habitat occupancy can have a profound influence on macroevolutionary dynamics, and a switch in major habitat type may alter the evolutionary trajectory of a lineage. In this study, we investigate how evolutionary transitions between marine and freshwater habitats affect macroevolutionary adaptive landscapes, using needlefishes (Belonidae) as a model system. We examined the evolution of body shape and size in marine and freshwater needlefishes and tested for phenotypic change in response to transitions between habitats. Using micro‐computed tomographic (µCT) scanning and geometric morphometrics, we quantified body shape, size, and vertebral counts of 31 belonid species. We then examined the pattern and tempo of body shape and size evolution using phylogenetic comparative methods. Our results show that transitions from marine to freshwater habitats have altered the adaptive landscape for needlefishes and expanded morphospace relative to marine taxa. We provide further evidence that freshwater taxa attain reduced sizes either through dwarfism (as inferred from axial skeletal reduction) or through developmental truncation (as inferred from axial skeletal loss). We propose that transitions to freshwater habitats produce morphological novelty in response to novel prey resources and changes in locomotor demands. We find that repeated invasions of different habitats have prompted predictable changes in morphology.     

Ontogeny of osmoregulation in the crayfish Astacus leptodactylus

Osmoregulation was studied during the postembryonic development of Astacus leptodactylus Eschscholtz 1823 in juvenile stages 1-8 and in adults. Juveniles hatch and later stages develop in freshwater or in moderately saline waters. The time of acclimation from freshwater to a saline medium increased from early juveniles to adults. At all stages, it was longer than in comparable stages of marine crustaceans, reflecting the high impermeability of the teguments to water and ions. All stages were able to hyperisoosmoregulate. In freshwater, the ability to hyperosmoregulate was established at hatching and increased during development. The hemolymph osmolality increased from 286 mosm kg-1 in stage 1 juveniles to 419 mosm kg-1 in adults. All stages also hyperregulated at low salinities (7 per thousand and 13 per thousand salinity) and were osmoconformers at higher salinities up to 21 per thousand salinity. The lowest isosmotic salinity tended to increase with the developmental stages. The ability to osmoregulate at hatch and throughout postembryonic development is probably a key physiological adaptation in this and other freshwater crayfish.     

Adaptation and constraint in a stickleback radiation

The evolution of threespine sticklebacks in freshwater lakes constitutes a well‐studied example of a phenotypic radiation that has produced numerous instances of parallel evolution, but the exact selective agents that drive these changes are not yet fully understood. We present a comparative study across 74 freshwater populations of threespine stickleback in Norway to test whether evolutionary changes in stickleback morphology are consistent with adaptations to physical parameters such as lake depth, lake area, lake perimeter and shoreline complexity, variables thought to reflect different habitats and feeding niches. Only weak indications of adaptation were found. Instead, populations seem to have diversified in phenotypic directions consistent with allometric scaling relationships. This indicates that evolutionary constraints may have played a role in structuring phenotypic variation across freshwater populations of stickleback. We also tested whether the number of lateral plates evolved in response to lake calcium levels, but found no evidence for this hypothesis.     

Loci under selection during multiple range expansions of an invasive plant are mostly population specific,but patterns are associated with climate

Identifying the genes underlying rapid evolutionary changes, describing their function and ascertaining the environmental pressures that determine fitness are the central elements needed for understanding of evolutionary processes and phenotypic changes that improve the fitness of populations. It has been hypothesized that rapid adaptive changes in new environments may contribute to the rapid spread and success of invasive plants and animals. As yet, studies of adaptation during invasion are scarce, as is knowledge of the genes underlying adaptation, especially in multiple replicated invasions. Here, we quantified how genotype frequencies change during invasions, resulting in rapid evolution of naturalized populations. We used six fully replicated common garden experiments in Brazil where Pinus taeda (loblolly pine) was introduced at the same time, in the same numbers, from the same seed sources, and has formed naturalized populations expanding outward from the plantations. We used a combination of nonparametric, population genetics and multivariate statistics to detect changes in genotype frequencies along each of the six naturalization gradients and their association with climate as well as shifts in allele frequencies compared to the source populations. Results show 25 genes with significant shifts in genotype frequencies. Six genes had shifts in more than one population. Climate explained 25% of the variation in the groups of genes under selection across all locations, but specific genes under strong selection during invasions did not show climate‐related convergence. In conclusion, we detected rapid evolutionary changes during invasive range expansions, but the particular gene‐level patterns of evolution may be population specific.     

Heterogeneity within the native range: population genetic analyses of sympatric invasive and noninvasive clades of the freshwater invading copepod Eurytemora affinis

Invasive species are often composed of highly differentiated populations or sibling species distributed across their native ranges. This study analysed patterns of distribution and the evolutionary and demographic histories of populations within the native range of the copepod species complex Eurytemora affinis. Genetic structure was analysed for samples from 17 locations from both the invaded and native ranges in the St Lawrence River drainage basin, using 652 base pairs of the mitochondrial cytochrome oxidase subunit I gene. This study revealed a high degree of heterogeneity in genetic structure and habitat type in the native range, as well as a bias in the sources of invasive populations. Two genetically distinct clades showed a pattern of niche partitioning within the St Lawrence basin. The noninvasive North Atlantic clade primarily occupied the central portion of the St Lawrence Middle Estuary, whereas the invasive Atlantic clade was more prevalent along the margins, in the upstream reaches of the estuary and downstream salt marshes. Habitat partitioning and genetic subdivision was also present within the Atlantic clade. The freshwater populations were genetically more proximate to the Atlantic clade populations in the estuary than to those in the salt marsh, suggesting the estuary as the source of the invasive populations. The freshwater invading populations showed evidence of a modest population bottleneck. Populations from both clades showed genetic signatures of demographic population expansions that preceded the timing of the last glacial maximum, supporting the St Lawrence as a secondary contact zone between the two clades. Additional analyses on physiological and evolutionary properties of populations in the native range, along with analysis of the selection regime within native habitats, might yield insights into the evolutionary potential to invade.     

RAPID AND REPEATED INVASIONS OF FRESH WATER BY THE COPEPOD EURYTEMORA AFFINIS

Invasions of fresh water by marine organisms have been of great interest to evolutionary biologists and paleontologists because they typically constitute major evolutionary transitions. Recent (< 200 years) invasions of fresh water by brackish or marine species offer an opportunity to understand mechanisms underlying these events, but pathways of invasion from salt water have not been confirmed using genetic data. This study employed mitochondrial DNA sequences (652 base pairs from the cytochrome oxidase I (COI) gene) to reconstruct the geographic and evolutionary history of freshwater invasion by the common estuarine and saltmarsh crustacean Eurytemora affinis (Copepoda; Poppe 1880). Phylogenetic analysis of populations from North America, Europe, and Asia revealed at least eight independent invasions of fresh water from genetically distinct lineages. At least five of these freshwater invasions most likely arose independently in different river drainages, recently from saltwater sources within each river drainage. An analysis of molecular variance (AMOVA) was performed at three geographic scales (among continents, among drainages, and within drainages) to assess the hierarchical distribution of genetic variance. Results indicated that 52% of the genetic variance was explained by differences among drainages, 43% by differences among continents, but only 5% by differences within drainages, thus supporting geographic patterns of invasions inferred from the phylogeny. Physiological experiments were performed to determine whether adults and larvae from saltwater populations could tolerate freshwater conditions. Transfer to zero salinity resulted in high mortalities, but with some survival to the second generation in one population. This study provides genetic evidence and physiological support for rapid transitions from a saline life history into fresh water, with repeated invasions on a global scale.     

Adaptive patterns of osmotic and ionic regulation, and the invasion of fresh water by the palaemonid shrimps

To evaluate trends in the osmoregulatory behavior of neotropical, palaemonid shrimps, we investigated osmotic and ionic regulatory patterns in five species of Palaemon or Macrobrachium. The species' life histories depend on saline water to differing degrees, their habitats ranging from the marine/intertidal (P. northropi), through estuaries (P. pandaliformis) to coastal, freshwater streams (M. olfersii, M. potiuna) and inland, continental river systems (M. brasiliense). Hemolymph osmolality, chloride, sodium and magnesium concentrations were measured in shrimps exposed to experimental media ranging from fresh water (<0.5 per thousand ) to concentrated seawater (42 per thousand ) for up to 10 days. The marine and estuarine Palaemon species exhibit well-developed hyper/hypo-osmotic, sodium and chloride regulatory capabilities in mid-range salinities, tending to hyperconform in low salinities. The freshwater Macrobrachium species show variable hyperosmotic, sodium and chloride regulatory capacities, tending to hypoconform or unable to survive at higher salinities. All species hyper-regulate magnesium in fresh water, but hyporegulate strongly in saline media. Palaemonids from the saline habitats show the strongest osmoregulatory capabilities, and fresh water may have been gradually invaded by ancestral species with similar regulatory capacity. However, this regulatory plasticity has been lost to varying degrees in extant freshwater species.     

Gene expression plasticity evolves in response to colonization of freshwater lakes in threespine stickleback

Phenotypic plasticity is predicted to facilitate individual survival and/or evolve in response to novel environments. Plasticity that facilitates survival should both permit colonization and act as a buffer against further evolution, with contemporary and derived forms predicted to be similarly plastic for a suite of traits. On the other hand, given the importance of plasticity in maintaining internal homeostasis, derived populations that encounter greater environmental heterogeneity should evolve greater plasticity. We tested the evolutionary significance of phenotypic plasticity in coastal British Columbian postglacial populations of threespine stickleback (Gasterosteus aculeatus) that evolved under greater seasonal extremes in temperature after invading freshwater lakes from the sea. Two ancestral (contemporary marine) and two derived (contemporary freshwater) populations of stickleback were raised near their thermal tolerance extremes, 7 and 22 °C. Gene expression plasticity was estimated for more than 14 000 genes. Over five thousand genes were similarly plastic in marine and freshwater stickleback, but freshwater populations exhibited significantly more genes with plastic expression than marine populations. Furthermore, several of the loci shown to exhibit gene expression plasticity have been previously implicated in the adaptive evolution of freshwater populations, including a gene involved in mitochondrial regulation (PPARAa). Collectively, these data provide molecular evidence that highlights the importance of plasticity in colonization and adaptation to new environments.     

The origins and persistence of anadromy in brook charr

We synthesized the results of a coordinated study examining the spatial and temporal movements, genetic structure, and physiological characteristics of sympatric populations of resident and sea-run brook charr across eastern Canada. Our goal was to critically evaluate three working hypotheses that may explain anadromous behaviour in brook charr: (1) resident and anadromous forms have different phylogenic origins; (2) anadromy emerges from freshwater residents; and (3) freshwater residency emerges from anadromous individuals. Our synthesis is consistent with the conclusion that freshwater residency emerging from anadromous individuals is most probable. Overall, anadromy in brook charr is poorly developed and the tactic of sea-running may be described as partial, facultative anadromy if the species must be described in terms of anadromy. The sea-run tactic most probably results from the species’ propensity to move and disperse, the over-production of juveniles, an archetypical physiological ability to tolerate saline environments, and the persistence of critical habitats. When the spatial and temporal physical environments permit, the anadromous behaviour is expressed. The tactic creates an apparent fitness advantages related to growth, but it is not necessarily the only evolutionary stable strategy for a river. Most populations with sea-run forms are declining and successful conservation of sea-run forms will depend on managing harvests and more importantly, protection of the temporally and spatially complexity of critical habitats.     

Parallels between two geographically and ecologically disparate cave invasions by the same species,Asellus aquaticus (Isopoda,Crustacea)

Caves are long‐known examples of evolutionary replications where similar morphologies (troglomorphies) evolve independently as the result of strong natural selection of the extreme environment. Recently, this paradigm has been challenged based on observations that troglomorphies are inconsistent across taxa and different subterranean habitats. We investigated the degree of replicated phenotypic change in two independent cave invasions by the freshwater isopod Asellus aquaticus; the first in a sulphidic aquifer in Romania, the second in a sinking river in the Dinaric Karst in Slovenia. Both ancestral surface populations still live alongside the subterranean ones. Phylogenetic analyses show independence of the two colonization events, and microsatellite analysis shows no evidence of ongoing genetic exchange between surface and subterranean ecomorphs. The overall morphology has changed dramatically at both sites (50 of 62 morphometric traits). The amount of phenotypic change did not reflect differences in genetic diversity between the two ancestral populations. Multivariate analyses revealed divergent evolution in caves, not parallel or convergent as predicted by the current paradigm. Still, 18 traits changed in a parallel fashion, including eye and pigment loss and antennal elongation. These changes might be a consequence of darkness as the only common ecological feature, because Romanian caves are chemoautotrophic and rich in food, whereas Slovenian caves are not. Overall, these results show that morphologically alike surface populations can diverge after invading different subterranean habitats, and that only about one‐third of all changing traits behave as troglomorphies in the traditional sense.     

Parallel and nonparallel ecological,morphological and genetic divergence in lake–stream stickleback from a single catchment

Parallel phenotypic evolution in similar environments has been well studied in evolutionary biology; however, comparatively little is known about the influence of determinism and historical contingency on the nature, extent and generality of this divergence. Taking advantage of a novel system containing multiple lake–stream stickleback populations, we examined the extent of ecological, morphological and genetic divergence between three‐spined stickleback present in parapatric environments. Consistent with other lake–stream studies, we found a shift towards a deeper body and shorter gill rakers in stream fish. Morphological shifts were concurrent with changes in diet, indicated by both stable isotope and stomach contents analysis. Performing a multivariate test for shared and unique components of evolutionary response to the distance gradient from the lake, we found a strong signature of parallel adaptation. Nonparallel divergence was also present, attributable mainly to differences between river locations. We additionally found evidence of genetic substructuring across five lake–stream transitions, indicating that some level of reproductive isolation occurs between populations in these habitats. Strong correlations between pairwise measures of morphological, ecological and genetic distance between lake and stream populations supports the hypothesis that divergent natural selection between habitats drives adaptive divergence and reproductive isolation. Lake–stream stickleback divergence in Lough Neagh provides evidence for the deterministic role of selection and supports the hypothesis that parallel selection in similar environments may initiate parallel speciation.     

Branchial carbonic anhydrase activity and ninhydrin positive substances in the Pacific white shrimp, Litopenaeus vannamei, acclimated to low and high salinities

The Pacific white shrimp, Litopenaeus vannamei, acclimated to 30 ppt salinity, was transferred to either low (15 and 5 ppt), or high (45 ppt) salinity for 7 days. Hemolymph osmolality, branchial carbonic anhydrase activity, and total ninhydrin-positive substances (TNPS) in abdominal muscle were then measured for each condition. Hemolymph osmotic concentration was regulated slightly below ambient water osmolality in shrimp acclimated to 30 ppt. At 15 and 5 ppt, shrimp were strong hyper-osmotic regulators, maintaining hemolymph osmolality between 200 and 400 mOsm above ambient. Shrimp acclimated to 30 ppt and transferred to 45 ppt salinity were strong hypo-osmotic and hypo-ionic regulators, maintaining hemolymph osmolality over 400 mOsm below ambient. Branchial carbonic anhydrase (CA) activity was low (approximately 100 micromol CO(2) mg protein(-1) min(-1)) and uniform across all 8 gills in shrimp acclimated to 30 ppt, but CA activity increased in all gills after exposure to both low and high salinities. Anterior gills had the largest increases in CA activity, and levels of increase were approximately the same for low and high salinity exposure. Branchial CA induction appears to be functionally important in both hyper- and hypo-osmotic regulations of hemolymph osmotic concentrations. Abdominal muscle TNPS made up between 19 and 38% of the total intracellular osmotic concentration in shrimp acclimated to 5, 15, and 30 ppt. TNPS levels did not change across this salinity range, over which hemolymph osmotic concentrations were tightly regulated. At 45 ppt, hemolymph osmolality increased, and muscle TNPS also increased, presumably to counteract intracellular water loss and restore cell volume. L. vannamei appears to employ mechanisms of both extracellular osmoregulation and intracellular volume regulation as the basis of its euryhalinity.     

Global analysis of genes involved in freshwater adaptation in threespine sticklebacks (Gasterosteus aculeatus)

Examples of parallel evolution of phenotypic traits have been repeatedly demonstrated in threespine sticklebacks (Gasterosteus aculeatus) across their global distribution. Using these as a model, we performed a targeted genome scan--focusing on physiologically important genes potentially related to freshwater adaptation--to identify genetic signatures of parallel physiological evolution on a global scale. To this end, 50 microsatellite loci, including 26 loci within or close to (<6 kb) physiologically important genes, were screened in paired marine and freshwater populations from six locations across the Northern Hemisphere. Signatures of directional selection were detected in 24 loci, including 17 physiologically important genes, in at least one location. Although no loci showed consistent signatures of selection in all divergent population pairs, several outliers were common in multiple locations. In particular, seven physiologically important genes, as well as reference ectodysplasin gene (EDA), showed signatures of selection in three or more locations. Hence, although these results give some evidence for consistent parallel molecular evolution in response to freshwater colonization, they suggest that different evolutionary pathways may underlie physiological adaptation to freshwater habitats within the global distribution of the threespine stickleback.     

Contrasting patterns in genetic diversity following multiple invasions of fresh and brackish waters

Biological invasions may combine the genetic effects of population bottlenecks and selection and thus provide valuable insight into the role of such processes during novel environmental colonizations. However, these processes are also influenced by multiple invasions, the number of individuals introduced and the degree of similarity between source and receiving habitats. The amphipod Gammarus tigrinus provides a useful model to assess these factors, as its invasion history has involved major environmental transitions. This species is native to the northwest Atlantic Ocean, although it invaded both brackish and freshwater habitats in the British Isles after introduction more than 65 years ago. It has also spread to similar habitats in Western Europe and, most recently, to Eastern Europe, the Baltic Sea, and the Laurentian Great Lakes. To examine sources of invasion and patterns of genetic change, we sampled populations from 13 native estuaries and 19 invaded sites and sequenced 542 bp of the mitochondrial COI gene. Strong native phylogeographical structure allowed us to unambiguously identify three allopatrically evolved clades (2.3-3.1% divergent) in invading populations, indicative of multiple introductions. The most divergent clades occurred in the British Isles and mainland Europe and were sourced from the St Lawrence and Chesapeake/Delaware Bay estuaries. A third clade was found in the Great Lakes and sourced to the Hudson River estuary. Despite extensive sampling, G. tigrinus did not occur in freshwater at putative source sites. Some European populations showed reduced genetic diversity consistent with bottlenecks, although selection effects cannot be excluded. The habitat distribution of clades in Europe was congruent with the known invasion history of secondary spread from the British Isles. Differences in salinity tolerance among lineages were suggested by patterns of habitat colonization by different native COI clades. Populations consisting of admixtures of the two invading clades were found principally at recently invaded fresh and brackish water sites in Eastern Europe, and were characterized by higher genetic diversity than putative source populations. Further studies are required to determine if these represent novel genotypes. Our results confirm that biological invasions need not result in diminished genetic diversity, particularly if multiple source populations, each with distinctive genetic composition, contribute to the founding populations.