Genetic patterns across multiple introductions of the globally invasive crab genus Carcinus

The European green crab Carcinus maenas is one of the world's most successful aquatic invaders, having established populations on every continent with temperate shores. Here we describe patterns of genetic diversity across both the native and introduced ranges of C. maenas and its sister species, C. aestuarii, including all known non‐native populations. The global data set includes sequences from the mitochondrial cytochrome c oxidase subunit I gene, as well as multilocus genotype data from nine polymorphic nuclear microsatellite loci. Combined phylogeographic and population genetic analyses clarify the global colonization history of C. maenas, providing evidence of multiple invasions to Atlantic North America and South Africa, secondary invasions to the northeastern Pacific, Tasmania, and Argentina, and a strong likelihood of C. maenas × C. aestuarii hybrids in South Africa and Japan. Successful C. maenas invasions vary broadly in the degree to which they retain genetic diversity, although populations with the least variation typically derive from secondary invasions or from introductions that occurred more than 100 years ago.     

Population genetics,gene flow,and biogeographical boundaries of Carcinus aestuarii (Crustacea: Brachyura: Carcinidae) along the European Mediterranean coast

Carcinus aestuarii Nardo, 1847 is a widespread coastal crab species throughout the Mediterranean Sea with a pelagic larval phase. This species tolerates a wide range of environmental conditions and typically inhabits fragmented habitats, such as embayments, lagoons and estuaries. It is therefore a good candidate species for studying and testing different phylogeographical hypotheses in the Mediterranean Sea. By contrast to its Atlantic sister species, Carcinus maenas, studies on the population genetic structure of C. aestuarii in its native range are still scarce. In the present study, specimens from along the European Mediterranean Sea were collected and DNA‐sequenced and analyses were applied to discriminate between present day and historical factors influencing the population genetic structure of this species. The results obtained demonstrate the existence of two genetically distinct geographical groups, corresponding to the eastern and western Mediterranean, with further subdivision within the East Mediterranean Basin. A strong asymmetric gene flow was recorded toward the Eastern Basin, which may play a crucial role in shaping the present day biogeographical patterns of this species and potentially other sympatric ones with pelagic larvae. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109 , 771–790.     

Immunocytochemical identification of structures containing putative red pigment-concentrating hormone in two species of decapod crustaceans

Summary By use of an antiserum raised against the Nterminal sequence pGlu-Leu-Asn-Phe..., common to red pigment-concentrating hormone (RPCH) of Pandalus borealis and three structurally similar insect neuropeptides, putative RPCH-immunopositive structures were revealed in the eyestalks of Carcinus maenas and Orconectes limosus and in the brain and thoracic ganglion (TG) of C. maenas. In the eyestalks, complete neurosecretory pathways were demonstrated, consisting of perikarya, axons and terminals in the neurohemal organ, the sinus gland (SG). In C. maenas approximately 20 small RPCH cells are present as a distinct group adjacent to the medulla terminalis ganglionic X-organ (MTGXO, XO). They are morphologically different from the larger XO perikarya, which contain the crustacean hyperglycemic hormone (CHH). The occurrence of both neuropeptides in distinct neurosecretory pathways was ascertained by immunologic double staining (PAP/gold) or by analysis of consecutive sections. In addition, a group of two to four larger RPCH cells is located in the proximal part of the MT. In O. limosus, RPCH cells are found in the XO. Cells corresponding to the proximal MT cells of C. maenas were not found. In both species, a few more weakly staining immunopositive perikarya were observed in clusters of cell somata of the optic ganglia. It is uncertain whether these are connected to the SG.In the brain of C. maenas, several smaller and three larger perikarya were consistently observed in the dorsal lateral cell somata adjacent to the olfactory lobes. In the optic nerve, two axons that project into the eyestalk were stained. Some axons were also observed in the ventral median neuropil of the brain. In the TG, RPCH cells were found in small numbers in median positions, i.e., in clusters of somata between the ganglia of the appendages.HPLC analysis of the red pigment-concentrating activity from the SG of C. maenas revealed that the retention time of the neuropeptide is similar but not identical to that of Pandalus borealis RPCH.     

The role of the transbranchial potential difference in hyperosmotic regulation of the shore crabCarcinus maenas

When isolated gills of the shore crabCarcinus maenas were bathed and perfused with identical solutions on both sides (50 % sea water), a spontaneous transepithelial potential difference (PD) of some millivolts (hemolymph side negative) was established. This PD is of active nature and requires the metabolism of the living cell, since it uses its own sources of energy in addition to organic nutrients offered in the flow of artificial hemolymph. Addition of sodium cyanide and dinitrophenole to bathing and perfusion medium resulted in reversible breakdown of PDs in a concentration-dependent mode. In posterior gills ofC. maenas, the potential differences were more negative compared to data measured in anterior gills of the same individuals. These results are correlated with higher specific activities of Na-K-ATPase in posterior gills. Experiments with triamterene indicate that sodium uptake inC. maenas is sensitive to this diuretic drug, when applied on the apical side of the epithelial cell. The results obtained show that active uptake of sodium from medium to blood across the gills is performed by a complex mechanism including participation of several basal and apical transport steps.     

Episodic global dispersal in shallow water marine organisms: the case history of the European shore crabs Carcinus maenas and C. aestuarii

Aim This paper evaluates global collection records, evidence of anthropogenic transport methods, and experimental and distributional data relative to temperature requirements to understand the historical and potential dispersal of a well‐known genus of estuarine crab. Location The records analysed are from temperate and tropical coastal ocean areas. Methods The study is based primarily on literature analysis and examination of museum specimens. Results The human‐mediated successful global dispersal of the European shore crabs Carcinus maenas (Linnaeus, 1758) and C. aestuarii (Nardo, 1847) occurred in three major episodes: around 1800, in the 1850s–70s, and in the 1980s–90s. The nineteenth century introductions occurred through transport by ships (probably in hull fouling or in solid ballast), while the introductions in the 1980s could have occurred through a greater variety of dispersal mechanisms (ships’ hull fouling and seawater system fouling; fouling on semisubmersible drilling platforms; ballast water; transport with fisheries products intended for food or bait; scientific research; releases from aquaria maintained for educational or scientific purposes; or intentional non‐governmental releases for human food production). These introductions have resulted in Carcinus’ establishment in five temperate regions outside of its native Europe in Atlantic North America, Australia, South Africa, Japan and Pacific North America, while releases into tropical regions have not established populations. C. maenas’ range in both its native and introduced regions appears to be regulated by similar temperature parameters, enabling an assessment of its potential distribution. Main conclusions The second episode of Carcinus’ global dispersal, the period from the 1850s to 1870s, may be part of a broader surge of world‐wide invasions caused by an increase in shipping.     

GENETIC DIFFERENTIATION IN THE FLORIDA SUBSPECIES OF HELIANTHUS DEBILIS (ASTERACEAE)

Techniques of horizontal starch gel electrophoresis were employed to estimate levels of genetic variation within and genetic differentiation among populations of the three Florida subspecies of Helianthus debilis. The subspecies are H. d. debilis, H. d. vestitus, and H. d. tardiflorus. These taxa are very similar with respect to levels of genetic variability. The average values across all populations for proportion of polymorphic loci (25.5%) and number of alleles per polymorphic locus (2.35) are comparable to those found in other outcrossing plants. The average frequency of heterozygotes per locus (0.05) is lower than that found in other outcrossers. Local populations within each subspecies are genetically very similar. The average genetic distance between local populations is D = 0.010 ± 0.001. Subspecies vestitus and subspecies tardiflorus are also genetically very similar (D = 0.015 ± 0.001). However, subspecies debilis is differentiated from both subspecies vestitus (D = 0.121 ± 0.006) and subspecies tardiflorus (D = 0.103 ± 0.005). This genetic differentiation parallels morphological differentiation. The average genetic distance among all three subspecies is D = 0.080 ± 0.003. A moderate amount of genetic differentiation accompanies the process of subspeciation in Helianthus debilis.     

SMi biomarkers summit, 2–3 February 2009, London,UK

As part of a programme to develop biomarker assays for polycyclic aromatic hydrocarbons (PAHs) in marine invertebrates, two species of crabs, Carcinus maenas and Carcinus aestuarii were exposed to benzo(a)pyrene (B(a)P) or crude oil. Microsomes were prepared from the midgut gland (hepatopancreas), examined by gel electrophoresis and Western blotting and assayed for B(a)P monooxygenase activity. In early experiments there was evidence of protein degradation and results were inconsistent and inconclusive. However, when steps were taken to minimize this in subsequent experiments, including the inclusion of four protease inhibitors in the homogenization buffer, there was consistent evidence for an increase of proteins of estimated molecular weight 45-60 kDa, and particularly of a distinct band at c. 48 kDa, following exposure to PAH at levels down to 0.1 ppm in ambient water. In C. aestuarii the increase in this band was found to coincide with an 8-12-fold increaseof B(a)P monooxygenase activity in midgut gland microsomes. These results suggest that one or more forms of cytochrome P450 may be induced by PAHs in these species. However, Western blotting using antibodies raised to vertebrate P450s, and representing four different gene families, failed to recognize any proteins in either the PAH-treated samples or in the controls. The isolation and characterization of induced protein, and the production of antibodies may provide the basis for a biomarker assay to measure a response to environmental PAHs in crabs.     

Development and Application of Microsatellites in Carcinus maenas: Genetic Differentiation between Northern and Central Portuguese Populations

Carcinus maenas, the common shore crab of European coastal waters, has recently gained notoriety due to its globally invasive nature associated with drastic ecological and economic effects. The native ubiquity and worldwide importance of C. maenas has resulted in it becoming one of the best-studied estuarine crustacean species globally. Accordingly, there is significant interest in investigating the population genetic structure of this broadly distributed crab along European and invaded coastlines. Here, we developed polymerase chain reaction (PCR) primers for one dinucleotide and two trinucleotide microsatellite loci, resulting from an enrichment process based on Portuguese populations. Combining these three new markers with six existing markers, we examined levels of genetic diversity and population structure of C. maenas in two coastal regions from Northern and Central Portugal. Genotypes showed that locus polymorphism ranged from 10 to 42 alleles (N = 135) and observed heterozygosity per locus ranged from 0.745 to 0.987 with expected heterozygosity ranging from 0.711 to 0.960; values typical of marine decapods. The markers revealed weak, but significant structuring among populations (global F_ST = 0.004) across a 450 km (over-water distance) spatial scale. Combinations of these and existing markers will be useful for studying population genetic parameters at a range of spatial scales of C. maenas throughout its expanding species range.     

The ultrastructure of nerve endings containing pigment-dispersing hormone (PDH) in crustacean sinus glands: Identification by an antiserum against a synthetic PDH

Summary A high-liter antiserum has been obtained from two rabbits immunized with a glutaraldehyde conjugate of synthetic pigment-dispersing hormone (PDH) from Uca pugilator and bovine thyroglobulin. The antiserum blocked melanophore-dispersing activity of the peptide in vivo. In sinus glands (SG) of Carcinus maenas, Cancer pagurus, Uca pugilator and Orconectes limosus, electron-microscopic immunocytochemistry revealed sparsely distributed axon endings containing a distinct PDH-immunoreactive type of neurosecretory granules (diameter 90–130 nm). Exocytotic figures indicating release of the content of these granules into hemolymph lacunae were occasionally observed. Preservation of fine structure and antigenicity of the PDH granules were markedly dependent on the fixation procedure used. A preliminary experiment with C. maenas showed that preterminal axon dilatations near the basal lamina seemed to accumulate PDH-granules when animals were kept in complete darkness for three days. Immunodot blotting of fractions after high pressure liquid chromatography (HPLC) of extracts from SGs of C. maenas and O. limosus revealed a strongly immunoreactive substance at a retention time very similar to those of synthetic PDHs of Uca pugilator and Pandalus borealis. It is also coincident with a zone of biological activity. Thus, the antigen demonstrated by immunocytochemistry is identical or very similar to one of the known PDHs.     

Allozyme diversity in Chinese,Seguin and American chestnut (Castanea spp.)

Allozyme genetic variability in three chestnut (Castanea) species was investigated using 19 loci from ten enzyme systems. G-tests of heterogeneity of isozymic allele distribution showed significant differences between the three species at 15 of the 19 loci, and between the 13 C. mollissima populations at 13 of the 19 loci examined. C. mollissima was found to possess a significantly-higher value of mean gene heterozygosity (H=0.3050±0.0419), the percentage of polymorphic loci (P=84.21%) and the average number of alleles per locus (A=2.05), than any other species in the Castanea section Eucastanon. When the genetic variability of populations of C. mollissima from four regions in China was investigated, the population from the Changjiang river region showed a markedly higher mean gene heterozygosity (H=0.3480±0.0436) than populations from the other regions. Genetic relationships among the four regions were assessed by Nei's genetic identity I and standard genetic distance D. An approximately-identical distance between the population from the Changjiang river region and populations from the three other regions was observed, while populations from the latter regions showed almost the same genetic distance from each other. These data, when considered with information existing prior to this study, contribute to an understanding of the possible origin and progenitor of the chestnut species.     

A hitchhiker’s guide to the Maritimes: anthropogenic transport facilitates long‐distance dispersal of an invasive marine crab to Newfoundland

Aim To determine timing, source and vector for the recent introduction of the European green crab, Carcinus maenas (Linnaeus, 1758), to Newfoundland using multiple lines of evidence. Location Founding populations in Placentia Bay, Newfoundland, Canada and potential source populations in the north‐west Atlantic (NWA) and Europe. Methods We analysed mitochondrial and microsatellite genetic data from European and NWA populations sampled during 1999–2002 to determine probable source locations and vectors for the Placentia Bay introduction discovered in 2007. We also analysed Placentia Bay demographic data and shipping records to look for congruent patterns with genetic analyses. Results Demographic data and surveys suggested that C. maenas populations are established and were in Placentia Bay for several years (c. 2002) prior to discovery. Genetic data corroboratively suggested central/western Scotian Shelf populations (e.g., Halifax) as the likely source area for the anthropogenic introduction. These Scotian Shelf populations were within an admixture zone made up of genotypes from both the earlier (early 1800s) and later (late 1900s) introductions of the crab to the NWA from Europe. Placentia Bay also exhibited this mixed ancestry. Probable introduction vectors included vessel traffic and shipping, especially vessels carrying ballast water. Main conclusions Carcinus maenas overcame considerable natural barriers (i.e., coastal and ocean currents) via anthropogenic transport to become established and abundant in Newfoundland. Our study thus demonstrates how non‐native populations can be important secondary sources of introduction especially when aided by human transport. Inference of source populations was possible owing to the existence of an admixture zone in central/western Nova Scotia made up of southern and northern genotypes corresponding with the crab’s two historical introductions. Coastal vessel traffic was found to be a likely vector for the crab’s spread to Newfoundland. Our study demonstrates that there is considerable risk for continued introduction or reintroduction of C. maenas throughout the NWA.     

Phylogeography of the white-throated dipper Cinclus cinclus in Europe

Cytochrome b gene sequence variation (902 bp) was used to investigate phylogenetic relationships and genetic differentiation among the four subspecies of European white-throated dipper Cinclus cinclus (Aves; Cinclidae). Six cytochrome b haplotypes were revealed, the most common one (H 13–4) was shared by all seven populations studied. Central European C. c. aquaticus was genetically more diverse than the other subspecies (although not significantly so, due to small sample sizes), whereas our sample of the nominate subspecies from the French Pyrenees showed no haplotype variation. Pairwise comparisons of the four subspecies showed significant genetic differentiation between the taxa except for a lack of differentiation of C. c. hibernicus versus C. c. aquaticus and C. c. cinclus. The genetic structure and the phylogeographic pattern were not related to the geographic distribution of the four subspecies thus disputing the validity of the current infraspecific taxonomy of C. cinclus at least in Europe.Béatrice Lauga and Christine Cagnon contributed equally to this paper.     

Stimulus selection (light) in four subspecies of deermice (Peromyscus maniculatus)

Abstract

Four subspecies of deermice (Peromyscus maniculatus) were selected from habitats along a gradient from mesic hardwood forests (gracilis), tall grass prairie (bairdi), short grass prairie (nebrascensis), to desert grassland (labecula) in effort to relate the light preferences of the mice to the habitat of their origin. Six individuals of each subspecies were maintained in dark chambers for 25 days, during which they could turn on a dim light for 30 seconds on ten of these days. Mice from the mesic habitats turned the lights on more frequently (light reinforcement) than mice from xeric habitats. The same subspecies that exhibited light reinforcement when raised in laboratory light conditions failed to show light reinforcement when raised in total darkness. The circadian periodicities also differed significantly among the subspecies. Conclusions drawn from the results were: (1) selection for light is genetically influenced as indicated by subspecific differences, (2) some genotypes are readily modified by environmental light conditions than others, and (3) natural selection in each environment appears to contribute to the degree of genetic fixation or genetic plasticity.     

Biochemical markers in rats: Linkage relationships of aconitase (Acon-1), aldehyde dehydrogenases (Ahd-2 and Ahd-c), alkaline phosphatase (Akp-1), and hydroxyacid oxidase (Hao-1)

We have examined the linkage relationships between five biochemical markers, Acon-1, Ahd-2, Ahd-c, Akp-1, and Hao-1, and 19 other genetic loci in five breeding combinations. The genetic locus that codes for a recently described aldehyde dehydrogenase in the liver (Ahd-c) has been assigned to linkage group X (LG X). Hydroxyacid oxidase is coded for by a locus (Hao-1) that is linked to genes that encode agouti coat color and seminal vesicle proteins in linkage group IV. Alkaline phosphatase (Akp-1) was linked to the locus that encodes the C6 component of complement and this association provisionally defines a new linkage group (LG XI) in the rat. The locus Acon-1 could not be positively assigned to a specific linkage group but the results from one breeding combination suggest that this locus may be included in linkage group II. No linkage relationship could be detected for the aldehyde dehydrogenase coded for by Ahd-2.This work was supported by Grant GM 32580 from the National Institutes of Health, United States Public Health Service.     

European green crabs (Carcinus maenas) in the northeastern Pacific: genetic evidence for high population connectivity and current-mediated expansion from a single introduced source population

Aim The European green crab (Carcinus maenas) expanded dramatically after its introduction to the west coast of North America, spreading over 1000 km in < 10 years. We use samples of Carcinus maenas collected over time and space to investigate the genetic patterns underlying the species’ initial establishment and spread, and discuss our findings in the context of the species’ life history characteristics and demography. Location The central west coast of North America, encompassing California, Oregon, and Washington (USA) and British Columbia (Canada). Methods We collected 1040 total samples from 21 sites representing the major episodes of population establishment and expansion along the west coast of North America. Microsatellite markers were used to assess genetic diversity and structure at different time points in the species’ spread, to investigate connectivity between embayments and to estimate both short‐term effective population sizes and the number of original founders. Assignment testing was performed to determine the likely source of the introduction. Results Carcinus maenas in western North America likely derived from a single introduction of a small number of founders to San Francisco Bay, CA from the east coast of North America. Throughout its western North American range, the species experiences periodic migration between embayments, resulting in a minor loss of genetic diversity in more recently established populations versus the populations in the area of initial establishment. Main conclusions Low genetic diversity has not precluded the ability of C. maenas to successfully establish and spread on the west coast of North America. An efficient oceanographic transport mechanism combined with highly conducive life history traits are likely the major drivers of C. maenas spread. Evidence for a single introduction underscores the potential utility of early detection and eradication of high‐risk invasive species.     

Polymorphic microsatellite loci for tiger salamanders,Ambystoma tigrinum

Microsatellites have been developed for few amphibian species. However, developing genetic markers for population genetic studies in amphibians is critical because amphibians are declining globally. The tiger salamander, Ambystoma tigrinum, is widespread throughout the United States and includes the endangered subspecies, A. t. stebbinsi. We present primers and amplification conditions for 10 polymorphic microsatellite loci that have produced successful results in three subspecies of A. tigrinum. Number of alleles per locus ranged from one to 11 and heterozygosity ranged from 0 to 0.815 depending on the subspecies and locus analysed. These markers should prove useful for future studies of genetic diversity and population subdivision.     

Biotic resistance experienced by an invasive crustacean in a temperate estuary

Communities high in species diversity tend to be more successful in resisting invaders than those low in species diversity. It has been proposed that the biotic resistance offered by native predators, competitors and disease organisms plays a role. In Yaquina Bay, Oregon, we observed very little overlap in the distribution of the invasive European green crab, Carcinus maenas, and the larger red rock crab, Cancer productus. C. productus dominates the more saline, cooler lower estuary and C. maenas, the less saline, warmer upper estuary. Because caged C. maenas survive well in the lower estuary, we decided to test the hypothesis that C. productus prey on C. maenas and thus contribute to their exclusion from the more physically benign lower estuary. A laboratory species interaction experiment was designed to determine whether C. productus preys on smaller C. maenas at a higher rate than on smaller crabs of their own species. Crabs of both species were collected and sorted by weight into three size classes: small, medium and large. Small and medium crabs of both species were paired with C. maenas and C. productus of various sizes. When conspecifics were paired, mortality was less than 14%, even in the presence of larger crabs. Smaller C. productus survived well in the presence of larger C. maenas, but the reverse was not true. When small C. maenas (60–67 mm carapace width) were matched with medium and large C. productus, their mortality increased to 52% and 76%, respectively. A less dramatic pattern was observed for medium C. maenas (73–80 mm) in the presence of medium and large C. productus. Thus on the West Coast of North America, the more aggressive red rock crab, C. productus, has the potential to reduce the abundance of C. maenas in the more saline and cooler lower estuaries.     

Polymorphic microsatellite markers for the goosander (Mergus merganser)

To investigate the population genetic structure of the goosander (Mergus merganser) in Europe and to identify populations with a significant conservation value, we isolated nine microsatellite loci, and screened them in the subspecies Mergus merganser merganser and Mergus merganser americanus. All markers were polymorphic with two to 15 alleles per locus. Average observed and expected heterozygosity values were 0.422 and 0.624, respectively, for the European and 0.497 and 0.667, respectively, for the North American subspecies. Only one marker departed significantly from Hardy–Weinberg expectations in both subspecies. This marker was highly variable but homozygous in all females, suggesting a sex‐linked inheritance.     

Genetic variation in the midcontinental population of sandhill cranes,Grus canadensis

Three subspecies of sandhill crane (Grus canadensis) are recognized in the Midcontinental population, the lesser (Grus c. canadensis), Canadian (G. c. rowani), and greater (G. c. tabida). Blood samples collected on the population's primary spring staging area in Nebraska, U.S.A., were used to resolve the genetic relationship among these subspecies. Phylogenetic analysis of 27 G. canadensis, by DNA sequencing of a 675 bp region of the mtDNA, supports the subspecies designations of G. c. canadensis and G. c. tabida. G. c. rowani individuals were intermediate with each of the other two subspecies. Genetic divergence ranged from 6.5 to 14.5% between G. c. canadensis and G. c. tabida, 0.5 to 6.6% within G. c. canadensis, and 0.1 to 6.0% within G. c. tabida. Sufficient DNA for analysis was obtained from shed feathers indicating a source of genetic material that does not require the capture or sacrifice of the birds. Other genetic markers and methods, including satellite telemetry, are required for obtaining detailed information on crane distributions as needed to establish effective management units for the MCP.