Isolation and characterization of eight polymorphic microsatellite markers from pink conch (Strombus gigas)

Many marine organisms have pelagic larvae, and these are often important agents of dispersal. The larval phase and the multiple paternity that occur in marine gastropods such as Strombus gigas are crucial for the success of this species throughout the Caribbean Sea. To analyse these factors, we developed eight microsatellite loci specific to S. gigas. On the same set of individuals, the microsatellite loci exhibited a greater level of polymorphism than previously studied allozyme markers and thus, will permit fine‐scale analysis and larval pool studies.     

Genetic diversity of Typha latifolia (Typhaceae) and the impact of pollutants examined with tandem-repetitive DNA probes

Genetic diversity at variable-number-tandem-repeat (VNTR) loci was examined in the common cattail, Typha latifolia (Typhaceae), using three synthetic DNA probes composed of tandemly repeated “core” sequences (GACA, GATA, and GCAC). The principal objectives of this investigation were to determine whether: (1) the previously reported almost complete lack of polymorphism at allozyme loci in this species was indicative of a reduced amount of genetic diversity at VNTR loci as well; (2) VNTR markers were informative about possible clonal propagation; and (3) significant differences in genetic structure of sampling sites were associated with differences in environmental levels of pollutants at those sites. Previously, widespread sampling across the eastern United States, surveying across ten allozyme loci, has detected only two genotypes, involving a difference at a single locus, among 104 populations. In this study, the amount of genetic diversity detected at VNTR loci: (1) among ramets (N = 40; 40 genotypes detected) collected at ∼8-km intervals along a 320-km transect; (2) among ramets (N = 220; 117 genotypes detected) from five study sites separated by 50–3000 m; and (3) even among ramets within each study site [N = 44 per site; from 13 to 34 genotypes detected per site (270 m²)] exceeds that previously found in those more geographically widespread allozyme surveys. Among the 260 ramets analyzed here, the mean number of bands scored per individual was 48.61 (SD = 2.80). Mean genetic similarity among ramets collected along the 320-km transect was 0.91, which was within the range of mean genetic similarity within the five study sites (range: 0.89–0.95). Among the five study sites, 61% of the samples analyzed appeared to be clonal ramets, with up to 12 clones detected for 44 ramets sampled within a site. Clones grew intermingled and ranged up to 39 m in extent. Permutation tests of genetic similarity revealed significant genetic differentiation between each of the five study sites. Consistent with the previous allozyme studies, T. latifolia was characterized by extremely low genetic variation relative to levels of polymorphism detected at VNTR loci in other plant species. Estimated heterozygosity among ramets along the 320-km transect ranged from 0.11 to 0.13, while that within the five study sites ranged from 0.05 to 0.12. Estimates of F_st (0.32–0.41) also indicated considerable genetic subdivision among these stands. Significantly higher genetic diversity was detected at the two study sites that chemistry and toxicity data indicate to be the most severely impacted by pollutants. Although this correlation does not establish cause and effect, the results of this study indicate that the analysis of genetic diversity at VNTR loci may be a useful tool for monitoring anthropogenic-induced changes in the genetic structure of natural populations of plants.     

Allozyme Loci Selection in the Pacific Mussel Mytilus trossulus under Stressful Conditions

The allozyme variability for 15 loci in two samples of the Pacific mussel Mytilus trossulus collected from a single giant cluster was investigated using the method of gel electrophoresis. One sample was subjected to short-term anaerobic stress and then to a longer aerobic stress, leading to the death of about 85% of the individuals. At some of the loci, significant differences in the genotypic and allele frequencies were found between the samples. The results are suggestive of the differential survival of mussels with different genotypes and alleles at some of the surveyed loci under stress. Our data are in agreement with the hypothesis of the adaptive significance of allozyme polymorphism.     

Allozyme diversity among local populations of the pacific mussel <Emphasis Type="Italic">Mytilus trossulus</Emphasis> (Bivalvia: Mytilidae) from polluted areas of Peter the Great Bay (Sea of Japan)

Allozyme variability was assessed, using starch gel electrophoresis, at 15 polymorphic loci in two samples of the Pacific mussel Mytilus trossulus collected from local populations in heavily (Golden Horn Bay) and slightly polluted areas (Sukhoputnaya Bay) of Peter the Great Bay. Significant differences between samples were found in the genotypic frequencies at nine loci and in allele frequencies, at six loci. The results are suggestive of the differential survival of individuals having different genotypes and alleles at some of the surveyed loci under conditions of pollution. Our data are not in conflict with the hypothesis of the adaptive significance of allozyme polymorphism.     

Microsatellites for the net‐spinning caddisfly Plectrocnemia conspersa (Polycentropodidae)

Six polymorphic microsatellites were developed for the caddisfly Plectrocnemia conspersa from two enriched partial genomic libraries. These represent the first microsatellites published for the order Trichoptera. We show that, whilst it is possible to develop a highly enriched library, isolation of polymorphic microsatellites is difficult for this species, as has been found in some other of invertebrate groups. The genotypes of 160 individuals were determined using these new loci. Observed heterozygosities ranged from 0.013 to 0.788. Despite their problematic isolation, polymorphic microsatellite loci show greater levels of variation than previously studied allozyme markers.     

The effects of diet and physiological stress on the evolutionary dynamics of an enzyme polymorphism

In the northern acorn barnacle Semibalanus balanoides, polymorphism at the mannose-6-phosphate isomerase (Mpi) locus appears to be maintained by distinct selection regimes that vary between intertidal microhabitats. The goal of the present experiment was to elucidate the mechanism of selection at the Mpi locus by examining the relationship between genotype and fitness-related life-history traits in laboratory manipulations. When barnacles were cultured on a mannose-supplemented diet and exposed to thermal stress, different Mpi genotypes exhibited differences in the rate of growth that predicted survivorship. In contrast, no such relationship was observed in control or fructose-supplemented dietary treatments either in the presence or in the absence of stress. Similarly, the phenotype and survivorship of genotypes at another allozyme locus and a presumably neutral mitochondrial DNA marker were homogeneous across all treatments and unaffected by experimental manipulations. These results suggest that the differential survivorship of Mpi genotypes in the field and laboratory results from a differential ability to process mannose-6-phosphate through glycolysis. The widespread polymorphism at Mpi observed in marine taxa may reflect the interaction between dietary composition and environmental heterogeneity in intertidal habitats.     

STRUCTURE OF AN ASYMMETRIC HYBRID ZONE BETWEEN TWO WATER STRIDER SPECIES (HEMIPTERA: GERRIDAE: LIMNOPORUS)

The water stricter species Limnoporus dissortis and L. notabilis hybridize across a broad zone in western Canada. Body length and alleles at four allozyme loci show a steep cline along the east slope of the Rocky Mountains in western Alberta, while in central British Columbia the parental phenotypes coexist without merging fully. One sex-linked locus shows little introgression, while there is apparently considerable gene flow at three autosomal loci. Although the hybrid zone has characteristics of a broad tension zone, the spatial distribution of introgression suggests that habitat patchiness and differential habitat associations of the two species also contribute to the pattern of hybridization. Asymmetry in interspecific mating success and incompatibilities of sex chromosomes with each other or with cytoplasmic factors appear to account for the occurrence of L. dissortis genotypes within the range of L. notabilis, and the lack of L. notabilis genotypes within the range of L. dissortis. The genetic structure of this hybrid zone supports the importance of sex-linked traits in maintaining the integrity of species, while its spatial structure suggests that extrinsic habitat features can combine with intrinsic genetic incompatibilities to produce complex hybrid interactions.     

Isolation and characterization of highly polymorphic microsatellite markers from the marine invasive species Crepidula fornicata (Gastropoda: Calyptraeidae)

Many marine organisms have pelagic larvae and these are often important agents of dispersal and establishment of exotic species. The larval phase and the hermaphroditic reproductive system of the invasive marine gastropod Crepidula fornicata are crucial in determining the success of this species along the European coasts. To analyse these factors, we developed seven microsatellite loci specific to C. fornicata. On the same set of individuals, the microsatellite loci exhibited a greater level of polymorphism than previously studied allozyme markers and, thus, will permit fine‐scale analysis and larval pool studies.     

Protein polymorphisms in six species of the genus Calanus

Isoelectric focusing was used to study total protein patterns and allozyme variations of six species of the genus Calanus. Each species could be characterized by total protein patterns. The results of the allozyme study indicated, in agreement with previous morphological studies, that the six Calanus species belong to three different groups: the C. finmarchicus group C. finmarchicus, C. glacialis and C. marshallae the C. helgolandicus group (C. helgolandicus, C. pacificus), and C. hyperboreus, which stands apart. There is no indication that there are more loci coding for the proteins studied in species with larger genome sizes. Nor is the degree of enzyme polymorphism related to genome size in these species.     

Intraspecific Genetic Diversity in the Marine Shrimp Penaeus vannamei: Multiple Polymorphic Elongation Factor-1α Loci Revealed by Intron Sequencing

Intron sequences from the elongation factor-1α (EF1α) gene from the marine shrimp Penaeus vannamei reveal extensive variation even among inbred populations of hatchery-raised shrimp. Among 44 individuals analyzed, we found 13 alleles varying by up to 7.5% sequence differences, and including several allele-diagnostic insertions and deletions. High heterozygosity contrasts with low genetic variation at allozyme loci, but we observed up to four alleles per individual, suggesting that we have identified two separate, polymorphic loci. We partitioned the observed alleles into two groups representing hypothetical duplicated loci. However, the alleles are so similar to one another that a phylogenetic analysis does not cluster them into monophyletic groupings. A possible explanation is that concerted evolution is acting to homogenize genetic variation among these two putative loci. Received July 17, 1998; accepted November 23, 1998.     

Genetic distinctness of three widespread and morphologically variable species of Drupella (Gastropoda,Muricidae)

Corallivorous gastropods of the genus Drupella have caused considerable damage to corals at widely separated reefs in the Indo-Pacific. Morphological variability of Drupella species within and between areas has caused taxonomic confusion. To clarify the relationships, we examined allozyme variation at 16 gene loci in samples from Western Australia, Queensland and Japan. Within sites, the species D. cornus, D. rugosa and D. fragum were distinguishable individually by each of 9 to 11 loci, with average genetic identities of about 0.25. The differences extended across sites, whereas the conspecific genetic identities over distances up to 6000 km were 0.86 to 1.00, supporting the view that there are three widespread species of Drupella. Nevertheless, there is much variation within species for allozymes, size, shape and colour.     

Extreme morphological diversity between populations of Littorina obtusata (L.) from Iceland and the UK

Marine gastropods which do not disperse larvae in the plankton exhibit a relatively high degree of interpopulation morphological variability. This phenomenon has been the root of considerable taxonomic confusion, particularly in the Littorinidae. In the present study, specimens of Littorina obtusata from the UK were compared morphologically and genetically with two samples of a northern high-spired form from Iceland which has been referred to by some workers as Littorina palliata. A multivariate discriminant function analysis based on three measured shell dimensions clearly separated the three samples, correctly predicting the origin of shells on the basis of morphology alone in 96% of cases. However, genetic analysis revealed that the most distant relationship, based on allozyme data at 13 loci, was surprisingly close (Nei's I = 0.983) providing no evidence to suggest that L. palliata is not conspecific with L. obtusata.     

Adaptation at Specific Loci. III. Field Behavior and Survivorship Differences among Colias Pgi Genotypes Are Predictable from IN VITRO Biochemistry

Previous work on the phosphoglucose isomerase (PGI) polymorphism of Colias butterflies led to predictions concerning aspects of differential survivorship and fecundity among the polymorphic genotypes in the wild. Explicit assumptions underlying these predictions were that functional differences among genotypes at the in vitro biochemical level reflected roughly corresponding differences in vivo, and that the interaction of such differences with the thermal dependence of flight capacity was correctly understood. All those predictions tested were confirmed. We now report experimental designs for testing three more of these predictions. They concern both differential survivorship and the flight activity component of differential fecundity. We find, as predicted: (1) certain heterozygotes, kinetically most effective at low temperature, begin flight earlier in the day than do other genotypes (six replicates); (2) among the three most common genotypes, the order of kinetic effectiveness, i.e., 3/4 > 3/3 >> 4/4, is reflected in asymmetric order of heterotic advantage, 3/4 > 3/3 >> 4/4, in time of flight initiation, breadth of flight time and/or overall flight density through the day (six replicates); (3) under high temperature stress, the usual survivorship advantage of kinetically favored genotypes is reversed, and the three most thermally stable genotypes show better survivorship.——These results strengthen further the case for direct natural selection on this locus. Implications for population sampling practices, for studies of the adaptive organization of metabolism, and for studies of the interaction of genetic variation with patterns of environmental variability are discussed.     

Genetic analysis of ecological relevant morphological variability in Plantago lanceolata L.

Summary Morphological variability was analysed in an F₂-generation derived from crosses between two ecotypes of Plantago lanceolata L. Six allozyme loci, localised in five linkage groups, were used as markers. For two marker loci, Got-2 and Gpi-1, segregations did not fit monogenic ratios. In the linkage groups to which these two loci belonged, male sterility genes appeared to be present. In these crosses, male sterility (type 3, as described by Van Damme 1983) may be determined by two recessive loci located in the linkage groups of Got-2 and of Gpi-1. Many correlations of morphological and life history characters with allozyme markers were observed. The quantitative trait loci did not appear to be concentrated in major gene complexes. Often many loci were involved, sometimes with effects opposite to those expected from the population values. Main effects of the linkage groups appeared to be more important than interaction effects in determining variability. It also appeared that there is a positive correlation between the number of heterozygous allozyme loci and generative growth.Grassland Species Research Group Publication No. 115     

Morphological and molecular tools in identifying the Mediterranean limpets Patella caerulea, Patella aspera and Patella rustica

Allozyme electrophoresis, a partial nucleotide sequence of a mitochondrial gene for cytochrome oxidase I (COI) and discriminant analysis of shell morphometric characters were used to study the relationships among the Sicilian marine gastropods of the Patella genus. Allozyme and mtDNA markers unequivocally distinguished the species and were very useful markers in correctly classifying the different species when morphological characters overlapped each other. Several allozyme loci and many nucleotide positions were diagnostic of species. In contrast, the discriminant analysis of simple morphometric shell characters failed to adequately discriminate the species, suggesting that environmental factors influence colouration and morphological patterns in the Patella species. Our results underline the importance of a genetic approach, as compared to a morphological approach, in discriminating the Mediterranean Patella species.     

Genetic heterogeneity among intertidal habitats in the flat periwinkle, Littorina obtusata

Comparisons among patterns exhibited by functionally distinct genetic markers have been widely used to infer the impacts of demography and selection in structuring genetic variation in natural populations. However, such multilocus comparisons remain an indirect evaluation of selection at particular candidate loci; ideally, the identification of a candidate gene by comparative genetic methodologies should be complemented by functional analyses and experimental manipulations of genotypes in the laboratory or field. We examined genotype frequency variation among replicated intertidal habitats at two spatial scales in the grazing snail Littorina obtusata. Both of the candidate allozyme markers varied predictably with environment, and these patterns were consistent at both spatial scales. Three of four reference loci were spatially homogeneous, but one microsatellite exhibited significant structure at both geographical and mesoscales. To initiate a direct examination of whether the observed genotype frequency variation at one of the candidate markers, mannose-6-phosphate isomerase (MPI), was impacted by differential survivorship of genotypes, we conducted a series of laboratory-based thermal stress assays using snails from two geographically disparate source populations. When snails were exposed to bouts of thermal/desiccation stress, patterns of mortality were nonrandom with respect to MPI genotype. Furthermore, patterns of mortality in the laboratory manipulation coincided with the observed distribution of genotypes in the field. The data suggest the operation of selection at the Mpi or a linked locus, but functional studies and further experimentation are required to establish the relationship between MPI genotype and fitness across heterogeneous intertidal environments.     

Antibiosis resistance to cereal leaf beetle,Oulema melanopus (L.) (Coleoptera: Chrysomelidae), in Central Asian wheat germplasm

The cereal leaf beetle, Oulema melanopus (Coleoptera: Chrysomelidae), is a new invasive pest of cereals in western Canada. Host plant resistance is a potential option in the integrated management of this insect. Wheat (Triticum aestivum L.) genotypes are known to possess antixenosis against O. melanopus; however, mechanisms involving antibiosis are relatively less explored. We present results of an investigation exploring antibiotic resistance in wheat germplasm of Central Asian origin with putative resistance to O. melanopus. Our laboratory assessment of development and survivorship of O. melanopus on test genotypes indicated antibiotic properties in four of the six genotypes evaluated. Antibiosis effects were reflected through lower survivorship, extended developmental periods and low adult fitness. However, two genotypes were excellent hosts, and beetles had higher survivorship and fitness on them. The performance of O. melanopus on these two genotypes was comparable to that on the susceptible genotype CDC GO included as a check. The genotypes with antibiotic resistance identified by this research can now serve as sources of genetic resistance for breeding cultivars adapted to western Canadian conditions.     

Peroxisomal proteomic approach for protein profiling in blue mussels (Mytilus edulis) exposed to crude oil

Peroxisomal proteomic protein profiles of exposure to marine pollution have been recently introduced in biomonitoring experiments. However, laboratory experiments to study the independent effect of common pollutants are needed to define a minimal protein expression signature (PES) of exposure to a specific pollutant. The aim of this study was to obtain PESs in blue mussels (Mytilus edulis) exposed to two different crude oil mixtures for future application in biomonitoring areas affected by oil spills. In the study, peroxisome-enriched fractions from digestive gland of M. edulis (L., 1758) were analysed by two-dimensional fluorescence difference electrophoresis (DIGE) and mass spectrometry (MS) after 3 weeks of exposure to crude oil mixtures: crude oil or crude oil spiked with alkylated phenols (AP) and extra polycyclic aromatic hydrocarbons (PAH) in a laboratory flow-through system. A minimal PES composed by 13 protein spots and unique PESs of exposure to the two different mixtures were identified. A total of 22 spots from the two-dimensional maps that had shown a significant increase or decrease in abundance in each of the exposed groups exposed were analysed. The hierarchical clustering analysis succeeded in discriminating the exposed groups from the control groups based on the unique PES. The PESs obtained were consistent with protein patterns obtained in previous field experiments. The results suggest that the protein profiles obtained by peroxisomal proteomics could be used to assess oil exposure in marine pollution assessments.     

DISEQUILIBRIUM BETWEEN DISEASE-RESISTANCE VARIANTS AND ALLOZYME LOCI IN AN ANNUAL LEGUME

Polymorphism existed at 58% of the enzyme loci examined (11/19) in one population of the highly self-pollinated annual legume Amphicarpaea bracteata. Due to extreme gametic disequilibrium among loci, genetic variation in this population was structured into a small number of multilocus genotypes. Over 97% of the plants sampled could be grouped into two classes (biotypes “A” and “B”), each consisting of a few highly similar genotypes. The two classes had mutually exclusive sets of alleles at nine loci. These classes differed sharply in their disease resistance toward one isolate of the specialist fungal pathogen Synchytrium decipiens from their native habitat. All biotype A plants were strongly susceptible, and all biotype B plants were resistant. When plants of both biotypes were exposed to this pathogen in a greenhouse, the resistant biotype (B) exhibited a significantly higher growth rate. The strong association between plant disease-resistance phenotypes and allozyme variants implies that pathogen attack could be a major selective agent influencing the evolution of neutral or near-neutral alleles at enzyme loci in this plant.     

Isolation and characterization of 10 polymorphic microsatellite loci from small yellow croaker (Pseudosciaena polyactis)

Small yellow croaker (Pseudosciaena polyactis) is an economically important marine fish species. About 43 microsatellite loci were isolated from two enriched genomic library of Pseudosciaena polyactis. Ten of these loci were polymorphic in a test population with alleles per locus ranging from two to six, and observed and expected heterozygosities per locus from 0.3750 to 0.8750 and from 0.3112 to 0.8121, respectively. No loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction and no significant linkage disequilibrium was found between pairs of loci. These polymorphic microsatellite loci would be useful for investigating genetic diversity of Pseudosciaena polyactis.