Isolation of microsatellite loci from the scleractinian corals,Montastraea cavernosa and Porites astreoides

The ability to assess genetic variation is critical for determining genetic diversity and population structure. In corals, slow evolutionary rates in mitochondrial genomes have left allozymes as the only markers presently available to investigate patterns of intraspecific genetic variation. Characteristics of microsatellites render them more informative than allozymes for such analyses; however, few coral microsatellites are available. This study describes polymorphic microsatellite loci isolated from two scleractinian coral species. Most loci exhibit significant heterozygote deficiencies, likely due to nonrandom mating or Wahlund effects. These markers are being used to investigate gene flow among populations, providing insight into reef connectivity.     

The genetic structure of endangered populations in the Cranberry Fritillary,Boloria aquilonaris (Lepidoptera,Nymphalidae): RAPDs vs allozymes

The genetic population structure of the Cranberry Fritillary Boloria aquilonaris was studied using both RAPDs (random amplified polymorphic DNA) and allozymes. In Belgium, B. aquilonaris has a naturally fragmented distribution that has been accentuated due to human activity during the last century. The genetic population structure of this butterfly was analysed at the regional (several Ardenne uplands) and at the landscape level (several populations within an Ardenne upland). Both population genetic markers confirmed results from a previous CMR study at the landscape scale. At the regional scale however, important incongruences were observed between RAPDs and allozymes. The average gene diversity for the RAPD data was twice that of the allozyme data. The degree of population subdivision was also much greater for RAPDs than for allozymes. The UPGMA clusters produced by each of these markers differed significantly. We believe that, given the higher rate of mutation of RAPDs and the greater number of loci assayed by this method, RAPDs reveal a more accurate and recent population genetic structure than allozymes.     

Concordance of genetic divergence among sockeye salmon populations at allozyme, nuclear DNA, and mitochondrial DNA markers

Abstract.— We examined genetic variation at 21 polymorphic allozyme loci, 15 nuclear DNA loci, and mitochondrial DNA in four spawning populations of sockeye salmon ( Oncorhynchus nerka ) from Cook Inlet, Alaska, to test for differences in the patterns of divergence among different types of markers. We were specifically interested in testing the suggestion that natural selection at allozyme loci compromises the effectiveness of these markers for describing the amount and patterns of gene flow among populations. We found concordance among markers in the amount of genetic variation within and among populations, with the striking exception of one allozyme locus ( sAH ), which exhibited more than three times the amount of among-population differentiation as other loci. A consideration of reports of discordance between allozymes and other loci indicates that these differences usually result from one or two exceptional loci. We conclude that it is important to examine many loci when estimating genetic differentiation to infer historical amounts of gene flow and patterns of genetic exchange among populations. It is less important whether those loci are allozymes or nuclear DNA markers.     

Development of microsatellite markers as a molecular tool for conservation studies of the Mediterranean reef builder coral Cladocora caespitosa (Anthozoa, Scleractinia)

Cladocora caespitosa is a reef-building zooxanthellate scleractinian coral in the Mediterranean Sea. Mortality events have recurrently affected this species during the last decade. Thus, knowledge of its genetic structure, population diversity, and connectivity is needed to accomplish suitable conservation plans. In order to obtain a better understanding of the population genetics of this species, 13 highly variable microsatellites markers were developed from a naturally bleached colony. The developed primers failed to amplify zooxanthella DNA, isolated from C. caespitosa, verifying that these markers were of the coral and not algal symbiont origin. The degree of polymorphism of these loci was tested on tissue samples from 28 colonies. The allele number for each loci ranged from 2 to 13 (mean N(a) = 5.4), with an average observed heterozygosity of 0.42 (H(e) = 0.43) and all loci were in Hardy-Weinberg equilibrium. These new markers should be useful in future conservation genetic studies and will help to improve the resolution of the individual identification within this coral species. Primers were also tested in Oculina patagonica, with successful amplifications of several loci.     

Diversity of symbiotic algae of the genus Symbiodinium in scleractinian corals of the Xisha Islands in the South China Sea

Symbiotic algae （Symbiodinium sp.） in scleractinian corals are important in understanding how coral reefs will respond to global climate change. The present paper reports on the diversity of Symbiodinium sp. in 48 scleractinian coral species from 25 genera and 10 families sampled from the Xisha Islands in the South China Sea, which were identified with the use of restriction fragment length polymorphism （RFLP） of the nuclear ribosomal DNA large subunit gene （rDNA）. The results showed that： （i） Symbiodinium Clade C was the dominant zooxanthellae in scleractinian corals in the Xisha Islands; （ii） Symbiodinium Clade D was found in the corals Montipora aequituberculata, Galaxea fascicularis, and Plerogyra sinuosa; and （iii） both Symbiodinium Clades C and D were found simultaneously in Montipora digitata, Psammocora contigua, and Galaxeafascicularis. A poor capacity for symbiosis polymorphism, as uncovered by RFLP, in the Xisha Islands indicates that the scleractinian corals have low adaptability to environmental changes. Further studies are needed to investigate zooxanthellae diversity using other molecular markers.     

Regional genetic differentiation among northern high-latitude island populations of a broadcast-spawning coral

Knowledge of genetic connectivity is useful for understanding of the recovery potential of coral populations after various disturbances, such as coral mass bleaching. Population genetic studies in corals are mostly restricted to Australian and Caribbean species; studies in the northern Pacific are relatively limited. Using microsatellite markers, the population genetics of Acropora sp. 1 was examined between two regions in Japan, the Okinawa-Aka and Bonin Islands, which are separated by approximately 1,500 km of open water in a high-latitude area. Statistically significant but small genetic differentiation in Acropora sp. 1 was detected between and within these regions. Genetic diversity was not obviously reduced in populations of the Bonin Islands, which are relatively isolated. Thus, some level of connectivity appears to be maintained between the two regions, likely because of the high dispersal ability of this broadcast spawner.     

Differentiation between natural and cultivated populations of Medicago sativa (Leguminosae) from Spain: analysis with random amplified polymorphic DNA (RAPD) markers and comparison to allozymes

The conservation of a crop's wild relatives as genetic resources requires an understanding of the way genetic diversity is maintained in their populations, notably the effect of crop-to-wild gene flow. In this study, the amount of differentiation between natural and cultivated populations of Medicago sativa was analysed using random amplified polymorphic DNA (RAPD) markers and an extension of the AMOVA procedure adapted to autotetraploid organisms. Simulations of structured populations were performed to test whether AMOVA provides estimates of population structure in autotetraploids that can be directly compared to those obtained for allozyme data. Simulations showed that straight phi-statistics allow a good estimation of population differentiation when unbiased allelic frequencies are used to correct the conditional expectations of squared genetic distances. But such unbiased estimates can not be practically guaranteed, and population structure is notably overestimated when some populations are fixed for the presence of amplified fragments. However, removing fixed loci from the data set improves the statistical power of the test for population structure. The genetic variation of 15 natural and six cultivated populations of M. sativa was analysed at 25 RAPD loci and compared to estimates computed with allozymes on the same set of populations. Although RAPD markers revealed less within-population genetic diversity than allozymes, the quantitative and qualitative patterns of population structure were in full agreement with allozymes. This confirmed the conclusions drawn from the allozymic survey: crop-to-wild gene flow occurred in many locations, but some other mechanisms opposed cultivated traits to be maintained into natural populations.     

Allozyme and microsatellite loci provide discordant estimates of population differentiation in the endangered dusky grouper (Epinephelus marginatus) within the Mediterranean Sea

The dusky grouper, Epinephelus marginatus, inhabits coastal reefs in the Mediterranean Sea and Atlantic Ocean. A decline in the abundance of this long-lived protogynous hermaphrodite has led to its listing as an endangered species in the Mediterranean, and heightened management concerns regarding its genetic variability and population substructure. To address these concerns, we analysed genetic variation at seven microsatellite and 28 allozyme loci in dusky groupers sampled from seven areas (for microsatellites) and three areas (for allozymes) in the west-central Mediterranean. Levels of genetic variability were higher for microsatellites than for allozymes (mean H(E) = 0.78 and 0.07, respectively), but similar to those observed in other marine fishes with comparable markers. Both microsatellites and allozymes revealed significant genetic differentiation among all areas analysed with each class of marker, but the magnitude of differentiation revealed by allozymes over three locales (F(ST) = 0.214) was greater than that detected with microsatellites over seven areas, or over the three areas shared with the allozyme analysis (F(ST) = 0.018 and approximately 0, respectively). A large proportion of the allozyme differentiation was due to a single locus (ADA*) possibly influenced by selection, but allozyme differentiation over the three areas was still highly significant (F(ST) = 0.06, P < 0.0001), and the 95% confidence intervals for allozyme and microsatellite F(ST) did not overlap when this locus was excluded. There was no evidence of isolation by distance with either class of markers. Our results lead us to conclude that dusky groupers are not panmictic in the Mediterranean Sea and suggest that they should be managed on a local basis. However, more work is needed to elucidate genetic relationships among populations.     

Assessment of population genetic structure in common wild rice Oryza rufipogon Griff. using microsatellite and allozyme markers

The genetic structure of five natural populations of common wild rice Oryza rufipogon Griff. from China, was investigated with 21 microsatellite loci and compared to estimates of genetic diversity and genetic differentiation detected by 22 allozyme loci. Microsatellite loci, as expected, have much higher levels of genetic diversity (mean values of A = 3.1, P = 73.3%, Ho = 0.358 and He = 0.345) than allozyme loci (mean values of A = 1.2, P = 12.7%, Ho = 0.020 and He = 0.030). Genetic differentiation detected by microsatellite loci ( FST = 0.468, mean I = 0.472) was higher than that for allozyme loci ( FST =0.388, mean I = 0.976). However, microsatellite markers showed less deviation from Hardy-Weinberg expectation (Wright's inbreeding coefficient FIS = -0.069) than do allozymes ( FIS = 0.337). These results suggest that microsatellite markers are powerful high-resolution tools for the accurate assessment of important parameters in population biology and conservation genetics of O. rufipogon, and offer advantages over allozyme markers.     

Population structure and genetic diversity of metamorphic and paedomorphic populations of the tiger salamander,Ambystoma tigrinum

Genetic relationships, population subdivision and genetic diversity were estimated from mtDNA and allozyme data for two subspecies of tiger salamander, one of which is obligately metamorphic and the other polymorphic for paedomorphosis (larval reproduction). Far greater genetic differentiation exists between subspecies than within subspecies, suggesting that the subspecies have evolved in allopatry. Values of F_st calculated from both mtDNA and allozymes were greater than 0.400 for each subspecies. Significant population subdivision was detected even on a microgeographic scale. This extensive population subdivision indicates that populations can respond to extremely localized selection pressures. In the case of paedomorphosis, populations in permanent water should evolve paedomorphosis as long as the appropriate genes exist. For both mtDNA and allozymes, comparisons of population structure within the polymorphic subspecies and between polymorphic and metamorphic subspecies reveal no discernible effects of paedomorphosis. However, a comparison of paedomorphic and metamorphic populations of the polymorphic subspecies showed significantly higher mtDNA diversity in paedomorphic populations. The discrepancy between the allozyme and mtDNA results may be due to the lower effective population size of mtDNA compared to autosomal genes.     

Genetic structure and mating system of Euterpe edulis Mart. Populations: a comparative analysis using microsatellite and allozyme markers

A comparative study between microsatellite and allozyme markers was conducted on the genetic structure and mating system in natural populations of Euterpe edulis Mart. Three cohorts, including seedlings, saplings, and adults, were examined in 4 populations using 10 allozyme loci and 10 microsatellite loci. As expected, microsatellite markers had a much higher degree of polymorphism than allozymes, but estimates of multilocus outcrossing rate ( = 1.00), as well as estimates of genetic structure (F(IS), G(ST)), were similar for the 2 sets of markers. Estimates of R(ST), for microsatellites, were higher than those of G(ST), but results of both statistics revealed a close agreement for the genetic structure of the species. This study provides support for the important conclusion that allozymes are still useful and reliable markers to estimate population genetic parameters. Effects of sample size on estimates from hypervariable loci are also discussed in this paper.     

Long distance dispersal and connectivity in amphi-Atlantic corals at regional and basin scales

Among Atlantic scleractinian corals, species diversity is highest in the Caribbean, but low diversity and high endemism are observed in various peripheral populations in central and eastern Atlantic islands and along the coasts of Brazil and West Africa. The degree of connectivity between these distantly separated populations is of interest because it provides insight into processes at both evolutionary and ecological time scales, such as speciation, recruitment dynamics and the persistence of coral populations. To assess connectivity in broadly distributed coral species of the Atlantic, DNA sequence data from two nuclear markers were obtained for six coral species spanning their distributional ranges. At basin-wide scales, significant differentiation was generally observed among populations in the Caribbean, Brazil and West Africa. Concordance of patterns in connectivity among co-distributed taxa indicates that extrinsic barriers, such as the Amazon freshwater plume or long stretches of open ocean, restrict dispersal of coral larvae from region to region. Within regions, dispersal ability appears to be influenced by aspects of reproduction and life history. Two broadcasting species, Siderastrea siderea and Montastraea cavernosa, were able to maintain gene flow among populations separated by as much as 1,200 km along the coast of Brazil. In contrast, brooding species, such as Favia gravida and Siderastrea radians, had more restricted gene flow along the Brazilian coast.     

Effects of life history traits and sampling strategies on genetic diversity estimates obtained with RAPD markers in plants

A compilation of studies using RAPD markers for evaluating population differentiation resulted in 78 estimates of AMOVA-derived Φ_ST and 31 estimates of Nei's G_ST, as well as in 41 estimates of Nei's within-population diversity. In outcrossing taxa, estimates of between-population diversity were closely correlated with maximum geographic distance between sampled populations. A corresponding association was not found in selfing taxa. These results suggest that RAPD can be a sensitive method for detection of genetic structuring according to the isolation-by-distance model. However, it also means that sampling strategies, as applied in individual studies, can seriously influence the resulting estimates of between-population diversity. Other sampling strategies, like number of plants per population and number of scored polymorphic markers, do not seem to impart any serious artefacts. As previously verified with allozyme data, RAPD markers showed that long-lived, outcrossing, late successional taxa retain most of their genetic variability within populations. By contrast, annual, selfing and/or early successional taxa allocate most of the genetic variability among populations. Estimates for between- and within-population diversity, respectively, proved to be negatively correlated, as previously reported for allozyme data. The only major discrepancy between allozymes and RAPD markers concerns geographic range; within-population diversity was strongly affected by distributional range of the investigated species in the allozyme data but not in the RAPD data. Moreover, RAPD-based values for between-population diversity increased with increasing distributional range whereas the opposite has been reported in a large allozyme data compilation. Contrary to allozymes, RAPD marker-derived within-population diversity is probably therefore not a very good predictor of total species genetic diversity.     

Genetic markers in population studies of Atlantic salmon Salmo salar L.: Karyotype characters and allozymes

The review, which consist of two parts, summarizes literature data on all genetic markers used in population studies of Atlantic salmon. The first part of the review concerns karyotype features and allozyme markers of Salmo salar. The latter are effectively used for distinguishing populations and subpopulations of Atlantic salmon, as well as for genetic monitoring of its populations. It is shown that the distribution of alleles of some allozymes may be related to selection for resistance to certain environmental conditions.     

Genetic markers in population studies of Atlantic salmon Salmo salar L.: karyotype characters and allozymes

The review, which consist of two parts, summarizes literature data on all genetic markers used in population studies of Atlantic salmon. The first part of the review concerns karyotype features and allozyme markers of Salmo salar. The latter are effectively used for distinguishing populations and subpopulations of Atlantic salmon, as well as for genetic monitoring of its populations. It is shown that the distribution of alleles of some allozymes may be related to selection for resistance to certain environmental conditions.     

Detection of highly polymorphic microsatellite loci in a species with little allozyme polymorphism

Microsatellite loci are regions of DNA containing tandem repeats of a short sequence motif; they occur abundantly in all eukaryotic genomes and have been shown to be a rich source of highly polymorphic genetic markers in humans and other mammals. These loci are particularly suitable for population studies because they can be relatively easily scored using a combination of polymerase chain reaction (PCR) amplification of each locus followed by electrophoresis to separate alleles. This paper details a method for finding these loci in any species. This method demonstrates that trinucleotide microsatellite loci are abundant and highly polymorphic in the social wasp Polistes annularis , whereas allozyme electrophoresis reveals very little polymorphism. The first six loci examined were all polymorphic with a mean observed heterozygosity of 0.62; in comparison average heterozygosity of 33 allozymes was 0.035. We suggest that this method can be used to detect variation where other methods have failed, making it an ideal tool for population and conservation geneticists who must deal with populations lacking other types of genetic variability.     

Amplified fragment length polymorphism (AFLP) analysis of the genetic structure of the zebra mussel, Dreissena polymorpha, in the Mississippi River

1. We predicted that zebra mussel, Dreissena polymorpha (Pallas), genetic structure in the Mississippi River would follow a model of invasive species genetics, which predicts low genetic structure among populations of recently established species. This prediction was upheld in our previous genetic study using allozymes, however, one locus yielded anomalous results. 2. We employed amplified fragment length polymorphism (AFLP) analysis as a neutral marker to assess the amount of genetic structure within and among populations, and as a test of expected population structure from both invasion genetic theory, and the results from our previous study. 3. There was greater spatial differentiation, as measured by F_st, observed using AFLP's than for allozymes (P < 0.001). There was no evidence that AFLP variation conformed to an isolation by distance model, and genetic relationships of populations, as measured by AFLP markers, were not similar to those detected in our allozyme survey. 4. The lack of concordance between these two genetic marker systems probably reflects their differential responses to drift, migration, and selection occurring during this rapid invasion. Strong population structure is counter to predictions that populations of invasive species will not be differentiated, as with observations based on allozyme markers. Therefore, newly established species may require genetic surveys using multiple marker systems to evaluate population structure.     

Spatial heterogeneity of mitochondrial DNA and allozymes among populations of white-tailed deer and mule deer.

A white-tailed deer (Odocoileus virginianus) population in northeastern Minnesota and a mule deer (O. hemionus) population in the Bridger Mountains Montana, have previously been shown to be spatially subdivided into contiguous subpopulations. We assessed the degree of genetic differentiation among subpopulations and tested the hypothesis that differentiation will be greater for mitochondrial DNA (mtDNA) than for nuclear-encoded allozymes. Differentiation of the white-tailed deer subpopulations was significant for two allozyme loci but not for mtDNA, and the overall degree of differentiation was low. Gene flow, recent founding of the subpopulations, and polygamous breeding structure may all have contributed to this pattern. Greater differentiation was evident among disjunct populations than between the contiguous subpopulations of white-tailed deer. The contiguous mule deer subpopulations were significantly differentiated for mtDNA and one allozyme locus. Differentiation was greater for mtDNA than for allozymes. These results are consistent with demographic data that indicate mule deer males disperse more than do females. Disjunct mule deer populations may be similar or dramatically different in mtDNA haplotype frequencies that do not necessarily vary with geographic distance. Current and historical gene flow and breeding structure will influence population genetic patterns.     

Genetic variation at allozyme and RAPD loci in sessile oak Quercus petraea (Matt.) Liebl.: the role of history and geography

The nuclear genetic variation within and among 21 populations of sessile oak was estimated at 31 RAPD loci in conjunction with previous estimates of variation at eight allozyme loci. The aim of the study was to assess the relative role of isolation-by-distance and postglacial history on patterns of nuclear variation. Because of its small effective population size and maternal transmission, the chloroplast genome is a good marker of population history. Both kinds of nuclear variation (RAPD and allozyme) were therefore compared, first, to the geographical distances among populations and, secondly, to chloroplast DNA restriction polymorphism in the same populations. Multiple Mantel tests were used for this purpose. Although RAPDs revealed less genetic diversity than allozymes, levels of genetic differentiation ( G _ST) were identical. The standard genetic distance calculated at all RAPD loci was correlated with geographical distances but not with the genetic distance calculated from chloroplast DNA data. Conversely, allozyme variation was correlated with chloroplast DNA variation, but not with geography. Possibly, divergent selection at two allozyme loci during the glacial period could explain this pattern. Because of its greater number of loci assayed, RAPDs probably provided a less biased picture of the relative role of geography and history.     

The clonal and population structure of a rare endemic plant, Wyethia reticulata (Asteraceae): allozyme and RAPD analysis

Genetic structure arises when limited gene flow between populations favours the development of distinct arrays of genetic characters within each population. Determining the spatial scale at which this differentiation occurs is critical to our understanding of population biology and microevolution of species. The genetic structure and spatial pattern of genetic variation in an endemic, clonal perennial, Wyethia reticulata E. Greene, was investigated using random amplified polymorphic DNA (RAPD) markers and allozyme alleles. Large stands (250–360 m²) were found to contain few genetic individuals. Despite the small population sizes and endemism of the species, W. reticulata was highly diverse genetically, with most of the variation (75–81%) distributed within populations. A population structure in full agreement with spatially defined populations was achieved only by combining RAPD and allozyme markers. Analysis using both types of markers appeared to provide estimates of genetic similarity between individuals that were most consistent with empirical data on plant distributions. We postulated that large, long-lived clones dominated genetic relationships within populations but also provided opportunities for gene flow between populations on a longer time scale. The two marker types yielded different estimates of between-individual similarity and revealed disparate patterns of population structure. This result will arise because allozymes and random DNA segments have dissimilar evolutionary dynamics with respect to mutation and selection.