Comparative allozyme and microsatellite population structure in a narrow endemic plant species, Centaurea corymbosa Pourret (Asteraceae)

Centaurea corymbosa Pourret (Asteraceae) is a narrow endemic species known only from six populations located in a 3-km2 area in the south of France. Earlier field experiments have suggested that pollen and seed dispersal were highly restricted within and among populations. Consistent with the field results, populations were highly differentiated for five allozyme loci and among-population variation fitted an isolation-by-distance model. In the present study, we investigated the genetic structure of C. corymbosa using six microsatellite loci. As with allozymes, microsatellites revealed no within-population structure and a large differentiation among populations. However, allozyme loci were less powerful than microsatellites in detecting the extent of gene flow assessed by assignment tests. The patterns of structuration greatly varied among loci for both types of marker; we suggest that differences in single-locus pattern could mainly be an effect of stochastic variation for allozymes and an effect of variation in mutation rate for microsatellites. In contrast to the multilocus results, the two most polymorphic microsatellite loci did not show any isolation-by-distance pattern. Our results suggest that highly variable loci might not always be the best suited markers to quantify levels of gene flow among populations.     

High-resolution typing of Toxoplasma gondii using microsatellite loci.

High-resolution typing of Toxoplasma gondii is essential to understand the effect of genetic differences among strains on the variation in disease manifestation and transmission patterns. Current typing methods discern 3 lineages with minimal within-lineage variation. Described here are 6 new variable loci. These loci, including a minisatellite and 5 microsatellites, were more polymorphic than allozymes, restriction fragment length polymorphisms, and sequence variation in introns. Most importantly, these loci revealed, for the first time, substantial within-lineage variation that was over 6-fold higher than that detected by other markers. Genotyping at these loci facilitates classification of isolates beyond the lineage level.     

Do discrepancies between microsatellite and allozyme variation reveal differential selection between sea and lagoon in the sea bass (Dicentrarchus labrax)?

In the present study the genetic structure of Dicentrarchus labrax (14 samples from the Mediterranean) was analysed at six microsatellite loci, in order to test the hypothesis that some enzymatic loci undergo selection between marine and lagoon habitat. Eight of the 14 samples were analysed at both microsatellite and allozyme markers. The analysis of the genetic variation among the Mediterranean samples showed that (i) &Fcirc;ST values obtained with the six microsatellite loci were much smaller than those obtained with the 28 allozymes and (ii) microsatellite loci seemed to reflect more the geographical proximity than an ecological one. Thirteen enzymatic loci exhibited moderate to high values compared with microsatellites. This was interpreted as evidence that these allozymes are non-neutral. However, only six loci seemed to be implicated in differentiation between marine and lagoon samples, the causes of selection being unknown for the others. A possible scenario of population dynamics of the sea bass between marine and lagoon habitat is suggested.     

Comparative Assessment of Genetic Variability in the Populations of Endemic and Endangered Yellow Catfish, Horabagrus brachysoma (Teleostei: Horabagridae), Based on Allozyme, RAPD, and Microsatellite Markers

The comparative assessment of genetic diversity using allozymes, random amplified polymorphic DNA (RAPD), and microsatellite markers was conducted in endemic and endangered yellow catfish (Horabagrus brachysoma) sampled from three locations in Western Ghats river systems of India. Among the three markers, microsatellites show more polymorphism, having 100% polymorphic loci, whereas allozymes show the least (56%). In RAPD, 60.5% of fragments were polymorphic. Observed heterozygosity and F(ST) values were very high in microsatellites, compared with the other markers. Microsatellite and RAPD markers reported a higher degree of genetic differentiation than allozymes among the populations depicted by pairwise F(ST)/G(ST), AMOVA, Nei's genetic distance, and UPGMA dendrogram. The three classes of markers demonstrated striking genetic differentiation between pairs of H. brachysoma populations. The data emphasize the need for fishery management, conservation, and rehabilitation of this species.     

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.     

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.     

Growth rate correlates to individual heterozygosity in the European eel, Anguilla anguilla L

Heterozygosity-fitness correlations (HFCs) have been reported in populations of many species. We provide evidence for a positive correlation between genetic variability and growth rate at 12 allozyme loci in a catadromous marine fish species, the European eel (Anguilla anguilla L.). More heterozygous individuals show a significantly higher length and weight increase and an above average condition index in comparison with more homozygous individuals. To a lesser extent, six microsatellite loci show a similar pattern, with positive but not significant correlations between heterozygosity and growth rate. The HFCs observed could be explained by an effect of either direct allozyme over-dominance or associative overdominance. Selection affecting some of the allozyme loci would explain the greater strength of the HFCs found at allozymes in comparison with microsatellites and the lack of correlation between MLH at allozymes and MLH at microsatellites. Associative overdominance (where allozyme loci are merely acting as neutral markers of closely linked fitness loci) might provide an explanation for the HFCs if we consider that allozyme loci have a higher chance than microsatellites to be in linkage disequilibrium with fitness loci.     

Low genetic variation in muskoxen (Ovibos moschatus) from western Greenland using microsatellites

Muskoxen are large herbivores living in Arctic environments. Lack of genetic variation in allozymes has made it difficult to study the social and genetic structure of this species. In this study, we have tried to find polymorphic microsatellite loci using both cattle-derived microsatellite primers and primers developed from a genomic plasmid library of muskoxen. Only limited variation was found for both sets of microsatellite loci. We conclude that this consistent low genetic variation is probably due to demographic features of the muskoxen populations rather than to methodological constraints caused by the transfer of microsatellites between species.     

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.     

Genetic structure of avian populations--allozymes revisited

Selection on allozymes has sometimes been advanced as one explanation for the low levels of population differentiation detected in avian populations by the use of enzymatic markers. Comparisons of the amount of population subdivision (estimated by FST values or analogous indices) measured by enzymatic and mitochondrial DNA (mtDNA) markers in birds were seen as evidence for this because mtDNA typically produces a more structured picture of population subdivisions. In fact, when taking into account the smaller effective population size of mtDNA, nuclear and mitochondrial markers give concordant results. Some discrepancies still exist, but I suggest that some might originate from different amounts of nuclear vs. mitochondrial gene flow due to partial reproductive isolation. Variable number of tandem repeat (VNTR) loci do not provide a dramatically different picture of population structures in birds compared to allozymes. Although more tests are needed, such as comparing the amount of genetic structure detected in the same populations with allozymes and microsatellites, the low levels of population subdivision measured with allozymes in birds seem to reflect historical and demographic processes and would not appear to result from any peculiarities of bird enzymatic loci.     

Concordance of allozyme and microsatellite differentiation in a marine fish, but evidence of selection at a microsatellite locus

Previous studies have reported higher levels of divergence for microsatellites than for allozymes in several species, suggested to reflect stabilizing selection on the allozymes. We compared the differentiation patterns of 11 allozyme and nine microsatellite loci using 679 spawning Atlantic herring (Clupea harengus) collected in the Baltic and North Seas to test for differential natural selection on these markers. Observed distributions of F statistics for the two types of markers are conspicuously dissimilar, but we show that these differences can largely be explained by sampling phenomena caused by different allele frequency distributions and degrees of variability. The results show consistently low levels of differentiation for both marker types, with the exception of one outlier microsatellite locus with a notably high F(ST). The aberrant pattern at this locus is primarily due to two alleles occurring at markedly high frequencies in the Baltic, suggesting selection at this locus, or a closely linked one. When excluding this locus, the two marker types show similar, weak differentiation patterns with F(ST) values between the Baltic and the North Seas of 0.001 and 0.002 for allozymes and microsatellites, respectively. This small heterogeneity, and weak isolation by distance, is easier to distinguish statistically with microsatellites than with allozymes that have fewer alleles and skewed frequency distributions. The allozymes, however, also detect surprisingly low levels of divergence. Our results support suggestions that previously described differences between marker types are primarily caused by a small number of outlier loci.     

Genetic structure of northwestern Spanish brown trout (Salmo trutta L.) populations, differences between microsatellite and allozyme loci

Genetic variation in nine wild brown trout (Salmo trutta L.) populations was studied by means of allozyme and microsatellite markers. All brown trout populations were clearly separated into two clusters that represented the Sil and Duero basins. Although both markers revealed a strong genetic differentiation between basins, microsatellite loci resulted much more accurate when population structure at the intrabasin level was analysed. Also pairwise multilocus FST estimates and assignment tests of individual fish to the set of sampled populations demonstrated a much higher efficiency of microsatellites compared to allozymes. The analysis of both markers provides new insights in defining the conservation units at this local area and confirms the existence of a recognized sub-lineage in the Duero basin. The management implications of these findings are discussed and changes in trout release activity are recommended to avoid mixing of trout gene pools mainly in the Sil basin.     

Assessing genetic structure with multiple classes of molecular markers: a case study involving the introduced fire ant Solenopsis invicta.

We used 30 genetic markers of 6 different classes to describe hierarchical genetic structure in introduced populations of the fire ant Solenopsis invicta. These included four classes of presumably neutral nuclear loci (allozymes, codominant random amplified polymorphic DNAs (RAPDs), microsatellites, and dominant RAPDs), a class comprising two linked protein-coding nuclear loci under selection, and a marker of the mitochondrial DNA (mtDNA). Patterns of structure revealed by F statistics and exact tests of differentiation were highly concordant among the four classes of neutral nuclear markers, although the microsatellites were the most effective markers for detecting structure. The results from the mtDNA complemented those from the neutral nuclear markers by revealing that strong limitations to female-mediated gene flow were the cause of the local structure registered by the nuclear markers. The pattern of structure inferred from the selected nuclear loci was markedly different from the patterns derived from the other sets of markers but was predictable on the basis of the presumed mode of selection acting on these loci. In general, the results for all six classes of markers can be explained by known features of the social and reproductive biology of fire ants. Thus, the results from these diverse sets of markers, combined with detailed natural history data, provide an unusually complete picture of how the fundamental evolutionary forces of gene flow, drift, and selection govern the distribution of genetic variation within and between fire ant populations.     

Spatiotemporal genetic differentiation of Cuban natural populations of the pink shrimp <Emphasis Type="Italic">Farfantepenaeus notialis</Emphasis>

We analyzed the spatiotemporal genetic structure of Farfantepenaeus notialis populations using five microsatellites loci in order to understand the influence of natural events such as hurricanes on the genetic drift/migration balance as the main cause for the variation of allele frequencies over time. The results were compared with the previous ones obtained from allozymes and mtDNA. High and stable genetic diversity levels (He=0.879+/-0.0015) were found over eight years for the populations that inhabit the south Cuban platform, however significant changes of allele frequencies were detected over time. The F(ST) estimates, albeit low, revealed significant differences among populations inside the Ana Maria Gulf for 1995 but not for the 1999 and 2003 samples. The F(ST), AMOVA and the genetic distance analysis revealed the instability of the genetic structure over time in accordance with allozymes results. The correspondence of the microsatellite results with those obtained from allozymes confirm the effects of migration enhanced by natural events as the main cause of the temporal variation of allele frequencies. The genetic drift effect was discarded through the evaluation of Ne and the M ratio, while natural selection effects were rejected because of the lowest probability of microsatellite loci being under selective pressures. The microsatellite data are also consistent with the results obtained with mtDNA in detecting significant and persistent genetic differences between the Gulfs of Ana María and Batabanó for the years 1995 and 2003.     

A consolidated linkage map for rainbow trout (Oncorhynchus mykiss)

Androgenetic doubled haploid progeny produced from a cross between the Oregon State University and Arlee clonal rainbow trout (Oncorhynchus mykiss) lines, used for a previous published rainbow trout map, were used to update the map with the addition of more amplified fragment length polymorphic (AFLP) markers, microsatellites, type I and allozyme markers. We have added more than 900 markers, bringing the total number to 1359 genetic markers and the sex phenotype including 799 EcoRI AFLPs, 174 PstI AFLPs, 226 microsatellites, 72 VNTR, 38 SINE markers, 29 known genes, 12 minisatellites, five RAPDs, and four allozymes. Thirty major linkage groups were identified. Synteny of linkage groups in our map with the outcrossed microsatellite map has been established for all except one linkage group in this doubled haploid cross. Putative homeologous relationships among linkage groups, resulting from the autotetraploid nature of the salmonid genome, have been revealed based on the placement of duplicated microsatellites and type I loci.     

Rise of the machines--recommendations for ecologists when using next generation sequencing for microsatellite development

Next generation sequencing is revolutionizing molecular ecology by simplifying the development of molecular genetic markers, including microsatellites. Here, we summarize the results of the large-scale development of microsatellites for 54 nonmodel species using next generation sequencing and show that there are clear differences amongst plants, invertebrates and vertebrates for the number and proportion of motif types recovered that are able to be utilized as markers. We highlight that the heterogeneity within each group is very large. Despite this variation, we provide an indication of what number of sequences and consequent proportion of a 454 run are required for the development of 40 designable, unique microsatellite loci for a typical molecular ecological study. Finally, to address the challenges of choosing loci from the vast array of microsatellite loci typically available from partial genome runs (average for this study, 2341 loci), we provide a microsatellite development flowchart as a procedural guide for application once the results of a partial genome run are obtained.     

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.     

Allozyme electrophoresis still represents a powerful technique in the management of coral reefs

Understanding genetic variability and gene flow between populations of scleractinian corals separated by one to several hundred kilometers is crucially important as we head into a century of climate change in which an understanding of the connectivity of populations is a critically important question in management. Genetic methods that directly use molecular variance in the DNA should offer greater precision in detecting differences among individuals and populations than the more traditional allozyme electrophoresis. However, this paper highlights the point that the limited number of DNA markers that have been identified for scleractinian coral genetic studies do not necessarily offer greater precision than that offered by allozymes. In fact, at present allozyme electrophoresis yields greater information than the eight different DNA markers used in this study. Given the relative ease of use of allozymes and the wealth of comparable data sets from numerous previously published studies, allozyme electrophoresis should not be dismissed for population structure and connectivity studies on coral reefs. While continued effort should be placed into searching for new DNA markers, until a more sensitive DNA marker becomes available for scleractinian corals, allozyme electrophoresis remains a powerful and relevant technique for understanding the connectivity of coral population studies.     

Microsatellite polymorphism and genetic differentiation in three Norwegian populations of Elymus alaskanus (Poaceae)

 Variation at seven microsatellite loci was investigated in three local E. alaskanus populations from Norway and microsatellite variation was compared with allozyme variation. The percentage of polymorphic loci was 81%, the mean number of alleles per polymorphic locus was 5.7 and expected heterozygosity was 0.37. An F-statistic analysis revealed an overall 48% deficit of heterozygotes over Hardy-Weinberg expectations. Gene diversity is mainly explained by the within population component. The averaged between population differentiation coefficient, F _st , over 7 loci is only 0.13, which accounts for only 13% of the whole diversity and was contrary to allozyme analysis. The mean genetic distance between populations was 0.12. However, a χ² -test showed that allele frequencies were different (p < 0.05) among the populations at 5 of the 7 loci. In comparison with the genetic variation detected by allozymes, microsatellite loci showed higher levels of genetic variation. Microsatellite analysis revealed that population H10576 possesses the lowest genetic variation among the tested three populations, which concur with allozyme analysis. The dendrogram generated by microsatellites agreed very well with allozymic data. Our results suggest that natural selection may be an important factor in shaping the genetic diversity in these three local E. alaskanus populations. Possible explanations for deficit heterozygosity and incongruence between microsatellites and allozymes are discussed. Received November 6, 2001; accepted April 24, 2002 Published online: November 14, 2002 Addresses of the authors: Genlou Sun (e-mail: Genlou.sun@STMARYS.CA), Biology Department, Saint Mary's University, Halifax. Nova Scotia, B3H 3C3, Canada. B. Salomon, R. von Bothmer, Department of Crop Science, The Swedish University of Agricultural Sciences, P.O. Box 44, SE-230 53, Alnarp, Sweden.     

Experimental evaluation of the usefulness of microsatellite DNA for detecting demographic bottlenecks

Evolutionary and conservation biologists often use molecular markers to evaluate whether populations have experienced demographic bottlenecks that resulted in a loss of genetic variation. We evaluated the utility of microsatellites for detection of recent, severe bottlenecks and compared the amounts of genetic diversity lost in bottlenecks of different sizes. In experimental mesocosms, we established replicate populations by releasing 1, 2, 4 or 8 pairs of the western mosquitofish, Gambusia affinis (Poeciliidae). Using eight polymorphic microsatellite loci, we quantified seven indices of genetic diversity or change that have been used to assess the effects of demographic bottlenecks on populations. We compared indices for the experimentally bottlenecked populations to those for the source population and examined differences between populations established with different numbers of founders. Direct count heterozygosity and the proportion of polymorphic loci were not very sensitive to genetic changes that resulted from the experimental bottlenecks. Heterozygosity excess and expected heterozygosity were useful to varying degrees in the detection of bottlenecks. Allelic diversity and temporal variance in allele frequencies were most sensitive to genetic changes that resulted from the bottlenecks, and the temporal variance method was slightly more correlated with bottleneck size than was allelic diversity. Based on comparisons to a previous study with allozymes, heterozygosity, temporal variance in allele frequencies and allelic diversity, but not proportion of polymorphic loci, appear to be more sensitive to demographic bottlenecks when quantified using microsatellites. We found that analysis of eight highly polymorphic loci was sufficient to detect a recent demographic bottleneck and to obtain an estimate of the magnitude of bottleneck severity.