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A new set of microsatellite loci (simple sequence repeats, SSRs) from the overexploited mangrove crab Ucides cordatus is described in this study. Microsatellite isolation used a highly simplified and inexpensive protocol based on (i) multiple enzyme digestion/ligation; (ii) mixed biotin-labeled probes and streptavidin-coated magnetic bead hybridization capture strategy, and (iii) a double-repeat-enrichment procedure. A genomic library, double-enriched for inserts containing tetranucleotide repeat motifs [(GACA)6, (GATA)7, (GGAT)5 and (GTAT)5], was constructed to increase the chance of recovering SSR-containing sequences within DNA fragments. Amplified enriched DNA was cloned and transformed into competent E. coli. Then, positive clones were identified by 'white/blue plaque selection’. One hundred and five colonies were PCR-screened for sequencing, and 72 of these were found to have unique SSR inserts. Microsatellite motifs contained more than five repeats, and most loci were found to have perfect tandem repeats (51.4%), of which 94.4% were dinucleotide and 5.5% trinucleotide. Only 20% of all loci were compound and 28.6% were imperfect repeats containing di-, tri- and/or tetranucleotides. The high frequency of perfect repeat motifs after enrichment is additional evidence of the importance of adopting this procedure for the isolation of SSR. The novel 34 SSRs described in this study are expected to be highly polymorphic and, therefore, useful in population/stocks discrimination of this valuable mangrove species throughout its range, currently subjected to excessive fishing efforts.

Background: The mangrove crab Ucides cordatus is considered a key species for the ecological balance of mangrove forests and a major source of employment and income for traditional crab collectors in Brazil. Several studies evidenced weak genetic variation among populations due to an efficient larval transport. However, gene flow patterns of the species is poorly understood, with no information about migration rates. The influence of the two main Brazilian currents in larval dispersion is also not clear. In order to provide baseline information for conservation, planning and management of this important fishery resource, the present study aimed to estimate and evaluate spatial distribution of genetic diversity, migration rates and gene flow directivity among populations of U. cordatus in Brazil. Methods: Nine microsatellites were used to resolve population structure of 319 crabs collected from six sites located along the Brazilian coast. The degree of geographical differentiation included estimates of genetic diversity, population structure and gene flow models, with spatial analysis of shared alleles (SASHA), isolation by distance tests, AMOVA, discriminant analysis of principal components (DAPC) and Bayesian clustering. We estimated the amount of ongoing gene flow between clusters using the coalescent-based method implemented in Migrate-N. Results: Loci were highly polymorphic (average of 12.4 alleles per locus) evidencing high genetic variability. There was significant differentiation among localities, despite of the low value of FST (= 0.019; P < 0.001). FST and Jost's D indexes were also estimated in pairwise comparisons and showed significant differences between most of the surveyed site pairs (P < 0.05). Structure evidenced a single genetic group among samples, however SASHA pointed to a non-panmictic condition (P = 0.011). AMOVA detected four statistical significant clusters with low level of differentiation (FCT = 0.037; P = 0.023). The gene flow model that best described the population connectivity was the island model, with ~24 crabs being exchanged among localities per generation. Discussion: The high migration rates found among localities seem to be the main force acting to sustain the distribution of the genetic diversity of U. cordatus. Despite the high gene flow and the weak population structure among samples, the significant genetic differences found suggest that gene flow alone does not bypass the effects of genetic drift, natural selection and/or human exploitation. These findings are vital for the establishment of a database to be used in the development of conservation programs. [ABSTRACT FROM AUTHOR]

For a snapshot assessment of the genetic diversity present within Melipona subnitida, an endemic stingless bee distributed in the semi-arid region of northeastern Brazil, populations separated by over 1,000 km distance were analyzed by ISSR genotyping. This is a prerequisite for the establishment of efficient management and conservation practices. From 21 ISSR primers tested, only nine revealed consistent and polymorphic bands (loci). PCR reactions resulted in 165 loci, of which 92 were polymorphic (57.5%). Both ΦST (ARLEQUIN) and θB (HICKORY) presented high values of similar magnitude (0.34, p<0.0001 and 0.33, p<0.0001, respectively), showing that these two groups were highly structured. The dendrogram obtained by the cluster analysis and the scatter-plot of the PCoA corroborate with the data presented by the AMOVA and θB tests. Clear evidence of subdivision among sampling sites was also observed by the Bayesian grouping model analysis (STRUCTURE) of the ISSR data. It is clear from this study that conservation strategies should take into account the heterogeneity of these two separate populations, and address actions towards their sustainability by integrating our findings with ecological tools. [ABSTRACT FROM AUTHOR]

The isolation and characterization of the first polymorphic microsatellite markers for the mangrove crab Ucides cordatus are described. The number of alleles at each locus ranged from three to 25, mean of nine alleles, in 46 crabs captured in two Brazilian mangroves. The markers averaged high levels of observed (0.709  ±  0.183) and expected (0.716  ±  0.170) heterozygosities. Departures from Hardy–Weinberg equilibrium were observed at two loci. Linkage disequilibrium tests were not significant and no evidence of null alleles was detected. All these microsatellite loci are expected to be useful in estimating fine-scale population processes of this valuable mangrove species currently subjected to excessive fishing efforts. [ABSTRACT FROM AUTHOR]