Materials and Methods Between 1998 and 2012, a total of 66 tissue samples were collected from Chilean dolphins at 8 localities along the Chilean coast ranging from 35°20′S to 52° 40'S (). Skin samples were obtained from free-swimming adult dolphins by skin swabbing (n = 10) [] and biopsy darting (n = 41) []. Additionally skin samples from different parts of the body of freshly entangled animals were also collected (n = 15, ). Samples were stored in 90% ethanol and DNA extraction followed the salt extraction method []. The skulls from entangled individuals were reviewed to confirm the species identification, based on skull diagnostic characteristics []. Samples were collected under permit from the Chilean Under Fisheries RES 665/2009, RES 67/2010 and RES 334/2012 and approved by the Bioethical Committee at the Universidad de Chile.

Genotyping Samples were genotyped at 22 dinucleotide microsatellite loci: EV1, EV14, EV37, EV94, EV104 []; KWM12 []; MK5, MK6 []; PPH110, PPH130, PPH137, PPH142 []; GT023, GT211, GT575 []; TtruGT51, TtruGT142 []; Sgui02, Sgui03, Sgui06, Sgui17 [] and TexVet5 []. Amplification reactions contained 25–50 ng DNA, PCR buffer, 5 mM MgCl2, 0.4 μM of each primer, 0.2 mM deoxynucleoside triphosphate (dNTP) and 0.5 units Platinum Taq DNA polymerase (Invitrogen Life Technologies). Cultures 2 The Gates Of Asgard Free Full Download.

The thermal cycler profile consisted of a preliminary denaturing period of 3 min. At 94°C followed by 30 cycles of denaturation for 30 s at 94°C, primer annealing for 30 s with variable temperature (45°C to 55°C) depending of primer used, polymerase extension for 30 s at 72°C. A final extension period for 10 min. At 72°C was included. Following amplification and genotyping, allele peaks were visually verified using Gene Marker (Softgenetics). To minimize genotyping error each amplification and size run included a negative control to detect contamination and at least two internal control samples to ensure comparable allele sizing across all runs.

Additionally three blind replicates were independently run for 10 loci. Microsatellite analyses The dataset was initially checked for genotyping or scoring errors caused by null alleles, stuttering and short allele dominance using Microchecker v. Re-sampled individuals were identified by comparing genotypes in Cervus v. 0 [] and Pedigree v. To avoid false exclusion, initial matching allowed for up to five mismatching loci, re-examining visually all the cases.

Sex was subsequently compared to support our confidence in correctly identifying re-samples. To determine spatial population boundaries we used Geneland [], a Bayesian-based program that uses genotypes and spatial coordinates of individuals to cluster them into populations at approximately Hardy-Weingberg equilibrium, considering linkage equilibrium between loci. An allele frequency uncorrelated model was set, with 1,000,000 MCMC iterations and thinning of 100. Population structure was also evaluated through the Bayesian clustering method implemented in Structure v. The admixture model with correlated allele frequencies was used without specifying sampling locations.

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The model was run with the likely number of clusters (K) set to values from 1 to 9 using a burn-in period of 100,000 iterations followed by 500,000 Markov chain Monte Carlo (MCMC) iterations. Five independent runs were conducted for each value of K to check for convergence of results. The number of clusters or populations (K) was inferred from the posterior probability distribution Pr (K/X) calculated from the posterior probability of the data Log Pr (X/K). Additionally, in order to corroborate and visualize the number of populations identified, the Evanno method was implemented in the Structure Harvester program [,]. Assignment of individuals to their putative populations identified by Geneland and Structure programs was verified by the Bayesian method of Rannala and Mountain [] with an alpha of 0.01 and 10,000 repetitions of the Paetkau et al. [] MCMC re-sampling algorithm implemented in Geneclass v. Fisher Exact test conducted in Genepop [] was performed to evaluate the population structure between populations identified through the Bayesian clustering methods, considering all the individuals and also males and females separately in order to test sex-biased dispersal.