## Similar protocols

## Protocol publication

[…] Twenty-two localities (11 from Algeciras and 11 from Tangier) where E. australis and E. arborea co-occurred, plus one locality in Algeciras where only E. australis was found, were sampled (, ). Population sampling consisted of fresh leaves from up to 30 individuals per population (spaced at least 10 m from each other), except in four populations of E. australis and three populations of E. arborea where this number could not be reached (). Thus, the total sampling included 676 individuals of E. australis (357 from Algeciras and 319 from Tangier) and 631 individuals of E. arborea (308 from Algeciras and 323 from Tangier). Samples were dried in silica gel and stored at room temperature until DNA extraction. Dry leaf material, approximately 100 mg per sample, was reduced to fine powder using stainless steel beads on a Mixer Mill MM400 cell disrupter (Retsch, Llanera, Spain). DNA was extracted using SpeedTools plant DNA extraction kit (**Biotools**, Madrid, Spain), and eluted in 50 µl in Tris-EDTA 0.1×buffer.Amplification of microsatellite loci followed Segarra-Moragues et al. , . Seven microsatellite loci (Ecoc108, Ecoc117, Ecoc132, Ecoc137, Ecoc142, Ecoc431 and Ecoc446) were amplified in E. australis, and eight (the same as for E. australis plus Ecoc115) in E. arborea. These microsatellite loci were unlinked and polymorphic in both species. Fifteen percent of the 1307 genotyped individuals were included as duplicates to check for possible genotyping errors. All of them showed identical allelic profiles to their corresponding original samples.PCR products were electrophoresed in an ABI3730 automated sequencer (Applied Biosystems, Madrid, Spain) using LIZ500 as internal lane size standard. Assignment of fragments to allele classes was carried out with **Genemarker** version 1.97 software (Softgenetics, State College, PA). Genotypic matrices were deposited at DRYAD (http://datadryad.org/) under accession number doi:10.5061/dryad.bj70k. [...] GENETIX v. 4.05 was used to estimate allele frequencies, mean number of alleles per locus (A), and observed (H
O) and unbiased expected (H
E) heterozygosities . Wright’s F-statistics were estimated according to Weir & Cockerham using **GENEPOP**′007 and tested for significance using Fisher’s exact tests. This latter software was also used to estimate the Maximum likelihood frequency of null alleles and 95% confidence intervals for null allele frequencies according to Dempster et al. .To evaluate, for each species, whether population diversity indices differed between Algeciras and Tangier groups of populations, average allelic richness per locus (A
*) estimated according to the rarefaction method of Hurlbert adapted by El Mousadik & Petit , average observed heterozygosity (H
O), average genetic diversity within populations (H
S), and inbreeding coefficient (F
IS), were compared using FSTAT version 2.9.3.2 and tested for significance using 10,000 permutations. This same software was used to check for significant differences in population differentiation (i.e. average F
ST values) for each species between Algeciras and Tangier.Bayesian analyses in STRUCTURE v. 2.1 , were used to estimate the population genetic structure and to infer the most likely number of genetic clusters (K) in both heath species. STRUCTURE assigns individuals to the K different genetic clusters based on allele frequencies at each locus. Estimated number of K clusters ranged from 1 to 21, and analyses were based on an admixture ancestral model with correlated allele frequencies. In each run of the program Monte Carlo Markov Chain (MCMC) and burn-in period length consisted of 1.2×106 and 8×105 iterations, respectively. The amount of variation of the likelihood was evaluated by carrying out ten runs for each K. The most likely number of genetic clusters (K) was estimated following Evanno et al. , which uses an ad hoc parameter (ΔK) to estimate the rate of change of likelihood values between successive K values. POPULATIONS version 1.2.3. beta was used to compute pairwise D
A genetic distances between populations of each species. These D
A genetic distance matrices were used to compute eigenvalues and eigenvectors to perform Principal Coordinates Analyses (PCoA) for each species. Minimum Spanning Trees (MST) using D
A distance matrices were constructed with **NTSYSpc** version 2.1 and were superimposed onto the 2D-PCoA plots. MST computes minimum-length pairwise connections between points. When superimposed onto PCoA plots it helps detecting local distortions (i.e. pairs of points which look close together in the PCoA but actually are far apart if other dimensions). Isolation by distance (IBD) was estimated separately for E. arborea and E. australis at each side of the Strait by matrix correlation analyses using a matrix of log-transformed pairwise geographical distances between populations and a matrix of a pairwise linearized F
ST values (i.e., F
ST/(1 - F
ST); ) computed with **ARLEQUIN** v. 3.11 . Significance of the correlation was tested for each species and side of the Strait with Mantel test (1000 permutations) using NTSYSpc. Then, ANCOVAs were used to test for parallelism of the resulting IBD slopes in Algeciras and Tangier within each species. Finally, gene flow between population pairs was also estimated separately for each species at each side of the Strait as the effective number of migrants per generation (N
m), obtained directly from F
ST values by using Wright’s island model. Although these values should not be taken as accurate estimates of numbers of migrants, the comparison of average N
m values between populations at each of the two sides of the Strait may still be useful to explore for each species the existence of a decrease in gene flow associated with higher patchiness. For the sake of this study, we focused on geographical distances separating pairs of populations smaller than 30 km in order to factor out or minimize possible geographical effects other than sandstone patchiness. […]

## Pipeline specifications

Software tools | Biotools, GeneMarker, Genepop, NTSYSpc, Arlequin |
---|---|

Application | Population genetic analysis |

Organisms | Eubalaena australis |