Computational protocol: Phylogeography of a Marine Insular Endemic in the Atlantic Macaronesia: The Azorean Barnacle, Megabalanus azoricus (Pilsbry, 1916)

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Protocol publication

[…] The intraspecific analysis was carried out with the complete mitochondrial control region sequences flanked by the 12S rRNA and tRNA-Ile genes. Moreover, the partial mitochondrial 16S rRNA and COX1 gene sequences were obtained in a subset of the sample for the interspecific comparative analysis of sequences in phylogenetic reconstruction and DNA barcoding. The near complete mitochondrial 16S rRNA was amplified with Cirr-16S-5F (5′-CAACCTGGCTYACGCCGGTCT-3′) and Cirr-Val-3R (5′-GCTGTTTAAGCATTTCATTTACACTGAAAAGG-3′) primers. The fragment in the COX1 mitochondrial gene was amplified using the LCO1490 and HCO2198 primers []. Finally, amplification of a fragment containing the complete mitochondrial control region and flanking sequences was carried out with the primers Mazo-12S-1F (5′-CTATCAAAGTAATCCTTTTGTCAGGCA-3′) and Mazo-Ile-2R (5′-′GATAACAACACGGACCTCAACGAT-3′).Amplifications were performed in a 15 μL reaction volume using GeneAmp 9700 thermocyclers (Applied Biosystems, Carlsbad, CA). PCR reactions comprised 1X AmpliTaq 360 buffer (Applied Biosystems), 3.5mM MgCl2, 200 μM each dNTP, 0.13 μM each primer, 0.15 units of AmpliTaq 360 (Applied Biosystems), and 10–50 ng of total DNA. However, amplification within the COX1 gene was performed with 2.5mM MgCl2. The consensus PCR profile was 94°C for 3 min, followed by 36 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 60s, then 72°C for 7 min.The PCR products were digested with Exo-SAP-It (Affimetrix, Santa Clara, CA), to remove primers and to deactivate unused dNTPs, and sequenced in both directions using the BigDye 3.1 sequencing kit (Applied Biosystems). The extension products were purified with DyeEx columns (Qiagen, Hilden, Germany) and separated and detected in a 3730xl Genetic Analyzer (Applied Biosystems). Following chromatogram revision and trimming with Sequence Scanner software (Applied Biosystems), the sequences were aligned with ClustalX [] implemented in BioEdit []. [...] The external morphology of the specimens was verified for compliance with previous descriptions of the Azorean barnacle [, , ]. A meta-analysis of the phylogenetic relationships among Megabalanus taxa based on the available homologous 16S rRNA partial gene data was performed to corroborate the phylogenetic singularity of the sampled M. azoricus and define their phylogenetic relationships in a limited but informative data set. Additionally, our dataset (N = 54) was compared with the available Megabalanus COX1 sequences through BLAST ( and BOLD [] to find homologous sequences with maximum similarity. The ribosomal phylogenetic inference was based in the Bayesian criterion in MrBayes v.3.2 []. The HKY model [] was selected for 16S rRNA alignment based on AIC estimations in jMoldeltest v.2.1.1 [] and implemented in MrBayes with an α parameter of the γ distribution = 0.32 and the proportion of invariable sites = 0.57 sets as priors. Markov chain Monte Carlo (MCMC) analysis was run twice for 10,000,000 generations with default parameters being obtained the 50% majority rule tree. [...] Genetic diversity estimations for the control region and COX1 sequences were obtained in Arlequin v. 3.5 [] and DnaSPv.5.10 [], these included nucleotide diversity (π), haplotype diversity (h), and Tajima’s D in populations and observed haplogroups.The demographic expansion hypothesis within this species was tested with alternative tests based on different criteria. First, using the distribution of the observed number of differences between sequence pairs obtained with Arlequin v3.5 [], the so called mismatch distributions, which are usually unimodal in populations under recent demographic expansion [, ]. The mismatch distributions were obtained for both populations and haplogroups. The adequacy of the expansion model was assessed using a parametric bootstrap approach [], by means of i) the sum of squared deviations (SSD) between the observed and expected distributions, and ii) computing the raggedness index (H’s Ri) from the observed distribution as suggested by Harpending []. The corresponding estimators of time of expansion (τ) and mutational parameters θ 0 and θ 1, before and after the expansion event, were also obtained in Arlequin by parametric bootstrapping [].We also performed the Tajima’s [] and the Fu’s [] tests for neutrality. Moreover, the significant values for the Tajima’s D statistic can be related to demographic dynamics [] and the Fu’s Fs statistic shows large negative values when estimated from a population undergoing demographic expansion []. For small sample sizes the R 2 test is superior in detecting population growth [].The pairwise F ST among the four sampled populations and their significance were computed in Arlequin v.3.5 []. The significance of the partition of genetic diversity within a jerarquized structure including populations and their groups was tested with AMOVA, also in Arlequin v.3.5 []. The population size dynamics over time were reconstructed by Bayesian coalescent inference []. A Bayesian skyline plot of effective breeding population size (N e) through time was obtained with BEAST (version 1.7.5) [] under the HKY model [] with site rate heterogeneity, specifying an estimated fixed mean substitution rate value, using a linear model, and 50,000,000 generations for the MCMC algorithm.Relationships among the sampled haplotypes, their relative frequencies, and geographic locations were graphically described by means of a median-joining network [] in Network v.4.6 (Fluxus Technology Ltd., Suffolk, UK). Additionally, the phylogenetic relationships and assemblage of haplotypes were corroborated in a neighbor-joining optimal mid-point rooted tree inferred with MEGA 5 [], with the branch support estimated by bootstrapping (2,000 replicates). Considering the available models of sequence evolution for Arlequin and MEGA calculations, the model that best fit the control region sequence data based on AIC estimations from jMoldeltest v2.1.1 [] was the Tamura-Nei [], with an α value = 0.2220 for the gamma distribution. […]

Pipeline specifications

Software tools Clustal W, BioEdit, MrBayes, Arlequin, BEAST, MEGA
Applications Phylogenetics, Population genetic analysis
Organisms Serinus canaria