Computational protocol: Molecular phylogeny of the bivalve superfamily Galeommatoidea (Heterodonta, Veneroida) reveals dynamic evolution of symbiotic lifestyle and interphylum host switching

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[…] Total DNA was isolated following a previously described method []. Adductor muscle tissue was homogenized in 800 μl lysis buffer and incubated at 55°C overnight, after which 80 μl saturated potassium chloride was added to the lysate. This solution was incubated for 5 min on ice and then centrifuged for 10 min. The supernatant (700 μl) was transferred to a new tube, cleaned once with a phenol/chloroform solution, and precipitated with an equal volume of 2-propanol. The DNA pellet was rinsed with 70% ethanol, vacuum-dried, and dissolved in 100 μl TE buffer.We sequenced the fragments of the nuclear 18S and 28S ribosomal RNA (rRNA), H3 and the mitochondrial COI genes. Polymerase chain reactions (PCRs) were used to amplify ~1700 bp of 18S rRNA, ~1000 bp of 28S rRNA, ~350 bp of H3 and ~700 bp of COI. Amplifications were performed in 20 μl mixtures consisting of 0.4 μl of forward and reverse primers (primer sequences are provided in Additional file ), 1.6 μl of dNTP, 2.0 μl of ExTaq buffer, 0.1 μl of ExTaq polymerase (TaKaRa, Otsu, Japan), and 15.1 μl of distilled water. Thermal cycling was performed with an initial denaturation for 3 min at 94°C, followed by 30 cycles of 30 s at 94°C, 30 s at a gene-specific annealing temperature (Additional file ), and 2 min at 72°C, with a final 3 min extension at 72°C. The sequencing reaction was performed using the PCR primers and internal primers (Additional file ) and the BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA) and electrophoresed on an ABI 3130 sequencer (Applied Biosystems). The obtained sequences have been deposited in the DDBJ/EMBL/GenBank databases with accession numbers AB714745–AB714907 (Additional file ). [...] Sequences of the 18S, 28S, H3 and COI genes were aligned using Muscle [] as implemented in the software Seaview [,] under default settings. The alignments of H3 and COI sequences did not require insertion of gaps and was therefore unambiguous. We used the software Gblocks v0.91b [-] to delimit ambiguously aligned regions in the 18S and 28S alignments (Additional file ), and the following phylogenetic analyses were conducted with and without alignment-ambiguous regions. The full 18S and 28S alignments contained 474 and 282 variable sites, respectively, and 453 and 249 variable sites when alignment-ambiguous regions were excluded. The H3 and COI alignments contained 87 and 297 variable sites, respectively, indicating that despite their short sequence lengths, they contain comparable amount of information as the 18S and 28S partitions. Because the initial phylogenetic analyses of individual genes did not produce essentially different results (Additional file ), we focused our analyses on the combined four-gene data set, which will be described below.Phylogenetic trees were obtained by the maximum-likelihood (ML) and Bayesian methods. For the ML analysis, model selection and tree search were conducted using the TreeFinder program [,]. The robustness of the ML tree was validated by bootstrap analysis with 1000 replications using the same program.Bayesian analyses were performed using MrBayes 3.1.2 [] with substitution models chosen using MrModeltest 2.3 []. In the combined data set, substitution parameters were estimated separately for each gene using the ‘unlink’ command. Two independent runs of Metropolis-coupled Markov chain Monte Carlo were performed simultaneously, sampling trees every 100 generations and calculating the average standard deviation of split frequencies every 1000 generations. Using the ‘stoprule’ option, analyses were continued until the average standard deviation of split frequencies dropped below 0.01, at which point the two chains were considered to have achieved convergence. Because the average standard deviation of split frequencies was calculated based on the last 75% of the samples, we discarded the initial 25% of the sampled trees as burn-in. We confirmed that analyses reached stationarity well before the burn-in period by plotting the ln-likelihood of the sampled trees against generation time. […]

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