Computational protocol: Pursuing the quest for better understanding the taxonomic distribution of the system of doubly uniparental inheritance of mtDNA

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Protocol publication

[…] Cox1 sequences were edited and aligned using MEGA 6 (version6.06; ). Amino acid sequences were deduced using the invertebrate mitochondrial genetic code. Calculations of nucleotide and amino acid p-distances were performed with MEGA 6 (with 1,000 bootstrap replicates) (version6.06; ).Following a similar approach than to look for the presence of two intraspecific “F and M” clades in S. plana and Y. hyperborea, maximum likelihood (using RAxML version 8.2.8; Stamakis, 2014) with bootstrap analyses (1,000 replicates) and Bayesian phylogenies (using MrBayes v3.2.6; ; ; ) were performed on cox1 nucleotide sequences of both species with Soletellina virescens(Bivalvia, Veneridae, Genbank accession number: JN859944) and Yoldia eightsii(Bivalvia, Nuculanida, Genbank accession number: KJ571167) as outgroups for S. plana and Y. hyperborea, respectively (i.e., closest sequences according to BLAST search). Bayesian Information Criterion (BIC) () implemented in PartitionFinder (v1.1.1; ) was used to estimates the best-fitting models of evolution. Figtree (v1.4.2; ) was used to edit the phylogenetic trees.S. plana and Y hyperborea were included in an expanded phylogenetic analyses designed to verify molecular relationships among DUI species in general. Maximum likelihood (ML), maximum parsimony (MP) and Bayesian phylogenies were thus performed on F and M cox1 nucleotide and amino acid sequences from all DUI species known to date and Octopus vulgaris(Mollusca: Octopoda) and Aplysia californica(Mollusca: Gastropoda) were used as outgroup taxa. F and M cox1 sequences other than those obtained in the present study for S. plana and Y. hyperborea were retrieved from Genbank; the complete phylogenetic dataset is shown in . Cox1 sequences were aligned using MEGA 6 (version6.06; ) and the best-fitting models of DNA evolution were selected using PartitionFinder (v1.1.1; ) according to BIC values (Schwartz,1978). Best models were applied whenever possible. Data were partitioned according to nucleotide position and gaps were treated as missing data.ML analyses were conducted with RAxML (version 8.2.8; ). A non-parametric bootstrap () analysis was performed, using 1,000 bootstrap replicates and 20 ML searches, to assess nodal support for both trees. Outgroups were set to be paraphyletic to the monophyletic ingroup. MP analyses were carried out using PAUP software (v 4.0a147; ). To optimize the chance of having the best topology, 100 random stepwise additions under tree-bisection reconnection branch swapping were applied (). Reliability of the internal nodes was evaluated by 1,000 pseudoreplicates using the heuristic search algorithm. Bayesian analyses were conducted using MrBayes (v3.2.6; ; ; ). Each analysis consisted of two independent runs of 4 MC3 chains that were run for 10,000,000 generations. Convergence was estimated through the log likelihood value of trees, potential scale reduction factor (PSRF) and standard deviation of average split frequencies sampled every 1,000 generations (). Trees were sampled every 100 generations and a majority-rule consensus tree was computed after discarding the first 25% as burn-in. Fidelity of the topology was evaluated with the posterior probabilities from the consensus tree. All phylogenetic trees were edited for easier readability using FigTree (v1.4.2; ).All the alignments used for phylogenetic reconstruction are available here (https://dx.doi.org/10.6084/m9.figshare.3798789.v1). […]

Pipeline specifications

Software tools MEGA, RAxML, MrBayes, PartitionFinder, FigTree, PAUP*
Application Phylogenetics