Computational protocol: Screening of spider mites (Acari: Tetranychidae) for reproductive endosymbionts reveals links between co infection and evolutionary history

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Protocol publication

[…] All sequences were aligned and manually corrected using BioEdit. Prior to phylogenetic analysis, the best-fitting nucleotide models were determined by jModeltest version 2. Wolbachia phylogeny was determined by reconstructing Bayesian Inference (BI) and Maximum-Likelihood (ML) trees of the concatenated data set of MLST genes from this study and PubMLST database (http://www.pubmlst.org/wolbachia/). Bayesian analyses were performed in MrBayes 3.1 under the GTR + I + G model. Four Markov chains were run for 15,000,000 generations with sampling every 100 generations, and the first 37,500 generations were discarded. Convergence of runs was assumed when split frequencies reached <0.01. ML analysis was also conducted for the concatenated data set of MLST genes in MEGA 5.0 under the GTR + I + G model, bootstrap pseudoreplicates were calculated 1,000 times.The phylogenetic analyses of Cardinium, Spiroplasma and Rickettsia were performed using Bayesian Inference (BI) and Maximum-Likelihood (ML) estimation for the sequences of gyrB gene, rpoB gene and gltA gene, respectively. The evolutionary models used were as follows: gyrB- GTR + I + G, rpoB-GTR + I + G, and gltA-HKY+G. For each Bayesian analysis, four Markov chains were run 1,000,000 generations with sampling every 100 generations, and the first 25% of samples were discarded as burn-in. Convergence of runs was assumed when split frequencies reached <0.01. ML analyses were conducted in MEGA 5.0, bootstrap pseudoreplicates were calculated 1,000 times.In addition, Bayesian and ML phylogenetic trees of spider mite were constructed using a supermatrix that consisting of the COI, 18SrRNA and 28SrRNA sequences. Analyses were performed in MrBayes 3.1 and MEGA 5.0 under the GTR+G+I model, respectively. [...] Endosymbiont infection patterns were first compared on the level of genera and species of spider mites. Two extensively sampled species, T. truncatus and A. viennensis were selected to test the differences of Wolbachia and Cardinium infections. Because co-infection was common in spider mites, co-infection levels were evaluated with a Spearman correlation analysis of the presence/absence of each endosymbiont within spider mite individuals against the presence/absence of other endosymbionts. The analysis was performed with Stata 11.0. In addition, we tested for correlation between host genetic distance and the corresponding Wolbachia, Cardinium strains genetic divergence to infer their infection patterns. Correlation analyses were performed using a Mantel test in Arlequin 3.1 with 1000 permutations. Genetic distance matrices of the concatenated MLST dataset for Wolbachia, gyrB genes for Cardinium and the concatenated nuclear and mitochondrial loci for spider mites were calculated with the Kimura 2-parameter model in MEGA 5.0. […]

Pipeline specifications

Software tools BioEdit, jModelTest, MrBayes, MEGA, Stata, Arlequin
Databases PubMLST
Applications Phylogenetics, Population genetic analysis
Organisms Wolbachia endosymbiont of Armadillidium vulgare str. wVulC, Escherichia coli
Diseases Rickettsia Infections, Coinfection