Computational protocol: Reticulate evolution in stick insects: the case of Clonopsis (Insecta Phasmida)

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Protocol publication

[…] Total genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen). Partial sequence of cox2 was amplified and directly sequenced according to []. The primers utilized were TL2-J-3034 and TK-N-3785 []. Sequencing covered 639 bp coding for 213 aminoacids of the Cytochrome Oxidase subunit 2 and corresponds to the gene region sequenced in several insect orders []. All sequences were aligned with the Clustal algorithm of MEGA 3.1 []. A Bayesian analysis was performed using MrBayes 3.1 (20,000,000 generations; []) (Figure ). A Templeton's network [] was obtained with TCS 1.21 [] (Figure ). Bacillus grandii grandii, Bacillus atticus atticus and Bacillus rossius redtenbacheri were utilized as outgroups in phylogenetic reconstructions based on cox2. [...] DNA fingerprinting with selectively neutral AFLP markers was produced according to [], using EcoRI/TaqI as restriction enzymes. After a preliminary screening, three highly polymorphic primer pairs carrying ACA/AAC, ACA/AAG and ATG/AAC as selective nucleotides were assayed on 100 ng of total genomic DNA. AFLP fragments were separated by electrophoresis on 8% polyacrylamide gels and the polymorphisms were visually scored as dominant markers, coding with 1 the presence and with 0 the absence of the band. Markers with more than 5% of missing data were removed from the definitive dataset.The genetic relationships at individual level were assessed by the Factorial Correspondence Analysis (FCA), a statistical method enabling analysis and description graphically and synthetically of two-way or multi-way contingency tables. For this, the software GENETIX 4.05 was used [] (see additional file ).To assess the molecular structure of Moroccan Clonopsis we used a model-based bayesian procedure as implemented in the software STRUCTURE 2.2 [,]. This model enables identification of the K (unknown) populations of individuals, and the probabilistic assignment of each individual to one or several populations if its genotype indicates that it is admixed. The model assumes that the loci are unlinked and at linkage equilibrium. STRUCTURE version 2.2 calculates a logarithmic probability for the data being assigned to a given number of clusters, based on minimizing linkage between clusters, and maximizing linkage within.A Minimum Evolution tree (ME, Figure ) based on AFLP markers was calculated using PAUP 4.0 []; support for each node was obtained using bootstrap (1000 replicates, []). Given the results of cox2, C. maroccana has been used as an outgroup in the AFLP-based tree, since more phylogenetically distant species (such as Bacillus ssp.) would not be easily comparable using AFLP markers, which are more suitable for genetic analyses of population strains or closely related species [], and have been proved to be resolving for phylogenetic structure in rapidly evolving systems []. […]

Pipeline specifications

Software tools MEGA, MrBayes, PAUP*
Application Phylogenetics