Computational protocol: A new endogean, anophthalmous species of Parazuphium Jeannel from Northern Morocco (Coleoptera, Carabidae), with new molecular data for the tribe Zuphiini

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Protocol publication

[…] Total genomic DNA for the single specimen of Parazuphium aguilerai sp. n. was extracted using QIAGEN Dneasy tissue kit (Qiagen, Hilden, Germany). To characterize the new species we amplified fragments of six genes, four mitochondrial and two nuclear: 3' end of cytochrome c oxidase subunit (cox1); a single fragment including the 3' end of the large ribosomal unit (rrnL), the whole tRNA-Leu gene (trnL) and the 5' end of the NADH dehydrogenase 1 (nad1); 5' end of the small ribosomal unit, 18S rRNA (SSU); and an internal fragment of the large ribosomal unit, 28S rRNA (LSU). Primers used are given in . Additionally, we extracted DNA from one specimen of Parazuphium cf. baeticum (K. and J. Daniel 1898), Zuphium olens Rossi 1790, Ildobates neboti Español 1966 and several other outgroups among Carabidae (), which were amplified for the same molecular gene fragments. PCR reactions were made using PuReTaq Ready-To-Go PCR beads (GE Healthcare, UK) and standard conditions [39 cycles using 48–50°C as annealing temperature]. New sequences have been deposited in GenBank (NCBI) with Acc. Nos JF778779-JF778845. Each individual gene matrix was aligned in MAFFT with the Q-ins-i option and default parameters. The four genes fragments were concatenated to get a final dataset of 20 taxa and 3376 bp that was employed in phylogenetic analyses. shows taxa information, source and accession number for each DNA sequence. [...] Bayesian phylogenetic analyses (BA) were performed with MrBayes v.3.1. (, ), partitioning by gene with a GTR+G model applied to each partition. Two independent runs of 20,000,000 generations were conducted, each with three hot and one cold chain, whereby trees were sampled every 100 generations. Sampled trees were analysed with Tracer v.1.5 () and their half compact consensus tree was calculated with a burning value of 10% with node posterior probabilities used as support values, checking for an appropriate degree of convergence between chains with the effective sample size in Tracer v.1.5. MrBayes was run on-line at the freely available computational service of Bioportal ( Trees were visualized in FigTree v.1.3.1 (). […]

Pipeline specifications

Software tools MAFFT, MrBayes, FigTree
Databases BioPortal
Application Phylogenetics
Organisms Serinus canaria