Computational protocol: Aphanius arakensis, a new species of tooth-carp (Actinopterygii, Cyprinodontidae) from the endorheic Namak Lake basin in Iran

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Protocol publication

[…] Total genomic DNA was extracted according to phenol/chloroform procedures (). A 900 base pairs (bp) fragment of the cytochrome b gene was successfully amplified via PCR using the primers (forward: Glu-F, 5’ - AACCACCGTTGTATTCAACTACAA-3’; reverse: ThrR, 5’-CCTCCGATCTTCGGATTACAAGACCG-3’ (). Amplification was performed in a thermal cycler programmed as follows: initial 94 °C for 3 min, 35 cycles at 94 °C for 50 s, 56 °C for 45s, 72 °C for 1 min, followed by a final extension at 72 °C for 5 min. Sequencing was performed by Macrogen company, South Korea. Cytochrome b nucleotide sequences were edited with BioEdit and aligned through Geneious pro v5.4 (). Additional Aphanius sequences were obtained from the NCBI GenBank ( and included in the analyses (see above). The achieved cytb sequences for the here studied Aphanius populations were deposited in GenBank under numbers JX154880–JX154898. Maximum likelihood-based phylogenetic relationships were estimated by using the program SeaView version 4 (). The best-fit model of nucleotide substitution was obtained using the program JmodelTest 0.1.1 (). Accordingly, the GTR + I + G model (= General Time Reversible model + proportion of Invariable sites + Gamma-shaped distribution of rates across sites) was chosen. Maximum parsimony based phylogenetic relationships were estimated using the program SeaView version 4 () with 100 heuristic searches using random additions of sequences and implementing the Close-Neighbor-Interchange (CNI) on random tree algorithm. To test this phylogeny, bootstrap method using 2000 replication was used. To document the degree of homoplasy and degree to which potential synapomorphy is exhibited on the tree, the Consistency Index (CI) and the Retention Index (RI) were calculated by using the parsimony model within the Mesquite system for phylogenetic computing (). The Neighbor Joining (NJ) distance-based phylogenetic relationships were estimated by using the computer program Geneious pro v5.4 (). The HKY85 model () of molecular evolution was used with gamma distributed among site rate variation. There were a total of 771 positions in the final dataset. […]

Pipeline specifications

Software tools BioEdit, Geneious, SeaView, jModelTest, Mesquite
Application Phylogenetics
Organisms Cyprinus carpio, Hemisus marmoratus, Dipturus trachyderma
Diseases Focal Nodular Hyperplasia