Computational protocol: Distribution of endangered Italian gudgeonRomanogobiobenacensis(Cypriniformes,Cyprinidae, Gobioninae) withremarks on distinguishing morphological characters

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[…] Measurements were made according to . All measurements were made point-to-point with a digital calliper and recorded to the nearest of 0.1 mm. Vertebrae counts are given according to . Last two rays in dorsal and anal fins based on a single pterygiophore were counted as 1½ ray. Unbranched rays in dorsal and anal fins were counted from radiographs. In total, 30 morphometric indices were used for descriptions and statistical analyses as in Table and 18 meristic characters as in Table were examined. All characters were obtained from specimens of both sexes and combined in analyses and tables. A Mann-Whitney U Test and a Discriminant Function Analysis (DFA) were performed using STATISTICA v6.0 and PRIMER v6.1.9 to identify the most important characters that contribute to the differentiation of the two species and visualise the classification of the Reka and Mirna specimens into one of them.(Abbreviations: SL, standard length; HL, lateral head length including skin fold; HDBI, Croatian Biological Research Society; NMW, Naturhistorisches Museum Wien; PZC, private collection of Primož Zupančič) [...] Besides species identification based on morphological features, species were characterised using sequences of cytochrome b gene (cytb), which is commonly used mtDNA genetic marker for species affiliation of European cyprinids (e.g. , , ). Total DNA was extracted from pectoral fin tissue with the Qiagen DNeasy Blood and Tissue Kit (Qiagen, Germany) following the manufacturer protocol. After extraction, total genomic DNA was stored on -20°C until the polymerase chain reaction (PCR) was conducted. The primers used for cytochrome b were GluF and ThrR (). The PCR was carried out with the HotStarTaq Master Mix Kit (Qiagen). PCR reactions were prepared in a total volume of 50 µL comprised of 2.5 U HotStarTaq DNA Polymerase, 1.5 mM MgCl2, 200 µM each dNTP, 0.2 µM of each primer and 20 ng of DNA template. The amplification process was conducted with the same conditions as described in . Purification and sequencing of the PCR products were prepared by Macrogen Inc. (Seoul, South Korea) using the same primers used for gene amplification. Purified PCR products were sequenced on ABI 3730XL DNA Analyzer (Applied Biosystems, Foster City, USA). Sequence chromatograms were analysed using SEQUENCHER (version 5.3; Gene Codes Corp., Ann Arbor, USA) and aligned by eye.The obtained sequences in this study (1141 base pairs long, bp) were examined using Nucleotide Basic Local Alignment Search Tool (Nucleotide BLAST; to screen for the most similar sequences in the GenBank nucleotide database (National Center for Biotechnology Information, U.S. National Library of Medicine, USA).The Median-Joining (MJ) haplotype network () was used to infer the intraspecific relations in R. benacensis with cytb sequences obtained in this study and 342 bp sequences which were downloaded from the GenBank (). The MJ network was computed using PopART (Population Analysis with Reticulate Trees) v1.7 ().Phylogenetic tree reconstructions were conducted using the cytb sequences obtained in this study and the available sequences belonging to Gobio and Romanogobio (1141 bp) from the GenBank (, , , , , , , , ; Table ). Sequences originating from the tench Tinca tinca (Linnaeus), the European bitterling Rhodeus amarus (Pallas) and the stone moroko Pseudorasbora parva (Temminck & Schlegel) were used as an outgroup. Newly obtained sequences in this study were deposited in the GenBank under accession numbers shown in Table (will be available for publication). Phylogenetic reconstructions were inferred using three methods (Maximum Likelihood – ML, Bayesian Inference – IB and Maximum Parsimony – MP). The best-fit evolutionary model used in ML and IB was computed using jModelTest2 (version 2.1.6; ) with the Bayesian information criterion (BIC) as implemented on the Cipres Science Gateway (version 3.1;; ). Best-fit model of nucleotide substitution was Generalised Time Reversible (GTR) () with a gamma distributed rate variation among sites (+G) and a significant proportion of invariant sites (+I). The ML was run using RAxML-HPC2 Workflow on XSEDE (version 8.2.8; ) on the Cipres Science Gateway with optimized parameters. For ML analysis, 200 search replicates to find the ML tree and 1000 nonparametric bootstrap replicates under the GTRGAMMA model were applied. The IB was run in MrBayes 3.2 () on the Cipres Science Gateway. Two independent runs with four MCMC chains were run for 50 million generations and sampled every 5000 generations, with temperature parameter set to 0.2 and the first 12.5 million generations discarded as burn-in. The convergence of runs was screened using AWTY () while effective sample sizes of parameters were checked using TRACER 1.5 (). The MP analysis was performed in MEGA 6.06 (). The MP tree was obtained using the Subtree-Pruning-Regrafting algorithm () with search level 1 in which the initial trees were obtained by the random addition of sequences (10 replicates). Nodes in phylogram which have bootstrap values P ≥ 70 in ML and MP, and posterior probabilities (pp) values ≥ 0.95 in IB were considered supported.Three user trees (“Tree 1: R. benacensis sister taxon for R. kesslerii and R. banaticus”, “Tree 2: R. benacensis sister taxon for all Romanogobio”, and “Tree 3: R. benacensis sister taxon for all Gobio”) were analysed using tree topology tests [1sKH – one sided KH test based on pairwise SH tests (, , ); SH – Shimodaira-Hasegawa test (2000); ELW – Expected Likelihood Weight (Strimmer-Rambaut 2002); 2sKH – two sided Kishino-Hasegawa test (1989)]. All topology tests were computed in TREE-PUZZLE v5.3rc16 (). […]

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