Computational protocol: Molecular characteristics of representatives of the genus Brachylecithum Shtrom, 1940 (Digenea, Dicrocoeliidae) with comments on life cycle and host specificity

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[…] For molecular analysis, DNA was extracted from ethanol-fixed, single specimens of adults, metacercariae, and sporocysts (with cercariae) using the Qiagen DNeasy™ tissue kit and Genoplast Tissue Genomic DNA Extraction Mini Kit according to the manufacturer’s instructions. The partial nuclear ribosomal 28S rDNA (D1–D3) gene was amplified using the forward primer DLS1 and the reverse primer 1500R. The thermocycling profile was as follows: 3 min denaturation at 94 °C; 35 cycles of 30 s at 94 °C, 1 min at 55 °C, and 1 min at 72 °C; and 10 min extension at 72 °C. The partial mitochondrial cytochrome c oxidase subunit 1 (cox1) gene was amplified using the forward primers COIA3/COIDF1 and the reverse primers COITR1/COIDR1 with the following thermocycling profile: 3 min denaturation at 94 °C; 35 cycles of 30 s at 94 °C, 30 s at 50 °C, and 1 min at 72 °C; and 5 min extension at 72 °C. PCR and sequencing primers are listed in Table . The amplified products were purified using a PCR purification kit (Genoplast, Poland) according to the manufacturer’s instruction and sequenced directly on an ABI Prism 373xl automated sequencer (Applied Biosystems, USA) using ABI BigDye™ (Applied Biosystems) by Genomed (Poland). DNA products were sequenced in both directions using the PCR primers and sequencing primers (lsrDNA). Forward and reverse sequences were assembled and aligned using Vector NTI Advance 11.0 (Invitrogen, Life Technologies) software and submitted to GenBank. Accession numbers of adults and larvae are listed in Table . [...] The first phylogenetic analysis was based on partial 28S rDNA gene with the newly generated sequences of Brachylecithum and selected sequences of dicroceoliids from GenBank (Table ). The nucleotides were aligned with AlignX (Vector NTI Advance 11.0), with default settings. Regions that could not be unambiguously aligned were excluded from the analysis. JModelTest version 2.1.4 (Darriba et al. ; Guindon and Gascuel ) was used to select models of evolution using the Akaike information criterion (AIC). The chosen parameters of the substitution model were GTR + G. Phylogenetic trees were constructed using Bayesian inference (BI) as implemented in the MrBayes program version 3.2 (Ronquist and Huelsenbeck ) with Macvicaria magellanica (Opecoelidae) (Laskowski et. al. ) as an outgroup.The sequences of a partial region of the cytochrome c oxidase subunit 1 gene were obtained for adults of Brachylecithum and Lyperosomum as well as from sporocysts, cercariae, and metacercariae isolated from their snail hosts (Table ). All generated mitochondrial DNA sequences were used in the next analysis using the same parameters and the same software as in the 28S rDNA analysis, with the HKY + G + I models of evolution.Subsequently, partial 28S rDNA sequences were concatenated with partial mitochondrial cox1 sequences in the second dataset for independent phylogenetic analysis. In mixed analyses (28S + cox1), the data were partitioned into two character sets: (1) 28S and (2) mt data as nucleotides. The phylogenetic analysis was carried out using the BI in the MrBayes program. Likelihood settings were set to the GTR + G model (28S) and HKY + G (cox1) in accordance with the AIC results. In concatenated datasets, parameters were estimated separately. The analysis of combined sequences of 28S + cox1 was provided with Lyperosomum collurionis as an outgroup (based on 28S rDNA analysis). Phylogenetic trees were visualized using the TreeView software (Page ). The evolutionary divergence between the sequences was estimated using MEGA6 software (Tamura et al. ); analyses of the number of nucleotide substitutions were conducted using the maximum composite likelihood model (Tamura et al. ). […]

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