Gyrodactylus undetermined

Gyrodactylus specimens were subjected to DNA amplification and sequencing. Specimens stored in 96% ethanol were dried using an Eppendorf 5301 Concentrator. Total genomic DNA was extracted using the DNeasy ® Blood and Tissue Kit (QIAGEN) following the protocol for the purification of total DNA from animal tissues. Two nuclear ribosomal DNA markers suitable for the differentiation of Gyrodactylus spp. were used (for instance, see [10, 33, 60, 75, 113]). A fragment spanning ITS 1, 5.8S and ITS 2 (ITS regions) was amplified using forward primer ITS 1F (5 0 -GTTTCCGTAGGTGAACCT-3 0 ) [90], complementary to the sequence at the 3 0 end of the 18S rRNA gene, and reverse primer ITS 2 (5 0 -TCCTCCGCTTAGTGATA- 3 0 ) [17], complementary to the sequence at the 5 0 end of the 28S rRNA gene [17]. A partial fragment of 18S rDNA containing the V 4 region, which exhibits intraspecific variation in Gyrodactylus [16, 60], was amplified using the primer pairs PBS18SF (5 0 -CGCGCAACTTACCCACTCTC-3 0 ) and PBS18SR (5 0 -ATTCCATGCAAGACTTTTCAGGC-3 0 ) [13]. Polymerase chain reactions (PCRs) for the 18S rDNA gene and ITS region were performed in a final volume of 30 µL, containing 1xPCR buffer, 1.5 mM MgCl 2, 200 µM of each dNTP, 0.5 µM of each primer, 1 U of Taq DNA Polymerase (Thermo Scientific) and 5 µL of template DNA. The PCRs were carried out in the Mastercycler ep gradient S (Eppendorf) using the following steps: i) ITS regions: an initial denaturation at 96 ° C for 3 min, followed by 39 cycles of denaturation at 95 ° C for 50 s, annealing at 52 ° C for 50 s, and an extension at 72 ° C for 50 s, and a final elongation at 72 ° C for 7 min; and ii) 18S region: an initial denaturation at 95 ° C for 3 min, followed by 39 cycles of denaturation at 94 ° C for 1 min, annealing at 54 ° C for 45 s, and an extension at 72 ° C for 1 min 30 s, and a final elongation at 72 ° C for 7 min. PCR products were electrophoresed on 1.5% agarose gels stained with Good View (SBS Genetech, Bratislava, Slovakia) and then purified using EPPiC Fast (A&A Biotechnology, Gdynia, Poland), following the manufacturer’ s protocol. The purified PCR products were sequenced directly in both directions using the PCR primers. Sanger sequencing was carried out using a BigDye ® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and an Applied Biosystems 3130 Genetic Analyzer. Newly-generated DNA sequences were assembled and edited using Sequencher software v. 5.0 (Gene Codes, Ann Arbor, MI, USA) and aligned using ClustalW [101] as implemented in MEGA v. 11 [99]. Sequences were further checked by the nBLAST Search Tool (https://blast.ncbi.nlm. nih.gov/Blast.cgi: blastn, default settings, access date: 11/09/ 2023) to assess any similarity to available congeners, then deposited in GenBank under accession numbers indicated in the Results section. Genetic divergences were estimated using uncorrected p -distances in MEGA v. 11 [99].

A phylogenetic tree was reconstructed based on the newly generated ITS sequences of G. crysoleucas and G. mediotorus, together with 31 DNA sequences representing 29 Gyrodactylus spp. retrieved from the GenBank database. The dataset included Gyrodactyloides bychowskii Albova, 1948, Ieredactylus rivuli Schelkle et al., 2011, and Laminiscus gussevi (Bychowsky and Polyansky, 1953) Pálsson and Beverley-Burton, 1983 as the outgroup following Rahmouni et al. [81] (Table 1). Nucleotide sequences were aligned by multiple alignments using MAFFT v7.505 [45] and trimmed using trimAl v1.2rev57 [11] through plugins installed in PhyloSuite v1.2.3 [108, 110]. The plugin of ModelFinder v2.2.0 [44] in PhyloSuite v1.2.3 [108, 110] was used to determine the best-fit substitution model for the dataset. A final alignment of 33 ITS sequences composed of 927 bp was used to infer the phylogenetic relationships using Maximum Likelihood (ML) and Bayesian Inference (BI). ModelFinder v2.2.0 [44] indicated GTR+F+G4 as the bestfitting evolutionary models for ML analysis based on the corrected Akaike Information Criterion (AICc) [40, 97]. ML trees were inferred using IQ-TREE v1.5.5 [69] based on the selected model employing a sub-tree pruning and re-grafting (SPR) branch-swapping algorithm. The branch support (bootstrap support, BS) was estimated using ultrafast bootstrap approximation [64] with 1,000 replicates. BI analysis was performed using MrBayes v3.2.1 [91] and applying the GTR+I +G evolutionary model with two independent Markov Chain Monte Carlo (MCMC) simulations (six chains, 10 6 generations, sampling frequency 100, 25% burn-in to obtain the consensus tree and posterior probability values (PP)). Chain stationarity and parameter convergence were assessed in TRACER v1.7.1 [84], with effective samples sizes (ESS) always>200 for all parameters, and via the average standard deviation of split frequencies (always well below 0.01), and post burn-in trees were summarized in a 50% majority rule consensus tree. The resulting ML and BI trees were visualized in FigTree v1.4.4 (http:// tree.bio.ed.ac.uk) and manually edited on Photoshop v13.0.