Gyrodactylus spp.

Rahmouni, Chahrazed, Seifertová, Mária, Bean, Megan G. & Šimková, Andrea, 2024, Intraspecific variation in Gyrodactylus mediotorus and G. crysoleucas (Gyrodactylidae) from Nearctic shiners (Leuciscidae): evidence for ongoing speciation, host-switching, and parasite translocation, Parasite (Paris, France) 31 (29), pp. 1-14 : 4

of Gyrodactylus spp.

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.