Computational protocol: Host-specific Dactylogyrus parasites revealing new insights on the historical biogeography of Northwest African and Iberian cyprinid fish

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Protocol publication

[…] Dactylogyrus species collected from cyprinids in Africa and Europe were sequenced to obtain partial sequences of 28S rDNA and partial sequences of 18S rDNA and the ITS1 region. Dactylogyrus specimens were individually removed from ethanol and dried by using a vacuum centrifuge. Genomic DNA extraction was performed following a standard protocol (DNeasy Blood & Tissue Kit, Qiagen, Hilden, Germany). Partial 28S rDNA was amplified using the forward primer C1 (5′-ACC CGC TGA ATT TAA GCA-3′) and the reverse primer D2 (5′-TGG TCC GTG TTT CAA GAC-3′) []. PCR followed the protocol included in Šimková et al. []. Partial 18S rDNA and the entire ITS1 region were amplified in one round using the primers S1 (5′-ATT CCG ATA ACG AAC GAG ACT-3′) and IR8 (5′-GCT AGC TGC GTT CTT CAT CGA-3′) [] that anneal to 18S and 5.8S rDNA, respectively. Each amplification reaction for partial 18S rDNA and the ITS1 region was performed in a final volume of 15 μl, containing 1.5 U of Taq polymerase, 1× buffer, 1.5 mM MgCl2, 0.2 mM of each dNTP, 0.5 μM of each primer, and 2.5 μl of DNA (20 ng/μl). PCR was carried out using the following steps: 2 min at 94 °C, followed by 40 cycles of 1 min at 94 °C, 1 min at 53 °C and 1 min 30 s at 72 °C, and 10 min of final elongation at 72 °C. The PCR products were checked on 1.5% agarose gel, purified using ExoSAP-IT kit (Ecoli, SK) following a standard protocol and directly sequenced using the PCR primers and BigDye Terminator Cycle sequencing kit (Applied Biosystems, Foster City, CA). Sequencing was carried out using an ABI 3130 Genetic Analyser (Applied Biosystems). Sequences were analysed using Sequencher 4.7 (Gene Codes Corp., Ann Arbor, MI, USA), and new sequences were deposited in GenBank (see Table  for accession numbers). The sequences of other Dactylogyrus species parasitizing European and Asian cyprinid species were retrieved in GenBank (Table ) and were used for phylogenetic analyses. [...] The first alignment included the partial 28S rDNA sequences of 55 Dactylogyrus species. Among them, 36 were newly sequenced for this study. The sequences of the other 19 Dactylogyrus species as well as the sequences of three species of the Dactylogyridae (Euryhaliotrematoides pirulum Plaisance & Kritsky, 2004, Euryhaliotrematoides triangulovagina Yamaguti, 1968 and Aliatrema cribbi Plaisance & Kritsky, 2004 with accession numbers AY820618, AY820619 and AY820612, respectively), used as the outgroup in the phylogenetic analyses, were retrieved from GenBank. The second alignment included the partial 18S rDNA sequences and the ITS1 region of 26 Dactylogyrus species belonging to Dactylogyrus lineage III. D. vistulae Prost, 1957 and D. sphyrna Linstow, 1978 were used as the outgroup in the phylogenetic analyses based on the 18S rDNA and ITS1 sequences.All sequences of a given dataset were aligned using ClustalW multiple alignments [] in Bioedit v. 7.2.5 []. The phylogenetic analyses were performed using unambiguous alignments. Gaps and ambiguously aligned regions were removed from alignments using GBlocks v. 0.91 []. The best-fit DNA evolution model was determined using the Akaike’s information criterion (AIC) in JmodelTest 2.1.10 [, ]. Phylogenetic trees were inferred using minimum evolution (ME) analysis using PAUP* 4b10 [], maximum likelihood (ML) analysis using PhyML 3.0 [], and Bayesian inference (BI) analysis using MrBayes 3.2 []. Supports for internal nodes were computed from a bootstrap re-sampling procedure [] with 1000 pseudoreplicates for ME, and 500 pseudoreplicates for ML using the TBR algorithm. A search for the best ML tree was performed using the TBR branch-swapping algorithm. Bayesian inference (BI) analyses were performed using four Monte Carlo Markov chains running on 1000,000 generations for each data set, with trees being sampled every 100 generations. The “burn-in” asymptote was estimated by plotting the number of generations against the log likelihood scores for the saved trees, and all the trees (25%) before stationarity were discarded as “burn-in”. The posterior probabilities of the phylogeny and its branches were determined for all trees left in the plateau phase with the best ML scores.The mapping of characters was performed in Mesquite 3.2 []. Prior to the mapping of characters, a new alignment was prepared using partial 28S rDNA sequence data from 55 Dactylogyrus species. Phylogenetic reconstruction using BI analysis was performed as described above. Dactylogyrus bicornis Malewitzkaja, 1941 was used for rooting the phylogenetic tree following the output of phylogenetic analyses using the external outgroup. The first character mapped onto the phylogenetic reconstruction represents fish lineages, i.e. different fish families as applied in Yang et al. [] (Acheilognathinae, Xenocyprinae, Gobioninae, Leuciscinae and Cyprininae as different character states). The second character represents fish lineages including the branching within Cyprininae, the target group of our study (Cyprinini, Labeonini, Torini, Smiliogastrini, Barbini including the genus Barbus Cuvier & Cloquet, 1816, and Barbini including the genus Luciobarbus were used as the character states). The revised classification of the subfamily Cyprininae by Yang et al. [] was adopted for this mapping. The last character represents the distribution of host species with the following character states applied: southern Asia including Southeast Asia, a large part of Eurasia, Europe with only West Asia, the Iberian Peninsula, the Balkan Peninsula, Northwest Africa and West Africa. The distribution of cyprinid species follows Froese & Pauly []. […]

Pipeline specifications

Software tools Sequencher, Clustal W, BioEdit, Gblocks, jModelTest, PhyML, MrBayes
Application Phylogenetics
Organisms Toxoplasma gondii, Danio rerio