All samples were analyzed in order to determine DNA content and to sequence two mtDNA markers: the control region (CR) and the cytochrome c oxidase subunit I (COI).
Genomic DNA was extracted both from muscle and hepatopancreas tissues using the Wizard® Genomic DNA Purification Kit (Promega Corporation, Madison, WI, USA) and DNeasy® Tissue Kit di Qiagen (Hilden, Germany, EU) and a good quality of DNA was obtained from both.
The primers used for mtDNA control-region amplification were previously published [34 (link)], and the reaction conditions were as follows: initial denaturation at 95 °C for 2 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 1 min and elongation at 72 °C for 1 min, followed by final extension at 72 °C for 10 min. Besides, a fragment of the mitochondrial gene coding for the cytochrome c oxidase subunit I was amplified using the primers published by Folmer et al., 1994 [40 (link)].
The PCR reactions contained 1X Buffer GoTaq, 2.5 mM of each deoxyribonucleotide triphosphate (dNTP), 0.3 µM of each primer, 0.03 U/µL of GoTaq DNA polymerase (Promega Corporation; Madison, WI, USA), 30 ng of genomic DNA and H2O to a final volume of 25 µL. The PCR amplification was carried out as follows: 94 °C for 2 min, followed by 35 cycles of 95 °C for 30 s, 55 °C for 1 min, 72 °C for 1 min and then 72 °C for 10 min. The PCR fragments were purified using exonuclease I and alkaline phosphatase (ExoSAP-IT enzymatic system-USB Corporation, Cleveland, OH, USA) and subsequently Sanger-sequenced with the following primers: LCO1490 and HCO2198 [40 (link)] for COI sequencing and a specifically designed primer (5′-CTTCTAAAAATGTTCCCCCC-3′) for CR region, by using Primer3 software. Sequences were aligned to the sequence considered as reference mitogenome (JN991197, [41 (link)]) for the haplotype (HT) annotation through the Sequencher software (www.genecodes.com (accessed on 12 August 2022)). In order to ensure the uniformity of all sequences, they were trimmed at the same range, from the nucleotide position (np) 4717 to np 5434 for the mtDNA CR marker, and from np 78 to np 651 for the COI region.
The COI region was investigated in 53 samples, selecting at least 2 samples for each CR HT from each basin.
Mitochondrial DNA sequence variation parameters were estimated by using DnaSP 5.1 software (www.ub.edu/dnasp/index_v5.html (accessed on 12 August 2022)). Haplotype number codes were assigned to each sample as previously classified in Dörr et al., 2021 [35 (link)] and with increasing numbering only for our convenience.
The evolutionary relationships among the haplotypes here identified were evaluated for each single mtDNA marker through median-joining trees built using Network software v.10.2.
In order to graphically display and summarize the mitogenetic relationships among the analyzed populations, a Principal Component Analysis (PCA) based on concatenated mtDNA (COI and CR) haplotypes was performed using the Excel software implemented by XLSTAT. Haplotype frequencies were used as input data.
Finally, to construct a Bayesian phylogeny for the two combined mtDNA regions, we used BEAST.v2.7.3 and the analyses were run for 100,000,000 generations [42 (link)].
Genomic DNA was extracted both from muscle and hepatopancreas tissues using the Wizard® Genomic DNA Purification Kit (Promega Corporation, Madison, WI, USA) and DNeasy® Tissue Kit di Qiagen (Hilden, Germany, EU) and a good quality of DNA was obtained from both.
The primers used for mtDNA control-region amplification were previously published [34 (link)], and the reaction conditions were as follows: initial denaturation at 95 °C for 2 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 1 min and elongation at 72 °C for 1 min, followed by final extension at 72 °C for 10 min. Besides, a fragment of the mitochondrial gene coding for the cytochrome c oxidase subunit I was amplified using the primers published by Folmer et al., 1994 [40 (link)].
The PCR reactions contained 1X Buffer GoTaq, 2.5 mM of each deoxyribonucleotide triphosphate (dNTP), 0.3 µM of each primer, 0.03 U/µL of GoTaq DNA polymerase (Promega Corporation; Madison, WI, USA), 30 ng of genomic DNA and H2O to a final volume of 25 µL. The PCR amplification was carried out as follows: 94 °C for 2 min, followed by 35 cycles of 95 °C for 30 s, 55 °C for 1 min, 72 °C for 1 min and then 72 °C for 10 min. The PCR fragments were purified using exonuclease I and alkaline phosphatase (ExoSAP-IT enzymatic system-USB Corporation, Cleveland, OH, USA) and subsequently Sanger-sequenced with the following primers: LCO1490 and HCO2198 [40 (link)] for COI sequencing and a specifically designed primer (5′-CTTCTAAAAATGTTCCCCCC-3′) for CR region, by using Primer3 software. Sequences were aligned to the sequence considered as reference mitogenome (JN991197, [41 (link)]) for the haplotype (HT) annotation through the Sequencher software (
The COI region was investigated in 53 samples, selecting at least 2 samples for each CR HT from each basin.
Mitochondrial DNA sequence variation parameters were estimated by using DnaSP 5.1 software (
The evolutionary relationships among the haplotypes here identified were evaluated for each single mtDNA marker through median-joining trees built using Network software v.10.2.
In order to graphically display and summarize the mitogenetic relationships among the analyzed populations, a Principal Component Analysis (PCA) based on concatenated mtDNA (COI and CR) haplotypes was performed using the Excel software implemented by XLSTAT. Haplotype frequencies were used as input data.
Finally, to construct a Bayesian phylogeny for the two combined mtDNA regions, we used BEAST.v2.7.3 and the analyses were run for 100,000,000 generations [42 (link)].
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