BOLD TaxonID Tree of Madeiran water mites (BOLD Dataset “DS-MADWM”)
Description
Compact Neighbour-Joining tree of all analysed water mite species from Madeira Island (Portugal) based on Kimura 2-parameter distances. BINs are based on the barcode analysis from 3 March 2025. The analyses involved all 584 COI nucleotide sequences.
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Molecular analysis was conducted at the Institute of Biology, University of Szczecin (IoB-UoS) and in University of Florence (UNIFI), Florence, Italy. In both cases DNA was extracted using a non-destructive protocol. At the IoB-UoS, DNA was extracted using the GeneMATRIX Tissue DNA Purification Kit (EURX Sp. z o.o., Gdańsk, Poland). The cytochrome oxidase subunit I (COI) gene fragment was amplified by PCR with the universal primers LCO1490-JJ and HCO2198-JJ (Astrin & Stüben 2008). The PCR amplification was carried out in a final volume of 10.1 μl with 5.0 μl of DreamTaq PCR Master Mixes and 2.4 μl of nuclease-free water (Thermo Fisher Scientific Baltics UAB), 0.8 μl of 5 µM forward and reverse primers and 1.1 μl of total DNA. The PCR reactions were performed in a T100TM Thermal Cycler (Bio-Rad, California, USA) using the HOU 2007 program for 3 hours in 35 cycles. According to the following thermal profile: initial denaturation at 94 °C for 3 minutes, then at 94 °C – 0.5 minute, annealing primers at 45 °C (for COI) – 1.30 minute, elongation at 72°C – 1 minute, and final elongation at 72°C for 5 minutes. The PCR products were separated on a 1% agarose gel with Midori Green Advance DNA Stain (NIPPON Genetics EUROPE GmbH, Düren, Germany) in TBE buffer. To visualise and document the obtained results, Gel DocTM XR+ system was used (Bio-Rad, California, USA). DNA sequencing of all obtained PCR products was performed at the Faculty of Biology and Environmental Protection, University of Lodz, Poland. At the UNIFI, samples were digested using 95 ul of extraction buffer (100mM Tris-HCl, 5mM EDTA, 100mM NaCl, 0.5%SDS, pH 8) and 5 ul of proteinase K. Dilutions (1:10) of crude digested samples were used as template for the amplification of the mitochondrial cytochrome c oxidase subunit I (COI). Amplicons were amplified and barcoded in a single-step PCR using a cocktail of two barcoded primer pairs, namely Folmer primers (LCO1490, HC02198; Folmer et al., 1994) and Lep primers (LepF1, LepR1; Herbert et al. 2004). PCR was performed using the Kapa3G Plant PCR Kit according to the manufacturer’s protocol and with the following thermal profile: initial denaturation step of 3 min at 94 °C, 35 cycles of 20 s at 95 °C, annealing for 15 s at 52 °C and extension for 30 s at 72 °C, and a final extension for 1 min at 72 °C. Amplicons were checked on a 1.2 % agarose gel and pooled in a single tube. The amplicon mix was used to prepare a PacBio library with the SMRTbell prep kit 3.0 according to the manufacturer’s protocol. The library was sequenced on an 8M ZMW SMRT cell on a PacBio Sequel IIe platform. Raw reads were demultiplexed using the Pacific Biosciences SMRT Link software. Consensus sequences were generated with the PacBio Amplicon Analysis (pbaa) tool. Primer trimming, translation and stop codon checking were performed using Geneious Prime 2024.0.1.