Computational protocol: Deep intraspecific DNA barcode splits and hybridisation in the Udeaalpinalis group (Insecta, Lepidoptera, Crambidae) – an integrative revision

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Protocol publication

[…] The study is based on adult specimens of Udea alpinalis, U. austriacalis, U. cretacea, U. rhododendronalis, and U. uliginosalis, collected at different localities in Europe and Central Asia. The genetic dataset was complemented with sequences of U. bourgognealis, U. carniolica, U. murinalis, and U. nebulalis. Udea ruckdescheli Mally, Segerer & Nuss, 2016 of the U. numeralis species group (sensu ) served as outgroup. The morphologically investigated material is summarised in the ‘material examined’ sections of the respective species in the taxonomic results, the genetic data are summarised in Table .Molecular data from three different genes were used for the dataset: the 5’ half of the mitochondrial Cytochrome c oxidase subunit 1 (COI) gene (the “DNA Barcode”), 657 base pairs (bp) in length, the 5’ part of the nuclear Elongation factor 1-alpha (EF1a) gene (780 bp), and the nuclear Wingless gene (372 bp). In addition, a screening for molecular traces of Wolbachia infections was done by amplifying the bacterial markers Wolbachia surface protein (wsp), aspartyl/glutamyl-tRNA(Gln) amidotransferase subunit B (gatB) and Filamenting temperature-sensitive mutant Z (ftsZ).COI Barcode sequences and specimen data for the Udea species of interest were obtained from ongoing Barcoding projects of PH and MN on the Barcoding of Life Database (BOLD, www.boldsystems.com), Version 4. The DNA lab protocols at the Canadian Centre for DNA Barcoding (CCDB) are available at http://www.ibolproject.org/resources.php. Barcodes with less than 500 bp were excluded, and public records retrieved from NCBI GenBank were included. In addition, DNA Barcode sequences were obtained for several specimens through PCR and sequencing in the DNA labs of the Senckenberg Natural History Collections Dresden, Germany (SNSD) and the Institute of Biology at the University of Bergen, Norway (UiB).For DNA lab protocols at SNSD see . The DNA lab protocols at UiB are as follows: The abdomen was detached from the dried specimen and DNA was extracted using the DNeasy Blood & Tissue kit (Qiagen) according to the manufacturer’s protocol. Gene sequences were amplified in 25 µl reactions from 2 µl DNA extract using 400 nM of each primer, 800 µM dNTP mix, 2.5 µl Taq buffer (incl. MgCl2), 0.75u TaKaRa Ex Taq DNA Polymerase and distilled water added up to 25 µl in total per reaction. COI primers were HybLCO (forward) and HybNancy (reverse) (, ), EF1a primers were HybOscar-6143 (forward) and Bosie-6144 (reverse) (, ), and Wingless primers were HybLepWg1 (forward) and HybLepWg2 (reverse) (). Those primers contained the universal primer tail pair T7/T3 (‘Hyb’ in the primer names; ), which were used for sequencing. The wsp gene was amplified using the primers WspecF and WspecR (). In cases of lacking amplification success the internal primers INTF1 and INTR2 () were used. The genes gatB and ftsZ were amplified using the primers of in combination with the universal forward (T7 promoter) and reverse (T3) tails ().The PCR programme for COI was: initial phase at 95 °C for 5 min, 38 cycles with 95 °C for 30 s, 50 °C for 30 s and 72 °C for 60 s, final phase at 72 °C for 10min and cooling at 8 °C. For EF1a and Wingless a touchdown PCR was performed: 24 cycles at 95 °C for 30 s, 55 °C with -0.4 °C/cycle for 30 s and 72 °C for 60 s +2s/cycle, then 12 cycles at 95 °C for 30 s, 45 °C for 30 s and 72 °C for 120 s +3 s/cycle, final phase at 72 °C for 10min and cooling at 8 °C. The PCR protocol of was used for For wsp, and the protocol of for gatB and ftsZ.PCR results were examined via gel electrophoresis on a 1 % agarose gel and GelRed as dye agent. Successful PCR samples were cleaned with ExoSAP and subsequently amplified in Sanger-sequencing PCR reactions for both primers using the BigDye kit and this setup: 0.5–3.0 µl of PCR sample (depending on the sample’s band thickness on the agarose gel), 160 nM primer, 1 µl buffer, 0.5 µl BigDye, and adding up distilled water to 10 µl in total per reaction. Sequencing was conducted at the sequencing facility of UiB, Dept. of Molecular Biology. PCR and sequencing PCR were performed on a Bio-Rad 1000 thermal cycler; ExoSAP clean-up was done on a MJ Research PTC-200 thermal cycler.The three gene datasets (COI, Wingless, EF1a) were aligned with PhyDE 0.9971 () and analysed individually with raxmlGUI v. 1.5b2 (; ), using a Maximum Likelihood (ML) search under the GTRGAMMA model () and with a thorough bootstrap of 1,000 Bootstrap replicates. Phylograms were edited in TreeGraph version 2.13.0-748 beta (). The corresponding alleles and supergroups of successful Wolbachia sequences were sought in the BIGSdb database ().Dissection of genitalia was performed according to , with modifications. In order to preserve the tympanal organ, the abdomen was cut open longitudinally along one pleural membrane, detached from the genitalia, cleaned, and embedded under a separate cover slip next to the cover slip with the genitalia. Morphological structures were investigated using a Leica M125 stereomicroscope. Photographic documentation of imagines was done with a Canon EOS 60D in combination with a Canon EF 100mm 1:2,8 Macrolens and Canon EOS Utility Version 2.10.2.0 on a Windows PC. A Leica CTR6000 microscope in combination with a Leica DFC420 camera and Leica Application Suite programme (Version 3.8.0) on a Windows PC was used for documentation of the genitalia. Images were edited in GIMP 2.8.6. The distribution maps were generated with DIVA-GIS version 7.5.0.0 () and SRTM 90 m digital elevation data (). […]

Pipeline specifications

Software tools raxmlGUI, TreeGraph, BIGSdb, DIVA-GIS
Applications Phylogenetics, De novo sequencing analysis
Organisms Wolbachia endosymbiont of Armadillidium vulgare str. wVulC