Computational protocol: Species limits and phylogeography of Newportia (Scolopendromorpha) and implications for widespread morphospecies

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[…] Thirty-four specimens of Newportia from Mexico, Guatemala, Honduras, and Costa Rica were sorted mostly from collections made by the LLAMA (Leaf Litter Survey of Mesoamerica) project, deposited in the Museum of Comparative Zoology (MCZ), Harvard University, Cambridge Massachusetts, USA and accessible through the dedicated data base MCZbase (http://mczbase.mcz.harvard.edu). All tissues were fixed in absolute ethanol and thus were amendable to DNA sequencing.Identifications were made using the most recent key for Newportia (Newportia) (Schileyko, 2013), supplemented with taxonomic descriptions in modern literature (; ), standard monographs (), original descriptions, and examination of type material designated by R. I. Pocock in The Natural History Museum (London) and or by R. V. Chamberlin in the MCZ.LLAMA specimens keyed to either Newportia (Newportia) monticola Pocock, 1890, Newportia (Newportia) stolli (Pocock, 1896), Newportia (Newportia) oreina Chamberlin, 1915, or Newportia (Newportia) divergens Chamberlin, 1922. All LLAMA specimens were sequenced for two mitochondrial loci: 16S rRNA and cytochrome c oxidase subunit I (COI). These loci were selected because they vary both within and between species, and even between individuals from geographically close populations. The 34 LLAMA samples were supplemented with Newportia (Newportia) and Newportia (Ectonocryptoides) sequences from our previous work (), nine new Newportia specimens from five localities in Amazonas and Roraima, Brazil, and novel sequences for an individual of Newportia (Newportia) pusilla Pocock, 1893, from Ecuador (see Table for morphospecies determinations and locality data).Total DNA was extracted from the legs utilizing the NucleoSpin®Tissue kit (Macherey-Nagel). Samples were incubated overnight. PCR amplifications were performed with illustra TM PuReTaq TM Ready-To-GoTM PCR Beads (GE Healthcare). The COI fragments were amplified using primer pair HCO1490 (Folmer et al. 1994) and HCOout () and the 16S rRNA fragments using primer pair 16Sa/16Sb (; ). The normal amplification cycle for COI consisted of an initial denaturation step (2 min at 95 °C), followed by 35 cycles of denaturation (1 min at 95 °C), annealing (1 min at 43 °C) and extension (1.5 min at 72 °C), followed by a final extension step (4 min at 72 °C). For the 16S rRNA fragment the cycle consisted of an initial denaturation step (2 min at 94 °C), followed by 35 cycles of denaturation (30 s at 94 °C), annealing (30 s min at 43 °C) and extension (1 min at 72 °C), followed by a final extension step (7 min at 72 °C). Visualization of the PCR products was done by 1 % agarose electrophoresis using Midori Green Advanced DNA Stain and FastGene® GelPic LED Box (Nippon Genetics, GmbH).Samples were purified using ExoSAP-IT (Affymetrix) and sent to FIMM (Institute for Molecular Medicine Finland) for sequencing. Chromatograms were visualized and assembled using Sequencer 5.0.1 (Gene Codes Corp., Ann Arbor, Michigan, USA). Sequence alignment editor Se-Al (Rambaut 1996) was used to visualize the sequences simultaneously. GenBank registrations for new sequences are listed in Table .Parsimony analysis was conducted with POY ver. 5.1.1 () run in 16 nodes in the high-performance supercluster Taito at CSC (IT-Center of Science), Finland. A timed search of three hours was first performed on the unaligned data set. The resulting tree was used as the starting tree for the next round in which an additional timed search of six hours was performed. Parameter set 111 (indel/transversion and transversion/transition costs all equal) was used throughout the searches and branch lengths were reported using the newly implemented command “report (“file_name.tre”, trees:(total, branches:true))”. Nodal support was calculated using parsimony jackknifing ().Additional analyses used a probabilistic approach with the maximum likelihood program RAxML ver. 8.0.22 (). For these, multiple sequence alignments (MSA) were first estimated with MUSCLE ver. 3.6 () and then trimmed using Gblocks ver. 0.91b (; ) to remove areas of ambiguous alignment. Since COI sequences showed no length variation, they were not trimmed in Gblocks. The amount of 16S rRNA data that remained after trimming was 59% of the original 585 positions. The two data sets were concatenated using SequenceMatrix () and the concatenated data were analyzed with RAxML in the CIPRES Science Gateway (). A unique general time reversible (GTR) model was specified for each partition independently. Nodal support was estimated using the rapid bootstrap algorithm (applying the Majority Rule Criterion) using the GTR-CAT model (). […]

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