Computational protocol: Three new species in the harvestmen genusAcuclavella (Opiliones, Dyspnoi, Ischyropsalidoidea), including description of maleAcuclavella quattuor Shear, 1986

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[…] Bi-directional Sanger reads were assembled into contiguous sequences using Sequencher v4.5 (Gene Codes Corporation, MI). EF-1α haplotypes were reconstructed in PHASE () using an ingroup-only matrix with the following parameter settings: 100 iterations, thinning interval of 1, burn-in of 100, probability cut-off set to 0.70 (). Haplotypes with nucleotides inferred with probability values < 0.70 were left ambiguous. The PHASE input file was converted from a FASTA alignment using SeqPHASE (). COI, EF-1α exon, and Wnt2 gene regions were unambiguously aligned using amino acid translations in MacClade v4.06 (). EF-1α intron and 28S rRNA data were aligned using MAFFT (), utilizing the Q-INS-i alignment algorithm strategy with parameters: BLOSUM62 alignment scoring matrix, 200PAM / K=2 scoring matrix value, an opening gap penalty of 1.53, and an offset value of 0.1. Regions of alignment uncertainty were removed from the 28S matrix with Gblocks () with the following parameter settings: minimum length of a block = 3, allowed gap position with half, minimum number of sequences used to identify a conserved position and a flanking position 11 and 17 respectively, and 8 as the maximum number of contiguous non-conserved positions.Individual gene trees were reconstructed using maximum likelihood and Bayesian inference. Bayesian analyses were implemented using MrBayes v3.1.2 () run on an XSEDE utilizing the CIPRES portal (). Models of DNA sequence evolution used in Bayesian analyses were determined using jModelTest v0.1.1 (, ). Likelihood scores were computed with three substitution schemes (24 models), unequal base frequencies, proportion of invariable sites, rate variation among sites, and used the BIONJ algorithm of . The goodness of fit of alternative models was determined using AIC () with CI set to 100%. Bayesian gene tree analyses were run for 5 X 106 generations sampling every 1000 trees, using best-fit models of molecular evolution. At this many generations, the average standard deviation of split frequencies between runs was < 0.01. The first 40% of the sampled trees were discarded as burn-in, and stationarity was confirmed visually using Tracer v2.4 (). Maximum likelihood gene tree analyses were conducted using RAxML v7.2.8 (), also on the CIPRES portal using the GTR + GAMMA model for tree inference and bootstrapping (). The EF-1α data set was partitioned by intron and exon, and the COI data was partitioned by codon position. The Wnt2 and 28S matrices were not partitioned. Outgroup sequences were used to root all gene trees.All available DNA sequences, with the exception of apparently nuclearized copies of COI (see Results), were concatenated for phylogeny reconstruction. The concatenated matrix was analyzed using both maximum likelihood (RAxML) and Bayesian approaches (MrBayes v3.1.2) using a seven-partition strategy (EF-1α intron + exon, Wnt2, 28S, individual COI codon positions). The Bayesian analysis was run for 1 X 107 generations; tree sampling and burn-in were as above. Outgroup sequences for five of six ischyropsalidoid genera (Ceratolasma, Taracus, Hesperonemastoma, Sabacon, and Ischyropsalis) and two genera from Troguloidea (Dendrolasma and Ortholasma) were used in concatenated phylogenetic analyses. These sequences were generated in-house or downloaded from GenBank (see ). [...] A cybertaxonomic approach was undertaken for enhanced dissemination of this work (e.g., ). This work is available as an open access PDF (doi: 10.3897/zookeys.311.2920). Molecular sequence data has been uploaded to GenBank (http://www.ncbi.nlm.nih.gov/Entrez; see for accession numbers). Some species of Acuclavella are difficult to diagnose using morphological characters alone. An effort has been made to facilitate DNA barcoding by uploading COI sequences to the Barcode of Life Data Systems (BOLD) v. 2.5 (http://boldsystems.org). Aligned matrices and all phylogenetic trees have been deposited in the Dryad Digital Depository (doi: 10.5061/dryad.16737). Anatomical images included in this publication and many additional images have been deposited in MorphBank (http://www.morphbank.net/?id=822371). A Keyhole Markup Language file (KML) for interactive viewing of species distributions and collection information in Google Earth (http://earth.google.com) is available as a supplementary file (Appendix VI). All nomenclatural acts have been registered with ZooBank (http://www.zoobank.org). New species described in this study are provided to Encyclopedia of Life (http://www.eol.org) where interconnection of information is linked back to this open access, peer-reviewed work. […]

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