Computational protocol: Integrative taxonomy of Metrichia Ross (Trichoptera: Hydroptilidae: Ochrotrichiinae) microcaddisflies from Brazil: descriptions of twenty new species

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[…] Specimens were collected manually (larvae or diurnal active adults) or using Malaise or light traps, and then fixed in 96% ethanol. Collecting permits in Brazil were issued by Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio) (SISBIO 43047 and 14591). To observe genital structures, abdomen of males were removed and cleared in a heated solution of 10% KOH for 20 min. Then, abdomens were mounted in temporary slides, which were used to draw pencil sketches with compound microscope equipped with camera lucida. Vector graphics were traced in Adobe Illustrator CS6 (Adobe Systems Inc.) using pencil sketches as templates. Descriptions provided here were made with DELTA software (Description Language for Taxonomy) (). Terminology used throughout this paper follows that provided by and . Types for newly described species are deposited at Coleção Entomológica Prof. José Alfredo Pinheiro Dutra, Departamento de Zoologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro (DZRJ); Museu Nacional, Universidade Federal do Rio de Janeiro, Rio de Janeiro (MNRJ); Instituto Nacional de Pesquisas da Amazônia, Manaus (INPA); Coleção Zoológica do Maranhão (CZMA), Caxias; and Museu de Zoologia da Universidade Federal da Bahia, Salvador (MZUFBA).The electronic version of this article in Portable Document Format (PDF) will represent a published work according to the International Commission on Zoological Nomenclature (ICZN), and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix The LSID for this publication is: The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central and CLOCKSS. [...] Forward and reverse sequences of each sample were assembled and manually edited in Sequencher 4.1 (Gene Codes, Ann Arbor, Michigan, USA). Sequences were verified with the Blast tool in GenBank to check for contamination. Subsequently, COI sequences were aligned with ClustalW implemented in MEGA 6 () and translated into amino-acid sequences to check for stop codons. The final alignment resulted in a matrix with 577 bp ().COI sequences were used to explore putative species limits with four different methodologies: (1) lineages recovered in neighbor-joining tree; (2) lineages recovered with Bayesian Inference; (3) ABGD; and (4) GMYC. The neighbor-joining tree was calculated in MEGA 6 using Kimura 2-Parameter (K2P) distances (), with partial deletion of missing information. Although the use of K2P distances in DNA barcoding is debated (), to allow comparison with previous works we also used this evolutionary model because it is frequently used in studies of species delimitation based on COI sequences. Branch support of neighbor-joining tree was assessed with 1,000 pseudoreplicates of non-parametric bootstrap ().BI analysis was conducted with MrBayes v. 3.2.2 () with four independent runs, each one with four MCMC chains running for 50,000,000 generations, with sample frequency of 5,000. Convergence of sampled parameters was checked in Tracer v. 1.5 () and the first 10% of sampled trees and parameters discarded as burnin. GTR + I + G was the best fit model selected by Akaike Information Criterion (AIC) with jModeltest v. 0.1.1 () and it was applied in BI analysis in MrBayes. Branch support was assessed by posterior probability (PP), presented on a 50% majority consensus tree.ABGD analysis was run using the on-line version available in, where the COI alignment was uploaded. The analysis was conducted with the following settings: Pmin = 0.001; Pmax = 0.1; steps = 20; relative gap width = 1.0, also based on K2P model. This method statistically infers the DNA barcode gap in a single locus alignment, partitioning the data based on this gap in putative species ().The GMYC analysis (; ) was performed in R () using the SPLITS package () with single-threshold method. Basically, the method estimates branching patterns on an ultrametric tree, identifying the most likely transition point from coalescent to speciation branching. The ultrametric tree used here was obtained with BEAST v. 1.8 () under a relaxed uncorrelated molecular clock (). The node including Ochrotrichia species was calibrated based on fossil evidence with a lognormal distribution offset at 20 mya and log(mean) = 2.8 to represent the possible range of 20–140 mya (); and the divergence of Ochrotrichiinae was calibrated based on with a normal distribution with mean 82.17 ± 12 mya. The BEAST analysis ran for 200,000,000 generations, sampled every 10,000 generations. Convergence was verified with Tracer and a maximum credibility tree was written using TreeAnotator, discarding the first 10% as burnin. […]

Pipeline specifications

Software tools Adobe Illustrator, Sequencher, Clustal W, MEGA, ABGD, MrBayes, jModelTest, BEAST
Applications Miscellaneous, Phylogenetics, Population genetic analysis
Organisms Musa acuminata, Metaphire vulgaris