Computational protocol: A new BrazilianPassiflora leafminer:Spinivalva gaucha, gen. n., sp. n. (Lepidoptera, Gracillariidae, Gracillariinae), the first gracillariid without a sap-feeding instar

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Protocol publication

[…] High-quality DNA was purified from larval tissue using the organic method of Cetyl Trimethyl Ammonium Bromide (CTAB) to investigate (i) levels of genetic variation within Spinivalva specimens collected in different localities and from different host plants (Passiflora misera, Passiflora suberosa and Passiflora actinia) and (ii) reconstruct phylogenetic relationships of this new genus among and within the Parectopa group of gracillariids. A total of nine field-collected specimens from three populations: 1) Porto Alegre, RS, from Passiflora suberosa and Passiflora misera (Pop. 1); 2) São Francisco de Paula, RS, from Passiflora actinia (Pop. 2) and 3) Curitiba, PR, also from Passiflora actinia (Pop. 3). They were used to amplify 1.5 kb of mitochondrial genes cytochrome c oxidase subunit I (CO-I), transfer RNA (tRNA-Leu), and cytochrome c oxidase subunit II (CO-II). For the PCR amplification we used the primer pairs Jerry + Pat II for the first segment (700 bp), and Patrick + Eva for the second (800 bp), following the procedure described by . Additionally, we amplified genetic material from three specimens of Spinivalva, using the universal barcode primers LCO1490 (5’-ggtcaacaaatcataaagatattgg-3’) and HCO2198 (5’-taaacttcagggtgaccaaaaaatca-3’), following the procedure of . We obtained variants that exactly matched the region previously sequenced in 6 representative taxa of the Parectopa group of gracillariids, downloaded from GenBank and incorporated into our analysis (). The remaining PCR products were treated with exonuclease I and shrimp alkaline phosphatase (ExoSAP) (Fermentas Inc.), sequenced using the BigDye sequencing kit and analyzed in an ABI 3730XL DNA Analyzer (Applied Biosystems Inc.). Sequences were aligned and visually inspected using the algorithm Clustal X in MEGA 5 () running in full mode with no manual adjustment. The dataset of 1.5 kb generated for specimens of Spinivalva from three different localities was deposited in GenBank and BOLD, under the accession numbers KC512114 to 512123 and GRABR001-13 to 010-13, respectively. The phylogenetic tree was reconstructed based on Bayesian inference and implemented in BEAST 2.0 () to recover (i) the evolutionary distance within Spinivalva taxa from different localities and host plants, and (ii) relationships of Spinivalva among the lineages of gracillariids surveyed in this study. In both trees, the HKY85 model of sequence evolution () was used with empirical base frequencies and 4 gamma categories. A relaxed uncorrelated log-normal clock was used, with no fixed mean substitution rate and a Yule prior on branching rates. We used four independent runs of 10 million generations, with the first 500,000 of each run discarded as burn-in. Posterior probabilities were used as an estimate of branch support. The species-level tree was unrooted, while the genus-level was rooted with a species of Bucculatricidae (Bucculatrix ulmella Zeller, 1848). […]

Pipeline specifications

Software tools Clustal W, MEGA, BEAST
Application Phylogenetics
Organisms Nitrosomonas sp., Homo sapiens, Drosophila melanogaster, Caenorhabditis elegans