Computational protocol: Fast-Evolving Homoplastic Traits Are Best for Species Identification in a Group of Neotropical Wasps

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Protocol publication

[…] We inferred the relationships of 95 species of doryctine wasps, focusing on Costa Rican Heterospilus and related genera, using 4.3kB of sequence data from 5 loci: nuclear protein coding gene fragments from Alpha Spectrin, RNA Polymerase II, and Carbamoyl Phosphate Synthetase (CAD), the nuclear ribosomal gene 28S, and the mitochondrial protein-coding gene COI. Specimen data and Genbank accessions are listed in , and primer sequences are provided in . This taxon sample is necessarily smaller than the 350 species in the Costa Rican fauna, as most species are known only from older collections with degraded DNA, while some are represented by single specimens. Thus, we limited our phylogenetic sample to all available specimens collected into ethanol within the past 8 years. In addition to Costa Rican collections, we included seven freshly-collected specimens from Ecuador and two from warm temperate North America. Additional doryctine genera were included in the analysis because of potential paraphily of Heterospilus [].Genomic DNA was non-destructively extracted from the intact mesothorax, metathorax, and mesosoma after removing the head and prothorax. Specimens were soaked for 4-12 hours in a proteinase K solution, and the DNA was isolated using a Qiagen DNeasy kit according to the manufacturer’s protocol. Each sampled specimen was then scored for the Lucid key characters. The morphological matrix was >95% complete, as species sampled only from males and specimens with damaged antennae precluded observation of ovipositor and antennal characters for several taxa. Voucher specimens are deposited in the Illinois Natural History Survey (INHS) collections.DNA was amplified in a polymerase chain reaction using Takara Ex Taq and the manufacturer’s reagents under the recommended protocol. The nuclear protein-coding genes were amplified using a 2-stage nested PCR (described in []), with extension times adjusted to suit the length of the target fragment and annealing temperatures in accordance with primer Tm. PCR product was purified using Qiagen Qiaquick elution columns per the manufacturer’s protocol, and the DNA was sequenced using Sanger sequencing on an ABI 3730XL capillary sequencer. Chromatograms were edited in BioEdit [] and aligned in Mesquite 2.7 using Opal [].Phylogenies were inferred for individual loci and for the concatenated data using MrBayes 3.1 [], with substitution models selected using MrModeltest []. We employed a 5-partition set for the final analyses as follows: 28S, COI codon positions 1 & 2; COI codon position 3; nuclear protein-coding genes codon positions 1 & 2; and nuclear protein-coding genes codon position 3. We replicated the final MrBayes analysis twice, for over 2.5 x107 generations each time, and checked convergence among runs using AWTY [] and among parameter estimates using Tracer []. We obtained an ultrametric tree by reanalyzing the matrix using the same models and partition scheme in BEAST [] for 5 x 107 generations, using the MrBayes consensus as a starting tree and a relaxed molecular clock model. We did not specify any absolute age constraints. […]

Pipeline specifications

Software tools BioEdit, Mesquite, Opal, MrBayes, MrModelTest, BEAST
Application Phylogenetics