Computational protocol: Gelechiidae Moths Are Capable of Chemically Dissolving the Pollen of Their Host Plants: First Documented Sporopollenin Breakdown by an Animal

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Protocol publication

[…] For molecular phylogenetics and molecular clock dating, total DNA was extracted from wild-caught and lab-reared Deltophora adults and larvae using standard methods. We PCR-amplified a part of the mitochondrial cytochrome oxidase subunit I gene (COI) using the primers CO1-F 5′-ATA ATT TTT TTT ATA GTT ATA C-3′ and CO1-R 5′-GAT GGG CTC ATA CAA TAA ATC CTA-3′ . Sequences have been submitted to GenBank (GU247447-GU247452). Deltophora cytochrome c oxidase subunit I (COI) sequences were aligned with GenBank Gelechioidea sequences, with outgroup taxa chosen based on and . For time calibration (below), we included Tegeticula (Prodoxidae) and Galagete (Autostichidae). The tree was rooted on Prodoxidae.For each Phyllanthus species, we isolated total genomic DNA from silica-dried leaves material using the methods described in and sequenced the plastid maturase K (matK) gene for which over 130 other fully vouchered Phyllanthaceae sequences are available in GenBank –. The GenBank matK sequence of Bridelia ferruginea was renamed Bridelia sp. based on and that of Sauropus androgynus became S. garrettii based on . Primers were those described in . New sequences have been submitted to GenBank (GU263803 and GU263804). The matK matrix used to infer closest relatives consisted of 132 sequences of Phyllanthaceae plus a few Picrodendraceae, Euphorbiaceae, and Putranjivaceae for rooting purposes.Maximum likelihood (ML) analyses on the moth and plant matrices were performed under the GTR + G model of substitution using RAxML . Bootstrap support was estimated in RAxML with 100 replicate heuristic searches under the same model as used in the searches. Maximum likelihood trees with branch lengths were then used in relaxed molecular clock models that incorporated at least one fossil-based calibration point (given as a probability distribution). Specifically, we used the uncorrelated-rates Bayesian relaxed clock approach implemented in BEAST v.1.4.8 , which uses a Markov chain Monte Carlo (MCMC) method to co-estimate topology, substitution rates, and node ages. Posterior probability distributions of node ages were obtained from the cytochrome c oxidase subunit I or matK alignments, with the number of taxa slightly reduced by taking out identical or near-identical moth and plant sequences. Analyses used a speciation model that followed a Yule tree prior, with rate variation across branches lognormally distributed. MCMC chains were run for 10 million generations, with parameters sampled every 1000th step. Whether the chains had reached stationarity was checked using Tracer v. 1.4.1 ; this showed that the effective sample sizes for all estimated parameters were well above 200.For calibration of the moth phylogeny, we used a normal prior distribution with a mean of 3.3 million years (Ma) and a SD of 1 Ma for the Galagete crown group and a mean of 32.4 Ma (SD 1 Ma) for the Tegeticula crown group. The divergence time between Tegeticula synthetica and T. yuccasella has been dated to 32.4 Ma . The first radiation event on the Galápagos archipelago within the Galagete lineage has been dated to 3.3 Ma [27).For calibration of the plant phylogeny, the crown group age of Phyllanthus sensu lato (including Breynia, Glochidion, and Sauropus) was set to 55 million years (Ma) (SD = 1 Ma) based on pollen described from the Lower Eocene Woolwich bed in Kent, England . Although the pollen of Phyllanthus is heterogeneous, the Woolwich pollen appears to be correctly assigned. As an alternative minimal constraint, we could use fossil fruits that resemble those of Phyllanthus from the Maastrichtian, 65–70 Ma ago . […]

Pipeline specifications

Software tools RAxML, BEAST
Application Phylogenetics
Organisms Caenorhabditis elegans
Chemicals Ethanolamine