Methods for DNA Barcoding Photosynthetic Protists Emphasizing the Macroalgae and Diatoms
Why this work is in the frame
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Bibliographic record
Abstract
This chapter outlines the current practices used in our laboratory for routine DNA barcode analyses of the three major marine macroalgal groups, viz., brown (Phaeophyceae), red (Rhodophyta), and green (Chlorophyta) algae, as well as for the microscopic diatoms (Bacillariophyta). We start with an outline of current streamlined field protocols, which facilitate the collection of substantial (hundreds to thousands) specimens during short (days to weeks) field excursions. We present the current high-throughput DNA extraction protocols, which can, nonetheless, be easily modified for manual molecular laboratory use. We are advocating a two-marker approach for the DNA barcoding of protists with each major lineage having a designated primary and secondary barcode marker of which one is always the LSU D2/D3 (divergent domains D2/D3 of the nuclear ribosomal large subunit DNA). We provide a listing of the primers that we currently use in our laboratory for amplification of DNA barcode markers from the groups that we study: LSU D2/D3, which we advocate as a eukaryote-wide barcode marker to facilitate broad ecological and environmental surveys (secondary barcode marker in this capacity); COI-5P (the standard DNA barcode region of the mitochondrial cytochrome c oxidase 1 gene) as the primary barcode marker for brown and red algae; rbcL-3P (the 3' region of the plastid large subunit of ribulose-l-5-bisphosphate carboxylase/oxygenase) as the primary barcode marker for diatoms; and tufA (plastid elongation factor Tu gene) as the primary barcode marker for chlorophytan green algae. We outline our polymerase chain reaction and DNA sequencing methodologies, which have been streamlined for efficiency and to reduce unnecessary cleaning steps. The combined information should provide a helpful guide to those seeking to complete barcode research on these and related "protistan" groups (the term protist is not used in a phylogenetic context; it is simply a catch-all term for the bulk of eukaryotic diversity, i.e., all lineages excluding animals, true fungi, and plants).
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Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.005 | 0.001 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.001 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from it