Abstract
While a host of molecular techniques are utilized by evolutionary developmental (evo-devo) biologists, tools for quantitative evaluation of morphology are still largely underappreciated, especially in studies on microscopic animals. Here, we provide a standardized protocol for geometric morphometric analyses of 2D landmark data sets using a combination of the geomorph and Morpho R packages. Furthermore, we integrate clustering approaches to identify group structures within such datasets. We demonstrate our protocol by performing exemplary analyses on stomatal shapes in the model nematodes Caenorhabditis and Pristionchus. Image acquisition for 80 worms takes 3–4 d, while the entire data analysis requires 10–30 min. In theory, this approach is adaptable to all microscopic model organisms to facilitate a thorough quantification of shape differences within and across species, adding to the methodological toolkit of evo-devo studies on morphological evolution and novelty.
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Code availability
The entire code for an analysis based on geomorph can be copied from the different steps of the procedure. In addition, two ready-to-use R markdown files that contain the code for both geometric morphometrics packages (geomorph and Morpho) and a R routine that allows the generation of landmark files can be downloaded from the supplementary material of this paper (Supplementary Data 4–6).
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Acknowledgements
J. Claude (Institut des Sciences de l’Évolution-Montpellier, Montpellier, France) is gratefully acknowledged for his advice on geometric morphometrics using geomorph and Morpho in RStudio during a workshop given in Calgary (attended by B.S. in 2017). Earlier drafts of this manuscript were improved by advice on many steps of data analysis by F. Chan (Friedrich Miescher Laboratory, Tübingen, Germany) and critical comments on the general methodology of geometric morphometrics by P. Arnold (University of Potsdam, Potsdam, Germany). Furthermore, we would like to thank A. Rohrlach (Max Planck Institute for the Science of Human History, Jena, Germany) for his comments on statistical procedures and multivariate hypothesis testing using R. N. Kanzaki (Forestry and Forest Products Research Institute, Tsukuba, Japan) provided expertise on nematode stomatal morphology in an early phase of this project. Further thanks go to M. Riebesell for providing the TEM image of the P. pacificus stoma (Supplementary Fig. 1a). D. Sharma and J. de la Cuesta are to be thanked for general discussion. This work was funded by the Max Planck Society. T.T. was supported by the IMPRS ‘From Molecules to Organisms’.
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Contributions
Conceptualization: T.T., B.S. and M.S.W. Coding: B.S. and T.T. Data acquisition: T.T., S.S.W. and M.S.W. Data analysis: T.T. Writing the original draft: T.T. and S.S.W. Reviewing and editing the initial draft: B.S., M.S.W. and R.J.S. Revision of the original manuscript: T.T., B.S., S.S.W., M.S.W. and R.J.S. Supervision: M.S.W. and R.J.S.
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Related links
Key references using earlier versions of this protocol
Sieriebriennikov, B. et al. Mol. Biol. Evol. 34, 1644–1653 (2017): https://academic.oup.com/mbe/article/34/7/1644/3067497
Sieriebriennikov, B. et al. PLoS Genetics. 16, e1008687 (2020): https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1008687
Early paper from our lab using a similar approach but a different protocol
Susoy, V. et al. Elife 4, e05463 (2015): https://elifesciences.org/articles/05463
Supplementary information
Supplementary Information
Supplementary Figs. 1–12 and Supplementary Tables 1 and 2.
Supplementary Data 1
Landmark file for the P. pacificus dataset (including the two different morphs).
Supplementary Data 2
Landmark file for the Pristionchus dataset (including P. pacificus, P. bucculentus and P. elegans).
Supplementary Data 3
Landmark file for the Caenorhabditis dataset (including C. elegans and C. briggsae).
Supplementary Data 4
R markdown file containing the code for geometric morphometrics based on the geomorph package (main text code).
Supplementary Data 5
Alternative R markdown file containing the code for geometric morphometrics based on the Morpho package.
Supplementary Data 6
R routine to automatically reformat original landmark files to fit the requirements of the readlands.tps() function. Note that functionality might be affected by the text editor that was used to generate the ‘lands.txt’ file.
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Theska, T., Sieriebriennikov, B., Wighard, S.S. et al. Geometric morphometrics of microscopic animals as exemplified by model nematodes. Nat Protoc 15, 2611–2644 (2020). https://doi.org/10.1038/s41596-020-0347-z
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DOI: https://doi.org/10.1038/s41596-020-0347-z
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