Visualizing shape differences of van Leeuwenhoek’s ‘animalcules’

How do you bridge the molecular toolkit of small model organisms with 'evo-devo' and morphological studies?
Published in Protocols & Methods
Visualizing shape differences of van Leeuwenhoek’s ‘animalcules’

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By Tobias Theska & Michael S. Werner:

Evolutionary developmental biologists are united by their shared pursuit to understand the nature of phenotypic change. One major aim is to uncover the mechanistic basis of morphological innovation. This is typically addressed by investigating the evolution of developmental processes, which produce the phenotypes of interest, in a handful of selected model systems. Consequently, contemporary studies focusing on morphological evolution in animals follow three major objectives: (1) identifying candidate genes or mechanisms that produce the phenotypes of interest, (2) performing functional genetic experiments to establish a causal link between a candidate mechanism and an observed morphological difference, and (3) quantifying morphological changes which are either due to phenotypic evolution or experimental manipulation.

Surprisingly however, only a small number of research programs incorporate all of the aforementioned lines of inquiry. While functional genetic experiments and attempts to identify developmental mechanisms now routinely utilize the most sophisticated methods that are currently available (e.g. RNA-seq or CRISPR/Cas9), we don’t do the same when we acquire morphological data. Anatomical differences are still mainly captured in the form of tables that contain linear measurements of homologous elements, while more advanced quantification methods like geometric morphometrics, which allow us to obtain information on biological shapes, are still largely underappreciated. This might be explained by the steep ‘learning curve’ involved in implementing the sophisticated statistics and computation required for geometric morphometrics, precluding the full utility of the molecular and genetic toolkits available to model organisms in evo-devo research.

Within our own department at the Max Planck Institute for Developmental Biology in Tübingen (Germany), approaches to quantify shape changes varied over time and multiple students used their own combination of programs, statistical methods and landmark configurations to test hypothesis on shape differences among nematodes. Thus, the morphometric data sets that were generated in our department over the last five years were not comparable, let alone easily replicable by new students. So, before the last graduate student in our department who had experience with quantitative shape analysis left Germany for a post-doc in the US, a few of us sat down with him on a Wednesday morning to engage in a final crash course on the topic, in order to keep institutional knowledge in the lab. What was initially planed as a morning meeting turned out to be a day-long discussion, at the end of which we wondered: Wouldn’t it be much more useful to have a publicly available, ready-to-use R script that all members of our department, and more generally of research communities using small model organisms, could use? When we decided the answer was an unequivocal ‘yes’, we realized we should write a protocol.

With that in mind, we ‘brainstormed’ over the next few weeks which aspects of geometric morphometrics should fit into the protocol; what background information would be useful for novices to know, which statistical analyses to use, and what type of visualizations convey the most important results. As often happens in science, an idea for a short project expanded into an exhaustive study over the course of several months. Ultimately, we thought Nature Protocols was the perfect journal to convey the utility of geometric morphometrics to a broad community, with the same original concept of bridging the sophisticated molecular biology of model organisms with sophisticated quantitative morphological evaluation, even in structures as small as the ‘teeth’ of our favorite nematode P. pacificus.

The results of this investigation are summarized in our recent paper: Now, over 300 years after van Leeuwenhoek first described microorganisms from pond water using a homemade microscope, we hope that our protocol will provide evo-devo researchers the ability to take a closer look, and investigate quantitative differences in morphology in experimentally tractable small model organisms.


  1. Mallarino, R. & Abzhanov, A. Paths less traveled: evo-devo approaches to investigating animal morphological evolution. Annu. Rev. Cell Dev. Biol. 28, 743–763 (2012).
  2. Theska, T., Sieriebriennikov, B., Wighard, S.S. et al. Geometric morphometrics of microscopic animals as exemplified by model nematodes. Nat. Protoc.(2020).


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