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Digging out Kinorhynch Roots

Periodic Reporting for period 1 - KinoRoot (Digging out Kinorhynch Roots)

Reporting period: 2019-03-15 to 2021-03-14

Segmentation, i.e. a general body plan composed of repetitive units, has always played a central role in our understanding of animal evolution and phylogeny. The importance of segmentation is expressed in vertebrates and two of the most successful and diverse invertebrate groups, annelids and arthropods, which both follow a strictly segmented body plan. However, researchers tend to neglect the existence of a third highly segmented invertebrate group, the Kinorhyncha
or mud dragons. This small phylum contains about 300 described species, all less than 1 mm in length that inhabit marine sediments all around the world. Most of them are characterized by a clearly segmented trunk, which in adults is always composed of eleven segments. The kinorhynchs are related to the unsegmented groups Priapulida (penis worms) and Loricifera (girdle wearers), and together with these were recently suggested to constitute the sister clade to all other Ecdysozoans (molting animals) including the two extremely species-rich phyla Nematoda and Arthropoda. With Kinorhyncha and Arthropoda nested in different subgroups of Ecdysozoa and both presenting a segmented body plan, the key question arises: did segmentation arise before or after the diversification of Ecdysozoa?

Two alternative hypotheses will be addressed in order to answer this question:
a) Segmentation in kinorhynchs and arthropods is homologous and represents the ancestral ecdysozoan condition, to be found in the ecdysozoan ancestor.
b) Segmentation in kinorhynchs and arthropods evolved independently within Ecdysozoa, leaving the Kinorhyncha as a third example of convergently evolved invertebrate segmentation. This would support paleontological findings,
suggesting that the ecdysozoan ancestor was an unsegmented worm with a head resembling the kinorhynch head.

In both cases, the kinorhynchs represent the essential key to understand the early ecdysozoan evolution.

Main objectives:
- Examine traces of segmentation in three potentially early branching and unsegmented kinorhynch genera by the use of immunohistochemical staining, confocal laser scanning microscopy (CLSM), histology, and computational 3D reconstruction of muscular and nervous systems.
- Analyze phylogenetic relationships among a representative sample of kinorhynch genera, using Illumina RNAseq, in order to determine the positions of the less segmented kinorhynchs one or several of which may constitute the sister group to all remaining Kinorhyncha.
- Use the combined results of morphological examinations and phylogenetic analyses to reconstruct the ancestry of segmentation in Kinorhyncha and its evolution within Ecdysozoa.

- The muscular and nervous system in “less segmented” kinorhynchs clearly follows a segmental pattern with some modifications.
- Morphological and molecular data demonstrate that segmentation is an ancestral trait for kinorhynchs and the “less segmented” or aberrant forms are convergent.
- Segmentation most likely evolved independently in kinorhynchs and arthropods.
KinoRoot was divided into three main work packages:
- Study the morphology of weakly segmented kinorhynchs
We examined traces of segmentation in three potentially unsegmented kinorhynch genera through immunohistochemical, histological, and ultrastructural examinations of muscular and nervous systems combined with computational 3D reconstruction.
Previously collected, identified and fixed target species of the genera Cateria, Franciscideres, and Zelinkaderes were stained using specific markers for muscles (phalloidin) and to characterize different subsets of the nervous system we used antibodies against alpha-tubulin, serotonin, and FMRFamide combined with nuclear labeling (DAPI). Tracing and labeling muscle sets and three-dimensional models based on the CLSM scans were built for each of the species. Immunohistochemical analyses were complemented with additional histological, scanning electron microscopy, or transmission electron microscopy studies.
- Analysis of phylogenetic relationships in Kinorhyncha.
We collected 18 species from 15 key genera representing half of the diversity of the group, extracted RNA from single specimens, sequenced, de novo assembled their transcriptomes. Generate phylogenomic trees to explain the evolutionary relationships within the phylum.
- Reconstruct the evolution of segmentation.
Based on combined results from the morphological and molecular analyses the evolution of kinorhynch segmentation was reconstructed on the obtained phylogenomic tree.

Results and exploitation:
- Comprehensive morphological database (including 3D models) of the nervous system and musculature of five kinorhynch genera, three of them “less segmented”. These will be used as a reference for any future comparative morphological study within the phylum and outside by any researcher interested in the evolution of organ systems.
- Establishment and optimization of a protocol to successfully extract RNA from single individuals
- Generation of 18 new high-quality kinorhynch transcriptomes representing 15 genera inclusive 3 of the weakly segmented genera. This will be useful for testing hypotheses within Kinorhyncha and for future, large-scaled phylogenomic or comparative transcriptomic analyses.
- Production for the first time of a robust phylogenomic tree resolving most of the internal relationships in kinorhynchs inclusive the position of aberrant, less segmented genera.
- Resolved evolution of segmentation within kinorhynchs and proposal of a scenario for the evolution of segmentation in Ecdysozoa. Understanding the evolution of this body plan is pivotal for understanding the evolution of most other organ systems. Therefore, KinoRoot results are relevant and useful for a broad range of evolutionary scientists, paleontologists, and invertebrate zoologists.

To date I have published 2 research papers during the development of this action, one more is under review and I am currently in the process of publishing a fourth one. I attended one international conference during the action, another one just before the start of the action introducing my project, and I intend to attend an additional one (which was postponed) after the action to present the final results. Additional dissemination of my results was through participation as a speaker in several seminars at the Natural History Museum of Demark and The BIO department at the University of Copenhagen.
The output of this project contributed to fill a gap of knowledge combining advanced morphological studies for Kinorhyncha with state-of-the-art molecular sequencing that no other research group would have the capability to produce nowadays. The availability of a high-quality transcriptomic dataset will benefit any evolutionary biologist answering broad questions across metazoans, ecdysozoans, or scalidophorans; and the comprehensive morphological data will be of interest to any biologist interested in the evolution of the organ systems.
In addition, the results produced in KinoRoot shed light on the origin of segmentation within Ecdysozoa, but also broaden our knowledge on segmentation patterns, their deviations, and evolutionary transformations throughout ancient history. With humans being segmented vertebrates, any new knowledge on the formation and evolution of segmentation is of relevance for understanding our own body pattern and the mechanisms controlling its formation.
Anatomy of a segmented (A,C) and less-segmented (B,D) kinorhynch. A-B SEM. C-D 3D of the musculature