(C) PLOS One This story was originally published by PLOS One and is unaltered. . . . . . . . . . . Comparative embryogenesis of two salp species reveals rogue development and evolutionary divergence from sessile tunicates [1] ['Marie Lebel', 'Laboratoire De Biologie Du Développement De Villefranche-Sur-Mer', 'Lbdv', 'Cnrs', 'Sorbonne Université', 'Villefranche-Sur-Mer', 'Centre De Recherche En Biologie Cellulaire De Montpellier', 'Crbm', 'Université De Montpellier', 'Cnrs Umr'] Date: 2026-03 Tunicates are the closest living relatives of vertebrates. Recent phylogenies place the little-studied, free-swimming thaliaceans—including salps—within sessile ascidians, highlighting a remarkable ecological transition. Historical reports hinted at a parallel developmental shift. Salp embryogenesis diverges from that of ascidians and involves unique maternal cells called calymmocytes. Here, we provide foundational resources for two distantly related salp species, Salpa fusiformis and Thalia democratica. Using advanced microscopy, we generated developmental staging tables showing that while embryogenesis is stereotyped within species, it differs in cleavage patterns and blastomere positioning between them. We traced the origins of calymmocytes and confirmed their conserved role in separating blastomere clusters that form adult tissues. Apoptosis contributes to the progressive elimination of maternal calymmocytes. Finally, we show that calymmocytes express embryonic developmental regulators, suggesting co-option of an embryonic gene program. These findings provide an advanced framework for studying embryogenesis evolution in a previously underexplored chordate lineage. Funding: This work was supported by the ANR (ANR-22-CE02-0016) to AA. ML received salary support from ENS (École Normale Supérieure de Lyon). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Data Availability: Raw confocal images and reconstructed 3D images are available in https://octopus.obs-vlfr.fr/public/salps/ and in Zenodo ( https://doi.org/10.5281/zenodo.18165317 ). Sequences generated in this study are available in GenBank under the accession numbers PX826397, PX826398, PX833381, and PX833380. Copyright: © 2026 Lebel et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Decades after the last reports on salp embryogenesis (reviewed in Piette and Lemaire [ 5 ]), the present findings should act as a catalyst and constitute a valuable resource to encourage further developmental biology research on thaliacean embryology, the last group of chordates still in need of foundational embryological descriptions. Historical studies on thaliacean embryonic development are often contradictory, lacking information on intra- and interspecific reproducibility [ 5 ]. The paucity of intermediate developmental stages analyzed [ 27 ], limited interspecific comparisons, and lower-resolution imaging techniques may have contributed to these inconsistencies. In this study, we revisited salp embryology with confocal and biphoton microscopy to reconstruct early embryos and provide detailed developmental staging tables of two species that are relatively distant phylogenetically [ 11 , 28 ]: Salpa fusiformis and Thalia democratica. Our 3D reconstructions showed consistent developmental patterns within each species, while revealing notable interspecific differences in cleavage patterns and spatial relationships between maternal calymmocytes and embryonic blastomeres. Analysis of programmed cell death by TUNEL assay suggests that apoptosis contributes to the disappearance of calymmocytes in the developing oozooid (the individual produced sexually from the fertilized egg). Finally, we adapted whole-mount in situ hybridization (WMISH) to reveal the expression patterns of salp orthologs of Otx and Rar, two genes regulating early development in ascidians [ 29 – 31 ]. In both species, we detected expression of these genes mainly in calymmocytes, suggesting a potential contribution to the patterning mechanisms. The phylogenetic placement of thaliaceans suggests an evolutionary origin from a sessile, tadpole-forming ancestor, with an embryogenesis similar to that of modern ascidians ( Fig 1B ). Within this scenario, Piette and Lemaire [ 5 , 26 ] hypothesized that as thaliaceans transitioned to a pelagic lifestyle, the selective pressures to maintain a swimming tadpole larva likely lessened, resulting in deviations from the robust, conserved ascidian embryogenesis and yielding the variety observed in thaliaceans. An updated understanding of thaliacean embryogenesis is essential to explore these evolutionary shifts, as it could illuminate broader processes in animal evolution. A critical first step is to determine to what extent salps exhibit developmental stereotypy and cellular invariance, as seen in ascidians, along with a comparison in molecular signatures and their regulation, both within salps and with other tunicates. In ascidians, early embryogenesis is highly conserved with a stereotyped bilateral holoblastic cleavage and a very similar gastrulation pattern [ 4 , 14 ]. This determinate developmental process with cellular invariance usually produces a larva with a typical chordate body plan [ 4 , 15 ]. In contrast, thaliaceans embryogenesis varies greatly among groups (reviewed in [ 16 – 19 ]). Doliolids are oviparous, with early cleavages akin to ascidians and a “tadpole” larval stage [ 19 – 21 ]. Pyrosomes are ovoviviparous, bearing large yolky eggs with meroblastic cleavage and epibolic gastrulation [ 17 , 18 , 22 – 24 ]. Salps are viviparous, possessing a placenta, and, like pyrosomes, undergo direct development without a larval stage ( Fig 1 ) [ 5 , 17 , 25 ]. Salp embryogenesis is particularly distinct from that of other tunicates, largely due to a complex interplay between zygote-derived cells (blastomeres) and a specialized class of maternal cells known as calymmocytes. These calymmocytes actively invade the developing embryo, where they continue to divide and physically separate groups of blastomeres, playing a crucial role in shaping the overall embryonic architecture [ 4 , 17 ]. In salps, the term “embryo” encompasses both blastomeres and calymmocytes, reflecting the integrated nature of these two cell populations. Notably, salps are among the few animals that do not display clearly defined gastrula or neurula stages, underscoring the exceptional character of their developmental process. Recent phylogenetic analyses suggest that thaliaceans are a monophyletic group nested within the paraphyletic ascidians ( Fig 1B ) [ 1 , 2 , 6 – 13 ]. According to Delsuc and colleagues (2018) [ 1 ], the divergence between thaliaceans and closely related ascidian orders occurred around 300 million years ago. Despite the nested position of thaliaceans within benthic ascidians, and the general conservation of their respective adult zooid bauplan, ascidians and thaliaceans embryogenesis differ significantly. (A) Lifecycle of Thalia democratica. (B) Consensus phylogenetic tree of tunicates and table showing the state of some characters in each order: benthic or pelagic lifestyle of the adult, presence/absence of a developmental stage with a “tadpole” morphology (at the larva, juvenile or adult stages), and presence/absence of a stereotypic ascidian-like embryogenesis (bilaterally symmetric embryos developing in an invariant manner). Tunicates, the sister group to vertebrates, include ascidians, which transition from a short planktonic larval stage to a lifelong sessile phase, and the holopelagic appendicularians and thaliaceans [ 1 – 4 ]. The three orders of thaliaceans, doliolids, pyrosomes, and salps all form large planktonic colonies with complex life cycles (see Fig 1A for a salp life-cycle) [ 5 ]. In salps, this cycle alternates between a solitary oozooid, which develops from a fertilized egg, and a colonial blastozooid, which arises asexually and produces the next generation sexually. The blastozooid carries the developing embryo, making it the focus of embryological investigations. Results Developmental staging tables for Salpa fusiformis and Thalia democratica A developmental staging table is a fundamental tool to federate a community of researchers working on a model organism, as it standardizes anatomical descriptions and provides a framework to describe deviations from the wild-type and compare species. Previous work by Heider [32] in Salpa fusiformis led to a first table mostly relying on external morphology and macroscopic features of the embryo, such as the development of the incubation folds and the placenta, and complemented with drawings of embryo sections. No developmental table was available for Thalia democratica. Encouraged by the quality of confocal imaging of early embryos from both species, we set out to complement Heider’s Salpa fusiformis staging table (S1 Table and S3 Fig) and generate an equivalent Thalia democratica table (S2 Table and S4 Fig). In the case of Thalia democratica, the lack of high quality drawings to facilitate the rapid identification of the indicated stages in the lab led us to augment this table with brightfield microscopy pictures. To facilitate access to the developmental staging tables and associated figures for both Salpa fusiformis and Thalia democratica, we organized the data into an online web portal (https://octopus.obs-vlfr.fr/public/salps). This interface currently hosts searchable, stage-by-stage tables, representative images, and links to 3D reconstructions (when available). The portal is temporarily hosted on the LBDV’s Octopus server and is intended as a dynamic resource for the salp and broader tunicate research community. In S. fusiformis, 3D confocal stacks allowed us to refine Heider’s table (1895) with additional substages based on internal features, such as the formation of internal cavities by calymmocytes. This staging table includes the 11 main Heider stages (A to L) that can be recognized with a stereoscope. Two stages (E and F) were separated in substages that can only be distinguished from the confocal images (labeled by a lowercase letter). The developmental table begins with early cleavage (stage A) and ends with muscle band formation (stage L) (S1 Table and S3 Fig). For each stage and substage, S1 Table provides a characteristic feature used to identify the stage with a simple stereoscope, an estimate of the size of the embryo, of the number, size and chromatin appearance of blastomeres when known, a detailed description of the embryo, of its cavities, and a pointer to representative drawings or pictures. In T. democratica, a staging table was created de novo based on the observation and confocal imaging of 90 embryos. Seven stages (I to VII) were labeled with roman numbers and defined by morphological characteristics easily observable under a stereoscope. Seventeen sub-stages, labeled by a lowercase letter following the main stage number, were defined based mostly on the embryo’s internal morphology. This staging table covers development from the zygote (stage Ia) to stolon-producing embryo ready for release (stage VIIc) (S2 Table and S4 Fig). It provides a foundational framework for studying T. democratica development with greater precision and consistency. To enable interspecies comparisons and highlight both conserved and divergent aspects of salp development, we synthesized the staging tables of Salpa fusiformis and Thalia democratica into a unified comparative matrix. Rather than enforcing a strict one-to-one correspondence between individual stages, which is complicated by differences in cleavage geometry, calymmocyte organization, and heterochronies in formation of periembryonic structures, we grouped stages into broader developmental periods anchored around shared morphological transitions (e.g., onset of calymmocyte invasion, blastomere segregation, organogenesis). This period-based approach allowed us to propose developmental equivalences while preserving species-specific dynamics. The criteria used for each period are outlined in the table and supported by descriptive data and imaging presented in the following sections. Together, this synthesis (Table 1) provides a practical framework for comparative developmental analysis across salp species and highlights both homology and divergence in their embryogenesis. Note that as all samples were collected from the field and processed immediately upon return from the sea, the duration of each stage is unknown. PPT PowerPoint slide PNG larger image TIFF original image Download: Table 1. Comparative developmental periods in Salpa fusiformis (S.f) and Thalia democratica (T.d). https://doi.org/10.1371/journal.pbio.3003636.t001 These annotated staging tables constitute foundational resources for in-depth developmental analyses within each species, offering reproducible criteria to guide future investigations. However, comparative analyses between species remain challenging, particularly when attempting to identify stage homologies between S. fusiformis and T. democratica. Nonetheless, these tables establish a standardized framework, and the data presented in the following sections help address these challenges (see Discussion). [END] --- [1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3003636 Published and (C) by PLOS One Content appears here under this condition or license: Creative Commons - Attribution BY 4.0. via Magical.Fish Gopher News Feeds: gopher://magical.fish/1/feeds/news/plosone/