# The evolution of an individual-like dispersive stage in colonial siphonophores

Maciej K Mańko, Catriona Munro, Lucas Leclère

► To cite this version:

Maciej K Mańko, Catriona Munro, Lucas Leclère. The evolution of an individual-like dispersive stage in colonial siphonophores. Current Biology, 2025, 35 (20), pp.4946-4958.e4.
10.1016/j.cub.2025.08.068. hal-05381436

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<figure><img src="image_1.png" /></figure>
# Current Biology

## The evolution of an individual-like dispersive stage in colonial siphonophores

### Graphical abstract

<figure><img src="image_2.png" /></figure>

### Authors

Maciej K. Mańko, Catriona Munro, Lucas Leclère

### Correspondence

maciej.manko@ug.edu.pl

### In brief

Mańko et al. show that the life cycle of certain siphonophores involves programmed fragmentation, producing individual-like eudoxids. They identify a specialized muscle responsible for fragmentation and find that release induces eudoxid maturation. They also show that eudoxids evolved once within siphonophores and that eudoxids occupy different niches from parental colonies.

### Highlights

* Individual-like eudoxids form via controlled fragmentation of the siphonophore colony

* Specialized muscle releases a colony fragment, inducing its maturation to a eudoxid

* Eudoxids evolved once following stepwise zooid integration and bract simplification

* Eudoxids and colonies display the same behavior but differ in distributional niches

<figure><img src="image_3.png" /></figure>
Article

# The evolution of an individual-like dispersive stage in colonial siphonophores

Maciej K. Mańko,<sup>1,4,*</sup> Catriona Munro,<sup>2</sup> and Lucas Leclère<sup>2,3</sup>

## SUMMARY

Reproduction in sedentary colonial species often involves the release of a dispersive stage, such as a larva or individual zooids, which promotes gene flow and enables range expansion. In some species of pelagic colonial siphonophores (Cnidaria: Hydrozoa), reproduction occurs through colony fragmentation, an evolutionarily unique strategy in which the entire terminal zooid cluster, instead of releasing individual reproductive zooids, detaches as a dispersive unit known as a eudoxid. Although composed of multiple functionally specialized zooids, the eudoxid behaves as a single individual, suggesting an elusive evolutionary transition in individuality. The mechanisms and evolutionary origins of eudoxid production, however, remain unknown. Using live imaging, immunohistochemistry, and pharmacological inhibition, we provide a mechanistic understanding of eudoxid formation. We demonstrate that eudoxid release is controlled by a dedicated muscle and involves tissue remodeling, resulting in the formation of a physiologically integrated dispersive unit with distinct behaviors. Our analyses suggest that eudoxids evolved once, through the concomitant reorganization of colony architecture and the evolution of zooid structures. We also show that eudoxids and their parental colonies often have different distributions, suggesting niche partitioning. These findings reveal how muscle evolution and structural modifications of the colony enabled the emergence of a unique, individual-like dispersive stage, contributing to the ecological success of siphonophores in marine environments.

## INTRODUCTION

The hierarchical integration of lower-level entities into higher-level units of selection has led to remarkable cases of biological evolution, from the multicellular aggregations of cells to the colonial or social aggregations of individuals.<sup>1-3</sup> As the integration progresses, lower-level units may specialize and their individuality may become subordinated.<sup>4</sup> In clonal colonial animals, such as hydrozoans (Cnidaria), this evolutionary transition in individuality has likely been achieved by retention of asexually budded individuals, driving subsequent division of labor and polymorphism of colony-building modules,<sup>5</sup> followed by an evolutionary complexity drain.<sup>6</sup> This functional diversification of modules, integrated to collectively function as a single organism, blurs the distinction between the individual and the colony.

Siphonophores, a clade of entirely marine hydrozoan cnidarians, have unequivocally the most complex colony architecture and the highest degree of zooid functional specialization among animals.<sup>7</sup> The majority of siphonophore colonies are streamlined, with asexual budding of zooids, homologous to free-living polyps and medusae, restricted to either one or two localized growth zones.<sup>7,8</sup> Non-locomotory zooids are budded off from a single growth zone, giving rise to iterative clusters of

polymorphic zooids, called cormidia, dispersed in an ontogenetic sequence along an elongated stem.<sup>4,8</sup> Despite limited data on siphonophore reproduction and life cycles,<sup>9</sup> siphonophores are known to decouple sexual reproduction from parental colonies by detaching gonad-bearing reproductive individuals, gonophores, either individually or in clusters.<sup>10</sup>

Release of reproductive individuals is well documented across hydrozoans, where the canonical life cycle involves detachment of a sexually reproducing individual, medusa, from a benthic polyp. Siphonophore gonophores, being motile but not able to feed, fall centrally within hydrozoan continuum of medusa trunca tion, which ranges from fully independent medusa (moving and feeding) to sporosacs, which typically remain attached to polyps.<sup>11</sup> Some members of the most-speciose clade of siphonophores, Calycophorae, have taken the reproduction-related specialization one step further (Figure 1). Instead of releasing only the reproductive individuals (gonophores), their colonies undergo sequential fragmentation, detaching entire cormidia—called eudoxids after release—composed of gonophores along with other functionally specialized zooids. Eudoxids exhibit a distinct morphology compared with the cormidia still attached to the parental colony, suggesting the existence of unknown processes of post-release morphological change. If judged by their

<footnote n="1"><sup>1</sup>Laboratory of Plankton Biology, Department of Marine Biology and Biotechnology, University of Gdańsk, 46 Pilsudskiego Avenue, Gdynia 81-378, Poland</footnote>

<footnote n="2"><sup>2</sup>Sorbonne Université, CNRS, Laboratoire de Biologie du Développement de Villefranche-sur-Mer (LBDV), 181 Chemin du Lazaret, Villefranche-sur-Mer 06230, France</footnote>

<footnote n="3"><sup>3</sup>Sorbonne Université, CNRS, Biologie Intégrative des Organismes Marins (BIOM), 1 Avenue Pierre Fabre, Banyuls-sur-Mer 66650, France</footnote>

<footnote n="4"><sup>4</sup>Lead contact</footnote>

<footnote>*Correspondence: maciej.manko@ug.edu.pl</footnote>

<footnote>https://doi.org/10.1016/j.cub.2025.08.068</footnote>
<figure><img src="image_4.png" /><figcaption>**Figure 1.** Life cycle and colony-to-individual transition in calycophoran siphonophores</figcaption></figure>

A polygastric colony of *Chelophyes appendiculata* comprises nectophores (swimming bells), an elongated stem (longitudinal stolon connecting colony members) on which zooids are budded in cormidia (repetitive clusters). Each cormidium consists of a gastrozooid (feeding zooid), a gonophore (gonad-bearing zooid), and a bract (gelatinous zooid of unclear homology) containing the bracteal canal (an extension of the gastrovascular system). The release of a terminal cormidium from the polygastric colony gives rise to a dispersive stage, the eudoxid, which shows distinct morphology from that of the undetached cormidium. Specifically, its bract is of a different shape and it contains a phyllocyst (swollen branch of the bracteal canal). Eudoxid not drawn to scale.

multi-zooid composition, eudoxids could be considered a colony derived from another colony.¹² However, considering the functional integration of eudoxid components, they might also be viewed as individuals. The question of whether eudoxids are colonial or individual entities remains largely unresolved.

Despite over two centuries of siphonophore research,⁸ the fragility of siphonophore colonies and the inability to culture them⁹ have precluded a thorough characterization of eudoxid production. The sole attempt to describe this process, conducted in 1871,¹³ suggested that eudoxids detach at weak points along the stem. Here, we characterize the stages of eudoxid development and release using *in vivo* imaging and further investigate cellular dynamics through immunohistochemistry, electron microscopy, and pharmacological inhibition experiments. We also assess the ecological significance of eudoxid acquisition by reviewing the entire siphonophore literature, describe a newly observed eudoxid behavior, and reconstruct the evolution of the eudoxid using an improved siphonophore phylogeny.

## RESULTS

### Stages of cormidium development

We characterized eudoxid formation in *Chelophyes appendiculata*, a highly abundant, epipelagic siphonophore, known to release eudoxids.¹⁴ The formation of eudoxids comprises three spatially and temporally separated processes: cormidium development, cormidium release, and eudoxid maturation, the latter occurring only in released cormidia.

We categorized cormidium development into four stages based on size and morphology of bract and gonophore. Stage 1 (S1) begins when undifferentiated projection from the stem develops into a gastrozooid with a tentacle (Figures 2A, 2B, 2D, and 2E). Then, a bract buds off from the left side of the gastrozooid's peduncle (Figures 2A, 2B, 2C, and 2E), with actin-rich structures present at the base of the bracteal canal (Figure 2C). Similar structures in other connection sites (e.g., stem-nectophore,

Figure S1) suggest their role in zooid attachment. The bract then extends over the stem and migrates toward the stem's dorsal side (Figures 2A and 2B), while the bracteal canal elongates and reorients perpendicularly to the stem's anterior-posterior (A-P) axis. The transition into stage 2 (S2) is marked by bract growth so that it resembles a cone wrapped around the stem, with the right side overhanging the left one (Figure 2F); the development of an apical projection of the bracteal canal (Figures 2F and 2I); and the appearance of a gonophore bud from the gastrozooid's peduncle left side (Figures 2E and 2F). Stage 3 (S3) begins when the apical projection of the bracteal canal grows to twice the height of its lateral branches (Figure 2G) and the gonophore starts actively pumping. In stage 4 (S4; Figures 2H and 2I), the lateral branches of the bracteal canal retract, while its apical projection enlarges and protrudes deeper into the bract, likely due to the mesoglea buildup (Figure 2H). As a result, only two connections between the bracteal canal and the stem are maintained: the apical connection, joining the bracteal canal's apical projection with the stem, and the basal one. Simultaneously, the bract shortens, acquires a more triangular shape, and its two flaps retract, revealing the bracteal furrow, a groove through which the stem runs (Figure 2H).

### Muscle-dependent cormidium release

Cormidia are released one at a time (20/20 release events), consistently upon reaching S4 of development (14/14; Table S1). This process occurs in a fixed order, starting from the posterior end of the colony (20/20).

Cormidium release can be triggered by disrupting the connection between a stem fragment with several cormidia and the remaining colony (Table S2), 2-, 3-, and 4-cormidia-long stem fragments (CLSFs) at stage S3 ($n_{CLSFs} = 1, 8$, and 7, respectively), dissected from *C. appendiculata* colonies, consistently released all their cormidia ($n_{released cormidia} = 56$). When a stem from a single colony was cut into two 3-CLSFs ($n_{colonies} = 6$), the order of cormidia release was maintained within each fragment but not
<figure><img src="image_5.png" /><figcaption>**Figure 2. Stages of cormidium development in *Chelophyes appendiculata***</figcaption></figure>

(A) Immunostaining of tyrosinated tubulin (gray), anti-FMRFamide-positive neurons (yellow), and nuclei (magenta) in young stem. Arrowheads point to the youngest (top, anterior) and the oldest (bottom, posterior) bract visible. Scale bar: 100 µm.

(B) Schematic representation of early cormidium differentiation with color-coded zooids.

(C) Actin detection in a young stem section showing the junction of the bracteal canal and the gastrovascular system of a gastrozooid.

(D) Overview of colony.

(E-H) Stages S1-S4 of cormidium development, with color-coded zooids and other components of the colony. Black arrowhead points to the mesoglea buildup site between the bracteal canal and the stem. (F', G', and H') Schemes of bracteal canal progression through stages S2 (F), S3 (G), and S4 (H), with the extent of

(legend continued on next page)
between the two fragments. The two fragments started releasing their posterior-most fragments nearly simultaneously, suggesting that an inhibitor signal prevents the release of more anterior cormidia.

To characterize the sequence of events leading to cormidium release, time-lapse videos were obtained of immobilized 3-CLSFs. This revealed that the release of cormidia is preceded by a constriction of the stem at a specific site, hereafter referred to as the detachment ring. This constriction occurs immediately posterior to the gastrozooid's peduncle of a second-to-last cormidium of the colony (Figures 3A and 3B; Video S1). Although the detachment ring remains constricted, the posterior portion of the stem undergoes a series of longitudinal contractions, and the gonophore continues to pump, ultimately leading to the release of the terminal cormidium at the detachment ring. The detachment ring remains constricted even after the release, as indicated by actin staining (Figures 3D and 3E), and is resorbed by the preceding cormidium only later (Figure 3F). This likely serves to maintain separation of the gastrovascular system from the surrounding environment.

Electron microscopy images corroborate the muscular nature of the detachment ring (Figures 3C′–3C′′). This structure is made up of two layers of myofilaments, as indicated by the hexagonal lattice of myosin and actin filaments (Figure 3C′′′): ectodermal and endodermal, separated by an electron-dense layer of mesoglea (Figures 3C′ and 3C′′). On the ectodermal side, the mesoglea branches out, separating individual myofilaments, likely to provide additional support, while no such branching is observed on the endodermal side. Importantly, the two layers of myofilaments are perpendicular to each other (Figure 3C′′′), with the ectodermal layer running along the stem's length, while the endodermal layer forms circular bands. In the other parts of the stem, myofilaments were found only in the ectoderm (Figure 3C′′).

Pharmacological inhibition experiments on 2- and 3-CLSF demonstrate that muscle contractions play a role in cormidium release (Table S3). Under control conditions, release occurred relatively rapidly, with fully formed eudoxids appearing within 22 h in all cases (19/19). Treatment with the non-muscle myosin inhibitor slightly impaired the stem's ability to release cormidia, with effects more pronouncedly at higher concentrations (release success of 10/14 and 5/14, for 1 and 5 µM Blebbistatin, respectively). In contrast, blocking muscle contractions with an isotonic MgCl₂ solution consistently impaired the stem's ability to release cormidia (release success 2/44).

## Release competence correlates with cormidium development

Chelophyes appendiculata colonies with less-developed stems did not produce eudoxids (Table S1). To systematically test whether this ability correlates with cormidium maturation, we dissected 2-, 3- and 4-CLSFs ($n_{CLSFs} = 3, 16, \text{and } 12$, respectively) at S2 of development (Table S2). Of the 102 S2

cormidia in 31 stem fragments, none were released throughout the duration of the experiment, thus indicating that the competence to release arises during the transition from S2 to S3.

The competence to release eudoxids correlates with the development of the detachment ring and bracteal canal. The detachment ring is already discernible in cormidia within the growth zone (Figures 4A and 4A'), with a single detachment ring forming posteriorly to each newly budded gastrozooid. Initially, the detachment ring is shaped like a semi-ring, composed of a few myofilaments running perpendicularly to the A-P axis of the stem. It extends from the gastrozooid's base, to roughly the mid-width of the stem on both sides (Figures 4A and 4A'). As the cormidium develops, the detachment ring begins to encircle the entire stem (Figures 4B and 4B'). Upon entering S3, the detachment ring shifts posteriorly and widens (Figures 4C and 4C'), making it distinguishable even without actin staining (Figure 4D). Similarly, the bracteal canal changes shape between S2 and S3 (Figures 2E–2H) with a strengthening of its connection to the stem (Figure 4).

## Cormidium release induces ectomyosin-dependent eudoxid maturation

The process of release triggers eudoxid maturation, during which the bract undergoes distinct morphological changes (Figure S2). We used time-lapse videos to reconstruct the course of post-release maturation (Figure S2A; Videos S1, S2, and S3). The newly released cormidium initially displayed an elongated stem fragment, which it fully resorbed within 4 h (Figures S2A–S2G). As the stem resorbed, the bracteal canal elongated into the bract mesoglea, gradually narrowing down to form the phyllocyst—a swollen branch of the bracteal canal acting as nutrient reservoir (Figure S2). This process was concomitant with a shift in the A-P axis of the eudoxid, making the longer axis of the eudoxid's phyllocyst aligned with the A-P axis of the eudoxid (Figures S2A–S2H).

The most striking feature of eudoxid maturation is the remodeling of the bract, characterized by a significant increase in its volume (Figures S2A–S2H). This process is likely accompanied by an uptake of water into the bract mesoglea rather than active growth, as our experiments were conducted on 1-day-starved specimens in microfiltered seawater. The resulting increase in volume caused the bracteal furrow to close, which, in turn, led the bract's shape to change from the thin, leaf-like structure wrapped around the stem into a pyramidal one positioned on top of the eudoxid.

Following cormidium release, the stem undergoes a series of contractions (Figures S2A–S2C), likely facilitating its resorption (Figures 5A and 5B). A strong actin signal at the connection site between the stem and the bracteal canal (Figures 5A and 5B) indicates that it is an active site for the pulling of the remnant stem. In a fully mature eudoxid, the connection between the stem and bracteal canal was no longer visible, and the base of

<footnote>the stem and gastrozooid limited by the dashed line; note that the (H) photo shows the terminal S4 cormidium, while the corresponding scheme (H') documents S4 cormidium morphology in preceding cormidium (i.e., not terminal). Figures are aligned so that the anterior pole of the colony faces up, except for (D), where the colony's anterior pole faces left. Note that images (A), (C), and (E) were flipped for presentation purpose and that, in all schemes, the gastrozooid's tentacles were omitted for clarity. Labels: bc.a, bracteal canal attachment; gz, gastrozooid; s, stem.
See Figures S1 and S4.</footnote>
<figure><img src="image_6.png" /><figcaption>**Figure 3. Constriction at the detachment ring is necessary for cormidium release**</figcaption></figure>

(A) Stem fragment (from Video S1), with a constricted detachment ring at the second-to-last cormidium. Color coded as in Figure 2.

(B) Close up of the detachment ring shown in (A).

(C) Schematics of TEM ultra-thin sections shown in (C') and (C''). (C') Microanatomy of the detachment ring, with endodermal muscle myofilaments colored in orange and their ectodermal counterparts in purple. (C'') Stem microanatomy below the detachment ring, color scheme as in (C'). (C''') Close up of myofilaments across the mesoglea within the detachment ring.

(D) Actin (cyan) and nuclei (magenta) detection in the second-to-last cormidium, fixed right after the detachment of the posterior-most cormidium.

(E) Close up of the area marked in (D), depicting a constricted detachment ring (arrowhead) and the actin-rich structures associated with attachment sites.

(F) Actin (cyan) and nuclei (magenta) detection in the posterior-most cormidium without any visible remnants of the detachment ring. All figures are aligned so that the anterior pole of the colony faces up. Labels: bc, bracteal canal; bc.a, bracteal canal attachment; ec, ectoderm; en, endoderm; gon, gonophore; gon.a, gonophore attachment; gz, gastrozooid; s, stem; tent, tentillum; arrowhead: detachment ring. Scale bars: 100 µm (A, B, and D-F), 1 µm (C' and C''), and 200 nm (C''').

See Figure S2 and Videos S1, S2, and S3.

the phyllocyst was composed of circularly arranged actin-rich structures of unknown function (Figure 5C).

In contrast, the absence of actin detection on the bract's surface, before and after remodeling, along with a faint actin signal within the bracteal canal (Figures 5A and 5B), suggest that the bract remodeling is not driven by spatial differences in the tension of its surface epithelium or the bracteal canal. To assess whether myosin-dependent processes play a role in the bract remodeling, we revisited the previously mentioned pharmacological inhibition experiments (Figures 5D-5G; Table S3). Treatment

with magnesium chloride had no significant effect on bract remodeling, with only a small proportion of remodeled bracts displaying aberrant morphology (6/44; Figure 5D). Similarly, the development of the bracteal canal into a phyllocyst appeared unaffected, with only 10 out of 44 cases showing a retained apical connection with the bract's surface. Interestingly, although cormidium detachment was not observed in magnesium-chloride-treated 3-CLSF, the three cormidia still progressed through all stages of cormidium development and eudoxid maturation but remained interconnected by a narrow stem (Figures 5D and 5G).
<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /><figcaption>**Figure 4.** Detachment ring and stem-bracteal canal connection change during cormidium maturation</figcaption></figure>

(A–C) Actin (cyan in A–C, gray in A’–C’) and nuclei (magenta) detection in the colony of *C. appendiculata*. (A and A’) growth zone, (B and B’) S2 cormidium, (C and C’) S3 cormidium. Arrowheads point to the developing detachment rings (A–C).

(D) Tyrosinated tubulin (gray) and nuclei (magenta) detection in stem with four S3 cormidia, with a close up of one of detachment rings (inset). Arrows point to detachment rings. A-P depicts anterior and posterior axis in all figures. Labels: bc, bracteal canal; br, bract; gon, gonophore; gz, gastrozooid; s, stem. Scale bars: 100 µm.
See Tables S1 and S2.

Treatment with the non-muscle myosin inhibitor Blebbistatin resulted in malformed bracts and bracteal canals (n = 28/28; Figure 5D). Bracts in Blebbistatin-treated stem fragments continued to grow but failed at remodeling, which resulted in their pronounced flattening (Figure 5F). Bracteal canals expanded, similar to progression from S3 to S4 of cormidium development, but failed to elongate. In addition, in released cormidia, the bracteal canal remained connected to the bract's surface via the apical connection, causing it to bend toward the direction of the bracteal furrow (Figures 5F and 5G). The stem resorption was also affected, as remnants of the stem could be discerned both along the bracteal furrow and near the bracteal canal base (Figure 5G). Taken together, these results suggest that the massive reorganizations of the region connecting the stem and the bracteal canal—two key steps in eudoxid formation—are regulated by non-muscle myosin.

**Eudoxids were acquired once in siphonophore evolution**

Our phylogenetic analyses provided a comprehensive picture of the evolutionary relationships within Calycophorae, thanks to the

acquisition of new 16S and 18S sequences for previously unsampled species and genera (Figures 6A and S3; Table S5). This robust phylogeny, constrained on a phylogenomic backbone,¹⁵ allowed us to revise calycophoran families. We propose to dismiss the family Abyliidae L. Agassiz, 1862, as it is nested within Diphyidae Quoy and Gaimard, 1827, by transferring its constituent taxa therein, and to split the polyphyletic Clausophyidae Bigelow, 1913, into (1) Chuniphyidae Moser 1925 (type genus *Chuniphyes* Lens and van Riemsdijk, 1908), resurrected from synonymy with Clausophyidae Bigelow, 1913, and containing only eudoxid-producing genera, and (2) Clausophyidae Bigelow, 1913, now including only one genus, *Clausophyes* Lens and van Riemsdijk, 1908, which does not produce eudoxids. The new clade Eudoxida, defined by the presence of a eudoxid in the life cycle, includes the families Chuniphyidae, Sphaeronectidae Huxley, 1859, and Diphyidae. The inclusion of *Tottonophyes enigmatica* in this clade remains ambiguous, awaiting confirmation that it produces eudoxids.¹⁶

By reconstructing 26 characters on the siphonophore phylogeny (Data S1), we inferred that the eudoxid was acquired once
<figure><img src="image_9.png" /><figcaption>Figure 5. Cormidium release and eudoxid maturation depend on muscle activity</figcaption></figure>

(A) Actin (cyan) and nuclei (magenta) detection in newly released cormidium.

(B) Close up of (A) depicting actin detection in stem-bract connection site.

(legend continued on next page)
in the common ancestor of Eudoxida, following a stepwise truncation of cormidium complexity and subsequent simplification of the bract's internal morphology (Figure 6A). We also inferred a single loss of the eudoxid, in the ancestor *Sulculeolaria*, and loss of the bract in the common ancestor of Clausophyidae and Hippopodiidae. The ability of some prayid species to fragment their stem could not be linked to the origin of eudoxids, as this trait was found scattered among non-Eudoxida calycophorans.

Pronounced cormidium complexity decrease occurred in the common ancestor of calycophorans (Figures 6A and S3), marked by the presence of only one bract per cormidium. This was then followed, in Eudoxida, by an acquisition of traits likely involved in maintaining integrity (shift in bract attachment and stem resorption) and functional independence (e.g., phyllocyst) in released cormidia. A subsequent reduction in complexity led to gastrovascular canal system simplification within Diphyidae and Sphaeronectidae (Figure 6A; Data S1).

Cormidium release requires the presence of a detachment ring (Figure 6B), which we inferred to be a synapomorphy of the clade Eudoxida. We detected a detachment ring in the stem of five Eudoxida species (Figures 6B and 6C. *appendiculata*: Figure 3), while a “break” in the longitudinal fibers was observed at the corresponding location in four non-Eudoxida codonophorans (Figure 6B).

## Eudoxids and colonies occupy distinct spatiotemporal niches

Using an extensive literature review, we found evidence for distributional differences of eudoxids and polygastric colonies, across species and scales (temporal, horizontal, and vertical; Figure 7; Data S2), with about 43% of references supporting that claim. Vertical distribution of eudoxids differed from that of polygastric colonies in a species-specific pattern, with either broader (*Abylopsis tetragona*¹⁷) or narrower (*Muggiaea bargmanna*¹⁸) depth ranges of eudoxids, or their predominance at shallower (*Gilia reticulata*¹⁵) or greater (*Diphyes antarctica*¹⁹) depths. Eudoxids also exhibited a distinct diel distribution pattern from polygastric colonies (Figures 7A and 7E), suggesting differences in vertical migration, with one stage performing more pronounced migration than the other.²⁰

One factor contributing to the differences in vertical distribution between eudoxids and polygastric colonies could be the greater buoyancy of mature eudoxids compared with undetached cormidia, which may reduce overall colony buoyancy. We tested this hypothesis by comparing the vertical position of dissected stems spanning S3 and S4 cormidia (five replicates, total $n_{cormidia} = 89$) to that of the eudoxids they released (see STAR Methods). Dissected stems remained at the bottom of the test tank, but

within 24 h, all cormidia were released, matured to eudoxids, and simultaneously gained positive buoyancy (Table S5), likely through pronounced volume increase of the bract (Figure S2).

We also found evidence of significant horizontal distribution mismatch between eudoxid and polygastric colonies, with eudoxids being present despite the absence of polygastric colonies (Figures 7A and 7C). This was documented both in local studies (*Ceratocymba leuckarti*²³) and those spanning greater oceanic regions (*Chuniphyes multidentata*²¹; Figure 7C). These distributional differences often mirrored the hydrological (*Heteropyramis maculata*²⁴) and bathymetric (*Ceratocymba leuckarti*²³) setting of the area, thus corroborating the distinct environmental preferences of eudoxids and polygastric colonies. The lack of spatial overlap could also be indicative of a temporal mismatch. Although the fact that eudoxids are produced by polygastric colonies implies tight temporal coupling of their distributions, we found evidence to the contrary (Figures 7A and 7D), like the presence of eudoxids in the absence of polygastric colonies,²² suggesting an extended lifespan of eudoxids and an ecological mismatch between the two.

## Eudoxids and colonies share complex foraging behavior

Despite differences in distribution, eudoxids share morphological traits with undetached cormidia, notably, the type of tentillum,²⁵ suggesting that their diets are likely similar to that of entire colonies. In most calycophoran polygastric colonies, feeding occurs through veronica movement,²⁶ in which nectophores pump to swim in circles, while the stem and tentacles extend to form a feeding spiral that maximizes the prey capture area (Figure 7F). We observed similar behavior in eudoxids (Figure 7G; Video S4), despite it being achieved with different zooids. We documented, for the first time, *Chelophyes appendiculata* eudoxids displaying the foraging behavior of polygastric colonies, swimming in circles with gonophore pumping, while simultaneously extending their tentacles.

## DISCUSSION

Here, we describe the acquisition of an evolutionarily unique stage in the life cycles of colonial siphonophores, known as eudoxids. We provide evidence for the integration of zooids within a eudoxid, an evolutionary trajectory marked by a decreasing complexity of cormidia and zooids, and the acquisition of eudoxid-specific structures and processes (e.g., detachment ring, phyllocyst, and post-release maturation). Additionally, we documented distributional niche partitioning between the calycophoran life cycle stages and the display of a colony's complex foraging behavior by eudoxids, resulting from the cooperative

<footnote>(C) Actin detection at the base of mature eudoxid phyllocyst. The red rectangle on the inset marks the location of the close up.</footnote>

<footnote>(D) Results of a pharmacological inhibition experiment scoring released cormidia, aberrant bract morphology, and aberrant bracteal canal morphology across treatments, with number of observations per treatment shown in bold (Table S3; MFSW; DMSO; 1 µM Blebb, 1 µM Blebbistatin in DMSO; 5 µM Blebb, 5 µM Blebbistatin in DMSO; MgCl₂, isotonic 7.5% MgCl₂ solution in MFSW).</footnote>

<footnote>(E) Normal phenotype of eudoxid from control MFSW treatment. (E') Close up of (E) showing phyllocyst morphology.</footnote>

<footnote>(F) Eudoxid with flattened bract (arrow) from 5 µM Blebbistatin treatment. (F') Close up of (F) showing un-resorbed stem (right-hand arrows) and aberrant phyllocyst, connected with bracteal furrow (arrow on the left side).</footnote>

<footnote>(G) Three cormidia from MgCl₂ treatment remaining interconnected with narrow stem (arrows), each with remodeled bract and some aberrations to phyllocyst formation. (G') Close up of (G) with arrows pointing at narrow stem (left and right arrow) and bent phyllocyst (top arrow). Scale bars: 100 µm, except for (C) where scale bar is 50 µm.</footnote>

<footnote>See Figure S1, Table S3, and Extended Data 3.</footnote>
A

<figure><img src="image_10.png" /><figcaption>**Figure 6.** Single origin of eudoxids in siphonophore evolution</figcaption></figure>

(A) Simplified siphonophore phylogeny (see Figure S3) inferred from concatenated 18S and 16S sequences, with key morphological evolution events marked in blue. Dotted line represents polyphyletic relationship.

(B) Actin (gray) detection in stems of selected Eudoxida species (orange) shows detachment rings (orange arrowhead), while a “break” in the longitudinal fibers (green arrowheads) is observed at the corresponding location in non-Eudoxida species (green). Scale bars: 100 µm.

See Figures S3 and S5, Table S4, Data S1, and Extended data 1, 2, and 4

B

<figure><img src="image_11.png" /><figcaption>Abylopsis tetragona</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>Eudoxoides spiralis</figcaption></figure>

<figure><img src="image_13.png" /><figcaption>Muggiaea atlantica</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>Sphaeronectes koellikeri</figcaption></figure>

<figure><img src="image_15.png" /><figcaption>Lilyopsis medusa</figcaption></figure>

<figure><img src="image_16.png" /><figcaption>Hippopodius hippopus</figcaption></figure>

<figure><img src="image_17.png" /><figcaption>Nanomia bijuga</figcaption></figure>

<figure><img src="image_18.png" /><figcaption>Agalma elegans</figcaption></figure>

actions of zooids. Thus, we consider eudoxids to exhibit all the hallmarks of individuality—cooperative, functional, and evolutionary.<sup>3,27</sup>

## Evolution of siphonophore colony architecture

Calycophoran cormidia have been shown to be more modular than other siphonophores in that individual zooids are not dispersed along the stem but rather remain clustered.<sup>7</sup> Our data, however, indicate that *Chelophyes appendiculata* budding bypasses canonical probud subdivision in that the original bud develops directly into a gastrozooid, while subsequent zooids are budded directly from the base of the gastrozooid (Figure 2A). Alteration of probud subdivision might be a shared feature of Calycophorae, as it was indicated in two other species.<sup>28,29</sup> Future exploration of cormidium budding across calycophorans, especially among “prayid” siphonophores, will be necessary to better understand the evolution of siphonophore colony architecture.

The apparent increase in cormidia modularity in Calycophorae coincided with the loss of their complexity (Figures 2 and 6).

Aside from the reduction in zooid types and numbers, we also documented the alteration to the bract morphology following eudoxid acquisition (e.g., twisting of the bract attachment; Figure 6; Data S1). Given the likely muscular nature of these lamellae<sup>12,30</sup> (Figures 2C, 3E, 5C, and S1), such repositioning might have secured a stronger attachment of the bract, necessary for mature eudoxid functioning and complex behavior (Figure 7G). The subsequent evolutionary modifications of the bract included a reduction in the number of bracteal canal branches (Figure 6; Data S1), leading to the retention of only the one forming the phyllocyst after cormidium detachment.

## Mechanisms of siphonophore colony fragmentation

The A-P patterning of the linear colonies in siphonophores, and their directional growth with strongly localized budding and stem cell populations,<sup>31</sup> hampers module replacement and removal, as indicated by the limited regenerative potential of siphonophores.<sup>4,26,32</sup> One plausible outcome of the limited regenerative potential of siphonophores is the phylogenetically broad distribution of records of colony fragmentation<sup>33</sup> (Data S1). Some siphonophore colonies attain enormous lengths, notably apolemiid “physonects” (over 30 m<sup>12,33</sup>) and “prayid” Calycophorae (45.7 m in *Praya dubia*<sup>34</sup>), and stem fragmentation was documented in these two groups (apolemiids<sup>35</sup> and “prayids”<sup>36</sup>; Data S1). These freely drifting stem fragments vary in length, module composition, and reproductive capacity,<sup>12,35</sup> indicating the unprogrammed nature of their separation, likely driven by the injury to the colony. In contrast, we show here that colony fragmentation leading to eudoxid formation (i.e., cormidium release and eudoxid maturation) is a precisely timed process, relying on module (here cormidium) development (Figure 4; Tables S1 and S2) and loss of connectivity with the colony (Table S2).

The temporal and spatial separation of eudoxid and cormidium maturation suggests the presence of an inhibitory signal
<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /><figcaption>**Figure 7.** Ecological mismatch between eudoxid and polygastric life cycle stages</figcaption></figure>

(A) Results of literature review indicating percent of all references analyzed (numbers in bold; Data S2), containing evidence for distribution mismatch between two life cycle stages separated into four types of distributional data: vertical, horizontal, seasonal, and diel.

(B) Example of vertical distribution mismatch in *Abylopsis tetragona*, based on data from Grossmann et al.,¹⁷ depicting depth ranges and mean distribution (gray line) of two life cycle stages.

(C) Example of horizontal distribution mismatch in *Chuniphyes multidentata*, based on data from Hosia et al.,²¹ depicting sites where either both life cycle stages (light blue) or only eudoxids (dark blue) were present.

(D) Example of seasonal mismatch in eudoxid (dark green) and polygastric colonies (light green) of *Dimophyes arctica*, based on data from Hosia and Båmstedt.²²

(E) Example of diel mismatch in distribution ranges of eudoxids (Eud) and polygastric colonies (Poly) in *Lensia cossack*, based on data from Grossmann et al.¹⁷

(F) Photograph of *Chelophyes appendiculata* performing veronica movement captured by Alexander Semenov (White Sea Biological Station).

(G) Frame grab from video (Video S4) documenting *C. appendiculata* eudoxid performing veronica movement. Dotted gray line indicates extent of eudoxid tentacle to facilitate interpretation.

See also Table S5, Data S2, Extended Data 3, and Video S4.

suppressing bract remodeling, phyllocyst formation, and stem resorption before cormidium release (Figure S2). The nature of this signal remains to be determined, but our data on the role of the detachment ring in cormidium release (Figures 3A, 3B, and 3D), and on cormidium release from stem fragments (Table S2), suggest that it is the loss of connectivity with the colony that induces cormidium release and eudoxid maturation. It is

likely, given the complex coordination system present in *C. appendiculata* and other siphonophores,³⁷ that sensing of cormidium integration with the colony (presence of connectivity) may involve the nervous system. Supporting this claim, we documented the existence of transverse neuronal bands in stems of *C. appendiculata* (Figure 2A) that mirrored the position of detachment rings and remained connected with the two giant axons
running along the entirety of the stem (Figures 2A and S4A). Alternatively, sensing of connectivity may be related to the flow in the stem,³⁷ food availability, or a mix of both. Our observations on spatial distribution of the detachment rings (Figure 4D) and their constriction prior to the detachment (Figure 3) suggest that they play a role in restricting flow across the stem, potentially serving as a cue to initiate cormidium release. Interestingly, although we observed detachment rings only in siphonophore species releasing eudoxids (Figure 6), in non-Eudoxida we observed the presence of transverse actin-rich bands at corresponding sites (Figure 6), consistent with earlier studies that hypothesized their role in controlling local stem contractions.³⁰ This may indicate that eudoxid-releasing calycophorans have co-opted the neuro-muscular toolkit present in other siphonophores, using detachment rings to mediate intra-colony connectivity.

## Role of muscles in eudoxid production

Through multiple lines of evidence (Figures 4 and 5), we found that cormidium release depends on muscle contraction of the detachment ring, likely a Eudoxida-unique structure (Figure 6). Despite functional diversity of cnidarian muscles,³⁸ information on their involvement in reproductive processes and life cycle transitions is scarce. Neuro-muscular control of zooid detachment was suggested during the shedding of bracts and nectophores,³⁰ as well as reproductive release of gonophores.¹² Those three zooids share a similar structure of the actin-rich stem attachment at their base, called the lamella or “autotomy joints”¹²,³⁰ (Figures 2C, 3E, 5C, and S1), which could be contractile and participate in zooid detachment. We also show that eudoxid maturation is controlled by actomyosin (Figure 5), reminiscent of the role of non-muscle myosin in the remodeling of regenerating *Clytia hemisphaerica* jellyfish.³⁹

## Ecological success of eudoxids

Fishing postures in siphonophores are closely related to their trophic niche.⁴⁰ The so-called veronica movement, observed in both eudoxids and polygastric colonies (Figures 7F and 7G), appears to be restricted to members of Eudoxida. In contrast, other siphonophores, particularly those with longer and less-contrac tile stems, adopt a “long-line” fishing posture.¹²,⁴⁰ The mechanisms underlying the acquisition and coordination of eudoxid behavioral complexity remain to be determined. One possibility is that it involves nervous system rearrangement. Indeed, we found that the two giant axons of the stem disappear during stem resorption (Figure S4).

The common assumption regarding the distributions of eudoxids and parental colonies is that both co-occur within the same environment.²⁰,⁴¹ Through a comprehensive literature review, we found evidence challenging this widely held view (Figure 7; Data S2). Of 386 references related to calycophoran distribution, we identified 122 studies that counted abundances of both life cycle stages separately, and, among these, 43% documented distributional mismatches (horizontal, vertical, seasonal, and diel) between eudoxids and polygastric colonies (Figure 7; Data S2). These mismatches may help reduce intra-specific competition—likely a concern for Eudoxida, as veronica movement in both life cycle stages suggests similar dietary preferences (Figure 7). Moreover, the most abundant siphonophores

globally belong to the Eudoxida,¹²,⁴² and their high population densities increase the likelihood of competition for limited resources, reinforcing the ecological importance of spatiotemporal niche segregation. This is consistent with data from other marine taxa with complex life cycles, which show that differences in habitat use, trophic modes, and mobility across life stages are key drivers of ecological success.⁴³,⁴⁴

## RESOURCE AVAILABILITY

### Lead contact

Requests for further information and resources should be directed to, and will be fulfilled by, the lead contact, Maciej K. Mańko, maciej.manko@ug.edu.pl.

### Materials availability

This study did not generate any unique materials.

### Data and code availability

* Sequence data have been deposited at NCBI GenBank under the following accession numbers: 16S rRNA (GenBank: PV190272-PV190290) and 18S rRNA (GenBank: PV138272-PV138290). Extended Data 1–4 were archived on Figshare: https://doi.org/10.6084/m9.fishshare.29988385. All other data needed to evaluate the conclusions in the paper are provided as supplemental information.

* Custom code needed to generate plots in Figures 5 and 7 is provided as Extended Data 3, available from Figshare: https://doi.org/10.6084/m9.fishshare.29988385.

* Any additional information required to reanalyze the data reported in this work paper is available from the lead contact upon request.

## ACKNOWLEDGMENTS

We would like to thank numerous people who have made this work possible: for assisting us in sampling in the Villefranche Bay (Alexandre Alié, Institut de la Mer de Villefranche [IMEV], and Alexandre Jan, University of Bergen), for aiding in *C. appendiculata* maintenance (Alexandre Jan, University of Bergen), for helping with confocal (Sébastien Schaub, IMEV, and Michał Rychłowski, University of Gdańsk) and electron microscopy (Bastien Salmon, BIOM; Sophie Pagnotta, Université Nice Côte d'Azur; and Dorota Łuszczek and Magdalena Narajczyk, University of Gdańsk), for providing guidelines on histological analysis (Katarzyna Smolarz, University of Gdańsk), to Alexander Semenov (White Sea Biological Station) for providing us with his photograph of *C. appendiculata*, to Anna Panasiuk for providing samples of *Diphyes antarctica*, and to Samuel Church (Yale University) for commenting on an earlier version of manuscript. M.K.M. also thanks Steven Haddock (Monterey Bay Aquarium Research Institute) for the invitation to join the research cruise to the Central Pacific, where some of the animals were sampled, and the Laboretoire de Biologie du Développement at the IMEV, Clytia team in particular, for their hospitality during his numerous visits. We are grateful to the Imaging Platform (PIM), animal facility (CRB), and sailors (Jean-Yves Carval, in particular) of the IMEV, which are supported by EMBRC-France, whose French state funds are managed by the ANR within the Investments of the Future program under reference ANR-10-INBS-0. We also acknowledge the Electron Microscopy Facility CCMA (Centre Commun de Microscopie Appliquée) from the Université Côte d'Azur, part of the Microscopie Imagerie Côte d'Azur GIS IBISA-labeled platform, supported by Université Côte d'Azur, the Région Sud, and the Département 06.

This work received financial support from the following grants: National Science Centre in Poland, Preludium 15, 2018/29/N/ZN8/01305 (M.K.M. and L. L.); University of Gdańsk, UGrant-START 533-0000-GS10-23 (M.K.M.); and European Marine Biological Resource Centre, Access, OOV-EMBRC FR-AAP2018-2180 (M.K.M. and L.L.).

## AUTHOR CONTRIBUTIONS

M.K.M., conceptualization, methodology, investigation, formal analysis, funding acquisition, visualization, writing – original draft, and writing – review and
editing; C.M., investigation and writing - review and editing; L.L., conceptualization, methodology, investigation, formal analysis, funding acquisition, writing - review and editing, and supervision.

### DECLARATION OF INTERESTS

The authors declare no competing interests.

### STAR★METHODS

Detailed methods are provided in the online version of this paper and include the following:

* **KEY RESOURCES TABLE**

* **EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS**

* Collection and maintenance of siphonophores

* **METHOD DETAILS**

* Initial processing of specimens

* *In vivo* experiments

* Competence to release

* Immunostaining and actin detection

* Pharmacological inhibition experiments

* Electron microscopy

* **QUANTIFICATION AND STATISTICAL ANALYSIS**

* Experimental data

* Phylogenetic analyses

* Siphonophore ecology review

### SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.cub.2025.08.068.

Received: May 2, 2025

Revised: July 17, 2025

Accepted: August 28, 2025

Published: September 19, 2025

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## STAR★METHODS

### KEY RESOURCES TABLE

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3"><strong>Antibodies</strong></td></tr><tr><td>Anti-Tyrosinated Tubulin YL1/2</td><td>Abcam</td><td>Cat#6160; RRID:AB_305328</td></tr><tr><td>Anti-FMRFamide antibody</td><td>Immunostar</td><td>Cat#20091; RRID:AB_572232</td></tr><tr><td>Goat anti-Rat IgG (H+L) Secondary Antibody, Rhodamine</td><td>Thermo Fisher Scientific</td><td>Cat#31680; RRID:AB_228357</td></tr><tr><td>Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Cyanine5</td><td>Thermo Fisher Scientific</td><td>Cat#A10523; RRID:AB_2534032</td></tr><tr><td colspan="3"><strong>Biological samples</strong></td></tr><tr><td>Chelophytes appendiculata</td><td>This paper</td><td>N/A</td></tr><tr><td colspan="3"><strong>Chemicals, peptides, and recombinant proteins</strong></td></tr><tr><td>Phusion™ High-Fidelity DNA Polymerase</td><td>Thermo Fisher Scientific</td><td>Cat#F530S</td></tr><tr><td>Sylgard 184</td><td>Dow Corning Corporation</td><td>Cat#4019862</td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma-Aldrich</td><td>Cat#P4333</td></tr><tr><td>Blebbistatin</td><td>Sigma-Aldrich</td><td>Cat#B0560</td></tr><tr><td>Alexa Fluor 488-conjugated phalloidin</td><td>Thermo Fisher Scientific</td><td>Cat#10125092</td></tr><tr><td>Hoechst 33258</td><td>Sigma-Aldrich</td><td>Cat#94403</td></tr><tr><td colspan="3"><strong>Critical commercial assays</strong></td></tr><tr><td>DNeasy DNA Blood and Tissue Kit</td><td>Qiagen</td><td>Cat#69504</td></tr><tr><td>QIAquick PCR Purification Kit</td><td>Qiagen</td><td>Cat#28104</td></tr><tr><td colspan="3"><strong>Deposited data</strong></td></tr><tr><td>16S rRNA sequences</td><td>This paper</td><td>NCBI GenBank: PV190272-PV190290</td></tr><tr><td>18S rRNA sequences</td><td>This paper</td><td>NCBI GenBank: PV138272-PV138290</td></tr><tr><td>Nexus file for the morphological evolution analysis with Mesquite</td><td>This paper</td><td>FigShare: https://doi.org/10.6084/m9.fishshare.29988385</td></tr><tr><td>Concatenated alignment of 16S and 18S sequences</td><td>This paper</td><td>FigShare: https://doi.org/10.6084/m9.fishshare.29988385</td></tr><tr><td>Code used to generate plots in Figure 5 and 7</td><td>This paper</td><td>FigShare: https://doi.org/10.6084/m9.fishshare.29988385</td></tr><tr><td>Topology used to constrain the IQTree ML analyses</td><td>This paper</td><td>FigShare: https://doi.org/10.6084/m9.fishshare.29988385</td></tr><tr><td colspan="3"><strong>Software and algorithms</strong></td></tr><tr><td>ImageJ</td><td>Schneider et al.<sup>45</sup></td><td>https://imagej.nih.gov/ij/</td></tr><tr><td>MAFFT v7.271</td><td>Katoh et al.<sup>46</sup></td><td>https://mafft.cbrc.jp/alignment/software/</td></tr><tr><td>IQTree v1.5.5</td><td>Nguyen et al.<sup>47</sup></td><td>http://www.iqtree.org</td></tr><tr><td>Mesquite</td><td>Maddison & Maddison<sup>48</sup></td><td>http://www.mesquiteproject.org</td></tr><tr><td colspan="3"><strong>Other</strong></td></tr><tr><td>17L Kreisel tank</td><td>Exotic Aquaculture</td><td>Cat#EA-JCT-17</td></tr><tr><td>Angled micro knife, 22.5°</td><td>Fine Science Tools GmbH</td><td>Cat#10316-14</td></tr></tbody></table>

## EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

### Collection and maintenance of siphonophores

Live specimens of *Chelophytes appendiculata* used in all experiments were collected in spring (2018–2022) from the Villefranche Bay (northern Mediterranean Sea), either in the vicinity of the Point B (43.6830 N, 7.3170 E) monitoring station or near the entrance of the bay. Sampling occurred in the early morning hours onboard RV Pelagia (Institute de la Mer de Villefranche, France). To collect siphonophores, a plankton net (650 µm mesh) was dropped below the surface (1-2 m) and was slowly towed behind the vessel to avoid damage to specimens. Then, the cod-end was gently emptied into a 15L bucket (CAMBRO®) prefilled with natural seawater. In case of large concentrations of plankton (e.g., during salp bloom) colonies of *C. appendiculata* were immediately transferred to a separate bucket with ladles. Additional specimens were scooped directly from the surface waters with a plastic 2 L beaker.
After collection, specimens were moved to a temperature-controlled room (18°C) with a 12 h: 12 h light cycle. Colonies designated for experiments were maintained unfed, in 15L buckets filled with filtered sea water, for no longer than 3 days. All other specimens (e.g., for immunostaining) were kept in 17L Kreisel tanks (Exotic Aquaculture) for 1-2 weeks, were fed daily with a mix of zooplankton, collected with 180 µm mesh plankton net from the Villefranche Bay and were starved only 1 day prior to use.

## METHOD DETAILS

### Initial processing of specimens

Specimens designated for subsequent analyzes were individually placed in a Petri dish coated with Sylgard 184 (Dow Corning Corporation) filled with microfiltered sea water (MFSW) and relaxed by dropwise addition of isotonic 7.5% solution of magnesium chloride hexahydrate in Mili-Q water at 1/3 MgCl₂ to 2/3 MFSW ratio. As tentacles tended to entangle and thus obscure observations, they were trimmed with an angled-micro knife (22.5°; Fine Science Tools GmbH). When stem fragments were needed, they were dissected from colonies also using angled micro-knife.

### In vivo experiments

Ethics approval is not required for experimentation on cnidarians. The following *in vivo* experiments were run on 1-day starved *C. appendiculata*: Competence to release (Table S1), Stem fragmentation (Table S2; Videos S1, S2, and S3), Buoyancy (Table S6) and Eudoxid behavior (Video S4). They were all run in a temperature-controlled room (18°C).

### Competence to release

*Chelophyes appendiculata* colonies (nectophore and entire stem) used were initially relaxed (as described above) in order to stage and photograph (NEX-5R Sony camera mounted on an Olympus SZ61 stereomicroscope) the posteriormost cormidium. To avoid specimen damage due to tentacle entanglement, all tentacles were trimmed. After staging, colonies were transferred to one of two treatments: unperturbed or immobilized. In unperturbed treatment, specimens were transferred to 1L glass beakers filled with MFSW. In case of immobilized treatment, specimens were individually placed in Sylgard 184 coated Petri dish, prefilled with solution of 7.5% magnesium chloride hexahydrate in Mili-Q mixed with MFSW in 1:2. The anterior nectophore of a colony was then pinned down to Sylgard using ethanol-washed *Opuntia* sp. spines. Usually, three spines were used per nectophore, with one driven through nectophore tissues near the ostium, and two placed on both sides of the anterior tip of the nectophore, which successfully limited nectophore movement. Then, the magnesium chloride solution was gently replaced with MFSW with antibiotics (1:2000 dilution of a penicillin [10000 units stock] and streptomycin [10 mg per ml] solution; Sigma-Aldrich, #P4333), which was replaced daily. The experiments were run for either 24 h or 48 h, with observations every 2-3 h, except for nights. Upon every release event, the stage of released cormidium was noted down (Table S1).

### Stem fragmentation

Stems of *C. appendiculata* colonies were relaxed and their tentacles trimmed as described above. They were then cut into 2-, 3- or 4-cormidia long stem fragments (once a single cormidium was dissected off, Video S3), and the stage of development of individual cormidia was noted down. The fragments were then individually transferred either into 6-well plates or glass micro-chambers. In case of 6-well plates, they were prefilled with 6 ml of MFSW with antibiotics (as above; replenished daily), and fragments were observed under Olympus SZ61 stereo-microscope with NEX-5R Sony camera at varying frequency (Table S2), with the picture taken at every time point and observations of cormidium release noted down. The glass micro-chambers were made by breaking glass microscope slides and positioning resulting pieces in a Petri dish (3 cm diameter) to form a channel, roughly 1 mm in width. The chambers were then placed directly under Zeiss Axio Observer, prefilled with MFSW with antibiotics to the upper level of glass slide upper surface, and the stem fragment was put directly to the channel. The movement of the stem fragment within the channel was then constrained by placing *Opuntia* spines on either side of the channel. Zeiss's ZEN Microscopy Software was then used to set automatic picture capture every 1 or 2 minutes, for about 24 h, across the full z-extent of the channel. To minimize file size, these pictures were then exported as movies (Videos S1, S2, and S3) at high frames-per-second rating and at a single z-plane, chosen based on stem fragment position throughout the experiment.

### Eudoxid behavior

Five experiments were run, each with a single *C. appendiculata* colony. Each colony was first relaxed (as described in the Initial processing of specimens section), all tentacles were trimmed, and a single stem fragment was dissected off that spanned all S3 cormidia. The stem fragment was then placed in a 50 ml measuring cylinder, prefilled with MFSW with antibiotics (as described above), and the position of cormidia was recorded as either laying on the bottom (0) or floating (1). Then the subsequent recording occurred after 24 h (Table S5).

### Eudoxid behavior

Twenty eudoxids were taken from Kreisel tank where polygastric colonies were kept and were transferred to a 1 L rectangular tank filled with MFSW. Eudoxid behavior was recorded using Canon EOS 6D camera with Canon 100 mm f/2.8 L EF Macro IS USM lens (Video S4).
**Immunostaining and actin detection**

Specimens designated for immunohistochemistry were processed as in Maríko et al.⁹ Briefly, after initial processing (as described above), specimens were fixed in cold MFSW containing 0.5% glutaraldehyde and 3.7% paraformaldehyde for 2 min at room temperature (RT) or overnight at 4°C. They were then washed three times, 10 min each wash, in 0.2% 100X Triton in 1X PBS (PBSTr), permeabilized in PBStr overnight at 4°C, and blocked in 3% bovine serum albumin and 3% goat serum albumin in PBSTr (PBSTr-BSA-GSA) for 1 h at RT. Then specimens were incubated for 2 h at RT with anti-tyrosinated tubulin primary antibody (YL1/2, 1:50, rat, Abcam, #6160) and/or anti-FMRFamide primary antibody (1:800, rabbit, Immunostar, #20091) in PBSTr-BSA-GSA. Afterwards, they were washed three times, 10 min each, in PBSTr and incubated overnight at 4°C with secondary antibodies (Goat anti-Rat coupled with rhodamine, 1:500, Thermo Fisher Scientific, #31680, and/or Goat anti-Rabbit coupled with Cyanine5, 1:1000, Thermo Fisher Scientific, #A10523), and Alexa Fluor 488-conjugated phalloidin (1:50, solubilized in methanol; Thermo Fisher Scientific, #10125092). Then, specimens were washed two times, 10 min each, in PBSTr at RT, incubated 15 min at RT in Hoechst (Sigma-Aldrich, #94403, 1:2000) in 1X PBS, washed again three times in PBSTr, incubated 30 min in 1X PBS at RT and gradually (10%-20%-40%) transferred to Citifluor AF-1 antifade mountant for imaging. Preparations were then visualized under Leica's SP5 and SP8 confocal microscopes with. Given the large size of imaged animals, multiple tiles spanning all-z planes were often taken. These were automatically aligned using Leica Application Suite X Life Science Microscope software, while z-stacks were projected using standard deviation projection in ImageJ.⁴⁵

**Pharmacological inhibition experiments**

2- and 3-cormidia long stem fragments (Table S3) were individually transferred to 6-well plates prefilled with 10 ml of solutions corresponding to treatments analyzed. These included: 1 or 5 µM blebbistatin (Sigma-Aldrich, #B0560; solution in MFSW, diluted from a 34 mM stock solution in DMSO); MgCl₂ (solution of 7.5% magnesium chloride hexahydrate in Mili-Q mixed with MFSW in 1:2 proportion); DMSO (1.47 µL dimethyl sulfoxide in MFSW, corresponding to DMSO concentration in 5 µM Blebbistatin treatment); MFSW. Experiments were run in temperature-controlled conditions at 18°C, and antibiotics were added to each treatment as described above. The development stage of each cormidium was determined for each stem fragment analyzed and individually photographed either under Zeiss Axio Observer or using Sony camera (NEX-5R) mounted on an Olympus SZ61 stereomicroscope. They were imaged again after 22 h and their phenotype was scored in the following categories: released cormidia (number of cormidia released from stem fragment); remodeled bracts (number of bracts that whose morphology matches that of released eudoxid bract); aberrant bract morphology (number of bracts whose morphology differs from that of eudoxid bract); aberrant bracteal canal (number of bracts showing alteration of bracteal canal morphology); stems resorbed (number of cormidia not showing signs of presence of loose stem).

**Electron microscopy**

Two samples (3-cormidia long stem fragment and two eudoxids of C. appendiculata) were preprocessed (relaxing and dissections) as described in Initial processing of specimens section. Specimens were then transferred separately to 1.5 ml Eppendorf tube and were fixed overnight at 4°C in a solution of 0.05 M sodium cacodylate buffer (pH 7.2-7.4), 0.025% MgCl₂, 2.5% glutaraldehyde, 0.8% paraformaldehyde and 4.45 M NaCl in deionized water. Subsequent steps leading to resin embedding were conducted at the Plateforme Commune de Microscopie Electronique, Université Nice Côte d'Azur. Transmission electron microscopy images of ultrathin sections were acquired on Tecnai G2 Spirit BioTWIN (FEI) at 120 kV at the Electron Microscopy Section at the Faculty of Biology at the University of Gdańsk.

**DNA Extraction and sequencing**

DNA was extracted from nectophores or a portion of the siphosome stored in ethanol at -20°C. DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen), following kit instructions. As much ethanol was removed from samples as possible before adding buffer ATL. Samples were incubated at 56°C for 3 hours. PCR of 16S and 18S was done with the following primers (16S: SHA 5' ACGGAATGAACTCAAATCATGT 3', SHB 5' TCGACTGTTTACCAAAAACATA 3', 74; 18S MitchA 5' AACCTGGTTGATCC TGCCAGT 3', MitchB 5' TGATCCTTCTGCAGGTTCACCTAC 3', 75), using the Phusion™ High-Fidelity DNA Polymerase (ThermoFisher) and annealing temperatures of 50°C and 55°C respectively (for cycling profiles see 16S:33, 18S:76). PCR products were cleaned up using the QIAquick PCR Purification Kit (Qiagen) following kit instructions and were sent to Eurofins Genomics GmbH (Germany) for Sanger sequencing. In addition to using the same forward and reverse PCR primers for sequencing, the following internal primers were used for 18S sequencing: 18SR1028 5'CTGCGAAAGCATTTGCCAAG 3' and 18SF970: 5'CTAGGACGGTATCTGATCGTCTTCG 3',77). All new sequences generated here were deposited at GenBank (accession numbers: PV190272- PV190290, and PV138272-PV138290; Table S4).

**QUANTIFICATION AND STATISTICAL ANALYSIS**

**Experimental data**

Quantitative and qualitative observations were recorded per time point across replicates in each of experiments following methods described above. No inferential statistics were performed beyond summary counts.
## Phylogenetic analyses

Available 16S and 18S rDNA sequences of siphonophores were retrieved from NCBI, except for *Physophora gilmeri* and *Thermopalia taraxaca*, whose 18S sequences were partial, and *Rosacea flaccida* and *Gymnopraia lapislazula*, whose phylogenetic positions were poorly supported. Additionally, because the positions of *Cordagalma ordinatum* and *Frillagalma vityazi* in rDNA-based phylogenies<sup>25,49</sup> were incongruent with those in phylogenomic-based phylogenies,<sup>15</sup> these species were also excluded from the analyses. Four species included in the molecular phylogeny (*Dendrogramma enigmatica*, *Tottonophyes enigmatica*, *Sphaeronectes haddocki*, and *Sphaeronectes christiansonae*) were excluded from the analyses due to the lack of information about their eudoxids.

We added new sequences for 18 calycophoran species for both ribosomal markers, as well as the 18S of *Lilyopsis medusa*. The outgroup included five hydrozoan species: *Ectopleura dumortieri*, *Hydra circumcincta*, *Staurocladia wellingtoni*, *Porpita porpita*, and *Velella velella*. We aligned the sequences using MAFFT v7.271 L-INS-I algorithm<sup>46</sup> and conducted constrained and unconstrained Maximum Likelihood (ML) analyses on the concatenated 16S-18S alignment using IQTree v1.5.5<sup>47</sup> with 1000 bootstrap replicates (Figure S5). ModelFinder implemented in IQTree was used to assess the relative model fit, selecting GTR+R4 for having the lowest Bayesian Information Criterion score. The constrained tree topology was derived from Munro et al.<sup>15</sup> (Extended Data S2 and S4).

A matrix including 26 morphological characters coded for all described siphonophore species was assembled from an extensive literature review (Data S1, Extended Data S2). For each species included in the phylogeny, we first analyzed the original species description, and in case of absence of information on a given trait we subsequently checked information in morphological reviews (Data S1). We then encoded additional information stemming from present work. The evolution of these characters was inferred by parsimony onto the ML phylogeny using Mesquite.<sup>48</sup>

## Siphonophore ecology review

Information on distribution mismatch between polygastric colonies and eudoxids were retrieved from a review of siphonophore reference database curated by late Philip R. Pugh (National Oceanographic Centre), including 1655 positions, spanning years 1725-2023.

First, the type of each reference was verified, and only scientific papers and books were retained, removing technical reports, regional species checklists, theses, and grey literature. Then we inferred the field of study for each of those by analysis of the title and abstract, retaining only references containing information on species distribution (vertical, horizontal, seasonal, or diel; *n*=520). We then verified if these contain information on distribution of Calycophorae, excluding references dealing either collectively with siphonophores, or not mentioning calycophores (*n*=386). From these we then kept only those that include information on eudoxids (*n*=122, Data S2), and these were then read thoroughly and checked for indication of distribution mismatch. Distribution was classified as pointing at mismatch when: for horizontal data, eudoxids present at sites when no polygastric stages were present, for vertical data, eudoxids showing different depth range or mean depth distribution than polygastric, for seasonal data, eudoxids not following seasonal abundance cycle of polygastric stages (e.g., presence in seasons when no or almost no polygastric stages were recorded), for diurnal data, eudoxids showing different vertical distribution between day and night as compared to polygastric stages.

Example plots were generated using raw data provided in the papers (*Chuniphyes multidentata* horizontal distribution,<sup>21</sup> *Abylopsis tetragona* vertical and *Lensia cossack* diel distributions<sup>17</sup>) or data were retrieved through digitization of published plots using *plotdigitizer.com* (*Dimophyes arctica* seasonal distribution<sup>22</sup>).
Supplemental Information

The evolution of an individual-like dispersive
stage in colonial siphonophores

Maciej K. Mańko, Catriona Munro, and Lucas Leclère
<figure><img src="image_21.png" /><figcaption>Figure S1. Actin-rich structures in the stem-nectophore connection site in *Chelophyes appendiculata*. Related to Figures 2 and 5C.</figcaption></figure>

Actin (cyan) and nuclei (magenta) detection at the connection site between stem and nectophore (nectophore somatocyst indicated with label). The arrowhead points at the actin-rich structures located at the connection site. Scale bar: 100 µm.
<figure><img src="image_22.png" /><figcaption>Figure S2. Eudoxid maturation comprises stem resorption, phyllocyst formation and bract remodeling. Related to Figure 3 and Videos S2-S3.</figcaption></figure>

Individual frame grabs (A-H) with their timestamps (hours and minutes after dissection) from time-lapse video depicting post-release modifications to cormidium morphology. Stem shaded in blue and bracteal canal/phyllocyst in red. Scale bar (shown only in A): 100 µm.
<figure><img src="image_23.png" /><figcaption>**Figure S3.** Siphonophore phylogeny based on 16S and 18S rDNA. Related to Figure 6.</figcaption></figure>

ML phylogeny inferred from concatenated 18S and 16S sequences, constrained by the phylogenomic tree from Munro et al.⁵¹ (Figure S7). Morphological evolution related to eudoxids is indicated on the phylogeny based on parsimony analysis. Red dots mark constrained nodes, black dots represent maximal bootstrap support.
<figure><img src="image_24.png" /><figcaption>Figure S4. Nervous system architecture in *Chelophyes appendiculata*. Related to Figure 2A.</figcaption></figure>

(A) Immunostaining of anti-FMRFamide positive neurons (yellow), actin (cyan) and nuclei (magenta) in C. appendiculata stem. (B) Immunostaining of FMRFamide (yellow) and nuclei (magenta) in newly released eudoxid with resorbing stem. Arrowheads point at the stems' giant axons. Scale bars 100 µm.
<figure><img src="image_25.png" /><figcaption>Figure S5. Maximum likelihood IQTree inference. Related to Figure 6.</figcaption></figure>

(A) Unconstrained phylogeny. (B) Phylogeny constrained by the phylogenomic tree from Munro et al.⁵¹. Numbers in red indicate bootstrap support values.
<table><thead><tr><th>ID</th><th>Treatment</th><th>Stage of<br>posterior-<br>most<br>cormidium</th><th>Stage of 1<sup>st</sup><br>cormidium<br>released</th><th>Stage of 2<sup>nd</sup><br>cormidium<br>released</th><th>Eudoxids<br>released</th><th>Observation<br>duration</th></tr></thead><tbody><tr><td>2021.3</td><td>unperturbed</td><td>S2</td><td>-</td><td>-</td><td>No</td><td>48h</td></tr><tr><td>2021.3</td><td>unperturbed</td><td>S2</td><td>-</td><td>-</td><td>No</td><td>48h</td></tr><tr><td>2021.5</td><td>immobilized</td><td>S2</td><td>-</td><td>-</td><td>No</td><td>48h</td></tr><tr><td>2021.5</td><td>immobilized</td><td>S2</td><td>-</td><td>-</td><td>No</td><td>48h</td></tr><tr><td>2021.Col2</td><td>unperturbed</td><td>S2</td><td>-</td><td>-</td><td>No</td><td>24h</td></tr><tr><td>2021.8</td><td>immobilized</td><td>S2</td><td>-</td><td>-</td><td>No</td><td>24h</td></tr><tr><td>2021.8</td><td>immobilized</td><td>S2</td><td>-</td><td>-</td><td>No</td><td>24h</td></tr><tr><td>2021.16</td><td>immobilized</td><td>S2</td><td>-</td><td>-</td><td>No</td><td>24h</td></tr><tr><td>2021.16</td><td>immobilized</td><td>S2</td><td>-</td><td>-</td><td>No</td><td>24h</td></tr><tr><td>2021.18</td><td>unperturbed</td><td>S2</td><td>-</td><td>-</td><td>No</td><td>24h</td></tr><tr><td>2021.18</td><td>unperturbed</td><td>S2</td><td>-</td><td>-</td><td>No</td><td>24h</td></tr><tr><td>2021.14.1</td><td>immobilized</td><td>S3</td><td>S4</td><td>S4</td><td>Yes</td><td>48h</td></tr><tr><td>2021.14.1</td><td>immobilized</td><td>S3</td><td>S4</td><td>S4</td><td>Yes</td><td>48h</td></tr><tr><td>2021.14.1</td><td>immobilized</td><td>S3</td><td>S4</td><td>S4</td><td>Yes</td><td>48h</td></tr><tr><td>2021.14.2</td><td>immobilized</td><td>S3</td><td>S4</td><td>S4</td><td>Yes</td><td>48h</td></tr><tr><td>2021.14.2</td><td>immobilized</td><td>S3</td><td>S4</td><td>S4</td><td>Yes</td><td>48h</td></tr><tr><td>2021.15</td><td>immobilized</td><td>S3</td><td>NA</td><td>NA</td><td>Yes</td><td>48h</td></tr><tr><td>2021.15</td><td>immobilized</td><td>S3</td><td>NA</td><td>NA</td><td>Yes</td><td>48h</td></tr><tr><td>2021.15</td><td>immobilized</td><td>S3</td><td>NA</td><td>NA</td><td>Yes</td><td>48h</td></tr><tr><td>2021.Col1</td><td>unperturbed</td><td>S3</td><td>S4</td><td>S4</td><td>Yes</td><td>48h</td></tr><tr><td>2021.6</td><td>unperturbed</td><td>S3</td><td>S4</td><td>S4</td><td>Yes</td><td>48h</td></tr></tbody></table>

Table S1. Observations of cormidium release from entire *Chelophyes appendiculata* colonies. Related to Figure 4.

For details see Methods section. NA - not available.
<table><caption>Table S2. Stem fragmentation experiments. Related to Figure 4.</caption><thead><tr><th>ID</th><th>Colony ID</th><th>CLSF type</th><th>Cormidium staging at T0 [posterior to anterior]</th><th>Number of eudoxids</th><th>Observation frequency</th></tr></thead><tbody><tr><td>2021.18</td><td>2021.18.1</td><td>4</td><td>S3-S3-S3-S3</td><td>4</td><td>after 24h</td></tr><tr><td>2021.18</td><td>2021.18.2</td><td>4</td><td>S3-S3-S3-S3</td><td>4</td><td>after 24h</td></tr><tr><td>2021.19</td><td>2021.19.1</td><td>4</td><td>S3-S3-S3-S3</td><td>4</td><td>after 24h</td></tr><tr><td>2021.19</td><td>2021.19.2</td><td>4</td><td>S3-S3-S3-S3</td><td>4</td><td>after 24h</td></tr><tr><td>2021.19</td><td>2021.19.3</td><td>4</td><td>S3-S3-S3-S3</td><td>4</td><td>after 24h</td></tr><tr><td>2021.19</td><td>2021.19.4</td><td>4</td><td>S3-S3-S3-S3</td><td>4</td><td>after 24h</td></tr><tr><td>2021.19</td><td>2021.19.5</td><td>4</td><td>S3-S3-S3-S3</td><td>4</td><td>after 24h</td></tr><tr><td>2021.2</td><td>2021.2.1</td><td>4</td><td>S2-S2-S2-S2</td><td>0</td><td>after 24h</td></tr><tr><td>2021.2</td><td>2021.2.1</td><td>4</td><td>S2-S2-S2-S2</td><td>0</td><td>after 24h</td></tr><tr><td>2021.2</td><td>2021.2.2</td><td>4</td><td>S2-S2-S2-S2</td><td>0</td><td>after 24h</td></tr><tr><td>2021.2</td><td>2021.2.3</td><td>4</td><td>S2-S2-S2-S2</td><td>0</td><td>after 24h</td></tr><tr><td>2021.2</td><td>2021.2.4</td><td>4</td><td>S2-S2-S2-S2</td><td>0</td><td>after 24h</td></tr><tr><td>2021.2</td><td>2021.2.5</td><td>4</td><td>S2-S2-S2-S2</td><td>0</td><td>after 24h</td></tr><tr><td>2021.2</td><td>2021.2.6</td><td>4</td><td>S2-S2-S2-S2</td><td>0</td><td>after 24h</td></tr><tr><td>2021.2</td><td>2021.2.7</td><td>4</td><td>S2-S2-S2-S2</td><td>0</td><td>after 24h</td></tr><tr><td>2021.3</td><td>2021.3.1</td><td>4</td><td>S2-S2-S2-S2</td><td>0</td><td>every 24h for 3 days</td></tr><tr><td>2021.3</td><td>2021.3.2</td><td>4</td><td>S2-S2-S2-S2</td><td>0</td><td>every 24h for 3 days</td></tr><tr><td>2021.4</td><td>2021.4.1</td><td>4</td><td>S2-S2-S2-S2</td><td>0</td><td>after 2, 8, 18, 23, 25h</td></tr><tr><td>2021.4</td><td>2021.4.2</td><td>4</td><td>S2-S2-S2-S2</td><td>0</td><td>after 2, 8, 18, 23, 25h</td></tr><tr><td>2022.1</td><td>2022.1.1</td><td>2</td><td>S4-S4</td><td>2</td><td>every 2h for 8h, then occasionally</td></tr><tr><td>2022.1</td><td>2022.1.2</td><td>3</td><td>S2-S2-S2</td><td>0</td><td>every 2h for 8h, then occasionally</td></tr><tr><td>2022.1</td><td>2022.1.3</td><td>3</td><td>S4-S4-S4</td><td>3</td><td>every 2h for 8h, then occasionally</td></tr><tr><td>2022.1</td><td>2022.1.3</td><td>3</td><td>S4-S4-S3</td><td>3</td><td>every 2h for 8h, then occasionally</td></tr><tr><td>2022.1</td><td>2022.1.4</td><td>3</td><td>S4-S3-S3</td><td>3</td><td>every 2h for 8h, then occasionally</td></tr><tr><td>2022.1</td><td>2022.1.4</td><td>3</td><td>S3-S3-S3</td><td>3</td><td>every 2h for 8h, then occasionally</td></tr><tr><td>2022.4</td><td>2022.4.1</td><td>3</td><td>S3-S3-S3</td><td>3</td><td>every 1h for 5h, then every 24h</td></tr></tbody></table>

Data acquired from stem fragmentation experiments, listing experiment identifier (ID), colony identifier (ColonyID), type of fragments expressed as CLSF (cormidium-long-stem-fragment), staging of individual cormidia within the fragment, number of eudoxids released and observation frequency.
<table><caption>Table S3. Pharmacological inhibition experiment. Related to Figure 5.</caption><thead><tr><th>ID</th><th>CLSF type</th><th>Treatment</th><th>Cormidium staging at T0 [posterior to anterior]</th><th>Released cormidia</th><th>Remodeled bracts</th><th>Aberrant bract morphology</th><th>Aberrant bracteal canal</th><th>Stems resorbed</th></tr></thead><tbody><tr><td>2022.5.inh.</td><td>3</td><td>1 µM B</td><td>S4-S4-S4</td><td>3</td><td>0</td><td>3</td><td>3</td><td>0</td></tr><tr><td>2022.5.inh.</td><td>3</td><td>1 µM B</td><td>S3-S3-S3</td><td>3</td><td>0</td><td>3</td><td>3</td><td>0</td></tr><tr><td>2022.5.inh.</td><td>3</td><td>1 µM B</td><td>S3-S3-S3</td><td>1</td><td>0</td><td>3</td><td>3</td><td>0</td></tr><tr><td>2022.bb.inh.</td><td>3</td><td>1 µM B</td><td>S4-S4-S4</td><td>3</td><td>0</td><td>3</td><td>3</td><td>0</td></tr><tr><td>2022.fs.inh.</td><td>2</td><td>1 µM B</td><td>S3-S3</td><td>0</td><td>0</td><td>2</td><td>2</td><td>0</td></tr><tr><td>2022.5.inh.</td><td>3</td><td>5 µM B</td><td>S4-S4-S4</td><td>1</td><td>0</td><td>3</td><td>3</td><td>0</td></tr><tr><td>2022.5.inh.</td><td>3</td><td>5 µM B</td><td>S3-S3-S3</td><td>1</td><td>0</td><td>3</td><td>3</td><td>0</td></tr><tr><td>2022.5.inh.</td><td>3</td><td>5 µM B</td><td>S3-S3-S3</td><td>0</td><td>0</td><td>3</td><td>3</td><td>0</td></tr><tr><td>2022.bb.inh.</td><td>3</td><td>5 µM B</td><td>S4-S4-S4</td><td>3</td><td>0</td><td>3</td><td>3</td><td>0</td></tr><tr><td>2022.fs.inh.</td><td>2</td><td>5 µM B</td><td>S3-S3</td><td>0</td><td>0</td><td>2</td><td>2</td><td>0</td></tr><tr><td>2022.5.inh.</td><td>3</td><td>DMSO</td><td>S4-S4-S4</td><td>3</td><td>3</td><td>0</td><td>0</td><td>3</td></tr><tr><td>2022.5.inh.</td><td>3</td><td>DMSO</td><td>S3-S3-S3</td><td>3</td><td>3</td><td>0</td><td>0</td><td>3</td></tr><tr><td>2022.fs.inh.</td><td>2</td><td>DMSO</td><td>S3-S3</td><td>2</td><td>2</td><td>0</td><td>0</td><td>2</td></tr><tr><td>2022.5.inh.</td><td>3</td><td>MFSW</td><td>S4-S4-S4</td><td>3</td><td>3</td><td>0</td><td>0</td><td>3</td></tr><tr><td>2022.5.inh.</td><td>3</td><td>MFSW</td><td>S3-S3-S3</td><td>3</td><td>3</td><td>0</td><td>0</td><td>3</td></tr><tr><td>2022.bb.inh.</td><td>3</td><td>MFSW</td><td>S4-S4-S4</td><td>3</td><td>3</td><td>0</td><td>0</td><td>3</td></tr><tr><td>2022.fs.inh.</td><td>2</td><td>MFSW</td><td>S3-S3</td><td>2</td><td>2</td><td>0</td><td>0</td><td>2</td></tr><tr><td>2022.2.inh.</td><td>2</td><td>MgCl<sub>2</sub></td><td>S3-S3</td><td>0</td><td>2</td><td>0</td><td>0</td><td>0</td></tr><tr><td>2022.2.inh.</td><td>2</td><td>MgCl<sub>2</sub></td><td>S3-S3</td><td>0</td><td>2</td><td>0</td><td>0</td><td>0</td></tr><tr><td>2022.2.inh.</td><td>3</td><td>MgCl<sub>2</sub></td><td>S3-S3-S3</td><td>0</td><td>3</td><td>0</td><td>0</td><td>0</td></tr><tr><td>2022.4.inh.</td><td>3</td><td>MgCl<sub>2</sub></td><td>S3-S3-S3</td><td>0</td><td>3</td><td>0</td><td>0</td><td>0</td></tr><tr><td>2022.4.inh.</td><td>3</td><td>MgCl<sub>2</sub></td><td>S3-S3-S3</td><td>0</td><td>3</td><td>0</td><td>0</td><td>0</td></tr><tr><td>2022.4.inh.</td><td>3</td><td>MgCl<sub>2</sub></td><td>S3-S3-S3</td><td>0</td><td>3</td><td>0</td><td>0</td><td>0</td></tr><tr><td>2022.4.inh.</td><td>3</td><td>MgCl<sub>2</sub></td><td>S4-S4-S4</td><td>0</td><td>3</td><td>0</td><td>0</td><td>0</td></tr><tr><td>2022.4.inh.</td><td>3</td><td>MgCl<sub>2</sub></td><td>S3-S3-S3</td><td>0</td><td>3</td><td>0</td><td>0</td><td>0</td></tr><tr><td>2022.6.inh.</td><td>3</td><td>MgCl<sub>2</sub></td><td>S4-S4-S4</td><td>0</td><td>3</td><td>0</td><td>0</td><td>0</td></tr><tr><td>2022.bb.inh.</td><td>3</td><td>MgCl<sub>2</sub></td><td>S4-S4-S4</td><td>0</td><td>3</td><td>0</td><td>2</td><td>0</td></tr><tr><td>2022.fs.inh.</td><td>2</td><td>MgCl<sub>2</sub></td><td>S3-S3</td><td>0</td><td>2</td><td>2</td><td>2</td><td>0</td></tr><tr><td>video5</td><td>2</td><td>MgCl<sub>2</sub></td><td>S4-S4-S4</td><td>1</td><td>2</td><td>0</td><td>1</td><td>0</td></tr><tr><td>video6</td><td>3</td><td>MgCl<sub>2</sub></td><td>S4-S3-S3</td><td>1</td><td>3</td><td>3</td><td>3</td><td>0</td></tr><tr><td>video7</td><td>3</td><td>MgCl<sub>2</sub></td><td>S3-S3-S3</td><td>0</td><td>3</td><td>1</td><td>1</td><td>0</td></tr><tr><td>video8</td><td>3</td><td>MgCl<sub>2</sub></td><td>S4-S4-S4</td><td>0</td><td>3</td><td>0</td><td>1</td><td>0</td></tr></tbody></table>

Summary of results of pharmacological inhibition experiments, experiment identifier (ID), type of fragments expressed as CLSF (cormidium-long-stem-fragment), staging of individual cormidia within the fragment and five scored phenotypes: released cormidia, remodeled bracts, aberrant bract morphology,
aberrant bracteal canal, stem resorbed. Treatments: MFSW, microfiltered sea water; DMSO, dimethyl sulfoxide; 1 µM B, 1 µM Blebbistatin in DMSO; 5 µM B, 5 µM Blebbistatin in DMSO; MgCl2, 7.5% magnesium chloride solution in MFSW.
<table><thead><tr><th>Sub-order</th><th>Family</th><th>Species</th><th>16S<br>accession<br>number</th><th>18S<br>accession<br>number</th></tr></thead><tbody><tr><td>Calycophorae</td><td>Abylidae</td><td>Abyla bicarinata</td><td>PV190272</td><td>PV138272</td></tr><tr><td>Calycophorae</td><td>Abylidae</td><td>Abylopsis tetragona</td><td>AY935303</td><td>AY937345.1</td></tr><tr><td>Calycophorae</td><td>Abylidae</td><td>Bassia bassensis</td><td>PV190273</td><td>PV138273</td></tr><tr><td>Calycophorae</td><td>Clausophyidae</td><td>Chuniphyes moserae</td><td>PV190276</td><td>PV138276</td></tr><tr><td>Calycophorae</td><td>Clausophyidae</td><td>Chuniphyes multidentata</td><td>AY935293</td><td>AY937335.1</td></tr><tr><td>Calycophorae</td><td>Clausophyidae</td><td>Clausophyes moserae</td><td>PV190277</td><td>PV138277</td></tr><tr><td>Calycophorae</td><td>Clausophyidae</td><td>Clausophyes tropica</td><td>PV190278</td><td>PV138278</td></tr><tr><td>Calycophorae</td><td>Clausophyidae</td><td>Crystallophyes amygdalina</td><td>KX374466</td><td>KX421850.1</td></tr><tr><td>Calycophorae</td><td>Clausophyidae</td><td>Kephyes ovata</td><td>AY935294</td><td>AY937336.1</td></tr><tr><td>Calycophorae</td><td>Diphyidae</td><td>Chelophyes appendiculata</td><td>PV190274</td><td>PV138274</td></tr><tr><td>Calycophorae</td><td>Diphyidae</td><td>Chelophyes contorta</td><td>PV190275</td><td>PV138275</td></tr><tr><td>Calycophorae</td><td>Diphyidae</td><td>Diphyes antarctica</td><td>PV190280</td><td>PV138280</td></tr><tr><td>Calycophorae</td><td>Diphyidae</td><td>Diphyes bojani</td><td>MZ230468.1</td><td>MZ230443.1</td></tr><tr><td>Calycophorae</td><td>Diphyidae</td><td>Diphyes chamissonis</td><td>MZ230470.1</td><td>MZ230445.1</td></tr><tr><td>Calycophorae</td><td>Diphyidae</td><td>Diphyes dispar</td><td>AY935276</td><td>AY937318.1</td></tr><tr><td>Calycophorae</td><td>Diphyidae</td><td>Eudoxoides mitra</td><td>MZ230478.1</td><td>MZ230453.1</td></tr><tr><td>Calycophorae</td><td>Diphyidae</td><td>Eudoxoides spiralix</td><td>PV190281</td><td>PV138281</td></tr><tr><td>Calycophorae</td><td>Diphyidae</td><td>Gilia reticulata</td><td>KX374469.1</td><td>KX421854.1</td></tr><tr><td>Calycophorae</td><td>Diphyidae</td><td>Lensia conoidea</td><td>AY935318</td><td>AY937360.1</td></tr><tr><td>Calycophorae</td><td>Diphyidae</td><td>Lensia cossack</td><td>MZ230483.1</td><td>MZ230458.1</td></tr><tr><td>Calycophorae</td><td>Diphyidae</td><td>Lensia hardy</td><td>PV190282</td><td>PV138282</td></tr><tr><td>Calycophorae</td><td>Diphyidae</td><td>Lensia subtilis</td><td>PV190283</td><td>PV138273</td></tr><tr><td>Calycophorae</td><td>Diphyidae</td><td>Muggiaea atlantica</td><td>AY935295</td><td>AY937337.1</td></tr><tr><td>Calycophorae</td><td>Diphyidae</td><td>Muggiaea kochii</td><td>PV190285</td><td>PV138286</td></tr><tr><td>Calycophorae</td><td>Diphyidae</td><td>Sulculeolaria chuni</td><td>PV190287</td><td>PV138288</td></tr><tr><td>Calycophorae</td><td>Diphyidae</td><td>Sulculeolaria quadrivalvis</td><td>PV190288</td><td>PV138289</td></tr><tr><td>Calycophorae</td><td>Hippopodiidae</td><td>Hippopodius hippopus</td><td>AY935299</td><td>AF358069.1</td></tr><tr><td>Calycophorae</td><td>Hippopodiidae</td><td>Vogtia glabra</td><td>AY935308</td><td>AY937350.1</td></tr><tr><td>Calycophorae</td><td>Hippopodiidae</td><td>Vogtia pentacantha</td><td>AY935320</td><td>AY937362.1</td></tr><tr><td>Calycophorae</td><td>Hippopodiidae</td><td>Vogtia serrata</td><td>PV190289</td><td>PV138290</td></tr><tr><td>Calycophorae</td><td>Hippopodiidae</td><td>Vogtia spinosa</td><td>PV190290</td><td>PV138291</td></tr><tr><td>Calycophorae</td><td>Prayidae</td><td>Craseoa lathetica</td><td>AY935297</td><td>AY937339.1</td></tr><tr><td>Calycophorae</td><td>Prayidae</td><td>Desmophyes haematogaster</td><td>DQ080006</td><td>KX421851.1</td></tr><tr><td>Calycophorae</td><td>Prayidae</td><td>Desmophyes villafrancae</td><td>PV190279</td><td>PV138279</td></tr><tr><td>Calycophorae</td><td>Prayidae</td><td>Lilyopsis fluoracantha</td><td>SRR1548373</td><td>AY919607.1</td></tr><tr><td>Calycophorae</td><td>Prayidae</td><td>Lilyopsis medusa</td><td></td><td>PV138284</td></tr><tr><td>Calycophorae</td><td>Prayidae</td><td>Maresearsia praeclara</td><td>PV190284</td><td>PV138285</td></tr><tr><td>Calycophorae</td><td>Prayidae</td><td>Nectadamas diomedea</td><td>AY935306</td><td>AY937348.1</td></tr><tr><td>Calycophorae</td><td>Prayidae</td><td>Nectopyramis natans</td><td>AY935307</td><td>AY937349.1</td></tr><tr><td>Calycophorae</td><td>Prayidae</td><td>Praya dubia</td><td>AY935285</td><td>AY937326.1</td></tr></tbody></table>
<table><caption>Table S4. List of sequences used in this study, with their GenBank accession numbers. Novel sequences generated in this study are written in bold. Related to Figure 6.</caption><thead><tr><th></th><th></th><th></th><th>PV190286</th><th>PV138287</th></tr></thead><tbody><tr><td>Calycophorae</td><td>Sphaeronectidae</td><td>Sphaeronectes bougisi</td><td>AY935301</td><td>AF358070.1</td></tr><tr><td>Calycophorae</td><td>Sphaeronectidae</td><td>Sphaeronectes koellikeri</td><td>AY935284</td><td>AF358065.1</td></tr><tr><td>Cystonectae</td><td>Physaliidae</td><td>Physalia physalis</td><td>AY935309</td><td>AY937351.1</td></tr><tr><td>Cystonectae</td><td>Rhizophysidae</td><td>Rhizophysa eysenhardtii</td><td>AY935286</td><td>AY937327.1</td></tr><tr><td>Cystonectae</td><td>Rhizophysidae</td><td>Rhizophysa filiformis</td><td>AY935270</td><td>AY937312.1</td></tr><tr><td>Physonectae</td><td>Agalmatidae</td><td>Agalma clausi</td><td>AY935271</td><td>AY937313.1</td></tr><tr><td>Physonectae</td><td>Agalmatidae</td><td>Agalma elegans</td><td>AY935272</td><td>AY937314.1</td></tr><tr><td>Physonectae</td><td>Agalmatidae</td><td>Agalma okenii</td><td>AY935274</td><td>AY937316.1</td></tr><tr><td>Physonectae</td><td>Agalmatidae</td><td>Athorybia rosacea</td><td>AY935316</td><td>AY937323.1</td></tr><tr><td>Physonectae</td><td>Agalmatidae</td><td>Halistemma rubrum</td><td>DQ080009</td><td>SRR1548374</td></tr><tr><td>Physonectae</td><td>Agalmatidae</td><td>Lychnagalma utricularia</td><td>DQ080007</td><td>SRR1548375</td></tr><tr><td>Physonectae</td><td>Agalmatidae</td><td>Marrus claudanielis</td><td>AY935296</td><td>AY937338.1</td></tr><tr><td>Physonectae</td><td>Apolemiiidae</td><td>Nanomia bijuga</td><td>KF214712</td><td>KF214714.1</td></tr><tr><td>Physonectae</td><td>Apolemiiidae</td><td>Apolemia lanosa</td><td>KF214713</td><td>KF214715.1</td></tr><tr><td>Physonectae</td><td>Erennidae</td><td>Erenna laciniata</td><td>KX752722</td><td>KX752704.1</td></tr><tr><td>Physonectae</td><td>Erennidae</td><td>Erenna richardi</td><td>KX752723</td><td>KX752705.1</td></tr><tr><td>Physonectae</td><td>Erennidae</td><td>Erenna sirena</td><td>KX752725</td><td>AY937361.1</td></tr><tr><td>Physonectae</td><td>Forskaliidae</td><td>Forskalia asymmetrica</td><td>AY935277</td><td>AY937319.1</td></tr><tr><td>Physonectae</td><td>Forskaliidae</td><td>Forskalia edwardsii</td><td>AY935278</td><td>AY937320.1</td></tr><tr><td>Physonectae</td><td>Forskaliidae</td><td>Forskalia formosa</td><td>AY935302</td><td>AY937344.1</td></tr><tr><td>Physonectae</td><td>Forskaliidae</td><td>Forskalia tholoides</td><td>AY935279</td><td>AY937321.1</td></tr><tr><td>Physonectae</td><td>Physophoridae</td><td>Physophora hydrostatica</td><td>AY935300</td><td>AY937342.1</td></tr><tr><td>Physonectae</td><td>Pyrostephidae</td><td>Bargmannia amoena</td><td>AY935292</td><td>AY937333.1</td></tr><tr><td>Physonectae</td><td>Pyrostephidae</td><td>Bargmannia elongata</td><td>AY935321</td><td>AY937334.1</td></tr><tr><td>Physonectae</td><td>Resomiidae</td><td>Resomia ornicephala</td><td>SRR1548382</td><td>SRR1548382</td></tr><tr><td>Physonectae</td><td>Rhodaliidae</td><td>Stephalia dilata</td><td>AY935315</td><td>AY937357.1</td></tr><tr><td>Physonectae</td><td>Stephanomidae</td><td>Stephanomia amphytridis</td><td>AY935280</td><td>AY937322.1</td></tr><tr><td>Outgroup</td><td></td><td>Ectopleura dumortieri</td><td>EU305474</td><td>EU272616.1</td></tr><tr><td>Outgroup</td><td></td><td>Hydra circumcincta</td><td>GU722773</td><td>EU876568.1</td></tr><tr><td>Outgroup</td><td></td><td>Porpita porpita</td><td>AY935322</td><td>AF358086.1</td></tr><tr><td>Outgroup</td><td></td><td>Staurocladia wellingtoni</td><td>AJ580934</td><td>AF358084.1</td></tr><tr><td>Outgroup</td><td></td><td>Velella velella</td><td>AY935323</td><td>AF358087.1</td></tr></tbody></table>
<table><caption>**Table S5. Summary of buoyancy experiments. Related to Figure 7.**</caption><thead><tr><th rowspan="2">Colony ID</th><th rowspan="2">Number of cormidia</th><th colspan="2">Position at T = 0h</th><th colspan="2">Position at T = 24h</th></tr><tr><th>% at the bottom</th><th>% floating</th><th>% at the bottom</th><th>% floating</th></tr></thead><tbody><tr><td>col.1</td><td>25</td><td>100</td><td>0</td><td>0</td><td>100</td></tr><tr><td>col.2</td><td>27</td><td>100</td><td>0</td><td>0</td><td>100</td></tr><tr><td>col.3</td><td>13</td><td>100</td><td>0</td><td>0</td><td>100</td></tr><tr><td>col.4</td><td>14</td><td>100</td><td>0</td><td>0</td><td>100</td></tr><tr><td>col.5</td><td>10</td><td>100</td><td>0</td><td>0</td><td>100</td></tr></tbody></table>

Data were summarized to show the number of cormidia within each tested colony (ColonyID) and the outcome of experiment as percent of cormidia at a given position (bottom or floating) at the beginning of the experiment (T = 0h) and after 24h.
Supplemental reference

S1. Munro, C., Siebert, S., Zapata, F., Howison, M., Serrano, A.D., Church, S.H., Goetz, F.H., Pugh, P.R., Haddock, S.H.D., and Dunn, C.W. (2018). Improved phylogenetic resolution within Siphonophora (Cnidaria) with implications for trait evolution. Mol Phylogenet Evol 127, 823–833.