Origin of ecdysis: fossil evidence from 535-million-year-old scalidophoran worms

Abstract

With millions of extant species, ecdysozoans (Scalidophora, Nematoida and Panarthropoda) constitute a major portion of present-day biodiversity. All ecdysozoans secrete an exoskeletal cuticle which must be moulted periodically and replaced by a larger one. Although moulting (ecdysis) has been recognized in early Palaeozoic panarthropods such as trilobites and basal groups such as anomalocaridids and lobopodians, the fossil record lacks clear evidence of ecdysis in early scalidophorans, largely because of difficulties in recognizing true exuviae. Here, we describe two types of exuviae in microscopic scalidophoran worms from the lowermost Cambrian Kuanchuanpu Formation (ca 535 Ma) of China and reconstruct their moulting process. These basal scalidophorans moulted in a manner similar to that of extant priapulid worms, extricating themselves smoothly from their old tubular cuticle or turning their exuviae inside out like the finger of a glove. This is the oldest record of moulting in ecdysozoans. We also discuss the origin of ecdysis in the light of recent molecular analyses and the significance of moulting in the early evolution of animals.

1. Introduction

Ecdysozoans, defined literally as ‘the moulting animals', comprise three well-supported clades, namely Scalidophora (Loricifera, Priapulida and Kinorhyncha), Nematoida (Nematoda and Nematomorpha) and Panarthropoda (Arthropoda, Tardigrada and Onychophora) [1–8]. Together, these groups form an essential part of the present-day biodiversity [9,10], with millions of species living in terrestrial and aquatic environments. Ecdysozoans secrete an exoskeletal cuticle which must be shed during growth and replaced by a larger one [11,12], a process called ecdysis. Periodic moulting occurs in all extant representatives of Ecdysozoa and is a key diagnostic feature of the group [10]. Most previous studies on ecdysis focus on physiological and mechanical aspects such as secretion and hardening (e.g. tanning and biomineralization) of new cuticle, as well as shedding of exuvia [13–17] and the neuroendocrine process responsible for it [18]. Recently, genomic and transcriptomic studies have shed new light on the molecular pathways involved in ecdysis, which development has paved the way for the formulation of detailed evolutionary scenarios [18,19]. The two critical steps of ecdysis are separation of the old cuticle from the underlying epidermal cells and splitting of the cuticle along predetermined lines of weakness, a process which allows the animal to extricate itself from its old exoskeleton. In contrast to moulting in arthropods (e.g. crustaceans and insects), the moulting process in other ecdysozoan groups such as scalidophorans and nematoidans is much less well understood, except for certain parasitic nematoidan worms of the human body [20–24] and the larval stages of some scalidophorans [25–28]. Ecdysis is relatively well documented in early Palaeozoic panarthropods such as trilobites [10,29–31], and has also been recognized in more basal groups such as anomalocaridids [32] and lobopodians [33]. A recent study describes the apparent moulting process of a fuxianhuiid euarthropod from the early Cambrian Xiaoshiba Lagerstätte of China [34].

Scalidophoran worms (Priapulida, Kinorhyncha and Loricifera) occur in several exceptional fossil deposits of Cambrian age, either as complete body fossils, incomplete fragments or isolated cuticular sclerites [35–39]. Although some of these cuticular remains have been interpreted provisionally as exuviae [36,40,41] (electronic supplementary material, table S2), uncertainty remains as to whether such fossils are true exuvial fragments discarded by the animal after moulting or incomplete decayed body fossils [3,35,42–44]. Convincing evidence for ecdysis in early Cambrian scalidophorans is provided by a nearly complete specimen of a stem-group loriciferan from the Sirius Passet Lagerstätte that is closely associated with a deformed lorica interpreted as its exuvia [41]. Especially, in macrobenthic scalidophorans [26], the cuticle of modern scalidophoran worms is extremely thin [45] compared with that of arthropods and is prone to mechanical alteration (e.g. tearing, folding, wrinkling and scattering of fragments) after being discarded. This characteristic probably contributed to the scarcity and poor preservation of fossil exuviae and to difficulty in recognizing them. Here, we describe two types of exuviae in scalidophoran worms from the Lower Cambrian Kuanchuanpu Formation (Fortunian Stage, Terreneuvian Series) of Shaanxi Province [46,47], China. These fossils provide the earliest evidence of moulting in ecdysozoans.

2. Material and methods

Specimens examined in the present study were collected from Bed 2 of the Kuanchuanpu Formation in the Zhangjiagou Section near Kuanchuanpu, Xixiang County [47–49]. The fossil-bearing bed belongs to the Anabarites trisulcatus-Protohertzina anabarica Assemblage zone [48], which correlates with the Nemakit-Daldynian interval in Siberia [48]. The absolute age of this bed is estimated to be about 535 Ma [46,50]. Phosphatic fossils were obtained by digesting rock samples in 8–10% acetic acid [51] followed by picking under a binocular microscope. The most common elements are globular spherical microfossils, specifically olivooids [46,52], metazoan embryos [47], cnidarians [53,54], bacteria [55], possible deuterostomes [51] and problematic fossils [56]. Ecdysozoans are rare and consist of scalidophoran remains, most of which are deformed, cylindrical cuticular fragments secondarily mineralized in calcium phosphate; the rest consist of nearly complete specimens [37,38,57]. External ornament is preserved in three dimensions (e.g. sclerites) with remarkable fidelity.

The 13 best-preserved specimens (ELIXX86-112, ELIXX63-29, ELIXX94-84, ELIXX99-492, ELIXX40-125, ELIXX85-284, ELIXX90-221, ELIXX85-429, ELIXX57-467, ELIXX98-179, ELIXX78-205, ELIXX85-16 and ELIXX85-120) were mounted on stubs, coated with gold and imaged with a scanning electron microscope (SEM) (FEI Quanta 400 FEG, high vacuum, secondary electron modes). Synchrotron radiation X-ray tomographic microscopy (SRXTM) was conducted at SPring-8 in Hyogo, Japan (23 KeV, 0.51 × 0.51 × 0.51 µm³ pixel⁻¹) to study the internal anatomy of two specimens (ELIXX85-429 and ELIXX90-221). SRXTM data were processed with Dragonfly 4.0 software for microtomographic analysis and three-dimensional (3D) visualization. Specimens of Ottoia prolifica Walcott, 1911 from the middle Cambrian Burgess Shale were examined in the National Museum of Natural History (Smithsonian Institution), Washington, DC, USA and the Royal Ontario Museum, Toronto, Canada, and photographed using a NIKON D3X digital SLR camera fitted with a macro lens under polarized light. Live specimens of Priapulus caudatus were collected near the Kristineberg Marine Station, Sweden (see details of locality in [58,59]), observed in the laboratory and photographed. All fossil specimens from the Kuanchuanpu Formation are deposited in the palaeontological collections of the Shaanxi Key Laboratory of Early Life and Environments, Northwest University, Xi'an, China (ELIXX numbers). Specimens from the Burgess Shale are housed in the collections of the National Museum of Natural History, Washington DC (USNM numbers) and the Royal Ontario Museum, Toronto, Canada (ROMIP numbers).

3. Results

(a). Cuticular fragments showing positive or negative relief

We describe here two types of ornamented cuticular remains from the Kuanchuanpu Formation, based on 73 3D preserved specimens. The first type (n = 66) consists of crumpled and compressed cylindrical fragments exhibiting positive external relief (PER). Their length and diameter range from approximately 800 µm to 3000 µm and 600 µm to 1200 µm, respectively. They display up to 14 evenly spaced annuli (longitudinal length approx. 140 µm to 360 µm) separated by a deep furrow (figure 1a,b,e,f; electronic supplementary material, figure S4(h)-(i) and table S5). The annuli bear numerous small, evenly distributed sclerites having a broadly rounded or elliptical base extending into a strong apical hollow spine which is often broken. The length and width of the sclerites range from approximately 100 to 290 µm and approximately 30–140 µm, respectively. Sclerites are distributed radially and are slightly curved (figure 1a,b,e,f; electronic supplementary material, figure S4(a)-(i) and table S5). Each annulus bears between eight and 24 closed-packed sclerites separated by a very narrow gap occupied by crumpled cuticular material (electronic supplementary material, table S5). The small sclerites are not aligned in longitudinal columns but instead display a staggered arrangement. Most PER fragments exhibit at least one or two much larger (approx. 500 µm long) sclerites with a circular or elliptical base of the same size. These stout sclerites extend over three or four adjacent annuli and seem to have been symmetrically distributed in pairs around the tubular structure (figure 1a,b,e,f; electronic supplementary material, figure S4(a)–(d), (f)–(i) and table S5). Both sclerite types and other cuticular areas display a fine reticulated ornament with a mesh size varying from approximately 4 µm to 16 µm (electronic supplementary material, table S5). This ornament is particularly well developed in the proximal half of the sclerites (figure 1c,d), but elsewhere it has a wrinkled and more irregular appearance (electronic supplementary material, figure S4(e)). PER fragments display the same external morphology as that of 3D-preserved scalidophoran worms described from the same biota by previous authors (especially Indeterminate Form 1) and consisting of an annulated trunk lined with small and large scalids, a proboscis, a mouth opening and a tail part [38,60].

Figure 1.

Scalidophoran cuticular fragments from the lowermost Cambrian Kuanchuanpu Formation, Shaanxi Province, China. (a–f) Specimens with positive external relief (PER; see explanations in text). (g–p) Specimens with negative external relief (NER). (a–m) Specimens assigned to Indeterminate Form 1 (see the electronic supplementary material, S3). (n–p) A specimen assigned to Indeterminate Form 2 (electronic supplementary material, S3). (a–c) ELIXX86–112, general views from two opposite sides showing small sclerites on annuli and a large sclerite, and details of reticulated ornament along basal part of a sclerite (c). (d–f) ELIXX63-291, reticulated ornament (d) and general views from two opposite sides showing sclerites on annuli. (g–j) ELIXX90-221, general view showing sclerites pointing inwards towards the longitudinal axis of the tubular structure, longitudinal virtual section (microtomographic image) showing sclerites with hollow conical structure (white triangle), details of upper part with sclerites pointing inwardly and outwardly, and reticulated micro-ornament along the inner surface of a sclerite. (k–l) ELIXX85-429, general view and oblique virtual section showing sclerites pointing inwards (white triangle). (m) ELIXX85-16, compressed fragment with elongated end. (n–p) ELIXX85-120, general view from two opposite sides showing small spinose sclerites on annuli, pointing inwards, and details of reticulated ornament of inner surface of a sclerite. All SEM images except (h) and (l) obtained by using synchrotron radiation X-ray tomographic microscopy. Anterior–posterior orientation based on complete specimens described by Eopriapulites sphinx [37], Eokinorhynchus rarus [38], Qinscolex spinosus and Shanscolex decorus [60]. an, annulus; ant, anterior part; fu, furrow between annuli; ls, large spinose sclerite; pos, posterior part; ro, reticulated ornament; ss, small spinose sclerite; vo, void. Scale bars: 500 µm (a,b,e,f,g–i,k–o); 10 µm (c); 20 µm (d) and (j); and 50 µm (p). (Online version in colour.)

The remaining fragments (n = 6) exhibit the same size, crumpled cylindrical shape, small and large sclerites and reticulated ornament as PER fragments, but differ from them in showing inverted relief. Sclerites, instead of projecting outwards, point inwards radially, thus exposing the hollow interior (figure 1g–m); electronic supplementary material, figure S4(j)–(o)). Negative external relief (NER) and PER fragments possess the same type of small and large sclerites and the same reticulated micro-ornament (electronic supplementary material, S3 and table S5). The NER type appears to be an invaginated version of the PER type, having resulted from the inward folding of a flexible, cylindrical cuticular structure about its longitudinal axis, in a manner similar to the reversal of a glove finger.

Both NER and PER cuticular fragments undoubtedly belong to scalidophorans; however, their fragmentary nature allows neither generic nor specific determination. At least four virtually complete scalidophoran worms (Eopriapulites sphinx [37,57], Eokinorhynchus rarus [38], Qinscolex spinosus and Shanscolex decorus [60]) and 12 unnamed forms [38,43,60] have been reported from the Kuanchuanpu Formation. Our specimens may represent two distinct forms, predominantly Indeterminate Form 1 and Indeterminate Form 2 (figure 1n–p; electronic supplementary material, figure S3 and table S5).

4. Discussion

(a). Evidence for exuviae in the lowermost Cambrian

It is generally difficult to distinguish moults (exuviae) from carapaces unless the animal is preserved in the act of shedding its old exoskeleton [34,41]. Suggested criteria for the identification of fossil moults are mainly applicable to arthropods having a relatively hard carapace such as trilobites [10], and include the presence of suture lines as well as repetitive associations and configurations of specific exoskeletal elements [10].

Unlike the cuticle of arthropods, that of modern scalidophoran worms is extremely thin and fragile (electronic supplementary material, figure S1(a)–(g)) [59]. Exuviae are, therefore, prone to dispersal, tearing, folding and (eventually) decay, thus greatly reducing their preservation potential. Certain cuticular fragments from Cambrian and younger strata have been interpreted as possible exuviae (electronic supplementary material, table S2). Potential criteria for positive identification of exuviae (versus decayed body fossils) include: (i) a highly distorted and folded appearance [3,35,42,43]; (ii) the absence of remains of internal tissues and organs [41,44]; and (iii) dislocation of cuticle elements (e.g. isolated proboscis and trunk fragments detached from the proboscis) [36,41]. However, worm bodies (e.g. Priapulus) that have undergone selective decay (e.g. cuticle versus soft tissues) [61], post-mortem transportation and disarticulation [10] may exhibit configurations similar to those observed in putative fossil exuviae [61]. For example, Ottoia prolifica, from the middle Cambrian Burgess Shale is represented both by complete body fossils with a typical swollen shape (electronic supplementary material, figure S6) and preserved intestine, and by a variety of distorted and incomplete specimens produced by more or less advanced decomposition [36]. A very small percentage of these incomplete specimens, though folded, exhibit distinct outlines and seem to represent compressed, empty cuticular envelops that may confidently be interpreted as exuviae (electronic supplementary material, figure S6). Another example is a single loriciferan from the lower Cambrian Sirius Passet Lagerstätte that appears to be preserved in the process of emerging from its empty exuvia (loricae, [41]). Similarly, Müller & Hinz-Schallreuter [40] interpreted palaeoscolecid cuticular fragments with double-layer ornamentation as belonging to worms buried shortly before ecdysis. Aside from these rare cases, the identification of fossil exuviae is uncertain [3,42–44]. Indeed, PER fragments from the Kuanchuanpu Formation may represent either exuviae or body fragments in which soft parts are entirely decomposed. The same is definitely not true, however, for NER fragments (figure 1g–p; electronic supplementary material, figure S4(j)–(n)), which as argued above resulted from the invagination of a flexible, tube-like cuticular envelop. Such specimens could not have been produced by simple disarticulation or by external physical forces such as bottom currents. We conclude therefore that NER fragments are exuviae that underwent mechanical inversion during moulting. These unusual tubular fossils with inverted relief provide the solid confirmation of ecdysis in fossil scalidophorans, and extend the origin of moulting back to the lowermost Cambrian (ca 535 Ma).

(b). Moulting process in extant scalidophorans

The moulting process in extant scalidophorans was described by Lang [62] from Priapulus caudatus, which undergoes separation of the cuticle from the epidermis and breakup of the old cuticle in the oral region, allowing the worm to emerge from its exuvia through undulating movements and repeated friction with sediment [25,26,62]. Recent observations on Priapulus caudatus under laboratory conditions [62] (J.V., figure 2) revealed the operation of three successive steps: (i) gradual separation of the old cuticle from the body along the trunk (figure 2b); (ii) detachment of the cuticle lining the pharynx from the anteriormost part of the gut followed by evagination of this cuticle to form an empty, fragile envelope which remains attached to the proboscis temporarily (figure 2b–d); and (iii) splitting of the old cuticle into two parts around the proboscis, allowing the pharyngeal cuticle to detach (figure 2e,f). During the final stage, the worm is in the process of hardening a new cuticle and is able to extend and retract its proboscis within the tubular structure formed by the exuvia (figure 2g). How the newly moulted worm eventually extricates itself completely from its exuvia could not be observed. Nevertheless, we now know that at least one part of the exuvia (pharynx) can evaginate. Also, exuviae exhibiting negative relief similar to NER type in fossil worms (figure 1) were not observed under laboratory conditions (Petri dish with seawater [26–28,62] and present paper). More detailed studies comparing the moulting process of Priapulus with that of other priapulid worms such Halicryptus are needed to confirm these preliminary observations, and such observations must take into account the role of sediment in ecdysis (e.g. friction, anchoring and pressure exerted by sediment in burrows).

Figure 2.

Moulting process in the extant priapulid worm Priapulus caudatus from Sweden, as observed under laboratory conditions (no sediment). (a) Before moulting. (a–c) Evaginated pharyngeal cuticle, general view and details (showing pharyngeal teeth pointing upwards). (d) Folded pharyngeal cuticle still attached to proboscis. (e–f) Exuvia split into two parts (pharyngeal cuticle discarded) and newly moulted worm protruding beyond its old cuticle; general view and details of pharyngeal cuticle. (g) Newly moulted worm retracted into its old cuticle. Light photographs of live specimen in seawater. an, annulation; atg, attachment to gut; atp, attachment to proboscis; cp, caudal process; epc, evaginated pharyngeal cuticle; ex(1) anterior part of exuvia; ex(2) posterior part of exuvia; gu, gut; m, mouth; nmw, newly moulted worm; pr, proboscis; pt, pharyngeal teeth; tr, trunk. Scale bars: 5 mm. (Online version in colour.)

(c). Moulting process in lowermost Cambrian scalidophorans

Two types of scalidophoran exuviae coexist in the Kuanchuanpu biota. The most frequent type is represented by a deformed, cylindrical cuticular structure showing PER expressed by pointed sclerites (figure 1a,b,e,f; electronic supplementary material, figure S4(a)–(i)). We hypothesize that this type resulted from the uniform separation of the new cuticle, allowing the newly moulted worm to leave its old cuticle smoothly (figure 3a). The external cuticular ornament (e.g. scalids), by promoting friction and anchoring to sediment, may have helped the worm to withdraw from its exuvia, assuming that ecdysis occurred within the sediment (infaunal lifestyle as in extant priapulids). The second type has a comparable cylindrical shape but an NER expressed by sclerites pointing inwardly (figure 1g–p; electronic supplementary material, figure S4(j)–(o)). Two different mechanical processes may explain NER exuviae: (i) frictional forces between the cuticular ornament (e.g. sclerites) and sediment causing the cuticle to evaginate as the worm moves forwards (figure 3b); or (ii) local attachment of the cuticle to the body plus pressure from surrounding sediment causing the cuticle to break apart, with the posterior portion being released and the anterior part remaining attached to the body, invaginating, and then being dragged along as the worm moves forwards (figure 3c). Both mechanisms are capable of producing an exuvia exhibiting negative relief. PER and NER moulting processes have analogues in extant scalidophorans (figure 2) [26]. The PER type clearly corresponds to the moulting of the trunk of Priapulus (the newly moulted worm extricates itself smoothly from the old cuticle when loosened), while the NER type strongly recalls the evagination of the pharyngeal cuticle (figure 2b–f). An interesting analogue of cuticular invagination is also found in snakes (electronic supplementary material, figure S7—compare with figure 3b), in which the external layer of the skin is peeled away. The snake crawls out of it, turning it inside out and thus exposing scales with inverted relief. As in Cambrian scalidophorans, this process is initiated by local attachments of the old skin to the body and is associated with the splitting of the exuvia into tubular sections of various lengths.

Figure 3.

Hypothesized moulting processes in scalidophoran worms from the lowermost Cambrian Kuanchuanpu Formation, Shaanxi Province, China. Worm body and exuvia represented in light and dark blue, respectively. (a) Ecdysis of type 1 (PER); (1) the old cuticle detaches from body, (2) the worm extricates itself from its tubular exuvia via muscular contractions and (3) has shed its old cuticle which shows positive relief (expressed by sclerites pointing outwards). (b) Ecdysis of type 2 (NER); (1) friction or local anchoring to sediment (red arrows) cause the cuticle to withdraw and fold up posteriorly, (2) to be eventually evaginated (3 and 4) as the worm moves forwards (green arrows), leading to the exuvia being turned inside out (5). (c) Ecdysis with both types (PER and NER); (1) the cuticle remains attached locally (e.g. sclerite) to the body and breaks apart (owing to straining forces), (2) the posterior part is released as a PER exuvia whereas the anterior portion is dragged along and invaginated as the worm goes forward (3 and 4), leading to a NER exuvia (5). an, annulus; ant, anterior part; epc, evaginated pharyngeal cuticle; ex, exuvia; ls, large spinose sclerite; NER, negative external relief; nmw, newly moulted worm; PER, positive external relief; pos, posterior part; pr, proboscis; ss, small spinose sclerite; tr, trunk; white dots represent sediment. Not to scale. (Online version in colour.)

(d). Origin and evolution of ecdysis

Molecular phylogenies suggest that ecdysozoans have a very remote origin and ramified during the Ediacaran Period (approx. 635–543 Ma) [4,63–66]. However, uncertainties remain concerning the chronology of divergence of the major ecdysozoan clades (Nematoida, Scalidophora and Panarthropoda) and the phylogenetic relationships between subgroups (e.g. Priapulida, Kinorhyncha and Loricifera for Scalidophora). Alternative evolutionary hypotheses require proper testing using fossil data, more accurate calibration (e.g. [67]) and a critical approach to molecular clock dating (e.g. [66]).

Although scalidophorans and a variety of panarthropods including lobopodians [68] and iconic arthropods such as trilobites [69] are abundantly represented in early Cambrian marine communities (e.g. the 520-Ma-old Chengjiang biota [70] and the 518-Ma-old Qingjiang biota [39]), no ecdysozoan fossils have been found thus far in late Precambrian (Ediacaran) rocks [65]. Burrow systems attributed to priapulid-like worms are used to define the Precambrian–Cambrian boundary [58,71,72], but they are not associated with their assumed trace-makers. The oldest confirmed occurrence of ecdysozoans in the lowermost Cambrian (Fortunian Stage, ca 535 Ma) is the complete body fossils [37,38,57] and exuviae (this paper) of scalidophorans from the Kuanchuanpu Formation. Our findings also corrobarate hypotheses based on fossil evidence that younger Cambrian scalidophorans such as Ottoia prolifica (electronic supplementary material, figure S6) and other forms [3,35] shed their cuticle during growth.

The occurrence of moulting in early scalidophorans suggests that these animals had already acquired a genetic tool-kit and possibly a neuroendocrine system to achieve ecdysis. Although little is known about the molecular control of moulting in scalidophoran worms, a recent molecular study has shed light on key evolutionary aspects of ecdysis [18]. Scalidophorans possess genes that are known to play a key role in the moulting process of other ecdysozoan taxa (e.g. homologues of Early genes and Receptor genes (EcR, USP/RXR) [18]), but they lack genetic elements essential to the ecdysteroid biosynthesis of arthropods and their relatives (e.g. Halloween genes [18]). This raises the question of whether priapulids, kinorhynchs and loriciferans use steroid hormones such as 20-hydroxyecdysone (20E) or other molecules (e.g. dafachronic acid in free-living nematodes [73]) for moulting. The moulting hormone 20E also occurs in non-ecdysozoan animals (e.g. platyhelminthes, annelids and molluscs) and may have been present in the last common ancestor (LCA) of protostomes [18,74–77]. This set of recent molecular data suggests that the early evolution of ecdysis includes a suite of molecular innovations possibly starting with the occurrence of Early genes in the LCA of protostomes and continuing throughout the three ecdysozoan clades (acquisition of the EcR/USP complex and Halloween genes; figure 4).

Figure 4.

Evolution of moulting. Simplified tree showing phylogenetic relationships of the major bilaterian taxa, including ecdysozoans, lophotrochozoans and deuterostomes (modified after [18,19,78]). Numbers at nodes represent possible genetic innovations in the early evolution of ecdysis. Blue dots indicate the presence of the moulting hormone 20-hydroxyecdysone (20E), which is present in parasitic nematodes and has been not found in free-living forms to date. Green dot indicates the origin of ecdysone-induced moulting. Orange dots with white numbers indicate the early fossil record of ecdysis in basal scalidophorans (1; this paper), priapulids (2; [36,40]), loriciferans (3; [41]), and arthropods (4; fuxianhuiids, [34]). (Online version in colour.)

Fossil evidence confirms that several groups of moulting animals, including scalidophorans (this paper), loriciferans [41] and various panarthropods (e.g. stem-group arthropods, [34]), were present in the early Cambrian. The time of origin (whether Ediacaran or later) and general morphology of the first moulting animal remain an open question. We here hypothesize that this animal was a worm-like, possibly infaunal organism with a hydrostatic skeleton [79], the forces governing locomotion having been transmitted not through rigid skeletal elements but rather through internal pressure controlled by muscle contractions and relaxation as in extant priapulid worms [59]. The scalidophorans from the Kuanchuanpu Formation have high-relief sclerites, suggesting that thicker and more complex cuticular features developed among early ecdysozoans for greater protection against abrasion, and/or predation, or for more efficient locomotion (e.g. anchoring features for burrowing [7,68]). This set of innovations may have been of major importance in the rise of ecdysis, as an ornamented and relatively rigid exoskeleton is hardly compatible with continuous growth (e.g. extant crustaceans) and must be replaced by a larger one. Thus, ecdysis may have been selected as the most adequate solution to overcome these mechanical constraints, and may have opened up new evolutionary opportunities. Ecdysis implies molecular innovations probably inherited from genetic elements were already present in non-moulting ancestors (figure 4), an epidermal structure compatible with ecdysis, and close correlations between body plans and the moulting process (table 1). Ecdysis seems to have appeared in the context of the profound environmental changes that characterized the Precambrian–Cambrian transition, including increasing oxygen levels [87–89] and interactions among animals [90–92], and thus the onset of more complex marine ecosystems (e.g. food webs and colonization of infaunal niches [90,91]).

Table 1.

Ecdysis in extant Ecdysozoa and Annelida: relationships between body plan and moulting process. (*1: some parasitic nematodes have a reduced cuticle or no cuticle at all. *2: external segmental cuticular elements. *3: a stiff cuticular case with plate-like elements. *4: parapodia act as limbs in most polychaetes. *5: moult in pre-adult stages and females (e.g. Panagrellus silusiae). *6: moulting in adult males (e.g. Panagrellus silusiae). *7: friction with sediment may facilitate ecdysis. *8: observed in Polychaeta (e.g. Lumbrineris crosslandi; Marphysa sanguinea; Nereis diversicolor). *9: observed in jaws from some Polychaeta (e.g. Diopatra aciculata).)

clade	taxa	body plan	moulting process	refs
Panarthropoda	Arthropoda	articulated chitinous exoskeleton; jointed limbs	exuvia splits open through various ways, where cuticle is thinner	[80]
	Onychophora	unarticulated chitinous cuticle; unjointed limbs	ecdysis starts from mid-dorsal split line	[81]
	Tardigrada	unarticulated chitinous cuticle; unjointed limbs	ecdysis starts from anterior end	[15]
	Nematomorpha	worm-shaped; no limbs; no circular muscles	ecdysis starts from anterior end	[82]
Nematoida	Nematoda (free-living)	worm-shaped; no limbs; cuticle with collagen; no circular muscles	cuticle sheds in tatters (*5) or peels off from anterior end (*6)	[83]
	Nematoda (parasitic)	worm-shaped; no limbs; cuticle (*1) with collagen; no circular muscles	ecdysis starts from anterior end	[84]
	Priapulida	worm-shaped; no limbs; thin chitinous cuticle; circular and longitudinal muscles	ecdysis starts from anterior end; friction with sediment (*7)	[26,62]
Scalidophora	Kinorhyncha	worm-shaped; no limbs; segmented trunk (*2); thin chitinous cuticle; circular and longitudinal muscles	ecdysis starts from anterior end	[28]
	Loricifera	no limbs; trunk covered by lorica (*3); thin chitinous cuticle; circular and longitudinal muscles	ecdysis starts from anterior end	[27,85]
Lophotrochozoa	Annelida	segmented body; cuticle with collagen; parapodia (*4)	cuticle sheds in flakes (*8) or partial ecdysis (*9)	[74,86]

Data accessibility

All fossil specimens from the Kuanchuanpu Formation are deposited in the collections of the Shaanxi Key Laboratory of Early Life and Environments, Northwest University, Xi′an, China (ELIXX numbers), those from the Burgess Shale, in the collections of the National Museum of Natural History, Washington DC (USNM numbers) and the Royal Ontario Museum, Toronto, Canada (ROMIP numbers).

Authors' contributions

J.H. conceived the project. D.W., J.H. and J.V. interpreted the fossil material; T.K. and K.U. conducted the microtomographic analyses (synchrotron). J.S. reconstructed the microtomographic data. D.W., J.H., J.V. and I.S. wrote the paper with input from the other authors. All authors read and approved the final manuscript.

Competing interests

We declare we have no competing interests.

Funding

This work was supported by the Strategic Priority Research Program of Chinese Academy of Sciences (grant no. XDB26000000), Natural Science Foundation of China (nos. 41621003, 41772010, 41672009, 41720104002), Ministry of Science and 111 project of Ministry of Education of China (no. D17013), the Most Special Fund from the State Key Laboratory of Continental Dynamics, Northwest University, China (BJ11060) and Northwest University Graduate Innovation and Creativity Funds (no. YZZ17195).