A middle Cambrian macroscopic tardigrade ancestor

A middle Cambrian macroscopic tardigrade ancestor | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Biological Sciences - Article A middle Cambrian macroscopic tardigrade ancestor Marc Mapalo, Javier Ortega-Hernández This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8911114/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Tardigrades, commonly known as water bears, are a major phylum of microinvertebrates best known for their ability to withstand extreme environmental conditions 1 . As one of the three groups that comprise the megadiverse Panarthropoda, alongside onychophorans and euarthropods, tardigrades represent an emerging model system for understanding the origin of metazoan body plans 2 , the evolution of segmentation 3 , and the genetic machinery responsible for their formidable cryptobiotic capabilities 4 . However, the early history of the group remains elusive because fossil record is heavily biased against small-bodied aquatic invertebrates, with the only unequivocal tardigrade fossils known from Cretaceous-aged or younger amber deposits 5–8 . Here, we comprehensively revise the morphology and affinities of the claw-bearing lobopodian Aysheaia pedunculata from the mid-Cambrian (Wuliuan) Burgess Shale. Previously regarded as a possible relative of onychophorans based on their similar body size and general organization, we demonstrate several derived morphological characters shared between Aysheaia and extant tardigrades including supernumerary scythe-like claws, a bulbous pharynx, and reversed hind leg orientation. We employ a developmentally informed dataset to test between three different competing scenarios on the homology of anterior appendages found in the head of Aysheaia and extant tardigrades. The results consistently recover Aysheaia as a stem-group tardigrade and inform the likely morphology of the tardigrade last common ancestor prior to the main radiation of the crown-group. Our findings strengthen the evo-devo hypothesis that modern tardigrades evolved from a macroscopic ancestor through overall miniaturization 9 and loss of the intermediate region in the trunk and legs 3,10 . Biological sciences/Evolution/Palaeontology Biological sciences/Evolution/Taxonomy Biological sciences/Evolution/Phylogenetics Figures Figure 1 Figure 2 Figure 3 Figure 4 Main The leg-bearing panarthropods are the most diverse group of molting animals and include the extant phyla Euarthropoda (e.g. arachnids, myriapods, pancrustaceans, insects), Onychophora (velvet worms), and Tardigrada (water bears) 11 , which are estimated to have diverged from each other around 600 Mya 12 . Although panarthropods have a lengthy evolutionary history extending to the base of the Cambrian 13 , most of the available information on their macroevolution comes from early and mid-Cambrian sites with exceptional soft-tissue preservation thanks to the high fossilization potential of their chitinous cuticular exoskeleton. Lobopodians are the earliest branching panarthropods, diverse ecdysozoans resembling worms with legs that include stem lineage members of all extant panarthropod phyla 14 . Lobopodians inform critical early steps in the evolution of euarthropods, such as the origin of arthropodized legs, biramous appendages and a multisegmented head (e.g. 15–17 ), and onychophorans, whose marine Cambrian relatives exhibited substantial morphological and ecological disparity relative to modern terrestrial forms (e.g. 18–20 ). However, the Cambrian fossil record is comparatively silent with regard to the early history of tardigrades, which ultimately results in an incomplete view of panarthropod origins and macroevolution. Tardigrades are microinvertebrates (~50 um – 1.2mm) with a segmented body plan and four pairs of lobopodous legs that typically terminate in claws 21 , and which have the notable ability to survive extreme conditions, such as space vacuum, ionizing radiation, and extremely low temperatures 1 . The phylum is subdivided into the heterotardigrades, which, predominantly the marine ones, have high morphological disparity, including various external cuticular structures (e.g., cirri, clavae, dorsal plates), and the eutardigrades, which are predominantly limnoterrestrial and have a simpler plump-like appearance without external cuticular specializations 21 . Extant tardigrade diversity comprises over 1500 described species 22–24 , but extinct taxa are only known from four known crown-group representatives ranging from the early Cretaceous to the Miocene 5–8 . Fossil calibrated molecular clock estimates suggest that crown-group tardigrades diverged around the mid to late Cambrian 7 , implying the existence of Cambrian stem-group tardigrades. Although some Cambrian fossils have been regarded as potential members of total-group Tardigrada, there are issues with these interpretations that restrict their ability to reconstruct the early evolution of this phylum. Middle Cambrian Orsten-type Siberian microfossils have been interpreted as juvenile tardigrades due to their size and overall morphology 25,26 . These fossils differ from extant tardigrades in having only three pairs of claw-bearing legs, which led to the interpretation that the Cambrian forms sequentially added body segments during early ontogeny. However, embryological studies demonstrate that tardigrade legs develop simultaneously 27,28 , and Hox gene expression patterns indicate that four leg-bearing segments would still be expected even if terminal addition had been lost 3 . Thus, the tardigrade affinity of these Siberian fossils remains uncertain. A recent reappraisal of Cambrian lobopodians suggested that luolishaniids, a derived group typified by the presence of setulose anterior limbs used for suspension feeding, might be part of the tardigrade stem lineage based on the anteroposterior differentiation of the legs and presence of paired sensory structures on the head 29 . However, these shared characters appear superficial since the structures themselves are not morphological similar to tardigrades. Lastly, another Cambrian fossil that was proposed to have an affinity to tardigrades is Aysheaia pedunculata. Initially misidentified as a polychaete 30 , Aysheaia has the distinction of being the first described lobopodian chronologically. Although subsequent systematic work suggested possible affinities with tardigrades based on the reverse orientation of the hind legs and morphology of the terminal claws, the precise position of Aysheaia remains uncertain due to similarities with onychophorans including the overall morphology and macroscopic body size 31 . The most recent extensive redescription of this fossil also acknowledged its possible relationship to tardigrades but decided not to relegate it to any specific extant higher taxon 32 . Furthermore, although various studies have incorporated Aysheaia in phylogenetic analyses of lobopodians (e.g. 19,20,33,34 ) this taxon has never been the main focus and its position has proven unstable among the stem lineages of panarthropods, euarthropods and tardigrades. Here, we redescribe the morphology of the mid-Cambrian lobopodian Aysheaia pedunculata from the Burgess Shale in British Columbia, report new characters based on the type material housed at the Smithsonian Institution (USNM) and undescribed material housed at the Royal Ontario Museum (ROMIP), and reassess its phylogenetic affinities in the context of panarthropod evolution. Morphological redescription of Aysheaia pedunculata Studied material includes 31 specimens including new collections from the ROMIP and the type material 32 (Table S1,S2) (see Supplementary Text for remarks and discussion). Specimens consist of highly compacted macrofossils composed of organic carbon films typical of Burgess Shale-type preservation 35,36 ; with 45% of specimens preserved in lateral or oblique view, and 55% preserved in dorsal or ventral orientations (Table S1,S2). The body is elongate, with the length of complete specimens ranging from 1.06 to 5.4 cm (Table S3) and can be divided into the head region with the mouth part and a pair of spine-bearing frontal-most differentiated appendages, and the trunk region consisting of 10 pairs of walking legs (Figs 1A, S1A-D). Except for the mouth region, the entire body is annulated and features conical structures found at the dorsal side on top of the ridges separated by the annulations (Figs 1B,C,S2). This dorsal ornamentation is only observed in larger specimens (longer than ~2 cm) (Table S3). The anterior-facing mouth has a terminal position, and the opening is surrounded by six elongated papillae (Figs 1D, S1E-F). The circumoral papillae are semi-flexible (rather than rigid) and their position at the lateral sides (e.g., Fig S2) suggest that a portion of the mouth is at least partially retractable (Fig 1D). The frontal appendages are annulated (Fig 1D) and attached laterally at either side of the head region (Figs 1A, S1A-D). There are three to five spines running along the anterior side of the frontal appendages and three distal spines at their tips (Figs 1A,D, S1F, S3A,B). The spines have a rigid appearance, particularly when compared to the more malleable mouth papillae. The walking legs are also annulated, attached ventro-laterally to the trunk, and terminated with claws (Figs 1C, S2). The first nine pairs of legs face posteriorly, while the posteriormost 10 th leg pair faces anteriorly based on the orientation of the claws (see Figs 1C, S1A,C). The bases of the 10 th leg pair define the end of the trunk region with no recognizable posterior extension beyond this pair (Figs S4A,C). All the walking legs feature two spines – one near the tip of the leg and another more proximally situated (Figs 1C-E, S4B-D). These leg spines have similar morphology to the spines on the frontal appendages. The bases of these spines are found at the outer side of the legs and lateral with respect to the claws (Fig S4B). Five to eight claws with evident straight, long stalks are found in each leg and have a scythe-like morphology (Figs 1E, S4E-G). The alimentary canal is preserved as a dark film running along the body (Fig 1A,E). New material from ROMIP demonstrates that the alimentary canal has a distinctly widened anterior portion connected to the mouth opening, which we interpret as a bulbous pharynx (Fig 1E). Comparison of Aysheaia pedunculata to tardigrades Based on our redescription of A. pedunculata we identify several shared features with the major groups of extant tardigrades (Fig 2A; see 21 for review of tardigrade morphology). A. pedunculata resembles heterotardigrades in the claw and walking leg morphology (Figs 2A-E). The distinctive scythe-like claws of A. pedunculata (Fig 2B) are also found in heterotardigrades and the primary branch of apochelan claws. The presence of supernumerary claws, defined as four or more per leg, with this particular claw shape are characteristics that are only found in marine echiniscoidids, such as Echiniscoides (Fig 2C). The walking leg spines of A. pedunculata (Fig 2D) resemble the heterotardigrade leg sensory organs, such as in arthrotardigrades (e.g., Styraconyx ) (Fig 2E) and echiniscioideans (e.g., Echiniscus ), in terms of their rigid appearance and similar position on the legs. Features shared between A. pedunculata and eutardigrades include the circumoral papillae and dorsal sculpturing (Figs 2F-I). The mouth papillae of A. pedunculata (Fig 2F) resemble the six papillae surrounding the buccal opening of apochelans (e.g., Milnesium ) (Fig 2G), and the dorsal conical structures (Fig 2H) are reminiscent of the tubercles found in some parachelans with sculptured dorsal cuticles (i.e., Fractonotus ) (Fig 2I). A. pedunculata alsohas characteristics that are present across all tardigrades. The bulbous pharynx (Fig 2J) is a fundamental feature observed in both eutardigrades (Fig 2K) and heterotardigrades (Fig 2L). The orientation of the last pair of legs facing anteriorly and having a rotated orientation compared to the rest of the anterior pairs of legs is also shared between A. pedunculata (Fig 2M)and most tardigrades (Figs 2N,O). Lastly, the trunk of A. pedunculata lacks a posterior extension beyond the last pair of legs (Fig 2M), as in the majority of tardigrades (Figs 2N,O). In conclusion, the revised morphology of A. pedunculata reveals a mixture of ancestral and derived characters found across major tardigrade groups. Phylogenetic affinities of Aysheaia pedunculata Given the uncertainty regarding the precise homology of the protocerebral appendage pair of extant tardigrades with those of other panarthropods (see File S1 for details), we tested three different hypotheses and explore their phylogenetic implications in the context of the new data on A. pedunculata . The first (stylet) and second (sensorial) hypotheses regard that the protocerebral appendage pair is expressed as either the stylet apparatus or the lateral cephalic sensorial appendages, respectively. The third hypothesis reflects the complete loss of the protocerebral appendages in extant tardigrades (see Material and Methods). Our results based on Bayesian inference (BI) recovered A. pedunculata in a sister-group relationship relative to crown-group tardigrades (Figs 3B, S11-S13) under all three hypotheses (Fig 3A); the “sensorial hypothesis” produced the highest support values, whereas the “stylet hypothesis” recovered the lowest. Trees recovered using the maximum parsimony (MP) criterion had different topology depending on the character weighting strategy. Under equal weights and all hypotheses, A. pedunculata is recovered in a polytomy together with other lobopodians based on the strict consensus of the most parsimonious trees (MPTs) (Fig S14). However, A. pedunculata is recovered in a sister-group relationship with crown-group tardigrades under implied weights, congruent with the BI results, based on the strict consensus trees of the MPTs (Fig S15). The phylogenetic relationships among crown-group tardigrades under BI differ slightly depending on the tested hypothesis (Figs S11-S13). Under the “stylet hypothesis”, eutardigrades were consistently recovered as a strongly supported clade, but monophyletic heterotardigrades show weak support (Fig S11). In the other two hypotheses (Figs S12,S13), the echiniscoidids ( Echiniscoides and Isoechiniscoides ) were recovered separately from the other tardigrades, but this non-echiniscoidid clade (i.e., eutardigrades + other heterotardigrades) is weakly supported. The same relationship of the echiniscoidids and the rest of the tardigrades were observed in the MP trees using implied weights in all the hypotheses tested (Fig S15). Ultimately, our phylogenetic results indicate that A. pedunculata belongs to the tardigrade stem lineage, regardless of the precise hypothesis of homology of between the protocerebral appendages of extant tardigrades and other panarthropods. Discussion The morphological reappraisal of Aysheaia pedunculata (Fig 4)reveals informative derived characters exclusively shared with heterotardigrades and eutardigrades, including the scythe-like supernumerary claws, circumoral papillae, bulbous pharynx, anterior-facing hind legs, and the lack of posterior extensions (Fig 2). These features collectively provide evidence for the phylogenetic position of A. pedunculata as a stem-group tardigrade, which is further supported by our phylogenetic analyses. Critically, our results do not find support for the recently proposed phylogenetic position of Cambrian luolishaniid lobopodians ( contra 29 ), a highly derived group of armored lobopodians with setulose anterior appendages (e.g., 20,37,38 ), as early tardigrades. We find that the proposed shared characters between luolishaniids and tardigrades, namely the differentiation of anterior and posterior lobopodous limbs batches and the presence of paired cephalic sensory organs, do not represent conclusive synapomorphies. The morphology of all four pairs of the tardigrade legs essentially shares the same external morphology (see illustrated legs in (Fontoura et al. 2017a) and only differ in the reverse orientation of the fourth pair of legs. This contrasts with luolishaniids appendage batches, which are extremely different, consisting of elongate anterior limbs with series of setae and stout and spine-less claw-bearing posterior limbs [e.g. 33,38 ]. The fact that our phylogenetic analyses do not find support for a close relationship between luolishaniids and tardigrades, despite incorporating these proposed characters, indicates that A . pedunculata is the only Cambrian lobopodian that conclusively features numerous complex synapomorphies shared with modern tardigrades. The phylogenetic position of Aysheaia as a stem-group tardigrade provides direct paleontological support for the miniaturization of as suggested by developmental, genomic and morphological evidence 3,9,40 . According to this hypothesis, tardigrades evolved from a macroscopic ancestor that secondarily lost multiple leg-bearing segments from the body the mid-section, which is reflected in the corresponding loss of trunk Hox genes such as Antennapedia , Ultrabithorax , Abdominal-A 3 . Recent developmental work suggests that the walking legs of tardigrades also experienced shortening of their mid-section based on gap gene expression patterns 40 , and that tardigrade legs have a distal identity compared to other panarthropod legs 10 . This evolutionary shortening of the walking legs could explain the presence of two spines on the legs of A. pedunculata whereas heterotardigrades only have one 21 . The resolved affinity of A. pedunculata provides insights into the likely ancestral morphology of crown-group tardigrades prior to their major diversification. First, the body organization would have featured the same number of body segments as modern tardigrades, (i.e. head and four leg-bearing trunk segments), and homonomous walking legs with claws. The overall size would likely be at least as large than the biggest tardigrades alive today (~1.2 mm 21 , and miniaturization among the different groups of tardigrades would have been largely lineage dependent. Second, the last common ancestor most likely had cephalic sensorial appendages, such as cirri or clavae, that also underwent different degrees of reduction in different tardigrade lineages 41,42 , producing the disparity observed between the anterior morphology of heterotardigrades and eutardigrades. Lastly, the ancestral state for the tardigrade claw was likely scythe-shaped as observed in echiniscoids, some arthrotardigrades (e.g., coronarctids, stygarctids) and apochelans (i.e., primary branch of the claw). However, it is uncertain whether the supernumerary phenotype (i.e., more than four claws) is an ancestral state, as suggested by A. pedunculata , or independently derived in different heterotardigrade lineages (e.g., Echiniscoides , Batillipes ). These observations also suggest that the fusion of claws is derived and evolved within eutardigrades, specifically in parachelans, potentially to be able to cling to sediments more efficiently. Current efforts to reconstruct the ancestral states of tardigrades using extant species is inhibited by the unresolved phylogeny of the heterotardigrades 43,44 , which highlights the significance of A. pedunculata for polarizing major morphological characters that define this phylum. Methods Material provenance and digital imaging All studied specimens of Aysheaia pedunculata are housed at the collections of the Smithsonian National Museum of Natural History (NMNH) and the Royal Ontario Museum (ROMIP) (Table S1,S2). All specimens were collected from the Stephen Formation in British Colombia, Canada. Fossil specimens were photographed under cross polarized light, either dry or wet, with an Olympus DSX100 digital microscope, or a Nikon D80 fitted with a Nikkor 50mm macro lens. Digital photographs were adjusted using Adobe Lightroom 11.5. Body lengths were measured using FIJI 2.0. Slides of extant tardigrades mounted on Hoyer’s medium were imaged using an Axioscope 5 compound microscope (Zeiss) with Axiocam208 color camera (Zeiss). Figures were assembled using Adobe Illustrator 26.5. Phylogenetic analysis We tested the phylogenetic position of Aysheaia pedunculata in the broader context of lobopodian and panarthropod relationships using an updated developmentally informed character matrix. We produced three versions of the character matrix from Yang et al (2016) 45 to account for different hypotheses regarding the serial homology between the legs of tardigrades relative to the cephalic appendages of Cambrian lobopodians (see File S1 for details). The “stylet hypothesis” (File S2) posits that the first (protocerebral) appendage pair of lobopodians became internalized into the tardigrade stylet apparatus, based on the leg claws and stylet being produced by glands 21 and similarities in the muscle attachment sites between the styles and legs 46 . The “sensorial hypothesis” (File S3) posits that the first (protocerebral) appendage pair of lobopodians corresponds to the lateral cephalic cuticular extensions (primary clava and cirri A in heterotardigrades and sensory fields in eutardigrades) based on morphological and topological similarities 21 and developmental genetics studies 10 . The “secondary loss hypothesis” (File S4) posits that the first (protocerebral) appendage pair of lobopodians is completely lost and has not direct homologue in extant tardigrades. The dataset includes 71 taxa and 93 discrete characters for the sensorial and secondary loss hypotheses matrices, whereas the stylet hypothesis matrix has 94 discrete characters (See File S1 for the list of characters). In addition to Cambrian lobopodians, the taxonomic sampling covers tardigrade species representing all heterotardigrade families and eutardigrade superfamilies. Two extant (priapulids Tubiluchus troglodytes and Priapulus caudatus ),and two fossil scalidophorans (archaeopriapulid Ottoia prolifica and palaeoscolecid Cricocosmia jinningensis ) species were used as outgroups. The character matrices were then subjected to parsimony analysis and Bayesian inference (BI). Parsimony searches were run in TNT 1.5 47 under New Technology Search, using Driven Search with Sectorial Search, Ratchet, Drift and Tree fusing options activated with standard settings 48,49 . The analysis was set to find the minimum tree length 100 times and to collapse trees after each search. All characters were treated as unordered. For comparative purposes, analyses were performed under equal and different implied weights (k = 5, 10, 15, 20). The Bayesian analysis was done in MRBAYES 3.2 50 using the Mk model 51 + Gamma with the coding set to ‘variable’, which excluded two invariant characters. The analysis was run for at least 3 000 000 generations sampling every 500 generations and with 25% relative initial burn-in. Two runs were simultaneously done with each having one cold and three heated chains. Convergence was assessed by checking that the average deviation of split frequencies of the two runs was less than 0.01, effective sample size values were greater than 200 and the potential scale reduction factor was approximately = 1. A consensus tree was then obtained to summarize the resulting analysis. Declarations Data Availability All data used and generated are provided in either the main text or in the electronic supplementary material. Competing Interest We declare we have no competing interests. Funding JOH was funded by the Systematics Association and MCZ Ernst Mayr Travel Grant. References Møbjerg, N. et al. Survival in extreme environments - on the current knowledge of adaptations in tardigrades. Acta Physiol. (Oxf). 202 , 409–420 (2011). Smith, F. W. et al. 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Homology of the head sensory structures between Heterotardigrada and Eutardigrada supported in a new species of water bear (Ramazzottiidae: Ramazzottius ). Zoological Lett. 9 , 1–17 (2023). Fujimoto, S., Jørgensen, A. & Hansen, J. G. A molecular approach to arthrotardigrade phylogeny (Heterotardigrada, Tardigrada). Zool. Scr. 46 , 496–505 (2017). Grollmann, M. M., Jørgensen, A. & Møbjerg, N. Actinarctus doryphorus (Tanarctidae) DNA barcodes and phylogenetic reinvestigation of Arthrotardigrada with new A. doryphorus and Echiniscoididae sequences. Zootaxa 5284 , 351–363 (2023). Yang, J. et al. Fuxianhuiid ventral nerve cord and early nervous system evolution in Panarthropoda. Proc. Natl. Acad. Sci. U. S. A. 113 , 2988–2993 (2016). Halberg, K. A., Persson, D., Møbjerg, N., Wanninger, A. & Kristensen, R. M. Myoanatomy of the marine tardigrade Halobiotus crispae (Eutardigrada: Hypsibiidae). J. Morphol. 270 , 996–1013 (2009). Goloboff, P. A., Farris, J. S. & Nixon, K. C. TNT, a free program for phylogenetic analysis. Cladistics 24 , 774–786 (2008). Goloboff, P. A. Analyzing large data sets in reasonable times: Solutions for composite optima. Cladistics 15 , 415–428 (1999). Nixon, K. C. The parsimony ratchet, a new method for rapid parsimony analysis. Cladistics 15 , 407–414 (1999). Ronquist, F. et al. Mrbayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Syst. Biol. 61 , 539–542 (2012). Lewis, P. O. A likelihood approach to estimating phylogeny from discrete morphological character data. Syst. Biol. 50 , 913–925 (2001). Additional Declarations There is NO Competing Interest. Supplementary Files FileS1CharacterCoding.docx File S1 FileS2stylethypothesischaractermatrix.txt File S2 FileS3sensorialhypothesischaractermatrix.txt File S3 FileS4losthypothesischaractermatrix.txt File S4 SupplementaryTextFiguresTables.pdf Supplementary Text, Figures, and Tables Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8911114","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Biological Sciences - Article","associatedPublications":[],"authors":[{"id":593482365,"identity":"1328d4c9-0311-4344-b55c-d286b45a7589","order_by":0,"name":"Marc Mapalo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAUlEQVRIiWNgGAWjYDADPgbmAwyMDRCOBAMDM2EtbAxsCSRr4TEgTotu+9mHn27U3JFnY+/5/IJxh12+wfGzD28wVFgnNuDQYnYm3Vg659gzwzaes9ssGM8kW24AilgwnEnHreVAGhtzDtthxjaJ3G0GjG3MBpINaWwSjG2HcWs5/wyo5d9h+zaJnGdALfUGkv3PgFr+4dFyA2hLLtBMoBbmB0DDDfglQLY04NPyjFk6t+9ZchvPMTOGxLbjQC3PmC0SjqUb43ZYGuPnnG93bPvZmx9/+NhWbcDGn8Z440ONtSwuLVBwAESwSSTA+Ak41KFrYf5AWOEoGAWjYBSMRAAAUxFYc6NuFMkAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-7653-0508","institution":"Smithsonian National Museum of Natural History","correspondingAuthor":true,"prefix":"","firstName":"Marc","middleName":"","lastName":"Mapalo","suffix":""},{"id":593482366,"identity":"49f80636-9e30-4460-900b-9cfb19453ea8","order_by":1,"name":"Javier Ortega-Hernández","email":"","orcid":"https://orcid.org/0000-0002-6801-7373","institution":"Harvard University","correspondingAuthor":false,"prefix":"","firstName":"Javier","middleName":"","lastName":"Ortega-Hernández","suffix":""}],"badges":[],"createdAt":"2026-02-18 16:58:18","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-8911114/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8911114/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103193093,"identity":"c6503258-e146-4de7-b799-b1ad177e9b78","added_by":"auto","created_at":"2026-02-23 02:52:10","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1112094,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNew specimens of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAysheaia pedunculata \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eWalcott, 1911.\u003c/strong\u003e (A) ROMIP 61108, ventral view showing all 10 pairs of legs, anterior appendages, and digestive tract running along the body seen as a dark film. (B) ROMIP 68043, ventro-lateral view with evident conical structures on the dorsal cuticle. (C) ROMIP 68040, dorsal view and evident claws on the legs. (D) ROMIP 68035, ventral view, retracted mouth surrounded by six papillate structures. (E) ROMIP 68037, part, lateral view with the bulbous pharynx at the anterior end. Specimens photographed using cross-polarized light dry (A,B,D) or under water (C,E). Yellow arrowheads – leg spines. Scale bars: 1 mm.\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8911114/v1/9a03c1b71179da4e022cc1e3.jpeg"},{"id":103193101,"identity":"a3e18ca8-f027-4d05-ac06-e2905d04bcab","added_by":"auto","created_at":"2026-02-23 02:52:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1355895,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphological similarities between \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAysheaia pedunculata \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eWalcott, 1911 and tardigrades.\u003c/strong\u003e (A) Phylogenetic relationships of major tardigrade orders; “Arthrotardigrada” is paraphyletic and is not considered a valid taxonomic group\u003csup\u003e44\u003c/sup\u003e. (B,C) Claws: (B) USNM 235880, (C) \u003cem\u003eEchiniscoides\u003c/em\u003e. (D,E) Leg spines/sensory organs (yellow arrowheads): (D) ROMIP 68039, (E) \u003cem\u003eStygarctus\u003c/em\u003e. (F,G) Circumoral papillate structures: (F) ROMIP 63052, (G) \u003cem\u003eMilnesium\u003c/em\u003e. (H,I) Dorsal cuticular ornamentation: (H) ROMIP 68043, (I) \u003cem\u003eFractonotus\u003c/em\u003e. (J-L) Bulbuous pharynx: (J) ROMIP 68037 part, (K) \u003cem\u003eMilnesium\u003c/em\u003e, (L) \u003cem\u003eClaxtonia\u003c/em\u003e. (M-O) Hind leg orientation: (M) USNM 83942a, (N) \u003cem\u003eStygarctus\u003c/em\u003e, (O) \u003cem\u003eMinibiotus\u003c/em\u003e; white arrow – possible mouth cone\u003cem\u003e. \u003c/em\u003eSpecimens photographed using cross-polarized light dry (B,D,F,H,M) or under water (J). Scale bars: \u003cem\u003eA. pedunculata\u003c/em\u003e – 1 mm; tardigrades – 10 μm.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8911114/v1/adc2581804011c2e9d23b7ad.png"},{"id":103505787,"identity":"114837b6-b86d-4d3e-a053-51fa325ff2c5","added_by":"auto","created_at":"2026-02-26 13:33:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":304776,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenetic relationship of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAysheaia pedunculata \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eWalcott 1911. \u003c/strong\u003e(A) Three different hypotheses for the first pair of tardigrade limbs. (B) Tree reconstruction using Bayesian inference in MrBayes; values above the node represent the posterior probability values for the tree obtained under the (1) stylet hypothesis, (2) sensorial hypothesis, and (3) lost hypothesis, respectively; only values that are above 0.4 in all scenarios are shown at each node.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8911114/v1/46531f400b3f64adb6027294.png"},{"id":103193096,"identity":"c6ea91bf-4747-4eaa-81f1-322a7423478f","added_by":"auto","created_at":"2026-02-23 02:52:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":260538,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRepresentation of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAysheaia pedunculata \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eWalcott, 1911. \u003c/strong\u003e(A) Schematic diagram at lateral view. (B) Possible scenario of how the papillate structures are displaced laterally if a mouth cone is present and exposed. (C) Scythe-like morphology of the \u003cem\u003eA. pedunculata \u003c/em\u003eclaws. (D). Artistic reconstruction\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8911114/v1/4faf4f8a68a49c48def0a429.png"},{"id":106961252,"identity":"31e0f9bf-05c4-44f2-91da-2044a0719cfe","added_by":"auto","created_at":"2026-04-15 09:24:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3550638,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8911114/v1/4f46c829-0979-48c8-9a1a-7f03538377dc.pdf"},{"id":103193095,"identity":"634b0f94-d7f5-450c-8fc7-832c43d62d33","added_by":"auto","created_at":"2026-02-23 02:52:10","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1236922,"visible":true,"origin":"","legend":"File S1","description":"","filename":"FileS1CharacterCoding.docx","url":"https://assets-eu.researchsquare.com/files/rs-8911114/v1/a41ac543858b0a4fb69f5246.docx"},{"id":103193094,"identity":"d4a00d1a-f79b-433f-9d5a-8aa90cba7964","added_by":"auto","created_at":"2026-02-23 02:52:10","extension":"txt","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":21736,"visible":true,"origin":"","legend":"File S2","description":"","filename":"FileS2stylethypothesischaractermatrix.txt","url":"https://assets-eu.researchsquare.com/files/rs-8911114/v1/692a7ac3bc1981e118a1f3fe.txt"},{"id":103504994,"identity":"b2dcd471-a83f-47ea-9ef9-3788fc24c647","added_by":"auto","created_at":"2026-02-26 13:22:22","extension":"txt","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":21627,"visible":true,"origin":"","legend":"File S3","description":"","filename":"FileS3sensorialhypothesischaractermatrix.txt","url":"https://assets-eu.researchsquare.com/files/rs-8911114/v1/35cf22cfbdb1548fbcf7aace.txt"},{"id":103193099,"identity":"4c49afa5-7a2c-4666-be2e-b3d5b2bac726","added_by":"auto","created_at":"2026-02-23 02:52:10","extension":"txt","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":21628,"visible":true,"origin":"","legend":"File S4","description":"","filename":"FileS4losthypothesischaractermatrix.txt","url":"https://assets-eu.researchsquare.com/files/rs-8911114/v1/41746b5f6e16b341e474f27a.txt"},{"id":103193100,"identity":"77488354-fa40-4484-9e39-5c191f196099","added_by":"auto","created_at":"2026-02-23 02:52:10","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":4650714,"visible":true,"origin":"","legend":"Supplementary Text, Figures, and Tables","description":"","filename":"SupplementaryTextFiguresTables.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8911114/v1/abeba74e589722963ca0e910.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"A middle Cambrian macroscopic tardigrade ancestor","fulltext":[{"header":"Main","content":"\u003cp\u003eThe leg-bearing panarthropods are the most diverse group of molting animals and include the extant phyla Euarthropoda (e.g. arachnids, myriapods, pancrustaceans, insects), Onychophora (velvet worms), and Tardigrada (water bears)\u0026nbsp;\u003csup\u003e11\u003c/sup\u003e, which are estimated to have diverged from each other around 600 Mya\u0026nbsp;\u003csup\u003e12\u003c/sup\u003e. Although panarthropods have a lengthy evolutionary history extending to the base of the Cambrian\u0026nbsp;\u003csup\u003e13\u003c/sup\u003e, most of the available information on their macroevolution comes from early and mid-Cambrian sites with exceptional soft-tissue preservation thanks to the high fossilization potential of their chitinous cuticular exoskeleton. Lobopodians are the earliest branching panarthropods, diverse ecdysozoans resembling worms with legs that include stem lineage members of all extant panarthropod phyla\u0026nbsp;\u003csup\u003e14\u003c/sup\u003e. Lobopodians inform critical early steps in the evolution of euarthropods, such as the origin of arthropodized legs, biramous appendages and a multisegmented head (e.g.\u0026nbsp;\u003csup\u003e15\u0026ndash;17\u003c/sup\u003e), and onychophorans, whose marine Cambrian relatives exhibited substantial morphological and ecological disparity relative to modern terrestrial forms (e.g.\u0026nbsp;\u003csup\u003e18\u0026ndash;20\u003c/sup\u003e). However, the Cambrian fossil record is comparatively silent with regard to the early history of tardigrades, which ultimately results in an incomplete view of panarthropod origins and macroevolution. \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTardigrades are microinvertebrates (~50 um \u0026ndash; 1.2mm) with a segmented body plan and four pairs of lobopodous legs that typically terminate in claws\u0026nbsp;\u003csup\u003e21\u003c/sup\u003e, and which have the notable ability to survive extreme conditions, such as space vacuum, ionizing radiation, and extremely low temperatures\u0026nbsp;\u003csup\u003e1\u003c/sup\u003e. The phylum is subdivided into the heterotardigrades, which, predominantly the marine ones, have high morphological disparity, including various external cuticular structures (e.g., cirri, clavae, dorsal plates), and the eutardigrades, which are predominantly limnoterrestrial and have a simpler plump-like appearance without external cuticular specializations\u0026nbsp;\u003csup\u003e21\u003c/sup\u003e. Extant tardigrade diversity comprises over 1500 described species\u0026nbsp;\u003csup\u003e22\u0026ndash;24\u003c/sup\u003e, but extinct taxa are only known from four known crown-group representatives ranging from the early Cretaceous to the Miocene\u0026nbsp;\u003csup\u003e5\u0026ndash;8\u003c/sup\u003e. Fossil calibrated molecular clock estimates suggest that crown-group tardigrades diverged around the mid to late Cambrian\u0026nbsp;\u003csup\u003e7\u003c/sup\u003e, implying the existence of Cambrian stem-group tardigrades. Although some Cambrian fossils have been regarded as potential members of total-group Tardigrada, there are issues with these interpretations that restrict their ability to reconstruct the early evolution of this phylum.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMiddle Cambrian Orsten-type Siberian microfossils have been interpreted as juvenile tardigrades due to their size and overall morphology\u0026nbsp;\u003csup\u003e25,26\u003c/sup\u003e. These fossils differ from extant tardigrades in having only three pairs of claw-bearing legs, which led to the interpretation that the Cambrian forms sequentially added body segments during early ontogeny. However, embryological studies demonstrate that tardigrade legs develop simultaneously\u0026nbsp;\u003csup\u003e27,28\u003c/sup\u003e, and Hox gene expression patterns indicate that four leg-bearing segments would still be expected even if terminal addition had been lost\u0026nbsp;\u003csup\u003e3\u003c/sup\u003e. Thus, the tardigrade affinity of these Siberian fossils remains uncertain. A recent reappraisal of Cambrian lobopodians suggested that luolishaniids, a derived group typified by the presence of setulose anterior limbs used for suspension feeding, might be part of the tardigrade stem lineage based on the anteroposterior differentiation of the legs and presence of paired sensory structures on the head\u0026nbsp;\u003csup\u003e29\u003c/sup\u003e. However, these shared characters appear superficial since the structures themselves are not morphological similar to tardigrades. Lastly, another Cambrian fossil that was proposed to have an affinity to tardigrades is \u003cem\u003eAysheaia pedunculata.\u0026nbsp;\u003c/em\u003eInitially misidentified as a polychaete\u0026nbsp;\u003csup\u003e30\u003c/sup\u003e, \u003cem\u003eAysheaia\u0026nbsp;\u003c/em\u003ehas the distinction of being the first described lobopodian chronologically. Although subsequent systematic work suggested possible affinities with tardigrades based on the reverse orientation of the hind legs and morphology of the terminal claws, the precise position of \u003cem\u003eAysheaia\u0026nbsp;\u003c/em\u003eremains uncertain due to similarities with onychophorans including the overall morphology and macroscopic body size\u0026nbsp;\u003csup\u003e31\u003c/sup\u003e. The most recent extensive redescription of this fossil also acknowledged its possible relationship to tardigrades but decided not to relegate it to any specific extant higher taxon\u0026nbsp;\u003csup\u003e32\u003c/sup\u003e. Furthermore, although various studies have incorporated \u003cem\u003eAysheaia\u0026nbsp;\u003c/em\u003ein phylogenetic analyses of lobopodians (e.g.\u0026nbsp;\u003csup\u003e19,20,33,34\u003c/sup\u003e ) this taxon has never been the main focus and its position has proven unstable among the stem lineages of panarthropods, euarthropods and tardigrades.\u003c/p\u003e\n\u003cp\u003eHere, we redescribe the morphology of the mid-Cambrian lobopodian \u003cem\u003eAysheaia pedunculata\u0026nbsp;\u003c/em\u003efrom the Burgess Shale in British Columbia, report new characters based on the type material housed at the Smithsonian Institution (USNM) and undescribed material housed at the Royal Ontario Museum (ROMIP), and reassess its phylogenetic affinities in the context of panarthropod evolution.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMorphological redescription of\u003cem\u003e\u0026nbsp;Aysheaia pedunculata\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStudied material includes 31 specimens including new collections from the ROMIP and the type material\u0026nbsp;\u003csup\u003e32\u003c/sup\u003e (Table S1,S2) (see Supplementary Text for remarks and discussion). Specimens consist of highly compacted macrofossils composed of organic carbon films typical of Burgess Shale-type preservation\u003csup\u003e35,36\u003c/sup\u003e; \u0026nbsp;with 45% of specimens preserved in lateral or oblique view, and 55% preserved in dorsal or ventral orientations (Table S1,S2). The body is elongate, with the length of complete specimens ranging from 1.06 to 5.4 cm (Table S3) and can be divided into the head region with the mouth part and a pair of spine-bearing frontal-most differentiated appendages, and the trunk region consisting of 10 pairs of walking legs (Figs 1A, S1A-D). Except for the mouth region, the entire body is annulated and features conical structures found at the dorsal side on top of the ridges separated by the annulations (Figs 1B,C,S2). This dorsal ornamentation is only observed in larger specimens (longer than ~2 cm) (Table S3). The anterior-facing mouth has a terminal position, and the opening is surrounded by six elongated papillae (Figs 1D, S1E-F). The circumoral papillae are semi-flexible (rather than rigid) and their position at the lateral sides (e.g., Fig S2) suggest that a portion of the mouth is at least partially retractable (Fig 1D). The frontal appendages are annulated (Fig 1D) and attached laterally at either side of the head region (Figs 1A, S1A-D). There are three to five spines running along the anterior side of the frontal appendages and three distal spines at their tips (Figs 1A,D, S1F, S3A,B). The spines have a rigid appearance, particularly when compared to the more malleable mouth papillae. The walking legs are also annulated, attached ventro-laterally to the trunk, and terminated with claws (Figs 1C, S2). The first nine pairs of legs face posteriorly, while the posteriormost 10\u003csup\u003eth\u003c/sup\u003e leg pair faces anteriorly based on the orientation of the claws (see Figs 1C, S1A,C). The bases of the 10\u003csup\u003eth\u003c/sup\u003e leg pair define the end of the trunk region with no recognizable posterior extension beyond this pair (Figs S4A,C). All the walking legs feature two spines \u0026ndash; one near the tip of the leg and another more proximally situated (Figs 1C-E, S4B-D). These leg spines have similar morphology to the spines on the frontal appendages. The bases of these spines are found at the outer side of the legs and lateral with respect to the claws (Fig S4B). Five to eight claws with evident straight, long stalks are found in each leg and have a scythe-like morphology (Figs 1E, S4E-G). The alimentary canal is preserved as a dark film running along the body (Fig 1A,E). New material from ROMIP demonstrates that the alimentary canal has a distinctly widened anterior portion connected to the mouth opening, which we interpret as a bulbous pharynx (Fig 1E).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComparison of \u003cem\u003eAysheaia pedunculata\u003c/em\u003e to tardigrades\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on our redescription of \u003cem\u003eA. pedunculata\u003c/em\u003e we identify several shared features with the major groups of extant tardigrades (Fig 2A; see\u0026nbsp;\u003csup\u003e21\u003c/sup\u003e for review of tardigrade morphology). \u003cem\u003eA. pedunculata\u0026nbsp;\u003c/em\u003eresembles heterotardigrades in the claw and walking leg morphology (Figs 2A-E). The distinctive scythe-like claws of \u003cem\u003eA. pedunculata\u0026nbsp;\u003c/em\u003e(Fig 2B) are also found in heterotardigrades and the primary branch of apochelan claws. The presence of supernumerary claws, defined as four or more per leg, with this particular claw shape are characteristics that are only found in marine echiniscoidids, such as \u003cem\u003eEchiniscoides\u003c/em\u003e (Fig 2C). The walking leg spines of \u003cem\u003eA. pedunculata\u0026nbsp;\u003c/em\u003e(Fig 2D) resemble the heterotardigrade leg sensory organs, such as in arthrotardigrades (e.g., \u003cem\u003eStyraconyx\u003c/em\u003e) (Fig 2E) and echiniscioideans (e.g., \u003cem\u003eEchiniscus\u003c/em\u003e), in terms of their rigid appearance and similar position on the legs. Features shared between \u003cem\u003eA. pedunculata\u0026nbsp;\u003c/em\u003eand eutardigrades include the circumoral papillae and dorsal sculpturing (Figs 2F-I). The mouth papillae of \u003cem\u003eA. pedunculata\u0026nbsp;\u003c/em\u003e(Fig 2F) resemble the six papillae surrounding the buccal opening of apochelans (e.g., \u003cem\u003eMilnesium\u003c/em\u003e) (Fig 2G), and the dorsal conical structures (Fig 2H) are reminiscent of the tubercles found in some parachelans with sculptured dorsal cuticles (i.e., \u003cem\u003eFractonotus\u003c/em\u003e) (Fig 2I). \u003cem\u003eA. pedunculata\u0026nbsp;\u003c/em\u003ealsohas characteristics that are present across all tardigrades. The bulbous pharynx (Fig 2J) is a fundamental feature observed in both eutardigrades (Fig 2K) and heterotardigrades (Fig 2L). The orientation of the last pair of legs facing anteriorly and having a rotated orientation compared to the rest of the anterior pairs of legs is also shared between \u003cem\u003eA. pedunculata\u0026nbsp;\u003c/em\u003e(Fig 2M)and most tardigrades (Figs 2N,O). Lastly, the trunk of \u003cem\u003eA. pedunculata\u0026nbsp;\u003c/em\u003elacks a posterior extension beyond the last pair of legs (Fig 2M), as in the majority of tardigrades (Figs 2N,O). In conclusion, the revised morphology of \u003cem\u003eA. pedunculata\u003c/em\u003e reveals a mixture of ancestral and derived characters found across major tardigrade groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhylogenetic affinities of \u003cem\u003eAysheaia pedunculata\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven the uncertainty regarding the precise homology of the protocerebral appendage pair of extant tardigrades with those of other panarthropods (see File S1 for details), we tested three different hypotheses and explore their phylogenetic implications in the context of the new data on \u003cem\u003eA. pedunculata\u003c/em\u003e. The first (stylet) and second (sensorial) hypotheses regard that the protocerebral appendage pair is expressed as either the stylet apparatus or the lateral cephalic sensorial appendages, respectively. The third hypothesis reflects the complete loss of the protocerebral appendages in extant tardigrades (see Material and Methods). Our results based on Bayesian inference (BI) recovered \u003cem\u003eA. pedunculata\u0026nbsp;\u003c/em\u003ein a sister-group relationship relative to crown-group tardigrades (Figs 3B, S11-S13) under all three hypotheses (Fig 3A); the \u0026ldquo;sensorial hypothesis\u0026rdquo; produced\u0026nbsp;the highest support values, whereas the \u0026ldquo;stylet hypothesis\u0026rdquo; recovered the lowest. Trees recovered using the maximum parsimony (MP) criterion had different topology depending on the character weighting strategy. Under equal weights and all hypotheses, \u003cem\u003eA. pedunculata\u0026nbsp;\u003c/em\u003eis recovered in a polytomy together with other lobopodians based on the strict consensus of the most parsimonious trees (MPTs) (Fig S14). However, \u003cem\u003eA. pedunculata\u0026nbsp;\u003c/em\u003eis recovered in a sister-group relationship with crown-group tardigrades under implied weights, congruent with the BI results, based on the strict consensus trees of the MPTs (Fig S15).\u003c/p\u003e\n\u003cp\u003eThe phylogenetic relationships among crown-group tardigrades under BI differ slightly depending on the tested hypothesis (Figs S11-S13). Under the \u0026ldquo;stylet hypothesis\u0026rdquo;, eutardigrades were consistently recovered as a strongly supported clade, but monophyletic heterotardigrades show weak support (Fig S11). In the other two hypotheses (Figs S12,S13), the echiniscoidids (\u003cem\u003eEchiniscoides\u0026nbsp;\u003c/em\u003eand \u003cem\u003eIsoechiniscoides\u003c/em\u003e) were recovered separately from the other tardigrades, but this non-echiniscoidid clade (i.e., eutardigrades + other heterotardigrades) is weakly supported. The same relationship of the echiniscoidids and the rest of the tardigrades were observed in the MP trees using implied weights in all the hypotheses tested (Fig S15). Ultimately, our phylogenetic results indicate that \u003cem\u003eA. pedunculata\u0026nbsp;\u003c/em\u003ebelongs to the tardigrade stem lineage, regardless of the precise hypothesis of homology of between the protocerebral appendages of extant tardigrades and other panarthropods.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe morphological reappraisal of \u003cem\u003eAysheaia pedunculata\u0026nbsp;\u003c/em\u003e(Fig 4)reveals informative derived characters exclusively shared with heterotardigrades and eutardigrades, including the scythe-like supernumerary claws, circumoral papillae, bulbous pharynx, anterior-facing hind legs, and the lack of posterior extensions (Fig 2). These features collectively provide evidence for the phylogenetic position of \u003cem\u003eA. pedunculata\u0026nbsp;\u003c/em\u003eas a stem-group tardigrade, which is further supported by our phylogenetic analyses. Critically, our results do not find support for the recently proposed phylogenetic position of Cambrian luolishaniid lobopodians (\u003cem\u003econtra\u0026nbsp;\u003c/em\u003e\u003csup\u003e29\u003c/sup\u003e), a highly derived group of armored lobopodians with setulose anterior appendages (e.g., \u003csup\u003e20,37,38\u003c/sup\u003e), as early tardigrades.\u0026nbsp; We find that the proposed shared characters between luolishaniids and tardigrades, namely the differentiation of anterior and posterior lobopodous limbs batches and the presence of paired cephalic sensory organs, do not represent conclusive synapomorphies. The morphology of all four pairs of the tardigrade legs essentially shares the same external morphology (see illustrated legs in (Fontoura et al. 2017a)\u0026nbsp; and only differ in the reverse orientation of the fourth pair of legs. This contrasts with luolishaniids appendage batches, which are extremely different, consisting of elongate anterior limbs with series of setae and stout and spine-less claw-bearing posterior limbs [e.g.\u0026nbsp;\u003csup\u003e33,38\u003c/sup\u003e]. The fact that our phylogenetic analyses do not find support for a close relationship between luolishaniids and tardigrades, despite incorporating these proposed characters, indicates that \u003cem\u003eA\u003c/em\u003e\u003cem\u003e. pedunculata\u003c/em\u003e is the only Cambrian lobopodian that conclusively features numerous complex synapomorphies shared with modern tardigrades.\u003c/p\u003e\n\u003cp\u003eThe phylogenetic position of \u003cem\u003eAysheaia\u0026nbsp;\u003c/em\u003eas a stem-group tardigrade provides direct paleontological support for the miniaturization of as suggested by developmental, genomic and morphological evidence\u0026nbsp;\u003csup\u003e3,9,40\u003c/sup\u003e. According to this hypothesis, tardigrades evolved from a macroscopic ancestor that secondarily lost multiple leg-bearing segments from the body the mid-section, which is reflected in the corresponding loss of trunk Hox genes such as \u003cem\u003eAntennapedia\u003c/em\u003e, \u003cem\u003eUltrabithorax\u003c/em\u003e, \u003cem\u003eAbdominal-A\u003c/em\u003e \u003csup\u003e3\u003c/sup\u003e. Recent developmental work suggests that the walking legs of tardigrades also experienced shortening of their mid-section based on gap gene expression patterns \u0026nbsp;\u003csup\u003e40\u003c/sup\u003e, and that tardigrade legs have a distal identity compared to other panarthropod legs\u0026nbsp;\u003csup\u003e10\u003c/sup\u003e. This evolutionary shortening of the walking legs could explain the presence of two spines on the legs of \u003cem\u003eA. pedunculata\u0026nbsp;\u003c/em\u003ewhereas heterotardigrades only have one\u0026nbsp;\u003csup\u003e21\u003c/sup\u003e .\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe resolved affinity of \u003cem\u003eA. pedunculata\u0026nbsp;\u003c/em\u003eprovides insights into the likely ancestral morphology of crown-group tardigrades prior to their major diversification. First, the body organization would have featured the same number of body segments as modern tardigrades, (i.e. head and four leg-bearing trunk segments), and homonomous walking legs with claws. The overall size would likely be at least as large than the biggest tardigrades alive today (~1.2 mm\u0026nbsp;\u003csup\u003e21\u003c/sup\u003e, and miniaturization among the different groups of tardigrades would have been largely lineage dependent. Second, the last common ancestor most likely had cephalic sensorial appendages, such as cirri or clavae, that also underwent different degrees of reduction in different tardigrade lineages\u0026nbsp;\u003csup\u003e41,42\u003c/sup\u003e, producing the disparity observed between the anterior morphology of heterotardigrades and eutardigrades. \u0026nbsp;Lastly, the ancestral state for the tardigrade claw was likely scythe-shaped as observed in echiniscoids, some arthrotardigrades (e.g., coronarctids, stygarctids) and apochelans (i.e., primary branch of the claw). However, it is uncertain whether the supernumerary phenotype (i.e., more than four claws) is an ancestral state, as suggested by \u003cem\u003eA. pedunculata\u003c/em\u003e, or independently derived in different heterotardigrade lineages (e.g., \u003cem\u003eEchiniscoides\u003c/em\u003e, \u003cem\u003eBatillipes\u003c/em\u003e). These observations also suggest that the fusion of claws is derived and evolved within eutardigrades, specifically in parachelans, potentially to be able to cling to sediments more efficiently. Current efforts to reconstruct the ancestral states of tardigrades using extant species is inhibited by the unresolved phylogeny of the heterotardigrades\u0026nbsp;\u003csup\u003e43,44\u003c/sup\u003e, which highlights the significance of \u003cem\u003eA. pedunculata\u0026nbsp;\u003c/em\u003efor polarizing major morphological characters that define this phylum.\u0026nbsp;\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cem\u003eMaterial provenance and digital imaging\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll studied specimens of \u003cem\u003eAysheaia pedunculata\u003c/em\u003e are housed at the collections of the Smithsonian National Museum of Natural History (NMNH) and the Royal Ontario Museum (ROMIP) (Table S1,S2). All specimens were collected from the Stephen Formation in British Colombia, Canada. Fossil specimens were photographed under cross polarized light, either dry or wet, with an Olympus DSX100 digital microscope, or a Nikon D80 fitted with a Nikkor 50mm macro lens. Digital photographs were adjusted using Adobe Lightroom 11.5. Body lengths were measured using FIJI 2.0. Slides of extant tardigrades mounted on Hoyer\u0026rsquo;s medium were imaged using an Axioscope 5 compound microscope (Zeiss) with Axiocam208 color camera (Zeiss). Figures were assembled using Adobe Illustrator 26.5.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePhylogenetic analysis\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eWe tested the phylogenetic position of \u003cem\u003eAysheaia pedunculata\u0026nbsp;\u003c/em\u003ein the broader context of lobopodian and panarthropod relationships using an updated developmentally informed character matrix. We produced three versions of the character matrix from Yang et al (2016)\u003csup\u003e45\u003c/sup\u003e to account for different hypotheses regarding the serial homology between the legs of tardigrades relative to the cephalic appendages of Cambrian lobopodians (see File S1 for details). The \u0026ldquo;stylet hypothesis\u0026rdquo; (File S2) posits that the first (protocerebral) appendage pair of lobopodians became internalized into the tardigrade stylet apparatus, based on the leg claws and stylet being produced by glands\u003csup\u003e21\u003c/sup\u003e\u0026nbsp; and similarities in the muscle attachment sites between the styles and legs \u003csup\u003e46\u003c/sup\u003e. \u0026nbsp;The \u0026ldquo;sensorial hypothesis\u0026rdquo; (File S3) posits that the first (protocerebral) appendage pair of lobopodians corresponds to the lateral cephalic cuticular extensions (primary clava and cirri A in heterotardigrades and sensory fields in eutardigrades) based on morphological and topological similarities \u003csup\u003e21\u003c/sup\u003e and developmental genetics studies \u003csup\u003e10\u003c/sup\u003e. The \u0026ldquo;secondary loss hypothesis\u0026rdquo; (File S4) posits that the first (protocerebral) appendage pair of lobopodians is completely lost and has not direct homologue in extant tardigrades. \u0026nbsp;The dataset includes 71 taxa and 93 discrete characters for the sensorial and secondary loss hypotheses matrices, whereas the stylet hypothesis matrix has 94 discrete characters (See File S1 for the list of characters). In addition to Cambrian lobopodians, the taxonomic sampling covers tardigrade species representing all heterotardigrade families and eutardigrade superfamilies. Two extant (priapulids \u003cem\u003eTubiluchus troglodytes\u0026nbsp;\u003c/em\u003eand \u003cem\u003ePriapulus caudatus\u003c/em\u003e),and two fossil scalidophorans (archaeopriapulid \u003cem\u003eOttoia prolifica\u003c/em\u003e and palaeoscolecid \u003cem\u003eCricocosmia jinningensis\u003c/em\u003e) species were used as outgroups.\u003c/p\u003e\n\u003cp\u003eThe character matrices were then subjected to parsimony analysis and Bayesian inference (BI). Parsimony searches were run in TNT 1.5 \u003csup\u003e47\u003c/sup\u003e under New Technology Search, using Driven Search with Sectorial Search, Ratchet, Drift and Tree fusing options activated with standard settings \u003csup\u003e48,49\u003c/sup\u003e. The analysis was set to find the minimum tree length 100 times and to collapse trees after each search. All characters were treated as unordered. For comparative purposes, analyses were performed under equal and different implied weights (k = 5, 10, 15, 20). The Bayesian analysis was done in MRBAYES 3.2 \u003csup\u003e50\u003c/sup\u003e using the Mk model \u003csup\u003e51\u003c/sup\u003e\u0026nbsp; \u0026nbsp;+ Gamma with the coding set to \u0026lsquo;variable\u0026rsquo;, which excluded two invariant characters. The analysis was run for at least 3 000 000 generations sampling every 500 generations and with 25% relative initial burn-in. Two runs were simultaneously done with each having one cold and three heated chains. Convergence was assessed by checking that the average deviation of split frequencies of the two runs was less than 0.01, effective sample size values were greater than 200 and the potential scale reduction factor was approximately = 1. A consensus tree was then obtained to summarize the resulting analysis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data used and generated are provided in either the main text or in the electronic supplementary material.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe declare we have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJOH was funded by the Systematics Association and MCZ Ernst Mayr Travel Grant.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eM\u0026oslash;bjerg, N. \u003cem\u003eet al.\u003c/em\u003e Survival in extreme environments - on the current knowledge of adaptations in tardigrades. \u003cem\u003eActa Physiol. 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Biol.\u003c/em\u003e \u003cstrong\u003e50\u003c/strong\u003e, 913\u0026ndash;925 (2001).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8911114/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8911114/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTardigrades, commonly known as water bears, are a major phylum of microinvertebrates best known for their ability to withstand extreme environmental conditions \u003csup\u003e1\u003c/sup\u003e. As one of the three groups that comprise the megadiverse Panarthropoda, alongside onychophorans and euarthropods, tardigrades represent an emerging model system for understanding the origin of metazoan body plans \u003csup\u003e2\u003c/sup\u003e, the evolution of segmentation \u003csup\u003e3\u003c/sup\u003e, and the genetic machinery responsible for their formidable cryptobiotic capabilities \u003csup\u003e4\u003c/sup\u003e. However, the early history of the group remains elusive because fossil record is heavily biased against small-bodied aquatic invertebrates, with the only unequivocal tardigrade fossils known from Cretaceous-aged or younger amber deposits \u003csup\u003e5–8\u003c/sup\u003e. Here, we comprehensively revise the morphology and affinities of the claw-bearing lobopodian \u003cem\u003eAysheaia pedunculata\u003c/em\u003e from the mid-Cambrian (Wuliuan) Burgess Shale. Previously regarded as a possible relative of onychophorans based on their similar body size and general organization, we demonstrate several derived morphological characters shared between \u003cem\u003eAysheaia \u003c/em\u003eand extant tardigrades including supernumerary scythe-like claws, a bulbous pharynx, and reversed hind leg orientation. We employ a developmentally informed dataset to test between three different competing scenarios on the homology of anterior appendages found in the head of \u003cem\u003eAysheaia \u003c/em\u003eand extant tardigrades. The results consistently recover \u003cem\u003eAysheaia\u003c/em\u003e as a stem-group tardigrade and inform the likely morphology of the tardigrade last common ancestor prior to the main radiation of the crown-group. Our findings strengthen the evo-devo hypothesis that modern tardigrades evolved from a macroscopic ancestor through overall miniaturization \u003csup\u003e9\u003c/sup\u003e and loss of the intermediate region in the trunk and legs \u003csup\u003e3,10\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e","manuscriptTitle":"A middle Cambrian macroscopic tardigrade ancestor","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-23 02:52:05","doi":"10.21203/rs.3.rs-8911114/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bdde36e0-b2d8-4b0a-8d7c-dff5a5ee3ca6","owner":[],"postedDate":"February 23rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":63154275,"name":"Biological sciences/Evolution/Palaeontology"},{"id":63154276,"name":"Biological sciences/Evolution/Taxonomy"},{"id":63154277,"name":"Biological sciences/Evolution/Phylogenetics"}],"tags":[],"updatedAt":"2026-04-13T16:17:50+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-23 02:52:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8911114","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8911114","identity":"rs-8911114","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}