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. 2025 Aug 4;23:238. doi: 10.1186/s12915-025-02335-3

Quantification of leg mobility in the Burgess Shale Olenoides serratus indicates functional differences between trilobite and xiphosuran appendages

Sarah R Losso 1,✉, Karma Nanglu 1,2,3, Walker C Weyland 1, Javier Ortega-Hernández 1,✉
PMCID: PMC12320380  PMID: 40754578

Abstract

Background

Euarthropod appendages are specialized for diverse roles including feeding, walking, and mating, which require precise morphologies and ranges of motion. Cambrian fossils preserve exceptional details of extinct euarthropod appendages that can illuminate their anatomy and ecology. However, fossils are typically restricted by small sample sizes or incomplete preservation, and thus functional studies of the appendages usually rely on idealized reconstructions. The Burgess Shale Olenoides serratus is unique among trilobites owing to the availability of numerous specimens with soft tissue preservation that allow us to quantify its appendages’ functional morphology.

Results

We measured the range of motion of the legs in Olenoides serratus and the extant horseshoe crab Limulus polyphemus. Despite repeated ecological comparisons between trilobites and xiphosurans, we find significant differences in the appendages’ mobility between these taxa, with Limulus showing greater flexibility between the podomeres.

Conclusions

O. serratus legs have a more restricted range of motion relative to L. polyphemus, particularly in their distal region. Flexure between the protopodite played a critical role in allowing the endopodite to create known trilobite trace fossils and bring food toward the ventral groove.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12915-025-02335-3.

Keywords: Cambrian, Functional morphology, Limulus, Trilobite

Background

Trilobites are a group of euarthropods that were a major component of the Paleozoic macrofossil record thanks to their over 22,000 described species and a 270 million year stratigraphic range [1], with possible trace fossils from the Terreneuvian [2]. Unlike their calcitic dorsal exoskeleton, the non-biomineralized ventral appendages of trilobites are only known from Konservat-Lagerstätten such as the Chengjiang biota [3–6], Guanshan biota [7], Emu Bay Shale biota [8], Tatelt Formation [9], Burgess Shale biota [10, 11], Kinzers Formation [12], Fezouata Shale biota [13–15], Walcott-Rust Quarry [16], Beecher’s Bed [17–21], Herefordshire biota [22], and the Hunsrück Slate [23–26]. Similar to extant euarthropods, trilobite appendages were highly specialized and played a key role in the group’s autecology given their versatility and functional diversity including roles in feeding [27, 28], walking [29–31], mating [11, 32–34], sensing the environment [35, 36], and respiration [21, 37]. Despite their importance for reconstructing trilobite ecology and functional morphology, specimens with well-preserved appendages are typically few in number per species, making studies beyond descriptive work more challenging (but see [38, 39]). Basic parameters of limb flexure/extension and structural strength, and how these affect modes of feeding or locomotion, have been studied in select extinct euarthropods through various types of three-dimensional computer modeling including kinematics and finite element analysis. This approach has been applied to phylogenetically disparate Paleozoic taxa, including trilobites and arachnomorphs [40, 41], radiodonts [42], eurypterids [38], and hymenocarines [43]. These studies have the advantage of leveraging various analytical methods to test ecological assumptions based on the availability of exceptional fossils with a high quality of morphological preservation. However, such informative specimens are rare and available in small sample sizes, and thus this type of methodology mostly relies on the information produced from a reconstructed three-dimensional virtual model rather than direct measurements of the fossil specimens themselves.

Trilobite appendages have been subject of several reconstructions based on qualitative interpretations of their mobility, attachment site to the body, and assumed orientation. Variations of trilobite appendage stances include laterally splayed [44, 45], ventrally curved [21, 39, 41], or recurved [26, 46] (Fig. 1). The attachment between the protopodite and body, a crucial component of understanding limb position and function, has also been subject of diverse interpretations. The protopodite has been proposed to attach dorsally near the distal margin [44, 45], dorsally near the medial margin [10, 15, 26, 45, 46], or dorsolaterally angled [8, 11, 16, 47] (Fig. 1). Reconstructions of the attachment of the exopodite have also been highly variable, either along the distal margin of the protopodite [10, 19, 26, 44–46, 48] or more medial [8, 11, 16, 21]. More recently, it has also been proposed that the exopodites of Triarthrus eatoni [49] and Olenoides serratus [50] attach ventrally to the protopodite and dorsally directly to the body [21]. Accurately reconstructing the limb position of trilobites requires understanding the connection between the protopodite and the body wall as well as the degree of flexure between podomeres, all of which carry direct implications for understanding the functional morphology of these euarthropods. In the absence of a more robust understanding of the range of motion and positioning of the appendages in trilobites, fundamental questions such as how these animals could gather and process food [38, 41], walk [39], and groom their gills [51] remain open.

Fig. 1.

Fig. 1

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Morphological reconstructions of limb attachment and stance in selected Trilobita. a Reconstructions of Olenoides serratus (Burgess Shale; Cambrian). b Reconstructions of Triarthrus eatoni (Beecher’s Trilobite Bed; Ordovician). c Reconstructions of Ceraurus pleurexanthemus (Walcott Rust Quarry; Ordovician). d Reconstructions of Chotecops sp. (Hunsrück Slate; Devonian). e Reconstructions of Redlichia rex (Emu Bay Shale; Cambrian). All reconstructions modified from their original publications to match color coding

In this study, we formally redescribe the biramous appendages of the corynexochid trilobite Olenoides serratus from the mid-Cambrian (Wuliuan) Burgess Shale in British Columbia (Fig. 1). O. serratus is unique among Cambrian trilobites due to the large number of specimens with preserved appendages (79 specimens; Additional File 1: Table S1). Here, we studied a total of 28 well-preserved specimens of O. serratus to directly quantify and analyze the range of motion of the limbs ( [10, 52]; Additional File 1: Table S1). The endopodites of O. serratus occur in various stages of flexure and extension that reflect their natural mobility, and partly due to the action of turbidity flows that resulted in their burial [53, 54]. These data illuminate the range of motion of O. serratus’ appendages and allow us to produce the first quantitatively informed reconstruction of the functional morphology and mobility of trilobite limbs to test several questions: Could O. serratus use the lateral spines to groom the gill of the appendage anterior to it? Were the endopodites able to reach medially to the ventral groove? Can the limbs of O. serratus create different trace fossils attributed to trilobites? Could the limbs vary the stance enough to traverse complex surfaces? We make further comparisons with the walking legs of the Atlantic horseshoe crab Limulus polyphemus(Xiphosura) [75] to further contextualize our results, particularly since these taxa have been repeatedly suggested to have a similar benthic ecology [11, 41, 55]. We demonstrate that despite the superficial similarities between trilobites and horseshoe crabs, O. serratus and L. polyphemus show significant differences in the degree of joint flexure and extension of their limbs that results in distinct functional adaptations. Finally, our study demonstrates that despite the more limited degrees of flexure and extension in O. serratus, the endopodites could create various kinds of trace fossils attributed to trilobites but would have limited utility in grooming the gills with the lateral spines.

Results

Protopodite morphology and body wall articulation in Olenoides serratus

The protopodite of Olenoides serratus is subtriangular in outline from the anterior–posterior view (Fig. 2b, c). The dorsal margin is straight, and the ventral margin is bowed and studded with gently curved spinose endites. The protopodite’s medial margin (which delineates each side of the food groove) is divided into upper and lower sections that are separated by an inflection point (ca. 98°), with the upper medial margin connecting to the body wall through a flexible arthrodial membrane (Fig. 2b, c). Endites become progressively longer and thicker proximally, with the largest being at or near the inflection point between the ventral and lower medial margin. The lower medial margin is studded by short, thorn-like endites forming the gnathobase (Fig. 2b, c). Remains of the arthrodial membrane can be observed in GSC 34692b and USNM PAL 65515 as a wrinkled dark film extending from the body wall into the protopodite through the upper section of the medial margin (Fig. 2b, c). In both specimens, the arthrodial membrane is shrunken and does not extend the full length of the upper medial margin. It is not possible to resolve the inflection point between the upper medial margin and dorsal margin because this particular feature is not observed in any specimens.

Fig. 2.

Fig. 2

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Protopodites and exopodite preservation in the trilobite Olenoides serratus from the Burgess Shale. a USNM PAL 65510, dorsal view of complete specimen with antennae, endopodites, and cerci. b GSC 34692b, attachment of protopodite to the body wall. c USNM PAL 65515, attachment of protopodite to body wall. d USNM PAL 188574, dorsal view of isolated biramous limb. e USNM PAL 65514, posterior view of exopodites showing dumbbell-shaped cross section of lamellae. Magenta arrowheads mark curved adaxial margin of exopodite proximal lobe. Abbreviations: ant, antenna; ar, arthrodial membrane; cer, cerci; dar, distal article; en, endopodite; end, endites; ey, eye; gn, gnathobase; lm, lamellae; low, lower medial margin of protopodite; par, proximal article; pt, protopodite; up, upper medial margin of protopodite

Exopodite morphology in Olenoides serratus

The exopodite is composed of two articles, the proximal being wide and elongate while the distal being small and teardrop shaped. Specimen USNM PAL 188574 represents an isolated biramous appendage and shows that the overall exopodite morphology is exceptional detail, particularly its proximal organization and attachment to the protopodite (Fig. 2d). The medial third of the anterior margin in the proximal article of the exopodite is disrupted by arthrodial membrane, whereas the outline is smooth for the distal portion. The proximal article of the exopodite is gently curved adaxially relative to the body wall, and widens distally, being broadest at 3/4s of the length before tapering slightly in the articulation with the distal article (Fig. 2d). The proximal article of the exopodite bears ca. 55–65 lamellae, each ca. 3–8 mm long, ca. 0.3 mm in height (dorsoventrally), ca. 0.1 mm across the widest part, and spaced 0.1 mm apart, as measured on both USNM PAL 188574 (Fig. 2d) and USNM PAL 65514 (Fig. 2e). USNM PAL 188574 shows that the lamellae follow the adaxial curvature of the proximal article of the exopodite (Fig. 2d), gently curve towards the mid-section, and become nearly straight along the rest of the article (Fig. 2d). Critically, this disposition demonstrates that the adaxial margin of the exopodite is not in direct contact with the body wall.

Although exopodites are typically observed in a flattened lateral orientation, specimen USNM PAL 65514 shows the exopodite preserved in posterior view with the lamellae facing perpendicularly relative to the bedding plane; this orientation reveals the transverse morphology of the lamellae of O. serratus for the first time, which have a dumbbell-shaped cross section (Fig. 2e).

Endopodite morphology in Olenoides serratus

The endopodite consists of seven podomeres, including a tripartite terminal claw (Figs. 3, 4). Podomeres become more elongate and narrower distally, with a row of short and delicate (ca. 0.5–0.8 mm length and ca. 0.09 mm width) lateral spines on podomeres 5 and 4 (Figs. 3b, 4a, b). Podomere 7 is square to subrectangular in outline with a bulge along the ventral margin that carries a cluster of endites (Fig. 3c, d). Podomere 6 is rectangular, dorsoventrally widest distally with a large endite surrounded by smaller spines (Fig. 3d). Podomere 5 is rectangular with a pair of laterally projecting endites that diverge from each other (Figs. 3d, 4b). In addition to the paired endites of podomere 5, the distal margin bears a fringe of short spines, slightly shorter than the endites (Figs. 3d, 4a, b). Podomere 4 closely resembles podomere 5 but is more elongate and narrower and displays a fringe of short spines around its distal margin (Figs. 3d, 4b). Podomere 3 has an hourglass outline with a pair of distal endites (Fig. 3b, c, e). Podomere 2 is rectangular and dorsoventrally widest distally (Figs. 3, 4a, b). The three terminal claws are ventrally curved, frequently with darker tips suggesting higher carbon concentration, possibly due to more robust sclerotization (Figs. 3d, e, 4a, b). Pairs of endites project laterally from the distal margins of podomeres 6–4 (Figs. 3, 4). Distal endites project at an angle of 30–50° from the ventral margin of the distal more podomere.

Fig. 3.

Fig. 3

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Flexed endopodites in the trilobite Olenoides serratus from the Burgess Shale. a USNM PAL 65520, cephalic appendage. b USNM PAL 65519, cephalic appendages. c GSC 34694a, pygidial appendages. d USNM PAL 58589, thoracic and pygidial appendages. e USNM PAL 188573, thoracic appendages. Abbreviations: fs, fringe of distal spines; lm, lamellae; ls, lateral spines; pn, podomere number; pt, protopodite

Fig. 4.

Fig. 4

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Extended endopodites in the trilobite Olenoides serratus from the Burgess Shale. a GSC 34695a, thoracic and pygidial appendages. b GSC 34697, thoracic appendages. c ROMIP 38601, cephalic appendages. d USNM PAL 57656, thoracic and pygidial appendages. Abbreviations: cl, claw; end, endite; fs, fringe of distal spines; ls, lateral spines; pn, podomere number

Endopodite flexure and extension in Olenoides serratus

The endopodites of Olenoides serratus are flexed at various degrees of gentle ventral curvature (Fig. 3c–e), but several individuals also show joints in an extended position (e.g., Fig. 3d, e, 4a, c). Angle F, between the protopodite and podomere 7, has the highest degree of flexure (78–160° with a range of 82°; Fig. 7a). No measured specimens showed angle F in an extended position (Additional File 1: Table S1). All measured specimens of Angle E are in the flexed position (119–180°), with only one in the straight position (Fig. 7a). Angle D showed one of the most flexed joints (79°) of all the limbs measured, but also the most extended (211°), with a range of 132° (Fig. 7a). Angles C and B have comparable values for flexure (119° and 129° respectively) and extension (208° and 210° respectively; Fig. 7a). Angle A has similar maximum flexure (115°) but a lower value of maximum extension (192°) than angles D–B (Fig. 7a).

Fig. 7.

Fig. 7

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Quantitative comparison of podomere flexibility between endopodites of trilobite Olenoides serratus and xiphosuran Limulus polyphemus. Horizontal line marks 180° where podomeres would be in a straight position. a Variability of flexure and extension in endopodites from O. serratus measured on 138 limbs from 28 specimens. b Variability of flexure and extension in endopodites from L. polyphemus measured from one specimen with limbs in maximally extended and maximally dorsally rotated positions

Cephalic, thoracic, and pygidial appendages of O. serratus have broadly similar degrees of endopodite flexure and extension (Fig. 7a; Additional File 2: Fig. S1a). The highly modified thoracic claspers observed in ROMIP 66299 (see [11]) have the highest degree (78–79°) of flexure at angle F (Fig. 7a). The substantial flexure of the claspers at angle F is only matched by a single instance of similarly high flexure (79°) at joint angle D found in one of the endopodites in USNM PAL 65520 (Fig. 3a). These observations indicate that despite the functional specialization of the claspers in O. serratus, they showcase comparable degrees of motion that can also be expressed on other endopodites with a conventional morphology.

Protopodite morphology and body connection in Limulus polyphemus

The protopodite of Limulus polyphemus is dorsally elongate with a broad lateral insertion point (Fig. 5) which connects to the body wall (Fig. 6). The protopodite of the pusher leg (Fig. 5e, f) has a subrectangular outline relative to the laterally slender and elongate morphology of the pedipalps (Figs. 5a, b, 6a) or the subtriangular outline of the walking legs (Fig. 5c, d). Both the pedipalps and walking legs display a similar degree of flexure between the ventral side of the exoskeleton and lateral margin of the protopodite (ca. 53–96°; Fig. 6g). Although all limbs have a similar range of motion between the body wall and protopodite (30–43°; Additional File 1: Table S1), the pusher leg has an obtuse angle when ventrally flexed (Figs. 5e, f, 6e).

Fig. 5.

Fig. 5

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Comparison between full extension and flexure in endopodites of the Atlantic horseshoe crab Limulus polyphemus. a and b Left pedipalps (limb I). a Maximum extension. b Maximally dorsally rotated. c and d Left walking leg (limb III). c Maximum extension. d Maximally dorsally rotated. e and f Pusher leg (limb V). e Maximum extension. f Maximally dorsally rotated. Brackets on a, c, e indicate attachment point to the body. All scale bars 10 mm. Abbreviations: ar, arthrodial membrane; fac, facet

Fig. 6.

Fig. 6

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Flexure between protopodite and body wall in prosomal appendages of Limulus polyphemus. a, b Pedipalps. a Maximally ventrally flexed connection between protopodite and body. b Maximally dorsolaterally extended connection between protopodite and body. c, d Walking leg (limb III). c Maximally ventrally flexed connection between protopodite and body. d Maximally dorsolaterally extended connection between protopodite and body. e, f Pusher leg (limb V). e Maximally ventrally flexed connection between protopodite and body. f Maximally dorsolaterally extended connection between protopodite and body. g Degree of flexure between lateral margin of protopodite and the body wall. All scale bars 10 mm. Abbreviation: pt, protopodite

Appendage flexure and extension in Limulus polyphemus

Limulus polyphemus endopodites have four podomeres in the walking leg, five podomeres in the male pedipalps, six podomeres in the pushing leg, and terminate in chelae in the walking leg and pushing leg, and a claw in the male pedipalp (Fig. 5; see also Bicknell et al. [40]); therefore, all the endopodites in L. polyphemus have fewer podomeres than those in Olenoides serratus. The endopodites of L. polyphemus exhibit a broad range of motion between podomeres enabling them to lift the body above the sediment through extension (Fig. 5a, c, e) or dorsally rotate the limb with the prosoma resting along the substrate (Fig. 5b, d, f). Although each joint displays a unique range of motion (Fig. 7b), all endopodites show a broadly similar pattern of flexure and extension (Additional File 2: Fig. S1b). Angle F exhibits a high degree of flexure with no extension (41–123°; Fig. 7b). Angle E has moderate flexure (94°), but the highest degree of extension in the entire endopodite reaching 278° (Fig. 7b). Angle D has a similar degree of flexure as angle F, although more acute (20–132°; Fig. 7b) and cannot extend. Angle C ranges from moderate flexure to extension (109–211°; Fig. 7b).

The high degree of extension in Limulus polyphemus endopodites is achieved not only through movement in the transverse plane, but also through sagittal rotation. The protopodite of all appendages has a lateral facet on either the anterior (pedipalps and pusher leg; Fig. 5c, e) or posterior face (walking legs; Fig. 5a). When the endopodite is dorsally curled, podomere 4 fits into the facet (Fig. 5b, d, f). The facet of the pusher leg occupies over 50% of the posterior surface of the protopodite (Fig. 5e). In the dorsally curled position, the dorsal margin of podomeres 4 and 3 touches the medial margin of the protopodite (Fig. 5f).

Discussion

Morphological reconstruction of the biramous appendages in Olenoides serratus

The new anatomical data on Olenoides serratus available through the exhaustive study of available museum collections allow us to produce a new morphological reconstruction of the limbs and further revise their functional variability (Fig. 8). The biramous appendages of O. serratus have been previously reconstructed with a dorsal attachment to the body wall from a rectangular protopodite close to the sagittal line [19, 21, 44, 52] (Fig. 1a), which has since been modified to more accurately reflect its subtriangular morphology [41]. The angled upper medial margin of the protopodite (Fig. 2b) suggests a more lateral connection between the limb and the body wall (Fig. 8), rather than the entirely dorsal attachment from earlier reconstructions (Fig. 1a) [10, 21, 44, 52]. Consequently, the angled attachment would allow for larger muscle insertion into the protopodite and greater force to be applied to masticate food with the gnathobases (Fig. 8a) [41].

Fig. 8.

Fig. 8

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Morphological reconstruction of biramous appendages in Olenoides serratus. a Anterior view. b Posterior view. c Dorsal view. d Medial view. Abbreviations: ar, arthrodial membrane; dar, distal article; end, endites; fs, fringe of distal spines; gn, gnathobase; hin, hinge; low, lower medial margin of protopodite

The precise attachment between the exopodite and protopodite in Olenoides serratus has also been controversial, varying from distal [10, 44, 52], hinge like with a medial attachment [57, 58] or complete medial attachment [21] (Fig. 1a). The isolated appendage of USNM PAL 188574 shows a wrinkled margin along the medial third of the anterior margin of the proximal article and lamellae extending from the posteriormost edge (Fig. 2d). This suggests the exopodite connected to the protopodite along the medial third of the anterior margin, with the proximal article and lamellae extending posteriorly (Fig. 8c). Recent work [21] has suggested that the exopodite of O. serratus connected both to the protopodite and dorsally to the body wall, concluding this was an intermediate state relative to the condition where the exopodite and endopodite are separated by body wall observed in the Silurian synzhiphosurine Dibasterium durgae, and the crustacean biramous limb with distally attached exopodites [57, 59]. However, the ventrally hanging attachment style (Fig. 1a) shown by [21] is difficult to reconcile with the angled upper medial margin of the protopodite based on our new observations (Fig. 2b, c). Instead, comparisons with the three-dimensionally preserved appendages of Protolenus (Hupeolenus) sp. from the Tatelt Formation (Cambrian Series 2, Stage 4; Morocco) [9] demonstrate a similar exopodite morphology and dorsolaterally angled attachment style to our interpretation for O. serratus (Fig. 8a). This attachment style has also been recently reconstructed for Eoredlichia intermediata [60], Redlichia rex [8], Hongshiyanaspis yiliangensis [47], and Anacheirurus adserai [15], Ceraurus pleurexanthemus, and Flexicalymene senaria [16]. In conclusion, trilobite exopodites with a sagittally broad proximal lobe display dorsolaterally angled attachment style regardless of their chronostratigraphic age or phylogenetic affinity.

The endopodites of Olenoides serratus have been well documented [10, 11, 51, 52]. The hourglass shape of podomere 3 is distinct from the squarer and more rectangular proximal podomeres (Fig. 8a). The fringe of distal spines on podomeres 4 and 5 (Figs. 4b, 8a) is similar in size and shape to the lateral spines on the dorsal margin. Fringes of spines at the distal margin of podomeres have also been reported from Chotecops ferdinandi [26], but are longer than those in O. serratus and occur on all podomeres. The fringe of distal spines in O. serratus would not be in contact with the sediment (Fig. 8), and thus did not serve a tactile function unlike in C. ferdinandi [26] or contribute to trace fossils [61]. Instead, the short distal spines might have served sensory functions similar to endites [62, 63].

Functional and ecological implications of podomere mobility in Olenoides serratus

The abundance of exceptionally preserved fossils of Olenoides serratus allows us to reconstruct the range of motion of its appendages by using direct quantitative measurements (Fig. 9), which represents the first time that this approach is used for studying the functional morphology of any trilobite species or Cambrian euarthropod. The range of motion in O. serratus’s appendages would allow the endopodite to reach dorsally to the underside of the exoskeleton and ventrally to the food groove (Fig. 9d). The endopodites of O. serratus are most flexible and show the highest degree of extension between podomeres 6 and 3 (Fig. 6a), which also bear a short row of lateral spines that is uniquely known for this trilobite species (Fig. 4a, b). These lateral spines were used to groom the lamellae of the exopodite, and that they may also have aided in sidewise raking [10, 51]. Based on our measurements, maximal extension of the endopodite would certainly allow the lateral spines to reach dorsally above the lamellae (Fig. 9c). The offset location between the lateral spines and lamellae indicates that each endopodite would groom the exopodite of the preceding appendage pair [51]. However, even considering anterior and posterior rotation of the appendages during walking, the lateral spines would only directly interweave with the lateral half of the lamellae (Fig. 9e–g), and therefore a quantitative analysis of the flexure mechanics in O. serratus does not support gill grooming as the primary function of the lateral spines of the endopodite (contra 51). A grooming function would only be viable if most of the detrital matter on the lamellae would accumulate distally on the exopodite and thus fall within the narrow range of reach of the lateral spines at their points of maximum flexure (Fig. 9b). However, another possibility is that the lateral spines of O. serratus were used as sensory structures, similar to the socketed endites seen in Limulus appendages [63].

Fig. 9.

Fig. 9

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Quantitatively informed range of motion of biramous appendages in Olenoides serratus. a Neutral position. b Flexed protopodite-body wall joint with extended endopodites. c Flexed protopodite-body wall joint with extended endopodites. d All joints at maximum flexure. e–g sequence of limb movement to groom lamellae with lateral spines shown in dorsal view with pink region marking location of lateral spines on endopodites. e Limbs extending anteriorly, allowing raised lateral spines to be interwoven with lamellae. f Posterior more leg rotates laterally to move lateral spines through lamellae removed debris. g Continuation of lateral rotation to clear anterior more exopodites

The protopodite of O. serratus has a stud-like gnathobase along the lower portion of the medial margin and larger endites along the ventral side (Fig. 2b, c). Finite element analyses suggest that O. serratus was not well equipped for a durophagous diet because the elongate endites on the ventral margin of the protopodite would not have withstood the forces necessary to crush shells, and instead most likely favored soft-bodied prey and food items [41]. In this context, the endopodites of O. serratus would need to reach food items and move them towards the ventral groove for gnathobasic mastication, which would require a significant degree of ventral flexure. The large range of motion seen in all endopodite joints of O. serratus suggests that they were mechanically capable of manipulating food towards the gnathobasal edge, but not directly making contact (Figs. 7a, 9d; Additional File 1: Table S1). Whereas the substantial range of motion in Limulus polyphemus endopodites would allow podomeres 1 and 2 to reach directly to the gnathobasal edge (Fig. 7b) [38], the endopodites of O. serratus would only reach the middle of the protopodite (Fig. 9d).

Olenoides serratus displays a smaller range of motion at each joint (ca. 61–132°) compared to those reported for Limulus polyphemus (ca. 20–100°; see 38), which can curl its endopodites tightly (Fig. 5). This is accomplished through alternation of joints specialized for flexure (angles F and D) and extension (angles E and C) and exposure of arthrodial membrane between podomeres (Fig. 5). No specimens of O. serratus have endopodites extended or flexed enough that the podomeres are separated with arthrodial membrane exposed (Figs. 3, 4). Additionally, the paired distal endites of O. serratus project at a shallow angle from the ventral margin of the distal more podomere (Figs. 3, 4), which would limit the degree of ventral flexure. Taken together, this demonstrates a more restricted range of motion in O. serratus than modern horseshoe crabs.

The more limited flexure and extension of endopodite joints in Olenoides serratus and distinct patterning along the ramus indicates differences in the precise functionality of the appendages in this trilobite relative to those of xiphosurans. Limulus polyphemus can manipulate food directly into the food grove and hold it in place during protopodal mastication [38] thanks to the substantial flexibility of its podomeres (Fig. 7b). However, this would not be possible in O. serratus due to the comparatively more restricted range of motion of its appendages (Fig. 7a). Limulus polyphemus also differs in that the alternation of joint specialization allows the endopodite to dorsally curl, accommodate in the protopodite facet, and fit completely within the highly vaulted prosomal dorsal exoskeleton. In contrast, the dorsal exoskeleton of O. serratus is comparatively less convex with nearly flat pleural lobes, and lowering the margin of the exoskeleton could be accomplished instead by dorsally flexing the protopodite-body wall joint and extending the endopodite laterally.

Functional and ecological implications of protopodite mobility in Olenoides serratus

Locomotion across the seafloor requires both complex coordination and variation in limb position, especially when the substrate is uneven [64]. Trilobites could vary their gait and speed to walk or burrow in the sediment, producing different trace fossils [39]. Variation in limb position is important for adjusting to walking in different conditions. A broad, sprawling stance can help modern euarthropods safely maneuver over complex terrain [30, 31] or overcome fast water flow [31, 65]. The stance of Olenoides serratus could vary from laterally splayed with the protopodites close to the sediment to raising the body high above the sediment (Fig. 9b), similar to Limulus polyphemus [38] and what has been suggested for Chotecops ferdinandi [26]. The broad range of positions possible in trilobite legs would aid in traversing uneven sediment with debris or adjusting to different water flow.

Several trace fossils have been attributed to trilobites: Diplichnites, a fast-walking trace; Cruziana, a slow locomotion or burrowing trace; and Rusophycus, a resting trace (see Esteve and Rubio [39]). These trace fossils are widespread geographically and show the infaunalization of trilobites and an increase in bioturbation during the early Paleozoic Era [66]. Despite the widespread occurrence of these ichnofossils and study on gait and leg number [39], the changes in limb movement and stance to produce such a range of traces have not been thoroughly explored. Trilobites would walk using their appendages to produce a metachronal wave where each endopodite moves in sequence (see 39). Reconstructing the entire walking sequence would require a significantly more complex model that accounts for additional movements (e.g., anteroposterior) not considered in the present study because this information cannot be readily measured from the morphology preserved in studied fossils. Additional complexity in interpreting the creation of trace fossils comes from the different sediment types, whether the animal was walking on the substrate or if it was an infaunal trace. In this context, our model shows the range of motion that a single a pair of limbs in O. serratus could achieve and thus forms a foundation for establishing further comparisons with the trace fossil record, ultimately resulting in a more comprehensive model for trilobite locomotion that combines morphological and behavioral fossil data.

Diplichnites traces could be produced with various widths depending on the stance of the trilobite, wider with the protopodite held horizontal to the sediment (Fig. 9a) or narrower if the protopodite was flexed (Fig. 9b). Changes in stances may have impacted the stability to current flow along the sea floor or speed [31, 39, 65]. Comparisons with Cruziana and Rusophycus trace fossils (Fig. 10) support the range of motion shown by our work. Both traces have bilobed structures with paired scratch marks attributed to the segmented limbs of trilobites [67–70]. The central axis of both Cruziana and Rusophycus indicates the paired endopodites could curl to nearly meet at the sagittal line (Fig. 10c, d). Maximal flexure of the endopodites from the body wall through angle C with extension of the more distal joints would bring the terminal claws close together (Fig. 9d). Traces can exhibit variation in the depth of the lobes along the length of the trace maker (Fig. 10a) as well as substantial variation between different traces (Fig. 10b, c, e). More flexure at the protopodite body wall joint and extension of the endopodite would create deeper gouges (Fig. 10c) than if the endopodite curled from a sprawling position (Fig. 10i).

Fig. 10.

Fig. 10

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Trace fossils from the Cambrian of Laurentia. a, b USNM PAL 799141, convex Cruziana sp. a Ventral view with low angled lighting. b Tilted upwards to show depth of track. c Model showing limb movement required to produce USNM PAL 799141. d, e USNM PAL 008615, convex Cruziana rustic. d Ventral view with low angled lighting. e Tilted upwards to show depth of track. f Model showing limb movement required to produce USNM PAL 008615. g, h USNM PAL 66145, concave Cruziana sp. e Ventral view with low angled lighting. f Tilted upwards to show depth of track. i Model showing limb movement required to produce USNM PAL 66145. See Additional Files 3–6 for animations of movement

Conclusions

  • The protopodite of Olenoides serratus connected to the body wall through an angled upper portion of the medial margin, with the exopodite then hinged to the dorsal edge. The endopodite was composed of seven podomeres with endites, lateral spines, and a fringe of distal spines of podomeres 4 and 5.

  • The endopodite of O. serratus exhibited a range of motion at joints similar to, but more restricted than the Atlantic horseshoe crab Limulus polyphemus, and with distinct patterns of flexure and extension along the ramus. The endopodite of O. serratus had the greatest flexure in the middle of the ramus, whereas L. polyphemus displays an alteration of joints specialized for flexure or extension.

  • Three-dimensional modeling of the appendage articulations of O. serratus shows how the endopodites could extend dorsally, but the limited overlap between sequential appendages would limit its utility for grooming the lamellae. The distal end of the endopodites could reach the midpoint of the protopodite when flexed, but coupled with flexure at the body wall, and this would allow the animal to sweep food towards the ventral groove for feeding. Different patterns of motion could create various trace fossils attributed to trilobites such as Cruziana and Diplichnites through changes in patterns of flexure and extension. The mobility of the limbs would allow O. serratus to traverse complex, uneven surfaces and modify the height of the body to adjust to different current flows.

Methods

Fossil material

We studied 108 specimens of the trilobite Olenoides serratus housed at the Invertebrate Paleontology collections at the Smithsonian Institution (USNM; Washington, D. C., USA), the Royal Ontario Museum (ROMIP; Toronto, Ontario, Canada), the Geological Survey of Canada (GSC; Ottawa, Ontario, Canada), and Naturmuseum Senckenberg (SMF; Frankfurt, Germany) (Figs. 2, 3, and 4). Fossil specimens were photographed under cross-polarized light using a Nikon D850 DSLR camera fitted with a Macro Nikkor 60 mm lens, a Nikon D7500 DSLR camera fitted with a Macro Nikkor 40 mm lens, or a Canon Mark 5D camera. Podomeres are numbered from distal to proximal following [71–73] which is applicable across all euarthropods. We employ the term protopodite to address both the attachment of the appendage to the body wall in trilobites and Limulus polyphemus to facilitate comparison between these taxa [56]. In this context, we consider the trilobite protopodite as most likely homologous to the xiphosurid coxa [41, 57]. Figures were produced in Adobe Photoshop, Adobe Illustrator, RStudio, and Blender.

Modern material

One individual of Limulus polyphemus (male, cephalon measuring (sag.) 73 mm) was collected from the intertidal zone in Duxbury Bay in Duxbury, Massachusetts (USA) in September 2024 (Figs. 5, 6) in compliance with Massachusetts State Law for sampling this species (322 CMR, § 6.34 – Horseshoe Crab Management). The specimen was transported to the Museum of Comparative Zoology Laboratories in Harvard University on the same day and euthanized by immersion in clove oil overnight, then dissected the following morning before significant decay altered soft tissue structure and flexibility. The prosoma and opisthosoma were separated using a scalpel. To measure the flexure between the protopodite and dorsal exoskeleton, the specimen was placed dorsal side down and the limbs were manipulated and photographed in series from most flexed to most extended (Fig. 6). Appendages were removed from the body and photographed with the endopodite in the extended and dorsally contracted positions (Fig. 7).

Measurement of fossil and material

The degree of flexure (ventral bending) or extension (dorsal bending) between each podomere of Olenoides serratus was measured using ImageJ for a total of 156 appendages belonging to 28 individuals where at least two consecutive podomeres were completely visible. The angle was measured along the margin of the endopodite that was best exposed; in cases where both were visible, the ventral side was used (Figs. 3, 4). We observe that the measured angles on both the dorsal or ventral sides are broadly similar or complementary to each other. In O. serratus angles were labeled as A–F from distal to proximal starting at the joint between podomeres 2 and 3 (Additional File 2: Fig. 1a). Angle F was measured from the distalmost endite of the protopodite and the distal margin on the ventral edge of podomere 7 (Additional File 2: Fig. S1a). All other angles were measured from the proximal margin of the preceding podomere to the distal margin of the following, excluding the endites (Additional File 2: Fig. S1a). The endopodites of Limulus polyphemus contain fewer podomeres than O. serratus, so only angles F–C were measured (Additional File 2: Fig. S1b). Standard error was calculated for each joint using all observations treated independently since multiple limbs within a specimen can have different flexure (Additional File 1: Table S1).

3D reconstruction of appendages in Olenoides serratus

Articulating three-dimensional models were produced in the open source 3D computer graphic software Blender (https://www.blender.org, version 4.2.3). The protopodite morphology was updated from Bicknell et al. [41] and Losso and Ortega-Hernández [11], with a wedge-shaped cross section based on comparison with other trilobites [62]. Endopodite morphology updated herein with measurements from Losso and Ortega-Hernández [11]. Podomere cross sections were reconstructed as circular based on Losso et al. [62]. The exopodite was modeled on USNM PAL 188574 with lamellae that are dumb-bell shaped in cross section as seen in USNM PAL 65514, with inflation inferred from Losso and Ortega-Hernández [11]. During flexure of the endopodite (angles less than 180°), the hinge is located on the ventral margin between each podomere, whereas during extension (angles greater than 180°) it is located on the dorsal side. Limits on the flexure and extension of each joint were imposed via Limit Rotation Constraints in Blender based on the minimum and maximum angles measured. The range of motion of the limbs was reconstructed by contrasting the 3D model of the appendages with various bilobed trace fossils (ichnofossils) that have been attributed to trilobites, or at least trilobite-like producers, particularly Cruziana. The concave arc of the Cruziana lobes was delineated and reflected to create the movement necessary to produce the variable morphology expressed in the trace fossils showcasing different depth profiles. These concave arcs were then imported into Blender and the movements of the reconstructed limbs were calculated by tracing the arc with the distal most endopodite and all other motions were calculated by inverse kinematics. This cross section would allow for muscles to attach within the protopodite from the sternite as well as the dorsal exoskeleton [41, 56, 74] (Additional File 2: Fig. S3). Models are available on Sketchfab (https://sketchfab.com/3d-models/biramous-leg-of-olenoides-serratus-82bd4e6b5ae1402cbd8c4bcfd9a0d874 for single limb and https://sketchfab.com/3d-models/cross-section-of-olenoides-serratus-body-e7bfe004f8ee48d1a7bb9dc761cf39f6 for full model) and in Additional File 7.

Supplementary Information

12915_2025_2335_MOESM1_ESM.xlsx (27.8KB, xlsx)

Additional File 1: Table 1. xlsx – Podomere angle measurements in Olenoides serratus and Limulus polyphemus.

12915_2025_2335_MOESM2_ESM.docx (21.7MB, docx)

Additional File 2: Figures S1–S3.docx FigS1 – Measurement of podomere angle. Fig. S2 – Limb flexure along the body. Fig. S3 – Hypothesized muscle attachment in Olenoides serratus.

Download video file (11.5MB, mp4)

Additional File 3: Olenoides serratus limb movement. An additional movie showing articulating limbs.

Download video file (9.8MB, mp4)

Additional File 4: Olenoides serratus limb movement to create deep Cruziana. An additional movie showing limb articulations to make a deep trace fossil.

Download video file (7MB, mp4)

Additional File 5: Olenoides serratus limb movement to create moderately deep Cruziana. An additional movie showing limb articulations to make a moderately deep trace fossil.

Download video file (6.5MB, mp4)

Additional File 6: Olenoides serratus limb movement to create shallow Cruziana. An additional movie showing limb articulations to make a shallow trace fossil.

12915_2025_2335_MOESM7_ESM.blend (28.8MB, blend)

Additional File 7: Blend model of body with a pair of limbs in Olenoides serratus.

Acknowledgements

We thank Jessica Cundiff (Museum of Comparative Zoology, Cambridge, MA, USA), Jean-Bernard Caron and Maryam Akrami (Royal Ontario Museum, Toronto, Ontario, Canada), Michelle Coyne (Geological Survey of Canada, Ottawa, Ontario, Canada), Doug Erwin, Mark Florence, Gene Hunt and Nick Drew (Smithsonian Institution, Washington D.C., USA), Omar Rafael Regalado Fernández and Olaf Vogel (Naturmuseum Senckenberg, Frankfurt, Germany) for facilitating access to specimens; Madeleine Waskom for assistance in collecting specimens of Limulus polyphemus; Kenny Amici for discussing mechanics of the endopodites; Léo Laborieux for modelling the exopodite of O. Olenoides serratus; Jorge Esteve and Lukáš Laibl for their helpful comments during peer review.

Authors’ contributions

SRL and JOH conceived of the project. SRL and WCW collected specimens of Limulus polyphemus. SRL and KN photographed specimens. SRL measured specimens, analyzed and visualized data, produced all figures and drafted the first version of the manuscript. WCW produced the 3D model of Olenoides serratus. SRL, JOH and KN interpreted the results. All authors participated in the editing and revision of the final version of the manuscript.

Funding

This work was supported by a Research Grant from Human Frontier Science Program (Ref.-No: RGY0056/2022) and the National Science Foundation CAREER award No. 2047192 “Ecological turnover at the dawn of the Great Ordovician Biodiversification Event - quantifying the Cambro-Ordovician transition through the lens of exceptional preservation.”

Data availability

All data generated or analysed during this study are included in this published article and its supplementary information files.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Sarah R. Losso, Email: sarahlosso@g.harvard.edu

Javier Ortega-Hernández, Email: jortegahernandez@fas.harvard.edu.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

12915_2025_2335_MOESM1_ESM.xlsx (27.8KB, xlsx)

Additional File 1: Table 1. xlsx – Podomere angle measurements in Olenoides serratus and Limulus polyphemus.

12915_2025_2335_MOESM2_ESM.docx (21.7MB, docx)

Additional File 2: Figures S1–S3.docx FigS1 – Measurement of podomere angle. Fig. S2 – Limb flexure along the body. Fig. S3 – Hypothesized muscle attachment in Olenoides serratus.

Download video file (11.5MB, mp4)

Additional File 3: Olenoides serratus limb movement. An additional movie showing articulating limbs.

Download video file (9.8MB, mp4)

Additional File 4: Olenoides serratus limb movement to create deep Cruziana. An additional movie showing limb articulations to make a deep trace fossil.

Download video file (7MB, mp4)

Additional File 5: Olenoides serratus limb movement to create moderately deep Cruziana. An additional movie showing limb articulations to make a moderately deep trace fossil.

Download video file (6.5MB, mp4)

Additional File 6: Olenoides serratus limb movement to create shallow Cruziana. An additional movie showing limb articulations to make a shallow trace fossil.

12915_2025_2335_MOESM7_ESM.blend (28.8MB, blend)

Additional File 7: Blend model of body with a pair of limbs in Olenoides serratus.

Data Availability Statement

All data generated or analysed during this study are included in this published article and its supplementary information files.

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