Taphonomy of non-biomineralized trilobite tissues preserved as calcite casts from the Ordovician Walcott-Rust Quarry, USA

Losso, Sarah R.; Thines, Jennifer E.; Ortega-Hernández, Javier

doi:10.1038/s43247-023-00981-5

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Published: 19 September 2023

Taphonomy of non-biomineralized trilobite tissues preserved as calcite casts from the Ordovician Walcott-Rust Quarry, USA

Communications Earth & Environment volume 4, Article number: 330 (2023) Cite this article

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Abstract

Trilobites with appendages from the Rust Formation of New York State were discovered in the 1870s and represent one of the earliest known cases of exceptional preservation of non-biomineralized tissues. The Rust Formation trilobites feature three-dimensionally preserved walking legs and delicate respiratory lamellae, but the mechanism behind their fossilization remains unknown. Here we show that after burial, carcass decay produced framboidal pyrite, while fibrous calcite precipitated on the visceral side of the body, followed by the widespread formation of sparry calcite crystal replicating non-biomineralized morphological features. Trilobites and co-occurring calcite veins show no chemical or petrographic differences, rejecting the hypothesis that exceptional preservation was caused by a local microenvironment within enrolled trilobites. These results suggest that fine-grained sediment provided support for the appendages and facilitated their fossilization through calcite replacement. Our findings carry broader implications for understanding the exceptional three-dimensional preservation of animal Paleozoic body fossils through calcite casts.

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Introduction

The trilobites from the Walcott-Rust Quarry have been known for over 150 years^1,2,3,4,5 and represent an extremely rare case of exceptional three-dimensional preservation of non-biomineralized structures in the form of detailed biramous appendages fossilized as calcite casts within a micritic limestone (Fig. 1). Charles D. Walcott originally found and studied enrolled trilobite fossils in the 1870s, and further inspection of broken specimens led him to cut them into thin sections using steam-powered saws aided by his mother (Mary Walcott) and sister (Mary Josephine “Josie” Walcott)⁶. Preparing the specimens this way revealed exceptionally preserved three-dimensional appendages fossilized as calcite casts. Before this discovery, trilobite appendages had only been known from the endopodites (i.e., walking legs) of Isotelus latus⁷, whereas the material from the Walcott-Rust Quarry produced the first ever insights into the biramous organization of the limbs including the lamellae-bearing exopodite^1,2,3.

**Fig. 1: Exceptionally preserved trilobites from the Walcott-Rust Quarry.**

The Rust Formation outcrops near Trenton Falls, New York, and is named for the family that owned the land when Charles D. Walcott opened a quarry there to collect trilobite fossils⁶. Exceptionally preserved specimens are only found within Layer 3 of the Spillway Member⁵. Body fossils within the Spillway Member of the Rust Formation were deposited in an obrution event, and include completely articulated trilobites in various positions (fully enrolled, partially enrolled, prone) within the layers or on the surface⁵. Two trilobites, Flexicalymene senaria Conrad 1841 and Ceraurus pleurexanthemus Green 1832 are commonly found within Layer 3 with exceptionally preserved appendages that include fine anatomical details such as delicate respiratory lamellae⁵ (Fig. 1). Whereas the low-magnesium calcite exoskeletons of trilobites make them ubiquitous throughout the Paleozoic fossil record^8,9, their non-biomineralized appendages are much less frequently preserved. Given the rarity of trilobite appendages, only 40 species with known preserved appendages from an estimated diversity of 20,000 described species¹⁰, understanding the taphonomic processes responsible for soft tissue fossilization in multiple well-preserved taxa in the same locality (e.g., Ceraurus pleurexanthemus and Flexicalymene senaria in Walcott-Rust) is critical for reconstructing their broader paleobiological significance and the conditions necessary for new discoveries. Despite being known for over a century, the morphology of the appendages of the two most common trilobites from the Walcott-Rust, C. pleurexanthemus and F. senaria, remains controversial. This is particularly true for fine anatomical details, such as the presence of endites^3,4,11, that are important for understanding the autecology and feeding strategy of trilobites¹².

Despite their iconic historical context, the Rust Formation trilobites have not been thoroughly studied in terms of their taphonomy. Previous work suggested that the exceptional fossils from the Walcott-Rust Quarry were produced by a process involving carcass decay through sulfate-reducing bacteria within a local microenvironment facilitated by the partially enrolled trilobites⁵. This model would require the production of bicarbonate and increase the pH within the isolated cavity formed by the partially enrolled trilobites, leading to the precipitation of calcium carbonate that replicates the morphology of soft tissues⁵. Decay through sulfate-reducing bacteria would also play a key role in pyritization as it produces the sulfide which combines with iron¹³. Based on this parallel, previous authors⁵ proposed that there was little available iron in the sediment as the carcass decayed, preventing complete pyritization and the pyrite seen in the specimens (Fig. 1e) replaced calcite later in diagenesis. Although this hypothesis for exceptional preservation in calcite has remain unchanged for over 20 years, its veracity has not been corroborated through chemical analyses, and thus the precise mechanism responsible for the formation of these fossils remains unclear. In this study, we take a comprehensive analytical approach through scanning electron microscopy, micro-computed tomography, and Raman spectroscopy on new and historic museum specimens to examine the phases of calcite precipitation, the formation of pyrite framboids, and the crucial role of sediment in appendage preservation in the Walcott-Rust trilobites.

Results

Exceptionally preserved trilobites from the Walcott-Rust Quarry show a consistent mineralogy that comprises an isopachous rim of fibrous calcite associated with framboidal pyrite that completely fills small void spaces, and large sparry calcite crystals in the larger spaces (Fig. 1c–e). Fully enrolled specimens with the pygidium tucked into the cephalon are filled with calcite (e.g., MCZ:IP:201000) and have no sediment matrix within the specimen (Fig. 2a, b). Sparry calcite infills most commonly occur within the glabella and doublure (Fig. 2c, f), which are surrounded by the biomineralized exoskeleton. Three specimens from Layer 8, higher in the stratigraphic column⁵, have calcite infilling the doublure and glabella (Supplementary Fig. 2), but no appendages are visible in any of these fossils.

**Fig. 2: Trilobites filled with calcite crystals from the Walcott-Rust Quarry Layer 3.**

Pyrite occurs most commonly as framboids within the fossils and surrounding matrix (Fig. 3g, i). Fibrous calcite crystals form an isopachous rim ca. 150 μm long and grow inwards from the margin of the fossils (Fig. 1c, e–g). In small spaces, the fibrous crystals occupy the entire volume and are often colored with a faint orange hue that becomes more saturated towards the apex (Fig. 1e, g). Framboids are concentrated at the edges of fibrous crystals and frequently between the exoskeleton and the isopachous rim, but are also embedded within the crystals (Fig. 3g,) and seen throughout the matrix (Supplementary Figs. 3j, 4d). The sparry calcite crystals are clear, large, and blocky with the largest crystals near the center of the void space (Figs. 1c–g, 2a–c, f).

**Fig. 3: Elemental and mineralogical comparison between trilobites and veins from the Walcott-Rust Quarry.**

Fibrous crystals within veins are less well organized (Fig. 3h, j) as they nucleated off the uneven walls and grew inwards. Small syntaxial veins are filled by fibrous crystals with preserved median lines (Fig. 2c). In veins, pyrite framboids (Fig. 3i) fill large voids between fibrous or sparry crystals which are up to 1.5 mm long by 0.5 mm across (Fig. 3j). Comparisons indicate that trilobite fossils have noticeably less pyrite than veins, whereas the sparry calcite crystals are similar in appearance to those found in fossils and fill in larger spaces within veins (Fig. 3h, j).

Magnesium concentrations within the biomineralized calcitic exoskeleton vary from 1.2 wt% to concentrations below the limit of detection (<0.2 wt%; Fig. 4a, b; Supplementary Data 1). Fibrous and sparry crystals have overlapping ranges of magnesium, but compositions cluster based on specimens rather than crystal type (Fig. 4a, b). In MCZ:IP:198047, fibrous crystals have similar magnesium concentrations as the exoskeleton (Fig. 4a, b). Sparry crystals have decreasing magnesium towards the center of the calcite whereas most spots have non-detectable concentrations (Fig. 4a, Supplementary Data 1). The vein in MCZ:IP:199123 shows a similar pattern between fibrous and sparry crystals, although the concentrations are lower overall (Fig. 4a, b). Magnesium concentrations in sparry and fibrous calcite are similar within a single specimen but vary between specimens (Fig. 4a, b). The concentrations of calcium and carbon in the trilobite MCZ:IP:198048 and the vein MCZ:IP:199123 are similar and distinct from all other specimens (Fig. 4c). Iron concentrations are also consistent within specimens but vary between specimens. In MCZ:IP:198047, the sparry calcite has a higher iron concentration than the exoskeleton and fibrous calcite except for one spot (Fig. 4d, Supplementary Data 1, Supplementary Fig. 3). MCZ:IP:198048 has no detectable iron in the exoskeleton, fibrous, or sparry calcite. MCZ:IP:198046 has comparable values of iron in the sparry calcite as MCZ:IP:198047 (0.41–0.51 wt% and 0.41–0.59 wt%, respectively).

**Fig. 4: Comparative chemical analyses of Rust Formation trilobites and veins.**

The matrix surrounding MCZ:IP:198047 shows dark rims around blocky clasts of feldspar which is high in iron with trace amounts of barium (0.29–0.71 wt%; Fig. 3b, Supplementary Data 1). The matrix within the specimen has a similar composition of clasts but lacks the dark rims and barium (Fig. 3d). Two fully enrolled specimens have large masses of barite within the body (Fig. 4e–i). In MCZ:IP:8400, barite (Fig. 4i) occupies a third of the internal volume, centered within the axial lobe with repeating lobe-like structures of uncertain origin (Fig. 4h). Compaction lines are frequently found surrounding fossils and are concentrated around opposite sides depending on the original orientation of the trilobite (Fig. 1c, e). Compaction lines are not seen within the sediment inside partially enrolled specimens or in the flexed specimen MCZ:IP:158215 (Fig. 1d).

Close morphological examination of trilobite thin sections demonstrates the presence of small spinose endites that extend from the medial and ventral edges of the protopodite, as well as from the distal edges of the podomeres on MCZ:IP:110933 and MCZ:IP:112018 (Fig. 5a–d). The base of the endites is comparable in width to the lamellae (ca. 0.1 mm), although they taper to a terminal width of 0.03 mm. Both endites and lamellae are filled with minute fibrous calcite crystals (Fig. 5a, b), and the crystals within the endites continue into the endopodite (Fig. 5b).

**Fig. 5: Comparison of endites between *Ceraurus pleurexanthemus* and *Olenoides serratus* from the mid-Cambrian Burgess Shale.**

Discussion

Morphological identity of spinose endites in Ceraurus pleurexanthemus

The delicate quality of preservation of the Rust Formation trilobites includes fine morphological features (Fig. 1), but their appearance has caused some disagreement regarding their biological interpretation. For example, the spinose endites preserved on the ventral side of the limbs on Ceraurus pleurexanthemus have proven to be controversial (Fig. 5), with some authors regarding them as preservation artifacts¹⁴ while others interpreted them as the true morphology³. Given the significance of endite morphology for reconstructing feeding strategies in trilobites¹², it is pertinent to clarify their nature to better understand the functional morphology of Ordovician species. Two specimens (MCZ:IP:110933 and MCZ:IP:112018) preserve a transverse cross section of C. pleurexanthemus, showing in detail the attachment side of the proximal region of the biramous appendages relative to the body wall (Fig. 5a–d). Here, examination of the walking legs demonstrate that the features interpreted as spinose endites are exclusively found on the ventral side of the protopodite and endopodite podomeres (Fig. 5a–d). The spinose projections are morphologically identical in both specimens and show a consistent perpendicular orientation relative to the main limb axis. Critically, there is no evidence of randomly oriented spinose projections on the dorsal side of the limbs or other parts of the body, which would be expected if these features resulted from taphonomic processes rather than reflecting the original anatomical organization. The placement of the spinose endites in MCZ:IP:110933 and MCZ:IP:112018 is extremely similar to that observed in other exceptionally preserved trilobites^10,15, including an arrangement along the medial and ventral edges of the protopodite, and clusters in the mid-point of podomeres or at the distal margin (Fig. 5). Direct comparisons with the protopodite and endopodite morphology of the Burgess Shale trilobite Olenoides serratus clearly exemplifies these similarities in terms of their overall appearance, location, and distribution (Fig. 5e–h). We conclude that the endites are not artifacts of preservation but reflect the original morphology of the Rust Formation trilobites. The morphological similarity of the protopodite outline and spinose endites observed between C. pleurexanthemus and O. serratus (Fig. 5) suggest that the former Ordovician species was incapable of a durophagous diet, and instead most likely feed on soft-bodied organisms and/or food items¹².

Effect of sediment and enrollment

The findings of this study suggest that the fine-grained sediment played an important role in supporting the delicate appendicular structures and facilitating exceptional preservation (Figs. 1, 2, 5). The limbs are not preserved in fully enrolled specimens in which sediment was not able to directly encase the appendages, and the resulting cavity formed by the exoskeleton is instead filled with sparry calcite (Figs. 2a, 6b). Calcite is commonly found between the glabella and the hypostome (Fig. 2b, c, e, f) as well as within the doublure (Fig. 3d), which share the fact that these locations are surrounded by the rigid exoskeleton. The exoskeleton created sheltered voids that were easily infilled by calcite crystals without requiring support from the sediment. Our interpretation is that fine-grained micritic limestone created a mold of even the most delicate structures (e.g., lamellae and endites) that was maintained as the carcass decayed. In cases in which the appendages were damaged during transport within the turbidity flow¹⁶, the micritic mud mold was not able to form around the appendages, and thus calcite only formed within the exoskeleton-protected voids (Fig. 6c). Calcite is also found within the glabella and doublure of trilobites from Layer 8 which is higher in the section (Supplementary Fig. 2), but no appendages have been found from these specimens. Layer 8 is over twice as thick as Layer 3, and with the larger turbidity flow, the delicate appendages of trilobites may have been more prone to damage. When appendages were undamaged, the mud mold protected the external morphology of the limbs allowing for preservation of even the most delicate structures (Figs. 1c, 6d).

**Fig. 6: Taphonomic model for exceptional preservation through calcite replacement in the Walcott-Rust Quarry.**

Enrollment has a complex effect on exceptional preservation in the Rust Formation. Previous authors⁵ hypothesized that the partial enrollment created a microenvironment because of the protective barrier formed by the exoskeleton. Two specimens, MCZ:IP:158215 (Fig. 1d) and MCZ:IP:158238 (Fig. 2d), have preserved appendages, but critically these specimens are slightly dorsally flexed rather than enrolled. This indicates that enrollment was not a strict requirement for limb preservation. Evidence of compaction is commonly observed in the external sediment around the fossil (Fig. 1c, e), whereas there are no sediment compaction lines within the enrolled specimens. Interestingly, the protopodites of the prone specimen MCZ:IP:158215 (Fig. 1d) are not as well preserved as other specimens in a partially enrolled position, such as MCZ:IP:104956 (Fig. 1e), which suggests that the integrity of the mud mold around the appendages is important for their fossilization. Thus, although partial enrollment is not a prerequisite for appendage preservation, this position aided in the maintenance of the micritic mud mold and calcite cast during early diagenesis by decreasing the impact of compaction and resulting in a better preservation quality of ventral non-biomineralized structures. Such compaction could have distorted or damaged the appendages regardless of whether it occurred early or late during diagenesis.

No local microenvironment during decay

Our new data allows us to comprehensively test the traditional model for the exceptional preservation of soft tissues in the Rust Formation. Previous authors⁵ proposed that partially enrolled trilobites from the Walcott-Rust Quarry created a microenvironment where calcite precipitation was induced within the cavity produced by the specimen, and the exoskeleton acted as a physical protective barrier. Critically, this model would imply substantial physical and chemical discrepancies between the calcite formed within the trilobite fossil and the later-stage calcite veins found throughout the micritic rock matrix. However, we find no evidence of chemical differences between the calcite formed in fossils and veins, as both display the same crystal textures (i.e., a rim of fibrous crystals and large sparry crystals filling in larger voids) with no detectable differences in terms of elemental composition (Fig. 4). Four of the studied fossil specimens and veins (MCZ:IP:199124, MCZ:IP:199125, MCZ:IP:198046, MCZ:IP:198047) have similar calcite compositions, whereas two additional specimens (MCZ:IP:199123, MCZ:IP:198048) clustered together in terms of their magnesium and carbon content (Fig. 4b, c). Although MCZ:IP:199123 and MCZ:IP:198048 have similar values of calcium, MCZ:IP:198048 has higher magnesium, but both are within the range of the other veins and fossils (Fig. 4b). Original specimen locations within Layer 3 of the Rust Formation were not recorded during collection, so it is not clear if low-scale stratigraphic spatial distribution accounts for the similarities between MCZ:IP:199123 and MCZ:IP:198048. Although the calcite veins contain comparatively more pyrite than the trilobite fossils (Fig. 3j), both otherwise lack differences between composition and texture (Fig. 4c–e, h, j). Ultimately, we find no physical or chemical evidence for differences between the diagenetic formation of pyrite, fibrous calcite, and sparry calcite between trilobite body fossils, and the veins. We thus reject the hypothesis that enrolled trilobites produced a local microenvironment due to a barrier created by the exoskeleton that then facilitated exceptional preservation of soft tissues (contra previous authors⁵).

Decay studies of the caridean shrimp Crangon crangon have shown that a single carcass can produce chemical gradients of pH, sulfide, and oxygen into the surrounding substrate¹⁷. Although this experiment demonstrates that carcass decay will change the composition of the surrounding environment producing a chemical gradient, there is no evidence that the sclerotized exoskeleton functions as an impermeable barrier that would create a drastically different microenvironment within the carcass itself that then led to exceptional preservation. Likewise, although it has been shown that the endogenous bacteria within the digestive system of euarthropods can play an important role in the early diagenetic stabilization of the gut tract due to changes in the pH within the carcass¹⁸, the chemical gradients within the external environment alone are not sufficient to explain the exceptional preservation of other non-biomineralized tissues such as appendages.

Silurian fossils from the Herefordshire Lagersttäte are preserved in concretions that feature a halo with increased aluminum, silicon, iron, and magnesium, as well as depleted calcium¹⁹, suggesting that, in this case, the individual carcasses created chemical gradients within the surrounding matrix. Critically, such elemental halos are entirely absent from the Walcott-Rust fossils (Figs. 1–3, Supplementary Figs. 3–5), which combined with the lack of concretions, points towards important differences in terms of the lithification of the fossil-bearing strata between these localities. We hypothesize that for the Rust Formation, decay through sulfate-reducing bacteria took place within the carcasses, matrix, and veins as indicated by the presence of pyrite framboids, which may have aided in the precipitation of calcite, but that this process was not restricted to trilobite bodies.

Low iron environment

Previous authors⁵ proposed a low iron environment to explain the occurrence of calcite rather than pyrite within trilobite fossils, with the assumption that pyritization would follow a similar microbially-mediated pathway to calcite precipitation. In this work⁵ the pyrite observed in the Rust Formation fossils was interpreted as secondary, having replaced the original calcite during late diagenesis (Figs. 2a, 3d, g). In contrast, we demonstrate that pyrite framboids frequently occur around the calcite crystals in both the fossil and veins (Fig. 2a) and embedded within the crystals (Fig. 3d, f, g). Pyrite framboids have been shown to form through the production of hydrogen sulfide by sulfate-reducing bacteria during remineralization of organic matter²⁰.This suggests that the pyrite framboids within the Rust Formation fossils and veins formed at an early stage, likely mediated by microbial-induced decay, rather than during late diagenesis.

The presence of pyrite throughout the matrix and veins with large volumes of framboids indicates there was more iron within the sediment and pore water than previously suggested⁵, but not enough to completely pyritize the carcass as observed in other Ordovician deposits from New York, such as the famous Beecher’s Trilobite Bed²¹. Beecher’s Bed is located less than 15 miles from the Walcott-Rust Quarry, is similar in age (Late Ordovician), and also formed by turbidity flows, but is a dark shale that experienced extensive exposure to iron-rich pore water, leading to impressively pyritized macrofossils²¹. We hypothesize that fluid flow provided a continuous supply of iron to the veins and allowed for continued pyrite formation during compression and dewatering of the sediment at the Walcott-Rust Quarry, accounting for the large volumes of observed framboids (Fig. 3h–j). In this context, the rigid trilobite exoskeleton prevented internal compaction and vein growth within the carcasses, limiting the amount of available iron and hindering pyritization from sulfate-reducing bacteria within the fossils.

Presence of barite in Rust Formation trilobites

Barium found as rims around clasts in the matrix and large masses of barite within enrolled trilobites have not previously been reported from the Rust Formation. The presence of these features raises questions about the sequence of events leading to the exceptional preservation seen in trilobites (i.e., did the barite precipitate at the same time as calcite or during a later diagenetic event?). Barium is restricted to the external matrix of MCZ:IP:198047, an enrolled specimen found at the surface of Layer 3, suggesting the exoskeleton acted as a barrier to fluid flow after lithification and prevented alteration of the matrix within the specimen (Fig. 3b, d). The specimens with large masses of barite (MCZ:IP:8400 and MCZ:IP:8118) have cracks through which the fluid could have flowed (Fig. 4h). It is unclear if these specimens had original appendage preservation in calcite. In MCZ:IP:8400, the barite mass (light green) runs along the axis of the enrolled specimen with repeated lobe-like structures extending to the pleural lobe (Fig. 4h). It is unclear if this reflects original ventral morphology of the trilobite.

Barite forms in marine sediments when barium- and sulfate-rich waters mix, leading to precipitation²². The presence of barite in the Rust Formation implies that nearby barite deposits were dissolved by pore water, creating a barium-rich fluid that flowed through cracks within the lithified or consolidated sediment after the calcite cast fossils had fully formed. This enriched water then flowed between Layers 3 and 4 of the Rust Formation, and through cracks in the rock, such as those seen in MCZ:IP:8400 and MCZ:IP:8118. In some cases, such as the enrolled specimen MCZ:IP:198047, the exoskeleton prevented flow of the enriched water resulting in barium rims around clasts only in the external matrix and not within the fossil.

A new diagenetic model for soft tissue preservation in Rust Formation trilobites

We propose a new model for the exceptional preservation of non-biomineralized structures in the Walcott-Rust trilobites that can be subdivided into three major events: entombment, sediment stabilization, and crystallization (Fig. 6). During the initial entombment stage, turbidity flows transported live trilobites and other organisms such as bryozoans and crinoids (Supplementary Fig. 1d) downslope, ultimately killing them (Fig. 6a). Trilobites enrolled during transport as a defensive mechanism^23,24,25, with some specimens maintaining complete enrollment after death aided by interlocking co-adaptive devices (Fig. 6a). Depending on the final position during burial, sediment stabilization affected the specimens in different ways. Sediment did not encase the appendages in fully enrolled specimens (Fig. 6bi), and instead produced an empty void following the decay of the soft tissues (Fig. 6bii). In this case, the entire void space filled with calcite crystals without appendage preservation such as specimen MCZ:IP:201000 (Figs. 2a, b, 6biii). By contrast, in partially enrolled specimens the fine-grained sediment encased the non-biomineralized ventral side, including the appendages (Fig. 6c, d). If the appendages were damaged during transportation within the turbidity flow, they were likely destroyed by decay too rapidly before the fine-grained sediment created an external mold (Fig. 6cii). This would result in specimens like MCZ:IP:112030 (Fig. 2f) where calcite is only present in exoskeleton-protected spaces such as the glabella and doublure. In partially enrolled individuals with undamaged appendages, the fine-grained sediment completely encased the non-biomineralized ventral limbs (Fig. 6d). This process created a sediment-supported external mold that preserved exceptional morphological details, including the lamellae and endites, and provided structural protection that prevented collapse or further degradation (Fig. 6dii) such as in specimens MCZ:IP:110933 and MCZ:IP:112018 (Fig. 5a–d).

Regardless of the body position of the trilobites, pyrite framboid and calcite crystal formation started after entombment, and in some instances created an external mold of the appendages (Fig. 6e–h). Oxygen quickly depleted as a result of carcass decay and forced the switch to anoxic pathways, primarily sulfate-reduction through bacteria²⁶. Sulfate-reducing decay produced sulfide which combined with iron (II) in the pore water and led to the formation of pyrite framboids (Fig. 6l). Magnesium- and iron-bearing fibrous calcite crystals nucleated off the sediment, encasing the appendages or the exoskeleton, and grew inwards to incorporate framboids into the crystals (Fig. 6l). The originally low magnesium calcite trilobite exoskeleton⁸ was at least partially altered during this process, resulting in the observed high magnesium content in the Rust Formation specimens (Fig. 6f–h). Within the fossils, fluid flow limiting available iron (II) concentrations prevented complete pyritization and calcite precipitation dominated. Compression-induced fluid flow carried iron (II) through the veins (Fig. 6bii, cii, dii), facilitating continued pyrite formation within them during calcite precipitation (Fig. 6biii, ciii, diii). While the rigidity of the exoskeleton prevented compaction of the sediment within partially enrolled specimens and prevented loss of exceptional three-dimensional preservation of the appendages, it also inhibited flow of iron-rich fluid resulting in limited pyrite framboid formation. After the initial phase of fibrous calcite precipitation, large sparry calcite grew within larger void spaces such as the glabella, doublure, and axial lobe of the exoskeleton (Fig. 6biii, ciii, diii), producing various degrees of calcite infilling depending on the extent of the available voids (Fig. 2).

Implications for three-dimensional exceptional fossil preservation

Our findings provide new insights on the mechanisms responsible for the three-dimensional fossilization of non-biomineralized structures through calcite casts, which represents a highly informative but also extremely rare type of exceptional preservation. Calcite casts have been found in diverse conditions, ranging from Ediacaran fossils with crystals replicating the quilt-like body walls²⁷, and within plant cells^28,29. However, neither of these cases show evidence of the distinct two phases of calcite precipitation and pyrite framboids as seen in the Walcott-Rust fossils. The only close comparand in the entire Paleozoic is found in the Silurian Herefordshire biota of England, in which diverse organisms (e.g., sponges, euarthropods, mollusks) were encased in a volcaniclastic ash and later incorporated into carbonate concretions^19,30. Interestingly, both Walcott-Rust and Herefordshire fossils share the same occurrence of pyrite framboids and isopachous rims of fibrous and large sparry calcite crystals despite the different geologic settings (micritic limestones versus volcaniclastic sediment with carbonate concretions) and the order of magnitude difference in size of specimens (centimeters versus millimeters in width of calcite casts, respectively)¹⁹. Similarly to the Rust Formation trilobites, Herefordshire fossils also feature pyrite framboids around the margins of calcite infills, which are interpreted as co-precipitating because of their inclusion within calcite crystals¹⁹. The parallels with the Walcott-Rust Quarry suggest that the pyrite in Herefordshire fossils also formed early during diagenesis as the carcass was decaying and in tandem with calcite growth, rather than at a later stage as previously suggested (contra previous authors⁵). However, Herefordshire fossils display a fringe of clay-rich minerals and dolomitization of the concretions¹⁹ that are not observed in the Rust Formation. Herefordshire fossils displays a greater biodiversity with the preservation of entirely soft-bodied organisms such as annelids^31,32 (but see other work^33,34 for alternative interpretations) and aplacophoran mollusks³⁵. In these cases, it is likely that the volcaniclastic ash encasing the Herefordshire fossils was more effective at forming an external mold before decay than the micritic sediment. Further comparisons between the two localities are currently underway to better understand the processes responsible for the formation of these exceptional three-dimensional fossils in such different geologic settings.

Exceptional calcite cast preservation is difficult to discover in the field because it is not apparent in hand samples and requires cross sectional examination of large numbers of specimens. Calcite within the doublure of other trilobites is also known in the Silurian Bumastis? phix from the Dolyhir and Nash Scar Limestone Formation, Dolyhir, UK³⁶, indicating that similar conditions occurred elsewhere, but to date, appendages with similar preservation have not been found. However, the occurrence of fine-grained sediment flow with enough material to bury but not damage the appendages in a low iron environment is likely not unique to the Walcott-Rust Quarry in the geological record. We hypothesize that exceptional soft-tissue preservation through calcite casts might be more common in the fossil record than currently appreciated, and that the technical difficulties of recognizing it in the field represent the major obstacle in their discovery more broadly.

The calcite cast fossils from the Walcott-Rust Quarry provide a unique view of three-dimensional non-biomineralized trilobite appendages preserved in the original life position with fine detail. Calcite cast fossils, such as those found in Walcott-Rust Quarry and Herefordshire, have preserved delicate detail in three dimensions with little compaction or displacement^5,37 and occur in a variety of geological settings¹⁹. The spatiotemporally comparable Beecher’s Trilobite Bed from New York preserves fully pyritized organisms with comparable levels of morphological detail, including the well-known trilobite Triarthrus eatoni; however, non-biomineralized structures such as trilobite limbs are frequently displaced³⁸. The Walcott-Rust Quarry provides exceptional external detail comparable to well-known Lagerstätten such as Cambrian Burgess Shale-type sites but with the unique advantage of showing uncompacted appendages within enrolled trilobites, whereas even the three-dimensional fossils from Chengjiang show a degree of post-burial compaction^39,40,41.

Material and methods

Geological setting

The Spillway Member of the Rust Formation is exposed at the Walcott-Rust Quarry (New York, USA, 43.278533, −75.148761) (Supplementary Fig. 1). The Rust Formation is composed of thinly bedded skeletal wacke and packstones with some heavily bioturbated layers⁵. The quarry itself exposes the “prospect beds” deposited during a highstand, consisting of 14 limestone layers with shale interbeds. Trilobites, bryozoans, cephalopods, and crinoids from the Trenton shelf are frequently found in the quarry within the tempestitic limestone layers, having been deposited by turbidity currents moving from the northwest into a dysoxic foreland basin^5,42. At the surface of Layer 3, trilobites and crinoids are found both articulated and disarticulated (Supplementary Fig. 1), but only trilobite carcasses show evidence of fossilized non-biomineralized tissues. All specimens prepared as thin sections at the Museum of Comparative Zoology (MCZ) at Harvard University are from Layer 3, which corresponds to a 3–4 cm thick micritic limestone with little bioturbation (Supplementary Fig. 1). Layer 8 has a thickness between 8 and 10 cm, and no trilobites with preserved appendages have been reported to date (Supplementary Fig. 1).

Studied material, imaging, and analyses

Studied specimens of Ceraurus pleurexanthemus and Flexicalymene senaria are housed at the Museum of Comparative Zoology, Harvard University. Historic samples were collected from the Walcott-Rust Quarry by Charles Walcott in the 1870s, prepared as thin sections by hand, and affixed to glass slides using balsam sap⁶ (Figs. 1, 2). Over 180 kg of rock from Layers 3 and 8 were collected from the Walcott-Rust Quarry based on the presence of trilobites preserved at the surface and donated to the MCZ by Mr. Dan Cooper (Covington, Kentucky, USA) (Supplementary Fig. 1). Three new specimens from Layer 8 (Spillway Member, Rust Formation) were imaged as they showed evidence of calcite within the glabella and doublure (Supplementary Figs. 1, 2). Three trilobite specimens and three calcite veins from Layer 3 (Supplementary Fig. 1) were selected for chemical analyses (Figs. 3a, e, 4a–d) based on the presence of observable sparry calcite either in the hand sample or during grinding of the specimen. The three calcite veins from Layer 3 were chosen to compare minerology with the calcite within fossils to specifically test the hypothesis that enrolled trilobites produced a microenvironment for exceptional preservation⁵ (Figs. 3h–j, 4a–d). Veins were frequently found when large slabs (ca. 600 mm across) were broken into smaller pieces (150 to 300 mm across). The veins were selected based on area of calcite exposed and from separate slabs to ensure a broad spatial distribution. Specimens were prepared as one-inch epoxy mounts. Gold- or palladium/platinum-coated specimens were imaged at Williams College using a ThermoScientific Quattro S scanning electron microscope (SEM) fitted with an Everhart-Thornley, concentric backscatter, and EDAX Octane Elect detectors. Electron dispersive spectroscopy (EDS) spot analyses (30 second dwell) and mapping, and backscattered electron (BSE) and cathodoluminescence (CL) imaging were conducted in a high vacuum environment under 20kv operating conditions. Three-dimensional images of trilobite specimens were obtained using the Bruker SkyScan 1173 micro-CT scanner at the Harvard University Digital Imaging Facility with a voltage of 130 kv, wattage of 61 uA, and 0.25 mm brass filter. Scans were reconstructed as TIFF stacks in NRecon (Bruker Corporation) and visualized in Dragonfly 2019 4.0 (Object Research Systems, Montreal, Canada). Raman spectra were collected at the Center of Nanoscale Systems (CNS) at Harvard University using a Horiba XploRA Plus confocal Raman microscope with a 532 nm laser and 600 grooves/mm diffraction grating and spectral resolution of 1.5 cm⁻¹. Raman spectra were obtained using a 10 s integration time and a 50x Olympus long working distance microscope objective, which focused the beam to a spot size of 1 mm. The spectrometer was calibrated using a silicon wafer to the 520.7 wavenumber Si-Si peak. Two specimens of Olenoides serratus from the Burgess Shale (Wuliuan, Cambrian, British Columbia) were photographed for comparative purposes to study the endopodite morphology—one deposited at the Geological Survey of Canada (GSC; Ottawa) and the other at the invertebrate paleontology collections at the Smithsonian Institution (USNM; Washington, D.C., USA). Data was visualized using R. Figures were produced in Adobe Photoshop CS and Adobe Illustrator CS.

Data availability

The data generated and analyzed during this study are available in the Dryad repository, [https://doi.org/10.5061/dryad.1jwstqk16].

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Acknowledgements

The authors are deeply grateful to Dan Cooper for generously donating material from the Walcott-Rust Quarry to the MCZ as part of this study; Jessica Cundiff (Museum of Comparative Zoology, Cambridge, USA) and Michelle Coyne (Geological Survey of Canada, Ottawa, Canada) for facilitating specimen access; Phoebe Cohen (Williams College, North Adams MA, USA) for facilitated access to the SEM; Nancy Piatczyc (Williams College, North Adams MA, USA) for assistance with the SEM; Arthur McClelland (Harvard University, Cambridge MA, USA) for assistance collecting Raman spectra. This work is supported by the Palaeontological Association Sylvester-Bradley Award, PA-SB202001 and is published by a grant from the Wetmore Colles fund. The constructive comments of three reviewers helped us improve the manuscript.

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Authors and Affiliations

Museum of Comparative Zoology and Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA, 02138, USA
Sarah R. Losso & Javier Ortega-Hernández
Department of Geological Sciences, New Mexico State University, Las Cruces, NM, 88003, USA
Jennifer E. Thines

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Sarah R. Losso
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Contributions

S.R.L. and J.O.H. designed the research project. S.R.L. selected specimens for donation and processed material. S.R.L. and J.E.T. prepared specimens for analyses. S.R.L. photographed specimens, collected SEM and Raman data, and produced figures. S.R.L. and J.E.T. analyzed the data. S.R.L. wrote the manuscript with input from J.E.T. and J.O.H. All authors reviewed the final manuscript and gave approval for publication.

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Correspondence to Sarah R. Losso or Javier Ortega-Hernández.

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Losso, S.R., Thines, J.E. & Ortega-Hernández, J. Taphonomy of non-biomineralized trilobite tissues preserved as calcite casts from the Ordovician Walcott-Rust Quarry, USA. Commun Earth Environ 4, 330 (2023). https://doi.org/10.1038/s43247-023-00981-5

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Received: 24 April 2023
Accepted: 29 August 2023
Published: 19 September 2023
DOI: https://doi.org/10.1038/s43247-023-00981-5

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