Soft tissues of P. douvillei

For a long time, the hypostome and labrum of trilobites could not be clearly distinguished. A hypostome is generally recognized as a fusion of an anterior sclerite and the labrum (Park, Reference Park2023) or as a hypostome–labrum complex (Ortega-Hernández, Reference Ortega-Hernández2014, Reference Ortega-Hernández2015; Ortega-Hernández et al., Reference Ortega-Hernández, Janssen and Budd2017; Liu et al., Reference Liu, Ortega-Hernández, Zhai and Hou2020). Since El Albani et al. (Reference El Albani, Mazurier, Edgecombe, Azizi and Bakhouch2024) described the intricate mouthparts of Protolenus (Hupeolenus) and G. mauretanica preserved in volcanic ash, the hypostome and labrum can be differentiated: the hypostome is a biomineralized hard tissue, while the labrum is composed of non-mineralized soft tissue. The micro-CT results of P. douvillei show an obvious arc-shaped structure attached to the posterior edge of the hypostome, which is interpreted as the labrum. It is similar to that of Protolenus (Hupeolenus) and G. mauretanica; however, the lobes and furrows cannot be clearly distinguished. The upper sclerite corresponds to the hypostome (Fig. 4.6–4.10), which accommodates the antennal insertion (Figs. 5.2–5.4, 8.9, 8.11, 8.12, 10, 11). Although neither the esophagus nor the mouth could be identified, it is hypothesized that they are situated near the posterior part of the hypostome and are covered by it (Lerosey-Aubril et al., Reference Lerosey-Aubril, Hegna, Kier, Bonino, Habersetzer and Carré2012; Budd, Reference Budd2021; El Albani et al., Reference El Albani, Mazurier, Edgecombe, Azizi and Bakhouch2024). This could represent the typical mouth structure of the Ellipsocephaloidea, and even Trilobita, since some species have similar structures in the same position, which have been interpreted as the metastoma or postoral plate (El Albani et al., Reference El Albani, Mazurier, Edgecombe, Azizi and Bakhouch2024).

Multifaceted evidence from developmental biology, molecular biology, and paleontology indicates that the labrum has an appendicular origin, evolving from the protocerebral appendages to the plate-like labrum, with its varying morphologies being a consequence of diverse predation strategies or environmental adaptations (Budd, Reference Budd2021). The structure with a separated labrum and hypostome is similar to that of chelicerates (El Albani et al., Reference El Albani, Mazurier, Edgecombe, Azizi and Bakhouch2024). However, the difference is that the labrum reduces in the adults of chelicerates and megacheirans, e.g. Leanchoilia illecebrosa Hou, Reference Hou1987 (Liu et al., Reference Liu, Ortega-Hernández, Zhai and Hou2020). This could potentially be an adaptive evolution for active predation using chelicerae or for crushing and transporting food via gnathobases. P. douvillei requires the support and protection of the hypostome, and the labrum may have helped the cephalic appendages and gnathobases to transport, masticate, sieve, and ingest food. Such soft tissues might even function as receptors to identify food sources and possibly the physical and chemical conditions of seawater.

In contrast to the labrum, which is present in only a very limited number of specimens, appendages are observed in several trilobites (Hou et al., Reference Hou, Clarkson, Yang, Zhang, Wu and Yuan2008; Hu et al., Reference Hu, Zhu, Luo, Steiner, Zhao, Li, Liu and Zhang2013; Hou et al., Reference Hou, Hughes, Yang, Lan, Zhang and Dominguez2016; Zeng et al., Reference Zeng, Zhao, Yin and Zhu2017; Holmes et al., Reference Holmes, Paterson and García-Bellido2019; Hou and Hopkins, Reference Hou and Hopkins2024; Zhu et al., Reference Zhu, Zeng, Sun, Liu and Zhao2024). A complete individual of P. douvillei possesses a pair of long flagelliform uniramous antennae, four pairs of post-antennal biramous appendages in the cephalic region, 13–15 pairs on the trunk, and at least two pairs on the pygidium (Figs. 2.1, 2.6, 4.3–4.5, 5.2–5.4, 6, 7.3–7.5, 8, 10, 11). The four pairs of post-antennal cephalic appendages are similar to those of trilobites such as T. eatoni, O. serratus, Protolenus (Hupeolenus), and G. mauretanica. In addition, the presence of five pairs of reniform cephalic digestive glands in P. douvillei, coupled with the interpretation of S4 as a non-segmental exoskeletal structure (Park and Kihm, Reference Park and Kihm2017), suggests a mismatch between the number of cephalic segments and appendages in P. douvillei, which is similar to T. eatoni and O. serratus (Hou and Hopkins, Reference Hou and Hopkins2024). The segmental mismatch phenomenon is also prevalent among Cambrian arthropods, including fuxianhuiids (Yang et al., Reference Yang, Ortega-Hernández, Butterfield and Zhang2013, Reference Yang, Ortega-Hernández, Legg, Lan, Hou and Zhang2018), euthycarcinoids (Vaccari et al., Reference Vaccari, Edgecombe and Escudero2004), S. lunaris (Chen et al., Reference Chen, Ortega-Hernández, Wolfe, Zhai, Hou, Chen, Mai and Liu2019), and P. tetranodusa (Luo and Liu, Reference Luo and Liu2024). Its occurrence has been attributed to multiple factors, such as the expression of Hox genes (Eriksson et al., Reference Eriksson, Tait, Budd, Janssen and Akam2010), the configuration of the post-cephalic central nervous system (Yang et al., Reference Yang, Ortega-Hernández, Butterfield and Zhang2013), and the fusion of appendage muscle attachment sites (Hou and Hopkins, Reference Hou and Hopkins2024). This widespread phenomenon highlights the evolutionary flexibility of early arthropods.

The appendage morphology of P. douvillei shares its basic patterns with other Cambrian trilobites (Hou et al., Reference Hou, Clarkson, Yang, Zhang, Wu and Yuan2008; Hou et al., Reference Hou, Hughes, Yang, Lan, Zhang and Dominguez2016; Zeng et al., Reference Zeng, Zhao, Yin and Zhu2017; Holmes et al., Reference Holmes, Paterson and García-Bellido2019; Losso and Ortega-Hernández, Reference Losso and Ortega-Hernández2022; Hou and Hopkins, Reference Hou and Hopkins2024; El Albani et al., Reference El Albani, Mazurier, Edgecombe, Azizi and Bakhouch2024). The endopods are characterized by slender spines and lack enlarged protopodites or gnathobases, differing from species such as R. rex (Holmes et al., Reference Holmes, Paterson and García-Bellido2019), H. yiliangensis (Zeng et al., Reference Zeng, Zhao, Yin and Zhu2017), and E. intermedia (Hou et al., Reference Hou, Clarkson, Yang, Zhang, Wu and Yuan2008), which are considered predators with the ability to crush hard shells (Bicknell et al., Reference Bicknell, Holmes, Edgecombe, Losso, Ortega-Hernández, Wroe and Paterson2021). By contrast, the endopod morphology of P. douvillei resembles that of O. serratus from the Burgess Shale (Losso and Ortega-Hernández, Reference Losso and Ortega-Hernández2022; Whittington, Reference Whittington1975; Fig. 10), suggesting a diet that may have included soft-bodied animals or detritus.

The function of trilobite exopods has long been debated. While some researchers have proposed mechanical functions (e.g., Suzuki and Bergström, Reference Suzuki and Bergstrom2008; Suzuki et al., Reference Suzuki, Kondo and Bergstrom2008), others, including Whittington (Reference Whittington1980), Bruton and Haas (Reference Bruton and Haas1999), Hou et al. (Reference Hou, Clarkson, Yang, Zhang, Wu and Yuan2008), and Siveter et al. (Reference Siveter, Fortey, Briggs, Siveter and Sutton2021), have interpreted them as primarily respiratory structures. In P. douvillei, the exopods bear lamellae. Although “dumbbell-shaped” filaments typically associated with respiration in other taxa (Hou et al., Reference Hou, Hughes and Hopkins2021) were not found in P. douvillei, it can be inferred that although the exopods’ range of motion was likely limited (Hou et al., Reference Hou, Hughes and Hopkins2021), the exopods were capable of slight movement to agitate water flow. This agitation would facilitate oxygen renewal over the lamellar surfaces. Given that the flat, thin lamellae provide a substantial surface area for water contact—fulfilling the primary structural requirement for gas exchange—we interpret the exopods of P. douvillei as respiratory organs.

While distinct appendage specialization is well documented in various Cambrian arthropods, such as Fuxianhuia xiaoshibaensis Yang et al., Reference Yang, Ortega-Hernández, Butterfield and Zhang2013, Alacaris mirabilis Yang et al., Reference Yang, Ortega-Hernández, Legg, Lan, Hou and Zhang2018, and Isoxys curvirostratus Vannier and Chen, Reference Vannier and Chen2000 (Zhang et al., Reference Zhang, Liu, Ortega-Hernández, Wolfe, Jin, Mai, Hou, Guo and Zhai2023), it has been rarely reported in trilobites. Notable exceptions include O. serratus, some of whose endopods (En 10 and 11) are substantially different from other thoracic appendages and thus interpreted as serving a reproductive function (Losso and Ortega-Hernández, Reference Losso and Ortega-Hernández2022), and Protolenus (Hupeolenus) and G. mauretanica, whose specialized appendages include the antenniform exopod on C1 and the rudimentary or absent endopods on C1 and C2 (El Albani et al., Reference El Albani, Mazurier, Edgecombe, Azizi and Bakhouch2024). Although definitive appendage specialization has not been established for P. douvillei, its appendages exhibit a clear degree of functional differentiation (Zhang et al., Reference Zhang, Liu, Ortega-Hernández, Wolfe, Jin, Mai, Hou, Guo and Zhai2023). The shorter cephalic appendages were likely adapted for grasping small prey or food particles and transporting them to the mouth. By contrast, the longer and more robust endopods of the anterior trunk probably provided primary support and stability during locomotion, enabling rapid directional adjustments, and the appendages on the posterior part of the trunk assisted in locomotion (Figs. 10, 11). By promoting the functional differentiation and regionalization of arthropods, this phenomenon enabled appendages—protected by a selectively fused exoskeleton—to enhance feeding efficiency and locomotor flexibility and even to develop specialized functions. This adaptive innovation laid the foundation for the adaptive evolution of arthropods, facilitating their adaptation to diverse ecological niches and predation strategies.

In addition to the featured five pairs of cephalic digestive glands, the digestive system of P. douvillei further comprises the gut, crop, and several pairs of thoracic digestive glands (Fig. 9). Our μ-XRF results show that the gut lacks Si and Al elements (Fig. 8), suggesting that P. douvillei may not be a detritivore or filter feeder (Hopkins et al., Reference Hopkins, Chen, Hu and Zhang2017). Micro-CT failed to reveal the gut contents of P. douvillei, as was possible with B. incola (Kraft et al., Reference Kraft, Vaškaninová, Mergl, Budil, Fatka and Ahlberg2023), but considering the complex digestive system of P. douvillei, the above-mentioned appendage characters, and its species abundance in the Guanshan Biota, it is inferred that the complex digestive system is a result of intense intra- and inter-specific competition, which allows P. douvillei to significantly enhance food utilization efficiency. Although the main digestive systems of trilobites are generally divided into two categories—one with a gut and paired digestive glands and the other with a “crop” and a gut but without paired digestive glands (Chatterton et al., Reference Chatterton, Johanson and Sutherland1994; Lerosey-Aubril et al., Reference Lerosey-Aubril, Hegna and Olive2011)—both types can exist simultaneously in P. douvillei (Figs. 2.5, 4.1, 10). Since some trilobites and modern arthropods possessing both digestive glands and crop have been reported (Hopkins et al., Reference Hopkins, Chen, Hu and Zhang2017; Sun et al., Reference Sun, Zeng and Zhao2021; El Albani et al., Reference El Albani, Mazurier, Edgecombe, Azizi and Bakhouch2024), the co-existence of the crop and digestive glands is common. Although multiple factors such as burial conditions, food type, food quantity, and the digestive stage at the time of death and burial can all influence the complete preservation of the digestive system (Hopkins et al., Reference Hopkins, Chen, Hu and Zhang2017), and the disappearance of digestive glands may take place during the evolution of the crop (Lerosey-Aubril and Peel, Reference Lerosey-Aubril and Peel2018), it can still be clearly ascertained from the existing reports that the digestive system of trilobites already exhibited complexity in the early Cambrian. This was not an isolated case but rather the result of early adaptive evolution.

Phylogenetic analysis

The soft-tissue characters, labrum, appendages, digestive system, and so on are of great significance in the study of trilobite evolution. Historically, the construction of phylogenetic trees has depended heavily on exoskeletal features (Paterson and Edgecombe, Reference Paterson and Edgecombe2006; Paterson et al., Reference Paterson, Edgecombe and Lee2019). As the number of reported cases of soft-tissue preservation in trilobites grows, incorporating these data into phylogenetic analyses becomes increasingly important. When we added soft-tissue characters to the exoskeletal character set of Paterson et al. (Reference Paterson, Edgecombe and Lee2019), P. douvillei was not only recovered within the Palaeolenidae, but this placement also received strong statistical support (Figs. 12, S2–S4).

Figure 12.

Dated time-tree of Cambrian trilobites inferred from tip-dated Bayesian analyses of discrete, meristic, and continuous traits under a strict clock. Node bars indicate 95% highest posterior density intervals for divergence dates; node circles indicate posterior support for clade. Line drawings are from www.trilobites.info.

Figure 12. Long description

The diagram is a horizontal time-tree spanning from 538.8 to 486.85 million years ago, divided into Terreneuvian, Series 2, Miaolingian, and Furongian intervals. The vertical axis lists trilobite taxa, each at the tip of a branching structure. Branches split from left to right, indicating evolutionary divergence. Blue node bars represent 95 percent highest posterior density intervals for divergence dates, with blue circles at nodes indicating posterior support (darker blue means higher support). The bottom left legend explains these visual encodings. Several clades are highlighted, including Palaeolenidae in red with a corresponding trilobite illustration. Other line drawings are distributed to the right of major taxa, visually linking to their names. Key taxa such as Fallotaspis bondoni, Pagetia ocellata, Monakaspis draboviensis, and Corynexochus plumula are shown with their respective illustrations. The tree structure reveals the timing and relationships of major trilobite groups, with denser branching in Series 2 and Miaolingian. All taxon names and geological boundaries are labeled precisely as in the source.

The inclusion of these new characters and taxa did not significantly alter the overall topology of the dated-time trees compared with those trees inferred by Paterson et al. (Reference Paterson, Edgecombe and Lee2019). Major clades remained stable, with modifications occurring only in previously uncertain branches (Figs. 12, S2–S4). Furthermore, the estimated evolutionary rates for discrete and meristic characters (~0.010 and ~0.70, respectively) are highly consistent with previous research (Paterson et al., Reference Paterson, Edgecombe and Lee2019). These results reinforce the hypothesis that the Cambrian explosion represents a distinct, rapid evolutionary burst initiated in the Terreneuvian and largely concluding by Series 2 (Bowring et al., Reference Bowring, Grotzinger, Isachsen, Knoll, Pelechaty and Kolosov1993; Mángano and Buatois, Reference Mángano and Buatois2014; Na and Kiessling, Reference Na and Kiessling2015; Budd and Jackson, Reference Budd and Jackson2016; Paterson et al., Reference Paterson, Edgecombe and Lee2019), followed by a period of relatively constant morphological evolutionary rates in trilobites.

Bayesian analysis of the morphological dataset indicates that, irrespective of the clock model used, the clade comprising Palaeolenus, Gigoutella, and Megapalaeolenus forms a monophyletic group (e.g., Fig. 12, posterior probability PP = 0.99), with an estimated origin at approximately 517.9 Ma (95% HPD: 520–516.4 Ma). It should be noted, however, that this inference of monophyly is preliminary and requires verification through more comprehensive taxon sampling in future studies.

Within the family Palaeolenidae, the earliest divergence occurred at ~518 Ma, splitting P. douvillei from the other lineages in the family (Figs. 12, S2–S4). G. mauretanica is distinguished primarily by robust gnathobases and enlarged pleural spines on the second thoracic segment—a trait exhibited to an even greater extreme in P. longispinus Mansuy, Reference Mansuy1912, which displays pronounced spines both pleurally and genally. The resulting topology demonstrates that the traditional genus Palaeolenus is paraphyletic. Consequently, the key morphological features shared by P. longispinus and G. mauretanica (e.g., enlargement of the second thoracic segment) are likely the result of convergent evolution. Conversely, the unspecialized, large pleural spines observed in M. deprati may represent a secondary loss from their most recent common ancestor (shared with P. longispinus) or a retention of the ancestral state.

Given that M. deprati possesses a significantly enlarged frontal glabellar lobe and a smooth axial lobe lacking nodes, we concur with Zhao et al. (Reference Zhao, Liu and Bicknell2020) that P. douvillei and M. deprati should be treated as distinct species, countering earlier classifications that synonymized them (Lin and Peng, Reference Lin and Peng2004; Lin, Reference Lin2014; Luo et al., Reference Luo, Hu, Chen, Zhan and Lu2014; Lin and Peng, Reference Lin and Peng2016). Collectively, these data indicate that phenotypic similarity does not necessarily equate to close phylogenetic relationship. The evolutionary history of the Palaeolenidae reveals that, following the main phase of the Cambrian Explosion, this group continued to undergo morphological innovation and lineage differentiation at a relatively constant rate.