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Fuxianhuiid ventral nerve cord and early nervous system evolution in Panarthropoda Jie Yanga, Javier Ortega-Hernándezb,1, Nicholas J. Butterfieldb, Yu Liua,c,d, George S. Boyanc, Jin-bo Houa, Tian Lane, and Xi-guang Zhanga,2 a Yunnan Key Laboratory for Paleobiology, Yunnan University, Kunming 650091, China; bDepartment of Earth Sciences, University of Cambridge, Cambridge CB2 3EQ, United Kingdom; cDevelopmental Neurobiology Group, Biocenter, Ludwig-Maximilians-Universität, 82152 Martinsried, Germany; dGeoBio-Center Ludwig-Maximilians-Universität, Munich 80333, Germany; and eCollege of Resources and Environmental Engineering, Guizhou University, Guiyang 550003, China

Panarthropods are typified by disparate grades of neurological organization reflecting a complex evolutionary history. The fossil record offers a unique opportunity to reconstruct early character evolution of the nervous system via exceptional preservation in extinct representatives. Here we describe the neurological architecture of the ventral nerve cord (VNC) in the upper-stem group euarthropod Chengjiangocaris kunmingensis from the early Cambrian Xiaoshiba Lagerstätte (South China). The VNC of C. kunmingensis comprises a homonymous series of condensed ganglia that extend throughout the body, each associated with a pair of biramous limbs. Submillimetric preservation reveals numerous segmental and intersegmental nerve roots emerging from both sides of the VNC, which correspond topologically to the peripheral nerves of extant Priapulida and Onychophora. The fuxianhuiid VNC indicates that ancestral neurological features of Ecdysozoa persisted into derived members of stem-group Euarthropoda but were later lost in crown-group representatives. These findings illuminate the VNC ground pattern in Panarthropoda and suggest the independent secondary loss of cycloneuralian-like neurological characters in Tardigrada and Euarthropoda.

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stem-group Euarthropoda Onychophora Explosion Xiaoshiba Lagerstätte

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feature as the VNC. This interpretation is supported by comparisons with other preserved components of the internal anatomy. For instance, the VNC can be readily distinguished from the digestive tract of C. kunmingensis, which is expressed as a comparatively larger (maximum width, ∼860 μm) but fully compressed, linear structure running almost the entire length of the animal (23, figure 1 d and e). The VNC extends from at least the five anteriormost reduced trunk tergites (i.e., dorsal exoskeletal plates) to tergite T23 at the posterior end of the trunk (Figs. 1 and 2 and Figs. S1, S2, and S3). Although the VNC would have continued into the head region in life, our material does not preserve any anterior neurological structures that have been previously identified as the dorsal brain or nerves leading to the antennae and specialized postantennal appendages (8). The absence of fossilized brains in these otherwise exceptionally preserved specimens can be attributed to the small sample size and moderate postmortem disarticulation; likewise, other studies addressing neurological structures in Cambrian fossils rarely report the CNS preserved in its entirety (8–11). The VNC also expresses a distinctive dark/light color banding throughout its length, with the dark bands varying from black to reddish-brown Significance

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he nervous system represents a critical source of phylogenetic information and has been used extensively for exploring the evolutionary relationships of extant Panarthropoda (i.e., Onychophora, Tardigrada, Euarthropoda) (1–7). Identification of fossilized nervous tissues has provided a unique perspective on early euarthropod brain neuroanatomy and suggests that broad patterns of extant neurological diversity were already in place by the Cambrian (8–11). The ventral nerve cord (VNC) reflects fundamental aspects of panarthropod body organization that complement the organization of the brain and together illuminate the evolution of the CNS (1–3, 5, 7, 12–16). The early evolutionary history of the panarthropod postcephalic CNS, however, remains obscure due to the exclusive preservation of brains in most available fossils (8, 10, 11). Moreover, the unresolved phylogenetic relationships within Panarthropoda complicate accurate reconstruction of the CNS ground pattern (16–22). In this study, we demonstrate the exceptional preservation of postcephalic neurological features in the early Cambrian fuxianhuiid Chengjiangocaris kunmingensis, an upper stem-group euarthropod (17) from the Xiaoshiba Lagerstätte, South China (23). These fossils clarify the neurological organization of the VNC in early euarthropod ancestors, thereby polarizing the evolution of the panarthropod CNS. Results Five individuals of C. kunmingensis display a narrow (maximum width, ∼170 μm) and slightly convex rope-like structure with a metameric pattern that extends medially throughout the body (Figs. 1 and 2, Figs. S1 and S2, and Table S1); its segmental organization and position at the ventral midline identifies this www.pnas.org/cgi/doi/10.1073/pnas.1522434113

Understanding the evolution of the CNS is fundamental for resolving the phylogenetic relationships within Panarthropoda (Euarthropoda, Tardigrada, Onychophora). The ground pattern of the panarthropod CNS remains elusive, however, as there is uncertainty on which neurological characters can be regarded as ancestral among extant phyla. Here we describe the ventral nerve cord (VNC) in Chengjiangocaris kunmingensis, an early Cambrian euarthropod from South China. The VNC reveals extraordinary detail, including condensed ganglia and regularly spaced nerve roots that correspond topologically to the peripheral nerves of Priapulida and Onychophora. Our findings demonstrate the persistence of ancestral neurological features of Ecdysozoa in early euarthropods and help to reconstruct the VNC ground pattern in Panarthropoda. Author contributions: J.Y., N.J.B., and X.-g.Z. designed research; J.Y., J.O.-H., N.J.B., Y.L., J.-b.H., T.L., and X.-g.Z. performed research; Y.L. and G.S.B. contributed new reagents/ analytic tools; J.O.-H. analyzed data; J.O.-H. and N.J.B. wrote the paper; J.Y. collected and prepared all the fossil material; J.O.-H. performed light photography; N.J.B. and X.-g.Z. discussed and approved the manuscript; Y.L. and G.S.B. performed immunohistochemistry and living animal microscopy; and J.-b.H. and T.L. collected fossil material and performed photography. The authors declare no conflict of interest. This article is a PNAS Direct Submission. G.D.E. is a guest editor invited by the Editorial Board. 1

Present address: Department of Zoology, University of Cambridge, Cambridge CB2 3EJ, United Kingdom.

2

To whom correspondence should be addressed. Email: [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1522434113/-/DCSupplemental.

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Edited by Gregory D. Edgecombe, The Natural History Museum, London, United Kingdom, and accepted by the Editorial Board January 29, 2016 (received for review November 14, 2015)

Fig. 1. VNC in C. kunmingensis. Anterior at top. (A) YKLP 12023 (Holotype), complete specimen preserved in dorsal view with taphonomically dissected head shield (hs) showing internal organization of anterior region. (B) YKLP 12324, complete specimen preserved in dorsolateral view showing preserved VNC extending throughout the almost the entire length of the body. (C) YKLP 12324, magnification of VNC on the anterior trunk region showing the differential preservation of the condensed ganglia (ga) and longitudinal connectives (cn) as dark and light colored bands, respectively, and one-to-one correspondence between the ganglia and the walking legs (wl) (upper box in B). (D) YKLP 12024, magnification of the VNC on the posterior trunk region showing the progressive reduction of size of the ganglia and connectives toward the rear end of the body (lower box in B). Dotted lines highlight the anterior and posterior tergal borders of T15 for comparative purposes with the number of preserved ganglia. ant, antennae; SPA, specialized postantennal appendage; Tn, trunk tergites.

between specimens (Figs. 1 and 2 and Figs. S1 and S2C); this variability in color reflects differences in the extent of weathering between individual specimens. Raman spectroscopy indicates the presence of residual organic carbon associated with the darkcolored bands (Fig. S4) but not in the light ones, likely mirroring differences in their original histology and early diagenesis. In addition to the carbonaceous films, the VNC is typified by modest relief relative to adjacent exoskeletal features, attesting to a degree of early diagenetic permineralization before complete degradational collapse (24). Neurologically, the C. kunmingensis VNC consists of an interconnected series of separate metameric ganglia (dark colored bands) (Figs. 1 and 2 and Figs. S1 and S3). Longitudinal connectives are not morphologically discrete, but their presence is strongly suggested by the alternating light colored bands between the condensed ganglia. The distinct preservation style of these neurological structures likely stems from differences in their original histology; whereas the condensed ganglia are enriched in lipids due to the presence of abundant somata, the connectives consist of neurites and lack cell bodies (1, 3–5). The ganglia have an elongate subelliptic outline (maximum length, ∼600 μm; 3.5:1 length/width) and are roughly three times longer than the connectives (maximum length, ∼206 μm). The ganglia 2 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1522434113

remain separate throughout the entire VNC, with no indication of fusion or specialization. There is a progressive reduction in the size of individual ganglia along the body, with the anteriormost being ∼3 times longer and 1.5 times wider than the posterior ones (Fig. 1 C and D). The preserved proximal portions of the trunk endopods indicate that each ganglion was associated with a single pair of biramous appendages (Fig. 1C and Fig. S2 A and B), which, like the ganglia, become progressively smaller posteriorly (Fig. 1D). As such, each of the five anteriormost reduced tergites (Fig. 1A) correlates with an individual ganglion and leg pair, whereas the comparatively larger tergites T6–T26 overlie up to four ganglia and their corresponding appendages (Fig. 1D and Figs. S2 A and B and S3). This organization reflects the notable dorsoventral segmental mismatch that typifies the fuxianhuiid trunk (23, 25). The unique disarticulation pattern of the head shield in the Xiaoshiba fuxianhuiids (23)—aided by mechanical preparation— reveals submillimetric neurological detail in C. kunmingensis. In specimen YKLP 12026, the exposed VNC extends from the oral region into tergite T6, comprising a total of seven ganglia (Fig. 2 A–C and Fig. S1). Close inspection reveals the presence of delicate nerve roots (maximum length, ∼209 μm; width, 9 μm) emerging at both sides of the VNC. These nerve roots are Yang et al.

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Fig. 2. Fine neurological organization of the VNC in C. kunmingensis, YKLP 12026. Anterior is to the left. (A) Completely articulated specimen preserved in laterodorsal orientation with displaced head shield exposing VNC on anterior trunk region. (B) VNC showing the presence of seven sets of condensed ganglia (ga) linked by longitudinal connectives (cn) (box in A). (C) Composite fluorescence photograph of VNC (box in A). (D) Magnification of the VNC (box in B) showing regularly spaced peripheral nerve roots (arrowheads) emerging from the condensed ganglia and connective. (E) Composite fluorescence photograph magnification of the VNC (box in C).

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regularly spaced (∼17–20 μm) and originate from both the ganglia (i.e., segmental) and interganglia connectives (i.e., intersegmental) at acute angles (∼40–60°) relative to the VNC (Fig. 2 C–E and Figs. S1 B and C and S3). The emergent chevron-like pattern most likely resulted from moderate postmortem displacement of the distal portions of the peripheral nerves relative to the VNC. There are at least a dozen paired sets of roots associated with each ganglion and four to six nerve roots associated with the longitudinal connectives. Preferential preservation of the proximal bases of the nerve roots precludes the identification of precise relationships between these structures and the exoskeletal morphology, such as the walking legs. The lack of distal neurological preservation suggests that, similarly to 3D guts in Burgess Shale fossils (24), the VNC and the proximal nerve roots were permineralized during early diagenesis due to the chemically reactive composition of adjacent lipid-rich ganglia (8). Fluorescence microscopy corroborates the anatomical and compositional continuity of the regularly spaced nerve roots and VNC throughout the body (Fig. 2 C and E and Dataset S1). Discussion The Xiaoshiba fossils provide previously unidentified insights on the neurological diversity of Cambrian euarthropods. Within the

context of extant representatives, the postcephalic CNS of C. kunmingensis exhibits notable similarities with the thoracic VNC of the notostracan Triops cancriformis, which consists of a homonymous series of separate ganglia linked by short connectives that become progressively smaller toward the posterior end (1, 3). The overall morphology of T. cancriformis further resembles C. kunmingensis—and generally other fuxianhuiids—in the presence of dorsoventral trunk segmental mismatch expressed as appendage polypody (23, 25, 26). A similarly unspecialized VNC is also found in crustaceans typified by a trunk region with undifferentiated limb-bearing segments, such as the remipedes (5, 7) (Fig. S5C). Unlike C. kunmingensis and T. cancriformis, however, the body organization of remipedes does not evince any type of trunk segmental mismatch. Thus, the paleoneurological data available indicate that the fuxianhuiid CNS combines a relatively simple VNC without signs of functional specialization (Figs. 1–3) with a tripartite brain bearing both olfactory and optic lobes (8). Considering the position of fuxianhuiids within upper-stem Euarthropoda (17), this character combination could suggest the evolutionary convergence of either the VNC of Chengjiangocaris with Branchiopoda or the complex brain of Fuxianhuia with Malacostraca. Alternatively, the fuxianhuiid CNS may approximate the complex brain (sans

Fig. 3. Simplified cladogram showing the evolution of the postcephalic CNS in Panarthropoda. Detailed results of the phylogenetic analysis are provided in Fig. S6 and SI Text. The topology supports a single origin for the condensed ganglia (ga) in the VNC in a clade including Tardigrada and Euarthropoda; note that the presence of multiple intersegmental peripheral nerves (ipn) in C. kunmingensis represents an ancestral condition. Given the morphological similarity between peripheral and leg nerve roots, the presence of a single pair of leg nerves (lgn) in C. kunmingensis is hypothetical (dashed lines) and based on the condition observed in crown-group Euarthropoda. †, fossil taxa; ?, uncertain character polarity within total-group Euarthropoda. asn, anterior segmental nerve; cn, longitudinal connectives; co, commissure; dln, dorsolateral longitudinal nerve; ico, interpedal median commissure; irc, incomplete ring commissure; pn, peripheral nerve; psn, posterior segmental nerve; rc, ring commissure. Reconstruction of VNC in Onychophora adapted from ref. 13.

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1. Harzsch S, Waloszek D (2000) Serotonin-immunoreactive neurons in the ventral nerve cord of Crustacea: A character to study aspects of arthropod phylogeny. Arthropod Struct Dev 29(4):307–322. 2. Harzsch S, Wildt M, Battelle B, Waloszek D (2005) Immunohistochemical localization of neurotransmitters in the nervous system of larval Limulus polyphemus (Chelicerata,

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(31) and Onychophora (12, 13, 15, 29) but are otherwise greatly reduced in number or completely lost in crown-group Euarthropoda and Tardigrada, respectively (1, 5, 16, 28) (Fig. S5); the secondary reduction/loss is recognized as a result of parallel evolution between these lineages. The phylogenetic analysis also indicates that several neurological characters shared between Onychophora and Tardigrada are actually symplesiomorphic and likely reflect the broader ground pattern of Panarthropoda (Fig. 3); these include the median interpedal commissures, an orthogonallike organization, and paired segmental leg nerves (SI Text). A lateralized VNC is resolved as synapomorphic for Onychophora (SI Text). Taken together, these findings argue against the secondary loss of morphologically discrete segmental ganglia in the VNC of Onychophora (30). By contrast, the evolution of the VNC in crown-group Euarthropoda is the result of secondary simplification relative to the CNS of cycloneuralians and other panarthropods, as informed by the transitional neurological organization of C. kunmingensis (Fig. 3). The integration of paleoneurological data with the present understanding of the developmental biology of extant groups illuminates major issues pertaining the evolution of the nervous system in Panarthropoda. Our results indicate that the ground pattern of the panarthropod postcephalic CNS incorporates a paired, but not lateralized, VNC with median interpedal commissures, an orthogonal organization with complete ring-commissures, paired leg nerves, and intersegmental peripheral nerves (SI Text); in this context, an orthogonal VNC with complete ring commissures is a symplesiomorphy inherited from a cycloneuralian-like ancestor. The presence of condensed segmental ganglia, a lateralized organization, anteriorly displaced leg nerves following the parasegments, and a stomatogastric ganglion (SI Text) (Fig. 3), represent derived features that were acquired later in the evolutionary history of the different panarthropod phyla. Methods All of the described material is deposited at the Key Laboratory for Paleobiology, Yunnan University, Kunming, China (YKLP). Imaging. Fossils were photographed with a Nikon D3X fitted with a Nikon AF-S Micro Nikkor 105-mm lens. For close-up images, a LEICA M205-C stereomicroscope fitted with a Leica DFC 500 digital camera was used under directional illumination provided by a LEICA LED5000 MCITM. Geochemical analyses were performed with an inVia Raman microscope (Renishaw). Fluorescence photography was performed as described in ref. 32. Phylogenetic Analysis. The data matrix includes 50 taxa and 95 characters (Dataset S2 and SI Text). The analysis was run in TNT (33) under New Technology Search, using Driven Search with Sectorial Search, Ratchet, Drift, and Tree fusing options activated in standard settings (34, 35). The analysis was set to find the minimum tree length 100 times and to collapse trees after each search. All characters were treated as unordered. For an initial analysis, all characters were treated as equally weighted (Fig. S6A); subsequent repetitions with variable concavity values (k) were used to explore the effect of different degrees of homoplasy penalization to test the robustness of the dataset (Fig. S6 B–D) (36). ACKNOWLEDGMENTS. We thank K.-S. Du and J.-F. He for assistance with fossil collection. B. J. Eriksson (University of Vienna), G. Mayer (University of Leipzig), and G. Bicker (University of Veterinary Medicine Hannover) generously contributed photographic material for Fig. S5. This work was supported by National Natural Science Foundation of China (NSFC) Grants 41472022 and U1402232 (to J.Y. and X.-g.Z.), a research fellowship at Emmanuel College and a Herchel Smith fellowship (both University of Cambridge; to J.O.-H.), and a Ludwig Maximilians Universität München excellent Junior Researcher fund and NSFC Grant 41528202 (to Y.L.).

Xiphosura): Evidence for a conserved protocerebral architecture in Euarthropoda. Arthropod Struct Dev 34(3):327–342. 3. Fritsch M, Richter S (2010) The formation of the nervous system during larval development in Triops cancriformis (Bosc) (crustacea, Branchiopoda): An immunohistochemical survey. J Morphol 271(12):1457–1481.

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optic lobes) and unspecialized VNC of remipedes (5, 7). If the neurological organization of fuxianhuiids reflects an ancestral condition as suggested by their phylogenetic position, it would support the symplesiomorphic nature of a malacostracan-like brain for Pancrustacea/Tetraconata (6, 8) and also imply an independent increase in VNC complexity among extant crustacean lineages. However, the lack of consensus pertaining to the phylogenetic placement of Branchiopoda, Remipedia, and Malacostraca relative to Hexapoda based on molecular and neuromophological data (5–7, 27) complicates deciding between these hypotheses based on the available paleoneurological information. The neurological organization of the fuxianhuiid VNC leads to more informative comparisons with other panarthropod groups. Apart from Euarthropoda (1–5, 9), the presence of segmental ganglia is also characteristic of the CNS in Tardigrada (18, 28) (Fig. S5B) and has been used to advocate the sister-group relationship between these clades (16, 17, 20). Thus, the presence of segmental ganglia in C. kunmingensis provides a minimum phylogenetic threshold for the evolution of this feature in totalgroup Euarthropoda (SI Text). At a broader phylogenetic level, the neurological organization of C. kunmingensis also exhibits marked similarities with the VNC of Onychophora (Fig. 3 and Fig. S5A), namely the presence of numerous regularly spaced peripheral nerve roots (12–15, 29, 30). A major difference, however, is that the onychophoran postcephalic CNS is lateralized and lacks morphologically discrete segmental ganglia despite clearly having a segmented organization (SI Text). Although the proximal preservation of the peripheral nerves makes it uncertain whether the C. kunmingensis VNC possessed complete ring commissures like those in onychophorans (12–14), this feature could potentially add morphological support for the sister-group relationship between Euarthropoda and Onychophora suggested by molecular phylogenies (21, 22). Among crown-group euarthropods, there are only a restricted number of intersegmental peripheral nerves emerging from the longitudinal connectives in the VNC of some pancrustaceans (1, 4, 5) (SI Text, Fig. 3, and Fig. S5 C and D). Alternatively, it is possible that the presence of numerous peripheral nerves in the VNC of C. kunmingensis and Onychophora is symplesiomorphic, as comparable structures are also found in the orthogonal CNS of Priapulida (31) and other protostomes (12, 13, 15). In any event, the postcephalic CNS of C. kunmingensis reveals a unique combination of neurological characters otherwise unknown in any extant group within Panarthropoda (Fig. S3). To test these different hypotheses and to clarify the polarity of neurological characters in extant and extinct groups, we performed a comprehensive phylogenetic analysis incorporating the available fossil data on the postcephalic CNS of total-group Panarthropoda (SI Text and Dataset S2). The results support the sister-group relationship between Tardigrada and Euarthropoda (Fig. 3 and Figs. S6 and S7), corroborated by several unambiguous synapomorphies of the VNC. These synapomorphies include the presence of condensed segmental ganglia connected by commissures, the anterior shift of the leg nerves following a parasegmental organization, and the presence of a stomatogastric ganglion associated with the tritocerebral segment (SI Text) (14, 16, 18, 19, 28). In this context, the presence of numerous segmental and intersegmental peripheral nerves in C. kunmingensis (Fig. 2 B–E) is recognized as an ancestral condition that persisted in derived members of upper stem-group Euarthropoda (SI Text). Among extant groups, homologs of the intersegmental peripheral nerves are expressed in both Priapulida

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