Brain anatomy of the Cambrian fossil Jianfengia multisegmentalis informs euarthropod phylogeny

Domain: earth_science
Published: 2025-08-28
Editorial outcome: minor_revision

Abstract
Cambrian fossils from the Chengjiang biota demonstrate that over half a billion years ago early stem euarthropods existed coevally with representatives of already recognizable crown groups. Prominent stem taxa were Fuxianhuia protensa and Alalcomenaeus whose cerebral and ganglionic traits identify them as, respectively, stem mandibulates and stem chelicerates. Here we report on the visual systems and brain of the enigmatic lower Cambrian euarthropod Jianfengia multisegmentalis, which reveals neural traits suggestive of Pancrustacea despite its possession of ‘great appendages’. As occur in pancrustaceans, three nested optic neuropils are resolved in the eyestalks of Jianfengia, together with rostral ocelli and their associated nerves supplying a discrete forebrain region. Sutured eyestalks typifying crown Malacostraca provide compound eyes populated by ommatidia revealing structures suggesting cone-building cells. These and other neuroanatomical traits provide a powerful tool for resolving euarthropod relationships. Phylogenetic analyses deploying neural traits of Jianfengia, other Cambrian taxa, and extant Euarthropoda elucidate the status of Jianfengia as sister to total Mandibulata and reveal the short-bodied ‘great appendage’ Leanchoiliidae as sister to total Chelicerata. Together these data provide independent evidence for a 23 year-old proposition that ‘great appendage’ morphology defines the early stem from which derived the two branches of the euarthropod tree of life.

Introduction
FossilsoflowerCambrianstemeuarthropodsexhibitneuromorphological characters that typify the cerebra of crown Euarthropoda1–3. Examplesincludetheorganizationoftheoptictractsandneuropils subtending the compound eyes of Fuxianhuia protensa that correspond to visual centers defining certain mandibulates. Organization of the fuxianhuiid deutocerebrum supplying nerves to its antenniform appendages corresponds to mandibulate olfactory lobes and antennules. The cerebrum and visual pathways of the megacheiran Alalcomenaeus further reveal a cerebral organization corresponding to that of larval Limulus (Merostomata)2. Such distinctions relate directly to variations of neural organization within each of the three domains of the cerebrum that in extant euarthropods are genetically determined by the combinatorial activity of conserved homeobox transcription factors, thereby indicating their ancient origin4. Specific phenotypic distinctions of the internal organization of each cerebral domain further discriminate the euarthropod clades Chelicerata, Arachnida, and Myriapoda. Unrecognized until now are fossilized species that possessed cerebral traits corresponding to those that contribute to defining Pancrustacea, today’s most species-rich panarthropod group5.

Here we describe observations of six specimens of the genus Jianfengia multisegmentalis (Hou 1987) retrieved from the Cambrian (Series 2, Stage 3) Eoredlichia–Wutingaspis trilobite biozone, Yu’anshan Member, Chiungchussu Formation6. Jianfengia is a minute ‘great appendage’7 euarthropod (Fig. 1a), significantly smaller than other fossil taxa in which well-defined neural traces have been identified. To put this in perspective, the cerebrum of the mandibulate stem taxon Fuxianhuia protensa1 is at least three times broader than the cerebrum of Jianfengia. Accordingly, neural traces in Jianfengia are themselves microscopic and are in some specimens indicated by dense granular deposits rather than continuous dark profiles. We have here deployed strategies that utilize granularity to provide clear evidence of fossilized neuropil thereby providing empirical support for a more traditional approach of neuromorphological reconstruction. Together with phylogenetic analyses, our findings identify Jianfengia as a stem euarthropod sister to total Mandibulata exhibiting cerebral traits typifying Malacostraca and Branchiopoda that distinguish it from a cerebral organization defining the ‘great appendage’ Leanchoiliidae2,3.

Results
External attributes and diagnostic traits
Two Jianfengia specimens of similar size, YKLP11117 (Fig. 1a–e) and NIGPAS100123b (Supplementary Fig. 1), provide views of the external morphology of this species, with an emphasis on its rostral carapace and its associated skeletal attributes. Jianfengia multisegmentalis has a segmented trunk, approximately 2.5cm in length comprising 27-28 homonomous segments (Fig.1a). A rostral carapace 5mm in length and barely 3mm in width cover the cerebrum and the first three post-cerebral segments (Supplementary Table 1). Paired eyestalks extend laterally from the carapace immediately in front of a pair of uniramous postocular ‘great appendages’ that originate from beneath the carapace (Supplementary Fig. 1a–c). Each ‘great appendage’ comprises six podomeres of which the second and third provide an elbow-like articulation7. The elongated shaft of the third podomere terminates as three articulating blades that together furnish a stubby chela. These appendicular attributes conform to the general category of ‘great appendages’ ascribed to members of the paraphyletic clade Megacheira (Supplementary Table. 2), the significance of which is discussed later.

Rostrally, a pair of protrusions extending from the front of the carapace flank a substantial forward-projecting anterior sclerite, which arises from beneath the edge of the carapace with which it likely articulates (Figs. 1b, 2a–c). Despite considerable flattening typical of Chengjiang fossils, features of the anterior sclerite indicate that it is capped by three areas we interpret as ocellar-like lenses comparable to those described for the anterior sclerites of the deuteropodian Odaraia alata8, thereby corresponding to frontal eyes of trilobites9 and the nauplius eye/ocelli of extant pancrustaceans10,11. As shown in Fig. 2a-c, these putative ocelli overlie a palisade of rod-like components here interpreted as photoreceptors at the base of the anterior sclerite. Superimposition of neural traces from the anterior sclerite of Jianfengia specimen YKLP17299 onto the corresponding exoskeleton of YKLP11117 (Fig. 2d) further supports the presence of ocelli represented by a system of diverging axons from the anterior sclerite. These extend into the most rostral neuropil of the brain in a manner identical to axonal projections from a pancrustacean’s ocelli into its rostral cerebrum10,11.

Proven by developmental genetics of extant pancrustaceans, the combinatorial activity of Six3, FoxQ2 and hbn gene homologs is required for the development of rostral ocellar photoreceptors whose axons project into the prosocerebrum of the forebrain12–14. The corresponding organization in Jianfengia of axonal projections from the anterior sclerite (Fig. 2d) thus identifies the prosocerebral domain of its cerebrum. Conspicuously absent in extant Myriapoda (and in Fuxianhuiidae), the nauplius/ocellar visual system therefore appears to be restricted to certain lineages of Artiopoda8, Trilobita9, and Pancrustacea10,11. Jianfengia further possesses compound eyes that crown each of the paired eyestalks extending laterally from beneath the carapace (Fig. 1b–e). This organization corresponds to eyestalk morphology typifying eumalacostracan crustaceans15 and other members of Jianfengiidae16 whose eyestalks evince two articles connected by a suture (Fig. 1c–f). In extant taxa, the combinatorial activity of Six3, Otx and Pax6 defines the protocerebral domain of the pancrustacean forebrain12,17 and its nested optic neuropils, which receive information from the eye’s ommatidial array18. Compound eyes on stalks and the areas to which their associated fossilized optic neuropils project thus indicate the protocerebral domain of the jianfengiid cerebrum.

Reconstructing the brain of Jianfengia
Our initial procedure for reconstructing the cerebrum of Jianfengia (Fig. 3) followed a routine used previously on the large fuxianhuiid cerebrum where incomplete neural traces from several specimens were mapped as mirror symmetric profiles within an envelope representing the bilaterally symmetric fuxianhuiid head shield1. For Jianfengia, the neural traces, manifested as blue-black residues or as clusters of grey to near-black granules, were mapped onto a mirror-symmetric envelope for each of four specimens. The initial envelope was defined by the outline of the right frontal carapace and eyestalk of specimen YKLP11117 (from Fig. 1b). Flipping a copy of the outline across the animal’s midline provided the mirror symmetric envelope, as shown in Fig. 3b. The envelope was correspondingly adjusted to map neural traces in specimens that showed evidence of taphonomic misalignments, such as lateral displacements of the eyestalks or medial areas (e.g. specimen YKLP1367: Fig. 3c). These adjustments facilitate mapping of neural traces irrespective of such distortions, as shown for YKLP11368 (Fig. 3d). Likewise, specimen YKLP11369, in which only half the frontal volume could be retrieved, provided neural traces that mapped into the eyestalk and laterally alongside the esophageal foramen (Fig. 3e). To obtain the final reconstruction, the envelopes and their traces of each specimen were readjusted and registered in the summary envelope as shown in Fig. 3g. Finally, each tracing in one half of the envelope was mirrored in the other (Fig. 3h). The symmetrical profile from each of the four specimens was then filled and made 33% opaque. All four profiles were superimposed to provide a summed reconstruction of the Jianfengia cerebrum and its connection to the first three contiguous trunk ganglia T1 – T3 (Fig. 3h).

With manual tracing, however, it is impossible to exclude anticipatory bias19. To meet this challenge, we generated a second reconstruction using a minimum of human intervention. Two specimens were selected, YKLP11367 and YKLP17299, both of which provide evidence of bilaterally preserved neuropils and tracts. Unlike specimen YKLP17299, whose mirror-symmetric neural traces suggest it is preserved flat (Fig. 3o), the asymmetric disposition of traces in specimen YKLP11367 (Fig. 3i) indicates a slight taphonomic rotation around the fossil’s antero-posterior axis. Assuming that Jianfengia was a member of Bilateria, then these left and right traces in YKLP 11367 each represents a different depth within the specimen and are present on the contralateral side. These neural traces contribute to a mirror-symmetric organization as do the reflected outlines obtained by tracing. Reconstitution required four actions: the elimination of structures beneath a defined gray level (here 87% black of the CMYK scale), thus retaining the darkest puncta; inversion of the image to provide white profiles on a black background followed by the imposition of a Gaussian blur function (here radius expansion R=10px) that expands and runs together puncta (compare Fig. 3j, k). To counteract asymmetry around the midline (Fig. 3k), the right side of the processed image beneath its ipsilateral eyestalk was isolated (Fig. 3l), flipped to the other side and merged with the left half, guided by matching the perimeters of the esophageal foramen (Fig. 3m). The left half was then copied, flipped back to become the right half with the two halves joined at the midline (Fig. 3n). This provides a mirror-symmetric depiction of all traces present in the original specimen (Fig. 3n). Finally, tracings pertaining to the ocellar/naupliar system of specimen YKLP17299 (Fig. 3o) were identically processed (Fig. 3p). The mirror-symmetric image of YKLP17299 was superimposed onto that of YKLP11367, aligning their prominent ocellar features to provide the final reconstituted view of the jianfengiid cerebrum (Fig. 3q). This second procedure precludes subjective bias, yet the resultant image corresponds well with that obtained by tracing.

Corresponding branchiopod and malacostracan cerebral organization in Jianfengia
Both reconstruction methods resolved cerebral neuropils rostral to the esophageal foramen and fossilized nerve cords that extend around the esophageal foramen giving rise to neuropils disposed lateral to it. These nerves converge caudally to provide a synganglion immediately posterior to the foramen. The dispositions of these neuropils as well as neuropils defining the lateral expansion of the protocerebrum into the eyestalks correspond to traits typifying the brains described for the crown groups Branchiopoda and Decapoda5,18,20–22. As would be expected from observations of extant eucrustaceans, traces of neural tissue within the jianfengiid esophageal foramen (Fig. 3h, q) also align with the standard locations of the appendicular labrum15. In both reconstructions fossilized axon bundles diverge laterally from the anterior sclerite’s ocellar/nauplius eyes to supply the rostral neuropil of the prosocerebrum (Fig. 2d) where they merge with bilateral neuropils anterior and lateral to the esophageal foramen. These protocerebral areas receive the eyestalks’ nerves which connect a series of three nested neuropils originating from beneath the compound eyes. (Fig. 3h, q).

Next, we determined whether the reconstructed cerebra may correspond to the optic lobes and other neuropils that exist in today’s eucrustaceans. Both reconstructions of Fig. 3h, q allow an interpretive view of the jianfengiid brain (Fig. 4a–c). The lateral extension of the brain into the eyestalks comprises a small first optic neuropil (ON1) beneath the compound retina. This center is contiguous with a voluminous second optic neuropil (ON2) nestled against a smaller third neuropil (ON3). That the three neuropils are homologues of the lamina, medulla and lobula typifying pancrustaceans is supported by their alignment and similarity to the eyestalk neuropils exemplified hereby the extant decapod malacostracan Astacus astacus23 (upper left in Fig. 4a). The organization of the circumstomodeal nerve cords (Fig. 3i) indicates a slight taphonomic rotation around the fossil’s antero-posterior axis. Assuming that Jianfengia was a member of Bilateria, then these left and right traces in YKLP 11367 each represents a different depth within the specimen and are present on the contralateral side. These neural traces contribute to a mirror-symmetric organization as do the reflected outlines obtained by tracing. Reconstitution required four actions: the elimination of structures beneath a defined gray level (here 87% black of the CMYK scale), thus retaining the darkest puncta; inversion of the image to provide white profiles on a black background followed by the imposition of a Gaussian blur function (here radius expansion R=10px) that expands and runs together puncta (compare Fig. 3j, k). To counteract asymmetry around the midline (Fig. 3k), the right side of the processed image beneath its ipsilateral eyestalk was isolated (Fig. 3l), flipped to the other side and merged with the left half, guided by matching the perimeters of the esophageal foramen (Fig. 3m). The left half was then copied, flipped back to become the right half with the two halves joined at the midline (Fig. 3n). This provides a mirror-symmetric depiction of all traces present in the original specimen (Fig. 3n). Finally, tracings pertaining to the ocellar/naupliar system of specimen YKLP17299 (Fig. 3o) were identically processed (Fig. 3p). The mirror-symmetric image of YKLP17299 was superimposed onto that of YKLP11367, aligning their prominent ocellar features to provide the final reconstituted view of the jianfengiid cerebrum (Fig. 3q). This second procedure precludes subjective bias, yet the resultant image corresponds well with that obtained by tracing.

Corresponding branchiopod and malacostracan cerebral organization in Jianfengia
Both reconstruction methods resolved cerebral neuropils rostral to the esophageal foramen and fossilized nerve cords that extend around the esophageal foramen giving rise to neuropils disposed lateral to it. These nerves converge caudally to provide a synganglion immediately posterior to the foramen. The dispositions of these neuropils as well as neuropils defining the lateral expansion of the protocerebrum into the eyestalks correspond to traits typifying the brains described for the crown groups Branchiopoda and Decapoda5,18,20–22. As would be expected from observations of extant eucrustaceans, traces of neural tissue within the jianfengiid esophageal foramen (Fig. 3h, q) also align with the standard locations of the appendicular labrum15. In both reconstructions fossilized axon bundles diverge laterally from the anterior sclerite’s ocellar/nauplius eyes to supply the rostral neuropil of the prosocerebrum (Fig. 2d) where they merge with bilateral neuropils anterior and lateral to the esophageal foramen. These protocerebral areas receive the eyestalks’ nerves which connect a series of three nested neuropils originating from beneath the compound eyes. (Fig. 3h, q).

Next, we determined whether the reconstructed cerebra may correspond to the optic lobes and other neuropils that exist in today’s eucrustaceans. Both reconstructions of Fig. 3h, q allow an interpretive view of the jianfengiid brain (Fig. 4a–c). The lateral extension of the brain into the eyestalks comprises a small first optic neuropil (ON1) beneath the compound retina. This center is contiguous with a voluminous second optic neuropil (ON2) nestled against a smaller third neuropil (ON3). That the three neuropils are homologues of the lamina, medulla and lobula typifying pancrustaceans is supported by their alignment and similarity to the eyestalk neuropils exemplified hereby the extant decapod malacostracan Astacus astacus23 (upper left in Fig. 4a). The organization of the circumstomodeal nerve cords and their neuropils, however, align with those of branchiopod crustaceans21,24, such as Triops longicaudatus and Artemia salina, whose cerebral nervous systems, other than the optic lobes, are nearly identical to that reconstructed for Jianfengia (lower left, Fig. 4a). The jianfengiid brain thus features attributes of both the ground pattern of the malacostracan visual system18 and the deutocerebral-synganglion circumstomodeal nervous system of adult Branchiopoda, which can also be observed during early development of the decapod brain21,22.

Is Jianfengia a member of Tetraconata?
A trait claimed to be exclusive to pancrustaceans, and that inspired the term ‘Tetraconata’ as the alternative name for Pancrustacea25, is the presence of a quartet of Semper cells in each ommatidium of the compound eye. In living pancrustaceans each Semper cell sends four processes from the base of the ommatidium to secrete the transparent protein that builds the ommatidium’s light-focusing cone26,27. The presence of such an organization in Jianfengia could support its status as a protocrustacean. Specimen YKLP11117 shows a few ommatidia that appear to have lost their cuticular lenses, thus allowing a view beneath them. Combined UV and white light illumination reveal in four ommatidia a geometric arrangement of internal elements that are distinct from possible taphonomic artifacts (Supplementary Fig. 2). Resolving peak intensities in a defined chromatic range (see Methods) reveals grouped iridescent components in four ommatidia. Albeit a small sample, in two ommatidia the groups appear to be organized as a quartet, whereas in the other two ommatidia the resolution is ambiguous in suggesting more than four. Whereas the constrained tetrad arrangement is crucial for the patterning of the pancrustacean compound eye and its ommatidia26,27, in the Scutigeriidae—centipedes uniquely possessing compound eyes28—the Semper cells contribute at least eight prolongations that could provide the crystalline cone28,29. The implications that cone cell ambiguities exclude Jianfengia from Pancrustacea are considered next.

Neurocladistics identifies Jianfengiidae basal to total Mandibulata
Arrangements of putative Semper cells do not provide an unambiguous trait supporting pancrustacean affinity. To clarify the phylogenetic status of Jianfengia we used neural traits for cladistic determination of its relationship with representative mandibulates and chelicerates. This follows the strategy used for a previous neural cladistics study inferring phylogenetic relationships across extant euarthropods30. Here we assembled a matrix of 120 characters comprising mainly neural traits with additions pertaining to the carapace, cephalic appendages, and tagmatization (Supplementary Table 1). The traits were scored as present/absent across 17 living euarthropod species as well as the Chengjiang fossil Fuxianhuia protensa and Alalcomenaeus2,7, the Wuliuan stage Kaili fossil Leanchoilia5, the BST fossil Mollisonia symmetrica31 and the fossil limuliid Euproops danae from the Carboniferous Mazon Creek Konservat-Lagerstätte32. The cladistics analysis was rooted against three non-arthropodan outgroups, the spiralian Paranemertes peregrina, and the cycloneuralians Caenorhabditis elegans and Priapulus caudatus. For parsimony and likelihood analyses we employed PAUP* (Phylogenetic Analysis Using Parsimony*, version 4.0a168)33 to infer evolutionary relationships. To avoid bias, due to the uncertainty of Semper cell numbers that trait was excluded. For maximum parsimony analyses, all traits were unordered and initially considered under equal weighting (Fig. 5). We also performed Bayesian analyses on the same matrix in Mr. Bayes (version 3.2.7a), under the Markov k (Mk) model of character evolution34. The resultant phylogenetic trees and evolutionary relationships were largely congruent across all inference methods, with minor variation in likelihood and parsimony bootstrap support values and Bayesian posterior probabilities (see Supplementary Fig. 4). Notably, Jianfengia multisegmentalis was resolved as basal and sister to Mandibulata in all phylogenetic analyses with various levels of support (Supplementary Fig. 4), whereas the short body megacheirans Alalcomenaeus and Leanchoilia were resolved as basal and sister to total Chelicerata. Fuxianhuia protensa was resolved as a stem mandibulate closely allied with extant Myriapoda.

Methods
Material provenance
Descriptions herein refer to specimens of Jianfengia multisegmentalis retrieved from the Cambrian (Series 2, Stage 3) Eoredlichia-Wutingaspis trilobite biozone, Yu’anshan Member, Chiungchussu Formation, Haikou. The specimens used here YKLP11117, YKLP11367, YKLP11368, YKLP11369, YKLP17299 and NIGPAS100123b are curated at the Yunnan Key Laboratory for Palaeobiology (YKLP), Institute of Paleontology, Yunnan University, Yunnan, Kunming, China. Permission to access and study the material for this work was granted by Yunnan Key Laboratory for Palaeobiology Director.

Photomicroscopy
For light microscopy of fossil material, digital images were taken using a Nikon D3X attached to a Leica M205C photomicroscope (Leica Microsystems; Wetzlar, Germany). Images were transferred to Adobe Photoshop CS5 (Adobe Systems; San Jose, CA) and processed using the Photoshop camera raw filter plug-in to adjust sharpness, luminance, texture, and clarity. Colors were untouched and are those typical of Chengjiang fossils. For ultraviolet illumination, fossils were photographed using a Leica MZ10F stereomicroscope with appropriate filter blocks to evoke intense green fluorescence. Flexible fiberglass light guides were used to combine UV fluorescence and white-light illumination. Confocal reconstructions for Fig. 4a were made with an LSM3 Pascal confocal microscope (Zeiss, Oberkochen, Germany). From 10 to 30 images of 1,024 × 1,024 pixel resolution at 12-bit color depth were scanned by using 10X/0.3 plan Plan-Neofluar objectives. Light microscopy images of Bodian silver-stained neuropils were obtained with a Zeiss Axio Imager Z.2.

Interpretive drawings and tracings
Drawings were made using Adobe Illustrator for which tracings and reconstruction were generated using a Wacom Intuos Pen Tablet on projected photographic images.

Identification of putative semper cells
Enlargements of the compound eye of YKLP11117 shows its composition of about 120 facets. Patches of these at the eye margin and about 2/3rds across the eye’s surface resolve their hexagonal arrangement. (Fig. 1c, d; Supplementary Fig. 2a, b). Four ommatidia (numbered 1–4 in Supplementary Fig. 2c) show a loss of their capping lenses, thereby revealing structures compressed within the ommatidial shaft. Ultraviolet illumination reveals clusters of bright yellow-green images within four ommatidia. These configurations were then resolved at high magnification by isolating their computed maximum intensities). Between four and possibly seven profiles reside in a sampled ommatidium allowing their interpretation as arrangements of Semper (cone) cells that would have extended outwards to the crystalline cone (light gray) underlying its lens. Although we schematize quartets of Semper cells as their default organization typifying pancrustaceans, the sample is very small and two of the four ommatidia appear to have more than four units. This indicates the likelihood of variation across the retina.

Isolating neural traces across specimen YKLP11367
Imbalances of tone across this specimen were rectified by converting the image to its grey-scale mode and removing grey level densities responsible for imbalance (Supplementary Fig. 3). This provided approximate uniformity of grey scales across the specimen allowing adjustments of exposure and offset to provide a final image for analysis and reconstruction. Adobe Photoshop functions eliminating intensity levels beneath a defined threshold (here 87% black in the standard CMYK scale) provided an image that was grey scale inverted to provide white profiles on a black background. This was subjected to a Gaussian blur function (R=10px) to provide resolution of neural residues free of background noise (Fig. 3n, p). Description of the brain’s reconstruction is provided in the test and legend of Fig. 3.

Phylogenetic inference
Maximum parsimony and likelihood were performed using PAUP* (Phylogenetic Analysis Using Parsimony*, version 4.0a168) on a matrix of 120 characters scored in 26 extinct and extant taxa. Taxa include 17 extant euarthropod species, and 6 extinct euarthropods, including the Chengjiang fossils Fuxianhuia protensa and Alalcomenaeus, the leanchoiliid fossil Leanchoilia, the BST fossil Mollisonia symmetrica, and the fossil limuliid Euproops danae. Trees were rooted with three extant non-arthropodan taxa. All morphological characters were binary and scored as either present (1) or absent (0). Maximum parsimony was initially conducted with a heuristic search using unordered and unweighted characters employing 1,000 random-addition replicates with Tree-Bisection-Reconnection (TBR) branch swapping. Two most-parsimonious trees (tree length = 221 steps; CI = 0.54; RI = 0.78) provide the strict consensus tree shown in Fig. 5, with bootstrap support values shown in Suppl. Figure 4a. To examine the impact of homoplasy on parsimony trees, subsequent maximum parsimony searches varied character weights by implementing successive reweighting (Supplementary Fig. 4b). Successive reweighting was done using the rescaled consistency index until character weighting stabilized, which occurred after three successive rounds of reweighting. This approach increases support for J. multisegmentalis as sister to all Mandibulata (Supplementary Fig. 4b). Maximum likelihood was performed under the Mkv model, assuming a single substitution type, equal rates, and no invariant sites. Likelihood bootstrap analysis was performed on 1000 replicates with support values overlaid on the highest likelihood tree (Supplementary Fig. 4d). Bayesian analysis on the same matrix was performed using Mr. Bayes (version 3.2.7a), implementing the gamma rate variation model with unordered characters. Markov Chain Monte Carlo (MCMC) analysis was run for 100 million generations on four chains with the first 25% of trees removed as a conservative burn-in fraction. Runs converged well with the standard deviation of the split frequencies falling below 0.001. Trees resulting from all phylogenetic analyses were visualized in the Interactive Tree of Life (iTOL) viewer v654.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "See under review attachments", "reviewer_2": "This manuscript reports the putative findings of neural traces on a few specimens of a Cambrian great-appendage arthropod: Jianfengia multisegmentalis. This species has been described a few decades ago, even though their phylogenetic position within arthropods was not clear. This is not the first time that preservation of the neural tissue in Cambrian arthropod has been used to determine their phylogenetic position. To my knowledge, though, this is the first instance in which a great-appendage arthropods has been clustered together with the groups of crustaceans and hexapods based on these kind of data. Based on this and previous reports, the authors argue that neuroanatomical traits can be used as proxies to understand the placement of these fossils in the absence of external traits, assertion that I agree with. The authors present a brain and nervous system reconstruction of this species after having superimposed images of neural traces of different specimens and changing illumination parameters. I am not an expert in image treatment but I do not see any apparent introduce software artifact. I find this manuscript as well articulated and with conclusions grounded on the data presented. The images reporting the compound eyes and the neural organisation is consistent with pancrustacean neuroanatomy. I would like to see a bit more of discussion regarding the paraphyly of Megacheira. The phylogenetic placement of Jianfengia as an eucrustacean implies that great appendage arthropods can fall either within chelicerates or pancrustaceans. If that is the case, these large appendages were convergent? Lastly, I feel it migth be interesting to show in Fig. 3 or 4 stainings of the homologous cephalic area in one extant Eucrustacean species\nLines 57: typo \"barely1mm\"\nLines 391: typo \"Jiangfengia\"\nLine 445: typo \"(C)\" should be (D)", "reviewer_3": "PD Dr. Brigitte Schoenemann\nUniversity of Cologne\nDept. of Zoology (Neurobiology/Animal Physiology)\nBiocentre, Zülpicherstrasse 47b\n50674 Cologne/Germany\nB.Schoenemann@uni-koeln.de\nNature Communications\nA pancrustacean brain and its visual systems define a Cambrian “great appendage”\narthropod.\nNicholas J. Strausfeld, Xianguang Hou, and Frank Hirth\nAs its predecessors, this manuscript succeeds excellently in revealing fossilized nervous\nsystems, sometimes, as here, in very small organisms and more than half a billion years\nold. The authors aim to show that coevally lived stem and crown arthropods during the\nEarly Cambrian in the Chengjiang Biota by the example of Jianfengia multisegmentalis\n(Hou, 1987), a not (completely?) tagmatized arthropod. Two more characteristics should\nbe fulfilled to assign J. multisegmentalis to pancrustaceans– a tetraconate crystalline\ncone and a pancrustacean brain. We agree with the nervous system's interpretation but\nconsider the discussion of the eye systems as problematic. Finally, we come to a\nconclusion, different from the authors, as stated below:\nThis is an ambitious manuscript that aims to show that Early Cambrian fossils from the\nChengjiang Biota reveal recognizable crown groups, namely representatives of\ntertaconates/panarthropods, already existing coevally with early stem arthropods (e.g.\nFuxianhuia protensa Hou,1987 [stem mandibulate], Alalcomenaus [stem ch elicerate]) more\nthan half a billion years ago.\nThe authors describe 3 specimens of the genus Jianfengia multisegmentalis (Hou, 1987).\nTo investigate neural traces indicating shapes of brains, ganglia, and neural pathways\nwithin a carapace barely 1mm wide may be considered quite a challenge. Despite the\nabsence of trunk tagmatization, the latter would be typical for pancrustacea/tetraconates;\nin addition, the authors aim to demonstrate typical traits of pancrustaceans in J.\nmultisegmantalis to provide a 'stemward' identity towards Pancrustacea/Tetraconata as\nthere are:\na) Structured eye stalks (-> crown Malacostraca)\nb) Compound eyes with ommatidia, revealing tetradic structures (-> cone-\nbuilding Semper cells)\nc) nauplius-like eyes and associated nerves supplying a discrete forebrain region ->\npancrustaceans\nd) neuropils and tracts in the eye stalks -> nested optic neuropils as\noccur in eucrustaceans\ne) organization of the deuto- and tritocerebrum -> differentiation from stem\nChelicerata and extinct Myriapoda\nIf successful, the authors would show that neuroanatomical traits could provide a\npowerful tool for distinguishing characteristics to establish euarthropod relationships,\neven if external traits that differentiate identities are absent.\nThis is a difficult review because, of course, the manuscript presents its own\ninterpretation of the anatomical findings. Still, another view in the literature sheds a\nslightly different light on some aspects. In good academic tradition, I will not attack this\nmanuscript but briefly explain this different perspective. Based on this, I will discuss\nthe results presented in the manuscript, and ultimately, the reader may take his or her\nposition\nSo, what has been achieved?\na) Structured eye stalks (-> crown Malacostraca)\nThe joints of the articulated eye stalks can be seen very clearly. The authors indicate one,\nand there is another more distally (Fig. 1F). There are mentioned 2 (line 78), and an arrow\nshould also indicate the second one.\nIt is commonly accepted that Malacostraca possess stalked eyes, and at least among\nthe extinct and fossilized I know, the stalks are indeed always articulated. The other way\naround, I would expect that among the early arthropods, the artiopods, for example, we\nmay find articulated eye stalks. So, I would be slightly more cautious with the conclusion\ndrawn here [Meanwhile, these articulated stalked eyes have been found and described:\nSchmidt, Schoenemann, et al. 2025, Biology Communications, in press]. So probably\narticulated eye-stalks are typical for crustaceans (not hexapods, belonging to\nPancrustacea also!), and consequently, not all arthropods with articulated eye stalks are\npancrustaceans. Even the eye stalks of Leanchoilia and Alalcomenaeus are articulated,\nsee Fig 1 C,D.\n[FIGURE REDACTED]\nFigure 1 Articulated eye stalks of the artiopod Pygmaclypeatus daziensis\nZhang, Han & Shu 2000, of Leanchoilia, and Alalcomenaeus,\nb) Compound eyes with ommatidia, revealing tetradic structures (-> cone-building\nSemper cells)\nThe term semper cells defines cone cells of crustaceans and insects. The manuscript\ndevelops the idea of including J. multisegmentalis within the stem Pancrustacea. The\nmanuscript should take into account the initial and provisional nature of the use of this\nterm.\nThe question of the existence of tetradicly arranged cone-cells in J. multisegmentalis is\nan important, critical point. Indeed, J. multisegmentalis possesses compound eyes, and\nthe loss of cuticular lenses in specimen YKLP 11117 offers insights into the arrangement\nof internal structures of the ommatidia. Photoprocessing methods were applied to clarify\nthe individual elements. However, if I were to take the position of devil's advocate,\nI could say that the threshold can be adjusted until 'it fits' (I am sure the authors haven´t done\nthis.)\nIt is phenomenal that the authors succeed here in showing the cone cells. The problem\nfor me here is that none of the examples clearly shows 4 cone cells; one may find even\nmore at different sizes and numbers (Fig. 2). That the application of photo-processing\nbrings out the tetraconate pattern for me suggests, in conclusion, that there are more\nthan 4 (~6-8) cone-cells, but some of them , four in number, are more pronounced in\ntheir development than the others. This may indicate that we here are on a transitional\nway from a multiconate eye (-> Limulus-like (Xiphosura, Chelicarata), ~ 100 cone cells,\nFahrenbach 1968, Harzsch & Hafner 2006) to a tetraconate system. But still, it is not\nachieved yet.\nIn the manuscript, the system here is distinguished from the scutigerid myriapods by mentioning\none single extension of the cone cell contributing to the secretion of the crystalline cone in\ntetraconates, while the myriapod possesses two. I am pretty sure one may not find these delicate\nstructures in a fossil. Here, it may help with the argument that the scutigerid ommatidium is\ndeveloped secondarily from the myriapod ocelli, which in turn originated from original ommatidia\n(e.g. Paulus 1979, 2000).\nMüller et al. (2003) find for the myriapod Scutigera coleoptrata (Linnaeus, 1758), 8 cone\nsegments, produced by four crystalline cone cells; tripartite cones (Ascothoracida and\nCirripedia), and bipartite ones they suggested to have derived from the tetrapartite\nsystem. Thus, four cone cells alone may not be sufficient to postulate an assignment to\nTetraconata, even if it is the standard definition. At least this should be discussed more\ndifferentiated. It may well be that not all cone cells/segments can be found here in the\nspecimens of J. multisegmentalis; in one of the examples presented in the manuscript\n(Fig. 2,4), there may be evident even 8 elements (D4, another shows up 7, D3),\nsuggesting a tetrapartite origin as in Scutigera.\n[FIGURE REDACTED]\nFigure 2. The cone cells of genus Jianfengia multisegmentalis (Hou 1987) (from figure 2 of\nthe manuscript)\nc) nauplius-like eyes and associated nerves supplying a discrete forebrain region ->\npancrustaceans\nIt would be great to have them, and I am willing to find them in the figures of the main\ntext; I have tried hard ... I see the filamentous structures, and they may be relicts of a\npalisade of receptors, and I agree with the pigment. Still, please make the 3 nauplius-like\neye elements more evident in your main text figures. I find them in the UV (S1 C). A\nprobable explanation indeed would be that this was one organ consisting of three ocelli\ncovered by a membrane, which would explain the homogenous appearance of the whole\nsystem under white light. I agree with you that the organ may have been movable. The\nmovable character, I think, results from the not-fixed 'stalk,' which clearly protrudes from the\ncarapace. See the dark line (the margin of the carapace, indicated by the arrows in our Fig\n2c).\nThe small element in the reconstruction drawing (main text in Figure 4D) shows the same\ninterpretation as I gave it above and in Fig. C3, which is great, of course, but this finding\nshould be established more clearly in the main text.\nIf we accept that the filaments are relicts of receptors, please note that they do not\nshow any grouping. Typically, a Nauplius eye is a single median eye and consists of\nthree- or four-pigment cup ocelli. This nicely can be detected in your Figure S1C.\nI am not sure about the arthrodial membrane here, but I share the idea that this whole\nstructure could have been movable (see above).\nI think these results (3 ocelli, movable system) should be more emphasized and shifted\nfrom the Supplement to the main text.\nSo, we agree with a tripartite, nauplius-like eye, probably built by ocelli.\nFigure 3. The Nauplius-organ\nNeural organization\nd) neuropils and tracts in the eye stalks -> nested optic neuropils as occur in\neucrustaceans\ne) organization of the deuto- and tritocerebrum -> differentiation from stem Chelicerata and\nextinct Myriapoda\nTracing up the neural structures in this tiny organism, neuropils as pathways is\nfascinating, very skillful, and convincing. One should never forget that all these\ninvestigations start in a quarry, and we deal with 'rocks', fossils. The optic neuropils 1-3\ncan be discerned very clearly, the reconstruction of the entire brain for example by\noverlaying the results of different findings and the assumption of a bilateral organism, by\nthat, the methods of reconstruction for completing, for example, by flipping elements of\nthe one side to the other, are justified. The tables (S1, S2) are interesting, and the drawn\nreconstructions (Fig 5) are excellent and consequent. Unfortunately, the inscriptions in\nFigure 5 (as perhaps the whole figure) are too small and hardly readable/discernible.\nI am not an expert in arthropod brains and the genetic background of the genetic\norganization of an arthropod segmentation, especially the nervous system, but one\ncan follow the given reasoning, and a possible interpretation seems to have\nemerged.\nIn Figure 3 it is not clear what is from YKLP 11367 and YKLP 17299\nDiscussion\nThe discussion of the manuscript makes the point that the J. multisegmentalis among the\nstem-arthropods of the Chengjiang biota represents a true pancrustacean, because,\nalthough a trunk tagmatization still is missing, the eyes have a tetraconate ommatidium, J.\nmultisegmentalis possesses as typical for pancrustaceans a nauplius-like eye consisting of\nthree elements, and a brain that is organized as in pancrustaceans.\nWe may agree with the latter, but we see a problem with the interpretation of the eye\nsystems.\nNote that median eyes (-> Nauplius-like eyes, Nauplius eyes = Median eyes of\ncrustaceans) also exist in the megacheiran Leanchoilia! (Garcia-Bellido & Collins 2007,\nSchoenemann & Clarkson 2012, 2023), and indeed in Alalcomenaeus (see below).\n[FIGURE REDACTED]\nFigure 4 Median eyes of Alalcomanaeus cambricus Simonetta, 1970 and Leanchoilia\nsuperlata Walcott 1912\nThis has implications and should be considered and discussed.\nEven being aware of the work of Tanaka et al. 2013, one may be a bit careful with the\ninterpretation of the eyes of Alalcomenaeus and Leanchoilia. The specimens of\nAlalcomenaeus illustrated by Briggs & Collins (Fig 4.1, 4.4) or of Whittington (1981, Fig.\n126) clearly show that Alalcomenaeus has club-shaped, penduculate compound eyes\n(here Fig. 1D), surmounting articulated stalks; the same is true for Leanchoilia (here Figure 1 B,\nC). Here, the eyes and their stalks are much finer.\nNote that there are median eyes in the megacheiran Leanchoilia (e.g. Garcia- Bellido &\nCollins, 2007; Schoenemann Clarkson 2012) and in Alalcomenaeus also (e.g. Briggs &\nCollins, 2007, Fig. 4.3), as can be seen clearly here in Figure 4. This has implications and\nshould be considered and discussed - for example, consequently, Jianfenigia is NOT the\nonly Early Cambrian genus identified with median eyes (and crustacean-like eyestalks).\nPerhaps J. multisegmentalis is among the first with 3 median eyes. Still, there are\ncrustaceans with 4 ocelli in their median eye complex, e.g. among the branchiopoda (e.g.,\nReimann & Richter 2007) – so the number alone may is not game-changing here.\nWe agree, however, completely with the idea that genetically based the morphological\nvariance of homolog deutocerebral appendages is wide enough to comprise great\nappendages, antennules formed as 'pinchers' or pier-like antennules, or ...\nIn general, we also agree with the interpretations of the neural systems and the homologies\nexplained here. I am not an expert in arthropod nervous systems, but I think the deductions\nare reasonable.\nFinally, one of this manuscript's great conclusions is that not the Hox-gene-associated\ndivergences of the segmented trunk define ancestral and crown arthropods, but do the\nsegmental cerebral domains, the unique lineage-specific neuropils, and associated traits.\nThis is an important point, but our conclusion differs slightly from what the authors indicate\nhere.\nEven if one of the characteristics (like those of the nervous system as here) has passed\nthe 'finish-line' to reach a crown group state, while others have not (tagmatization of the\nbody), one has to be aware that as here, obviously boundaries of the stem-/crown\ngroups cannot always be rigidly defined, and that there are transitional situations (as\nrepresented in the eye-status of Jianfengia multisegmentalis (Hou 1987)). Here, one part\n(brain system) has reached that stage, and the other (tagmatization of the body) still has\nnot; the eye system of Jianfengia multisegmentalis (Hou 1987) is on its way. This may\nhave important implications for describing evolutionary processes in general.\nThe results here show that the formation of different crown-group characteristics\n(tagmatization of the trunk – crown–group-nervous-system – eye system) may evolve\nwith different velocities. An important point because one now will have to weigh when\nthe complete, or a 'sufficient' crown-group-state has been arrived. Probably it is not\nadequate if only one of the characteristics has passed the 'finish line'. Even if the focal\npoint in the considerations of this manuscript has been laid on the eyes and\nbrain/nervous structures, it may be an essential fact that the tagmatization of the body\nis not quite there yet.\nNevertheless, the authors are right to state that neuroanatomical traits could provide a\npowerful tool for distinguishing characteristics to establish euarthropod relationships, even\nif external traits that differentiate identities are absent.\nReferences\nButterfield, N. J. (2002). Leanchoilia guts and the interpretation of three-dimensional structures in Burgess\nShale-type fossils. Paleobiology, 28, 155-171.\nBriggs, D.E.G. & Collins, D. 1999: The arthropod Alalcomanaeus cambricus Simonetta, from the middle\nCambrian Burgess Shale of British Columbia. Palaeontology 42, 953–957.\nFahrenbach, W. H. (1968). The morphology of the eyes of Limulus: II. Ommatidia of the compound eye.\nZeitschrift für Zellforschung und Mikroskopische Anatomie 93, 451-483.\nGarcía-Bellido,D. C., & Collins, D. (2007). Reassessment of the genus Leanchoilia (Arthropoda,\nArachnomorpha) from the Middle Cambrian Burgess Shale, British Columbia, Canada. Palaeontology, 50,\n693-709.\nHarzsch, S., & Hafner, G. (2006). Evolution of eye development in arthropods: phylogenetic\naspects. Arthropod Structure & Development, 35, 319-340.\nHou, X., & Bergström, J. (1997). Arthropods of the Lower Cambrian Chengjiang fauna, southwest\nChina. Fossils and Strata 45, 1-177.\nMüller, C. H., Rosenberg, J., Richter, S., & Meyer-Rochow, V. B. (2003). The compound eye of Scutigera\ncoleoptrata (Linnaeus, 1758)(Chilopoda: Notostigmophora): an ultrastructural reinvestigation that adds\nsupport to the Mandibulata concept. Zoom orphology, 122, 191-209.\nPaulus, H. F. (1979) Eye structure and the monophyly of the arthropod eye. In Arthropod Phylogeny (ed.\nGrypta, A. P.) 299–383 (Nordstrand, 1979).\nPaulus, H. F. (2000). Phylogeny of the Myriapoda–Crustacea–Insecta: a new attempt using photoreceptor\nstructure. Journal of Zoological Systematics and Evolutionary Research, 38, 189-208.\nReimann, A., & Richter, S. (2007). The nauplius eye complex in ‘conchostracans’ (Crustacea,\nBranchiopoda: Laevicaudata, Spinicaudata, Cyclestherida) and its phylogenetic implications. Arthropod\nStructure & Development, 36, 408-419.\nRichter, S., Loesel, R., Purschke, G., Schmidt-Rhaesa, A., Scholtz, G., Stach, T., ... & Harzsch, S. (2010).\nInvertebrate neurophylogeny: suggested terms and definitions for a neuroanatomical glossary. Frontiers in\nZoology, 7, 1-49.\nSchmidt, M*., Schoenemann, B.*, Hou X.-g., Melzer & Liu, Y. (2025) A unique 1 Lower Cambrian\narthropod with two different compound eye systems. Communications Biology (2025), in press * shared\nfirst authorship\nSchoenemann, B., & Clarkson, E. N. (2012). The eyes of Leanchoilia. Lethaia, 45, 524-531.\nSchoenemann, B., & Clarkson, E. N. K. (2023). The median eyes of trilobites. Scientific Reports 13, 3917.\nTanaka, G., Hou, X., Ma, X., Edgecombe, G. D., & Strausfeld, N. J. (2013). Chelicerate neural ground pattern\nin a Cambrian great appendage arthropod. Nature, 502(7471), 364-367.\nWhittington H. B. (1981). Rare arthropods from the Burgess Shale, Middle Cambrian, British Columbia.\nPhilosophical Transactions of the Royal Society, Series B, 292, 329–357.\nA pancrustacean brain and its visual systems define a Cambrian “great appendage” arthropod.\nby Nicholas J. Strausfeld 2*, Xianguang Hou 1, and Frank Hirth 3*\nThis MS presents amazingly detailed comparisons between the neural structures of a\nCambrian megacheiran arthropod and those of modern crustaceans. The biological data are\nall of high quality. However, most interpretations of the fossil species are based on\nquestionable often very weak fossil evidence (see examples below). The extensive use of\nimage processing (e.g. Photoshop) is sometimes misleading. There is a huge difference\nbetween the idealized processed images and reconstructions, and what we actually see in the\nfossil specimen.\nJianfengia is interpreted as a lower stem pancrustacean, based on the assumed structure of\nits nervous system. However, its appendage structure (head appendages) is completely\ndifferent from that of pancrustacean. The authors do not take into account these key\nanatomical aspects.\nLine 45- What kind of digital adjustments ? This is a key point. Please give details.\nLine 48- The plural of retina is retinae (Latin)\nLine 61- I agree that the frontal rounded feature is the anterior sclerite (ASC). It appears to\nbe a sclerotized feature comparable with other hard parts of the exoskeleton (see figure B\nbelow). However, I see no convincing evidence of an underlying “system of lenses”. Clearly\nthis brownish feature is over-interpreted. I am saying that soft tissues (including sensory\norgans) do not occur underneath the sclerite but they are not visible in this fossil specimen.\nLine 62 + “Their detailed organization suggests structural homology to crustacean nauplius\neyes (8, 9) and insect ocelli (10, 11) (Fig. 1C, D; SI Appendix Fig. S1) comprising three lenses\noverlying a palisade of photoreceptors”\nThis statement is speculative considering that no detailed organization can be seen in\nthe fossil specimen in question.\nLine 65- Please explain what you mean by superimposition\nLine 68- This is perfectly true but what the readers would like to see here (in the\n“Results” paragragh) are more convincing fossil evidence.\nLine 76-77. The lateral eyes are well-preserved. However, this type of eyes occurs in numerous\nCambrian arthropods that clearly have no affinities with eumalacostracans (e.g. Artiopoda such\nas Acanthomeridion; also Isoxys, a basal megacheiran or even Tuzoia, that have no close affinities\nwith pancrustaceans)\nLine 85. The light spots seen in Fig. E are interpreted as possible ommatidia\nHowever, similar spots seem to occur elsewhere in the specimen. Unfortunately, I have no access\nto the high-resolution version of the text-figures. It is important that the authors provide comparative high-\nresolution images of the lateral eye and other areas of the fossil (see below). I would suggest to\nprovide back-scattered SEM images of the eye. It has been done with the eyes of Waptia from\nthe Burgess Shale and clearly revealed the outline of ommatidia that do not appear in light\nphotographs (see Vannier et al. 2018).", "reviewer_4": "This study analyses four specimens of the species Jianfengia multisegmentalis and provides new anatomical information about them thanks to the use of UV fluorescence and enhanced contrast methodology. This has led the authors to describe three main new characteristics of Jianfengia neuroanatomy, which establish a link to Pancrustacea, thereby improving our knowledge of pancrustacean neuroanatomy and the inner relationships of megacheirans. The three major findings of the paper are:\n1. The structural homology of Jianfengia anterior sclerite with crustacean nauplius eyes.\n2. The presence of Semper cells in the lateral stalked eyes.\n3. The nauplius-like eyes of Jianfengia and its nervous system are connected only in the forebrain region, in a manner similar to pancrustaceans.\nThe methodology, though I do not consider myself an expert on UV fluorescence, the results, and their interpretation and discussion are solid. UV optical techniques have been frequently used on similar materials to describe the nervous system and, once again, prove to be effective.\nOut of the three main results described, I have struggled only with identifying the triplet of lenses in the anterior sclerite (see below). However, due to the more easily identifiable radiated structure of the photoreceptors, which I found clear, I do not question the interpretation of the anterior sclerite as homologous to the nauplius eyes. Therefore, I concur with the three major findings of this work.\nI also believe that the contribution of this study to our knowledge of early euarthropod neuroanatomy, and its role in phylogenetic assessment, has the potential to significantly aid in resolving the relationships of major clades and in clarifying megacheiran classification. This contribution is more than significant and is likely to be useful for further studies. As such, I recommend its publication in Nature Communications.\nNevertheless, I suggest a minor revision to improve the exposition of the results and to enhance the discussion in some areas.\n-Lines 33-35: Add citations for Myriapoda, the oldest evidence of neuroanatomy.\n-Lines 51-52: I would smooth this phrase and say something like, “Together, these findings suggest Jianfengia as a stem euarthropod closer to pancrustaceans than to both Fuxianhuia and Alalcomenaeus.” Again, it is not that I am not convinced of the results, but rather that there is conflicting evidence in other characters.\n-Line 57: \"barely1mm\" should be \"barely 1 mm.\"\n-Lines 99-102: Rephrase and possibly split the sentence for better readability.\n-Line 391: Missing caption for le.\n-Figure 5: It would benefit from the addition of a schematic of Fuxianhuia. Also, consider using a hand-drawn cladogram based on your findings and the literature to illustrate the possible evolution of the anatomies depicted.\n-Figure S1: Please provide better contrast for the triplet of lenses, at least in the drawing. The structure is difficult to discern here and in Figure 1. Maybe add schematics to help the reader visualize what you see.\n-I strongly encourage the author to add a paragraph in the discussion about the possible implications of ontogeny for the anatomy of the nervous system and eyes in Jianfengia. Considering the possible life stage of the studied specimens, as well as of the other key taxa, could provide a clearer understanding of the evolutionary forces at play. Since many characters change during ontogeny, we do not always know whether the specimens represent fully developed adults, and character development may occur with shifts in relative timing among different groups. Addressing this aspect could help reconcile some of the conflicting signals observed in different traits (e.g., antennae/great appendage expression) and offer a clearer perspective on the mosaic evolution seen among Fuxianhuia, Alalcomenaeus, and Jianfengia itself.\nI do not wish to see the manuscript again, thus I will be happy to do so if the editors will consider it relevant. Lorenzo Lustri"}, "author_response": "Reply to Reviewer #1\nPD Dr. Brigitte Schoenemann\nA pancrustacean brain and its visual systems define a Cambrian “great appendage”\narthropod.\nNicholas J. Strausfeld, Xianguang Hou, and Frank Hirth\nAs its predecessors, this manuscript succeeds excellently in revealing fossilized nervous systems,\nsometimes, as here, in very small organisms and more than half a billion years old. The authors\naim to show that coevally lived stem and crown arthropods during the Early Cambrian in the\nChengjiang Biota by the example of Jianfengia multisegmentalis (Hou, 1987), a not\n(completely?) tagmatized arthropod. Two more characteristics should be fulfilled to assign J.\nmultisegmentalis to pancrustaceans– a tetraconate crystalline cone and a pancrustacean brain. We\nagree with the nervous system's interpretation but consider the discussion of the eye systems as\nproblematic. Finally, we come to a conclusion, different from the authors, as stated below:\nWe thank the reviewer for her positive appraisal of this work and much appreciate that she chose\nto be visible to the authors. That a reviewer doesn’t feel the need to hide behind the shield of\nanonymity makes discussions such as the present reply informative and relaxed.\nWe also agree with this reviewer that the section on cone cells needed serious reconsideration,\nwhich we have done here to the advantage of the work as a whole. Agreeing with the reviewer\nregarding ambiguities of the number of cone cells has led to our major revision which is a\ncladistic analysis of Jianfengia’s position, hence identity, within Euarthropoda using\nneuroanatomical and cephalic traits specifically omitting cone cell numbers for the presented\nphylogenetic analyses. This results in a phylogeny that positions Jianfengia basal to, but not\nwithin, Mandibulata. It follows that taxon-specific determination of cone cell number had not yet\nevolved within Jianfengiidae. This is now explained and discussed in this revision.\nThis is an ambitious manuscript that aims to show that Early Cambrian fossils from\nthe Chengjiang Biota reveal recognizable crown groups, namely representatives of\ntertaconates/panarthropods, already existing coevally with early stem arthropods (e.g.\nFuxianhuia protensa Hou,1987 [stem mandibulate], Alalcomenaus [stem chelicerate])\nmore than half a billion years ago.\nThe authors describe 3 specimens of the genus Jianfengia multisegmentalis (Hou, 1987).\nTo investigate neural traces indicating shapes of brains, ganglia, and neural pathways\nwithin a carapace barely 1mm wide may be considered quite a challenge. Despite the\n1\nabsence of trunk tagmatization, the latter would be typical for pancrustacea/ tetraconates;\nin addition, the authors aim to demonstrate typical traits of pancrustaceans in J. multisegmantalis to provide a 'stemward' identity towards Pancrustacea/Tetraconata as\nthere are: Structured eye stalks (-> crown Malacostraca): a) Compound eyes with\nommatidia, revealing tetradic structures (-> cone- building Semper cells); b) nauplius-\nlike eyes and associated nerves supplying a discrete forebrain region-> pancrustaceans;\nc) neuropils and tracts in the eye stalks -> nested optic neuropils as occur in\neucrustaceans organization of the deuto- and tritocerebrum -> differentiation from stem\nChelicerata and extinct Myriapoda. If successful, the authors would show that\nneuroanatomical traits could provide a powerful tool for distinguishing characteristics to\nestablish euarthropod relationships, even if external traits that differentiate identities are\nabsent.\nThe reviewer is correct in identifying the aims of this work, with this revision going significantly\nfurther. The over-arching question we addressed, and in this revision have resolved, is whether a\nbrain whose character traits are essentially eucrustacean but belonging to a group that lacks\nmandibles can be counted as indicating an upper stem eucrustacean or a basal taxon allied to\nMandibulata. It also addresses whether an euarthropod not equipped with antennules but with\nchelicerate ‘great appendages’ could be considered allied to Mandibulata (we show in the\nrevision that it can, as can a lack of mandibles).\nThus, the question what is Jianfengia? To resolve this, we undertook a major revision of the\noriginal study: an exploration of the phylogenetic relationship of Jianfengia within the context of\nChelicerata and Mandibulata. This new phylogenetic section of the manuscript demonstrates that\nJianfengia is indeed sister (thus basal) to total Mandibulata. The other “Great Appendage” clade,\nLeanchoiliidae, is basal and thus sister to Chelicerata. The explanatory power of neurocladistics\nreaching this result further emphasizes that consideration of cone cell numbers was not necessary\nto place Jianfengia where it is now is.\nGenetic studies, which we cited in the original manuscript and again here, indicate that the\ngenetic circuit underling the development of chelicera requires few substitutions to switch the\ncheliceral phenotype to a uniramous non-cheliceral phenotype. Thus, minor genetic innovations\nwould have provided a subsequent jianfengiid-like ancestor with bone fide antennules. But the\nmost interesting finding resolved by the now added neural cladistics is that it demonstrates that\nJianfengiidae being basal to Mandibulata means its ‘great appendage’ must have preceded the\nevolution of mandibulate antennules. (Likewise, Leanchoiliidae being basal to Chelicerata means\nits ‘great appendages’ were ancestral to the arachnid chela).\nThis is a difficult review because, of course, the manuscript presents its own interpretation\nof the anatomical findings. Still, another view in the literature sheds a slightly different\nlight on some aspects. In good academic tradition, I will not attack this manuscript but\nbriefly explain this different perspective. Based on this, I will discuss the results presented\nin the manuscript, and ultimately, the reader may take his or her position\nThis is rather ambivalent comment from someone who most definitely makes the point of\nthoroughly interpreting her own anatomical findings of Trilobite visual systems. We\nassume that Dr. Schoenemann shares with us the assumption (and hope) that readers\n2\nexpect descriptions of anatomy to include the authors’ interpretation of what is\nsignificance. Without authorial interpretations of empirical data all our work would be\nworth far less.\nSo, what has been achieved?\na) Structured eye stalks (-> crown Malacostraca)\nThe joints of the articulated eye stalks can be seen very clearly. The authors indicate\none, and there is another more distally (Fig. 1F). There are mentioned 2 (line 78), and an\narrow should also indicate the second one.\nThe other is of course the margin of the compound eye itself with the eyestalk cuticle but I\nam\nI am not sure if this is a true suture indicating the eye itself is derived from a modified\narticle.\nIt is commonly accepted that Malacostraca possess stalked eyes, and at least among the\nextinct and fossilized I know, the stalks are indeed always articulated. The other way\naround, I would expect that among the early arthropods, the artiopods, for example, we\nmay find articulated eye stalks. So, I would be slightly more cautious with the\nconclusion drawn here [Meanwhile, these articulated stalked eyes have been found and\ndescribed: Schmidt, Schoenemann, et al. 2025, Biology Communications, in press]. So\nprobably articulated eye-stalks are typical for crustaceans (not hexapods, belonging to\nPancrustacea also!), and consequently, not all arthropods with articulated eye stalks are\npancrustaceans. Even the eye stalks of Leanchoilia and Alalcomenaeus are\narticulated, see Fig 1 C,D.\nWe have enjoyed the Schmidt. et al paper. However, there is possibly a misunderstanding.\nWe don’t assert anywhere that arthropods with eyestalks must be indicative of pancrustaceans.\nConsider isopods, for example. Our own studies of Leanchoilia and Alalcomenaeus identified\npendulous eyestalks as did previous studies by others (e.g: doi.org/10.1016/j.asd.2015.07.005).\nWhether they were articulated, and thus had the ability to move (scan) in life is wholly\nunresolved for any fossil as it is for Jianfengia. We make no claims that any ancient eyestalk was\narticulated sensu Stomatopoda (which is the only substantiated example of individual eyestalk\nmotility in any arthropod, living or dead). Of phylogenetic interest is that the presence of sutures\nalong the length of a pronounced eyestalk suggests derivation from an articulated ancestry. That’s\nnot our original idea but was suggested much earlier, particularly by R.E. Snodgrass\n(Comparative Studies on the Head of Mandibulate Arthropods. Comstock, Ithaca, NY. (1951).\nWe do not contest Dr. Schoenemann’s observations of eyestalks in other fossil species. We are\nalso sure that Dr. Schoenemann would agree that fossils cannot provide convincing evidence for\nmotility. Afterall, data about eye stalks are from animals that were likely dead or dying, covered\nwith fine material, and then petrified in time. Who knows whether the moribund attitude of the\neyes displaced their angular disposition vis-à-vis the carapace. Trilobites of course don’t present\nthat problem, fortunately.\nFigure 1 Articulated eye stalks of the artiopod Pygmaclypeatus daziensis Zhang, Han & Shu\n3\n2000, of Leanchoilia, and Alalcomenaeus,\nYes, these are nice examples. In every case the postmortem attitude is that of a pendular\nattachment, which as Dr. Schoenmann has noted are distinct from laterally extended eyestalks\ntypifying Eucrustacea. All we are saying is that Jianfengia’s eyestalks conform to a\neumalacostracan type disposition. We don’t see any conflict with the paper cited above or\nwith Dr. Schoenemann’s own work or others she cites.\na) Compound eyes with ommatidia, revealing tetradic structures (-> cone-\nbuilding Semper cells)\nThe term semper cells defines cone cells of crustaceans and insects. The manuscript\ndevelops the idea of including J. multisegmentalis within the stem Pancrustacea. The\nmanuscript should take into account the initial and provisional nature of the use of\nthis term.\nThe question of the existence of tetradicly arranged cone-cells in J. multisegmentalis is an\nimportant, critical point. Indeed, J. multisegmentalis possesses compound eyes, and the\nloss of cuticular lenses in specimen YKLP 11117 offers insights into the arrangement of\ninternal structures of the ommatidia. Photoprocessing methods were applied to clarify the\nindividual elements. However, if I were to take the position of devil's advocate, I could say\nthat the threshold can be adjusted until 'it fits' (I am sure the authors haven´t done this.\nWe explain in the suppl. methods that obtaining the locations of maximum intensities in a\ncolor image requires not selecting a general “threshold” but choosing a narrow band of the\nimage’s color spectrum. It’s a matter of identifying that part of the color range that is\ndistributed across the sample and hence serves not to discriminate but to find inclusiveness\nin providing the empirical data set. It is not an “until it fits” procedure!\nIt is phenomenal that the authors succeed here in showing the cone cells. The problem\nfor me here is that none of the examples clearly shows 4 cone cells; one may find even\nmore at different sizes and numbers (Fig. 2). That the application of photo-processing\nbrings out the tetraconate pattern for me suggests, in conclusion, that there are more\nthan 4 (~6-8) cone-cells, but some of them , four in number, are more pronounced in\ntheir development than the others. This may indicate that we here are on a transitional\nway from a multiconate eye (-> Limulus-like (Xiphosura, Chelicarata), ~ 100 cone cells,\nFahrenbach 1968, Harzsch & Hafner 2006) to a tetraconate system. But still, it is not\nachieved yet.\nWe agree entirely (see below)\nIn the manuscript, the system here is distinguished from the scutigerid myriapods by\nmentioning one single extension of the cone cell contributing to the secretion of the\ncrystalline cone in tetraconates, while the myriapod possesses two. I am pretty sure one\nmay not find these delicate structures in a fossil. Here, it may help with the argument that\nthe scutigerid ommatidium is developed secondarily from the myriapod ocelli, which in\nturn originated from original ommatidia (e.g. Paulus 1979, 2000).\nthan 4 such prolongations in our own data which surely obviates skepticism.\nMüller et al. (2003) find for the myriapod Scutigera coleoptrata (Linnaeus, 1758), 8 cone\nsegments, produced by four crystalline cone cells; tripartite cones (Ascothoracida and\nCirripedia), and bipartite ones they suggested to have derived from the tetrapartite system. Thus,\nfour cone cells alone may not be sufficient to postulate an assignment to Tetraconata, even if it is\nthe standard definition. At least this should be discussed more differentiated. It may well be that\nnot all cone cells/segments can be found here in the specimens of J. multisegmentalis; in one of\nthe examples presented in the manuscript (Fig. 2,4), there may be evident even 8 elements (D4,\nanother shows up 7, D3), suggesting a tetrapartite origin as in Scutigera.\nWe appreciate Dr. Schoeneman’s expertise regarding the broader presence of cone cells, which\nwe agree must have very deep origins, preceding divergences of Myriapoda and Pancrustacea.\nThat is also stated clearly in the original manuscript and in this revision. Dr. Schoenemann’s\ncommentary above reflects her own scholarship, and we appreciate her request for caution in\nassigning the status of “crustacean” or “myriapod” cone cells. As already mentioned in this\nreply, for this revision we have down-played cone cells as reliable indicators. Instead, this has\npromoted our neurocladistic analysis based on neural or allied traits to determine Jianfengia’s\nphylogenetic position. Notably, to exclude bias we omit traits suggesting numbers or restrictions\nof cone cells. In the same spirit of caution, we have relegated the figure showing possible cone\ncells to the supplementary figures. The neurophylogenetic analysis reveals Jianfengia as sister to\ntotal Mandibulata, which includes Tetraconata and Myriapoda. This excludes any claim that\nJianfengia might be a Tetraconate.\nb) nauplius-like eyes and associated nerves supplying a discrete forebrain region -\n> pancrustaceans\nIt would be great to have them, and I am willing to find them in the figures of the main\ntext; I have tried hard ... I see the filamentous structures, and they may be relicts of a\npalisade of receptors, and I agree with the pigment. Still, please make the 3 nauplius-\nlike eye elements more evident in your main text figures. I find them in the UV (S1 C).\nA probable explanation indeed would be that this was one organ consisting of three\nocellic covered by a membrane, which would explain the homogenous appearance of\nthe whole system under white light. I agree with you that the organ may have been\nmovable. The movable character, I think, results from the not-fixed 'stalk,' which clearly\nprotrudes from the carapace. See the dark line (the margin of the carapace, indicated by\nthe arrows in our Fig 2c).\nWe are very glad that in view of her own expertise Dr. Schonemann agrees with our\nidentification of ocelli and ocellar nerves. We have modified the UV image accordingly\nto show the most plausible fit of three ocelli and this figure now appears where the\nreviewer suggests, namely as Fig. 2.\nThe small element in the reconstruction drawing (main text in Figure 4D) shows the\nsame interpretation as I gave it above and in Fig. C3, which is great, of course, but this\n5\nfinding should be established more clearly in the main text.\nIf we accept that the filaments are relicts of receptors, please note that they do not\nshow any grouping. Typically, a Nauplius eye is a single median eye and consists\nof three- or four-pigment cup ocelli. This nicely can be detected in your Figure S1C.\nI am not sure about the arthrodial membrane here, but I share the idea that this whole\nstructure could have been movable (see above).\nI think these results (3 ocelli, movable system) should be more emphasized and shifted\nfrom the Supplement to the main text.\nSo, we agree with a tripartite, nauplius-like eye, probably built by ocelli.\nWe agree that the evidence for the nauplius eyes is convincing and have now placed this as Fig. 2\nin the main narrative of the work. We thank the reviewer for the suggestion to do so and are glad\nwe concur on this important finding.\nNeural organization\nc) neuropils and tracts in the eye stalks -> nested optic neuropils as occur\nin eucrustaceans\nd) organization of the deuto- and tritocerebrum -> differentiation from\nstem Chelicerata and extinct Myriapoda\nTracing up the neural structures in this tiny organism, neuropils as pathways is\nfascinating, very skillful, and convincing. One should never forget that all these\ninvestigations start in a quarry, and we deal with 'rocks', fossils. The optic neuropils 1-3\ncan be discerned very clearly, the reconstruction of the entire brain for example by\noverlaying the results of different findings and the assumption of a bilateral organism, by\nthat, the methods of reconstruction for completing, for example, by flipping elements of\nthe one side to the other, are justified. The tables (S1, S2) are interesting, and the drawn\nreconstructions (Fig 5) are excellent and consequent. Unfortunately, the inscriptions in\nFigure 5 (as perhaps the whole figure) are too small and hardly readable/discernible.\nWe are glad that an expert on fossilized visual systems finds our identification of neural\ntraces convincing. The revised work now has an expanded analysis of these microscopic\nneural traces, using four specimens rather than just two. We have employed two\nstrategies for reconstructions: one using human tracing “by eye”; the other using\nGaussian amplification of traces comprising dark deposits. It is gratifying that the two\nstrategies converge. These two sets of results are now shown in Figure 3. We also\ninclude a supplementary figure (Supplementary Fig. S1) illustrating how Gaussian blur\ncan be utilized in revealing discrete structures.\nI am not an expert in arthropod brains and the genetic background of the genetic\norganization of an arthropod segmentation, especially the nervous system, but one\ncan follow the given reasoning, and a possible interpretation seems to have\nemerged.\nThat the character identity networks (ChINs) are shared across total Euarthropods\nsuggests a pre-Cambrian origin of the genetic ground pattern of the cerebrum. This will\n6\nbecome clearer as more fossilized brains are discovered and which show evidence of\nstructures specific to one or more of the genetic networks known to be shared across this\nphylum\nIn Figure 3 it is not clear what is from YKLP 11367 and YKLP 17299\nThank you for pointing this out. We have modified the text to clarify this.\nDiscussion\nThe discussion of the manuscript makes the point that the J. multisegmentalis among\nthe stem-arthropods of the Chengjiang biota represents a true pancrustacean, because,\nalthough a trunk tagmatization still is missing, the eyes have a tetraconate ommatidium,\nJ. multisegmentalis possesses as typical for pancrustaceans a nauplius-like eye consisting\nof three elements, and a brain that is organized as in pancrustaceans.\nWe may agree with the latter, but we see a problem with the interpretation of the\neye systems.\nNote that median eyes (-> Nauplius-like eyes, Nauplius eyes = Median eyes of\ncrustaceans) also exist in the megacheiran Leanchoilia! (Garcia-Bellido & Collins 2007,\nSchoenemann & Clarkson 2012, 2023), and indeed in Alalcomenaeus (see below).\nWe may have been too obscure in the original discussion. Our present revision makes it\nclear that when it comes to sorting out phylogenies, two visual systems are recognized\nand coded as two separate systems across Chelicerata and Mandibulata. The median eye\npairs of course exist in Megacheirans. And we note that Dr. Schoenemann has discovered\nthe same rule for Trilobites, thus aligning as an established fact that this arrangement\noccurs across Euarthropoda with the crucial exception of Myriapoda. Along with\nFuxianhuia, myriapods have no rostral eyes. There was no genotypic data available for\nPaulus when he was debating such matters. The absence of ocelli in myriapods appears to\nbe a genuine evolved loss. We refer to the relevant 2015 paper that further describes those\ntwo sets of eyes: the frontal pair belonging to the anterior part of the forebrain, the\nprosocerebrum (as do pancrustacean ocelli and nauplius eyes). The lateral pair (secondary\neyes in short body megacheirans) belongs to the posterior part of the forebrain, the\nprotocerebrum. The presence of two genetically distinct visual systems, one\nprosocerebral, the other protocerebral, has been widely recognized, including by Dr.\nSchoenemann and her co-author regarding the identification of frontal ocellar-like eyes\nand lateral compound eyes in Trilobites. It’s an important work confirming that two\ndifferent visual systems must very ancient. A number of studies of Radiodonta further\nsuggest the immense age of these two systems. We hope that our modification of the\nrevised manuscripts clarifies any ambiguities suggested by the original submission.\nFigure 4 Median eyes of Alalcomanaeus cambricus Simonetta, 1970 and Leanchoilia\nsuperlata Walcott 1912\nThis has implications and should be considered and discussed.\nEven being aware of the work of Tanaka et al. 2013, one may be a bit careful with the\ninterpretation of the eyes of Alalcomenaeus and Leanchoilia. The specimens of\nAlalcomenaeus illustrated by Briggs & Collins (Fig 4.1, 4.4) or of Whittington (1981,\nFig. 126) clearly show that Alalcomenaeus has club-shaped, penduculate compound eyes\n(here Fig. 1D), surmounting articulated stalks; the same is true for Leanchoilia (here\nFigure 1 B, C). Here, the eyes and their stalks are much finer.\nNote that there are median eyes in the megacheiran Leanchoilia (e.g. Garcia- Bellido &\nCollins, 2007; Schoenemann Clarkson 2012) and in Alalcomenaeus also (e.g. Briggs &\nCollins, 2007, Fig. 4.3), as can be seen clearly here in Figure 4. This has implications and\nshould be considered and discussed - for example, consequently, Jianfenigia is NOT the\nonly Early Cambrian genus identified with median eyes (and crustacean-like eyestalks).\nPerhaps J. multisegmentalis is among the first with 3 median eyes. Still, there are\ncrustaceans with 4 ocelli in their median eye complex, e.g. among the branchiopoda (e.g.,\nReimann & Richter 2007) – so the number alone may is not game-changing here.\nRegarding these two paragraphs: we are not sure why Dr. Schoenemann is emphasizing these very\nclear distinctions. They are well known to many working on arthropod vision including ourselves.\nSo, we do not know where or what in our manuscript would elicit those remarks. Our descriptions\nof the megacheiran visual system fully accords with Dr.Schoenemann’s views and the papers she\ncites. We have published elsewhere that Jianfengia’s visual system is described as one example\ncommon to Jianfengiidae, which includes Fortiforceps foliosa and Pseudoiulia (Sklerolibyon\nmaomima). Again, nowhere are we claiming that Jianfengia is the only Early Cambrian genus\nidentified with median eyes and crustacean-like eyestalks. That is explicit in the present work.\nIt is, however, crucial to distinguish eyes on robust eyestalks, which is a trait of all three species of\nJianfengiids. Further, we took care to describe the range of ocelli that occur across pancrustaceans,\nbearing in mind that in insects, and probably in eucrustaceans, two pairs of embryonic precursors\neach side of the midline provide either 4 ocelli, or multiples of four as in some reptantians, or three\nocelli as in Hexapoda, the middle ocellus being a developmental fusion of the two embryonic\nocelli either side of the midline. Thus, we see no differences of opinion or conclusions between\nour description and the works cited by Dr. Schoenemann or her own studies. The same goes for\nLeanchoilia and Alalcomenaeus except theirs’ are not strictly “median eyes” but are prosocerebral\nprincipal eyes, both of those genera being sister to Chelicerata.\nWe agree, however, completely with the idea that genetically based the morphological\nvariance of homolog deutocerebral appendages is wide enough to comprise great\nappendages, antennules formed as 'pinchers' or pier-like antennules, or ...\nIn general, we also agree with the interpretations of the neural systems and the homologies\nexplained here. I am not an expert in arthropod nervous systems, but I think the deductions\nare reasonable.\nWe are glad that the reviewer agrees. We suggest viewing Figure 4 of the revised\nmanuscript. It reveals striking correspondences of the jianfengiid cerebrum with the optic\nlobes and circum-stomodeal pathway of both malacostracan and brachiopod arrangements.\nFinally, one of this manuscript's great conclusions is that not the Hox-gene-associated\n8\ndivergences of the segmented trunk define ancestral and crown arthropods, but do the\nsegmental cerebral domains, the unique lineage-specific neuropils, and associated traits.\nThis is an important point, but our conclusion differs slightly from what the authors\nindicate here.\nIt is a pleasure to read that comment. It is one made by a biologist rather than a\npaleontologist who might not be so conditioned with regard to gene expression studies\nthat distinguish genetic determination of the trunk versus the cerebrum. We are not\nclaiming the cerebrum is segmented. Snodgrass clearly rejected that in his embryological\nstudies where he observed no true segments in the cephalon, his nomenclature for the\ncerebrum. Heymons, much earlier in 1901, published the same observation from\nstudying embryonic development of Scolopendra.\nEven if one of the characteristics (like those of the nervous system as here) has passed\nthe 'finish-line' to reach a crown group state, while others have not (tagmatization of the\nbody), one has to be aware that as here, obviously boundaries of the stem-/crown groups\ncannot always be rigidly defined, and that there are transitional situations (as represented\nin the eye-status of Jianfengia multisegmentalis (Hou 1987)). Here, one part (brain\nsystem) has reached that stage, and the other (tagmatization of the body) still has not; the\neye system of Jianfengia multisegmentalis (Hou 1987) is on its way. This may have\nimportant implications for describing evolutionary processes in general.\nExactly. The cardinal observation. Thank you.\nThe results here show that the formation of different crown-group characteristics\n(tagmatization of the trunk – crown–group-nervous-system – eye system) may evolve with\ndifferent velocities. An important point because one now will have to weigh when the\ncomplete, or a 'sufficient' crown-group-state has been arrived. Probably it is not adequate if\nonly one of the characteristics has passed the 'finish line'. Even if the focal point in the\nconsiderations of this manuscript has been laid on the eyes and brain/nervous structures, it\nmay be an essential fact that the tagmatization of the body is not quite there yet.\nNevertheless, the authors are right to state that neuroanatomical traits could provide a\npowerful tool for distinguishing characteristics to establish euarthropod relationships,\neven if external traits that differentiate identities are absent.\nIndeed, all these considerations are important. And they open interesting avenues down the\nroad. Hence the utility of providing neural phylogenetics that implies sequential branches of\ngradual change towards crown morphologies.\nReviewer #2 (Remarks to the Author):\nThis manuscript reports the putative findings of neural traces on a few specimens of a\nCambrian great-appendage arthropod: Jianfengia multisegmentalis. This species has been\ndescribed a few decades ago, even though their phylogenetic position within arthropods\nwas not clear. This is not the first time that preservation of the neural tissue in\nCambrian arthropod has been used to determine their phylogenetic position. To my knowledge,\nthough, this is the first instance in which a great-appendage arthropods has been clustered\ntogether with the groups of crustaceans and hexapods based on these kind of data. Based\non this and previous reports, the authors argue that neuroanatomical traits can be used as\nproxies to understand the placement of these fossils in the absence of external traits,\nassertion that I agree with.\nWe are very glad that the reviewer endorses that strategy.\nThe authors present a brain and nervous system reconstruction of this species after having\nsuperimposed images of neural traces of different specimens and changing illumination\nparameters. I am not an expert in image treatment but I do not see any apparent introduce\nsoftware artifact. I find this manuscript as well articulated and with conclusions grounded on\nthe data presented. The images reporting the compound eyes and the neural organisation is\nconsistent with pancrustacean neuroanatomy. I would like to see a bit more of discussion\nregarding the paraphyly of Megacheira. The phylogenetic placement of Jianfengia as an\neucrustacean implies that great appendage arthropods can fall either within chelicerates or\npancrustaceans. If that is the case, these large appendages were convergent? Lastly, I feel it\nmigth be interesting to show in Fig. 3 or 4 stainings of the homologous cephalic area in one\nextant Eucrustacean species.\nThese comments are much appreciated as is the suggestion that we provide homologous\ncephalic arrangement in extant eucrustaceans. This we have done, and it is now shown in\nFig. 4a, and discussed in the main narrative. Regarding the distinction of Megacheira:\naccepting the metric distinctions of the short bodied Leanchoiliidae and the\nmultisegmented Jianfengiidae, our identification of neural traits in the latter allows us to use\nthese and those already documented for Leanchoiliidae to explore Jianfengia’s phyletic\nposition within the euarthropod tree. Our neurophylogenetic analysis comprises one of the\nmajor revisions of this study as it allows us to put to one side the somewhat ambiguous\nimplications of the presence of cone cells. Those ambiguities, raised by one of the reviewers,\nrequired a phyletic reassessment in ascribing (or rejecting) a pancrustacean-like identity to\nJianfengia. Employing neural cladistics while omitting cone cell numbers as an unreliable trait\nprovides a crucial finding. Namely, cladistics places Jianfengia as sister to total Mandibulata\nwhereas Leanchoilia and Alalcomenaeus together representing short-bodied\nmegacheirans that occur as sister to total Chelicerata. This addition to the present study\nadds an important and possible novel observation regarding the evolutionary relationship\nbetween Jianfengiidae and Leanchoiliidae within the overall organization of the two major\nbranches of the euarthropod tree of life. It also raises the interesting possibility that\n“Megacheira’ should no longer be considered a clade but instead the two types of ‘great\nappendages’ as convergent character traits of deutocerebral appendages. We address this\npossibility in the revised discussion of the manuscript.\nLines 57: typo \"barely1mm\nOK\nLines 391: typo \"Jiangfengia\"\nResolved\nLine 445: typo \"(C)\" should be (D)\nResolved\nReply to Reviewer #3\nA pancrustacean brain and its visual systems define a Cambrian “great appendage”\narthropod.\nby Nicholas J. Strausfeld 2*, Xianguang Hou 1, and Frank Hirth\n3*\nThis MS presents amazingly detailed comparisons between the neural structures of a\nCambrian megacheiran arthropod and those of modern crustaceans. The biological data\nare all of high quality.\nWe appreciate that the reviewer approves the quality of the presented biological data.\nThis approval complete contradicts the ensuing criticisms which revolve around the\nmisconception that we claim that traits defining the jianfengiid brain correspond to\nthose of extant Eucrustacea. This was not the case in the submitted ms and is further\nemphasized in the revised manuscript.\nHowever, most interpretations of the fossil species are based on questionable often\nvery weak fossil evidence (see examples below). The extensive use of image\nprocessing (e.g. Photoshop) is sometimes misleading. There is a huge difference\nbetween the idealized processed images and reconstructions, and what we actually see\nin the fossil specimen.\nWe expect that a reviewer would consult extensive supplementary material for an\nunbiased assessment of the reported findings. There seems to be a misunderstanding\nhow fossil neural traces are imaged and reconstructed. The revised work now has an\nexpanded analysis of the microscopic neural traces, using four specimens rather than\njust two. We have employed two strategies for reconstructions: one using human\ntracing “by eye”; the other using Gaussian amplification of traces comprising dark\ndeposits. It is gratifying that the two strategies converge. These two sets of results are\nnow shown in Figure 3, their neuroanatomical interpretation in Figure 4 and their\ninterpretation in a phylogenetic context in Figure 5. Our detailed description of how\nGaussian blur can be utilized in revealing discrete structures is described in the\nmethods section and further detailed in the legend to Fig. 3. This was explained in the\noriginal submission, and has been understood by the three other reviewers, according\nto their commentaries.\nJianfengia is interpreted as a lower stem pancrustacean, based on the assumed structure\nof its nervous system. However, its appendage structure (head appendages) is\ncompletely different from that of pancrustacean. The authors do not take into account\nthese key anatomical aspects.\nNeither in the original submission nor in this revision do we claim Jianfengia as a lower stem\npancrustacean. Our now extended analyses showing correspondences with extant taxa identifies\ntwo neuropil arrangements, one malacostracan-like, the other branchiopod-like which may\nsuggest an intermediate evolutionary stage of the Jianfengiid cerebrum. This interpretation is\nfurther supported by newly added neurocladistics which focus on neural traits and cephalic\nappendages that resolve Jianfengia as sister to total Mandibulata. These correspondences are\n1\nnow provided in an amended Figure 4. The newly added phylogenetic inferences are presented in\nnew Figure 5 and supplementary Figure S7.\nLine 45- What kind of digital adjustments ? This is a key point. Please give details.\nThe methods applied were described in great detail in the original submission and again here in\nthe results section “Reconstructing the brain of Jianfengia” and in the methods section “Isolating\nneural traces” and in figures 3 and 4, as well as in the supplementary figures.\nLine 48- The plural of retina is retinae (Latin)\nThe conditions for using the word retina does not draw on the latinized plural in the context of\nstudies of visual systems. There the technical plural is ‘retinas’.\nLine 61- I agree that the frontal rounded feature is the anterior sclerite (ASC). It appears to be a\nsclerotized feature comparable with other hard parts of the exoskeleton (see figure B below).\nHowever, I see no convincing evidence of an underlying “system of lenses”. Clearly this\nbrownish feature is over-interpreted. I am saying that soft tissues (including sensory organs)\ndo not occur underneath the sclerite but they are not visible in this fossil specimen.\nTo support the allegation of over-interpretation the reviewer appends blurred out-of-focus copies\nof small images from our submission . The large, detailed figures available in the supplementary\nmaterial demonstrates that we interpret, not overinterpret. Hence the reviewer’s comments are at\nodds with the collective opinions of the three other reviewers. Also, we do not refer to an\n“underlying system” of lenses: lenses comprise a dioptric system overlying photoreceptors.\nThese are clearly indicated in the enlargement of what is now Figure 2.\nLine 62 + “Their detailed organization suggests structural homology to crustacean nauplius\neyes (8, 9) and insect ocelli (10, 11) (Fig. 1C, D; SI Appendix Fig. S1) comprising three lenses\noverlying a palisade of photoreceptors” This statement is speculative considering that no\ndetailed organization can be seen in the fossil specimen in question.\nDetails were provided in the original submission’s supplementary information, which is now Fig.\n2. We are confident in our identification of ocelli and their cognate central projections, as are the\nother reviewers of this work. Nor do we use the word “speculate” but use the word “suggest”\nindicating reasoned interpretation.\nLine 65- Please explain what you mean by superimposition\nThe Oxford Dictionary’s definition of the word is: “the action of placing or laying one thing over\nanother, typically so that both are still evident.” We note that three other reviewers all agree on\nour demonstration of ocelli. And following the recommendation of one reviewer who suggested\nthat this is such significant evidence that it ought to be placed in the main body of the paper, we\nhave accordingly done so (now Figure 2).\nparagragh) are more convincing fossil evidence.\nDirect demonstration of gene expression cannot be shown for a fossil. As explained in the\noriginal submission and again here, domains of the forebrain and midbrain are defined by the\nunique combinatorial activity of gene homologs as Character Identity Networks (CHINs). The\nChIN underlying ocelli formation and their nerve projections in extant pancrustaceans define the\nprosocerebrum. Thus, evidence that fossil ocelli send axon projections into the most anterior part\nof the Jianfengia brain suggests the same organization as in extant pancrustacea. It allows the\nassumption that the prosocerebral genetic program was already in action in the Cambrian (if not\nmuch earlier). This also applies to identifying the fossil protocerebrum: we ascribe terminals\nfrom the compound eyes as defining its protocerebrum because it receives inputs that underlie\nthe protocerebrally ChIN. This is (and was) explained in the narrative of our paper.\nLine 76-77. The lateral eyes are well-preserved. However, this type of eyes occurs in numerous\nCambrian arthropods that clearly have no affinities with eumalacostracans (e.g. Artiopoda such\nas Acanthomeridion; also Isoxys, a basal megacheiran or even Tuzoia, that have no close\naffinities with pancrustaceans)\nThis comment is factually incorrect. Compound multi-ommatidial eyes are not demonstrated in\ntaxa the reviewer cites. The eLife 2024 preprint of Acanthomeridion\n(doi.org/10.7554/eLife.93113) shows paired eyes, one enlargement of which appears to have lost\nits entire outer surface: no evidence or mention of facets provided. Searching for demonstrable\nfaceted eyes claimed by the reviewer failed us: Tuzoia as well as Odaraia have pedunculate\neyes; but facets are not demonstrated. Perfectly round profiles are crowed the perimeter of Isoxys\neyes; no crustacean-like hexagonal arrangement is demonstrable; there is no evidence of\nommatidia. Further, regarding affinities, there’s not yet a phylogenetic analysis of Tuzoia, Isoxys\nor Acanthomeridion based on neural/sensory characters that would resolve the systematic status\nof those taxa.\nLine 85. The light spots seen in Fig. E are interpreted as possible ommatidia However, similar\nspots seem to occur elsewhere in the specimen. Unfortunately, I have no access to the high-\nresolution version of the text-figures. It is important that the authors provide comparative high-\nresolution images of the lateral eye and other areas of the fossil (see below). I would suggest to\nprovide back-scattered SEM images of the eye. It has been done with the eyes of Waptia from\nthe Burgess Shale and clearly revealed the outline of ommatidia that do not appear in light\nphotographs (see Vannier et al. 2018).\nEnlarged images of the compound eye were included in the suppl. Data. High-res figures were\navailable with the initial submission and this one, as is the policy of the journal.\nBackscatter images in the cited Vannier work show no evidence that they belong to an eye. The\nimages provide dark patches claimed as hexagonally arranged. They do not appear to be which\nwould disqualify them as true compound eye. Nor could they be when none of the published\nWaptia specimens (doi.org/10.1098/rsos.172206) is preserved with a flattened ocular surface.\nBST preservation. But BSEM is inappropriate for the mudstone Chengjiang matrix. BSEM was\noriginally developed for analyzing crystalline/chemical organization in geological samples.\nUsing it on fossils risks misidentification crystalline deposits as organic traces. The reviewer is\nthus disingenuous in claiming that ommatidia in Waptia do not appear in light (sic) photographs\nbut only in SEM images.\nLine 87- I don’t think many readers will be convinced by images processed by Photoshop.\nIndeed, you cannot “determine if these are taphonomic artifacts or that they represent\nfossilized cellular elements of the ommatidia” by using image processing.\nPlease use other methods to reveal possible underlying retinal structures such as SEM. You may\nalso use a Keyence digital microscope. Although the preservation mode of your specimen is\ndifferent, SEM studies clearly revealed the inner structure of the eye of Jurassic\nthylacocephalans (see Vannier et al. in Nat. Comm)\nThe color of specimens of Jianfengia are typical of Chengjiang fossils. Digital color cameras\nrecord colors. The “Keyence” microscope cited by the reviewer is a proprietary name for one of\nseveral mid-high resolution digital microscopes available at the Yunnan Key Laboratory for\nPaleobiology (YKLP).\nBar one (Fig. 3f) images of fossilized neuropils in the present work were taken at the YKLP\nbetween 2012 and 2018 using instrumentation there that provided color figures shown in eight\npapers listed below published by NJS and coauthors. In challenging the veracity of images in\nthe present account, the reviewer challenges the veracity of images published in: Nature 2012,\n2013, 2014; Phil. Trans R. Soc, 2016; Current Biology 2015, 2016; and Science 2022, 2023.\nThe reviewer appears to assume that all fossil material is alike. It is not. Those working on\nChengjiang biota know that secondary weathering gives rise to coloration and that coloration is\noften fortuitously constrained to specific organ systems or exoskeletal attributes.\nWe would not partially destroy fossil material just for using SEM on a substrate that is wholly\nunsuitable for that technology. Chengjiang mudstone is not equivalent to BST preservation. Nor\nwould we want to split open a jianfengiid eye to emulate Vannier’s highly fortuitously fractured\nDolocaris specimen (Fig. 7: https://doi.org/10.1038/ncomms10320).\nLine 90 + Fig. 2C is supposed to show the inner structure of assumed ommatidia. Unfortunately\nthese processed images fail to provide evidence that a consistent 4-fold structure occurs in these\nassumed ommatidia. Honestly, I am not convinced. The outlines of these “light spots” are\nirregular and blurred. Again, I suggest the authors to use different methods (see above).\nAgain, one would not try and split open the retina of what is the best fossil of a species for\nwhich there are less than a dozen specimens.\nFigs D-F are also problematic. The risk with image processing is to make features appear as you\nwant them to appear.\nin the methods section “Isolating neural traces”; and in figures 3 and 4, as well as supplementary\nfigures S1 and S4. Suggesting that the authors “to make features appear as you want them to\nappear” is a comment unworthy a scholarly review.\nLine 95 Some spots seem to be more pentaradial than tetraradial ( see “3” below)\nOut of four visible groups of putative cone cells, one may be pentaradial, another may be\nseptaradial and two tetraradial. Applying Occam’s razor, our simplest interpretation is that what\nwe see are likely tetrads, accounting for two outliers not being coaxial with that of the\nmicroscope objective. However, we agree that there may be ambiguities that may be broadly\ninformative, promoting the question whether Jianfengia is ancestral to Myriapoda and\nPancrustacea. Thus, at this stage of jiangfengiid evolution there was no fixed number of Semper\ncell prolongations. Toward resolving this (and other aspects of the phylogenetic position of\nJianfengia) we modified the summary schematics and carried out phylogenetic analyses using\nprimarily neural traits (but not cone cells), following early accounts demonstrating their use for\nreconstructing brain evolution (doi:10.1098/rspb.2006.3536; also, doi.org/10.1016/j.asd.\n2011.02.002). This present revision (for data see Supplementary Information) has elucidated the\nphyletic position of Jianfengia as sister to total Mandibulata.\nThe reconstructions are unfortunately very speculative and based on no solid fossil evidence.\nWhat is said about the eyes of modern crustaceans is perfectly correct. The main concern with\nthis MS is the lack of strong fossil evidence.\nOur study is based on empirical data provided by extremely small fossils. That is quite an\naccomplishment in itself (see scale bar in Fig. 1a) and we use all the empirical information that\nthe specimens supply. To claim that the reconstructions are “very speculative” is without merit\nas is demonstrated in Fig. 3: this is not speculation but methodical analysis.\nI really have a problem with these two images. The upper one shows 5 “light areas” but only 4\nappear in the lower image. It looks very odd to me.\nIt is essential to understand the requirement of selecting a narrow band of the available color\nrange that would best transfer to intensity measurement across the image.\nLine 107 + I agree that there are ganglia (e.g. represented by dark area in B and F.\nHowever, the assumed paired labral ganglia are not visible. What we see in this area is a vague\ncluster of dark spots that could represent any kind of soft tissues (see Aria et al. in Bioessays).\nAgain, the weakness of this MS is the lack of fossil evidence and the overinterpretation of most\nfeatures.\nThis paper does not discuss ganglia. There is evidence of paired labral neuropil: not a pair of\nganglia.\nCondensed black deposits in the fossil cephalon comprise material evidence. This is what we\nmust work with and interpret in the light of what is known about the euarthropod cerebrum, its\nlabral neuropils and their evolution in euarthropods. Other studies demonstrate the ground\npattern location of the labral neuropils, explaining this feature and its occurrence (see Ortega-\nHernández and Wolfe: https://doi.org/10.1016/j.cub.2021.08.065).\nPlease provide details concerning the method used to obtain this image (below). Apparently\nit was obtained via the juxtaposition of one side of the head with its mirror image. This leads\nthe reader to believe that the structures are indeed perfectly symmetrical and paired. Again, there is a\nhuge difference between this idealized image and what we actually see in the fossil specimen.\nThis way of showing results is unfortunately somewhat (not intentionally) misleading.\nThe image shown by the reviewer is of the specimen YKLP 11367. The image is asymmetric due\nto its slight a-p rotation thereby revealing two different levels of traces. It is not “idealized.”\nEuarthropods are members of Bilateria so how could this image be a mirror image juxtaposition?\nIs there any trace of nerves that would belong to the great appendage?\nYes, there are, and these are indicated in Fig. 4a. This question is somewhat surprising\nconsidering that the reviewer earlier questioned why great appendages weren’t considered by us\nin this paper. They appeared as supplementary figure in the original submission (they are shown\nagain here as suppl. Fig. S1) and the possession of great appendages by Jianfengia occupied a\nconsiderable portion of the discussion as it does again in this revision.\nLine 163- The authors go too far in their interpretations (see discussion part) that are actually\nbased on very weak evidence (see my remarks above). I don’t think Jianfengia is a lower stem\npancrustacean. Please look at its appendages (see other papers; e.g. number and shape of head\nappendages). They have nothing to do with the appendages of a stem-crustacean (no mandible,\netc..)\nAs stated earlier, both in the original submission and in this revision, we nowhere claim(ed )\nJianfengia as a lower stem pancrustacean but as a possible evolutionary intermediate with\nmalacostracan-like and other branchiopod-like neuropil arrangements (see revised Figure 4).\nWe discuss the lack of mandibles and the absence of trunk tagmatization, in terms of the likely\nunderlying genetics and in the context of arthropod evolution. Both are consistent with our\ninterpretation of the reconstructed brain of Jianfengia and its evolutionary relationship within\nEuarthropoda as revealed by a newly added neurocladistics analysis.\nFIGURE 5 would really be an excellent one if the authors had sufficient fossil evidence.\nThe comparative drawings of extant species are perfectly correct and very clear.\nThe reviewer claims that the fossils are incorrect whereas the two extant species are perfectly\ncorrect. It is converse: the fossils are correctly depicted but there were two errors regarding\ngnathal segmentation of the two extant species. These have now been corrected.\nReply to Reviewer #4\nReviewer #4 (Remarks to the Author):\nThis study analyses four specimens of the species Jianfengia multisegmentalis and provides\nnew anatomical information about them thanks to the use of UV fluorescence and\nenhanced contrast methodology. This has led the authors to describe three main new\ncharacteristics of Jianfengia neuroanatomy, which establish a link to Pancrustacea, thereby\nimproving our knowledge of pancrustacean neuroanatomy and the inner relationships of\nmegacheirans. The three major findings of the paper are:\nWe appreciatie reviews such as this one that are open, thus obviating anonymity. We also\nappreciate that this reviewer endorses our use of UV fluorescence and contrast\nmethodology. In reaction to one anonymous reviewer who considered enhanced contrast\nreconstructions as “idealized overinterpretations” we have included in this revision a\nreconstruction using the conventional strategy of “tracing” to underline the difference of\nthe conventional method – which is unable to exclude “expectation bias” – with the\nenhanced method endorsed here, which is virtually “hands-off.”\n1. The structural homology of Jianfengia anterior sclerite with crustacean nauplius eyes.\n2. The presence of Semper cells in the lateral stalked eyes.\n3. The nauplius-like eyes of Jianfengia and its nervous system are connected only in\nthe forebrain region, in a manner similar to pancrustaceans.\nThe methodology, though I do not consider myself an expert on UV fluorescence, the\nresults, and their interpretation and discussion are solid. UV optical techniques have been\nfrequently used on similar materials to describe the nervous system and, once again, prove\nto be effective.\nAgain, we appreciate the endorsement of the methods used by us.\nOut of the three main results described, I have struggled only with identifying the triplet of\nlenses in the anterior sclerite (see below). However, due to the more easily identifiable\nradiated structure of the photoreceptors, which I found clear, I do not question the\ninterpretation of the anterior sclerite as homologous to the nauplius eyes. Therefore, I\nconcur with the three major findings of this work.\nWe are very glad to have your agreement with the main findings. We have now\nsuperimposed the outline of three lenses, commenting that they would be the most\nplausible arrangement with reference to what we know from extant examples of\nocellar/nauplius dioptrics.\nI also believe that the contribution of this study to our knowledge of early euarthropod\nneuroanatomy, and its role in phylogenetic assessment, has the potential to significantly\naid in resolving the relationships of major clades and in clarifying megacheiran\nclassification. This contribution is more than significant and is likely to be useful for further\nstudies. As such, I recommend its publication in Nature Communications.\nThe major revision of this work has been in identifying empirical evidence of neural and\ncephalic traits that can be used for neurocladistics to resolve evolutionary relationships\namong stem and crown Euarthropoda. As shown in Figure 5 of this revision, this has\nrevealed the position of two major subgroups of paraphyletic Megacheira: Jianfengiidae as\nsister to total Mandibulata; and Leanchoiliidae sister to total Chelicerata. This finding raises\nthe interesting possibility that “Megacheira’ should no longer be considered a clade but\ninstead the two types of ‘great appendages’ as convergent character traits of deutocerebral\nappendages. We address this possibility in the revised discussion of the manuscript in the\ncontext of euarthropod evolution.\nNevertheless, I suggest a minor revision to improve the exposition of the results and\nto enhance the discussion in some areas.\n-Lines 33-35: Add citations for Myriapoda, the oldest evidence of neuroanatomy.\nThe revision puts these elsewhere, but the citations are now present.\n-Lines 51-52: I would smooth this phrase and say something like, “Together, these findings\nsuggest Jianfengia as a stem euarthropod closer to pancrustaceans than to both Fuxianhuia\nand Alalcomenaeus.” Again, it is not that I am not convinced of the results, but rather that\nthere is conflicting evidence in other characters.\nWe wholly agree. And our implementation of neurocladistics (see Fig. 5) together with\nan amended discussion has, we hope, clarified this .\n-Line 57: \"barely1mm\" should be \"barely 1 mm.\"\nOK\n-Lines 99-102: Rephrase and possibly split the sentence for better readability.\nOK\n-Line 391: Missing caption for le.\nCorrected\n-Figure 5: It would benefit from the addition of a schematic of Fuxianhuia. Also, consider\nusing a hand-drawn cladogram based on your findings and the literature to illustrate the\npossible evolution of the anatomies depicted.\nThere are still some open questions regarding whether Fuxianhuiids possessed an\nintercalary (tritocerebral) ganglion, or whether their caliper-like paired postoral appendages\nrelate to the mandibular segment T2. Caution suggests that including Fuxianhuia in Figure 6\nmight be premature. However, the outcome of our neurocladistic analysis nevertheless\nresolves the phylogenetic position of Fuxianhuia as sister to Myriapoda within Mandibulata.\n-Figure S1: Please provide better contrast for the triplet of lenses, at least in the drawing.\nThe structure is difficult to discern here and in Figure 1. Maybe add schematics to help the\nreader visualize what you see.\nFigure S1 has now been shifted to Fig. 2 with a superimposed outline of what is the most\nplausible arrangement of the lenses, namely three arranged over a palisade of\nphotoreceptors. The original figure 2 has now been placed in the supplementary data.\n-I strongly encourage the author to add a paragraph in the discussion about the possible\nimplications of ontogeny for the anatomy of the nervous system and eyes in Jianfengia.\nConsidering the possible life stage of the studied specimens, as well as of the other key\ntaxa, could provide a clearer understanding of the evolutionary forces at play. Since many\ncharacters change during ontogeny, we do not always know whether the specimens\nrepresent fully developed adults, and character development may occur with shifts in\nrelative timing among different groups. Addressing this aspect could help reconcile some of\nthe conflicting signals observed in different traits (e.g., antennae/great appendage\nexpression) and offer a clearer perspective on the mosaic evolution seen among Fuxianhuia,\nAlalcomenaeus, and Jianfengia itself.\nWe agree and have included the question whether the two neuropil arrangements, one\nmalacostracan-like, the other branchiopod-like might suggest an intermediate\ndevelopmental stage of Jianfengia. The (here cited) 2014 paper by Liu et al. on development\nstages of Leanchoilia suggests that at least short-bodied megacheirans likely underwent\ndirect development. We now note that the much rarer Jianfengia specimens all suggest\nidentical segmentation and identical dimensions of the carapace/appendages: these imply\nthat the few specimens that exist likely represent at least the same stage of development\neven if not fully mature. But there is no evidence of “Orsten-like” intermediaries suggesting\nan anamorphic development nor have – thus far – small versions of Jianfengia been\ndescribed.\nI do not wish to see the manuscript again, thus I will be happy to do so if the editors will\nconsider it relevant.\nLorenzo Lustri\nResponses to final requests/remarks from reviewer 1,2:\nReviewer #1 (Remarks to the Author):\nThis has become an excellent paper now. With great respect, we note that following our\nsuggestions, this paper has been rewritten and has been changed in crucial points, as assigning\nJianfengia basal, but not within the Mandibulata, because the cone-cell number had not yet\nevolved within Jianggegiidae. Now Jianfengia’s position within Euarthropoda is established\nthrough a solid, diligent, and careful cladistic analysis, and in particular neuroanatomical and\ncephalic traits. The results are absolutely convincing and well exposed. Perhaps one could\nhave more emphasized that Jianfengia obviously has a transitional state between stem and\ncrown-group arthropod, because it bears features of both (number of cone cells vs, brain\nsystem). Great work – my congratulation.\nWe further thank the reviewer for her scholastic assessment and creative suggestions\nI found some small points:\nLine 32 end: a “t” too much\ncorrected\nLine 92, 93: This organization corresponds to eyestalk morphology typifying eumalocastracan\ncrustaceans and ...\nWe will find out later (l. 152 ff) why this is mentioned here, but at this point it may seem a\nbit suggestive. I would suggest to mention that these stalked eyes, as typical of other\nCambrian arthropods, also, correspond to those of modern eumalacostracan crustaceans ...\nWe have modified this to add clarity that may have escaped reviewer. Thus:\n“This organization corresponds to eyestalk morphology typifying eumalacostracan\ncrustaceans15...”\nLine 153: Both -> both\nThe reviewer seems to have mistaken the section heading as the introductory sentence of\nthe section, which indeed starts as Both...\nReviewer #2 (Remarks to the Author):\nThe authors addressed successfully all questions and concerns of the four reviewers. I already\nliked the previous version, but after applying the suggestions by different experts I consider this\nmanuscript as more robust, and I would like to see it published in its current form.\nResponse to reviewer\nWe thank this reviewer for their strengthened endorsement.\nReview\nPD Dr. Brigitte Schoenemann\nUniversity of Cologne\nDept. of Zoology (Neurobiology/Animal Physiology)\nBiocentre, Zülpicherstrasse 47b\n50674 Cologne/Germany\nB.Schoenemann@uni-koeln.de\nNature Communications\nA pancrustacean brain and its visual systems define a Cambrian “great appendage”\narthropod.\nNicholas J. Strausfeld, Xianguang Hou, and Frank Hirth\nAs its predecessors, this manuscript succeeds excellently in revealing fossilized nervous\nsystems, sometimes, as here, in very small organisms and more than half a billion years\nold. The authors aim to show that coevally lived stem and crown arthropods during the\nEarly Cambrian in the Chengjiang Biota by the example of Jianfengia multisegmentalis\n(Hou, 1987), a not (completely?) tagmatized arthropod. Two more characteristics should\nbe fulfilled to assign J. multisegmentalis to pancrustaceans– a tetraconate crystalline\ncone and a pancrustacean brain. We agree with the nervous system's interpretation but\nconsider the discussion of the eye systems as problematic. Finally, we come to a\nconclusion, different from the authors, as stated below:\nThis is an ambitious manuscript that aims to show that Early Cambrian fossils from the\nChengjiang Biota reveal recognizable crown groups, namely representatives of\ntertaconates/panarthropods, already existing coevally with early stem arthropods (e.g.\nFuxianhuia protensa Hou,1987 [stem mandibulate], Alalcomenaus [stem ch elicerate]) more\nthan half a billion years ago.\nThe authors describe 3 specimens of the genus Jianfengia multisegmentalis (Hou, 1987).\nTo investigate neural traces indicating shapes of brains, ganglia, and neural pathways\nwithin a carapace barely 1mm wide may be considered quite a challenge. Despite the\nabsence of trunk tagmatization, the latter would be typical for pancrustacea/tetraconates;\nin addition, the authors aim to demonstrate typical traits of pancrustaceans in J.\nmultisegmantalis to provide a 'stemward' identity towards Pancrustacea/Tetraconata as\nthere are:\na) Structured eye stalks (-> crown Malacostraca)\nb) Compound eyes with ommatidia, revealing tetradic structures (-> cone-\nbuilding Semper cells)\nc) nauplius-like eyes and associated nerves supplying a discrete forebrain region ->\npancrustaceans\nd) neuropils and tracts in the eye stalks -> nested optic neuropils as\noccur in eucrustaceans\ne) organization of the deuto- and tritocerebrum -> differentiation from stem\nChelicerata and extinct Myriapoda\nIf successful, the authors would show that neuroanatomical traits could provide a\npowerful tool for distinguishing characteristics to establish euarthropod relationships,\neven if external traits that differentiate identities are absent.\nThis is a difficult review because, of course, the manuscript presents its own\ninterpretation of the anatomical findings. Still, another view in the literature sheds a\nslightly different light on some aspects. In good academic tradition, I will not attack this\nmanuscript but briefly explain this different perspective. Based on this, I will discuss\nthe results presented in the manuscript, and ultimately, the reader may take his or her\nposition\nSo, what has been achieved?\na) Structured eye stalks (-> crown Malacostraca)\nThe joints of the articulated eye stalks can be seen very clearly. The authors indicate one,\nand there is another more distally (Fig. 1F). There are mentioned 2 (line 78), and an arrow\nshould also indicate the second one.\nIt is commonly accepted that Malacostraca possess stalked eyes, and at least among\nthe extinct and fossilized I know, the stalks are indeed always articulated. The other way\naround, I would expect that among the early arthropods, the artiopods, for example, we\nmay find articulated eye stalks. So, I would be slightly more cautious with the conclusion\ndrawn here [Meanwhile, these articulated stalked eyes have been found and described:\nSchmidt, Schoenemann, et al. 2025, Biology Communications, in press]. So probably\narticulated eye-stalks are typical for crustaceans (not hexapods, belonging to\nPancrustacea also!), and consequently, not all arthropods with articulated eye stalks are\npancrustaceans. Even the eye stalks of Leanchoilia and Alalcomenaeus are articulated,\nsee Fig 1 C,D.\n[FIGURE REDACTED]\nFigure 1 Articulated eye stalks of the artiopod Pygmaclypeatus daziensis\nZhang, Han & Shu 2000, of Leanchoilia, and Alalcomenaeus,\nb) Compound eyes with ommatidia, revealing tetradic structures (-> cone-building\nSemper cells)\nThe term semper cells defines cone cells of crustaceans and insects. The manuscript\ndevelops the idea of including J. multisegmentalis within the stem Pancrustacea. The\nmanuscript should take into account the initial and provisional nature of the use of this\nterm.\nThe question of the existence of tetradicly arranged cone-cells in J. multisegmentalis is\nan important, critical point. Indeed, J. multisegmentalis possesses compound eyes, and\nthe loss of cuticular lenses in specimen YKLP 11117 offers insights into the arrangement\nof internal structures of the ommatidia. Photoprocessing methods were applied to clarify\nthe individual elements. However, if I were to take the position of devil's advocate,\nI could say that the threshold can be adjusted until 'it fits' (I am sure the authors haven´t done\nthis.)\nIt is phenomenal that the authors succeed here in showing the cone cells. The problem\nfor me here is that none of the examples clearly shows 4 cone cells; one may find even\nmore at different sizes and numbers (Fig. 2). That the application of photo-processing\nbrings out the tetraconate pattern for me suggests, in conclusion, that there are more\nthan 4 (~6-8) cone-cells, but some of them , four in number, are more pronounced in\ntheir development than the others. This may indicate that we here are on a transitional\nway from a multiconate eye (-> Limulus-like (Xiphosura, Chelicarata), ~ 100 cone cells,\nFahrenbach 1968, Harzsch & Hafner 2006) to a tetraconate system. But still, it is not\nachieved yet.\nIn the manuscript, the system here is distinguished from the scutigerid myriapods by mentioning\none single extension of the cone cell contributing to the secretion of the crystalline cone in\ntetraconates, while the myriapod possesses two. I am pretty sure one may not find these delicate\nstructures in a fossil. Here, it may help with the argument that the scutigerid ommatidium is\ndeveloped secondarily from the myriapod ocelli, which in turn originated from original ommatidia\n(e.g. Paulus 1979, 2000).\nMüller et al. (2003) find for the myriapod Scutigera coleoptrata (Linnaeus, 1758), 8 cone\nsegments, produced by four crystalline cone cells; tripartite cones (Ascothoracida and\nCirripedia), and bipartite ones they suggested to have derived from the tetrapartite\nsystem. Thus, four cone cells alone may not be sufficient to postulate an assignment to\nTetraconata, even if it is the standard definition. At least this should be discussed more\ndifferentiated. It may well be that not all cone cells/segments can be found here in the\nspecimens of J. multisegmentalis; in one of the examples presented in the manuscript\n(Fig. 2,4), there may be evident even 8 elements (D4, another shows up 7, D3),\nsuggesting a tetrapartite origin as in Scutigera.\n[FIGURE REDACTED]\nFigure 2. The cone cells of genus Jianfengia multisegmentalis (Hou 1987) (from figure 2 of\nthe manuscript)\nc) nauplius-like eyes and associated nerves supplying a discrete forebrain region ->\npancrustaceans\nIt would be great to have them, and I am willing to find them in the figures of the main\ntext; I have tried hard ... I see the filamentous structures, and they may be relicts of a\npalisade of receptors, and I agree with the pigment. Still, please make the 3 nauplius-like\neye elements more evident in your main text figures. I find them in the UV (S1 C). A\nprobable explanation indeed would be that this was one organ consisting of three ocelli\ncovered by a membrane, which would explain the homogenous appearance of the whole\nsystem under white light. I agree with you that the organ may have been movable. The\nmovable character, I think, results from the not-fixed 'stalk,' which clearly protrudes from the\ncarapace. See the dark line (the margin of the carapace, indicated by the arrows in our Fig\n2c).\nThe small element in the reconstruction drawing (main text in Figure 4D) shows the same\ninterpretation as I gave it above and in Fig. C3, which is great, of course, but this finding\nshould be established more clearly in the main text.\nIf we accept that the filaments are relicts of receptors, please note that they do not\nshow any grouping. Typically, a Nauplius eye is a single median eye and consists of\nthree- or four-pigment cup ocelli. This nicely can be detected in your Figure S1C.\nI am not sure about the arthrodial membrane here, but I share the idea that this whole\nstructure could have been movable (see above).\nI think these results (3 ocelli, movable system) should be more emphasized and shifted\nfrom the Supplement to the main text.\nSo, we agree with a tripartite, nauplius-like eye, probably built by ocelli.\nFigure 3. The Nauplius-organ\nNeural organization\nd) neuropils and tracts in the eye stalks -> nested optic neuropils as occur in\neucrustaceans\ne) organization of the deuto- and tritocerebrum -> differentiation from stem Chelicerata and\nextinct Myriapoda\nTracing up the neural structures in this tiny organism, neuropils as pathways is\nfascinating, very skillful, and convincing. One should never forget that all these\ninvestigations start in a quarry, and we deal with 'rocks', fossils. The optic neuropils 1-3\ncan be discerned very clearly, the reconstruction of the entire brain for example by\noverlaying the results of different findings and the assumption of a bilateral organism, by\nthat, the methods of reconstruction for completing, for example, by flipping elements of\nthe one side to the other, are justified. The tables (S1, S2) are interesting, and the drawn\nreconstructions (Fig 5) are excellent and consequent. Unfortunately, the inscriptions in\nFigure 5 (as perhaps the whole figure) are too small and hardly readable/discernible.\nI am not an expert in arthropod brains and the genetic background of the genetic\norganization of an arthropod segmentation, especially the nervous system, but one\ncan follow the given reasoning, and a possible interpretation seems to have\nemerged.\nIn Figure 3 it is not clear what is from YKLP 11367 and YKLP 17299\nDiscussion\nThe discussion of the manuscript makes the point that the J. multisegmentalis among the\nstem-arthropods of the Chengjiang biota represents a true pancrustacean, because,\nalthough a trunk tagmatization still is missing, the eyes have a tetraconate ommatidium, J.\nmultisegmentalis possesses as typical for pancrustaceans a nauplius-like eye consisting of\nthree elements, and a brain that is organized as in pancrustaceans.\nWe may agree with the latter, but we see a problem with the interpretation of the\neye systems.\nNote that median eyes (-> Nauplius-like eyes, Nauplius eyes = Median eyes of\ncrustaceans) also exist in the megacheiran Leanchoilia! (Garcia-Bellido & Collins 2007,\nSchoenemann & Clarkson 2012, 2023), and indeed in Alalcomenaeus (see below).\n[FIGURE REDACTED]\nFigure 4 Median eyes of Alalcomanaeus cambricus Simonetta, 1970 and Leanchoilia\nsuperlata Walcott 1912\nThis has implications and should be considered and discussed.\nEven being aware of the work of Tanaka et al. 2013, one may be a bit careful with the\ninterpretation of the eyes of Alalcomenaeus and Leanchoilia. The specimens of\nAlalcomenaeus illustrated by Briggs & Collins (Fig 4.1, 4.4) or of Whittington (1981, Fig.\n126) clearly show that Alalcomenaeus has club-shaped, penduculate compound eyes\n(here Fig. 1D), surmounting articulated stalks; the same is true for Leanchoilia (here Figure 1 B,\nC). Here, the eyes and their stalks are much finer.\nNote that there are median eyes in the megacheiran Leanchoilia (e.g. Garcia- Bellido &\nCollins, 2007; Schoenemann Clarkson 2012) and in Alalcomenaeus also (e.g. Briggs &\nCollins, 2007, Fig. 4.3), as can be seen clearly here in Figure 4. This has implications and\nshould be considered and discussed - for example, consequently, Jianfenigia is NOT the\nonly Early Cambrian genus identified with median eyes (and crustacean-like eyestalks).\nPerhaps J. multisegmentalis is among the first with 3 median eyes. Still, there are\ncrustaceans with 4 ocelli in their median eye complex, e.g. among the branchiopoda (e.g.,\nReimann & Richter 2007) – so the number alone may is not game-changing here.\nWe agree, however, completely with the idea that genetically based the morphological\nvariance of homolog deutocerebral appendages is wide enough to comprise great\nappendages, antennules formed as 'pinchers' or pier-like antennules, or ...\nIn general, we also agree with the interpretations of the neural systems and the homologies\nexplained here. I am not an expert in arthropod nervous systems, but I think the deductions\nare reasonable.\nFinally, one of this manuscript's great conclusions is that not the Hox-gene-associated\ndivergences of the segmented trunk define ancestral and crown arthropods, but do the\nsegmental cerebral domains, the unique lineage-specific neuropils, and associated traits.\nThis is an important point, but our conclusion differs slightly from what the authors indicate\nhere.\nEven if one of the characteristics (like those of the nervous system as here) has passed\nthe 'finish-line' to reach a crown group state, while others have not (tagmatization of the\nbody), one has to be aware that as here, obviously boundaries of the stem-/crown\ngroups cannot always be rigidly defined, and that there are transitional situations (as\nrepresented in the eye-status of Jianfengia multisegmentalis (Hou 1987)). Here, one part\n(brain system) has reached that stage, and the other (tagmatization of the body) still has\nnot; the eye system of Jianfengia multisegmentalis (Hou 1987) is on its way. This may\nhave important implications for describing evolutionary processes in general.\nThe results here show that the formation of different crown-group characteristics\n(tagmatization of the trunk – crown–group-nervous-system – eye system) may evolve\nwith different velocities. An important point because one now will have to weigh when\nthe complete, or a 'sufficient' crown-group-state has been arrived. Probably it is not\nadequate if only one of the characteristics has passed the 'finish line'. Even if the focal\npoint in the considerations of this manuscript has been laid on the eyes and\nbrain/nervous structures, it may be an essential fact that the tagmatization of the body\nis not quite there yet.\nNevertheless, the authors are right to state that neuroanatomical traits could provide a\npowerful tool for distinguishing characteristics to establish euarthropod relationships,\neven if external traits that differentiate identities are absent.\nReferences\nButterfield, N. J. (2002). Leanchoilia guts and the interpretation of three-dimensional structures in Burgess\nShale-type fossils. Paleobiology, 28, 155-171.\nBriggs, D.E.G. & Collins, D. 1999: The arthropod Alalcomanaeus cambricus Simonetta, from the middle\nCambrian Burgess Shale of British Columbia. Palaeontology 42, 953–957.\nFahrenbach, W. H. (1968). The morphology of the eyes of Limulus: II. Ommatidia of the compound eye.\nZeitschrift für Zellforschung und Mikroskopische Anatomie 93, 451-483.\nGarcía-Bellido,D. C., & Collins, D. (2007). Reassessment of the genus Leanchoilia (Arthropoda,\nArachnomorpha) from the Middle Cambrian Burgess Shale, British Columbia, Canada. Palaeontology, 50,\n693-709.\nHarzsch, S., & Hafner, G. (2006). Evolution of eye development in arthropods: phylogenetic\naspects. Arthropod Structure & Development, 35, 319-340.\nHou, X., & Bergström, J. (1997). Arthropods of the Lower Cambrian Chengjiang fauna, southwest\nChina. Fossils and Strata 45, 1-177.\nMüller, C. H., Rosenberg, J., Richter, S., & Meyer-Rochow, V. B. (2003). The compound eye of Scutigera\ncoleoptrata (Linnaeus, 1758)(Chilopoda: Notostigmophora): an ultrastructural reinvestigation that adds\nsupport to the Mandibulata concept. Zoom orphology, 122, 191-209.\nPaulus, H. F. (1979) Eye structure and the monophyly of the arthropod eye. In Arthropod Phylogeny (ed.\nGrypta, A. P.) 299–383 (Nordstrand, 1979).\nPaulus, H. F. (2000). Phylogeny of the Myriapoda–Crustacea–Insecta: a new attempt using photoreceptor\nstructure. Journal of Zoological Systematics and Evolutionary Research, 38, 189-208.\nReimann, A., & Richter, S. (2007). The nauplius eye complex in ‘conchostracans’ (Crustacea,\nBranchiopoda: Laevicaudata, Spinicaudata, Cyclestherida) and its phylogenetic implications. Arthropod\nStructure & Development, 36, 408-419.\nRichter, S., Loesel, R., Purschke, G., Schmidt-Rhaesa, A., Scholtz, G., Stach, T., ... & Harzsch, S. (2010).\nInvertebrate neurophylogeny: suggested terms and definitions for a neuroanatomical glossary. Frontiers in\nZoology, 7, 1-49.\nSchmidt, M*., Schoenemann, B.*, Hou X.-g., Melzer & Liu, Y. (2025) A unique 1 Lower Cambrian\narthropod with two different compound eye systems. Communications Biology (2025), in press * shared\nfirst authorship\nSchoenemann, B., & Clarkson, E. N. (2012). The eyes of Leanchoilia. Lethaia, 45, 524-531.\nSchoenemann, B., & Clarkson, E. N. K. (2023). The median eyes of trilobites. Scientific Reports 13, 3917.\nTanaka, G., Hou, X., Ma, X., Edgecombe, G. D., & Strausfeld, N. J. (2013). Chelicerate neural ground pattern\nin a Cambrian great appendage arthropod. Nature, 502(7471), 364-367.\nWhittington H. B. (1981). Rare arthropods from the Burgess Shale, Middle Cambrian, British Columbia.\nPhilosophical Transactions of the Royal Society, Series B, 292, 329–357.\nA pancrustacean brain and its visual systems define a Cambrian “great appendage” arthropod.\nby Nicholas J. Strausfeld 2*, Xianguang Hou 1, and Frank Hirth 3*\nThis MS presents amazingly detailed comparisons between the neural structures of a\nCambrian megacheiran arthropod and those of modern crustaceans. The biological data are\nall of high quality. However, most interpretations of the fossil species are based on\nquestionable often very weak fossil evidence (see examples below). The extensive use of\nimage processing (e.g. Photoshop) is sometimes misleading. There is a huge difference\nbetween the idealized processed images and reconstructions, and what we actually see in the\nfossil specimen.\nJianfengia is interpreted as a lower stem pancrustacean, based on the assumed structure of\nits nervous system. However, its appendage structure (head appendages) is completely\ndifferent from that of pancrustacean. The authors do not take into account these key\nanatomical aspects.\nLine 45- What kind of digital adjustments ? This is a key point. Please give details.\nLine 48- The plural of retina is retinae (Latin)\nLine 61- I agree that the frontal rounded feature is the anterior sclerite (ASC). It appears to\nbe a sclerotized feature comparable with other hard parts of the exoskeleton (see figure B\nbelow). However, I see no convincing evidence of an underlying “system of lenses”. Clearly\nthis brownish feature is over-interpreted. I am saying that soft tissues (including sensory\norgans) do not occur underneath the sclerite but they are not visible in this fossil specimen.\nLine 62 + “Their detailed organization suggests structural homology to crustacean nauplius\neyes (8, 9) and insect ocelli (10, 11) (Fig. 1C, D; SI Appendix Fig. S1) comprising three lenses\noverlying a palisade of photoreceptors”\nThis statement is speculative considering that no detailed organization can be seen in\nthe fossil specimen in question.\nLine 65- Please explain what you mean by superimposition\nLine 68- This is perfectly true but what the readers would like to see here (in the\n“Results” paragragh) are more convincing fossil evidence.\nLine 76-77. The lateral eyes are well-preserved. However, this type of eyes occurs in numerous\nCambrian arthropods that clearly have no affinities with eumalacostracans (e.g. Artiopoda such\nas Acanthomeridion; also Isoxys, a basal megacheiran or even Tuzoia, that have no close affinities\nwith pancrustaceans)\nLine 85. The light spots seen in Fig. E are interpreted as possible ommatidia\nHowever, similar spots seem to occur elsewhere in the specimen. Unfortunately, I have no access\nto the high-resolution version of the text-figures. It is important that the authors provide\ncomparative high-resolution images of the lateral eye and other areas of the fossil (see below). I\nwould suggest to provide back-scattered SEM images of the eye. It has been done with the eyes\nof Waptia from the Burgess Shale and clearly revealed the outline of ommatidia that do not\nappear in light photographs (see Vannier et al. 2018)\nLine 87- I don’t think many readers will be convinced by images processed by Photoshop.\nIndeed, you cannot “determine if these are taphonomic artifacts or that they represent\nfossilized cellular elements of the ommatidia” by using image processing.\nPlease use other methods to reveal possible underlying retinal structures such as SEM.\nYou may also use a Keyence digital microscope. Although the preservation mode of\nyour specimen is different, SEM studies clearly revealed the inner structure of the eye\nof Jurassic thylacocephalans (see Vannier et al. in Nat. Comm)\nLine 90 +\nFig. 2C is supposed to show the inner structure of assumed ommatidia. Unfortunately, these\nprocessed images fail to provide evidence that a consistent 4-fold structure occurs in these\nassumed ommatidia. Honestly, I am not convinced. The outlines of these “light spots” are\nirregular and blurred. Again, I suggest the authors to use different methods (see above).\nFigs D-F are also problematic. The risk with image processing is to make features\nappear as you want them to appear.\nLine 95\nSome spots seem to be more pentaradial than tetraradial ( see “3” below)\nThe reconstructions are unfortunately very speculative and based on no solid fossil evidence.\nWhat is said about the eyes of modern crustaceans is perfectly correct. The main concern\nwith this MS is the lack of strong fossil evidence.\nI really have a problem with these two images. The upper one shows 5 “light areas” but\nonly 4 appear in the lower image. It looks very odd to me.\nLine 107 + I agree that there are ganglia (e.g. represented by dark area in B and F.\nHowever, the assumed paired labral ganglia are not visible. What we see in this area is a\nvague cluster of dark spots that could represent any kind of soft tissues (see Aria et al.\nin Bioessays). Again, the weakness of this MS is the lack of fossil evidence and the\noverinterpretation of most features.\nPlease provide details concerning the method used to obtain this image (below). Apparently\nit was obtained via the juxtaposition of one side of the head with its mirror image. This leads\nthe reader to believe that the structures are indeed perfectly symmetrical and paired. Again,\nthere is a huge difference between this idealized image and what we actually see in the fossil\nspecimen. This way of showing results is unfortunately somewhat (not intentionally)\nmisleading.\nIs there any trace of nerves that would belong to the great appendage ?\nLine 163- The authors go too far in their interpretations (see discussion part) that are actually\nbased on very weak evidence (see my remarks above).\nI don’t think Jianfengia is a lower stem pancrustacean. Please look at its appendages (see\nother papers; e.g. number and shape of head appendages). They have nothing to do with the\nappendages of a stem-crustacean (no mandible, etc..)\nFIGURE 5 would really be an excellent one if the authors had sufficient fossil evidence.\nThe comparative drawings of extant species are perfectly correct and very clear."}, {"round": 2, "reviewer_comments": {"reviewer_1": "This has become an excellent paper now. With great respect, we note that following our suggestions, this paper has been rewritten and has been changed in crucial points, as assigning Jianfengia basal, but not within the Mandibulata, because the cone-cell number had not yet evolved within Jianggegiidae. Now Jianfengia’s position within Euarthropoda is established through a solid, diligent, and careful cladistic analysis, and in particular neuroanatomical and cephalic traits. The results are absolutely convincing and well exposed. Perhaps one could have more emphasized that Jianfengia obviously has a transitional state between stem and crown-group arthropod, because it bears features of both (number of cone cells vs, brain system).\nGreat work – my congratulation.\nI found some small points:\nLine 32 end: a “t” too much\nLine 92, 93: This organization corresponds to eyestalk morphology typifying eumalocastracan crustaceans and ...\nWe will find out later (l. 152 ff) why this is mentioned here, but at this point it may seem a bit suggestive. I would suggest to mention that these stalked eyes, as typical of other Cambrian arthropods, also, correspond to those of modern eumalacostracan crustaceans ...\nLine 153: Both -> both", "reviewer_2": "The authors addressed successfully all questions and concerns of the four reviewers. I already liked the previous version, but after applying the suggestions by different experts I consider this manuscript as more robust and I would like to see it published in its current form."}, "author_response": "Reply to Reviewer #1\nThis has become an excellent paper now. With great respect, we note that following our\nsuggestions, this paper has been rewritten and has been changed in crucial points, as assigning\nJianfengia basal, but not within the Mandibulata, because the cone-cell number had not yet\nevolved within Jianggegiidae. Now Jianfengia’s position within Euarthropoda is established\nthrough a solid, diligent, and careful cladistic analysis, and in particular neuroanatomical and\ncephalic traits. The results are absolutely convincing and well exposed. Perhaps one could\nhave more emphasized that Jianfengia obviously has a transitional state between stem and\ncrown-group arthropod, because it bears features of both (number of cone cells vs, brain\nsystem). Great work – my congratulation.\nWe further thank the reviewer for her scholastic assessment and creative suggestions\nI found some small points:\nLine 32 end: a “t” too much\ncorrected\nLine 92, 93: This organization corresponds to eyestalk morphology typifying eumalocastracan\ncrustaceans and ...\nWe will find out later (l. 152 ff) why this is mentioned here, but at this point it may seem a\nbit suggestive. I would suggest to mention that these stalked eyes, as typical of other\nCambrian arthropods, also, correspond to those of modern eumalacostracan crustaceans ...\nWe have modified this to add clarity that may have escaped reviewer. Thus:\n“This organization corresponds to eyestalk morphology typifying eumalacostracan\ncrustaceans15...”\nLine 153: Both -> both\nThe reviewer seems to have mistaken the section heading as the introductory sentence of\nthe section, which indeed starts as Both...\nReviewer #2 (Remarks to the Author):\nThe authors addressed successfully all questions and concerns of the four reviewers. I already\nliked the previous version, but after applying the suggestions by different experts I consider this\nmanuscript as more robust, and I would like to see it published in its current form.\nResponse to reviewer\nWe thank this reviewer for their strengthened endorsement."}]

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