Report. The First Stalk-Eyed Phosphatocopine Crustacean from the Lower Cambrian of China

Current Biology 22, 2149 2154, November 20, 2012 ª2012 Elsevier Ltd All rights reserved http://dx.doi.org/10.1016/j.cub.2012.09.027 The First Stalk-Eyed Phosphatocopine Crustacean from the Lower Cambrian of China Report Xi-guang Zhang 1, * and Brian R. Pratt 2 1 Key Laboratory for Paleobiology, Yunnan University, Kunming 650091, China 2 Department of Geological Sciences, University of Saskatchewan, Saskatoon, SK S7N 5E2, Canada Summary Exhibiting Orsten-type preservation, specimens of the phosphatocopine Dabashanella sp. from the Lower Cambrian Stage 3 (about 520 million years ago) of southern China possess a single-fold shield and a set of appendages of crustacean design. More significantly, a pair of stalked eyes the earliest known visual structure in this group is attached to an ocular segment analogous to the anterior sclerite of various stem-group arthropods [1, 2]. Accordingly, a unique visual system must have been present among some, if not all, early phosphatocopines. In comparison with the ground pattern of later members of this group [3], the new phosphatocopine, which with its unique head segmentation and limb design is unlikely to be embraced within the previously proposed Labrophora [4, 5], demonstrates a remarkable modification and innovation in the appendages and visual system with time. Thus, this finding provides new data for the evaluation of the early evolutionary development and phylogenetics of the Crustacea and other related euarthropods. Results Phosphatocopina are a group of tiny uni- or bivalved crustaceans that inhabited seas of Cambrian age. In the fossil record, they are usually preserved as empty shields [6, 7], although rare specimens recovered from several successions containing Orsten concretions have soft parts so finely replicated by microcrystalline apatite [8] that they can be compared directly to living crustaceans. Such evidence has been used effectively to infer appendage differentiation and body segmentation associated with the evolution of early arthropods [3, 4, 9 11], but this has mostly relied on exquisite Orsten-type material from the Upper Cambrian Furongian Series (about 500 million years ago) of southern Sweden. Phosphatocopines are the most common component of this faunal assemblage with exceptionally preserved tissues and consequently have enormously advanced the understanding of the morphology, ontogeny, and early evolution of the Crustacea [2, 10, 11]. Even older, early Cambrian Stage 3 Orsten-type limestone nodules in the Heilinpu (formerly Yu anshan) Formation of southern China also contain phosphatized crustaceans [5, 12], and from these rocks we have obtained four phosphatocopine specimens that also display preservation of great fidelity of their ventral soft parts and appendages, especially evidence for their visual system. *Correspondence: xgzhang@ynu.edu.cn Material An almost complete specimen (YKLP 11955; Key Laboratory for Paleobiology, Yunnan University, Kunming, China) (Figures 1A 1F), a fragmentary one with soft parts (YKLP 11993) that may be a fecal pellet (Figure 1G), and another two with either labrum or appendages partially preserved (YKLP 11956 and 11957). Locality Xiaotan section, Yongshan, Yunnan, China (see Figure S1 available online); Heilinpu Formation, Eoredlichia-Wutingaspis Biozone (Chiungchussuan Stage [regional]), Cambrian Stage 3 (Figure S2). All phosphatocopines dealt with here co-occur with the eucrustacean Wujicaris muelleri [12], numerous specimens of the bradoriid Kunmingella douvillei, lingulid brachiopods, and various small shelly fossils in a thin-bedded nodular limestone about 5 m above the beds that have yielded material of Yicaris dianensis [5]. Description The bivalved shield is semicircular in lateral view, about 400 mm long, with outer surface either smooth or ornamented with a network of fine ridges. It has a straight dorsal rim and a single-fold articulation (not a true hinge) terminating in short anterocardinal and retrocardinal spikes (Figures 1B and 1E 1G). A wide duplicature is present along the inner free margin of each valve (Figures 1A and 1H 1J), which bears a very narrow marginal rim (Figures 1B, 1C, and 1H 1J). The body enveloped by the shield comprises at least ten segments, including those bearing the eye stalk, antennula, antenna, and mandible, followed by five postmandibular segments bearing limbs and an undifferentiated hind body. The paired eye stalks, w60 mm long, are located on the triangular anteriormost head segment, are ventrally directed, and taper gradually toward the visual surface at their distal tips. The outer surfaces of the eye stalk and the anterior (ocular) segment bear faint, fine annuli or annular zones, and the junction with the anterior segment is marked by a shallow ring-shaped furrow. The hypostome-labrum complex posterior to the anterior segment has a smooth surface (Figures 1A 1C and 1G). The rod-shaped antennula is w130 mm long, consisting of at least seven faintly defined annuli with a few long setae inserted on the terminal and on some of the distal annuli (Figures 1A and 1D). The paired antennae are positioned lateral to the hypostomelabrum complex (Figures 1A, 1D, and 1E), each consisting of (1) a coxa with a set of spine-like setae located near the posterior end of the labrum, (2) a subtriangular basipod bearing a median enditic elongation with setae, (3) a five-segmented endopod arising mediodistally from the basipod, (4) a multiannulated exopod consisting of at least 15 annuli each with a long seta inserted on its terminal, and some setae-free basal annuli. The mandible has a large coxa with a few short spinelike setae on its surface, a subtriangular basipod bearing a median enditic elongation with setae, a four-segmented endopod, and an annulated exopod with long, distally inserted setae and a setae-free basal part. The first postmandibular limb consists of a broad basipod with a set of spine-like setae, a seven-segmented endopod (Figures 1A and 1D),

Current Biology Vol 22 No 22 2150 Figure 1. Phosphatocopine Crustaceans (A J) Dabashanella sp. from Lower Cambrian Stage 3 Heilinpu Formation in Yongshan, Yunnan, China. (A F) YKLP 11955, open shield with all appendages nearly completely preserved: oblique lateroventral view (A); oblique laterodorsal view (B); anteroventral view displaying the stalked eyes (C). (D) (F) show enlargements of some appendage details. (G) YKLP 11993, fragment showing a well-preserved exopod (indicated by arrow) of a postmandibular limb and deformed labrum. (H and I) YKLP 11956, lateroventral and posteroventral views, open shield with hypostome-labrum complex, sternum, and limb setae (but no preserved combination of stalked eyes and ocular segment). (J) YKLP 11957, closed shield with broken left valve exhibiting two partially preserved appendages (indicated by arrows).

Cambrian Phosphatocopines with Stalked Eyes 2151 Figure 2. Reconstruction of Dabashanella sp. in Life Attitude (A) Anterior view. (B) Left-side view. Left valve is removed to show the soft body (light gray) behind, hypostome-labrum complex (dark gray), and all left appendages. Abbreviations are as in Figure 1, except as follows: pl1 pl5, 1 st 5 th postmandibular limbs; vs, visual surface. and a narrow paddle-like distally annulated exopod with fringing setae (Figures 1A, 1F, and 1G). The second, third, fourth, and fifth postmandibular limbs have the same general shape, consisting of an endopod and an exopod with progressively fewer segments and setae; only some basipods belonging to anterior limbs are visible. Because of incomplete preservation, it is not clear whether the hind body of the trunk bears paired furcal rami. Remarks The four specimens are assigned to Dabashanella sp. based on the particular features of the shield only (i.e., the singlefold articulation, narrow marginal rim, and wide duplicature), because species of Dabashanella were originally described from empty shields [7]. They are apparently distinguished from the type species, D. hemicyclica Huo, Shu, and Fu 1983, by having much more pointed cardinal spines and a slightly arched dorsal margin and thus may be a new species. Based on its limb number, the nearly complete specimen (YKLP 11955) represents a later growth stage. On this basis, it furnishes new data about the fundamental soft-body anatomy and reconstruction of this species (Figure 2), which on the whole is distinguishable from all other soft-bodied phosphatocopines reported hitherto. Discussion Like typical Orsten-type fossils, the apatite replacement in the figured specimens of Dabashanella sp. the oldest phosphatocopine with soft parts so far known displays high fidelity of preservation of the outer surface of its body but only incomplete relics of its internal structure, as revealed by a series of synchrotron-produced serial sections. For instance, the eye stalk is a hollow tube, and so the anterior (ocular) segment (Figures 3A and 3B), the body wall, and the inner lamellae, which are invisible under the scanning electron microscope, become traceable structures (Figures 3C 3F). The visual organ has been widely interpreted as belonging to the first head segment [9], and stalked eyes have been regarded as a key feature to characterize the node of Arthropoda sensu stricto, the sclerotized arthropods [13]. However, interpreting the anterior portion of the phosphatocopine body as a hypostome-labrum complex with median eyes anteroventrally inserted [3, 14] is difficult to reconcile with the eyebearing anteriormost body segment identified in other early arthropods, including Fuxianhuia and Anomalocaris [1, 2, 15]. Instead, with the stalked eyes on an isolated anterior segment with annulated outer surface, separate from and anteroventral to the hypostome-labrum complex, the head segmentation of Dabashanella sp. now appears to be the same as that of some stem arthropods [1, 2, 16]. Similar visual structure observed in a maxillopod crustacean (see Plate 2, Figure 2 and Plate 3, Figure 3 in [17]) may also imply the same style of head segmentation. Therefore, the ocular system seems to have been considerably modified in later phosphatocopines such as Hesslandona and Falites [3, 11, 14]. The shallow ring-shaped furrow at the base of each eye stalk of Dabashanella sp. was likely a flexible joint. On the visual surface, there is no sign of polygonal facets left by the ommatidia, because such tiny eyes, like those of juveniles of the univalved crustacean Henningsmoenicaris scutala from the Middle Cambrian (Series 3) [18], are probably too small to show details of the optical design. Because the blister-like eyes of some early instars of H. scutula became stalked (K) YKLP 11994, Hesslandona sp. from Cambrian Stage 3 Shuijingtuo Formation in Zhenba, Shaanxi, China, with interdorsum defined by a pair of folds (indicated by arrows). (L) YKLP 11995, Phosphatocopina gen. et sp. indet. from Lower Cambrian Stage 3 Shuijingtuo Formation in Pengshui, Chongqing, China with ventral spines (indicated by arrows). Scale bars represent 100 mm. The following abbreviations are used: a1, antennula; a2b, basipod of antenna; a2c, coxa of antenna; a2en, endopod of antenna; a2ex, exopod of antenna; acs, anterocardinal spike; as, anterior (ocular) segment; dp, duplicature; es, eye stalk; fsb, folded soft body; hb, hind body; hs, hypostome; id, interdorsum; la, labrum; ls, limb setae; lv, left valve; mdb, basipod of mandible; mdc, coxa of mandible; mden, endopod of mandible; mdex, exopod of mandible; mr, marginal rim; od, adhering organic debris; pl1b pl3b, basipod of 1 st 3 rd postmandibular limb; pl1en pl5en,, endopod of 1 st 5 th postmandibular limb; pl1ex pl5ex, exopod of 1 st 5 th postmandibular limb; rcs, retrocardinal spike; rf, ring-shaped furrow; rv, right valve; st, sternum.

Current Biology Vol 22 No 22 2152 Figure 3. Digital SRXTM Internal Sections of Dabashanella sp. YKLP 11955 (A F) Series of vertical transverse slices (from anterior to posterior) displaying hollow anterior sclerite and bifurcating stalked eyes (A and B), thin inner lamellae that connect the body and shield, well-defined digestive tract, and unevenly phosphatized outer layer of appendages that are hollow or filled with other material (C F). (G I) Horizontal slices (from dorsal to ventral) displaying hollow appendages and thin inner lamellae. (J L) Vertical longitudinal slices (from left to right) displaying the digestive tract and various appendages. Abbreviations are as in Figures 1 and 2, except as follows: bw, body wall; dt, digestive tract; il, inner lamella. compound eyes in later developmental stages (see Figures 5F and 5G in [18]), it is possible that Dabashanella sp. may have developed compound eyes with further growth as well. The presence of stalked eyes in both early phosphatocopines and other arthropods identifies this feature as a symplesiomorphy with limited value in the reconstruction of crown euarthropod phylogeny. Nonetheless, the stalked eyes of Dabashanella sp. are in contrast to the dome-like median eyes as present in some younger phosphatocopines, which are always closely associated with the hypostome. The absence of a separate ocular segment among later phosphatocopines, and possibly some other stem crustaceans [19, 20], appears to indicate the merging of the ocular segment to the immediately succeeding segment, a step that has also been inferred for other lineages among Euarthropoda [15]. On this basis, our finding implies a dramatic evolutionary development of the visual system, as well as head segmentation among the Cambrian phosphatocopines, but it is unclear how and why they evolved two seemingly disparate visual patterns. Unfortunately, the Orsten-type material can display mostly just external morphology, so little has been revealed to date about the internal anatomy of such tiny fossil eyes [18]. The shield of Dabashanella sp., like that of many other phosphatocopines [10, 21], has a single-fold articulation. Furthermore, some contemporaneous phosphatocopines display either an interdorsum (third valve), defined by a pair of folds (Figure 1K), or distinct marginal valve spines (Figure 1L). All these types of shields, along with a well-developed duplicature, marginal rim, and the likely inner lamellae that connect the soft body and shield (Figure 3), appear to exhibit stasis (relative stability) with time, being present in various younger lineages [3, 11]. Therefore, these persisting features characterize this arthropod clade. On the other hand, the varied or modified visual structure and detailed appendage components (e.g., proximal endite) can be regarded as autapomorphies and thus inapplicable character states [22] serving only for grouping some subsequently diversified phosphatocopine lineages, instead of the whole clade of Phosphatocopina. Appendages of some early crustaceans have been demonstrated to be sophisticated in design [23] and considerably more diverse [12] than had previously been expected. Such an appendage acquisition or diversification seems to have also occurred among phosphatocopines during their early evolution. Like Klausmuelleria salopensis a phosphatocopine recovered from the Lower Comley Limestone [4] the antenna and mandible of Dabashanella sp. also display distinct coxae and basipods. However, the endopods of the antenna and mandible of K. salopensis both bear three podomeres, whereas in Dabashanella sp. they bear five and seven podomeres, respectively. The difference is probably because the specimen of K. salopensis belongs to a younger growth stage. Notably, Dabashanella sp. is characterized by having a pair of rather large antennulae, in contrast to some late phosphatocopines that have tiny antennulae and fused coxa and basipod present either in the antenna or mandible [3, 11]. Moreover, although the postmandibular limbs in both taxa bear a distally annulated, paddle-like exopod, the corresponding endopod varies in number of podomeres. In particular, the proximal endite present in the first postmandibular limb of other previously described phosphatocopines [4, 11] is absent in that limb in Dabashanella sp., where the rather large basipod differs strikingly from all other endites. Such an appendage arrangement for Dabashanella sp. is clearly not in accordance with the autapomorphies previously envisaged for Phosphatocopina, which are based on younger taxa [3, 4, 21].

Cambrian Phosphatocopines with Stalked Eyes 2153 The Cambrian phosphatocopines have been regarded as stem-group crustaceans comprising the sister group of Eucrustacea, and both were also considered separate phyla of Labrophora [3 5]. The appendages of Dabashanella sp. share some major morphological attributes with the Eucrustacea, such as the medially setose exopods of the antenna and mandible. As the currently earliest known phosphatocopine, however, Dabashanella sp. not only demonstrates some apomorphies (e.g., the proximal endites on the postmandibular limbs) as well as dramatic modifications (e.g., the tiny antennula) in appendages that are shared by later representatives of the group but also retains some plesiomorphic characters mostly from the proposed ground pattern of the Labrophora. Thus, our discovery provides an important link for assessing the evolutionary morphological gap among these crustaceans. Nonetheless, as demonstrated here, Dabashanella sp. also possesses some unique features, especially the anterior segment with stalked eyes and the seven endopodal podomeres in postmandibular limbs, which are beyond the systematic criteria that characterize Labrophora [3, 4] but are clear evidence for a deep origin within the Euarthropoda. These features in turn substantially improve the understanding of the body plan of Phosphatocopina and provide fresh insight into the phylogenetic relationship between the Phosphatocopina and Labrophora, as well as other Euarthropoda. The inevitable modification of appendages over time makes it more difficult to trace their origins or likely homology; appendage-based analysis alone may fail to exactly mirror the evolutionary novelties that have led to the branching of an arthropod group of high taxonomic rank [24]. As an example, it remains unsettled whether the great-appendage arthropods, a hypothesized clade named from the sharing of a pair of highly modified anterior appendages, are genuinely monophyletic or merely paraphyletic [2]. In addition, agnostoids with crustacean-like appendages have been tentatively assumed to have arisen from the stem lineage of crustaceans [25, 26], but this appendage-based proposal has never been widely accepted simply because no other examples of detailed appendage preservation are known [27]. Thus, appendages should not be seen as the only features with fundamental taxonomic importance for determining the deep relationships within Arthropoda [24], even though this highly successful group is in fact characterized essentially by segmented body and appendages. Nevertheless, despite the uncertainties about their high-level affinity, the phosphatocopines are likely a monophyletic yet quite diverse clade [3]. As demonstrated by our discovery, they might be redefined by a set of synapomorphies, including the specialized visual structure associated with the first head segment, a set of modified appendages of crustacean design, and the seemingly simple shield with a unique type of articulation. Experimental Procedures The coarse outer surface of the shield, trunk, and hypostome-labrum complex in a specimen of Wujicaris muelleri (see Figures 1F and 1G in [12]) may be the result of overetching. To avoid such damage, for this study, 196 kg of limestone nodules collected from the same horizon were digested in acetic acid of even lower concentration (4%), and the insoluble residue was taken out of the acid solution more frequently (every day rather than every couple of days). The phosphatocopine specimens studied here, along with many phosphatized sclerites, were picked from the residue under a stereo microscope, and images were then acquired via scanning electron microscopy and synchrotron radiation X-ray tomographic microscopy (SRXTM). Supplemental Information Supplemental Information includes two figures and can be found with this article online at http://dx.doi.org/10.1016/j.cub.2012.09.027. Acknowledgments We thank N.J. Butterfield for critical comments on an earlier manuscript version; the manuscript referees for constructive suggestions and remarks; T.-Q. Xiao, Y. He, and J.-B. Hou for assistance in sample examination at the Shanghai Synchrotron Radiation Facility; and H.-L. Zhang for sample preparation. This study was supported by the National Natural Science Foundation of China (40972012) and the Natural Science Foundation of Yunnan Province (2008CC005). Received: June 21, 2012 Revised: August 9, 2012 Accepted: September 12, 2012 Published online: October 18, 2012 References 1. Budd, G.E. (2008). Head structure in upper stem-group euarthropods. Palaeontology 51, 561 573. 2. Budd, G.E., and Telford, M.J. (2009). The origin and evolution of arthropods. Nature 457, 812 817. 3. Maas, A., Waloszek, D., and Müller, K.J. (2003). Morphology, ontogeny and phylogeny of the Phosphatocopina (Crustacea) from the Upper Cambrian Orsten of Sweden. Fossils Strata 49, 1 238. 4. Siveter, D.J., Waloszek, D., and Williams, M. (2003). An early Cambrian phosphatocopid crustacean with three-dimensionally preserved soft parts from Shropshire, England. Spec. Pap. Palaeontol. 70, 9 30. 5. Zhang, X.G., Siveter, D.J., Waloszek, D., and Maas, A. (2007). An epipodite-bearing crown-group crustacean from the Lower Cambrian. Nature 449, 595 598. 6. Müller, K.J. (1964). Ostracoda (Bradorina) mit phosphatischen Gehäusen aus dem Oberkambrium von Schweden. Neues Jahrb. Geol. Palaontol. Abh. 121, 1 46. 7. Huo, S.C., Shu, D.G., Zhang, X.G., Cui, Z.L., and Tong, H.W. (1983). Notes on Cambrian bradoriids from Shaanxi, Yunnan, Sichuan, Guizhou, Hubei, and Guangdong. J. Northwest Univ. (Xi an) 13, 56 75. 8. Müller, K.J. (1979). Phosphatocopine ostracodes with preserved appendages from the Cambrian of Sweden. Lethaia 12, 1 27. 9. Waloszek, D., Maas, A., Chen, J.Y., and Stein, M. (2007). Evolution of cephalic feeding structures and the phylogeny of Arthropoda. Palaeogeogr. Palaeoclimatol. Palaeoecol. 354, 273 287. 10. Siveter, D.J. (2008). Ostracods in the Palaeozoic? Senckenb. Lethaea 88, 1 9. 11. Zhang, H.Q., Dong, X.P., and Xiao, S.H. (2011). Two species Hesslandona (Phosphatocopida, Crustacea) from the Upper Cambrian of western Hunan, South China and the phylogeny of Phosphatocopida. J. Paleontol. 85, 770 788. 12. Zhang, X.G., Maas, A., Haug, J.T., Siveter, D.J., and Waloszek, D. (2010). A eucrustacean metanauplius from the Lower Cambrian. Curr. Biol. 20, 1075 1079. 13. Kühl, G., Briggs, D.E.G., and Rust, J. (2009). A great-appendage arthropod with a radial mouth from the Lower Devonian Hunsruck Slate, Germany. Science 323, 771 773. 14. Müller, K.J. (1982). Hesslandona unisulcata sp. nov. with phosphatized appendages from Upper Cambrian Orsten of Sweden. In Fossil and Recent Ostracoda, R.H. Bate, E. Robinson, and L.M. Sheppard, eds. (Chichester, UK: Ellis Horwood), pp. 276 304. 15. Chen, J.Y., Zhou, G.Q., Edgecombe, G.D., and Ramsköld, L. (1995). Head segmentation in early cambrian fuxianhuia: implications for arthropod evolution. Science 268, 1339 1343. 16. Waloszek, D., Chen, J.Y., Maas, A., and Wang, X.Q. (2005). Early Cambrian arthropods New insights into arthropod head and structural evolution. Arthropod Struct. Dev. 34, 189 205. 17. Müller, K.J., and Walossek, D. (1988). External morphology and larval development of the Upper Cambrian maxillopod Bredocaris admirabilis. Fossils Strata 23, 1 70. 18. Castellani, C., Haug, J.T., Haug, C., Maas, A., Schoenemann, B., and Waloszek, D. (2012). Exceptionally well-preserved isolated eyes from

Current Biology Vol 22 No 22 2154 Cambrian Orsten fossil assemblages of Sweden. Palaeontology 55, 553 566. 19. Haug, J.T., Waloszek, D., Haug, C., and Maas, A. (2010). High-level phylogenetic analysis using developmental sequences: the Cambrian ymartinssonia elongata, ymusacaris gerdgeyeri gen. et sp. nov. and their position in early crustacean evolution. Arthropod Struct. Dev. 39, 154 173. 20. Stein, M., Waloszek, D., Maas, A., Haug, J.T., and Müller, K.J. (2008). The stem crustacean Oelandocaris oelandica revisited. Acta Palaeontol. Pol. 53, 461 484. 21. Maas, A., and Waloszek, D. (2005). Phosphatocopina ostracode-like sister group of Eucrustacea. Hydrobiologia 538, 139 152. 22. Beutel, R.G., Friedrich, F., Hörnschemeyer, T., Pohl, H., Hünefeld, F., Beckmann, F., Meier, R., Misof, B., Whiting, M.F., and Vilhelmsen, L. (2011). Morphological and molecular evidence converge upon a robust phylogeny of the megadiverse Holometabola. Cladistics 27, 341 355. 23. Harvey, T.H.P., and Butterfield, N.J. (2008). Sophisticated particlefeeding in a large Early Cambrian crustacean. Nature 452, 868 871. 24. Zhang, X.G. (2007). Phosphatized bradoriids (Arthropoda) from the Cambrian of China. Palaeontographica 281, 93 173. 25. Walossek, D., and Müller, K.J. (1990). Upper Cambrian stem-lineage crustaceans and their bearing upon the monophyly of Crustacea and the position of Agnostus. Lethaia 23, 409 427. 26. Stein, M., Waloszek, D., and Maas, A. (2005). Oelandocaris oelandica and the stem lineage of Crustacea. In Crustacea and Arthropod Relationships, S. Koenemann and R.A. Jenner, eds. (Boca Raton, FL: Taylor & Francis), pp. 55 71. 27. Cotton, T.J., and Fortey, R.A. (2005). Comparative morphology and relationships of the Agnostida. In Crustacea and Arthropod Relationships, S. Koenemann and R.A. Jenner, eds. (Boca Raton, FL: Taylor & Francis), pp. 95 136.