Electronic Supplement: CHENGJIANG DEUTEROSTOMES OTHER THAN THE VETULICOLIANS AND YUNNANOZOANS In this Supplement we briefly review the remainder of the Chengjiang deuterostomes. In comparison to the vetulicolians and yunnanozoans for the most part the imterpretations of the other deuterostomes are somewhat less controversial. A) First to the ambulacrarians. Perhaps surprisingly no bona-fide echinoderms are known, although the appearance of this group certainly pre-dates the Chengjiang Fossil-Lagerstätte. The vetulicystids, however, have been interpreted a pre-echinoderms (Shu et al. 2004; Shu 2008), and intriguingly have again a bipartite body with a tail (apparently segmented) and a globose anterior bearing at least three openings (Supplementary Figure. 2a,b). These openings were interpreted as anterior mouth and posterior anus, the latter flanked by a gill. These conclusions were treated with some scepticism by Smith (2004; also Swalla & Smith 2008). Thus, he queried the identification of the mouth, speculating it might be concealed at the anterior. Such is possible, but in the available material we see no conclusive evidence. So too Smith considered alternative possibilities for the functions of the various openings, noting that the identified gill may have served for a respiratory exchange rather than being a gill in the deuterostome sense. These discussions recall, of course, the protracted debate as to the correct interpretation of the various orifices found in the pre-radial echinoderms, and in this context it is worth emphasizing the remarkable diversity of the gill openings found in one group alone, the stylophorans (e.g. Jefferies 1986; Dominguez et al. 2002). Swalla & Smith (2008) commented that fossils of Vetulicystis “lack stereom and do not show a single echinoderm synapomorphy, so it is not clear why these were placed in the Ambulacraria” (p. 1560). This is certainly a valid point, but it goes to the heart of the question of how we might identify the transformations that lead to a given body-plan. Presumably at some point in the stem-group lineage of echinoderms the diagnostic calcitic stereom was acquired, just as subsequently other diagnostic characters, such as the water-vascular system and penta-radial symmetry, were acquired. Whilst provisional we would suggest that the vetulicystid group could well be ancestral to all these important additions. In his commentary Smith (2004) also mentioned the possibility that Phlogites (Supplementary Figure 3a-c) “might even be a hemichordate or part of the common ancestral lineage of echinoderms and hemichordates” (p. 412), a point that was cautiously endorsed in a subsequent paper (Swalla & Smith 2008). Further work on this possibility is in progress with Jean-Bernard Caron. B) Second the chordates. So far as they are concerned the record of a possible cephalochordate is restricted to Cathaymyrus (Shu et al. 1996b). The proposal by Chen and Li (1997) that it is a crushed specimen of Yunnanozoon seems difficult to reconcile with any yunnanozoan specimen, whatever its orientation (see also Smith et al. 2001, p. 74). Neither Cathaymyrus nor Pikaia from the Burgess Shale seem, however, to have a particular similarity to amphioxus. The story of the urochordates is more complicated. The 1 report by Chen et al. (2003) of a tunicate (Shankouclava) has been widely accepted (e.g. Smith 2004, Swalla & Smith 2008), but we suggest here that this may need reconsideration for two reasons. First, because of the taphonomic circumstances associated with the Chengjiang Fossil-Lagerstätte, which entails rapid burial by storm activity followed by early diagenesis, the tentacles of various taxa may be obscured in consolidated areas of sediment, and so require careful excavation. More specifically, a re-examination by some of us of the eight specimens originally included in Shankouclava (Chen et al. 2003) suggests that at least four of the specimens have remains of tentacle-like structures (unfortunately the relevant region is cropped in the illustrations of Chen et al. (2003)). Strong evidence in support of this view stems from the discovery of a shankouclavid (from the same locality and horizon at Shankou village) that has an almost identical main body, but in addition bears unequivocal distal tentacles (Figure 2g). For these reasons we suggest Shankouclava is unlikely to be a tunicate (see also Shu 2008, p. 231). An alternative proposal, that looks to Cheungkongella (Shu et al. 2001a), has met with some scepticism and this taxon has been regarded as a junior synonym of Phlogites (e.g. Chen et al. 2003). Whilst the single specimen of Cheungkungella (Supplementary Figure 3d) has a similar anatomy to Phlogites (Supplementary Figure 3a-c) in terms of the stalk and body, they differ fundamentally in the upper regions. Thus the former taxon appears to have no trace of the multiple branching tentacles that are characteristic of Phlogites. In addition the structure interpreted as an oral siphon (Shu et al. 2001a) is quadrate and so does not have the rounded shape seen in the inter-tentacular lobes of Phlogites (Caron et al. submitted and here Supplementary Figure 3a,b). The presence of definitive craniates in the Chengjiang Fossil-Lagerstätte, which are of course the earliest records of fish (Supplementary Figure 4), is perhaps the one area that has met with a general consensus (at least to the first approximation) (Janvier 1999, Valentine 2004, Halanych 2004, Dawkins 2004, Benton 2005, Prothero 2007). Three taxa are identified: Myllokunmingia fengjiaoa (Supplementary Figure 4k) (Shu et al. 1999a), Haikouichthys ercaicunensis (Supplementary Figure 4a-j) (Shu et al. 1999a, 2003; Hou et al. 2002; Zhang & Hou 2004), and Zhongjianichthys rostratus (Supplementary Figure 4l,n) (Shu 2003). The features that are visible make a telling contrast with the craniate interpretation of yunnanozoans, with unequivocal myomeres (except in the anguilliform Zhongjianichthys) which contra Hou et al. (2002) are W-shaped and not V-shaped (Supplementary Figure 4e; see also Zhang & Hou 2004), paired eyes and a nasal sac (Supplementary Figure 4a-d) (except in Myllokunmingia), and a prominent brachial basket (Supplementary Figure 4f) in Haikouichthys (Shu et al. 2003b, Shu 2003). In the last genus a notochord with equally-spaced vertebral elements has also been identified (Supplementary Figure 4f-j; Shu et al. 2003), but it is notable that in this fish material the notochord is more often indistinct, if not absent. Myllokunmingia (Supplementary Figure 4k) is only known from a single, albeit well-preserved specimen (Shu et al. 1999a). Although it has been regarded as synonymous with Haikouichthys (Hou et al. 2002), we suggest (see also Conway Morris 2006, Shu 2006) that this is unlikely for the following reasons. Thus, the two taxa have different body proportions (Myllokunmingia is less slender; e.g. Shu et al. 2003b, Fig. 1j, l), in Haikouichthys alone are dorsal fin rays evident (curiously these are anteriorly inclined; note the earlier suggestion that these rays are 2 artefacts (Hou et al. 2002) cannot be supported (see also Zhang & Hou 2004)), and most importantly in Myllokunmingia an absence of a branchial basket (along with a more ventral position of the gills) is in contrast to the situation in Haikouichthys. Moreover, Haikouichthys has as many as eight gill pouches, while Myllokunmingia has at most six. Despite the co-existence of a notochord and vertebral elements it is striking how these taxa, the most primitive of agnathans, differ from the extant cyclostomes. They provide an important reminder that invaluable as the latter are for molecular insights attempts to extrapolate the ecology and morphology of these living forms to the deep past can be problematic. 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Shu, D., Conway Morris, S. & Zhang, X-L. 1996b A Pikaia-like chordate from the Lower Cambrian of China. Nature 384, 156-157. 3 Shu, D-G., Conway Morris, S., Han, J., Zhang, Z-F. & Liu, J.N. 2004 Ancestral echinoderms from the Chengjiang deposits of China. Nature 430, 422-427. Shu, D-G., Luo, H-L., Conway Morris, S., Zhang, X-L., Hu, S-X., Chen, L., Han, J., Zhu, M., Li, Y. & Chen, L-Z. 1999a Lower Cambrian vertebrates from South China. Nature 402, 42-46. Shu, D-G. & Conway Morris, S. 2003 Response to comment on “A new species of yunnanozoan with implications for deuterostome evolution”. Science 300, 1372d. Smith, A.B. 2004 Echinoderm roots. Nature 430, 411-412. Smith, M.P., Sansom, I.J. & Cochrane, K.D. 2001 The Cambrian origin of vertebrates. In Major events in early vertebrate evolution (P.E. Ahlberg, ed.), pp. 67-84. London and New York: Taylor & Francis. Swalla, B.J. & Smith, A.B. 2008 Deciphering deuterostome phylogeny: molecular, morphological and palaeontological perspectives. Phil. Trans. R. Soc. B 363, 1557-1568. Valentine, J.W. 2004. On the origin of phyla. Chicago, IL: University of Chicago Press, Chicago. Zhang, X-G. & Hou, X-G. 2004 Evidence for a single median fin-fold and tail in the Lower Cambrian vertebrate, Haikouichthys ercaicunensis. J. Evol. Biol. 17, 1162-1166. Supplement Figures: Fig. 1 Vetulicola cuneata (a,b,d) and V. rectangulata (c); stem-group Deuterostomia. Details of gill slits. a, ELI-0000216, detail of gill slit viewed from exterior; note internal aperture (arrowed in blue) covered by filamentous structure,; b, ELI-0000212, detail of gill slit viewed from interior; note putative inhalant aperture (arrowed in blue); c, ELI-0000210, detail of two gill slits, with external apertures (arrowed in red) and flanking rhomboidal lappets; d, ELI-0000215, gill slits of left (lower level, with external aperture in red) and right (upper level) sides, in latter note connecting tube-like structures and also internal apertures (arrowed in blue). All scale bars millimetric. Abbreviation in this and Fig. 2: ELI, Early Life Institute, Northwest University, Xi’an, China. Fig. 2 Vetulicystids (a,b) and yunnanozoans (c,d); ?stem-group Ambulacraria. a,b ELI-Ech-04-001A, holotype of Vetulicystis catenata, with camera-lucida interpretation; note in particular the three prominent openings; c,d, Yunnanozoon lividum NWU93-1406A, although indifferently preserved this specimen shows clear evidence for partial detachment of the gill-bearing anterior from the more dorsal segmented section. All scale bars millimetric. Fig. 3 Phlogites (a-c); a, ELI-Phl-001, top view, note branching tentacles and inter-tentacular lobes; b, ELI-Phl-002, lateral view showing tentacles and lobes with 4 body containing gut and possible reproductive bodies; c, ELI-Phl-003, lateral view with tentacles and body with prominent gut; d, ELI-0000195, Cheungkongella, upper part of body; note absence of tentacles, but an oral siphonal structure unlike the inter-tentacular lobes of Phlogites. Fig. 4 The earliest fish Haikouichthys ercaicunensis (a-j), Myllokunmingia fengjiaoa (k) and Zhongjianichthys rostratus (l-n). a-d (respectively specimens ELI-Fish-273, 30B, 204, and 287): dorsal views, showing paired eyes, nostril, possible otic capsules, brain and/or spinal cord; e, ELI- Fish-107, mid- trunk, showing complex myomeres and probable heart; f-j (respectively specimens ELI- Fish-12B, 229, 251, 467, 97B, and 402): lateral views of the anterior, showing notochord with equally-spaced vertebral elements, eyes, dorsal fin, myomeres, and branchial arches. k, ELI-0000201, holotype, showing distinctive differences with Haikouichthys, including six gill pouches. l-n, (respectively specimens ELI-00001604, 1605,1601): m, dorsal views, l and n, lateral view, showing paired eyes and distinct antero-dorsal lobe. Abbreviations: Ba, branchial arches; B&Sc, possible brain and/or spinal cord; DF, Dorsal fin; H, Heart; Lap; Left anterior plate, LE, Left eye; M, Myomeres; N, Nostril; N&V, notochord with vertebral elements; ?Oc, possible otic capsules; Rap, Right anterior plate; RE, Right eye; G1,G2,G3,G4,G5,G6, gill pouches 1 to 6. 5