embryonic development of a middle cambrian

Journal of Paleontology, 85(5), 2011, p. 898–903 Copyright ’ 2011, The Paleontological Society 0022-3360/11/0085-0898$03.00

EMBRYONIC DEVELOPMENT OF A MIDDLE CAMBRIAN (500 MYR OLD) SCALIDOPHORAN WORM XI-GUANG ZHANG,1 BRIAN R. PRATT,2

AND

CEN SHEN1

1

Key Laboratory for Paleobiology, Yunnan University, Kunming, Yunnan 650091, China, ,[email protected].; and 2Department of Geological Sciences, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E2, Canada

ABSTRACT—Extraordinarily rare phosphatized embryos liberated from fossiliferous limestone of the Middle Cambrian (about 500 Myr old) Gaotai Formation of Duyun, southern China, are assigned to Markuelia qianensis n. sp. Several specimens with increasing numbers of blastomeres may represent four successive cleavage stages, seemingly exhibiting a radial holoblastic cleavage pattern. No subsequent stages showing gastrulation were observed. However, several specimens are late, pre-hatching stages, each with a vermiform shape coiled in either leftor right-handed directions within the fertilization envelope. These specimens indicate that the intuitively assumed difference in preservation potential between early cleavage and late pre-hatching stages is probably not valid. This new material clarifies the affinity of the first fossilized invertebrate embryos ever described, from the same rocks, which were originally attributed to arthropods, presumably trilobites. Instead, they belong to scalidophorans, and this finding infers likely diversified cleavage patterns for stem members of this group, and yields fresh insight into the embryogenesis of early metazoans as a whole.

INTRODUCTION

of a small number of tiny objects as invertebrate embryos from the Middle Cambrian (about 500 Myr old) of Duyun in southern China (Zhang and Pratt, 1994) at first seemed like a unique, fortuitous occurrence and thus merely a paleontological curiosity. Their significance only began to be more fully understood after a number of other fossilized embryo occurrences—albeit admittedly few—were discovered in Cambrian limestones (Bengtson and Yue, 1997; Kouchinsky et al., 1999; Dong et al., 2004, 2005; Steiner et al., 2004; Pyle et al., 2006; Chen et al., 2007). Moreover, based on this, putative embryos were identified in even older rocks belonging to the Ediacaran Doushantuo Formation (Li et al., 1998; Xiao et al., 1998; Yin et al., 2002; Yin et al., 2007). Such an extraordinary level of preservation by amorphous and finely crystalline apatite in these and other Cambrian invertebrate fossils (e.g., Waloszek, 2003; Zhang and Pratt, 2008) may be the result of microbial processes, as suggested by experimental evidence (Martin et al., 2005; Raff et al., 2008). These embryos have opened a fascinating window onto early metazoan evolution that is just beginning to be exploited. However, what hampers this effort is the fact that rock successions with the appropriate phosphatic preservation are so frustratingly rare and sporadic, and typically there is no preservation of co-occurring juveniles and adults reliably belonging to the same taxa. For example, the Doushantuo microfossils (about 570 Myr old), since being identified as embryos (Xiao et al., 1998), have attracted a great deal of interest. However, the presumed embryonic affinity has not been universally accepted (Bailey et al., 2007; Donoghue, 2007; Bengtson et al., 2010). During the early cleavage phase, with the repeated division an embryo contains more and more blastomeres in a regular way that can be expressed as 2n (where n 5 0, 1, 2, 3,…) without increasing in size. This pattern has long been established in the embyronic development of living animals and was recognized in two successive cleavage stages in the first reported fossil examples (Zhang and Pratt, 1994). Subsequently it has also acted as a criterion for the identification as embryos of both the Ediacaran (Xiao et al., 1998; Yin et al., 2002) as well as other Cambrian specimens

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HE RECOGNITION

(Steiner et al., 2004). It should be noted, however, that cleavage does not always involve synchronous cell division, so that the expected exponential growth may not happen in quite such an ordered fashion. Usually these embryos with blastomeres alone cannot be ascribed with certainty to individual taxa. One exception is a number of occurrences of coiled vermiform examples which, being late embryonic stages exhibiting key anatomical traits, provide more reliable criteria for their identification (Bengtson and Yue, 1997; Dong et al., 2004; Dong et al., 2005). These have been accorded taxonomic treatment, which is an important and necessary step to their future linking with adult forms and evaluating their phylogenetic significance. The first description of Cambrian embryos was based on a few embryos of cleavage stages, which Zhang and Pratt (1994) had originally attributed to arthropods, presumably trilobites because eodiscoid protaspides were the only fossil larvae recovered in the same rocks. Here, recovery of a larger collection from the same Middle Cambrian strata, containing specimens with both blastomeres and coiled vermiform bodies exhibiting ‘egg-larval’ features, constitutes a further contribution to the understanding of the paleo-embryological development and evolution of fossil lineages, and clarifies the affinity of the first-reported Cambrian embryos. MATERIAL AND METHODS

The Middle Cambrian (series 3) Gaotai Formation consists of light-gray coloured, limestone nodules separated by thinbedded shales. About 350 kg of the nodules were collected from the stratigraphic section near the village of Balang, in Duyun, Guizhou, southern China (Fig. 1). The nodules were digested in dilute (5 percent) acetic acid, essentially following the laboratory process introduced by Mu¨ller (1985). Phosphatized microfossils were then picked from the insoluble residue under a stereomicroscope. Along with numerous lingulate brachiopod valves and disarticulated eodiscoid trilobite exoskeletons, plus rarer bivalved bradoriid arthropod carapaces, sponge spicules, fragmentary exoskeletons of polymeroid trilobites, hyolithid molluscs, and various small shelly fossils of problematic affinities, 26 fossilized embryos were collected, amongst which are four pre-hatching stages

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FIGURE 1—Locality map. The fossil site at the Balang section in Duyun, Guizhou, southern China is indicated by a coiled shell symbol.

containing coiled vermiform animals. Of the 21 embryos with various numbers of blastomeres, only one specimen displays some blastomeres underneath the outermost layer of blastomeres (Fig. 2.2). For the others, it remains uncertain if there are more blastomeres inside. Even so, according to the rough size and number of visible cells around the periphery, the 21 embryos seem to represent four successive cleavage (probably corresponding to 32-cell, 64-cell, 128-cell, and 256-cell) stages of the same taxon. SYSTEMATIC PALEONTOLOGY

SCALIDOPHORA Lemburg, 1995 Genus MARKUELIA Val’kov, 1983 Type species.—Markuelia secunda Val’kov, 1984, from the Lower Cambrian (series 2) Pestrotsvet Formation of Siberia. MARKUELIA QIANENSIS n. sp. Figures 2.1–2.13, 4 Phosphatized arthropod embryos ZHANG AND PRATT, 1994, p. 638, fig. 1a–e. Diagnosis.—Species of Markuelia with embryo oblate spheroid. Late-stage embryo with tightly annulated body of about 110 annuli; tiny trunk spines sparse, irregularly distributed. At least two ranks of circum-oral spines arranged radially on longitudinally furrowed anterior region. Description.—All the embryos are at least partly enveloped by a smooth membrane about 1.3 mm thick (Fig. 2.2, 2.3, 2.13). Cleavage-stage embryos are oblate spheroids and vary in size, with a maximum length of 332–394 mm. Where the fertilization envelope has flaked off or disappeared before tissue replacement by apatite the blastomeres underneath display a tight packing with polygonal (hexagonal, rarely pentagonal and heptagonal) facets facing the exterior of the embryo. These consist of spheres surrounded by polygonal parietals (Fig. 2.3, 2.4). Where the spheres are not preserved, blastomeres appear as empty polyhedra (Fig. 2.1–2.3). Of the four cleavage stages, the embryo corresponding to the 32-cell stage displays a distinct size differentiation (76– 125 mm) in blastomere diameter (Fig. 2.1, 2.2), while those corresponding to the 64-cell (60–82 mm), 128-cell (54–64 mm) and 256-cell (23–34 mm) stages exhibit a subequal size range (Fig. 2.3–2.5). Late, pre-hatching stage embryos are also oblate spheroids with a maximum length 340–390 mm (Fig. 2.6–2.12). Within the fertilization envelope the vermiform body curls into a loop with anterior and posterior poles (head and tail ends) juxtaposed, exhibiting either a sinistral (Fig. 2.6) or dextral (Fig. 2.10) coiling. The tubular trunk is tightly annulated throughout with individual annuli 157–209 mm in width and 17–23 mm in length, but the annuli become slightly longer

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(33 mm) near the posterior end. In total the trunk bears about 110 annuli. Spines 27–60 mm in length are irregularly distributed on scattered trunk annuli (Fig. 2.8, 2.9). At the anterior end are short (,65 mm) longitudinal furrows and two ranks of about 20 circum-oral spines (25–34 mm long). Posterior spines may be present but the trunk termination is concealed (Fig. 2.6, 2.8). The surface of the trunk has a longitudinally oriented, fibrous-like texture (Fig. 2.12). The body stretched out would have been about 2.5 mm long (Fig. 4.3). Etymology.—After Qian, the abbreviated name for Guizhou Province, where these embryo specimens were collected. Holotype.—A vermiform embryo coiled within partially surviving fertilization envelope. Key Laboratory for Palaeobiology, Yunnan University (YKLP 11958; Fig. 2.6–2.9). Paratypes.—Twenty-four specimens belonging to either cleavage (21 specimens) or late pre-hatching stages (three specimens) (YKLP 11959–11982). Occurrence.—Middle Cambrian (series 3) Gaotai Formation, Balang section, Duyun, Guizhou province, southern China. Discussion.—It should be noted that species of Markuelia are based on embryos only. The taxonomy will no doubt change with the discovery of post-embryonic individuals. Markuelia qianensis is comparable in size and general morphology to M. hunanensis, but latter displays a spheroidal form, a spineless and more annulated trunk (with up to 130 annuli), and more ranks of oral spines tightly overlapped (Dong et al., 2004, fig. 2c, d; Dong et al., 2005, fig. 2E). In M. qianensis, further circum-oral spines may be hidden by a retracted part of the introvert, but even so it is unlikely to make the anterior end the same as that of M. hunanensis. Markuelia lauriei also displays an oblate-spheroid form, but has a relatively smaller size; more strikingly, it differs from the M. qianensis in having slender protuberances on trunk annuli (Haug et al., 2009). RESULTS

Cleavage Pattern.—Exhibiting detailed embryonic features, these embryos offer a glimpse for the first time nearly the entire cleavage pattern exhibited by Markuelia. Molecular sequence data indicate that the earliest ecdysozoan clade may have evolved radial cleavage (Valentine, 1997). Priapulus caudatus, as a ‘living fossil’ with a very ancient origin, does possess a stereotypical radial cleavage with larger cells at the vegetal pole (Wennberg et al., 2008). Similarly, the size differentiation in blastomeres of M. qianensis (Fig. 2.1, 2.2) may also result from this sort of cleavage, although the ever smaller blastomeres of subsequent stages are more uniform in size. In addition, the blastomeres seemingly inside the specimen of 32-cell stage may be evidence of holoblastic cleavage (Fig. 2.2). On this basis we

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FIGURE 2—Embryos of Markuelia qianensis n. sp. from the Middle Cambrian of Duyun, southern China. 1, embryo possibly corresponding to 32-cell stage (YKLP 11962), showing variably sized blastomeres, some of which had collapsed before phosphatization; 2, embryo possibly corresponding to 32cell stage (YKLP 11963) showing some inner blastomeres (arrowed) due to the collapse of some of the outermost blastomeres; 3, embryo possibly corresponding to 64-cell stage (YKLP 11964); 4, embryo possibly corresponding to 128-cell stage (YKLP 11965), showing secondary coatings (arrowed) on the outer surface of fertilization envelope; 5, embryo possibly corresponding to 256-cell stage (YKLP 11966); 6–9, sinistral vermiform embryo of late pre-hatching stage (holotype YKLP 11958): 6, posterior end (white-arrowed) to the left of anterior end (black-arrowed); 7, curved trunk to the right of head (arrowed); 8, posterior end (white-arrowed) with relatively fewer annuli, and irregularly located trunk spines (black-arrowed); 9, detail of trunk spine (arrowed) shown at lower left in 8; 10–12, dextral vermiform embryo of late pre-hatching stage (YKLP 11959): 10, circum-oral spines approximately arranged in ranks (white- and black-arrowed); 11, detail of oral region showing circum-oral spines of inner rank (white-arrowed) and outer rank (black-arrowed); 12, detail of rectangular area outlined in 10, showing equant, micrometre-sized apatite crystals and locally preserved fine fibrous-like structure of body wall; 13, detail of opposite side of 3, showing tightly packed blastomeres underneath thin fertilization envelope. Scale bar of 100 mm for 1–10.


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FIGURE 3—Embryo of late pre-hatching stage, belonging to a worm-like animal of unknown taxonomic affinity (YKLP 11983): 1, 2, oblique views showing annulated trunk and fine trunk spines (arrowed), with both ends contacting directly; 3, detail of opposite side of 1, showing trunk spine (arrowed).

tentatively interpret the Duyun embryos as possessing a radial, subequal, holoblastic cleavage pattern. The same cleavage strategy seems to be exhibited also by embryos of M. hunanensis with about 485 (Dong et al., 2004) and 630 (Dong, 2007) subequal-sized cells. Together, both display a hexagonal close packing of blastomeres. Because only one specimen shows any indication of its interior, it is unkown if other Duyun embryos also contain more blastomeres inside. Thus it cannot be ascertained if morula stages with fewer than 32 blastomeres are present or when the blastula and gastrula stages began. In the extant priapulids that have been studied, the blastocoel develops during the 16-cell stage, and gastrulation commences at the 64-cell stage (Wennberg et al., 2008). However, it may be that gastrulation took place at a later stage in both M. qianensis and M. hunanensis because their later cleavage stages still display large numbers of subequally-sized blastomeres. Coiling direction of egg-larvae.—The pre-hatching stages of all species of Markuelia, termed ‘egg-larvae’ by Haug et al. (2009), are similar in that the body inside forms an S-shaped coil and the ‘dorsal’ side is flattened against the fertilization envelope. Markuelia qianensis exhibits two directions of coiling, as mirror morphs. The same phenomenon is present also in M. sp. and M. lauriei and was referred to as ‘S-shaped’ and ‘inverted S-shaped’ (Donoghue et al., 2006), ‘S-curled’ and ‘anti-S-curled’ (Haug et al., 2009), and ‘L-form’ and ‘Rform’ (Dong et al., 2010). Although not specifically noted, it is probably also present in M. hunanensis (Dong et al., 2004, fig. 1c, e; Dong, 2007, fig. 1c–g, j–l). For an S-shaped body, the coiling trace from tail to head (or vice versa) proceeds

from left to right (Fig. 4.1). Accordingly, this style may be stated as sinistral (left-handed) and the inverted S-shaped one (Fig. 4.2) as dextral (right-handed). Markuelia hunanesis, M. lauriei and M. qianensis all exhibit both left- and righthanded coiling. Although M. secunda, M. spinulifera, and M. waloszeki have been known only exhibiting one direction of coiling (Dong et al., 2010), it seems likely that this feature is diagnostic at the generic level as being determined during embryogenesis. We recovered a fifth late, pre-hatching stage embryo (YKLP 11983). With a maximum diameter of 300 mm, its curved trunk bears at least 30 annuli and its head attaches directly to its tail without coiling (Fig. 3.1–3.3). It is clearly not conspecific with any known species of Markuelia but must belong to another annulated, worm-like taxon. DISCUSSION

Our new collection supports the claim that the preservation of embryos is temporally and taxonomically biased (Donoghue et al., 2006; Dong et al., 2010). Permineralization of soft tissues by authigenic apatite occurred sporadically during the Cambrian, but it cannot be stressed too much how exceedingly rare this kind of preservation is. It was undoubtedly related to a confluence of several factors, including elevated phosphorus in seawater, poor water circulation, restriction of bioturbation in the deeper marine setting in which formed nodular limestones (including ‘orsten’, as the fossil-bearing concretions are called in Swedish), and perhaps also specific properties of clay-bearing lime mud sediment, such as grain size and shape, that were conducive to concentration and precipitation of calcium phosphate (Waloszek, 2003; Zhang and Pratt, 2008).

FIGURE 4—Reconstruction of Markuelia qianensis n. sp. 1, a sinistral late pre-hatching stage embryo coiled left-handedly within its fertilization envelope; 2, mirror morph of 1, coiled dextrally; 3, late pre-hatching stage embryo unfurled. Abbreviations: an 5 annuli; cs 5 circum-oral spines; fe 5 fertilization envelope; lf 5 longitudinal furrow; m 5 mouth; ps 5 posterior spines; ts 5 trunk spine; tt 5 trunk termination.


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Probably the occurrences of such fossilized embryos involved microbial processes, as suggested by experimental evidence (Martin et al., 2005; Raff et al., 2008). As suggested by taphonomical experiments, embryos at various developmental stages can have differential preservation potential (Gostling et al., 2008). However, unlike the Middle and Late Cambrian embryos from Hunan which are mainly late embryonic stages, with just a few cleavage-stage ones (Dong et al., 2004), the Duyun beds yielded 21 specimens that correspond to cleavage stages and only five – each with a vermiform body inside – belonging to late, pre-hatching stages. This preponderance of early stages suggests that the presence of a tough cuticle may not have been as critical a factor for selective preservation as supposed by Donoghue et al. (2006). Other scenarios may be imagined, like selective phosphatization of embryos in some fecal matter or inside the gut of a dead and decaying predator or scavenger (cf. Butterfield, 2002), but such hypotheses cannot as yet be tested. Similarly, it remains unclear why no embryos of Markuelia species in the gastrula stage and in the stages prior to the prehatching form have been discovered. Perhaps the former is due to a short time span during which gastrulation lasted, a phenomenon that is seen in some living invertebrates (Davy and Turner, 2003). Although remote from the crown ancestor of Ecdysozoa (Harvey et al., 2010), priapulids with a well-preserved fossil record suggest they are a primitive lineage with little morphological change in the tubular body and spiny introvert over 500 million years (Webster et al., 2006). If so, it might be assumed that they have retained a conservative embryogenesis, such that their Cambrian ancestors may had already evolved a similarly early onset of gastrulation. However, the Duyun embryos suggest that gastrulation may have taken place at a later stage, since the ever larger number of more or less uniformly sized blastomeres in their subsequent cleavage stages still display a hexagonal close packing, and no sure indication of gastrulation at all. The same cleavage strategy seems to be exhibited also by embryos of M. hunanensis (Dong et al., 2004; Dong, 2007). A delayed occurrence of gastrulation in Markuelia may suggest a divergence of the two lineages early in the ‘Cambrian Explosion’. Alternatively, the cleavage pattern in extant priapulids could be a late-derived feature: there is no reason why embryogenesis could not have been subject to dramatic, perhaps non-adaptive evolutionary change too. Unlike the exceedingly rare Cambrian specimens, the Doushantuo embryo-like objects occur in astonishing abundance in shallow-water carbonate rocks (grainstones) deposited under high-energy conditions. Curiously, after over a decade of extensive investigation, no convincing post-embryonic counterparts have been confirmed. Our finding urges continued investigation of these intriguing organisms and their taphonomic history. ACKNOWLEDGMENTS

We thank L. Chen and B.-Q. Hao for assistance in fieldwork, H.-Q. Zhang and M. Tian for help in sample preparation, and S. Bengtson and P. C. J. Donoghue for comments on the manuscript. This study was supported by the National Natural Science Foundation of China (40972012), and Natural Science Foundation of Yunnan Province (2008CC005). REFERENCES

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