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Home , Evolutionary Fauna, Haplophrentis, Hyolitha, Hyolithida, Ovatoryctocara

1 New insight into the soft anatomy and shell microstructures of early 2 orthothecids () 3 4 Luoyang Li1,2, Christian B. Skovsted1,2, Hao Yun2, Marissa J. Betts2,3, Xingliang 5 Zhang2, 6 7 1Department of Palaeobiology, Swedish Museum of Natural History, Box 50007, SE- 8 104 05 Stockholm, Sweden. 9 2State Key Laboratory of Continental Dynamics, Shaanxi Key laboratory of Early 10 Life and Environments, Department of Geology, Northwest University, Xi’an 710069, 11 PR China. 12 3Palaeoscience Research Centre, School of Environmental and Rural Science, 13 University of New England, Armidale, NSW, Australia, 2351 14 E-mail for correspondence: [email protected] 15 16 Abstract 17 Hyoliths (hyolithids and orthothecids) were one of the most successful early 18 biomineralizing lophotrochozoans, and were a key component of the Cambrian 19 evolutionary fauna. However, the morphology, skeletogenesis and anatomy of earliest 20 members of this enigmatic clade, as well as its relationship with other 21 lophotrochozoan phyla remain highly contentious. Here we present a new orthothecid, 22 Longxiantheca mira gen. et sp. nov. preserved as part of the secondarily phosphatized 23 Small Shelly Fossil assemblage from the lower Cambrian Xinji Formation of North 24 China. Longxiantheca mira retains some ancestral traits of the clade with an 25 undifferentiated disc-shaped operculum and a simple conical conch with a two- 26 layered microstructure of aragonitic fibrous bundles. The operculum interior exhibits 27 impressions of soft tissues, including muscle attachment scars, mantle epithelial cells 28 and a central kidney-shaped platform in association with its feeding organ. Our study 29 reveals that the muscular system and tentaculate feeding apparatus in orthothecids 30 appear to be similar to that in hyolithids, suggesting a consistent anatomical 31 configuration among the total group of hyoliths. The new finding of shell secreting 32 cells demonstrates a mantle regulating mode of growth for the operculum. Taking all 33 these data into considerations, especially on the basis of shell microstructures, we 34 argue that hyoliths were an extinct sister group of molluscs. 35 36 Keywords: Cambrian, orthothecid, soft anatomy, shell microstructure 37 38 Introduction 39 The evolutionary emergence of metazoans during the Ediacaran–Cambrian transition, 40 the so called ‘’, led to the origination not only of most modern 41 phyla, but also some extinct groups with peculiar Baupläne [1,2]. Hyoliths are 42 such an enigmatic group of conical shelled , which thrived through Cambrian 43 to times, and then declined until their demise at the end of [3]. 44 They possess a deep conical conch and a lid-like operculum, and are commonly

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45 divided into two morphologically distinct subgroups (Orders): the Sysoev, 46 1957 [4] and the Orthothecida Marek, 1966 [5]. Hyolithids are characterized by a 47 ventral projecting shelf (ligula) at the aperture of the conch, a differentiated 48 operculum separated into cardinal and conical shield, and an additional pair of curved 49 lateral spines, named helens [6,7]. Orthothecids lack helens and ligula, but their 50 conchs generally display a high variability in transverse profile, e.g. they can have 51 circular, ovoid, kidney-shaped and trapezoid cross-sections [8]. Orthothecids are 52 widely considered to be sediment-feeders, while hyolithids are thought to have 53 developed a filter feeding lifestyle [9]. 54 In recent years, studies on hyoliths have mainly been focused on solving a 55 longstanding controversy regarding the biological affinity of the clade. It is generally 56 accepted that hyoliths belong to lophotrochozoans, yet a precise phylogenetic position 57 remains highly contentious. Three main hypotheses have been promoted historically: 58 1) the clade might represent an extinct group within the [3,12], 2) constitute 59 their own phylum [13], or 3) have a close relationship with sipunculid worms (peanut 60 worms) [14]. The proposed relationship of hyoliths with sipunculans has been more or 61 less abandoned, and the most popular molluscan affinities of hyoliths have also been 62 strongly challenged by several sensational discoveries of preserved soft tissues in 63 hyolith animals from -type Konservat-Lagerstätten. New data from the 64 Burgess Shale and the in North America have revealed a tentaculate 65 feeding apparatus in the hyolithid [15], and a supposedly coelomate, 66 pedicle-like apical attachment organ was reported in the orthothecid Pedunculotheca 67 from the Chengjiang Lagerstätte of South China [16]. These observations seemingly 68 point to a lophophorate (large group encompassing , and 69 bryozoans) affinity for hyoliths, specifically, a phylogenetic position close to 70 brachiopods [17, 18]. However, Liu et al., [19] has pointed out that the hyolith 71 tentaculate feeding organ may not be homologous with the of 72 lophophorates, and that the interpreted ‘pedicle’ very likely represents a partially 73 crushed portion of the shell. Additionally, study of hyolith shell microstructures 74 provides crucial evidence that hyoliths produce their biomineralized skeletons in a 75 strikingly similar way to molluscs, which tends to support a close relationship 76 between hyoliths and molluscs [20]. 77 The unusual character combination of a tentaculate feeding organ and typical 78 mollusc-like shell microstructures is center to the debate on hyolith affinity and 79 relationships. However, preservation of soft tissues vs. hard skeletons are usually 80 biased by different taphonomic pathways. The Burgess Shale-type preservation 81 involves the conservation of animal soft parts, preserved in extraordinary anatomical 82 detail [XXX], while delicate phosphatization processes in Small Shelly Fossil 83 assemblages can replicate very fine microstructural details of primary skeletons 84 [XXX]. In addition, question remains regarding the ancestral characters of the clade, 85 especially with respect to the orthothecid linage. Hyolithids and orthothecids are 86 phylogenetically closely related. A number of Cambrian hyolith taxa possess 87 morphological traits of the two orders, possibly representing an intermediate form 88 derived from orthothecids and leading to hyolithids [XXXX], or they indicated an

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89 ancestral state of hyoliths and through time evolved into the two well-differentiated 90 linages [XX]. 91 Here, we present a new orthothecid Longxiantheca mira gen. et. sp. nov. from 92 the lower Cambrian Xinji Formation of North China. The new material, collected as 93 part of secondarily phosphatized Small Shelly Fossil (SSF) assemblages, exquisitely 94 preserves not only primary aragonitic shell microstructures in the external wall of the 95 conch, but also information of soft tissues on the internal surface of the operculum. 96 This study provides important new data regarding the morphology, skeletogenesis and 97 soft anatomy, particularly the musculature, feeding apparatus and mantle system, of 98 early orthothecids. Taking all these evidence together, especially data of hyolith shell 99 microstructures from North China, we aim to contribute a clearer understanding on 100 hyolith affinity and its relationship with other lophotrochozoan groups. 101 102 Geological setting, material and methods 103 Precambrian–Cambrian strata along the southern margin of the North China Platform 104 include (in ascending order) the Luoquan, Dongpo, Xinji and Zhushadong formations 105 [21]. A disconformity occurs at the upper boundary of the Ediacaran Dongpo 106 Formation or Luoquan Diamictite, and the succeeding Xinji Formation yields the 107 oldest record of Cambrian deposits in North China, equivalent to upper Stage 3 or 108 lower Stage 4 [XXX]. The Xinji Formation is mainly composed of siliciclastic 109 sediments intercalated with carbonate intervals, and is conformably overlain by the 110 massive dolostones of the Zhushadong Formation (Fig. 1). Carbonates in the Xinji 111 Formation yield an abundant and diverse assemblage of SSF, including sponge 112 spicules, chancelloriids, brachiopods, hyoliths, micromolluscs, and 113 echinoderm ossicles, as well as other problematic fossils of uncertain biological 114 affinities and function [22–24]. Rock samples were collected and treated with 115 buffered, 10% acetic acid to retrieve acid-resistant microfossils. More than 140 116 specimens have been selected and studied from the acid-resistant residues. Specimens 117 were mounted, sputter-coated with gold for examination with a FEI Quanta 400 FEG 118 scanning electron microscope (SEM) and Zeiss Xradia 520 Versa Micro-CT at the 119 Northwest University as well as a Hitachi S4300 SEM at the Swedish Museum of 120 Natural History. Microfossils described below are deposited at the Early Life Institute 121 (ELI), Northwest University, Xian, China. 122 123 Results 124 Systematic paleontology 125 Class HYOLITHA Marek, 1963 126 Order ORTHOTHECIDA Marek, 1966 127 Longxiantheca Li in Li et al. gen. nov. 128 129 Etymology. Genus name Longxiantheca referring to the Longxian County where the 130 fossils were recovered; name mira meaning wonderful. 131 Type species. Longxiantheca mira Li in Li et al. sp. nov. 132 Diagnosis. Conch smooth, straight to slightly curved, with circular cross-section, low

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133 angle of divergence; Apertural margin planar and initial shell septate; Shell wall 134 consisting of two layers of aragonitic fibrous bundles. Operculum simple, disc-like 135 without cardinal processes and clavicles; marginal flange elevated; dorsal margin 136 distinct, flaring toward ventral side; internal surface bearing a distinct kidney-shaped 137 platform centrally. 138 Comparison. The conch of Longxiantheca is similar to that of , especially 139 Conotheca australiensis from contemporaneous strata of South Australia [25], North- 140 East Greenland [26] and North China [27], as well as Conotheca cf. mammilata and 141 Majatheca tumefacta from the Siberian Platform [28], Conotheca subcurvata from 142 France [9], Circotheca johnstrupi from Bornholm [10] and Petasotheca sp. from 143 Mexico [29]. All these taxa possess a similar conoidal conch with circular or 144 subcricular cross-section and a planar aperture. Moreover, the conch of 145 Longxiantheca is also reminiscent of Cupitheca in terms of its blunt apical 146 termination with characteristic dense tubules [30]. However, the older parts of 147 Cupitheca conchs are decollated successively during the life of the organism by a 148 specialized dissolution process [31], which is unknown in the septate initial parts of 149 Longxiantheca conchs. 150 Notwithstanding the obstacles in characterizing the conchs, the opercula tend to 151 be more informative. Opercula of Conotheca australiensis are well documented, and 152 it is clear that Longxiantheca opercula lack any trace of the cardinal processes and 153 clavicle-related structures seen in C. australiensis. Longxiantheca opercula can also 154 be easily distinguished from opercula of Cupitheca, which have prominent cardinal 155 processes on the dorsal margin of the internal surface [32]. Comparisons can also be 156 made with other early orthothecid taxa, including Ladatheca, Allatheca, Petasotheca 157 and disarticulated unidentified opercula from Greenland (orthothecid operculum A in 158 Peel et al.) [29,34,35] with similarly shaped opercula. The main difference 159 distinguishing Longxiantheca from all these taxa is that the Longxiantheca operculum 160 has a diagnostic kidney-shaped platform and a marginal flange, while the remaining 161 taxa only express a slightly elevated circumferential ridge. 162 163 Longxiantheca mira Li in Li et al. sp. nov. 164 165 LSID. urn:lsid:zoobank.org:act:911A5864-76A5-4B66-A697-483BEF2000D5 166 Holotype. ELI- LC06-23-03 from the Chaijiawa section of the Xinji Formation; Fig. 167 2, 3. 168 Material. Four conchs and 82 opercula, in addition to 23 articulated specimens from 169 the Chaijiawa section of Shaanxi Province; 40 conchs from the Sanjianfang section of 170 Henan Province, North China. 171 Description. The conch is straight to slightly curved with circular cross-section. The 172 angle of divergence is low (varying from 15° to 20°) and uniformly expanding (Fig. 173 2E–H). The aperture is straight in lateral profile with a diameter up to 1mm. The apex 174 is unknown as the conch usually exhibits a blunt termination in association with 175 breakage along internal septa (Fig. 2F, H). 176 The partially phosphatized shell material of the conch reveals shell microstructure

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177 in the form of aragonitic fibrous bundles that occur in a two-layered pattern (Fig. 2I). 178 In the outer layer (thickness is hard to determine owing to poor preservation), the 179 fibrous bundles are inclined longitudinally towards the conch apex (Fig. 3C), while 180 the fibrous bundles of the inner layer run transversely around the conch (Fig. 3A). The 181 inner layer (20μm in thickness) seems to be much thicker than the outer layer and 182 contains randomly distributed pores and elongated slits (Fig. 3B). Additionally, the 183 terminal ending of the conchs, corresponding to original transverse septa, consists of a 184 concentration of orthogonal tubules (Fig. 3D, E). 185 The operculum is circular in outline with a slightly elevated, central, rounded 186 apex (Fig. 2A). The external surface of the operculum is evenly convex, smooth or 187 ornamented with a combination of co-marginal growth lines and radial striations (Fig. 188 2A, B). The internal surface lacks cardinal processes and clavicles, is slightly concave 189 centrally, and is surrounded by an elevated circumferential marginal flange (Fig. 2C, 190 D). A kidney-shaped, symmetrically placed platform dominates the internal surface of 191 the operculum (Fig. 2M). This structure is also observable as a smooth impression on 192 the apertural surface of internal moulds (Fig. 2L). Replicas of soft tissues include 193 reticulate textures (each individual polygon ~10 μm wide) that are consistently 194 distributed within three distinct zones on the internal surface of phosphatized opercula 195 (Fig. 2P, Q). The most conspicuous zone follows the inner side of the marginal flange 196 where the reticulate microtexture is interpreted to be formed by mantle epithelial cells 197 (mec; Fig. 2P). The other two zones with reticulate textures are considered to 198 represent muscle attachment scars, and occur in the summit area and on the ventral 199 side of the operculum respectively (Fig. 2J, K, O). 200 Remarks. While Longxiantheca mira gen. et sp. nov. is very common in the studied 201 sections, articulated specimens only occur in the Chaijiawa section, probably owing to 202 unique taphonomic conditions inducing delicate phosphatization. Internal moulds of 203 the gastropod Pelagiella from this site also usually preserve very fine details of its 204 shell structures [XXX]. The apical portion of the conch is missing from all specimens 205 in our collections, presumably a consequence of breakage along internal septa. Some 206 specimens suggest that the operculum could be withdrawn some distances inside the 207 conch (Fig. 2E–G), as is frequently the case among orthothecids [36]. 208 Occurrence. The Xinji Formation in Shaanxi and Henan provinces, North China; 209 , Stages 3–4. 210 211 212 Discussion 213 214 (a) Ancestral morphology of hyoliths 215 The early evolution of hyoliths revolved around the origin and modification of an 216 ancestral state of anatomical configuration and skeletal architecture. However, the 217 earliest hyoliths are hard to identify, especially from the infamous ‘Tube World’ 218 period of the latest Ediacaran–earliest Cambrian interval, a time with diverse 219 enigmatic tube-like fossils [XXX]. The earliest unambiguous orthothecids occurred in 220 SSF assemblages in the early (middle Stage) of ,

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221 Mongolia and South China, represented by incomplete fossils of simple conoidal 222 conches with circular or sub-circular cross section, and their presumed opercula 223 exhibiting a simple disc shape [XXX]. The hyolithids appeared slightly later around 224 the beginning of Cambrian Series 2 [XXX]. The derivation of hyolithids from 225 ancestors within orthothecids is highlighted in a recent study as key characteristics of 226 hyolithids, i.e. the helens, have been demonstrated to be originated from rod-like units 227 of clavicles of some intermediate hyolith taxa [XXX]. 228 The exquisite preservation of Longxiantheca mira permits detailed 229 reconstruction of critical parts of the shell and soft anatomy. By comparison with 230 early orthothecid taxa discussed above, Longxiantheca mira appears to retain some 231 presumed ancestral traits of the clade: 1) simple conoildal conch with circular or sub- 232 cricular cross section; 2) presence of a loosely-organised fibrous bundle 233 microstructure; 3) undifferentiated operculum lacking cardinal processes and 234 clavicles, but possibly with circumferential deposits (elevated ridges or marginal 235 flange) on the internal surface; 4) soft body anatomy with three evolutionary 236 conserved muscles and a tentaculate feeding organ; 5) digestive tract was not 237 preserved in L. mira, but postulated to be similar to that of Conotheca subcurvata 238 from the Terreneuvian Heraultia Limestone of France which exhibits a sinuously- 239 folded gut tract, adapted to a deposit-feeding function [XXX]. According to this 240 model, the cardinal processes the operculum, clavicles, helens and a tentaculate 241 feeding apparatus adapted for filter feeding are all considered to be secondarily 242 evolved characteristics in later hyoliths. 243 244 (b) Soft tissues associated with the operculum 245 Tentaculate organs are widely distributed among lophotrochozoans and they 246 perform various functions including locomotion, respiration and suspension feeding 247 [37,38]. It has been demonstrated that the tentaculate feeding organ of hyoliths lacks 248 definitive apomorphies of a true lophophore [16]. In particular, the absence of cilia 249 and the arrangement of tentacles along two arms but not in a pattern surrounding a 250 central mouth, sets the hyolith feeding organs apart from a lophophore [16]. This has 251 cast doubt on the interpretation of the tentaculate structure in hyoliths as a 252 lophophore, and suggests that the hyolith animal is not a lophophorate. 253 Evidence from hyolithid Haplophrentis and orthothecid Triplicatella clearly 254 shows that the two hyolith linages had similar tentaculate feeding organs. The kidney- 255 shaped platform on the internal surface of L. mira opercula suggests the presence of a 256 similar tentaculate organ in this species, however no additional details of this 257 apparatus have been preserved. Such a kidney-shaped structure was probably a 258 support or attachment structure when the organism extends outside the shell for 259 feeding. 260 While the tentaculate feeding apparatus of hyoliths remains controversial, a 261 consensus has more or less been reached regarding the muscular system of the hyolith 262 soft body. The muscular system is best known in hyolithids, where a series of muscle 263 attachment scars have been identified from both conch and operculum, particularly in 264 Maxilites from the middle Cambrian and Gompholites from the late Ordovician of the

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265 Czech Republic [6]. A common muscular system in hyolithids include: (i) a pair of 266 large oval muscle scars (labeled ‘e’ in [6], also termed ‘adductor scars’ [39]) which 267 occurs on the conical shield of the operculum, and connects to the ventral side of 268 apertural wall of the conch, responsible for closure of the aperture; (ii) a single scar 269 close to the summit (labeled ‘c’ in [6]), located on the plane of symmetry of the 270 operculum and possibly associated with the feeding organ [40]; (iii) a series of smaller 271 muscle scars (labeled ‘a’, ‘b’, ‘d’ in [6]) that are associated with the cardinal process 272 and clavicles of the operculum. In orthothecids, a similar apical muscle attachment 273 site was recently observed in ‘Hyolith operculum A’ and Paramicrocornus 274 zhenbaensis of the Cambrian Shuijingtuo Formation, South China [40], as well as in 275 Triplicatella opimus from the Chengjiang Lagerstätte [16]. 276 Our new material shows that the arrangement of a summit muscle scar (sm) and 277 a pair of large oval adductor muscle scars (am) on the ventral side of the internal 278 surface of the L. mira operculum coincides closely with the ‘c’ and ‘e’ muscle scars of 279 hyolithids. These three shared muscle attachment sites thus appear to be 280 evolutionarily conservative and uniform in both orthothecid and hyolithid opercula, 281 which further suggests a common muscular system across the clade. The other smaller 282 muscle scars presumably evolved secondarily in association with the innovation of 283 cardinal processes and additionally in hyolithids, clavicles and helens (Fig. 4). 284 The reticulate texture along the inner side of the marginal flange, interpreted as 285 mantle epithelial cells, is of great importance for understanding the biomineralizing 286 mode of the operculum. The operculum of L. mira is morphologically similar to the 287 opercula of many operculate gastropods as both have a simple disc shape [41]. 288 However, the latter represents an accessory skeletal element secreted by a different 289 mechanism (by specific gland cells on the posterior foot) compared to the main shell 290 which is regulated by an underlying organic matrix, i.e. the mantle [42,43]. It is 291 generally proposed that the mineralized conch and operculum of hyolith skeletons are 292 secreted by the same shell secreting system. This idea is strongly supported by shared 293 microstructures including elongate tubules, aragonitic fibers and more advanced 294 foliated aragonite microstructures, as well as the fact that hyolith opercula generally 295 develop much more complex internal morphologies than the conch [20]. The new 296 material described herein not only confirms the presence of a mantle-regulating mode 297 of growth for the operculum, but also represents the most convincing evidence for, 298 and earliest record of the mantle system in orthothecids. 299 300 (c) Shell microstructures of the mineralized conchs 301 The shell microstructures of mineralized orthothecid conchs consist of various 302 fibrous microstructures, whose basic units are generally described as needle-like 303 crystalline aragonite or aragonitic fibers in the literature [44,45]. Such fibrous 304 microstructures are commonly replicated on the surface of phosphatized internal 305 moulds preserved as SSFs, and can be classified into three distinctive types according 306 to their morphological complexity, and orientation of fibers within the conchs [X]. 307 The first type, termed ‘fibrous bundles’, mainly occurred in some early 308 representatives of orthothecids that constructed simple conoidal conchs and disc-like

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309 opercula without cardinal processes but sometimes with circumferential deposits on 310 the internal surface, e.g. Majatheca tumefacta and Longxiantheca mira (herein) as 311 well as some other indeterminate allathecid and circothecid conchs [28,34]. These 312 fibrous bundles have a two-layered pattern with the crystalline aragonite oriented 313 transversely in the inner layer and longitudinally in the outer layer. The second form, 314 broadly termed ‘lamello-fibrillar’, occurred in some orthothecids whose operculum 315 interior bears a pair of cardinal processes and/or clavicle-like structures, as in 316 Cupitheca and some intermediate forms that possess combinations of characters 317 typical of both hyolithids and orthothecids, e.g. Paramicrocornus [27,30]. The 318 lamello-fibrillar structure is composed of elongated fibers of aragonite, amalgamated 319 into successive horizontal lamellae with aragonitic fibers in different orientations 320 from one stacked lamella to the next. In contrast to the lamello-fibrillar, the third form 321 is ‘parallel fiber’ (also described as fibrous foliated structure in the gastropod 322 Pelagiella madianensis) [45,46]. This structure consists of aragonitic fibers that are 323 parallel to the shell surface and are constant in orientation between adjacent lamellae. 324 Distribution of this type of shell microstructures in hyoliths is rarely known, but 325 seeming occurs in some derived orthothecids as well as in hyolithids [XX]. 326 Similar fibrous microstructures are also very common in our mollusc collections 327 from the same locality in North China, and show that the needle-like aragonite or 328 aragonitic fibers, especially in the lamello-fibrillar and fibrous foliated 329 microstructures, are actually shaped into elongate narrow laths [46]. Each individual 330 aragonitic micro-lath, approximately 2 μm in width and 10 μm in length, was 331 enveloped by organic membranes, and had its inferred crystallographic C-axis 332 perpendicular to the long axis of the lath. These micro-laths consist of numerous 333 elongate rod-like crystallites of aragonite, which are parallel to each other and have a 334 diameter of about 50–200 nm. According to these observations, we infer that similar 335 sub-structures and architectures also occur in L. mira and other orthothecids, whose 336 fibrous microstructures are amalgamated by a basic unit of micro-lath crystalline 337 aragonite and crystallite columns (Fig. 5). 338 In addition to various fibrous microstructures that occurred widely in early 339 orthothecids, the capability of building advanced shell microstructures evolved later in 340 numerous derived orthothecid and hyolithid taxa, such as foliated aragonite and 341 crossed foliated lamellar microstructures in Protomicrocornus and in the helens- 342 bearing hyolithids Microcornus, Parakorilithes and Parkula [16]. These taxa express 343 more complex morphological features of their opercula such as division into cardinal 344 and conical shields, clavicles, rooflets, teeth-like structures, lateral sinus, etc. [X]. 345 Crucially, the evolutionary trend of hyolith shell microstructures from loosely- 346 organized fibrous types in early orthothecids to more advanced forms in derived 347 orthothecids and hyolithids indicates increasing control over biomineralization by the 348 hyolith animal through time. 349 It is not surprising to us that orthothecid and hyolithid conchs are composed of a 350 variety of mollusc-like microstructures with complex hierarchical organization. 351 During the Ediacaran-Cambrian interval, mineralized skeletons evolved 352 independently in different biomineralizing animal phyla from their pre-existing

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353 organic counterparts respectively [X]. From this perspective, the commonalities of 354 shell microstructures in early Cambrian hyoliths and molluscs reflect a similar mantle 355 regulating system that independently derived from the same ‘biomineralization 356 toolkit’ of an unmineralized common ancestor of the two groups [46]. In this scheme, 357 the independent acquisition of skeletons is highlighted, and no mineralized common 358 ancestry exists between the two groups. 359 Even so, features of biomineralization provide crucial data for phylogenetic 360 studies, and have been widely and successfully applied to high-level classification, as 361 exemplified by tommotiids and linguliform brachiopods [XXX], and 362 aculiferan molluscs [X], bryozoans and calcareous brachiopods [X], etc., that a close 363 phylogenetic relationship is strongly implied by their similar shell microstructures. 364 This might be the same case for hyoliths and molluscs. The apparent similarities of 365 biomineralization in hyoliths and molluscs point to a much closer relationship of the 366 two groups than previously assumed. However,characteristic molluscan apomorphies 367 including the foot and radula are hitherto unknown in hyoliths, and that the hyolith 368 soft body possesses a unique muscular system and tentaculate feeding organ. 369 Therefore, we suggest that hyoliths may not belong to the Mollusca, but 370 phylogenetically may constitute a sister group to the molluscs. 371 372 Conclusion 373 There is convincing evidence suggesting that hyolithids derived from 374 orthothecids throughout numerous intermediate forms in the early Cambrian [XX]. 375 The new orthothecid Longxiantheca mira gen. et sp. nov. from North China retains 376 some ancestral morphologies of the clade. The mineralized skeletons of ancestral 377 hyoliths presumably encompass a simple conoidal conch with circular or sub-circular 378 crossed section and fibrous bundles microstructure, as well as a disc-shaped 379 operculum secreted by underlying mantle regulating system. Character combination 380 of soft anatomy appears to consist of three evolutionary conserved muscles, a deposit- 381 feeding tentaculate apparatus and a possible sinuously-folded digestive tract. 382 Mineralized conchs of early hyoliths, especially orthothecids, consists of three 383 main types of fibrous microstructures, amalgamated by basic units of micro-lath 384 crystalline aragonite and crystallite columns. The evolution of shell microstructures 385 from loosely-organised fibrous fabrics in early orthothecids to advanced fabrics with 386 complex hierarchical organization in derived hyolith taxa indicating increasing control 387 over biomineralization by the animal. Crucially, biomineralizing characters so far 388 provide the most reliable evidence for hyolith affinity, and a close relationship 389 between hyoliths and molluscs is strongly implied on the basis of their striking 390 similarities in shell microstructures, and accordingly, hyoliths are considered as a 391 sister group of molluscs. 392 393 Author contributions 394 L.L. conceived and prepared the manuscript. H.Y. assisted in to fossil collection and 395 fieldwork. C.B.S, X.Z. and M.J.B. provided input in the analysis and interpretation. 396 All authors contributed to subsequent revision of the manuscript.

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397 398 Competing interests 399 We declare we have no competing interests. 400 401 Acknowledgement. 402 We thank Sun Jie (Northwest University) for assistance in Micro-CT scanning 403 analysis. Thanks also goes to Pan Bing and Li Guoxiang (Nanjing Institute of 404 Geology and Palaeontology, CAS), Timothy P Topper (Swedish Museum of Natural 405 History), Fan Liu (Northwest University) for valuable discussions. We are grateful to 406 the three anonymous reviewers and the Editor for their constructive and encouraging 407 comments. 408 409 Funding. 410 This work was supported by the National Key Research and Development Program 411 [Grant 2017YFC0603101]; the Natural Science Foundation of China [Grants 412 41621003, 41890840, 41930319 and 41772011]; the Strategic Priority Research 413 Program of Chinese Academy of Sciences [Grant XDB26000000]; 111 Project 414 [D17013]; and the Swedish Research Council [Grant VR2016-04610]. 415 416 Reference 417 1. Shu D, Isozaki Y, Zhang X, Han J, Maruyama S. 2014 Birth and early evolution of 418 metazoans. Gondwana Res. 253, 884–895. (doi:org/10.1016/j.gr.2013.09.001) 419 2. Briggs DEG. 2015 The Cambrian explosion. Curr. Biol. 25, 864–868. 420 (doi:org/10.1016/j.cub.2015.04.047) 421 3. Malinky JM, Yochelson EL. 2007 On the systematic position of the Hyolitha 422 (Kingdom Animalia). Mem. Assoc. Australas. Palaeontol. 34, 521–536. 423 4. Sysoev AV. 1957 K morfologii, sistematicheskomu polozheniyu i sistematike 424 khiolotov [On the morphology, systematic position and systematics of 425 hyoliths]. Doklady Akademii nauk SSSR 116, 304–307. 426 5. Marek L. 1966 New hyolithid genera from the Ordovician of Bohemia. Časopis 427 národního muzea 135, 89–92. 428 6. Martı́ Mus M, Bergström J. 2005 The morphology of hyolithids and its functional 429 implications. Palaeontology 48, 1139–1167. (doi:org/10.1111/j.1475- 430 4983.2005.00511.x) 431 7. Martı́ Mus M, Jeppsson L, Malinky JM. 2014 A Complete Reconstruction of the 432 Hyolithid Skeleton. J. Paleontol. 88, 160–170. (doi:org/10.1666/13-038) 433 8. Malinky JM. 1987 Taxonomic revision of lower and middle Orthothecida 434 (Hyolitha) from North America and China. J. Paleontol. 61, 942–959. 435 (doi.org/10.1017/S0022336000029310) 436 9. Devaere L, Clausen S, Álvaro JJ, Peel JS, Vachard D. 2014 Terreneuvian 437 orthothecid (Hyolitha) digestive tracts from northern Montagne Noire, France; 438 taphonomic, ontogenetic and phylogenetic implications. PLoS ONE 9, e88583. 439 (doi:org/10.1371/journal.pone.0088583) 440 10. Berg-Madsen V, Valent M, Ebbestad JOR. 2018 An orthothecid hyolith with a

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485 26. Malinky JM, Skovsted CB. 2004 Hyoliths and small shelly fossils from the lower 486 Cambrian of north-east Greenland. Acta Palaeontol. Pol. 49, 551–578. 487 27. Pan B, Skovsted CB, Sun H, Li G. 2020 Biostratigraphical and 488 palaeogeographical implications of Early Cambrian hyoliths from the North 489 China Platform. Alcheringa. (doi:10.1080/03115518.2019.1577492) 490 28. Kouchinsky AV, Bengtson S, Clausen S, Vendrasco MJ. 2015 A lower Cambrian 491 fauna of skeletal fossils from the Emyaksin Formation, northern Siberia. Acta 492 Palaeontol. Po. 60, 421–512. (doi:org/10.4202/app.2012.0004) 493 29. Devaere L, Clausen S, Sosa-Leon JP, Palafox-Reyes JJ, Buitrón-Sánchez BE, 494 Vachard D. 2019 Early Cambrian Small Shelly Fossils from northwest 495 Mexico: Biostratigraphic implications for Laurentia. Palaeontol. Electr. 22, 1– 496 60. (doi:org/10.26879/880) 497 30. Vendrasco MJ, Checa AG, Porter SM. 2017 Periostracum and fibrous shell 498 microstructure in the unusual Cambrian hyolith Cupitheca. Span. J. 499 Palaeontol. 32, 95–108. 500 31. Sun H, Yin Z, Li G, Zhao F, Zeng H, Zhu M. 2019. Periodic shell decollation as 501 an ecology‐driven strategy in the early Cambrian Cupitheca. Palaeontology. 502 (doi:10.1111/pala.12463) 503 32. Skovsted CB, Pan B, Topper TP, Betts MJ, Li G, Brock GA. 2016 The operculum 504 and mode of life of the lower Cambrian hyolith Cupitheca from South 505 Australia and North China. Palaeogeogr. Palaeocl. 443, 123–130. 506 (doi:org/10.1016/j.palaeo.2015.11.042) 507 34. Dzik J. 1994 Evolution of ‘small shelly fossils’ assemblages of the Early 508 Paleozoic. Acta Palaeontol. Po. 39, 247–313. 509 35. Peel JS, Streng M, Geyer G, Kouchinsky AV, Skovsted CB. 2016 Ovatoryctocara 510 granulata assemblage (Cambrian Series 2-Series 3 boundary) of Løndal, 511 North Greenland. Mem. Assoc. Australas. Palaeontol. 49, 241–282. 512 36. Skovsted CB, Topper TP, Betts MJ, Brock GA. 2014 Associated conchs and 513 opercula of Triplicatella disdoma (Hyolitha) from the early Cambrian of South 514 Australia. Alcheringa 38, 148–153. (doi:org/10.1080/03115518.2014.843381) 515 37. Bonar DB. 1972 Feeding and tube construction in Chone mollis Bush (Polychaeta, 516 Sabellidae). J. Exp. Mar. Biol. Ecol. 9, 1–18. (doi:org/10.1016/0022- 517 0981(72)90002-0) 518 38. Strathmann RR. 2005 Ciliary sieving and active ciliary response in capture of 519 particles by suspension‐feeding brachiopod larvae. Acta Zool-Stockho. 86, 41– 520 54. (doi:org/10.1111/j.0001-7272.2005.00185.x) 521 39. Marek L. 1963 New knowledge on the morphology of Hyolithes. Sborník 522 geologických věd, řada Paleontologie 1, 53–72. 523 40. Zhang Z, Skovsted CB, Zhang Z. 2018 A hyolithid without helens preserving the 524 oldest hyolith muscle scars; palaeobiology of Paramicrocornus from the 525 Shujingtuo Formation (Cambrian Series 2) of South China. Palaeogeogr. 526 Palaeocl. 489, 1–14. (doi:org/10.1016/j.palaeo.2017.07.021) 527 41. Bandel K., 2008. Operculum shape and construction of some fossil Neritimorpha 528 () compared to those of modern species of the subclass. Vita

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529 Malacologica. 7, 19–36. 530 42. Houbrick RS. 1980. Observations on the anatomy and life history of Modulus 531 modulus (Prosobranchia: Modulidae). Malacologia 20, 117–142. 532 43. Checa AG, Jiménez-Jiménez AP. 1998 Constructional morphology, origin, and 533 evolution of the gastropod operculum. Paleobiology 24, 109–132. 534 (doi:org/10.1017/S0094837300020005) 535 44. Kouchinsky AV. 2000 Skeletal microstructures of hyoliths from the Early 536 Cambrian of Siberia. Alcheringa 24, 65–81. 537 (doi:org/10.1080/03115510008619525) 538 45. Moore J, Porter SM. 2018 Plywood‐like shell microstructures in hyoliths from the 539 middle Cambrian () Gowers Formation, Georgina Basin, Australia. 540 Palaeontology 61, 441–467. (doi:org/10.1111/pala.12352) 541 46. Li L, Zhang X, Yun H, Li G. 2017 Complex hierarchical microstructures of 542 Cambrian mollusk Pelagiella: insight into early biomineralization and 543 evolution. Sci. Rep. 7, 1935. (doi:org/10.1038/s41598-017-02235-9) 544 545 Figure Caption 546 547 Figure. 1. Map of North China outlining the geology, composite stratigraphical 548 column, and outcrops of studied sections. (a) geological map showing the 549 Precambrian-Cambrian rocks along the southern margin of the North China Block, the 550 distribution of Xinji Formation is marked in green. (b) composite stratigraphical 551 column of the Chaijiawa and Shangzhangwan sections. (c) outcrop of the studied 552 Chaijiawa section in Shaanxi Province. NP: Neoproterozoic; DP: Dongpo Formation; 553 E: Ediacaran; ZSD: Zhushadong Formation.

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554 555 556 Figure. 2. Anatomy and shell microstructure of Longxiantheca mira gen. et sp. nov. 557 from the Xinji Formation of North China. (a)–(d), Disc-shaped operculum. (a), ELI- 558 LC06-01-07, external view; (b), ELI-LC06-01-10, external view; (c), ELI-LC06-25- 559 05, internal view; (d), ELI-LC06-25-04, internal view. (e)–(h), articulated internal 560 mould combining operculum and conch. (e), ELI-LC06-29-06; (f), ELI-LC06-04-06; 561 (g), ELI-LC06-15-07; (h), ELI-LC06-17-05; (i), ELI-LC06-19-05, Micro-CT 562 scanning, showing the characteristic two-layered pattern of the conch. (j), ELI-LC06- 563 11-05, apertural end of conch, showing summit muscle scar (white arrow), adductor 564 muscle scars (black arrow); (k), ELI-LC06-10-03, apertural end of conch, (l), ELI- 565 LC06-23-03, internal view of operculum, showing kidney-shaped platform; (m), ELI- 566 LC06-25-03, internal view of operculum; (n), (o), ELI-LC06-28-03, summit muscle 567 scar (white arrow) and adductor muscle scars (black arrow); (o), mantle epithelial 568 cells (mec), (p), (q), polygonal texture. Scale bars represent 200μm (a, b, e, f, h); 569 100μm (c, d, g, j, m); 50μm (i, k, l, n, o); 20μm (p, q). 570

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571 572 Figure. 3. A two-layered pattern of fibrous bundle microstructure. (a andb) ELI-LC06- 573 19-06, (a) general view of the fibrous bundles of the conch; (b) magnification of (a) 574 showing the elongated slits and pores between adjacent fibrous bundles; (c) ELI- 575 LC06-31-04, outer layer of fibrous bundles along longitudinal direction; (d and e) 576 ELI-SJFH-28-3-11, showing terminal ending with vertical tubules; (f) ELI-SJFH-29- 577 3-06, tubules aligning in co-marginal rows on surface of internal moulds. (g) H, ELI- 578 LC06-19-05, inner layer of fibrous bundles with slits and pores. Scale bars represent 579 200μm (a and c); 100μm (d); 50μm (g and h); 20μm (b and e); 10μm (f).

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580 581 Figure. 4. A common muscular system and tentaculate feeding apparatus in 582 orthothecids and hyolithids. Sketch of orthothecid muscular system based on 583 Longxiantheca; hyolithid muscular system from Gompholites [6]. Abbreviations: sm, 584 summit muscle; am, adductor muscle; mec: mantle epithelial cells.

585 586 Figure. 5. Diagrammatic sketches of different fibrous microstructures in orthothecid 587 conchs. Fibrous bundles consist of two layers of bundles of aragonitic fibers. Parallel

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588 fibers consist of micro-lath crystalline aragonite parallel to shell surface and constant 589 in orientation. Lamello-fibrillae are composed of micro-lath crystalline aragonite that 590 are arranged in different directions between stacked lamellae.

591 592 593 Figure. 6. Hyolith intraphyletic relationships and suggested position within 594 lophotrochozoans. The tree topology is modified from [XXX] to account for the likely 595 relationship of some lophotrochozoan groups. Solid line indicates valid fossil data, 596 dotted lines represent molecular data and hypothesized pre-existing unmineralized 597 counterparts. Hyolith taxa in the diagram mainly from North China [XXX] with 598 Majatheca from Siberia [XXX], not all potential descendants on each branch are 599 indicated. The first acquisition of some key characteristics of molluscs and hyoliths 600 are denoted. 1: foot, radula, pre-existing organic counterpart. 1’: tentaculate feeding 601 organ, three conserved muscles, sinuously-folded digestive tract, pre-existing organic 602 counterpart. 2: mantle, skeleton inherited independently from the same 603 ‘biomineralization toolkit’ of unmineralized common ancestry. 3: sclerite, micro-lath 604 fabric. 4: shell, micro-lath fabric (fibrous microstructures). 5: lamello-fibrillar, 605 prismatic, cardinal process (orthothecids). 6: foliated inner layer, clavicle 606 (intermediate taxa). 7: helens (hyolithids). Abbreviations: CP: cardinal process, CL: 607 clavicle, R: rooflet, T: teeth-like structure, D: circumferential deposit, H: helens.

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608

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