1 New insight into the soft anatomy and shell microstructures of early Cambrian 2 orthothecids (Hyolitha) 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 ‘Cambrian explosion’, led to the origination not only of most modern 41 animal phyla, but also some extinct groups with peculiar Baupläne [1,2]. Hyoliths are 42 such an enigmatic group of conical shelled animals, which thrived through Cambrian 43 to Ordovician times, and then declined until their demise at the end of Permian [3]. 44 They possess a deep conical conch and a lid-like operculum, and are commonly 1 45 divided into two morphologically distinct subgroups (Orders): the Hyolithida 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 Mollusca [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 Burgess Shale-type Konservat-Lagerstätten. New data from the 64 Burgess Shale and the Spence Shale in North America have revealed a tentaculate 65 feeding apparatus in the hyolithid Haplophrentis [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 brachiopods, phoronids 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 lophophore 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 2 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, trilobites 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 Genus 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; Species 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 3 133 angle of divergence; Apertural margin planar and initial shell septate; Shell