HOMOLOGOUS SHELL MICROSTRUCTURES IN HYOLITHS AND

MOLLUSCS

by LUOYANG LI1,2, XINGLIANG ZHANG1*, CHRISTIAN B. SKOVSTED1,2*, HAO

YUN1, BING PAN2,3 and GUOXIANG LI3

1State Key Laboratory of the Continental Dynamics, Shaanxi Key Laboratory of Early Life and Environments, Department of Geology, Northwest University, Xi’an 710069, PR China; e-mail: [email protected], [email protected]

2Department of Palaeobiology, Swedish Museum of Natural History, Box 50007, SE-104 05

Stockholm, Sweden; [email protected], [email protected]

3State Key Laboratory of Palaeobiology and Stratigraphy, Nanjing Institute of Geology and

Palaeontology, Chinese Academy of Sciences, Nanjing 210008, PR China. [email protected], [email protected]

*Corresponding authors

Abstract: Hyoliths were one of the earliest biomineralizing metazoans in marine environments. They have been known for two centuries and widely assigned to lophotrochozoans. However, their origin and relationships with modern lophotrochozoan clades have been a longstanding paleontological controversy. Here, we provide broad microstructural data from hyolith conchs and opercula from the lower Cambrian Xinji

Formation of North China, including two hyolithid genera and four orthothecid genera as well as unidentified opercula. Results show that most hyolith conchs contain a distinct aragonitic lamellar layer that is composed of foliated aragonite except in the orthothecid

Protomicrocornus which has a crossed foliated lamellar microstructure. Opercula are mostly composed of foliated aragonite and occasionally foliated calcite. These blade or lath-like microstructural fabrics coincide well with biomineralization of Cambrian molluscs rather than lophophorates, as exemplified by the Cambrian members of the tommotiid- linage.

Accordingly, we propose that hyoliths and molluscs might have inherited their biomineralized skeletons from a non-mineralized or weakly mineralized common ancestor rather than as a result of parallel evolution or convergence. Consequently, from the view of biomineralization, the homologous shell microstructures in Cambrian hyoliths and molluscs strongly strengthen the phylogenetic links between the two groups.

Key words: hyoliths, lophotrochozoan, biomineralization, Cambrian, North China.

IN recent years, research on the origin and key transitions in the evolution of major lophotrochozoan clades (annelids, molluscs, “lophophorates”) have advanced remarkably in the light of numerous discoveries of crucial stem-group fossils in the Cambrian (Conway

Morris & Caron 2007; Zhang et al. 2014b) and new molecular phylogenetic investigations

(Kocot 2016). In many areas, consensus has been reached, for example regarding the placement of Myzostomida, Echiura and in the Annelida (Dunn et al. 2008); the divisions of the into two subgroups: Conchifera and Aculifera (Kocot et al. 2011;

Smith et al. 2011); the derivation of organophosphatic (Linguliformea) from a direct ancestor within tommotiids (Balthasar et al. 2009), etc. However, our current understanding especially surrounding the roots of the lophotrochozoans is far from complete, and a well-accepted tree of basal lophotrochozoan clades has not yet been established.

Intensive palaeontological studies have been focused on some controversial fossils in the

Cambrian including halwaxiids ( and wiwaxiids) (Conway Morris & Caron 2007;

Zhang et al. 2015), tommotiids (Skovsted et al. 2015) and hyoliths (Malinky & Yochelson 2007), which are conspicuous constituents of Cambrian marine communities and crucial breakthrough points for understanding the origin and radiation of lophotrochozoan during the .

Hyoliths were one of the earliest biomineralizing metazoans in Paleozoic marine environments (existing from the beginning of the Cambrian to end ), with two orders recognized (the Orthothecida and the ) (Malinky & Yochelson 2007). They produced a calcareous that consists of up to three parts: a conical conch, a lid-like operculum, and at least in the Hyolithida, an additional pair of elongate lateral spines or

“helens” protruding from the commissure between the conch and operculum (Martí Mus et al.

2014). Although hyoliths have been known for two centuries, and their affiliation with lophotrochozoans seems to be a widely accepted assumption, the precise phylogenetic position within the lophotrochozoan clade is still a subject of debate. Traditionally, they have most often been regarded as an extinct class within the Mollusca, mainly based on the presence of crossed lamellar microstructure in some younger taxa, or a type of aragonitic microstructural fabric only known from the Mollusca (Malinky & Yochelson 2007; Carter

1990). Alternatively, they have been assigned to sipunculan worms (Sun et al. 2016) or to constitute their own phylum (Runnegar et al. 1975; Runnegar 1980). The quintessence of the debate is whether the crossed lamellar microstructure is homologous or convergent between hyoliths and molluscs, which has been hard to assess (Malinky & Yochelson 2007). More recently, new discovery of soft-tissues from Cambrian -type Lagerstätten

(Moysiuk et al. 2017) and the Chengjiang Biota (Liu et al. unpublished data) revealed a tentaculate feeding structure and a distinct U-shaped gut, which suggest that they are closely related to the “lophophorates”, which include modern brachiopods, , and probably bryozoans (Dunn et al. 2014) as well as some fossil taxa such as tommotiids (Skovsted et al.

2008) and tentaculitoids (Vinn & Zatoń 2012). Early lophotrochozoan biomineralizers (including hyoliths, molluscs, brachiopods and tommotiids) acquired the capacity to produce mineralized external shells from their soft- bodied ancestors during the early Cambrian and exert exquisite controls over biomineralization (Murdock & Donoghue 2011; Kouchinsky et al. 2012). They generated a variety of biominerals, mainly calcium carbonate polymorphs and calcium phosphate mineral complex. The crystals, intercalated between macromolecules and secreted by the underlying tissues (the mantle), are used to construct an incredible diversity of shell patterns and macro- micro architectures (Carter 1990). Despite the fact that the phylogenetic significance of biomineralization in lophotrochozoans remains contentious, the most closely related groups generally share skeletal homologies and high morphological similarities, but each linage has evolved a distinctive array of characterization of their own in mineralogical composition, microstructural fabrics and crystallographic orientations, etc. For instance, nacre (“mother of pearl”), one of the most common types of shell microstructures known in molluscs, is generally composed of crystallites of aragonite arranged in a sheet or brick-wall appearance in bivalves (Cartwright et al. 2009), and a tower pattern in gastropods and cephalopods (Checa et al. 2009a). From this perspective, we expect that biomineralization can provide additional information towards a better resolution of deep lophotrochozoan phylogeny in general and that detailed shell microstructural investigations may shed new light on relationships between hyoliths and other lophotrochozoans, particularly with molluscs and “lophophorates”.

Here, we provide new microstructural data of hyolith conchs and opercula based on abundant and exceptionally well-preserved specimens from the lower Cambrian Xinji

Formation of North China, which can be assigned to two hyolithid genera (Microcornus

Mambetov, 1972 and Parakorilithes He & Pei in He et al., 1984), four orthothecid genera

(Allatheca Missarzhevsky in Rozanov et al., 1969, Missarzhevsky in Rozanov et al., 1969, Cupitheca Duan in Xing et al., 1984, and Protomicrocornus Pan et al., unpublished data), as well as unidentified opercula. Our study shows that hyoliths are extremely similar to

Cambrian molluscs in terms of their distinct lamellar shell microstructures that are composed of blade- or lath-like crystallites of either aragonite or calcite.

GEOLOGICAL SETTING

The North China Block is bounded to the north by the Central Asian Orogenic Belt, to the south by the Qinling-Dabie Belt and Su-Lu fault against the South China Block, to the west by the Qilian Orogenic Belt against the Tarim Block (Stern et al. 2018). During the early

Cambrian, the North China Block was situated near the northern margin of the Gondwana supercontinent, particularly close to the Australia Block (Brock et al. 2000; Yun et al. 2016).

The lithological sequences and sedimentary patterns from the Ediacaran to lower Cambrian are consistent and can be precisely correlated along the southern margin of North China, with sequences in ascending order: Luoquan Formation, Dongpo Formation, Xinji Formation and

Zhushadong Formation. They are generally in conformable contact except a distinct disconformity at the base of the Cambrian, with strata of the being absent anywhere. The Xinji Formation is the oldest record of Cambrian deposits, ranging stratigraphically in the upper part of Stage 3 or lower part of Stage 4 of the Cambrian System

(Fig. 1A, B).

The Xinji Formation consists of siliciclastic rocks intercalated with carbonates, which rests disconformably on the upper Ediacaran Dongpo Shale and is conformably overlain by the massive dolostones of the Zhushadong Formation in studied areas. The carbonate unit of the

Xinji Formation yields an abundant, diverse assemblage of so-called Small Shelly Fossils

(SSFs) including sponge spicules, chancelloriid sclerites, hyoliths, micromolluscs, echinoderm ossicles and fossil sclerites of problematic systematic position. The hyolith specimens studied herein were collected from the Chaijiawa section (LC) in Longxian County,

Shaanxi Province (Fig. 1C; Li et al. 2016; Yun et al. 2016) and the Sanjianfang Section (SJF) in Yexian County, Henan Province, respectively (Fig. 1D; Skovsted et al. 2016).

Rock samples were treated with buffered 5% acetic acid to retrieve acid-resistant microfossils. Abundant well-preserved hyoliths were recovered from the acid-resistant residues. Selected specimens were mounted, sputter-coated with gold and examined with a

FEI Quanta 400 FEG scanning electron microscope at Northwest University (NWU) and the

Swedish Museum of Natural History (SMNH). Microfossils described below are deposited at the Department of Geology, Northwest University, Xian, China (Fig. 2).

RESULTS

Cambrian Small Shelly Fossils (SSFs) with a variety of mineralogical constituents, e.g. carbonate, phosphate and silica minerals, are often secondly replaced by calcium phosphate owing to the global phosphogenesis event in the Cambrian (Porter 2004). Phosphatization contributes great potential to preserve delicate pseudo-morphological features of primary shell microstructures (Brasier & Callow 2007). Even though the mechanism of phosphatization is still poorly understood, well-preserved microstructural imprints and replicas on the surface of phosphatized internal moulds of hyoliths are easily distinguished from diagenetic artefacts by:

(1) highly-organized fabrics with a complex hierarchical pattern; (2) consistent occurrences in specimens, localities and taxa; (3) high comparability with structures in living and fossil molluscs; and (4) the formation of characteristic straight growth fronts for aragonite and tooth-like patterns with interfacial angle of about 102 degrees for calcite (Vendrasco et al.

2015; Pérez-Huerta et al. 2018). The lamellar microstructural fabrics of the conchs described below are easily discernable in our collections, but mainly visible near the edge of the conch aperture. The tubercles or pillar-like infillings of shell pores may be visible anywhere on the shell, but are sometimes absent in other specimens of the same species owing to preservational differences.

Three main types of lamellar microstructures were recognized in the investigated hyolith conchs. Bidirectional aragonite folia microstructure (B-FOA) occurs in the hyolithid

Microcornus eximius Duan, 1984 (Fig. 2A, 3A–E) and the orthothecid Allatheca sp. (Fig. 2B,

4A–E). This type of shell microstructure exhibits a distinct lamellar microstructural fabric.

The replicas of the smallest recognizable elements, termed folia, develop straight growth fronts, a principle character for aragonite. Generally, they are arranged parallel or subparallel to the shell surface with the straight growth fronts perpendicular to the edge of the conch aperture. The folia grow by basal marginal accretions and overlap slightly with the next set, and thus form a distinct imbricated pattern. The imbricated pattern, however, points in opposite directions along the dorsal-ventral axial plane of the conch (mirror symmetry) (Fig.

3D, E), as the folia are arranged clockwise on the left side and counter clockwise on the right side in apertural view (Fig. 4D, E).

Unidirectional aragonite folia (U-FOA) occur in the hyolithid Parakorilithes mammillatus

He & Pei in He et al., 1984 (Fig. 2C, 5A–D) and the orthothecid Cupitheca sp. (Fig. 2F, 6A–

G). This type of shell microstructure is strikingly analogous to B-FOA by the characteristic imbricated gross appearance of the folia, growth mode of basal accretion and the straight growth fronts of aragonite that are perpendicular to the edge of the conch aperture. However, the organization of basic units (folia) is slightly different, as the imbricated pattern of folia remain constant in clockwise direction surrounding the apertural circumference, and thus is easily distinguishable from the mirror-symmetrical pattern of B-FOA (Fig. 5B–D, 6G).

Crossed foliated lamellar microstructure (CFL) was found in the orthothecid

Protomicrocornus triplicensis Pan et al., unpublished data (Fig. 2E, 7A–E), exhibiting a derived complex hierarchical organization. The smallest recognizable elements (folia) express a similar gross appearance to that of B-FOA and U-FOA, characterized by imbrication, straight growth fronts typical of aragonite, and arrangement almost perpendicular to the edge of the conch aperture. However, the folia are assembled into a higher order of organization termed lamellae. The lamellae are generally parallel or subparallel to the shell surface, slightly overlapping each other laterally. They form a distinct alternate pattern along the comarginal direction, as the folia are arranged in opposite direction in alternating lamellae (Fig. 7A). This alternate pattern of CFL is consistent in our specimens, which precludes the possibility that this pattern is a preservational artefact resulting from compression of several parallel laminae on the surface of internal moulds.

In addition to the new aragonitic lamellar microstructures, tubules or pore systems are common in the studied hyolith conchs. They tend to be preserved as tubercles or pillar-like infillings on the surface of internal moulds, ranging from 5–10μm in diameter as seen in

Microcornus eximius (Fig. 3C). These phosphatized infillings are often preserved as hollow tubes, and postulated to have been originally filled with organic material. The tubules show slight variations in distribution, size and density between different species, particularly in the apical areas of Cupitheca sp. (Fig. 6D–F) and Conotheca australiensis Bengtson in Bengtson et al., 1990 (Fig. 2D, 8A–E), where the tubules are thought to play a role in the characteristic decollation of older parts of the conch after the formation of transverse septa (Bengtson et al.

1990). In Cupitheca sp., the tubules are irregular, scattered and circular in cross-section (5μm average diameters) on the surface of conch moulds with a concentration of hollow tubules surrounding the transverse septum representing the terminal end of the conch after decollation

(Fig. 6E). One specimen shows that the septum may also consist of a second set of tubules, orthogonally directed beneath the common parallel-orientated ones (Fig. 6F). In Conotheca australiensis, the tubercles are regular, robust, arranged in comarginal rows and elongated ovoid in cross-sections (7x10μm) on the conical wall. Additionally, the transverse septa are penetrated by a concentration of orthogonal tubules (Fig. 8D–F).

Possible casts of cells or cell aggregates of the outer mantle epithelium are discernible on one specimen of Microcornus eximius (Fig. 3B) and one specimen of Protomicrocornus triplicensis (Fig. 7E), which almost cover the whole surface of the conch. They are rounded or polygonal in outline, convex and 10μm in average diameter. Similar polygonal textures, concave, convex or with tubercles, are common on the internal moulds of Cambrian helcionelloids, e.g. Mackinnonia rostrata Zhou & Xiao, 1984 (Fig. 10H, I).

Interestingly, some internal moulds of opercula also preserve very fine replicas of primary lamellar shell microstructures. The opercula of Parakorilithes mammillatus and Conotheca australiensis have similar foliated aragonite microstructure (FOA) identified by the distinct imbricated organization and straight growth fronts of aragonite lamella (Fig. 9A–F). The opercula grow in a concentric pattern by basal marginal accretion. In P. mammillatus, four unusual zones composed of narrow crystallites of aragonite close to the ventral margin of the operculum are visible, possibly representing specialized sites for muscular attachment (Fig.

9C, D). Additionally, one unidentified operculum grows in similar concentric direction with folia forming distinct imbricated patterns, but growth fronts of crystallites are arranged in accurate tooth-like forms (interfacial angle: 103°, N=10) (Fig. 9A, B), which is analogous to foliated calcite microstructure (FOC) of some molluscs (Fig. 9G).

DISCUSSION

Highlights

This study increases our understanding of hyolith biomineralization in several ways: (1)

Cambrian hyoliths were diverse in terms of shell microstructures with examples of highly- organized lamellar fabrics of foliated aragonite (B-FOA, U-FOA), foliated calcite (FOC) and crossed foliated lamellar (CFL) in addition to fibrous types, i.e. lamello-fibrillae (LF) described in previous studies; (2) the complex hierarchical organization of the new types of shell microstructures indicates tight biological control and rapid evolution of biomineralization in the Cambrian; (3) the finding of foliated calcite for the first time demonstrates that hyoliths could also generate calcite to construct their shells; (4) the occurrence of similar shell microstructure in both conch and operculum supports the proposition that these two biomineralized hard parts are homologous; (5) although our microstructural data shows no distinctiveness between hyolithid and orthothecid shells, we note that it may be too soon to draw broad conclusion on the utilization of shell microstructures for hyolith taxonomy based on the currently insufficient data and poor resolution of the phylogeny of Cambrian hyoliths; and (6) more importantly, we emphasize that in contrast with the evolutionarily derived crossed lamellar and widespread lamello- fibrillar microstructures, the new types of blade or lath-like shell microstructures demonstrated herein have a more specific distribution within lophotrochozoan biomineralizers, and thus represents more convincing phylogenetic signals relevant for the phylogenetic position of hyoliths.

Brief review of hyolith shell microstructures

The macro-micro architectures of hyolith skeletons (conch and operculum) have been poorly understood in previous studies due to the limits of preservation, and only various types of fibrous microstructures were sporadically reported in some hyolithids and orthothecids (see review by Moore & Porter 2018). These fibrous microstructures, broadly termed lamello- fibrillae, are composed of elongated fibers of aragonite, amalgamated into successive horizontal lamellae with the fibers in different orientations from one stacked layer to the next

(Kouchinsky 2000; Vendrasco et al. 2015). Furthermore, the lamello-fibrillar layer may be penetrated by pores throughout the shell wall (Vendrasco et al. 2017). Unfortunately, these two well-known characteristics of hyolith skeletons are very widespread among lophotrochozoans and thus in them selves insufficient to solve the puzzle of hyolith affinities

(Vendrasco et al. 2017; Moore & Porter 2018). Specifically, lamello-fibrillae has been widely documented among serpulid tubes (annelids) (Vinn et al. 2008) and many Cambrian helcionelloid molluscs in addition to hyolith shells (Feng & Sun 2003; Li et al. 2017; Zhang et al. 2018). Meanwhile, the organic-filled pore system is an even more common character among lophotrochozoan biomineralizers and is found in bryozoans, molluscs, brachiopods, tommotiids and tentaculitoids (Taylor et al. 2010). So, while these observations can be used to demonstrate that hyoliths exhibit generalized characters of lophotrochozoan skeletons, they contribute less towards a precise position for where hyoliths nest in the lophotrochozoan tree.

The hypothesis of an affinity of hyoliths with molluscs and their relatives was previously based on the shared crossed lamellar microstructure. This homology, however, is hard to prove. The crossed lamellar shell structure is a derived microstructural fabric which has not yet been confirmed in early molluscs or hyoliths before the (Vendrasco et al.

2017). Although its evolutionary origin is uncertain, this type of shell microstructure has become the most common and successful biomaterial structure in modern bivalves, gastropods, chitons and scaphopods (Carter 1990). But in addition to molluscs, the crossed lamellar microstructure has only been found in two post-Cambrian hyoliths: Theca lanceolata

Morris in Strzelecki, 1845, and ‘Hyolithes’ columbanus Cowper Reed, 1909, which both provide strong comparative similarities with mollusc shells. However, this does not preclude the possibility of parallel evolution of these shell structures in the two groups. Instead we propose that specific attention should be directed towards comparison of shell structures of early representatives (Cambrian) of biomineralizing lophotrochozoans as these would be evolutionarily closer to their last common ancestor.

Microstructural comparison with molluscs and lophophorates

As demonstrated here, the similarities between Cambrian hyolith and molluscan shell microstructures are notable. Broadly speaking, the two common microstructural variations (B-

FOA and U-FOA) can be regarded as representing a general type of foliated aragonite (FOA).

The folia of FOA are supposed to be composed of sub-structures of elongated blade- or lath- like crystallites of aragonite, but these elements have not been identified in our specimens due to limits of preservation. This type of shell microstructure was initially described from extant monoplacophorans (Checa et al. 2009c), but recent work revealed that it is common in

Cambrian molluscs such as the helcionelloid Pelagiella madianensis Zhou & Xiao, 1984 (Fig.

10C) and the early bivalves Pojetaia runnegari Jell, 1980 and Fordilla troyensis Barrande,

1881 (Fig. 10F, G) (Vendrasco et al. 2011; Li et al. 2017). Furthermore, foliated aragonite has been proposed as a primitive type of shell microstructure and is probably the precursor that gave rise to nacre, mother of pearl, in molluscs during the Great Ordovician Biodiversification

Event (Vendrasco et al. 2013). The crossed foliated lamellar (CFL) is a type of shell microstructure first described in the Cambrian anisometric-coiled helcionelloid Pelagiella madianensis (Fig. 10A, B) (Li et al. 2017). More recently, it has also been observed from

Anabarella australis Runnegar in Bengtson et al., 1990, a Cambrian laterally compressed helcionelloid (Li et al. submitted). Presently, CFL is the most complicated microstructural fabric known in Cambrian molluscs, and its finding in Protomicrocornus triplicensis strongly highlights the skeletal similarities between Cambrian hyoliths and molluscs. As for the foliated calcite microstructure (FOC), this type of shell microstructure also occurs in the middle Cambrian molluscs Eotebenna pontifex Runnegar and Jell, 1976, and Pseudomyona queenslandica Runnegar and Jell, 1976 (Vendrasco et al. 2010), but being widely distributed among modern limpets (patellogastropods) and bivalves as well as some calcitic brachiopods

(Carter 1990; Checa et al. 2007, 2009b), the importance of this type of microstructure is difficult to evaluate.

In contrast, the finding of aragonitic lamellar microstructures provides little support for the placement of hyoliths among crown-group lophophorates. Of particular importance is the fact that brachiopods never secrete aragonite, except in some poorly understood Ordovician and

Silurian trimerellid brachiopods which might have secreted aragonitic shells (Balthasar et al.

2011). Instead, brachiopod shells generally consist of either calcite (Subphyla

Rhynchonelliformea and Craniiformea) (Pérez-Huerta & Cusack, 2008) or Ca-phosphate

(Subphylum Linguliformea) (Cusack et al. 1999). The calcitic brachiopods produce multi- layered shells that consist of a primary layer with acicular calcite, a secondary layer composed of semi-nacre in the Craniiformea and calcitic fibers in the Rhynchonelliformea, and sometimes a tertiary prismatic layer (Checa et al. 2009b; Gaspard & Nouet 2016). Phosphatic brachiopods were more common in the Cambrian than today and exhibited remarkably distinctive and complex shell microstructures, as being columnar (characteristic of acrotretids), bacculate (obolids), or laminar (paterinids) (Williams et al. 2004). In addition, intensive microstructural studies have suggested that stem-group phosphatic brachiopods such as

Mickwitzia Schmidt, 1888 might have evolved from a direct ancestor within tommotiids, a group of Cambrian problematic sclerite-bearing animals with composite tubular scleritomes, a hypothesis which is strongly supported by their homologous shell microstructures of organophosphatic lamina, setal tube system and distinct reticulate ornaments (Holmer et al.

2008; Balthasar et al. 2009; Skovsted et al. 2009).

Are hyolith and mollusc shells homologous?

The microstructural fabrics of hyolith shells, as documented in previous reports and by our new data, coincide well with biomineralization of Cambrian molluscs rather than lophophorates, as exemplified by the Cambrian members of the tommotiid-brachiopod linage.

However, a more important question is whether mollusc-type shell microstructures in

Cambrian hyoliths and molluscs are homologous or convergent, and how these skeletons originated. The growth and self-assembly of biominerals into highly-organized architectures by complex organisms involve a sophisticated physiological process regulated by a three- dimensional organic framework, composed of specialized macromolecules (Müller 2011).

The building of this complex crystal-regulatory system generally consumes more energy in comparison with the succeeding process of crystal deposition and patterning. It has been speculated that pre-existing crystal-regulatory system, i.e. secretome, might have evolved in non-mineralized or weakly mineralized ancestors of early metazoans (Wood et al. 2017). If this is true, then a shared primary mantle secretome (PMS) of the Cambrian lophotrochozoan biomineralizers might have evolved during Ediacaran time.

Various abiotic and biotic causes are hypothesized to have triggered the initial acquisition of the capacity to biomineralize specific mineral types, e.g. carbonate, phosphate or silica minerals, in particular metazoan clades by their non-mineralized ancestors during the

Ediacaran-Cambrian period (Knoll 2003; Porter 2010). Regardless of wide uncertainties, convincing fossil records indicate that early lophotrochozoans radiated rapidly in the

Terreneuvian (Cambrian Stage 1–2), represented by the occurrences of abundant and diverse skeletons of molluscs, hyoliths, tommotiids and brachiopods(Zhang et al. 2014a). In addition,

Terreneuvian molluscs and hyoliths were known to have had apparently exclusively aragonitic shells with characteristic fibrous shell microstructures in general (e.g. lamello- fibrillae) (Li et al. 2017), while the earliest members of the tommotiid-brachiopod lineage preferred to synthesize phosphate biocomposites within their shells (Balthasar et al. 2009). On this basis, early molluscs and hyoliths as well as their non-mineralized ancestors seem to have aragonite-specific primary mantle secretomes (A-PMS), which differ considerably from the phosphate-PMS (P-PMS) of the tommotiid-brachiopod linage in early Cambrian.

Furthermore, the shared LF, FOA, CFL, FOC and pore system in Cambrian hyolith and mollusc shells demonstrate that these two groups constructed their biomineralized shells with complex hierarchical architectures in a highly consistent mechanism, and that their mantle secretomes might be very similar to each other. To our knowledge, none of any distantly- related or unrelated biomineralizers may share the same secretome and high similarities of their skeletal macro-micro architectures by parallel evolution or convergence. Therefore, the striking skeletal similarities between Cambrian hyoliths and molluscs cannot be easily interpreted as the results of convergence, but are more likely derived from the same A-PMS inherited from a non-mineralized or weakly mineralized common ancestor.

Hard skeleton vs. soft anatomy for hyolith affinity

According to this hypothesis, from the view of biomineralization, the homologous shell microstructures in Cambrian hyoliths and molluscs strongly strengthen the assumption of phylogenetic links between the two groups. If so, then this argument focused on biomineralized hyolith skeletons seems to be incompatible with results of investigations based on soft anatomy of hyoliths from konservat-lagerstätten. The discovery of soft-tissues from

Cambrian Burgess Shale-type lagerstätten in Laurentia revealed that the hyolithid

Haplophrentis Babcock & Robison, 1988 had developed a tentaculate feeding organ, which was interpreted as a for filter-feeding as in lophophorates (Moysiuk et al. 2017). This feature, in combination with a distinct U-shaped gut, opposing bilateral sclerites and a deep ventral visceral cavity, lead to a suggested placement of hyoliths within the total group lophophorates, closely related to brachiopods (Moysiuk et al. 2017; Zhao et al. 2017).

However, recent investigation on feeding anatomy and functional morphology of the orthothecid Triplicatella Conway Morris in Bengtson et al., 1990 from the Cambrian

Chengjiang Biota of South China demonstrate that the tentaculate feeding organ of this hyolith does not represent a true lophophore or even a suspension feeding life mode (Liu et al. unpublished data). According to this interpretation the orthothecid feeding organs were adjusted to collect food particles directly from organic-rich substrates (Sun et al. 2018;

Kimmig et al. 2018, Liu et al. unpublished data) and suspension feeding in hyolithids must have evolved independently. However, the general morphology of the simplified feeding organ of and Triplicatella bears considerable similarities with the lophophore of derived lophophorates and may be homologous. Moreover, hyoliths lack key molluscan synapomorphies such as the radula and foot. Consequently, hyoliths would be better suited occupying a position on the stem of the lophophorate clade and are not close relatives of crown group brachiopods (Liu et al. unpublished data).

Although information based on hyolith skeletons and soft anatomy appear to indicate incompatible phylogenetic positions, that is not to say hyoliths can be easily assigned to either the Mollusca or the merely based on feeding structures or shell microstructures.

Actually, the remarkable mix of mollusc-type shell structures and a tentaculate feeding apparatus homologous with lophophorates provide more arguments for early lophotrochozoan evolution and indicate that hyoliths might represent a crucial intermediate between molluscs and lophophorates. It is important to note that biomineralization and feeding systems are very likely to have evolved asynchronously in early lophotrochozoans, with the shared primary mantle secretome (PMS) having a deep origin in the last common ancestor of various biomineralizing clades, predating the origin of specific features such as tentacle-bearing structures in hyoliths and the lophophore in derived lophophorates. In this respect, even if hyoliths occupy a basal position in the stem-group of lophophorates as indicated by the tentaculate feeding structures, it is still reasonable to assume that hyoliths share deeply homologous biomineralized shells with molluscs, and vice versa (Fig. 4).

Ultimately, the basal members of various lophotrochozoan clades, i.e. stem-group members, are likely to share considerable synapomorphies and their exact relationships will be difficult to clarify before their derived phyla attained distinctive body plans and characters such as the mantle, lophophore, foot, radula, etc. Thus, identification of the exact position of hyoliths depends on a more comprehensive record of early lophotrochozoan evolution and the sequence of character evolution of the hyolith body plan.

Acknowledgements. We are grateful for Zhang, Z-F. (Northwest University) for valuable discussions and suggestions. Great thanks also to Liu, W., Yang, Y-N., Wang, X., Zhang, Y.,

Cui, L-H., Wu, Y., Liu, C. and Pei, W-R. (Northwest University) for their assistance in fieldwork. Li, L-Y is supported by the China Scholarship Council (CSC, No. 201706970037) for one year research stay with C.B.S in Swedish Museum of Natural History. This research was supported by funds from the National Key Research and Development Program (Grant

No. 2017YFC0603101), Natural Science Foundation of China (Grant nos. 41621003 and zhongdaxiangmu etc.), the Strategic Priority Research Program of Chinese Academy of

Sciences (Grant No. XDB26000000), 111 Project (D17013), and Swedish Research Council

(Grant VR2016-04610).

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Figure captions

FIG. 1. Maps of North China outlining the geology, stratigraphical column and locations of studied sections. A, geological map showing the Precambrian-Cambrian rocks along the southern margin of North China Block. B, composite stratigraphical column of the Chaijiawa section and the Sanjianfang section. C, location of Chaijiawa section in Shaanxi Province. D, location of Sanjianfang section in Henan Province.

FIG. 2. Hyolith assemblages from the Xinji formation of North China. A, Microcornus eximius Duan, 1984, SJFH-23-3-08. B, Allatheca sp., SJFH-25-1-08. C, Parakorilithes mammillatus He & Pei in He et al., 1984, LC06-16-02. D, Conotheca australiensis Bengtson in Bengtson et al., 1990, SJF-H-02-02. E, Protomicrocornus triplicensis, SJFH-26-4-04. F,

Cupitheca sp., LC06-14-10. Scale bars represent: 50 μm (D); 100 μm (B, F); 200 μm (A, C,

E).

FIG. 3. Shell microstructures of hyolithid hyolith Microcornus eximius Duan, 1984. A, general view of Microcornus eximius Duan, 1984 in dorsal view; with specimen collected from Shangzhangwan (SZW) Section in Luonan, South Shaanxi, North China (Li et al. 2014,

Skovsted et al. 2016), SZW-14-02. B, internal mould of conch preserves the imprints of epithelial cells, SJFH-23-2. C, enlargement of the conch, shows the tubercles corresponding to the tubule system of outer shells, SJF-H-12-35. D, bidirectional aragonite folia microstructure (B-FOA) shows the distinct mirror-symmetrical pattern along the dorsal- ventral axial plane. The dorsal surface has crystallites of aragonite arranged in convergent directions; straight growth fronts of folia are perpendicular to conch aperture, SJF-H-12-30. E, ventral surface has crystallite of aragonite in opposite directions; with arrows indicate the growth directions of the folia, SJF-H-12-21. Scale bars represent: 20 μm (D); 50 μm (C); 100

μm (B, E).

FIG. 4. Shell microstructures of orthothecid hyolith Allatheca sp. A, general view of

Allatheca sp. in lateral view, shows distinct globular initial shell, SJFH-28-2-05. B, bidirectional aragonite folia microstructure (B-FOA) shows the distinct mirror-symmetrical pattern along the dorsal-ventral axial plane of the conch; the ventral surface has crystallites of aragonite arranged in opposite directions, SJF-H-14-33. C, enlargement of A, shows the shell microstructure in the lateral edge of the conch. D, dorsal surface has crystallites of aragonite arranged in convergent directions. SJFH-28-2-08. E, conch aperture of A shows the dorsal- ventral differentiation of the conch and distinct mirror-symmetrical pattern of B-FOA. Scale bars represent: 10 μm (D); 30 μm (C); 50 μm (B); 100 μm (A).

FIG. 5. Shell microstructures of hyolithid hyolith Parakorilithes mammillatus He & Pei in He et al., 1984. A, general view of P. mammillatus in lateral view, SJFH-23-4-06. B, enlargement of A, shows the unidirectional aragonite folia microstructure (U-FOA) with crystallites of aragonite (folia) in consistent clockwise direction in apertural view. C, D, folia have straight growth fronts, parallel to shell surface and perpendicular to apertural margin, ventral view,

SJF-H-09-18. Scale bars represent: 5 μm (C); 10 μm (B); 100 μm (A, D).

FIG. 6. Shell microstructures of orthothecid hyolith Cupitheca sp. A, general view of

Cupitheca sp. in frontal dorsal view, shows the characteristic flat terminal ending surrounded by a circumferential furrow, SJFH-26-3-02. B, enlargement of A, shows the internal mould of operculum with two prominent cardinal processes. C, articulated internal moulds in dorsolateral view, SJF-H-09-25. D, pillar-like phosphatic infillings of the primary tubule system of the external shell, SJF-H-01-06. E, concentration of hollow tubules surround the flat terminal end, SJF-H-10-39. F, hollow tubules is orientated orthogonally to parallel tubules,

SJF-H-01-06. G, unidirectional aragonite folia microstructure (U-FOA) with the folia in straight growth fronts, SJFH-25-1-02. Scale bars represent: 5 μm (D); 10 μm (F, G); 50 μm

(E); 100 μm (B); 300 μm (A, C).

FIG. 7. Shell microstructures of orthothecid hyolith Protomicrocornus triplicensis. A, crossed foliated lamellar microstructure (CFL), shows the second-order folia arranged in opposite directions between adjacent first-order lamellae, SJFH-25-4-07. B, general view of P. triplicensis in lateral view, articulated specimen of the conch and operculum, SJFH-1-5. C, apertural view of B, shows the operculum. D, oblique anterodorsal view of B, shows that the conch and operculum articulated dorsally by the cardinal process. E, internal mould of conch preserves the imprints of epithelia cells, SJFH-13-15. Scale bars represent: 4 μm (A); 20 μm

(E); 200 μm (B, C, D).

FIG. 8. Shell microstructures of orthothecid hyolith Conotheca australiensis Bengtson in

Bengtson et al., 1990. A, tubercles on surface of internal mould regularly aligned along the growth lines, SJFH-29-3-05. B, tubercles and distinct flat apical region, SJFH-29-3-02. C,

SJFH-28-4-03; D, SJFH-28-3-11, hollow tubules corresponding to the septum of conch and orthogonal to terminal end. E, tubercles and fibrous textures on surface of internal moulds,

SJFH-29-2-13. Scale bars represent: 20 μm (C, D, E); 100 μm (A, B).

FIG. 9. Shell microstructures of hyolith opercula from North China. A, operculum of unidentified genus and species, LC06-05-06. B, enlargement of A, shows the foliated calcite microstructures (FOC) with distinct tooth-like pattern; average interfacial angle 103°. C, internal mould, operculum of Parakorilithes mammillatus, LC061000. D, enlargement of C, shows the characteristic straight growth fronts of the folia of the foliated aragonite microstructure (FOA), and unusual zones with narrow crystallites of aragonite closed to the ventral areas. E, operculum of Conotheca australiensis, LC06-10-02. F, enlargement of E, shows the characteristic straight growth fronts of folia of the foliated aragonite microstructure.

G, foliated calcite microstructure (FOC) of living oyster, unidentified genus and species collected from China Bohai Sea, shows the distinct tooth-like pattern. H, foliated aragonite microstructure (FOA) of Cambrian helcionelloid mollusc Pelagiella madianensis Zhou &

Xiao, 1984, shows the characteristic straight growth fronts of aragonite. Scale bars represent:

5 μm (G, H); 10 μm (B); 20 μm (D, F); 40 μm (A); 100 μm (C, E).

FIG. 10. Shell microstructures of Cambrian mollusc shells from North China. A, B, crossed foliated lamellar microstructure (CFL) of Pelagiella madianensis, A, LC063047, B,

LC063087. C, foliated aragonite microstructure (FOA) of P. madianensis, LC063514. D, pore system of Figurina sp, LC03-31-02, E, enlargments of D, shows the nodes or tubercles on internal mould. F, lateral view of early bivalve Pojetaia runnegari, LC062920. G, enlargement of F, shows the foliated aragonite microstructure in distinct stepwise pattern and straight growth fronts. H, lateral view of the helcionelloid mollusc Mackinnonia rostrata

Zhou & Xiao, 1984, SJF-18-14. I, enlargement of H, shows the imprints of epithelia cells covering the whole surface of the internal mould. Scale bars represent: 5 μm (A, B, I); 10 μm

(C); 30 μm (E); 50 μm (G); 100 μm (F); 200 μm (D, H).

FIG. 11. A deep lophotrochozoan phylogeny showing possible placements of hyoliths. The tree topology is modified from Parkhaev (2017) and Vinther et al. (2017) to account for mollusc phylogeny, Geyer (2018) for hyolith phylogeny, Skovsted et al. (2008, 2009, 2015) and Zhao et al. (2017) for tommotiid–brachiopod phylogeny. The presence of biomineralized elements is denoted. Solid lines indicate valid fossil data, dotted lines represent molecular data and inferred relationships predating the fossil records in Cambrian. 1, primary mantle secretome (PMS); 2, aragonite-specific PMS (A-PMS) in hyoliths and molluscs; 3, phosphate-specific PMS (P-PMS) in tommotiid-brachiopod linage; 4, foot, radula and original aragonitic skeletons; 5, the tentaculate feeding organ indicates a slightly derived position of hyoliths possible on the stem of lophophorates; 6, origin of phosphatic tommotiid; 7, derivation of phosphatic brachiopods from tommotiids; 8, calcitic shells evolved independently in molluscs, hyoliths and brachiopods.