The Evolution of Mollusc Shells

Home , Gastropod shell

1 Article Title: The evolution of mollusc shells 2 3 Article Type: Focus article 4 5 First author 6 NAME: Carmel McDougall 7 ORCID: 0000-0002-2116-5651 8 AFFILIATION: Centre for Marine Sciences, School of Biological Sciences, The 9 University of Queensland, Brisbane 4072, Australia 10 PRESENT ADDRESS: Australian Rivers Institute, Griffith University, Brisbane 11 4111, Australia 12 EMAIL ADDRESS: [email protected] 13 CONFLICTS OF INTEREST: none 14 15 Second author 16 NAME: Bernard M. Degnan* 17 ORCID: 0000-0001-7573-8518 18 AFFILIATION: Centre for Marine Sciences, School of Biological Sciences, The 19 University of Queensland, Brisbane 4072, Australia 20 EMAIL ADDRESS: [email protected] 21 CONFLICTS OF INTEREST: none 22 23

This is the author manuscript accepted for publication and has undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/wdev.313

early larval shell

Trochophore larva Juvenile 25 26 Mollusc shells are fabricated by mantle epithelial cells, which develop from 27 the evolutionarily-conserved larval shell gland. The mantle secretome includes 28 high rates of gene co-option and loss and the rapid evolution of coding 29 sequences, and provides a molecular explanation for the development, 30 evolution and diversity of molluscan shells. 31

Page 2 This article is protected by copyright. All rights reserved. 32 Abstract 33 34 Molluscan shells are externally fabricated by specialised epithelial cells on the 35 dorsal mantle. Although a conserved set of regulatory genes appears to underlie 36 specification of mantle progenitor cells, the genes that contribute to the 37 formation of the mature shell are incredibly diverse. Recent comparative 38 analyses of mantle transcriptomes and shell proteomes of gastropods and 39 bivalves are consistent with shell diversity being underpinned by a rapidly- 40 evolving mantle secretome (suite of genes expressed in the mantle that encode 41 secreted proteins) that is the product of (i) high rates of gene co-option into and 42 loss from the mantle gene regulatory network, and (ii) the rapid evolution of 43 coding sequences, particular those encoding repetitive low complexity domains. 44 Outside a few conserved genes, such as carbonic anhydrase, a so-called 45 ‘biomineralisation toolkit’ has yet to be discovered. Despite this, a common suite 46 of protein domains, which are often associated with the extracellular matrix and 47 immunity, appear to have been independently and often uniquely co-opted into 48 the mantle secretomes of different species. The evolvability of the mantle 49 secretome provides a molecular explanation for the evolution and diversity of 50 molluscan shells. These genomic processes are likely to underlie the evolution of 51 other animal biominerals, including coral and echinoderm skeletons. 52 53

Page 3 This article is protected by copyright. All rights reserved. 54 Introduction 55 56 Biomineralisation refers to the process by which living organisms produce 57 mineralised structures (crystalline or amorphous) from inorganic precursor ions 58 via cellular processes 1, 2. Normally, the inorganic formation of analogous 59 minerals would require extreme temperatures and/or pressures. 60 Biomineralising organisms, however, produce various biomolecules that interact 61 with the mineral ions and allow these minerals to be formed under atmospheric 62 conditions 3. A distinction is often made between biologically-induced versus 63 biologically-controlled biomineralisation. The former generally refers to side- 64 products of metabolism that are dependent on environmental conditions and 65 often not significantly different to the inorganic minerals themselves, whereas 66 the latter are products of specialised, genetically-controlled metabolic processes, 67 are independent of environmental conditions, and are generally distinct from 68 their inorganic counterparts 2, 4. 69 70 Identifying the biomolecules involved in biomineralisation and understanding 71 how they interact with mineral ions is just one active area of study. 72 Biomineralisation research is diverse and includes investigating how the 73 biominerals observed today evolved 5, uncovering how the deployment of key 74 biomolecules is controlled by the genome 6, evaluating the effects of biomineral 75 production on ocean biochemistry 7, and predicting how increasing ocean 76 temperatures and acidification will impact biomineralising organisms and entire 77 ecosystems such as coral reefs 8. 78 79 Molluscs have long been used as models to study biomineralisation as they 80 produce an impressive diversity of shell types, are readily accessible, and have a 81 rich fossil record allowing for evolutionary inferences (Fig. 1). Mollusc shells are 82 unique in that the ontogeny of the biomaterial is recorded within the structure 83 itself, with the oldest part (the larval and juvenile shells) typically being visible at 84 the apex (‘spire’ or ‘umbo’) of the shell, and the newest part being at the shell 85 edge – a useful feature for developmental biologists. Additionally, the 86 predominant mineralising organ (the mantle 9) is easily accessible 1 and is

Page 4 This article is protected by copyright. All rights reserved. 87 separated into zones that are each responsible for secreting different layers of 88 the shell (see below), facilitating investigations into the differences in cell 89 biology and gene expression between these zones. From an applied perspective, 90 the shells of many molluscs have commercial value, particularly mother-of-pearl 91 (nacre), and this has fuelled research into the structure and formation of this 92 shell layer. Nacre is also one of the strongest known shell layers and has been 93 well-studied from a biomaterials perspective. Finally, molluscs produce the 94 highest diversity of biominerals of any animal group, both in terms of the 95 mineral type deposited and the function of the materials themselves 1, 4. 96 97 The evolution and diversity of biomineralised structures 98 99 The ability to mineralise is widespread throughout life on Earth and appeared 100 very early in evolution 1, 2, 10. The oldest known evidence is from stromatolites 101 (blue-green algae that perform biologically-induced biomineralisation), dating 102 back to 3,700 million years ago (mya) 11. The first examples of biologically- 103 controlled mineralisation may be the vase-shaped protist microfossils from the 104 Chuar Group, Grand Canyon (iron-based mineralisation, 742 mya) 12, or possibly 105 the scale microfossils of the Fifteenmile Group, Yukon (812-717 mya) 13. The 106 earliest known metazoan (animal) examples are represented by Cloudina and 107 associated forms (550 mya), which may be stem or crown group cnidarians 14. 108 Fossil remains of biomineralised structures from this time (the late Proterozoic) 109 are scarce, suggesting that biomineralisation, at least of large-scale skeletal 110 structures, was not widespread 10. The Cambrian (543-490 mya) saw a large 111 diversification of metazoan biomineralisation, with the first appearance of 112 mineralised skeletons of most phyla. Further diversification within phyla 113 occurred during the Ordovician radiation, at which time crown group members 114 of invertebrate phyla arose and grew numerous enough to impact on marine 115 carbonate and silica cycles 10. 116 117 Various hypotheses have been put forward to explain the rapid diversification of 118 biomineralisation during these periods. The Cambrian explosion occurred during 119 a period of high Ca2+ levels in seawater, prompting speculation that

Page 5 This article is protected by copyright. All rights reserved. 120 biomineralisation (biocalcification) evolved as a means of removing toxic levels 121 of intracellular calcium 15-17. However, this does not explain the concomitant 122 diversification of non-calcium biomineralisation (e.g, silica-based 123 biomineralisation) during the same period 10. The increased oceanic Ca2+ is 124 reported to be due to enhanced physical and chemical wearing of the continental 125 crust during terminal Ediacaran and Cambrian periods, a process that also

2+ - - + 126 triggered the release of additional ions, including Mg , HCO3 , H3SiO4 , K , and 127 Fe3+ 17. Therefore, the observed evolutionary wave of biomineralised structures 128 may have been trigged by the increased availability of these minerals, or perhaps 129 the overall increase in nutrient availability facilitated an acceleration of 130 diversification in general, with expansion of biomineralised forms just one 131 component. It is proposed that this was further driven as a defence against 132 increased predation pressure 10, 18. 133 134 The fossil record contains examples of numerous types of biominerals that are 135 also found in extant species. Calcium and silica-based minerals are the most 136 common, with aragonite and calcite (polymorphs of calcium carbonate) having 137 the broadest distribution across taxonomic groups 1. Other biominerals, 138 including iron and magnesium-based forms, are less common and have a more 139 restricted distribution 1, 4. The biomineralised structures produced are 140 functionally diverse and include skeletons (external and internal), teeth, spines, 141 gravity sensing organs, eye lenses and mineral storage units 1. Taking into 142 account the structures produced and the phylogenetic relationships of the 143 organisms making them, it is likely that many are the result of independent

144 evolutionary events, with calcium carbonate and silica biomineralisation 145 evolving at least 28 and 8 times in eukaryotes, respectively 10. 146 147 What biomineralised structures do molluscs make? 148 149 Most molluscs, although not all, produce external calcified shells. The phylum 150 Mollusca is composed of eight extant classes that are divided into two major 151 lineages (Fig. 2), the Conchifera (shell-bearers, including the gastropods, 152 bivalves, scaphopods, cephalopods and monoplacophorans) and the Aculifera

Page 6 This article is protected by copyright. All rights reserved. 153 (scale-bearers, including the polyplacophorans, solenogastres and 154 caudofoveates) 19-21. As the name implies, aculiferan molluscs do not produce a 155 single or paired shell, instead, their body is often covered in calcareous sclerites 156 (or spicules), scales, or plates. The aculiferans tend to be more difficult to find, 157 therefore the majority of research on molluscan biomineralisation has focussed 158 on the shells of conchiferan species. 159 160 Although the shells and sclerites are the most obvious biomineralised structures 161 synthesised by molluscs, members of the phylum also produce other mineralised 162 parts including the radula (teeth), statocysts (balance organs), the operculum 163 (trap-door for the shell aperture), the epiphragm (temporary shell-aperture 164 trap-door for terrestrial hibernators), egg capsules, love darts (reproductive 165 structures), gizzard plates (digestive system), and gill supports 4. While the shells 166 and sclerites are usually made of calcium carbonate - although, incredibly, there 167 is a deep sea hydrothermal vent gastropod that coats its shell with iron sulphide 168 22, 23 - many of these additional structures utilise other minerals including 169 magnetite, ferrihydrite, calcium phosphate and calcium oxalate 4. The radula, in 170 particular, of many species has strongly-bonded layers constructed of iron-based 171 minerals 24. In fact, the radula of the limpet Patella vulgata is currently the 172 strongest biological material known to exist 25. 173 174 Ontogeny of molluscan shells and sclerites 175 176 The shells of molluscs form early in development and display significant changes 177 in shape, ornamentation and patterning over the life of the organism (Fig. 3) 26. 178 In conchiferans, the initial stages of shell formation occur in early embryogenesis 179 and appear to be relatively conserved. An initial, and as yet incompletely 180 understood, induction event causes specification of an initial cluster of 181 biomineralising cells – the ‘shell field’ (reviewed in 27 28, 29). This shell field is 182 comprised of cells that are predominately descendants of the 2d micromere, with 183 descendants of 2a and 2c (and, very rarely, 2b) also contributing in some species 184 (28, 29 and references therein). After the induction event, the shell field 185 invaginates and the first organic shell secretions are issued from shell gland cells

Page 7 This article is protected by copyright. All rights reserved. 186 30; at this stage, the invagination may be sealed off from the external 187 environment 31, 32. The invaginated cells then evaginate, expand, and differentiate 188 to eventually form the larval and then juvenile mantle 27, 30. The timing of the 189 initial calcification of the shell appears to vary between species, but is generally 190 very early in the development of the shell field and may occur during the initial 191 invagination itself 27, 31, 32. The larval shell is, in the first instance, amorphous in 192 nature, at least in studied bivalves 33. As the shell field expands and forms the 193 larval mantle, so too does the shell expand so that the larva can be fully 194 accommodated inside. This larval shell is usually unsculptured and is called 195 protoconch I in gastropods or prodissoconch I in bivalves. In some species, a 196 second type of larval shell, the protoconch II or prodissoconch II, is also 197 produced – this is often more sculptured and ornate than the original shell 29. 198 The final shell, initiated at metamorphosis, is the teloconch. In most 199 conchiferans, the teloconch is maintained through to end of the animal’s life, but 200 it may also change a number of times in structure and pattern during juvenile 201 growth 26 (Fig. 3). Importantly, each shell is continuous with the one produced 202 before it, so that the entire history of an animal’s shell formation is visible. 203 204 In polyplacophorans (or chitons), the mantle tissue secretes both shell plates and 205 sclerites 34. Seven plate fields form within the dorsal ectoderm simultaneously 206 prior to settlement (an eighth plate forms later), however calcified valves are not 207 observed until after metamorphosis 27, 35, 36. Sclerite formation occurs towards 208 the lateral part of the mantle (=girdle) by secretions from a single cell; in some 209 species, additional cells contribute secretions once sclerite formation has been 210 initiated 34. In aplacophorans, sclerite formation appears to be very similar to 211 that in chitons, with the initial secretion occurring within an invagination of a 212 single cell 37-39. Whether or not the cells contributing to the shell field (and, 213 consequently, the mantle) and sclerite forming ectoderm can be considered 214 homologous across molluscan classes is unclear – cell lineage studies suggest 215 involvement of the 2d micromere and its descendants, however additional 216 micromeres can contribute to mineralising tissues in some taxa (reviewed in 29). 217 As cell lineage studies are sparse, more species need to be investigated in detail 218 before general developmental principles can be established for these mollusc

Page 8 This article is protected by copyright. All rights reserved. 219 classes. Given the apparent ease with which calcified structures can evolve, 220 homology of molluscan shells, plates, and sclerites should not be assumed. 221 222 Molluscan shell structure 223 224 Molluscan shells are diverse in architecture and pattern. In general, though, they 225 are composed of multiple layers of calcium carbonate polymorphs (usually 226 aragonite or calcite), as well as a proteinaceous outer layer, the periostracum 227 (Fig. 4). As well as displaying different polymorphs, the layers can also have 228 different microstructures, that is, different arrangements of individual calcium 229 carbonate nanometric units. These arrangements are classified based on their 230 morphology, and include prismatic, spherulitic, crossed-lamellar, homogenous 231 and nacreous forms (see 40 and 41 for descriptions). Nacre, or mother-of-pearl, is 232 arguably the best-studied microstructure. It is always aragonitic, is composed of 233 flattened polygonal or rounded tablets arranged in layers 40, 41, and is found in 234 several molluscan classes including bivalves, gastropods, cephalopods (Nautilus) 235 and monoplacophorans 42, 43. Significant differences exist in the nacre produced 236 by each class of mollusc. Gastropods possess columnar nacre in which the tablets 237 are stacked on top of each other, whereas in bivalves each tablet is offset from 238 the one below in a brick-like pattern (sheet nacre)44. Cephalopods produce nacre 239 that is an intermediate of the two 45. In addition, the tablets in gastropod, bivalve 240 and cephalopod nacre each display different crystallographic axes 46. Based on 241 these differences it has been hypothesised that nacre may have originated 242 several times independently 45, 47, 48, therefore caution needs to be exercised 243 when making generalisations relating to superficially similar shell layer 244 microstructures. 245 246 Aragonite is the mineral produced most frequently for the sclerites and scales of 247 aculiferans 38, 49, 50. In aplacophorans, the sclerites can be either solid or hollow, 248 and no layering is obvious within the structure 38. The shell plates of 249 polyplacophorans are complex. For instance, the plates of Tonicella marmorea 250 consist of six distinct layers, each with a unique microstructure 51. The structure 251 of polyplacophoran sclerites is substantially different, with the aragonite crystals

Page 9 This article is protected by copyright. All rights reserved. 252 more closely resembling those of the abiologically formed mineral, indicating 253 that these skeletal elements may be formed by a very different process to that of 254 the shell plates of the same animal 49. 255 256 In all the structures described above, the calcium carbonate nanometric units are 257 in close association with an organic matrix composed of proteins, 258 polysaccharides, and lipids 52. These organic components are secreted from the 259 underlying mantle tissue and are responsible for controlling shell formation. 260 Recrystallisation studies, whereby demineralised matrix is placed into a solution 261 containing saturated calcium carbonate, show that matrix from aragonitic shell 262 layers is capable of directing aragonite precipitation in a system that otherwise 263 produces calcite, and vice versa 53, 54. Proteomic studies have shown that the 264 matrices of shell layers with different polymorphs contain different proteins 55, 265 and biochemical analyses have found that polysaccharide (e.g., chitin)52 and lipid 266 56 content also differs. These studies indicate that the matrix can determine both 267 the polymorph and the microstructure formed in various shell layers. As more 268 molluscan species are studied at the ultrastructural level, it is becoming obvious 269 that the mantle itself is regionalised, and that unique secreted molecules 270 originating from different zones instruct the formation of the shell layer that 271 forms adjacent to it 26, 57-60. 272 273 The molluscan shell-formation gene regulatory network 274 275 The development of an animal is carefully orchestrated by a hierarchy of gene 276 expression. Co-ordinating this expression are regulatory genes that act at the 277 core of gene regulatory networks (GRNs) to precisely control the timing and 278 location of expression of downstream ‘effector’ genes 61. In molluscan 279 development the initiation of the shell field is likely under the control of its own 280 GRN; as the shell field develops into the mantle, and as shell formation is 281 generally continuous from the first secretions of the shell field through to the 282 final adult shell, the master regulators controlling the development of the shell 283 field are thus the core regulators of the entire process of shell formation 28. Sub- 284 circuits of the shell-formation GRN regulate the specification and maintenance of

Page 10 This article is protected by copyright. All rights reserved. 285 subsequent specialisations, for example, the different mantle zones responsible 286 for the secretion of different shell layers. A number of transcription factors and 287 signalling ligands that are involved in specification events during development of 288 other animals have demonstrated expression in discrete zones during molluscan 289 shell-field development, including engrailed, goosecoid, brachyury, 290 decapentaplegic, distal-less, and various Hox genes (reviewed in 28). However, 291 these genes are all likely to be under the regulation of those involved in the 292 initial induction event – a process that is not yet understood at the molecular 293 level. We also currently know nothing about the network architecture – except 294 that engrailed expression is not affected by knockdown of decapentaplegic 62. 295 296 The terminal branches of the shell formation GRN include genes encoding 297 proteins that are secreted from the mantle (the mantle secretome 59) and are 298 directly involved in the shell formation process 63, as well as genes that control 299 the deposition of polysaccharides and lipids in the shell. In the first models of 300 molluscan biomineralisation it was thought that the organic matrix was 301 comprised of relatively few components. For nacre, these components were 302 thought to be chitin, sulfated glycoproteins, and hydrophobic proteins 4. 303 Transcriptomic and proteomic approaches have demonstrated that in fact shell- 304 building appears to be complex, with at least 40-60 proteins with very different 305 characteristics being incorporated into a single shell (likely an underestimate 306 due to the limitations of proteomic techniques, see 64), and it is probable that 307 many more genes/proteins direct shell formation from within the mantle itself. 308 Therefore, there are numerous terminal outputs of the shell formation GRN. 309 310 The search for the molluscan biomineralisation toolkit 311 312 During the development of most metazoans, the organisation of the overall body 313 plan is specified through the action of a core set of highly conserved 314 developmental genes, often referred to as the ‘developmental’ or ‘genetic’ toolkit 315 65. Alteration in the expression of these genes can cause differences in 316 morphology on many different scales, from the conversion of one body segment 317 to another 66, to minute changes in the patterning of butterfly wings 67. The

Page 11 This article is protected by copyright. All rights reserved. 318 possibility that biomineralisation, given its widespread distribution in animals, 319 may also be controlled by a similar suite of conserved genes led Livingston and 320 colleagues 68 to introduce the concept of a ‘biomineralisation toolkit’. It was 321 suggested that a common set of genetic and developmental processes may 322 underlie skeletogenesis in divergent taxa, regardless of whether the structures 323 produced were strictly homologous. The authors found some similarities in the 324 early stages of the biomineralisation process in echinoderms and vertebrates, 325 including specification of skeletogenic cell types by the transcription factors ETS 326 and ALX. However, for the most part, proteins involved late in biomineralisation 327 were very different in the two taxa, although there were a few examples of 328 overlapping proteins, including members of the SPARC and cyclophilin gene 329 families. 330 331 To date, there is no evidence for involvement of ETS and ALX homologues in the 332 specification of the shell field or of the cells responsible for the secretion of any 333 other biomineralised structure in molluscs. However, the developmental 334 processes are by no means completely understood, so the existence of a widely 335 conserved metazoan biomineralisation toolkit cannot be discounted. Similar to 336 the case in deuterostomes, the analysis of transcriptomic and proteomic datasets 337 from mantles and shells (representing late biomineralisation events) has found 338 very little similarity between molluscan species 69-72, although, interestingly, 339 members of SPARC and cyclophilin gene families are expressed in the mantle in 340 several species 47. Only 15% similarity was observed between the nacre- 341 producing secretomes of an abalone and a pearl oyster 47, whereas only 1.1 to 342 7.7% similarity was detected between the proteomes of the gastropod Cepaea 343 nemoralis and other sequenced proteomes from two gastropod and three bivalve 344 species 69. This dissimilarity has led to the speculation that these shell types may 345 have evolved independently or that the secretome is very rapidly evolving 346 (reviewed in 73), and suggests that the changes observed in shell architectures 347 may be due to the actions of a large and diverse gene set, rather than the actions 348 of a core set of conserved developmental genes that are differentially expressed. 349

Page 12 This article is protected by copyright. All rights reserved. 350 Recently, Aguilera and colleagues 63 made use of existing mantle transcriptomes 351 to perform the most extensive comparative analysis of conchiferan secretomes 352 to date. By categorising the secretomes of eight bivalve and three gastropod 353 species into orthology groups and mapping them on to a phylogenetic tree, the 354 authors were able to establish which gene families were likely components of the 355 mantle secretome in the last common ancestor of bivalves and gastropods (i.e., 356 having at least one member in a bivalve and a gastropod; 782 families in total, 357 Fig. 5). This list includes gene families known to have other conserved functions 358 (e.g., many nervous system-related genes), as well as genes known to be involved 359 in biomineralisation. While these biomineralisation-related gene families could 360 be said to represent the ‘conchiferan biomineralisation toolkit’, dynamic patterns 361 of evolutionary gene loss have resulted in very few genes being present in all 362 investigated species. Therefore the majority of these gene families do not appear 363 to be necessary for biomineralisation. 364 365 Aguilera et al.63 found that the protein domain repertoire secreted from the 366 mantle was highly dynamic, resulting in each of the 11 species having a unique 367 domain profile. Nonetheless, some domains had been repeatedly and 368 independently co-opted into mantle secretome, including carbonic anhydrase, 369 glycoside hydrolase family 18, and polysaccharide deacetylase (Fig. 5). 370 Arivalagan and colleagues 74 performed a comparative proteomic analysis of the 371 shells of four bivalve species with different shell architectures. They found four 372 protein domains that were represented in each shell proteome, tyrosinase, 373 carbonic anhydrase, chitin-binding 2, and Von Willebrand factor-A, which they 374 propose were part of the ancestral conchiferan secretome. As mentioned above, 375 carbonic anhydrase domains appear to have been co-opted into the mantle 376 secretome independently, therefore it is unlikely that they were part of an 377 ancestral toolkit; although the molluscan secretome carbonic anhydrases were 378 found to form a single, well-supported clade in a previous study 75. For the 379 remaining domains, phylogenetic analysis has either not been performed or, in 380 the case of tyrosinase, the secretome members fall within a very large expansion 381 of the gene family in molluscs 76. As it is unknown whether all members have 382 biomineralisation roles, it cannot be determined whether this function is

Page 13 This article is protected by copyright. All rights reserved. 383 ancestral, and hence whether these could be considered true toolkit genes. 384 Regardless, it appears that these four domains represent core requirements for 385 molluscan biomineralisation. 386 387 As well as a core set of essential domains, molluscan biomineralisation may also 388 possess a number of other common principles (reviewed in 39). The most striking 389 is the preponderance of sequences containing repetitive, low complexity 390 domains (RLCDs) 47, which are generally involved in aggregation or binding 391 (reviewed in 73). Many of these RLCDs are glycine-rich, a feature commonly 392 observed in structural proteins that are generally involved in the production of 393 biological materials 77. Many proteins are also modular in nature, containing 394 multiple unrelated domains within the one sequence 78 75. Both of these 395 characteristics are likely to increase the evolvability of the secretomes via 396 unequal crossover and replication slippage, and are likely contributors towards 397 the rapid evolution of these genes 73. 398 399 Mechanisms of secretome evolution 400 401 The extent of novelty observed in mantle secretomes and shell proteomes has 402 been surprising, and raised questions regarding how this novelty was generated. 403 In the large scale secretome comparison study mentioned above, Aguilera and 404 colleagues estimated the age of gene families using a phylostratigraphic 405 approach and mapped these on to a phylogenetic tree. The analysis revealed 406 broad patterns of gene gain and loss in each species’ and lineage’s mantle 407 transcriptome. Large differences between the secretomes of these species were 408 observed, as seen in previous studies based on proteomes of fewer taxa 47, 69-72. It 409 was found that these differences were due not only to the gain of novel genes, 410 but also, more commonly, the differential co-option of ancient genes into 411 secretomes in different lineages. The uniqueness of the secretomes was further 412 reinforced by the loss of expression of these genes in a lineage-specific fashion. 413 414 Thus, genes that arose prior to bilaterian and molluscan cladogenesis, which 415 must have originally performed functions unrelated to the mantle as they pre-

Page 14 This article is protected by copyright. All rights reserved. 416 dated the evolution of this structure, are frequently recruited into the mantle 417 gene regulatory network. This indicates that the periphery of this gene network 418 is highly evolvable, allowing new genes to fall under and to escape control by the 419 core gene regulators that we can assume exist. Of all the genes found to be co- 420 opted into the secretome of conchiferan molluscs, an over-representation of 421 genes encoding a diverse suite of domains involved in processes including 422 protein-protein interactions, cell adhesion, recognition, immunity, extracellular 423 matrix functions, and carbohydrate metabolism, were found (Fig. 6). Other 424 proteomics-based studies have noted the presence of numerous proteins 425 containing many of these domains within the shell matrix 74, 79-81, suggesting that 426 this enrichment is likely to be found within genes directly involved in shell 427 formation, rather than those that are performing non-shell related functions 428 within the mantle. Thus, it seems that proteins with particular domains and/or 429 functions are favoured for co-option into the shell-formation process. We 430 currently do not know how these proteins are functioning within the shell. It is 431 possible that the domains have retained their ancestral function, meaning that 432 the proteins that contain them are pleiotropic (i.e., shell-forming proteins 433 contain immune-related domains that confer an immune function to the shell 434 itself). Alternatively, the domains may, either in their ancestral form or perhaps 435 via modification, be directly involved in aspects of the biomineralisation process, 436 for example, calcium binding or matrix formation. Within the organic matrix of 437 the shell, protein-protein interactions are likely to be critically important and to 438 directly impact the structure of the shell itself. Perhaps it is the case that the 439 precise nature of the interactions are not important, so long as the proteins 440 themselves are able to physically associate and to form a framework in which 441 biomineralisation can take place. In this case, it may have been relatively easy for 442 pre-existing proteins containing interaction domains to be recruited into the 443 mantle secretome – any additional characteristics that these particular proteins 444 possess can subsequently be acted on by natural selection to produce the variety 445 of shells observed today. 446 447 The relatedness of metazoan biomineralised structures 448

Page 15 This article is protected by copyright. All rights reserved. 449 Determining whether or not two biomineralised structures are homologous is 450 difficult, regardless of whether the comparison is being made within or between 451 phyla. Different interpretations will likely be made depending on whether the 452 assessment is being based on the final structure itself (i.e., in molluscs, the shells 453 of different classes, the shells and sclerites of a single animal), the cell lineage of 454 the cells that produce the structure, and/or the molecular processes underlying 455 its production. Adding to the confusion is the apparent ease by which 456 biomineralised structures can arise de novo. 457 458 Knoll 10 pointed out that all cells possess the ability to regulate calcium 459 metabolism, therefore many eukaryotes that do not possess biomineralising 460 ancestors nonetheless possess an underlying capacity to control calcium-based 461 mineralisations. This may explain the numerous independent evolutionary 462 events leading towards calcium-based biomineralised structures. Understanding 463 the evolutionary relatedness of these structures is therefore problematic. For 464 instance, although conchiferan shells are likely to be homologous and the 465 ontogeny of the shell-fabricating mantle appears to be shared, nacreous layers 466 appear to have evolved independently multiple times within this developmental 467 background. The questions that therefore arise are 1) what are the minimal 468 molecular requirements for the generation of biomineralised structures (i.e., 469 which proteins, protein domains, polysaccharides and lipids need to be present 470 to promote and direct the biomineralisation process), and 2) of the extant 471 biomineralising species, what are the shared molecular hallmarks that reflect a 472 single evolutionary event? 473 474 This is where a nested set of comparisons may prove useful. Elucidating which 475 features are shared between all biomineralised structures (if any), those shared 476 between all calcium-based biomineralised structures (most probably co-opted 477 from the general cell calcium metabolism machinery), and those shared between 478 more closely related animals will reveal the functional requirements for each 479 level of organisation. The analysis would also reveal the history of evolutionary 480 events, through shared similarities that, given the high level of gene co-option 481 and de novo gene gain, should be unique (i.e., a shared derived character, or

Page 16 This article is protected by copyright. All rights reserved. 482 novelty) 61 75. These core features will be integrated within the GRN governing 483 the biomineralisation process. The core genes of this GRN are likely to be broadly 484 conserved, should reflect the initial specification of biomineralising cells (and, 485 thus, cell lineage), and could be said to represent the biomineralisation 486 regulatory toolkit. Although our understanding of the number and types of genes 487 and proteins that are involved with biomineralisation is increasing at a rapid 488 rate due to the development of high-throughput ‘omic’ techniques, very little is 489 known about the non-protein components of shells (i.e., polysaccharides and 490 lipids). An additional challenge lies in gathering enough developmental, 491 morphological and functional data to be able to make the required comparisons 492 and inferences in a wide range of taxa. 493 494 Conclusions 495 496 Now that transcriptomic, proteomic and genomic data are accessible for non- 497 model organisms at reasonable cost, we can expect (and have already seen) a 498 dramatic increase in the sequence information available for molluscs, 499 particularly in relation to the shell and mantle. With this, and the recent 500 application of new tools for functional manipulation such as RNAi 62, 501 morpholinos 76, and CRISPR 77 to studies of molluscan development, it is likely 502 that we will see the elucidation of a molluscan skeletogenic GRN (in line with the 503 already established sea urchin GRN). In parallel, functional characterisation of 504 shell-building proteins will reveal the underlying mechanisms of shell formation, 505 enabling us to determine which proteins (or protein characteristics) are 506 required for each type of shell microstructure. 507 508 The integration of developmental and functional data from multiple molluscan 509 species will enable reconstruction of the evolution of the biomineralisation 510 process. Furthermore, the potential to sequence ancient proteomic data from 511 fossil shells provides a tantalising opportunity to test these reconstructions 2 78. 512 Just how far back these reconstructions can be pushed remains to be seen. 513

Page 17 This article is protected by copyright. All rights reserved. 514 Acknowledgements 515 516 The authors would like to thank two anonymous reviewers whose comments 517 and suggestions greatly improved the manuscript. Associated research is 518 supported by Australian Research Council grants to BMD. 519 520 521 References 522 523 1. Simkiss K, Wilbur KM. Biomineralization. San Diego: Academic Press; 524 1989. 525 2. Marin F, Le Roy N, Marie B, Ramos-Silva P, Bundeleva I, Guichard N, 526 Immel F. Metazoan calcium carbonate biomineralizations: 527 macroevolutionary trends - challenges for the coming decade. Bull Soc 528 geol Fr 2014, 185:217-232. 529 3. Veis A. Crystals and life: an introduction. Vol. 4. Chichester: John Wiley and 530 Sons; 2010. 531 4. Lowenstam HA, Weiner S. On biomineralization. New York: Oxford 532 University Press; 1989. 533 5. Murdock DJE, Donoghue PCJ. Evolutionary origins of animal skeletal 534 biomineralization. Cells, Tissues, Organs 2011, 194:98-102. 535 6. Ettensohn CA, Kitazawa C, Cheers MS, Leonard JD, Sharma T. Gene 536 regulatory networks and developmental plasticity in the early sea urchin 537 embryo: alternative deployment of the skeletogenic gene regulatory 538 network. Development 2007, 134:3077-3087. 539 7. Van Cappellen P. Biomineralization and Global Biogeochemical Cycles. In: 540 Dove PM, De Yoreo JJ, Weiner S, eds. Biomineralization. Vol. 54. 541 Washington DC: Mineralogical Society of America; 2003. 542 8. Ries JB, Cohen AL, McCorkle DC. Marine calcifiers exhibit mixed responses 543 to CO2-induced ocean acidification. Geology 2009, 37:1131-1134. 544 9. Beedham G. Observations on the mantle of the Lamellibranchia. Quart J 545 Mic Sci 1958, 3:181-197. 546 10. Knoll AH. Biomineralization and evolutionary history. In: Dove PM, De 547 Yoreo JJ, Weiner S, eds. Biomineralization. Washington: Mineralogical 548 Society of America; 2003, 329-356. 549 11. Nutman AP, Bennett VC, Friend CRL, Van Kranendonk MJ, Chivas AR. 550 Rapid emergence of life shown by discovery of 3,700-million-year-old 551 microbial structures. Nature 2016, 537:535-538. 552 12. Porter SM, Knoll AH. Testate amoebae in the Neoproterozoic Era: 553 evidence from vase-shaped microfossils in the Chuar Group, Grand 554 Canyon. Paleobiology 2000, 26:360-385. 555 13. Cohen PA, Knoll AH. Scale Microfossils from the Mid-Neoproterozoic 556 Fifteenmile Group, Yukon Territory. J Paleontology 2012, 86:775-800. 557 14. Germs GJB. New shelly fossils from Nama Group, South West Africa. Am J 558 Sci 1972, 272:752-761.

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Page 19 This article is protected by copyright. All rights reserved. 607 33. Weiss IM, Tuross N, Addadi L, Weiner S. Mollusc larval shell formation: 608 amorphous calcium carbonate is a precursor phase for aragonite. J Exp 609 Zool 2002, 293:478-491. 610 34. Haas W. Evolution of calcareous hardparts in primitive molluscs. 611 Malacologia 1981, 21:403-418. 612 35. Lord JP. Larval development, metamorphosis and early growth of the 613 gumboot chiton Cryptochiton stelleri (Middendorf, 1847) 614 (Polyplacophora: Mopaliidae) on the Oregon coast. J Mollus Stud 2011, 615 77:182-188. 616 36. Wanninger A, Haszprunar G. Chiton myogenesis: Perspectives for the 617 development and evolution of larval and adult muscle systems in 618 molluscs. J Morphol 2002, 251:103-113. 619 37. Woodland W. Studies in spicule formation. VI. The scleroblastic 620 development of the spicules in some Mollusca and in one Genus of 621 colonial ascidians. Quart J Mic Sci 1907, 51:45-53. 622 38. Kingsley RJ, Froelich J, Marks CB, Spicer LM, Todt C. Formation and 623 morphology of epidermal sclerites from a deep-sea hydrothermal vent 624 solenogaster (Helicoradomenia sp., Solenogastres, Mollusca). 625 Zoomorphology 2013, 132:1-9. 626 39. Kocot KM, McDougall C, Degnan BM. Developing perspectives on 627 molluscan shells part 1: introduction and molecular biology. In: Saleuddin 628 S, Mukai S, eds. Physiology of molluscs: A collection of selected reviews. Vol. 629 1: Apple Academic Press; 2016, 1-41. 630 40. Carter JC, Clark GRI. Classification and phylogenetic significance of 631 molluscan shell microstructure. In: Bottjer DJ, Hickman CS, Ward PD, eds. 632 Mollusks: notes for a short course. Vol. 13. Knoxville: University of 633 Tennessee Department of Geological Sciences Studies in Geology; 1985, 634 50-71. 635 41. Chateigner D, Hedegaard C, Wenk H. Mollusc shell microstructures and 636 crystallographic textures. J Struct Geol 2000, 22:1723-1735. 637 42. Marin F, Le Roy N, Marie B. The formation and mineralization of mollusk 638 shell. Frontiers in Bioscience 2012, 4:1099-1125. 639 43. Checa AG, Ramírez-Rico J, González-Segura A, Sánchez-Navas A. Nacre and 640 false nacre (foliated aragonite) in extant monoplacophorans 641 (=Tryblidiida: Mollusca). Naturwissenschaften 2009, 96:111-122. 642 44. Cartwright JHE, Checa AG. The dynamics of nacre self-assembly. J R Soc 643 Interface 2007, 4:491-504. 644 45. Vendrasco MJ, Checa AG, Kouchinsky AV. Shell microstructure of the early 645 bivalve Pojetaia and the independent origin of nacre within the Mollusca. 646 Palaeontology 2011, 54:825-850. 647 46. Hedegaard C, Wenk H. Microstructure and texture patterns of mollusc 648 shells. J Mollus Stud 1998, 64:133-136. 649 47. Jackson DJ, McDougall C, Woodcroft B, Moase P, Rose RA, Kube M, 650 Reinhardt R, Rokhsar DS, Montagnani C, Joubert C, et al. Parallel evolution 651 of nacre building gene sets in molluscs. Mol Biol Evol 2010, 27:591-608. 652 48. Marie B, Le Roy N, Marie A, Dubost L, Milet C, Bedouet L, Becchi M, 653 Zanella-Cléon I, Jackson D, Degnan B, et al. Nacre evolution : A proteomic 654 approach. Mater Res Soc Symp Proc 2009, 1187:1187-KK1101-1103.

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Page 22 This article is protected by copyright. All rights reserved. 751 Figures legends 752 753 Figure 1. “Prosobranchia”, a plate from Ernst Haeckel’s “Kunstformen der Natur” 754 (1904), is a beautiful depiction of the geometric beauty and diversity of sea 755 shells. 756 757 Figure 2. Current consensus of the relationships of molluscan classes from recent 758 studies 19-21. The position of Monoplacophora is unresolved. Images from 29. 759 760 Figure 3. The development of the shell of the abalone, Haliotis asinina. A. 761 Scanning electron micrograph (SEM) of a trochophore larva. sf., shell field. B. 762 SEM of the expanding protoconch (pc) in the larva. C. SEM of the complete larval 763 protoconch. D. SEM of early teloconch development in a recently settled juvenile. 764 The division between the protoconch and teloconch is indicated by an arrow. E. 765 Bright field image of a juvenile of a similar age to that in D. F. Eight week old 766 juvenile showing the beginnings of shell patterning. G. Juvenile shell. H. Adult 767 shell. Arrow shows the spire, where the juvenile shell is visible. Inset: higher 768 magnification of the spire of ‘F’. The location of the larval and early juvenile shell 769 is visible indicated by dotted lines. SEM images modified from 26. 770 771 Figure 4. Shell microstructure. A. Schematic of the organisation of the molluscan 772 shell and mantle. Redrawn from 9. B. SEM of a transverse section of the juvenile 773 shell of the abalone, Haliotis asinina. The top layer displays prismatic 774 microstructure (P), the lower layer is columnar nacre (N). Image by Kathryn 775 Green. 776 777 Figure 5. Evolution of the molluscan secretome. Modified from 63. A. Origin of 778 secreted mantle proteins from gastropods (top) and bivalves (middle), with 779 phylostrata (PS1-13) depicted below. The three main evolutionary periods are 780 indicated in red. The phylostrata indicated in grey are those that pre-date the 781 origin of Mollusca, these proteins must therefore have been co-opted into the 782 secretome. B. Organismal tree with pie charts showing the proportion of 783 secretome genes that have been gained (black), co-opted (red), lost (blue) and

Page 23 This article is protected by copyright. All rights reserved. 784 maintained from the last common ancestor of gastropods and bivalves (BGLCA, 785 brown). Examples of enriched domains that are inferred to have existed in 786 BGLCA (orange box), ancestral bivalve (green box), and ancestral gastropod 787 (blue box), are indicated. Three additional domains, carbonic anhydrase, 788 tyrosinase, and chitin-binding domain (white box), also existed in the BGLCA 789 secretome, these genes have also been identified in a study by Arivalagan and 790 colleagues 74. 791 792 Figure 6. Enrichment of domains in co-opted gene families. Modified from 63. 793 Broad functional categories are shown on the left vertical axis of the heat map, 794 and individual domain names are indicated on the right. Domains are 795 represented if they are significantly enriched in newly gained secreted gene 796 families from at least two lineages. On the horizontal axis of the heat map, 797 ancestral lineages are depicted on the left, current species on the right (refer to 798 the tree in Fig. 5 for the phylogenetic tree). The colour of the squares indicates 799 the level of enrichment, only enrichments with a P-value >0.05 are shown. 800

Subject: Re: Use of image? Date: Wednesday, 28 December 2016 at 3:39:22 AM Australian Eastern Standard Time From: Maik Scherholz To: Carmel McDougall Hi Carmel,

Sorry for my late response, but I am on vacaon. I remember you very well, and I am totally ﬁne with using my solenogaster pic for your review arcle. I wish you good luck with your arcle and a happy new year 2017!

Cheers,

Maik

Am 21.12.2016 01:21, schrieb Carmel McDougall: Hi Maik, I’m Carmel, a friend of Tim’s – I met you brieﬂy when I was in Vienna a couple of years ago! I’m emailing because I’m wring a review arcle, and was hoping to re-use some images from Andi and Tim’s Mollusca chapter, in 'Evoluonary developmental biology of invertebrates’. The solenogastre picture was originally from you, so would you be ok if I re-used it? A dra of the ﬁgure I’m proposing is aached. Thanks very much, Carmel

-- Dipl. Biol. Maik Scherholz Department of Integrave Zoology Center for Organismal Systems Biology Faculty of Life Sciences University of Vienna

Althanstraße 14 A-1090 Vienna (Austria) e-mail: [email protected]

Subject: Re: FW: Permission to re-use image Date: Monday, 23 January 2017 at 8:03:07 PM Australian Eastern Standard Time From: Schroedl, Dr. Michael To: Carmel McDougall Dear Carmel, sorry, I've overlooked your earlier request. Yes, you can use my image, thanks for asking twice. Good luck with your work, Cheers, Michael

Dear Michael,

I’m just checking to see if you received the email before, that I sent just before Christmas (may have been a bad me!). Please let me know whether you are happy for me to reuse the image.

Thanks very much,

Carmel

From: Carmel McDougall > Date: Wednesday, 21 December 2016 at 10:26 AM To: "[email protected] " > Subject: Permission to re-use image

Dear Michael,

I’m Carmel, a colleague of Andi Wanninger and Tim Wollesen. I’m currently wring a review of molluscan biomineralisaon, and am hoping to reuse some images from a ﬁgure in the Mollusca chapter of ‘Evoluonary developmental biology of invertebrates’. The image that Andi and Tim used of a monoplacophoran was originally taken by you. I was wondering if you would give permission for me to use it also? A dra of the ﬁgure is aached. I will be seeking permission from the publishers of the book as well.

Also, congratulaons on your Monoplacophoran mitochondrial genomes paper – it’s nice to see some data coming out!

Regards,

Carmel

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Questions? [email protected] or +1-855-239-3415 (toll free in the US) or +1-978-646-2777.

Subject: Re: Mollusc pictures Date: Monday, 19 December 2016 at 8:36:58 PM Australian Eastern Standard Time From: Tim Wollesen To: Carmel McDougall CC: [email protected] Dear Carmel, great idea! Yes, no problem from my side but yes, please ask Maik ([email protected]) and especially Michael ([email protected]) for permission to use those pics. All the best from Vienna, Tim

Am 19.12.2016 01:35, schrieb Carmel McDougall: Dear Andi and Tim, I hope that both of you are going well over there – and are keeping warm! I’m not having that problem here in Brisbane, that’s for sure! I’m emailing because Bernie and I are wring a review of molluscan biomineralisaon, and I’d really love to use some images from your Mollusca chapter in ‘Evoluonary developmental biology of invertebrates’. A dra of the ﬁgure is aached. I’ll obviously have to get permission from the publisher as well, but thought I’d ask you ﬁrst! I know that a couple of images were taken by others (the solenogastre from Maik, and the monoplacophoran from Michael Schrodl). Would you recommend I contact those guys separately? Have a great Christmas if I don’t hear from you before then! All the best, Carmel

-- Tim Wollesen (PhD) University of Vienna Department of Integrave Zoology Althanstr. 14 1090 Vienna Austria Phone: 00431427776313 Email: [email protected]

Subject: RE: Request permission for reuse of ﬁgure Date: Friday, 23 December 2016 at 12:26:48 AM Australian Eastern Standard Time From: permissions To: Carmel McDougall Dear Carmel,

Thank you for your enquiry. The Journal of Cell Science was formerly known as The Quarterly Journal of Microscopical Science. I believe if you search for this title you should find what you need.

Permission is granted with no charge. The acknowledgement should state "reproduced / adapted with permission" and give the source journal name - the acknowledgement should either provide full citation details or refer to the relevant citation in the article reference list - the full citation details should include authors, journal, year, volume, issue and page citation.

Where appearing online or in other electronic media, a link should be provided to the original article (e.g. via DOI).

Development: dev.biologists.org Disease Models & Mechanisms: dmm.biologists.org Journal of Cell Science: jcs.biologists.org The Journal of Experimental Biology: jeb.biologists.org Biology Open http://bio.biologists.org/

Please let me know if you need further assistance.

Kind regards

Richard

Richard Grove International Sales Manager The Company of Biologists Ltd Bidder Building, Station Road, Histon, Cambridge, CB24 9LF, UK T: +44 (0) 1223 653 874 |[email protected] | www.biologists.com

Registered office: The Company Of Biologists Ltd, Bidder Building, Station Road, Histon, Cambridge CB24 9LF, United Kingdom, Registered in England and Wales. Company Limited by Guarantee No 514735. Registered Charity No 277992 The information contained in this message and any attachment is confidential, legally privileged and is intended for the addressee only. Any dissemination, distribution, copying, disclosure or use of this message/attachment or its contents is strictly prohibited and may be unlawful. No contract is intended or implied, unless confirmed by hard copy. If you have received this message in error, please inform the sender and delete it from your mailbox or any other storage mechanism. The Company of Biologists Ltd cannot accept liability for any statements made which are clearly the senders' own and not expressly made on behalf of The Company of Biologists Ltd or one of their agents.

Page 1 of 2 This article is protected by copyright. All rights reserved. From: Carmel McDougall [mailto:[email protected]] Sent: 20 December 2016 01:01 To: permissions Subject: Request permission for reuse of figure

Hello,

I am hoping to reuse (with modiﬁcaon) a ﬁgure from the following arcle:

Beedham G. 1958. Observaons on the mantle of the Lamellibranchia. Quart. J. Mic. Sci. 3:181- 197.

The ﬁgure I would like to reuse is Figure 1a, for academic use in a review arcle for WIRES Developmental Biology. I would cite the original source and state that the image had been modiﬁed.

I had a look for the journal at the Copyright Clearance Centre, as per the instrucons for ‘The Journal of Cell Science’, but could not ﬁnd this older tle.

Please let me know how to proceed,

Thanking you

Carmel McDougall

OXFORD UNIVERSITY PRESS LICENSE TERMS AND CONDITIONS Nov 13, 2017

This Agreement between Carmel McDougall ("You") and Oxford University Press ("Oxford University Press") consists of your license details and the terms and conditions provided by Oxford University Press and Copyright Clearance Center.

License Number 4226950344032

License date Nov 13, 2017

Licensed Content Publisher Oxford University Press

Licensed Content Publication Molecular Biology and Evolution

Licensed Content Title Co-Option and De Novo Gene Evolution Underlie Molluscan Shell Diversity

Licensed Content Author Aguilera, Felipe; McDougall, Carmel

Licensed Content Date Jan 4, 2017

Licensed Content Volume 34

Licensed Content Issue 4

Type of Use Journal

Requestor type Author of this OUP content

Pharmaceutical support or No sponsorship for this project

Format Print and electronic

Portion Figure/table

Number of figures/tables 2

Will you be translating? No

Circulation/distribution 2000

Author of this OUP article

Order reference number

Title of new article The evolution of mollusc shells

Publication the new article is WIRES Developmental Biology in

Publisher of new article Wiley

Author of new article McDougall, Carmel and Degnan, Bernard

Expected publication date of Jan 2018 new article

Estimated size of new article 8 (pages)

Requestor Location Carmel McDougall School of Biological Sciences The University of Queensland

St Lucia, QLD 4072 Australia Attn: Carmel McDougall

Publisher Tax ID GB125506730

Billing Type Invoice

Billing Address Carmel McDougall School of Biological Sciences The University of Queensland

St Lucia, Australia 4072 Attn: Carmel McDougall

Total 0.00 AUD

Terms and Conditions

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granted. Use of materials as described in a revoked license, as well as any use of the materials beyond the scope of an unrevoked license, may constitute copyright infringement and Oxford University Press reserves the right to take any and all action to protect its copyright in the materials. 9. This license is personal to you and may not be sublicensed, assigned or transferred by you to any other person without Oxford University Press’s written permission. 10. Oxford University Press reserves all rights not specifically granted in the combination of (i) the license details provided by you and accepted in the course of this licensing transaction, (ii) these terms and conditions and (iii) CCC’s Billing and Payment terms and conditions. 11. You hereby indemnify and agree to hold harmless Oxford University Press and CCC, and their respective officers, directors, employs and agents, from and against any and all claims arising out of your use of the licensed material other than as specifically authorized pursuant to this license. 12. Other Terms and Conditions: v1.4

Questions? [email protected] or +1-855-239-3415 (toll free in the US) or +1-978-646-2777.