Biomineralization in cephalopods (Mollusca) : molecular processes and evolution

Les mollusques, l’un des embranchements les plus diversifiés au sein de des métazoaires, sont reconnus pour leur capacité à élaborer une structure minéralisée : la coquille. Chez ces organismes, le processus de biominéralisation est génétiquement contrôlé et réalisé en domaine extracellulaire. Il fait intervenir une matrice organique calcifiante. Cette dernière, partiellement occluse au sein de la structure coquillière, est composée de protéines, de glycoprotéines, de lipides et de polysaccharides sécrétés par l’épithélium externe calcifiant du manteau. Cette matrice constitue la « boîte à outils moléculaires » pour la minéralisation de la coquille. Depuis sa découverte, elle a surtout fait l’objet d’études chez les bivalves et les gastéropodes, laissant dans l’ombre une autre classe de mollusques tout aussi importante, les céphalopodes.Les céphalopodes représentent une classe majeure de mollusque, dont une partie seulement des représentants actuels possède une coquille minéralisée, interne ou externe. L’histoire macroévolutive du groupe indique, depuis les formes ancestrales jusqu’aux formes les plus dérivées, une tendance générale à la réduction de la coquille, à son internalisation, voire à sa disparition complète. Si les relations de parentés entre les formes minéralisantes du clade semblent plutôt bien établies, les mécanismes moléculaires de formation coquillière restent encore très mal connus. Il est donc opportun de se demander si les représentants à coquille du clade possèdent des « boîtes à outils moléculaire » similaires pour fabriquer leurs coquilles ? Ce projet propose de répondre à cette question en explorant la biominéralisation coquillière de trois céphalopodes actuels, par l’utilisation d’approches biochimiques et protéomiques de la matrice coquillière, couplée à des analyses microstructurales.Le premier modèle étudié est la spirule Spirula spirula (Spirulidae), petit céphalopode pélagique dont le cycle de vie demeure encore très mal documenté. Les analyses de RMN à l’état solide et de FT-IR suggèrent que les polysaccharides constituent une part importante de la matrice organique coquillière. La protéomique, et les analyses in silico sur des modèles métazoaires hétérologues et sur le transcriptome récemment acquis de S. spirula révèlent de nombreux peptides ; la majeure partie d’entre eux ne correspondant à aucune protéine coquillière déjà identifiée au sein du clade des mollusques et/ou des céphalopodes. Ces observations suggèrent que la spirule possède un répertoire coquillier unique qui ne semble pas porter un signal phylogénétique.Le deuxième modèle est le céphalopode Argonauta hians (Argonautidae), à coquille externe non-homologue de celle des autres céphalopodes/mollusques puisque sécrétée ici par la première paire de bras dorsaux de l’animal. La matrice acido-soluble apparaît comme majoritaire et semble essentiellement protéique. La faible proportion de sucre est majoritairement constituée de glycosaminoglycanes sulfatés. La protéomique génère de nombreuses séquences peptidiques et identifie quelques protéines, non partagées par d’autres mollusques, ce qui suggère le recrutement d’outils moléculaires uniques chez l’argonaute pour la calcification de sa coquille.Le troisième modèle, qui fait l’objet d’une étude toujours en cours, est la seiche commune Sepia officinalis (Sepiidae). Les premiers résultats montrent la prépondérance de la matrice acido-insoluble, à signature FT-IR chitineuse. L’observation microscopique d’os de seiche et de préparations histologiques montre un contact étroit entre tissus organiques et minéralisés, et suggère une minéralisation bidirectionnelle, avec pour origine la couche interne prismatique du bouclier dorsal.Nos résultats sur les trois modèles sont discutés dans le cadre de l’évolution de la biominéralisation des céphalopodes.Molluscs constitute one of the most diversified phyla within metazoans, known for their ability to mineralize a shell. The shell biomineralization is a genetically controlled process that is performed extracellularly via a calcifying organic matrix. This latter, which remains finally occluded (at least, partially) into the shell, is a mixture of proteins, glycoproteins, lipids and polysaccharides, that are collectively secreted by the external calcifying epithelium of the mantle. Today, the shell matrix is usually considered as the ‘molecular toolbox’ for constructing the shell. Since its discovery, it has been the focus of numerous studies, but mainly on bivalves and gastropods leaving cephalopods in the shadow.Cephalopods are an important class of molluscs, from which only part of the living representatives possess a calcified shell (internal or external). The macroevolutionary history of this clade suggests – from conserved forms to the most derived ones – a general tendency to shell reduction, internalization, up to its complete disappearance. In spite of rather well established phylogenetic relationships between living shell-bearing cephalopods, molecular mechanisms responsible for the shell formation remain almost entirely unknown. Thus, one question arises: do all of these mineralizing cephalopods share a common “molecular toolbox” to elaborate their shell? This project proposes to answer this puzzling question by exploring the shell biomineralization of three living cephalopods via biochemical and proteomic approaches on their organic matrices, combined to a microstructural investigation of their shells.The first organism is the Ram’s horn squid Spirula spirula (Spirulidae), a small pelagic cephalopod whose biology and life cycle remain still poorly documented. Solid-state nuclear magnetic resonance and infra-red analyses suggest that polysaccharides constitute a major part of the organic matrix. Proteomics and in silico searches on heterologous metazoans models and on the spirule’s transcriptome revealed several peptides, many of them not matching with already known molluscan or cephalopod shell proteins. These observations suggest that the Ram’s horn squid possesses a unique set of proteins and does not contain a phylogenetic signal.The second organism,the winged argonaut Argonauta hians (Argonautidae), possesses an external shell, not homologous to that of other cephalopods/molluscs since it is secreted by the female’s first dorsal arm pair. The shell matrix contains an important quantity of acid-soluble proteinaceous matrix. Sugars, which are in low proportion,, appear mostly as sulphated glycosaminoglycans. Proteomics identified several peptide sequences that match with a number of proteins, not shared with other molluscs. This finding may suggest the recruitment of unique molecular tools for the shell mineralization.The third model, the common cuttlefish Sepia officinalis (Sepiidae), is the subject of a an ongoing research. The first results reveal the high proportion of the acid-insoluble matrix, which is predominantly chitinous. Histological investigations of fresh specimens and SEM observations of the cuttlebone microstructures plead in favour of a close contact between both organic and mineralized tissues, and suggest in addition a bi-directional process of mineralization, starting from the inner prismatic layer of the dorsal shield.Our results on the molecular / structural aspects of the biomineralization of our three models are discussed in an evolutionary perspective

Biominéralisation chez les Céphalopodes (Mollusca) : processus moléculaires et évolution

Molluscs constitute one of the most diversified phyla within metazoans, known for their ability to mineralize a shell. The shell biomineralization is a genetically controlled process that is performed extracellularly via a calcifying organic matrix. This latter, which remains finally occluded (at least, partially) into the shell, is a mixture of proteins, glycoproteins, lipids and polysaccharides, that are collectively secreted by the external calcifying epithelium of the mantle. Today, the shell matrix is usually considered as the ‘molecular toolbox’ for constructing the shell. Since its discovery, it has been the focus of numerous studies, but mainly on bivalves and gastropods leaving cephalopods in the shadow.Cephalopods are an important class of molluscs, from which only part of the living representatives possess a calcified shell (internal or external). The macroevolutionary history of this clade suggests – from conserved forms to the most derived ones – a general tendency to shell reduction, internalization, up to its complete disappearance. In spite of rather well established phylogenetic relationships between living shell-bearing cephalopods, molecular mechanisms responsible for the shell formation remain almost entirely unknown. Thus, one question arises: do all of these mineralizing cephalopods share a common “molecular toolbox” to elaborate their shell? This project proposes to answer this puzzling question by exploring the shell biomineralization of three living cephalopods via biochemical and proteomic approaches on their organic matrices, combined to a microstructural investigation of their shells.The first organism is the Ram’s horn squid Spirula spirula (Spirulidae), a small pelagic cephalopod whose biology and life cycle remain still poorly documented. Solid-state nuclear magnetic resonance and infra-red analyses suggest that polysaccharides constitute a major part of the organic matrix. Proteomics and in silico searches on heterologous metazoans models and on the spirule’s transcriptome revealed several peptides, many of them not matching with already known molluscan or cephalopod shell proteins. These observations suggest that the Ram’s horn squid possesses a unique set of proteins and does not contain a phylogenetic signal.The second organism,the winged argonaut Argonauta hians (Argonautidae), possesses an external shell, not homologous to that of other cephalopods/molluscs since it is secreted by the female’s first dorsal arm pair. The shell matrix contains an important quantity of acid-soluble proteinaceous matrix. Sugars, which are in low proportion,, appear mostly as sulphated glycosaminoglycans. Proteomics identified several peptide sequences that match with a number of proteins, not shared with other molluscs. This finding may suggest the recruitment of unique molecular tools for the shell mineralization.The third model, the common cuttlefish Sepia officinalis (Sepiidae), is the subject of a an ongoing research. The first results reveal the high proportion of the acid-insoluble matrix, which is predominantly chitinous. Histological investigations of fresh specimens and SEM observations of the cuttlebone microstructures plead in favour of a close contact between both organic and mineralized tissues, and suggest in addition a bi-directional process of mineralization, starting from the inner prismatic layer of the dorsal shield.Our results on the molecular / structural aspects of the biomineralization of our three models are discussed in an evolutionary perspective.Les mollusques, l’un des embranchements les plus diversifiés au sein de des métazoaires, sont reconnus pour leur capacité à élaborer une structure minéralisée : la coquille. Chez ces organismes, le processus de biominéralisation est génétiquement contrôlé et réalisé en domaine extracellulaire. Il fait intervenir une matrice organique calcifiante. Cette dernière, partiellement occluse au sein de la structure coquillière, est composée de protéines, de glycoprotéines, de lipides et de polysaccharides sécrétés par l’épithélium externe calcifiant du manteau. Cette matrice constitue la « boîte à outils moléculaires » pour la minéralisation de la coquille. Depuis sa découverte, elle a surtout fait l’objet d’études chez les bivalves et les gastéropodes, laissant dans l’ombre une autre classe de mollusques tout aussi importante, les céphalopodes.Les céphalopodes représentent une classe majeure de mollusque, dont une partie seulement des représentants actuels possède une coquille minéralisée, interne ou externe. L’histoire macroévolutive du groupe indique, depuis les formes ancestrales jusqu’aux formes les plus dérivées, une tendance générale à la réduction de la coquille, à son internalisation, voire à sa disparition complète. Si les relations de parentés entre les formes minéralisantes du clade semblent plutôt bien établies, les mécanismes moléculaires de formation coquillière restent encore très mal connus. Il est donc opportun de se demander si les représentants à coquille du clade possèdent des « boîtes à outils moléculaire » similaires pour fabriquer leurs coquilles ? Ce projet propose de répondre à cette question en explorant la biominéralisation coquillière de trois céphalopodes actuels, par l’utilisation d’approches biochimiques et protéomiques de la matrice coquillière, couplée à des analyses microstructurales.Le premier modèle étudié est la spirule Spirula spirula (Spirulidae), petit céphalopode pélagique dont le cycle de vie demeure encore très mal documenté. Les analyses de RMN à l’état solide et de FT-IR suggèrent que les polysaccharides constituent une part importante de la matrice organique coquillière. La protéomique, et les analyses in silico sur des modèles métazoaires hétérologues et sur le transcriptome récemment acquis de S. spirula révèlent de nombreux peptides ; la majeure partie d’entre eux ne correspondant à aucune protéine coquillière déjà identifiée au sein du clade des mollusques et/ou des céphalopodes. Ces observations suggèrent que la spirule possède un répertoire coquillier unique qui ne semble pas porter un signal phylogénétique.Le deuxième modèle est le céphalopode Argonauta hians (Argonautidae), à coquille externe non-homologue de celle des autres céphalopodes/mollusques puisque sécrétée ici par la première paire de bras dorsaux de l’animal. La matrice acido-soluble apparaît comme majoritaire et semble essentiellement protéique. La faible proportion de sucre est majoritairement constituée de glycosaminoglycanes sulfatés. La protéomique génère de nombreuses séquences peptidiques et identifie quelques protéines, non partagées par d’autres mollusques, ce qui suggère le recrutement d’outils moléculaires uniques chez l’argonaute pour la calcification de sa coquille.Le troisième modèle, qui fait l’objet d’une étude toujours en cours, est la seiche commune Sepia officinalis (Sepiidae). Les premiers résultats montrent la prépondérance de la matrice acido-insoluble, à signature FT-IR chitineuse. L’observation microscopique d’os de seiche et de préparations histologiques montre un contact étroit entre tissus organiques et minéralisés, et suggère une minéralisation bidirectionnelle, avec pour origine la couche interne prismatique du bouclier dorsal.Nos résultats sur les trois modèles sont discutés dans le cadre de l’évolution de la biominéralisation des céphalopodes

Phylogeny of some Devonian trilobites and consequences for the systematics of Austerops (Phacopidae).

16 pagesInternational audienceA comprehensive phylogenetic hypothesis for Devonian phacopid trilobites of the genus Austerops has not previously been proposed. We carried out a cladistic analysis of the 13 species and subspecies assigned to Austerops, based on a data matrix of 63 characters. Two species of the morphologically very similar genus Chotecops, C. auspex and C. hoseri, and seven other close relatives (Reedops cephalotes hamlagdadianus, Boeckops stelcki, Morocops granulops, Paciphacops logani, Phacops latifrons, Phacops araw and Pedinopariops (Hypsipariops) vagabundus) were also included in the analysis in order to test their relationship with species of Austerops. Parsimony analyses using a heuristic method, with Calyptaulax callirachis and C. glabella as outgroup taxa, produced two most parsimonious trees of 341 steps. These trees are partly consistent with trees obtained from additional analyses performed with modified data sets (deletion of homoplastic characters, multistate characters, continuous characters carved into 5% increments, thoracic and pygidial characters, or taxa with numerous unknown characters). Results suggest that Austerops sp. B and A.? sp. D are close to Chotecops hoseri and C. auspex, and that these species constitute a monophyletic group. Austerops Austerops menchikoffi, A. speculator and A. punctatus are also a monophyletic group and reasonably constitute a sister group of that formed by the rest of Austerops and Chotecops sensu lato, while A. hottonensis seems phylogenetically distant from other representatives of Austerops. Chotecops including C. hoseri and C. auspex together with Austerops sp. B and A.? sp. D seem derived from A. legrandi. The relationship between Austerops and Chotecops remains partly unresolved but it seems likely that their recognition as separate taxa results in paraphyletic groups. The phylogenetic analysis suggests that the currently poorly known Austerops sp. B and A.? sp. D should be reassigned to Chotecops

Trilobites from the Red Fauna (latest Emsian, Devonian) of Hamar Laghdad, Morocco and their biodiversity

Trilobites are widespread in Early Devonian deposits of north Gondwana; some of the most emblematic ones were collected from the famous latest Emsian (Early Devonian) mudmound locality Hamar Laghdad in south-eastern Morocco. This locality is famous for its trilobites, especially for the conspicuous red-coloured remains of phacopid trilobites with often greenish eyes. Here, we present a taxonomic revision of the previously described trilobites from the so-called Red Fauna of Hamar Laghdad. We introduce the new taxa Harpes hamarlaghdadensis n. sp. and Morocops davidbrutoni n. sp. Phacopids dominate the trilobite assemblage from the Red Cliff at Hamar Laghdad in terms of the number of specimens (represented by Morocops) as a whole. Additionally, we focus on the description of trilobite diversity of this interval compared to Algerian assemblages

Supp. Table 1

Results of angular comparisons between species PLS1 and the common PLS for each species (sample size >20), on the cephalon (top of the table) and the pygidium (bottom of the table)

Supplementary Information - cephala procrusted landmarks

Procrusted landmarks from the cephalon of each individual

Supplementary Information - pygidia procrusted landmarks

Procrusted landmarks from the pygidium of each individual

Phylogeny of some Devonian trilobites and consequences for the systematics of <i>Austerops</i> (Phacopidae)

<p>A comprehensive phylogenetic hypothesis for Devonian phacopid trilobites of the genus <i>Austerops</i> has not previously been proposed. We carried out a cladistic analysis of the 13 species and subspecies assigned to <i>Austerops</i>, based on a data matrix of 63 characters. Two species of the morphologically very similar genus <i>Chotecops</i>, <i>C. auspex</i> and <i>C. hoseri</i>, and seven other close relatives (<i>Reedops cephalotes hamlagdadianus</i>, <i>Boeckops stelcki</i>, <i>Morocops granulops</i>, <i>Paciphacops logani</i>, <i>Phacops latifrons</i>, <i>Phacops araw</i> and <i>Pedinopariops</i> (<i>Hypsipariops</i>) <i>vagabundus</i>) were also included in the analysis in order to test their relationship with species of <i>Austerops</i>. Parsimony analyses using a heuristic method, with <i>Calyptaulax callirachis</i> and <i>C. glabella</i> as outgroup taxa, produced two most parsimonious trees of 341 steps. These trees are partly consistent with trees obtained from additional analyses performed with modified data sets (deletion of homoplastic characters, multistate characters, continuous characters carved into 5% increments, thoracic and pygidial characters, or taxa with numerous unknown characters). Results suggest that <i>Austerops</i> sp. B and <i>A.</i>? sp. D are close to <i>Chotecops hoseri</i> and <i>C. auspex</i>, and that these species constitute a monophyletic group. <i>Austerops Austerops menchikoffi</i>, <i>A. speculator</i> and <i>A. punctatus</i> are also a monophyletic group and reasonably constitute a sister group of that formed by the rest of <i>Austerops</i> and <i>Chotecops</i> sensu lato, while <i>A. hottonensis</i> seems phylogenetically distant from other representatives of <i>Austerops</i>. <i>Chotecops</i> including <i>C. hoseri</i> and <i>C. auspex</i> together with <i>Austerops</i> sp. B and <i>A</i>.? sp. D seem derived from <i>A. legrandi</i>. The relationship between <i>Austerops</i> and <i>Chotecops</i> remains partly unresolved but it seems likely that their recognition as separate taxa results in paraphyletic groups. The phylogenetic analysis suggests that the currently poorly known <i>Austerops</i> sp. B and <i>A</i>.? sp. D should be reassigned to <i>Chotecops</i>.</p

Data from: Functional integration for enrolment constrains evolutionary variation of phacopid trilobites despite developmental modularity

Modularity and integration are variational properties expressed at various levels of the biological hierarchy. Mismatches among these levels, for example developmental modules that are integrated in a functional unit, could be informative of how evolutionary processes and trade‐offs have shaped organismal morphologies as well as clade diversification. In the present study, we explored the full, integrated and modular spaces of two developmental modules in phacopid trilobites, the cephalon and the pygidium, and highlight some differences among them. Such contrasts reveal firstly that evolutionary processes operating in the modular spaces are stronger in the cephalon, probably due to a complex regime of selection related to the numerous functions ensured by this module. Secondly, we demonstrate that the same pattern of covariation is shared among species, which also differentiate along this common functional integration. This common pattern might be the result of stabilizing selection acting on the enrolment and implying a coordinate variation between the cephalon and the pygidium in a certain direction of the morphospace. Finally, we noticed that Austerops legrandi differs slightly from other species in that its integration is partly restructured in the way the two modules interact. Such a divergence can result from the involvement of the cephalon in several vital functions that may have constrained the response of the features involved in enrolment and reorganized the covariation of the pygidium with the cephalon. Therefore, it is possible that important evolutionary trade‐offs between enrolment and other functions on the cephalon might have partly shaped the diversification of trilobites

A Nature’s Curiosity: The Argonaut “Shell” and Its Organic Content

33 pagesInternational audienceMolluscs are known for their ability to produce a calcified shell resulting from a genetically controlled and matrix-mediated process, performed extracellularly. The occluded organic matrix consists of a complex mixture of proteins, glycoproteins and polysaccharides that are in most cases secreted by the mantle epithelium. To our knowledge, the model studied here-the argonaut, also called paper nautilus-represents the single mollusc example where this general scheme is not valid: the shell of this cephalopod is indeed formed by its first dorsal arms pair and it functions as an eggcase, secreted by females only; furthermore, this coiled structure is fully calcitic and the organization of its layered microstructures is unique. Thus, the argonautid shell appears as an apomorphy of this restricted family, not homologous to other cephalopod shells. In the present study, we investigated the physical and biochemical properties of the shell of Argonauta hians, the winged argonaut. We show that the shell matrix contains unusual proportions of soluble and insoluble components, and that it is mostly proteinaceous, with a low proportion of sugars that appear to be mostly sulfated glycosaminoglycans. Proteomics performed on different shell fractions generated several peptide sequences and identified a number of protein hits, not shared with other molluscan shell matrices. This may suggest the recruitment of unique molecular tools for mineralizing the argonaut's shell, a finding that has some implications on the evolution of cephalopod shell matrices