Biomineralization : its role in the Cambrian diversification of life
Symposium organized by André Adoutte, Andrew H. Knoll, and Peter Westbroek, May 5 to 11, 1997
André Adoutte (Université de Paris-Sud, Orsay), Guillaume Balavoine (University of Cambridge, Great Britain), Angela Belcher (University of California Santa Barbara), Stefan Bengtson (Swedish Museum of Natural History, Stockholm), Jo (van den) Biggelaar (Utrecht University, the Netherlands), Robert Bourrouilh (Université de Bordeaux-I), Simon Morris Conway (University of Cambridge, Great Britain), Paul Corsjtens (Leiden University, the Netherlands), Mikhaïl Fedonkin (Russian Academy of Sciences, Moscow), Andrew Knoll (Harvard University), Frédéric Marin (Leiden Institute of Chemistry, Gorlaeus Laboratory, the Netherlands), Sylvie Мazan (Université de Paris-Sud, Orsay), Alessandro Minеlli (University of Padova), Claus Nielsen (University of Copenhagen), Armand (de) Ricqlès (Collège de France), Adolf Seilacher (Institute of Geology and Palaeontology), Kenneth Simkiss (University of Reading), Peter Westbroek (Leiden University, the Netherlands).
Тhe Cambrian period, beginning ca. 544 million years ago, is marked by a remarkable diversification of skeleton-for-mining animals in the oceans. This spectacular proliferation of hard parts does not coincide with the first appearance of animals in the fossil record, nor does it provide the earliest evidence of mineralized skeletons. There is no doubt, nonetheless, that the evolution of highly organized shells, tests, bones and sclerites in at least eight phyla opened new functional and ecological vistas, at the same time introducing eukaryotic organisms as principal agents in the global cycles of carbonate and silica.
Many aspects of the Cambrian event remain controversial. Does the appearance of diverse body plans in latest Proterozoic and Cambrian rocks record the origin and rapid diversification of animal phyla, or did the principal clades diverge during an earlier interval of animal prehistory ? Was animal diversification driven principally by intrinsic biological factors, or was it facili- tated as well by extrinsic environmental events ? What role did biomineralization play in Cambrian diversification ? Did the principle clades acquire calcification independently, or was this feature already present in the common ancestor ?
Understanding Cambrian evolution requires multidisciplinary efforts in winch scientists with expertises as disparate as molecular phylogeny and sedimentary geochemistry work together. Through the generosity of the Fondation des Treilles, seventeen scientists representing ten countries and half a dozen disciplines gathered in Tourtour May 5-10, 1997, for a stimulating week of mutual education, discussion, and collaboration — all devoted to forging a new understanding of events more than 500 million years ago that gave rise to the modern world.
The acquisition, almost simultaneously in many animal stocks, of highly organized calсifiеd skeletons, is one obvious aspect of the Cambrian event. How could different, hitherto soft-bodied animais independently evolve skeletons ? One might argue that it is easy to produce minerals. Al that animals had to do was master the simple physicochemical rules of crys- tallization. But the more we learn about biomineralization, the more we are impressed with the complexity of this process. The building blocks — mineral ions and a complex mixture of macromolecules — are secreted by living tissue into the еxtra-cellular space, where they assemble into a densely packed three-dimensional filigree of micrometer-sized crystals and organic matrix. In bone formation, the correct expression of the matrix macromolecules and the recruitment and differentiation of the responsible cells is known to be organized in extensive cascades of gene regulation. These cascades are directed by a limited number of signalling proteins, of which the “bone morphogenetic proteins” (BMPs) are important representatives.
If biomineralization is such a complicated process, how are we to envisage the ease with which it was switched on in separate Cambrian animal stocks ? During the meeting, a great deal of attention was devoted to this problem. Peter Westbroek discussed the hypothesis that the calcifying apparatus is made up of components that were present prior to the Cambrian, but were adapted to functions unrelated to calcification. According to this pre-adaptation hypothesis, the ancient mechanisms merely had to be orchestrated in order for skeletal synthesis to proceed.
Frédéric Marin was the first to provide evidence for such a pre-adaptation. His argument was based on geological evidence that the level of oversaturation with respect to calcium carbonate was higher in the Neoproterozoic ocean than today. Marine organisms had to develop anti-calcifying mechanisms to inhibit spontaneous encrustation of their soft tissues with calcium salts. Likely candidates for these anti-calcifying materials were muci that are profusely secreted into the icean water by many animal tissues. Marin was able to show that muci of extant mollusks have retained their anti-calcifying macromolecules, whereas homologues of these materials are important constituents of the shell matrix. It appears then that inhibition of spontaneous precipitation is a major prerequisite for organized calcification, which may be triggered by controlled removal of the inhibitors in dedicated microenvironments. It is precisely this event which may have occurred in independent stocks during the Cambrian explosion.
Ken Simkiss pointed to the secretion of calcium out of the eukaryotic cytosol into organelles such as the Golgi apparatus and mitochondria as well as into intercellular space. This pumping would be another pre-adaptation to organized calcification. Originally required to permit phosphate-based energy metabolism, it later provided the driving force for calcification by providing oversaturated fluids inside the living tissues. Recent experiments where the gene for an acidic rnucopoly-saccharide was incapacitated in mice indicate the importance of anticalcification in these internal environments, as the target mice were hypercalcified. Calcification in the form of membrane-bound amorphous granules of calcium carbonate and phosphate is allowed in many extant metazoa however, and this mechanism may be considered as a further pre-adaptation to skeleton formation. The granules have a distinct fonction in detoxification both with respect to calcium and to heavy metals that are readily enclosed in the poorly organized packing. In some instances the granules were found to be intermediates in skeleton formation. They may be very ancient indeed, as they assume basic metabolic functions. Unfortunately, no trace of them has been found in the fossil record.
Angela Belcher described the structure of flat pearls, the formation of which was induced on glass slides inserted between the shell and the mantle of the abalone. These experiments showed that the mantle undergoes a distinct sequence in its secretory regime, mimicking the structure of the shell itself : first a layer of organic material is deposited, then calcite, then aragonitic nacre. Not inly glass, but also inorganic calcite aid aragonite forced the tissues to undergo the full sequence even if they had reached the final, nacre-secreting stage prior to experimentation. Only aragonitic biominerals were capable to induce nacre production right from the start, suppressing the expression of the previous stages. Belcher concluded that spe- cific signalling molecules incorporated within the biominerals induce the adjacent mantle tissue to assume a specific secretory regime.
Peter Westbroek discussed experiments by Evelyn Lopez, Сhristian Milet and co-workers of the Мuséum d’histoire naturelle in Paris, who showed a remarkable degree of compatibility between nacre and bone in the presence of human osteoblasts. Nacre was able to induce the secretion of bone from the ostеоblasts both in vitro and in vivo. Furthermore, the nacre was covered with new aragonite in the in vitro experiments. This work further confirmed the programming of secretory regimes in calcifying cels by specific signalling molecules contained in the biominerals. It also revealed a deep homology between biominerals that are widely separated both mineralogically and in their evolutionary position.
Note that all these results are in agreement with the pre-adaptation hypothesis. It is ikely then that, in spite of the differences between the various metazoan biominerals, they aIl have in common a number of basic traits derived from thеir common Neoproterozoic ancestors. Could we find similar, and maybe even homologous traits in non-metazoan stocks, such as the coccolithophore algae ? The common ancestors to these organisms and the metazoans appear to have lived around one billion years ago, and although recognizable coccolithophore skeletons (‘coccoliths’) do not appear before the Triassic, calсification in the form of loosely bound amalgamates of calcite rhombohedrons (‘holococcoliths’) may be much older. Corstjens discussed calcification in the coccolithophore Emiliania huxleyi, and showed that in this organism both calcification and anticalcification occur. These functions are mediated by a single, but highlу complex polysaccharide. Furthermore, sudden transitions between calcifying to non-calcifying cells, often occurring simultaneously in separate cultures, may be indicative of signalling molecules similar to the ones found in nacre and bone. Emiliania appears to be a promising outgroup for the study of calcification relationships.
Towards the end of the meeting, Simkiss proposed that one of the reasons why highlу organized calcification was held back for so long during Earth history was precisely the need to inhibit non-functional precipitation. The subtle feedback required for inducing the anti-calcifying system to adopt its regulatory function in organized calcification had to be fully developed before calcification could occur. This feedback may be related to the signalling system observed in the osteoblasts, the mantle cells, nacre and bone.
If comparative biology provides the basis for understanding the physiology and biochemistry of biomineralization, paleontology provides a historical record of early animal diversification, set in the context of Proterozoic-Cambrian environmental change. Andrew Knoll discussed the Proterozoic record of lifе and environments, stressing three points:
(1) The animal clade probably diverged from other principal groups of eukaryotic organisms 1200-1000 million years ago. This conclusion is based on three arguments. First, molecular phylogenies imply that the ancestors of animals diverged from the ancestors of various algal groups, fungi, ciliates, and a few others during a relatively brief burst of diversification. Second, unambiguous fossils of red algae, green algae, and photosynthetic stramenopiles occur in rocks older than 900 million years, placing a minimum contraint on the timing of this event.
(2) Sedimentological evidence from Neoproterozoic carbonates indicates that the oceans in which the first animals evolved were characterized by carbonate oversaturation levels much higher than those seen today. The abundant occurrence of carbonate sediments precipitated directly from seawater lends credence to the hypothesis that early animals would have required surface molecules capable of inhibiting spontaneous caldum carbonate nucleation and encrustation (anti-calcifica-tion).
(3) Isotopic geochemical data support the hypothesis that atmospheric oxygen concentrations increased rnarkedly just before the Ediacaran radiation of macroscopic animais, providing an environmental trigger for the evolution of large size and functional complexity in the Metazoa.
Mikhaïl Fedonkin reviewed the record of Ediacaran fossils, demonstrating the local density and diversity of unskeletonized animals during the thirty million years immediately before the start of the Cambrian Period. Diploblastic organisms appear to dominate Ediacaran faunas, but bilaterian animals are represented both by tracks and trails and by body fossils recently reinterpreted by Fedonkin and colleagues. The phylogenetic relationships of many Ediacaran organisms remain problematic, and it is possible that at least some forms represent early diverging clades now extinct. Andrew Knoll noted that in addition to the well known Ediacaran faunas, limestones deposited on latest Proterozoic shelves and platforms contain abundant and diverse — as well as problernatic — skeletons.
Simon Conway Morris continued the historical narrative into the Cambrian, demonstrating the rernarkable developmental, functional, and behavioral sophistication of animals that lived only 20 million years alter the acme of the Ediacarans. Cambrian faunas contain problematic remains to be sure, but many and perhaps most early Cambrian fossils can be interpreted as stem or crown taxa within extant phyla. Recent research on Cambrian fossils has yielded a few surprises with respect to the early history of biomineralization. For example, one group of phosphatic sclerites, long known from isolated elements can now be tied to priapulid worms. Conway Morris and Adoutte were both critical of recent molecular click data which suggest that animals not only diverged from other eukaryotes but diversified into several clades prior to 1000 million years ago. However, Adolf Seilaсher, taking a more empirical approach to unlocking the earliest record of animal evolution, argued that trace fossils in sandstones from India were made by coelomate animals more than 1000 million years ago. Armand de Ricqlès contributed a critical discussion of evidence for skeletonization in Paleozoic vertebrates.
Stefan, Bengtson capped the paleontological discussion by outlining the various factors that mаy have facilitated the evolution of skeletons in diverse animals. Еarly skeletons are mineralogically diverse, discouraging the simplistic view that changes in the chemistry of seawater drive skeleton evolution. Whatever other forces may have been in operation, the evolution of macrophagous predation was undoubtedly important. Following an economic fine of reasoning advanced by Ken Simkiss, Bengtson argued that natural selection would favor skeleton-forming organisms in cases where the physiological cost of construction was low relative to the reproductive benefits, improved locomotion and/or protection from predators. Bengtson also opened up a new avenue of paleontological research on early animal evolution, showing that early stages in development can be preserved by early diagenetic phosphorite emplacement. Finally, Robert Bourrouilh recapitulated the continental distribution around the Precambrian-Cambrian boundary, emphasizing the expansion of the shelves at that juncture. This might have created new opportunities for animal diversification.
Phylogeny and the evolution of ontogeny
The Cambrian explosion, as documented in the fossil record, mises two questions for the molecular evolutionist :
1) are presently availablе molecular phylogenies compatible with the notion of a ‘rapid’ diversification of the metazoan phyla?
2) how can this possibly rapid diversification in body plans be accounted for in terms of the modern understanding of developmental genetics ? These questions were addressed by five speakers at the Treilles meeting.
André Adoutte first summarized the present status of the molecular phylogeny of metazoans. Не еmphasizеd the fact that several phyla which were long held by traditional zoology to occupy an ‘intermediate’ position between diploblastic grade organisms (i.e. Cnidaria) and triploblastic coelomates, such as Platyhelminthes and varions Aschelminthes have now been rai-sed to much a higher position by molecular data, well within the protostomes. This drasticallу alters our views on the progressive nature of metazoan evolution and leaves us with a picture that he summarized, together with Guillaume Balavoine, as a triple explosion. Triploblastic metazoans now appear to be subdivided into three major clades. Two of them belong to the protostomes, the Lophotrochozoa on the one hand and the arthropods on the other. The last clade corresponds to the deuterostomes. The Lophotrochozoa as defined by Halanych et al., group the annelids, molluscs, platyhelmiths, nemerteans, etc. Quite recently, the arthropod clade was shown by Aguinaldo et al. to include nematodes as well as all the other cuticle bearing phyla, and it was therefore named Ecdysozoa. The important point is that resolution is difficult at the base of each of these three clades, suggesting a triple radiation sometime in the late Precambrian, from three stem lineages that might have diverged from each other earlier and remained poorly diversified, which would account for the difficulty in identifying them in the fossil record.
Before the meeting the molecular phylogenetical results appeared to be at odds with the views to be presented by Claus Nielsen, a specialist of animal evolution using morpho-anatomical and developmental characters. Nielsen was critical of the molecular phylogenies for their lack of consistency and for the fact that several undoubtedly monophyletic groups such as the molluscs often do flot emerge as such in molecular trees. The views were essentially reconciled at the meeting, however, on two grounds. Firstly, lack of monophyly, once artefacts in molecular trees are eliminated, can in fact be taken as evidence for an evolutionary radiation. Secondly, several of the phyla raised higher up in the tree through molecular phylogeny had indeed been suggested to occupy such a position by Nielsen through a thоughtful analysis of their anatomical and developmental characters.
In summary, molecular phylogeny as it presently stands is compatible both with traditional zoology and to a large extent with paleontology if one considers that the ‘Cambrian Explosion’ is not a single event but a triple one, preceeded by earlier deep splits. Molecular phylogeny, however, cannot date these splits accurately since this involves severe extrapolations from the well-dated vertebrate fossils to much earlier periods and several unrealistic assumptions on the linearity of the clock. It is interesting to note in addition, that the deep initial split in the metazoan stem lineages is fullу compatible with the independent emergence of calcification in different metazoan stocks.
Sylvie Mazan addressed the question of the evolutionary conservation of key developmental gene networks in animal evolution. She described the increasing number of homologous gene networks that have been found to pattern the early embryo in both Drosophila and the mouse. There now is good evidence that the antero-posterior axis (hox genes), the cephalic region (оtd/оtx and ems/emx genes), the dorso-ventral axis (sog-chordion/dpp-ВМР4) and the sensory organs, especially the eyes (eyeless/pax 6) are under the control of undoubdtedly homologous genes. It is therefore tempting to assume that the common ancestor to all triploblasts already used these gene networks for the same purposes and hence that it was quite an elaborate organism.
Further elaborating on this point, Guillaume Balavoine discussed to what extent the Hox gene complex, now known to have been present in the common ancestor of all triploblasts in the form of 6-7 genes, might have been involved in defining segmentation in this hypothetical ancestor. Whatever the case mау be, he emphasized a potential role of this complex in patterning the neuromeres.
Taking a rather different view, Jo van den Biggelaar stressed the différences in the basic process of mesoderm determination between protostomes and deuterostomes. On the basis of careful comparative observations of early development in molluscs, annelids and platyhelminths, he concluded that the process of mesoderm determination, although homologous in these groups was entirely different in the deuterostomes. He concluded that the mesoderm probably evolved twice independently. In a similar vein, Claus Nielsen, updating his Trochea theory, also reached the conclusion that bilateral symmetry originated twice independenly in the metazoans.
Finally, concentrating on the arthropods, Alessandro Minеlli discussed the process of `arthropodisation’ and suggested an interesting scenario starting from an unsegmented aschelminthgrade worm. The two key-points in his discussion were a reinterpretation of the origin of segmental appendages and the establishment of a relationship between the complexity of these appendages with that of the post-embryonic developmental schedule.
On the whole, then, although complete and unifying answers are not yet at hand, it was a very exciting experience to witness discussions among representatives of such different scientific denominations. То the best of our knowledge, it was for the first time that the Cambrian explosion was discussed from so many different angles. In this ongoing discussion, the paleontologists set the stage both in terms of the time frame and of the actual shape of Precambrian and early Cambrian organisms. In addition, they provide highlу relevant information on the environmental parameters prevailing in these distant periods. The evolutionary developmental biologists, on the other hand, have rapidly progressed in the inventory of the common gene pool and of developmental processes shared by all triploblasts, thus providing an increasingly sharper image of the genetic ‘toolbox’ that was available in the late Precambrian. This now allows us to make some specific hypotheses as to the internal parameters that may have allowed the rapid diversification of the animal phyla, rendering the whole episode less mysterious. It is through systematic confrontation of such extrinsic versus intrinsic factors that we can progress in understanding this key event in the history of lifе. This discussion now brings to the fore biomineralisation as a key parameter in understanding metazoan evolution. We are eagerly awaiting new information on the molecular details of this process. The important role presently played by metazoan calcification in the carbon cycle and in global climate regulation further points to the importance of this theme.
André Adoutte – Can the Cambrian explosion be seen through the molecular phylogeny ?
Guillaume Balavoine – Segmentation and the origin of the Hox cluster
Angela Belcher – Proteins controlling phase, CaCo3 crystal orientation and morphology in the process of abalone shell formation
Stefan Bengtson – The evolutionary significance of skeletons
Jo van den Вiggelaar – Ontogeny and phylogeny of protosomian animals
Robert Bourrouilh – Lifе development, place-techtonics and continental fits
Simon Conway Morris – Early metazoan phylogeny
Paul Corstjens – Coccolithoporid calcification : intracellular coccolith production by the unicellular algae
Мikhaïl Fedonkin – Animal life in proterozoic : long way towards Cambrian diversification
Andrew Knoll – The physical aid biological context of early animal evolution
Frédéric Marin – Genetic investigations on the caleifying system – Genetic homologies between mucins and skeletal matrices
Sylvie Mazan – The genetic control of pattern formation in triploblastic meta¬zoans : towards the common ancestor ?
Alessandro Minelli – Segments, body regions, or skeleton : what first, in the history of arthropods?
Claus Nielsen – Early metazoan phylogeny : a morphological approach
Armand de Ricglès – Hard tissues among early vertebrates
Adolf Seilacher – The ecologic side of the Cambrian revolution
Kenneth Simkiss – Membrane/mineral interactions in the formation of intracellular deposits – Amorphous minerals as storage and detoxification systems preadapted to function as initiators of biomineralization
Peter Westbroek – Appearance of biomineralization in the Cambrian : the preadaptation hypothesis