Announcement and Invitation   


The development and the evolution of the stomodeum and neurohypophysis in ascidians: insight into the origin of the olfactory and adenohypophysial placodes of vertebrates
Alberto Agnoletto, Lucia Manni,  Giovanna Zaniolo, Paolo Burighel
Dipartimento di Biologia, Universita di Padova, via U. Bassi 58/B,
I-35121 Padova, Italy

The ascidian larva has a central nervous system sharing basic characteristics with vertebrates. At metamorphosis, this nervous system regresses while the adult’s cerebral ganglion and associated neural gland form starting from two ectodermal structures, the neurohypophysial duct, derived from the embryonic neural tube, and the stomodeum, i.e. the rudiment of the mouth. For embryonic origin, anatomical position and cell derivatives these two ectodermal structures were proposed to contain cell populations homologous to the vertebrate olfactory/adenohypophysial placodes and hypothalamus. We have analysed embryos and metamorphosing larvae of Ciona intestinalis documenting the contribution of the neurohypophysial duct and the stomodeum to the formation of adult structures: the neurohypophysial duct differentiates into the neural gland rudiment and its dorsal wall proliferate neuroblasts, which migrate and converge to build up the cerebral ganglion. Anteriorly, the duct participates together with the stomodeum to formation of the aperture of the neural gland into the mouth. We hypothesise that the vertebrate pituitary gland did not evolve from any of the structures of ascidian larvae, but that the ascidian stomodeum/neurohypophysial duct complex shares a common origin and possesses cell populations homologous to components of the vertebrate olfactory/adenohypophysial placodes and hypothalamus.


Conodonts and the origin of the vertebrate skeleton
Richard J. Aldridge and Philip C. J. Donoghue
Department of Geology, University of Leicester, Leicester LE1 7RH, UK
Department of Earth Sciences, University of Bristol, Wills Memorial Building, Queens Road, Bristol BS8 1RJ, UK

Phylogenetic analyses based on existing knowledge of euconodont soft tissues consistently place conodonts as more derived than lampreys and, thereby, within the crown group vertebrates.  It has been argued that the phosphatic feeding apparatus of conodonts represents an independent evolutionary experiment in skeletonization from that of the remainder of the vertebrates, but, given that conodonts resolve as the sister group to other skeletonized vertebrates, this contention is not parsimonious.  There are very close histological similarities between the crown tissue of euconodont elements and enamel, and between euconodont basal bodies and dentine, and it is apparent that euconodont elements grew in an identical manner to the enamel-dentine system in other vertebrate taxa.  It has recently been demonstrated that this condition holds for the biomineralized elements of the most primitive of euconodonts.  Thus, palaeontological evidence indicates that the earliest condition of the mineralized vertebrate skeleton is as odontodes of dentine plus enamel or of dentine alone in the oro/pharyngeal cavities of euconodonts or of their putative ancestors, the paraconodonts.  Functional considerations demonstrate that the conodont feeding array was adapted for predation/scavenging, so the vertebrate skeleton was first employed for offensive, not defensive, purposes.

Apoptosis and recognition of senescent cells in a compound ascidian:
a comparison with Vertebrates
Francesca Cima, Stefano Tiozzo, Adams Menin, Riccardo Zacchia, Sara Zanarotti, Giovanna Zaniolo, Paolo Burighel, Elena Fortunato, Giuseppe Basso, Loriano Ballarin
Department of Biology and Department of Pediatrics, University of Padova,
via U. Bassi 58/B, I-35121 Padova, Italy

Programmed cell death by apoptosis is a fundamental process in the develop-ment and tissue homeostasis of Vertebrates. Although widespread among meta-zoans, few data are available on the role of apoptosis in invertebrates.
     Ascidians are tunicates belonging to the phylum Chordata and, therefore, closely related to Vertebrates. We studied the expression of markers related to apoptosis and recognition of senescent cells in this group of organisms.
     Adult zooids of the ascidian Botryllus schlosseri are cyclically resorbed and replaced by zooids of new blastogenic generations. Take-over lasts about 20 h and is characterised by massive apoptosis in zooid tissues. During this period, a significant increase in the amount of phagocytes engulfed with dying cells or cell debris, as compared to mid-cycle stages, occurs. In addition, a significant higher frequency of cells expressing Bax, Fas and FasL, and of Bcl-2-negative cells is observed. These observations are consistent with the occurrence of both mitochondria-independent and mitochondria-dependent pathways of cell death induction.

     The amount of cells expressing phosphatidylserine and anti-CD36 on their surface increased as colonies approach take-over. As both these markers are involved in the recognition of effete cells by phagocytes, results strongly suggest that fundamental recognition mechanisms are well conserved throughout Chordate evolution.

A cladogram for the Deuterostomia based on molecular-biological and fossil evidence
Patricio Dominguez
Dept. Paleontología, Fac. CC. Geologicas, Universidad Complutense de Madrid, Spain
Richard Jefferies
The Natural History Museum, London, UK

On molecular-biological evidence from extant animals,  the basic cladogram for deuterostomes reads: (((hemichordates + echinoderms) Xenoturbella) chordates). Moreover there is now molecular evidence that, if the likely effects of long-distance attraction are discounted, the basic cladogram for chordates should read:
(acraniates (tunicates + vertebrates)). Here we accept these results and attempt to place the most primitive deuterostome fossils in the stem groups implied by the cladograms.  In doing so, we  find that a stereomic calcitic skeleton of echinoderm type, with each plate a single crystal of calcite, is not a hallmark of echinoderms as commonly believed. Rather, in the latest common ancestor of all extant deuterostomes it would have existed  but  has been lost at least six times among the   descendants of that animal. In this light, a calcitic skeleton in a carpoid is a  symplesiomorphy - it  does not indicate an echinoderm but merely a deuterostome. We further conclude that the carpoids known as ctenocystoids are stem-group hemichordates, mainly because one  recently discovered ctenocystoid must on  functional-morphological grounds have been a burrower comparable to an extant enteropneust, and in particular to the newly described deep-sea enteropneust Torquarator. The carpoids called Cincta, which probably gave rise to the ctenocystoids, are also likely members of the hemichordate stem group. The echinoderm stem group is probably represented by the basically triradial fossils known as helicoplacoids and Tribrachidium. Their triradiality presumably evolved, in more crownward parts of the echinoderm stem group, into the standard quinqueradiality of extant echinoderms. The most primitive  (most "rootward") part of the chordate stem group was probably included in the solute carpoids, while more crownward members of the chordate stem group were the cornutes. The most primitive members of the chordate crown group were the mitrates. The most primitive known solute was attached by the end of its tail, whereas more crownward stem-group chordates, and the mitrates also, locomoted rearwards tail first.

Gene duplication, extinction, and vertebrate evolution
Philip C. J. Donoghue and Mark A. Purnell
Department of Earth Sciences, University of Bristol, Queens Road, Bristol BS8 1RJ, United Kingdom
Department of Geology, University of Leicester, Leicester LE1 7RH, UK,  

Vertebrate evolution has been punctuated by three episodes of widespread gene or genome duplication, each of which coincides with an apparent burst of character acquisition and increase in phenotypic complexity. The three duplications have been linked with the origin of vertebrates, gnathostomes and teleosts, respectively, three evolutionary events taken by many people to mark the establishment of new bodyplans within vertebrates. Despite what seems to be clear coincidence between the timing of gen(om)e duplications and evolutionary events, this does not necessarily imply causality. Furthermore the pattern of congruence is based on a dataset that encompasses living taxa alone. When palaeontological data are incorporated into the phylogenetic milieu, apparent bursts of character acquisition and diversity are diminished. Thus, while there may be grounds for ignoring extinct taxa in attempts to unravel genomic evolution, analyses of the phenotypic consequences should be based on total evidence, integrating data from both living and fossil taxa. From the perspective of total evidence, the association of phenotypic and genomic evolution is far from clear and it quite possible that the two are not associated, or that the retention of paralogues is an effect arising from – rather than the cause of – phenotypic complexity.

Earliest chordates in the fossil record
Jerzy Dzik
Instytut Paleobiologii PAN, Twarda 51/55, 00-818 Warszawa, Poland

The most ancient members of a clade are of importance because fossils closer in time to the ancestor are likely to be closer to it also morphologically and may reveal anatomical characters later lost. Affinities of the geologically oldest undoubted chordates, the conodonts, are supported by their V-shaped myomeres in laterally compressed body, and possible homology of the oral apparatus elements crown tissue with enamel. Such myomeres were also reported in Metaspriggina from the Burgess Shale. Though Pikaia from the same strata is more widely accepted as an ancient chordate, its similarity to the early Cambrian worm Myoscolex suggests that it was rather a polychate (as originally interpreted). Sinuous myomeres were proposed to occur in Myllokunmingia-Haikouichthys from the early Cambrian Chengjiang fauna, a still inadequately known organism that shares its overall body shape and the presence of gill arches with the much better documented Yunnanozoon-Haikouella from the same strata. These Chengjiang fossils show the original three-dimensional disposition of body organs, with the sediment fill of the oesophagus separating left and right series of gill arches and gonads. The gonads are usually dark-stained and preserved in a positive relief on some rock slabs in result of differential collapse of particular organs under the sediment load. The notochord was also resistant to collapse, but it shows a darkening only on its outline. This suggests the original presence of a collagenous envelope surrounding a hydraulic skeleton built of organic matter-poor, vacuolarized cells. Various specimens of Yunnanozoon show different representation of their anatomy depending on local taphonomic conditions. In the Haikou locality, blood vessels supporting the gill arches and the head are well preserved. Probably the collageneous basement membrane of the endothelial cells promoted the staining. In rare specimens complex head structures are represented, suggestive of large eyes. The most controversial and enigmatic aspect of the anatomy of Yunnanozoon is its serial, laterally flattened dorsal body unit. It seem that these were muscular chambers with a thin envelope of the basement membrane, filled with a fluid or fluid-rich tissue. Being located dorsally of the notochord, these chambers differ from myomeres of adult chordates, but resemble the embryonic Anlagen of the myomeres in amphioxus. There was an attempt to restore the ancestral chordate body plan with dorsally located serial muscular chambers; the gill slits proposed to originate from the intestinal caeca. The ancestral chordate depicted in this way fits thus the anatomy of Yunnanozoon in respect to the dorsal location of probable muscular chambers, but is even more similar to the body plan of the Ediacarian dipleurozoans. They show a dorsal series of paired muscular chambers, serial metameric intestinal caeca, and lateral rows of probable oval gonads. The proposed dipleurozoan-yunnanozoan-conodont evolutionary sequence, if true, places all the more recent deuterostomian taxa, traditionally believed to fill the space between the protostomes and typical vertebrates, on side branches of the main evolutionary lineage.

The main Metazoan Radiation: a Pre-Cambrian event
Mikhail A. Fedonkin and Alberto Simonetta
Paleontological Institut, Russian Academy of Sciences, Moscow, Russia
Department of Animal Biology, University of Florence, Italy, 

It is now certain that the pre-Cambrian (Ediacaran) Kimberella quadrata is a very primitive Mollusc. The occurrence of a true member of a living phylum proves that the radiation of the main phyla did not occur in the Cambrian, but not later than the middle Ediacaran. Moreover, as Molluscs are typical trochophorates and spiralians, either the separation between protostomians and deuterostomians is not valid or must have occurred even earlier, possibly even before the beginning of the Ediacarian, this strongly arguing against maintaining the reality of the "Cambrian Explosion".
     As far as the origin of Vertebrates is concerned, while we know now a variety of Cambrian Chordates, the development in vertebrates of their most typical tissue, i.e. bone, must have been a late Cambrian development, a plausible hypothesis being that it was strictly linked with the development of the placodes of the lateral line system.

Convergence and divergence in early embryologic stages of vertebrates and invertebrates
Frietson Galis
Leiden University

The early organogenesis stages in metazoans differ drastically between higher order taxa such as phyla and classes. The segmented germ band stage in insects, the nauplius stage of crustaceans, and the neurula/pharyngula stage in vertebrates are examples of this diversification. In striking contrast with this divergence, is the similarity of these stages within these taxa, i.e., within insects, crustaceans, and vertebrates. The early organogenesis stages, have, thus, remained very similar among most species within higher taxa since the evolutionary origin of the latter. These stages are considerably more similar to each other than to the earlier stages of cleavage and gastrulation. Cleavage and gastrulation stages display not only great variability, but also striking examples of apparent convergence among species in different phyla, for example in the many cases of epiblastic cleavage in yolk-rich eggs. This leads to the paradoxical situation that the overall similarity of cleavage and gastrulation stages is in general higher among metazoans than of the early stages of organogenesis, but within phyla and classes the situation is the reverse. I will discuss data and evaluate possible causes for conservation, homoplasy, and diversification in an attempt to throw light on this paradoxical situation.

Vascular regeneration in a tunicate recalls vertebrate angiogenesis
Fabio Gasparini, Federico Caicci, Fabrizio Longo, Giovanna Zaniolo Dept. Biology, University of Padova, via U. Bassi 58/B, I-35121 Padova, Italy

 Among chordates, the regenerative potentiality is particularly developed in the tunicate ascidians, which are able to regenerate tunic and the embedded blood vessels with a sort of process recalling the angiogenesis occurring during physiological or pathological conditions in vertebrates.
     The ascidian Botryllus schlosseri forms colony of numerous clonal individuals derived by asexual reproduction and all embedded in a common thin extracellular tunic containing numerous scattered cells. Tunic holds a network of vessels which are limited by a simple flat epithelium, and are in continuum with the vascular lacunae of zooids. During the life of colony the circulatory system extends and remodel accompanying the propagation of the colony.
     We studied the regeneration of this system in vivo, by EM, and immunohistochemistry. New tunic formation, sprouting, elongation and fusion of vessels were the mechanisms observed. The epithelium of growing vessels has columnar cells with features suggesting intense protein synthesis for tunic formation. Proliferative activity are at the apex of regenerated vessels and in blood cells crossing the vessel epithelium towards tunic. Regenerating vessels give positive response to antibodies against vertebrate angiogenetic factors FGF2, VEGF and VEGFR1. These data suggest that a similar mechanism occurs in vertebrate angiogenesis and B. schlosseri vascular regeneration.

Origin of the centralized Chordate nervous system
Neil J. Gostling (Seb Shimeld- Ph.D. supervisor)
Bristol University

The vertebrate nervous system is patterned by genes in the Zic and Gli gene families, however, the ortholgues of these genes play no part in patterning the Drosophila nervous system, instead Odd-paired (Zic), patterns visceral mesoderm, while Cubitus interruptus (Gli) regulates Hedgehog signaling. Here we show that Zic has a primitive role in patterning the neural cell lineages in the Metazoa.

Hatschek's work continues - fossils confirm that L-R asymmetry was important in chordate origins
Richard Jefferies and Patricio Dominguez
The Natural History Museum, Cromwell Rd., London, SW7 5BD, UK
Dept. Paleontología, Fac. CC. Geologicas, Universidad Complutense de Madrid, Spain, 

Hatschek and others demonstrated that the ontogeny of  the pharynx  in amphioxus is R-L assymmetrical with primary, secondary and tertiary sets of gill slits. The primary slits are left slits and are the only ones to  appear during  larval life; the secondary slits are right  slits and arise at metamorphosis; and the tertiary slits are left and right gill slits and arise during the post larval stage, posterior to the slits already there. All this is remarkable since the fossil cornutes, such as Cothurnocystis, had left gill slits only like the primary slits of amphioxus; while the fossil mitrates had right and left gill slits, the right slits corrsponding to the secondary slits of amphioxus; and, the mitrate Lagynocystis had median gill slits corresponding to the tertiary slits of amphioxus. The fossils therefore suggest that the L-R asymmetry of the gill slits in amphioxus is a striking case of Haeckelian recapitulation. The sudden origin of the secondary gill slits in ontogeny could well result from a macromutation in phylogeny and as such would repay further developmental research. R-L asymmetries may result from an episode in the ancestry of all deuterostomes when a bilaterally symmetrical ancestor lay down on the right.

Characterization of Eyes absent and sine oculis genes in amphioxus
Zbynek Kozmik, Jana Kreslova, Nicholas D. Holland and Linda Z. Holland
Institute of Molecular Genetics, Prague, Czech Republic
Scripps Institution of Oceanography, La Jolla, California, USA 

Cranial placodes are supposed to be unique vertebrate characteristics from which the paired sense organs of the eyes, ears and nose, in addition to the distal parts of some of the cranial sensory ganglia are formed. Vertebrate placodes arise in the cranial ectoderm. Some placodes, like the otic, nasal, and lens placodes, form as visible thickenings that subsequently invaginate. Others, like the trigeminal and epibranchial placodes, are not distinguishable morphologically. One of the key molecular characteristics of vertebrate placodes is the co-expression of Eyes absent/Eya and sine oculis/Six1/Six4 genes. Vertebrate Eya and Six genes encode proteins that function together in a transcriptional complex; Six protein provides DNA-binding function while. In  order to gain insight into the evolutionary origin of placodes, we have cloned and characterized Eya, Six1 and Six4 genes in the invertebrate chordate amphioxus (Branchiostoma floridae). We have found areas of co-expression of Eya/Six1 and Eya/Six4 during amphioxus embryonic development that might correspond to vertebrate placodes. In addition, we have found that amphioxus Eya and Six1/4 proteins require their direct protein-protein interaction for proper function. Our results suggest, based on regionalized expression of specific transcription factors and their properties, that the origin of at least some cranial placodes might predate the origin of vertebrates.

What does it take to be a jawed vertebrate?
Shigeru Kuratani

To understand how the jaw evolution took place, the lamprey, a jawless vertebrate, was used as a model. Hox gene cognates were isolated and the lamprey’s mandibular arch (MA) was found to be in the Hox code-default state (devoid of Hox transcripts), as is true in gnathostomes. Spatial colinearity was observed for Hox2 and Hox3 cognates in the pharyngeal ectomesenchyme. Thus, the gnathostome jaw and agnathan oral apparatus seem to have evolved independently on the shared embryonic patterns.  Some jaw-patterning-related genes were also isolated from the lamprey, and their expression patterns were compared with those in gnathostomes. In gnathostomes, the expression Dlx1 defines the MA domain in the rostral ectomesenchyme and rostral to the MA, there is a Dlx1-negative premandibular region that is not incorporated into the oral patterning. In contrast, the lamprey uses the entire rostral ectomesenchyme for oral patterning. From these data, it was concluded that heterotopic shift of the tissue interaction, based on the same molecular cascade, was a prerequisite for the patterning of the gnathostome jaw. It will also be discussed that, in the transition from agnathan to gnathostome states, the emergence of diplorhiny should have preceded the heterotopy of oral patterning, as an embryological prerequisite.

Stylophorans ("calcichordates"): not the ancestry of vertebrates
Bertrand Lefebvre and Oldøich Fatka
UMR Biogéosciences, Université de Bourgogne, Dijon, France
Institute of Geology and Palaeontology, Charles University, Prague, Czech Republic

Stylophorans (cornutes, mitrates) are a class of Palaeozoic (Middle Cambrian - Upper Carboniferous) calcite-plated, marine deuterostomes. They consist of two well-defined regions : a delicate appendage and an asymmetrical, flattened, polyplated body (test). Since more than 40 years, their precise phylogenetic placement within deuterostomes remains highly controversial, as there is no general agreement on the interpretation of several key anatomical features. The recent discovery of putative echinoderm-like fossils in the Early Cambrian of China (vetulicystids) was presented as a strong evidence supporting a relatively basal placement of stylophorans within deuterostomes, either as basal-most members of the echinoderm stem-group or as early chordates (calcichordates). These two closely-related scenarios are based on (1) the absence of the typical five-fold radial symmetry in stylophorans, (2) the interpretation as a tail of the stylophoran appendage, and (3) the interpretation as gill slits of respiratory structures present in some cornutes. However, first, it is risky to deduce the basal phylogenetic position of stylophorans based on the absence of one single character: five-fold symmetry is frequently, repeatedly, and independently lost in various groups of "undisputable" echinoderms in Palaeozoic times (e.g. crinoids, rhombiferans). In each case, the loss of pentaradial symmetry was correlated with the adoption of an epibenthic, free (unattached) mode of life, comparable to that of stylophorans. Consequently, the hypothesis that five-fold symmetry was secondarily lost in these fossils can not be ruled out. Second, examination of the stylophoran appendage shows that its distal portions consisted of two sets of delicate, movable cover plates (left and right) articulated to one series of massive, uniserial ossicles. The presence of articulatory facets on cover plates indicates that they could open in life. Moreover, the upper edges of cover plates are frequently rounded. These two observations are not compatible with the interpretation of the stylophoran appendage as a closed, tightly sutured organ (stem or tail). On the other hand, the presence of two sets of movable cover plates, and the numerous structures present on the internal surface of ossicles are very comparable to the situation in the feeding arms of many "normal" echinoderms (e.g. eocrinoids, crinoids, ophiuroids). Third, respiratory structures present in some cornutes were interpreted as gill slits, because they are preserved as holes in the body wall. However, many similar structures (holes through the body wall) occur in other "undisputable" Palaeozoic echinoderms (e.g. eocrinoids, diploporites), in which they are interpreted as respiratory structures, not as gill slits. "Cothurnopores" and "lamellipores" of cornutes are fundamentally sutural pores (epispires) comparable to those of many eocrinoids, and there is no scientific argument to interpret them differently. In conclusion, the three main arguments suggesting a basal placement of stylophorans within deuterostomes (asymmetry, tail, gill slits) are falsified by close examination of the fossils. Stylophorans more probably correspond to derived echinoderms, which have secondarily lost they five-fold symmetry.

Early Gnathostomes: Morphotypes or Stereotypes?
John G. Maisey
American Museum of Natural History, New York, NY 10024-5192, USA

Earlier theories about the evolution of the jaws and cranium in gnathostome vertebrates were largely morphotypic and invoked many preconceived notions about primitive and derived characters in early vertebrates.  Until recently, the earliest known fossil gnathostomes did not contradict or refute the traditional dogma of gnathostome phylogeny.  However, new fossil discoveries of "basal" gnathostomes are beginning to revolutionize our views on morphology and evolution in the earliest crown group gnathostomes (osteichthyans and chondrichthyans), while developmental studies also cast new light on fundamental questions surrounding vertebrate and gnathostome origins. Some of these discoveries and ideas are reviewed and their implications for future directions of research in lower vertebrate phylogeny are discussed.

Amphioxus, Cambrian fossil animal Haikouella, and the Origin of the Vertebrates
Jon Mallatt
Washington State University, Pullman WA, USA 99164-4236

The phylogenetic position of the Cambrian yunnanozoans, or Yunnazoon and Haikouella, is controversial, with strong disagreement over whether these animals were vertebrate-like chordates, or basal deuterostomes but not chordates, or even cephalochordates related to amphioxus (Chen JY et al. 1999, Nature 403:519; Mallatt J and Chen JY 2003, Journal of Morphology 258:1; Shu D. et al. 2003 Science 299:1380).  Fossils of the soft-bodied yunnanozoans have been found in numerous locations in the Maotianshan Shale of southern China, but the exceptional preservational detail of the original Haikouella lanceolatum specimens from Ercai village, Haikou, has not been fully appreciated.  In yunnanozoans, the trunk region of the body has generated most of the challenges to a chordate interpretation because its vertical segments (proposed myomeres) project so far dorsal to its rod-like structure (proposed notochord) - which ould be highly unusual for a chordate. Yet the body segments of numerous Ercai specimens show stacks of horizontal lines indicative of muscle lamellae in myomeres, indicating yunnazoans indeed had chordate myomeres. Turning to the pharynx region, the Ercai specimens were the first yunnanozoan material to show evidence of true gills ("gill rays") on the branchial bars, and true gills only occur in vertebrates, not in other deuterostomes.  The head region of Ercai Haikouella, impossible to interpret in any detail in other yunnanozoans, shows a well-preserved oral hood with large upper lips and a lower lip (resembling these structures in modern larval lampreys); a large brain that thins posteriorly into a spinal nerve cord dorsal to the notochord; and in four specimens, small lateral eyes or eye lenses (which appear as either a dark circle around a light spot or a light circle around a dark spot, depending on how the rock was split into part or counterpart).  All these characters - including others that were combined in a parsimony-based analysis of phylogenetic relationships - indicate that yunnanozoans were the sister group of vertebrates and were not basal deuterostomes or cephalochordates. 
     Interestingly, the Haikouella specimens also share some characters with amphioxus, sometimes with striking similarity.  Besides its lancet-shape and similar body size (up to 2.5 cm long), Haikouella has amphioxus-like branchial hearts, a similar duct of the atrium leading to the atriopore, anterosuperiorly tilted branchial bars, a similar food-filtering screen across the mouth opening, and apparently the same, trough-shaped endostyle. 
     Thus, Haikouella fits Northcutt and Gans' hypothesis of the origin of the vertebrates (Biological Reviews 1989, 64:221), which said vertebrates evolved from amphioxus-like suspension feeders that became more-active swimmers, leading to marked ventilatory changes in the pharynx and to more complexity in the head, for sensory and information processing.

Hair cell in ascidians and vertebrates: homology or convergent evolution?
Lucia Manni, Federico Caicci, Giovanna Zaniolo, Paolo Burighel
Dept. Biology, University of Padova, via U. Bassi 58/B, I-35121 Padova, Italy

Recently, we described a mechanoreceptor organ, the coronal organ, in the oral siphon of  ascidians of the group pleurogonids.  It is constituted of ciliated cells (hair cells) innervated by the cerebral ganglion, and forming a sensorial row, recalling the vertebrate lateral line. These sensory cells vary in morphology, and in some species  they strongly resemble vertebrate hair cells, since they bear cilia situated at one side of a crescent-shaped bundle of graded in length stereovilli. Because only primary sensory cells were known in ascidians and hair cells were considered exclusive to vertebrates for their morphology and origin from neurogenic placodes, the evidence of these mechanoreceptors in the protochordate ascidians opened a debate regarding the evolution of chordate sensory cells.
     Analysing the coronal organ in the group enterogonids, we have found that hair cells manifest differences from those found in pleurogonid species, as the absence of  graded stereovilli. We think that the secondary sensory cell represents a plesiomorphy of ascidians inherited from the ancestor of chordates. We discuss its possible homology or convergence with vertebrate hair cell and its possible differentiation from a pan-placodal field of ascidians.  

Molecular evidence from Ciona intestinalis for the evolutionary origin of vertebrate
sensory placodes
Françoise Mazet and Sebastian M. Shimeld
University of Oxford, The Tinbergen Building, South Parks Road, Oxford OX1 3PS, UK

Cranial sensory placodes have long been considered a key character of vertebrates. Despite their importance for understanding vertebrate origins, the evolutionary origin of placodes has remained obscure. We used a panel of molecular markers from the Six, Eya, Pax, Dach, and COE gene families to examine the tunicate Ciona intestinalis for evidence of structures homologous to vertebrate placodes. Our results identify two domains of Ciona ectoderm that are marked by the genetic cascade that regulates vertebrate placode formation. The first is just anterior to the brain, and we suggest this territory is equivalent to the olfactory/adenohypophyseal placodes of vertebrates. The second is a bilateral domain adjacent to the posterior brain and includes cells fated to form the atrial siphon of an adult Ciona. We show this bares most similarity to placodes fated to form the vertebrate acoustico-lateralis system. We also examine a panel of pro-neurogenic and neurogenic markers from the FoxI, ngn, phox2 and pouIV gene families. Surprisingly, their expression was not limited to the embryonic stages and in some cases was induced in the developping siphons at metamorphic stages.
     We therefore conclude that sensory placodes did not arise de novo in vertebrates, but evolved from pre-existing specialised areas of ectoderm that contributed to sensory organs in the common ancestor of vertebrates and tunicates. The implication of developmental heterochrony in the current chordates evolutionary scenarios will be discussed.

Getting a grip on the bare bones of skeletal origins
Moya Meredith Smith
King's College London

Incorporation of fossil and extant data gives the most complete perspective on the origin of the skeleton at the chordate vertebrate transition, but skeletal characters may be the only information available for some taxa. Both, a framework of topographic position of the skeletal tissue and rigorously tested phylogenies of relationships based on whole body information are necessary to propose the order of origin of skeletal characters and the polarity of change. The status of ‘primary mineralised skeleton’ is not established and can be any of the axial, neurocranial, visceral-splanchnic, or dermal. Priority based on origin from embryonic cell type, mesodermal versus neural crest, is part of that debate.  The recent demonstration of neural-crest-like cells in ascidian urochordates, although known not to have skeletogenic potential, may influence views on the earliest skeleton.
     Emphasis on pattern differences between dermal and pharyngeal denticles suggested that the latter offer a pre-pattern for tooth sets in dentitions of stem gnathostomes and that regulatory genes are in the endoderm, not ectoderm. These pharyngeal denticle sets are present in stem gnathostomes, both with and without jaws.  It is argued that co-option occurred from pharyngeal arch denticle sets to tooth sets on the jaw margins, contrary to canonical theory.

From 2R to 3R: evidence for a fish-specific genome duplication (FSGD)       Axel Meyer and Yves Van de Peer
University of Konstanz, Germany and Ghent University, Belgium

An important mechanism for the evolution of phenotypic complexity, diversity, and innovation and the origin of novel gene functions is the duplication of genes and entire genomes.  Recent phylogenomic studies suggest that, during the evolution of vertebrates, the entire genome was duplicated in two rounds (2R) of duplication. Later, ~350 mya, in the stem lineage of ray-finned (actinopterygian) fishes, but not in that of the land vertebrates, a third genome duplication occurred – the fish-specific genome duplication (FSGD or 3R), leading, at least initially, to up to eight copies of the ancestral deuterostome genome. Therefore, the sarcopterygian (lobe-finned fishes and tetrapods) genome possessed originally only half as many genes compared to the derived fishes, just like the most basal and species-poor lineages of extant fishes that diverged from the fish stem lineage before the 3R duplication. Most duplicated genes were secondarily lost, yet some evolved new functions. The genomic complexity of the teleosts might be causally related to their evolutionary success and astounding biological diversity.

The development and evolution of cranial muscles in amphibians
Lennart Olsson
Institut für Spezielle Zoologie, Universität Jena, Germany

Our goal is to describe the order of acquisition of innovations in selected phases of amphibian evolution. Both skull and cranial muscle development are in focus. I will present selected parts of this ongoing project including; cranial muscle differentiation and morphogenesis in the Australian lungfish and in the Mexican axolotl, cranial muscle and skeleton development in Xenopus laevis and its relative Hymenochirus boettgeri, as well as skull development and morphology in a caecilian, Ichthyophis kohtaoensis. To determine the onset of differentiation we use antibodies against desmin and optical sectioning using confocal laser scanning microscopy on whole-mount immunostained embryos. Antibodies against acetylated tubulin are used to clarify muscle innervation patterns, and collagen II staining gives an overview of developing cartilage. This technique makes it possible to document head development in three dimensions while keeping the specimens intact. To obtain an appreciation of complicated three-dimensional structures in the head, we use reconstructions based on serial sections (two different methods will be shown). The project provides a morphological foundation for further studies of head skeleton as well as cranial muscle cell fate and early differentiation in a comparative approach. The focus is on understanding the developmental origins of morphological innovations.

Gen(om)e duplication and vertebrate origins: the problems of stem vertebrates and the meaning of complexity
Mark A. Purnell and Philip C. J. Donoghue
Department of Geology, University of Leicester, University Road, Leicester LE1 7RH, United Kingdom
Department of Earth Sciences, University of Bristol, Wills Memorial Building, Queen's Road, Bristol BS8 1RJ, United Kingdom

Vertebrate evolution was punctuated by three episodes of widespread gene or genome duplication, the first of which, between amphioxus and hagfish, has been implicated in the origin of vertebrates. Unfortunately, of the three episodes, this is the most problematic. The gen(om)e duplication appears to be associated with an evolutionary jump in phenotypic complexity but, like the other duplications (see Donoghue and Purnell abstract), we suspect that the apparent congruence between duplication and evolution is a consequence of ignoring fossils. Only the pattern of character evolution through the vertebrate stem can reveal the true picture, but this is confused by preservational biases and difficulties inherent in assigning fossils to the stems of clades with deep divergence times. Exceptionally preserved fossils, such as Yunnanozoon, Haikouella, and Haikouichthys from the Chengjiang lagerstätte, provide clear illustrations of this. Although exquisitely detailed, these fossils are the products of capricious fossilization - carbonized residues and mineral replacements of partially, and variably, decomposed carcasses. Anatomy cannot simply be read directly from the fossil, and serious complications arise because interpretation is necessarily predicated on comparisons with extant organisms, which inevitably colour subsequent anatomical determinations and hypotheses of affinity.
     Another general problem with hypotheses linking genome duplication to the evolution of vertebrate complexity arises from the lack meaning of 'phenotypic complexity'. How it might be measured is also unclear. We cannot currently test for congruence between genomic duplications and increases in phenotypic complexity because complexity is defined too loosely for the hypothesis to be falsifiable.

Hatschek’s pit and the evolution of vertebrate placodes
Gerhard Schlosser
Brain Research Institute, University of Bremen, 28334 Bremen, Germany

The placodes of vertebrates give rise to different cell types and contribute to many cranial sensory organs and ganglia. Recent studies indicate that all placodes originate from a panplacodal primordium, defined by the expression of Six1, Six4 and Eya genes, which may promote generic placodal properties such as neuronal specification and cell shape changes. Our experiments in Xenopus suggest that this primordium is induced in rostral nonneural ectoderm by a combination of neural and mesodermal signals. The common developmental origin of placodes suggests that all placodes may have evolved – possibly in several steps - from a common precursor. Traditionally, placodes were considered to be evolutionary innovations of vertebrates, but recent studies in ascidians and amphioxus suggested that some placodes evolved earlier in the chordate lineage. Many cellular and molecular components of placodes (e.g., sensory and neurosecretory cell types, regionalized expression of transcription factors) indeed predate the origin of vertebrates. However, there is presently little evidence that these components are integrated into placodes in protochordates. I propose instead that all placodes evolved from an adenohypophyseal-olfactory protoplacode, which may have originated in the vertebrate ancestor from the anlage of a rostral neurosecretory organ retained in present day amphioxus as Hatschek’s pit.

Phylogeny of early deuterostomes and origin of vertebrates
Degan Shu
Early Life Institute, Northwest University, Xi’an, 710069 China
School of Earth Sciences and Resources, China University of Geosciences, Beijing, 100083, China

Knowledge from study of the Early Cambrian Chengjiang Largestatte from 1984 to 1994 was restricted to protostomes. More intensive investigation and careful examination in the following ten years have yielded many discoveries of deuterostomes, which include not only the earliest-known representatives of the major branches in all three extant phyla, such as vetulocystids, yunnanozoans, Cheungkongella, Cathaymyrus, Myllokunmingia and Haikouichtys belonging to the most primitive vertebrates, but also an extinct phylum Vetulicolia. This allows us to propose a tentative deuterostome phylogeny with vetulicolians at the bottom (Shu 2003; Shu et al. 2004). As the most recent common ancestor of all extant deuterostomes would have been a segmented animal with pharyngeal slits (Gee 2001), vetulicolians with both primitive segmentation and gill slits should represent such kind of ancestor. If R-L asymmetries have resulted from an episode in the ancestry of all deuterostomes, the bilaterally symmetrical vetulicolians would right be or very close to that ancestor. Since endostyle may be a primitive feature in deuterostomes, vetulicolians, having no notochord, should be less likely related to chordates. Yunnanozoon and Haikouella have no eyes and brain (Shu and Conway Morris 2003) and even a notochord (Valentine 2004). Instead they share similar boy-plan with vetulicolians and bear both dorsal and ventral neural cords, which is characteristic of living hemichordates. K. Halanych’s comments (2004) are: Paleontological work of Shu and collaborators (1999, 2001a, 2001b, 2003) is revising our understanding of early evolution of chordates.

Fox gene duplication in vertebrate evolution
Karl Wotton, Françoise Mazet, Sebastian M. Shimeld
University of Oxford

The genome of higher vertebrates has undergone many duplication events. Often these duplication events appear to be associated with phenotypic changes.
     For example, during the evolution of vertebrates, mesodermal patterning has been substantially elaborated into a variety of subtypes. How this complex layout evolved is poorly understood. However, this evolutionary event may coincide with the duplication of a group of mesodermally expressed genes in the vertebrate lineage, named FoxF and FoxC.
     To test this hypothesis I am identifying these genes in different vertebrate lineages. Particularly I am concentrating on the identification and characterisat-ion of these genes and their expression patterns in basal vertebrates such as sharks and lampreys.

Developmental basis for the origin of the vertebrate skeleton
GuangJun Zhang and Martin J. Cohn
Department of Zoology, University of Florida, Gainesville, FL 32611, USA

 Development of a cartilagenous endoskeleton was a pivotal innovation during vertebrate evolution.  The underlying molecular genetic mechanisms, however, remain poorly understood. We have taken a comparative approach to this problem by comparing the mechanisms of skeletal development in lamprey and shark embryos. Analysis of a suite of developmental control genes during skeletogenesis reveals that the molecular mechanisms of skeletal development are highly conserved across vertebrates. Comparison of lamprey and gnathostome gene expression patterns also showed evidence for subfunctionalization following gene duplication events, in which the ancestral expression pattern was partitioned among the two duplicated genes. The extent of conservation is surprising, given the reported structural differences between aganthan and gnathostome cartilage, and suggests a more ancient origin of the cartilage developmental  program than previously reported.