Primitive Chordates Research Articles

The role of population size, pleiotropy and fitness effects of mutations in the evolution of overlapping gene functions.

Sheltered from deleterious mutations, genes with overlapping or partially redundant functions may be important sources of novel gene functions. While most partially redundant genes originated in gene duplications, it is much less clear why genes with overlapping functions have been retained, in some cases for hundreds of millions of years. A case in point is the many partially redundant genes in vertebrates, the result of ancient gene duplications in primitive chordates. Their persistence and ubiquity become surprising when it is considered that duplicate and original genes often diversify very rapidly, especially if the action of natural selection is involved. Are overlapping gene functions perhaps maintained because of their protective role against otherwise deleterious mutations? There are two principal objections against this hypothesis, which are the main subject of this article. First, because overlapping gene functions are maintained in populations by a slow process of "second order" selection, population sizes need to be very high for this process to be effective. It is shown that even in small populations, pleiotropic mutations that affect more than one of a gene's functions simultaneously can slow the mutational decay of functional overlap after a gene duplication by orders of magnitude. Furthermore, brief and transient increases in population size may be sufficient to maintain functional overlap. The second objection regards the fact that most naturally occurring mutations may have much weaker fitness effects than the rather drastic "knock-out" mutations that lead to detection of partially redundant functions. Given weak fitness effects of most mutations, is selection for the buffering effect of functional overlap strong enough to compensate for the diversifying force exerted by mutations? It is shown that the extent of functional overlap maintained in a population is not only independent of the mutation rate, but also independent of the average fitness effects of mutation. These results are discussed with respect to experimental evidence on redundant genes in organismal development.

Retinoic acid perturbs Otx gene expression in the ascidian pharynx

In vertebrate embryos, ectopic application of all-trans retinoic acid (RA) alters the expression of Otx genes in the developing midbrain. In conjunction with RA-induced misexpression of other regulatory genes this leads to a loss of anterior CNS. In the ascidian Herdmania curvata, RA primarily inhibits the development of the juvenile pharynx. An ascidian Otx gene, Hec-Otx, is expressed largely in this tissue, associated stomodeal structures and the anterior endostyle of the juvenile. Treatment with 10-6 M RA reduces Hec-Otx mRNA levels in the juvenile to about 12% of normal and is correlated closely with the loss of pharyngeal structures. During embryogenesis the expression of Hec-Otx becomes restricted to cell lineages fated to give rise to the anterior-most nervous system and the stomodeal component of the primordial pharynx. In hatched larvae Hec-Otx transcripts are detected only in the sensory (brain) vesicle. RA reduces Hec-Otx expression in the tailbud stomodeal pharynx primordium/anterior nervous system cell line but not in the larval sensory vesicle, suggesting that RA regulation of Hec-Otx expression is restricted to pharyngeal tissues throughout embryonic and postlarval development. RA does not affect expression of Hec-Pax2/5/8, which is normally expressed within the developing nervous system immediately posterior to Hec-Otx at the tailbud stage, lending support to the proposition that RA does not impact CNS axial patterning. These data combined with those from other chordates suggest that RA regulation of Otx expression in the anterior nerve cord and pharynx is a primitive chordate feature which has been maintained predominantly in pharyngeal tissues in the ascidian.

Homologies in stylophora:A test of the ‘calcichordate theory’

Abstract Stylophora (cornutes and mitrates) are controversial Paleozoic fossils, variously interpreted as ‘bizarre’echinoderms or as ‘calcichordates’ (primitive chordates). The calcichordate theory is based on the identification of left/right asymmetries supposed to be homologous in stylophorans and modern chordates. This theory imposes the homology of the plano-concave surface of the cornute test with the convex surface of the mitrate test. A detailed analysis of numerous internal and external structures exhibited by the appendage and the test demonstrates the homology of the plano-concave surface of the test in all stylophorans. The basic assumption of the calcichordate theory is thus not valid. Comparison of left/right asymmetries in these organisms with those of modem chordates is not grounded. Stylophora do not represent `calcichordates' but actual echinoderms.

AmphiPax3/7, an amphioxus paired box gene: insights into chordate myogenesis, neurogenesis, and the possible evolutionary precursor of definitive vertebrate neural crest.

Amphioxus probably has only a single gene (AmphiPax3/7) in the Pax3/7 subfamily. Like its vertebrate homologs (Pax3 and Pax7), amphioxus AmphiPax3/7 is probably involved in specifying the axial musculature and muscularized notochord. During nervous system development, AmphiPax3/7 is first expressed in bilateral anteroposterior stripes along the edges of the neural plate. This early neural expression may be comparable to the transcription of Pax3 and Pax7 in some of the anterior neural crest cells of vertebrates. Previous studies by others and ourselves have demonstrated that several genes homologous to genetic markers for vertebrate neural crest are expressed along the neural plate-epidermis boundary in embryos of tunicates and amphioxus. Taken together, the early neural expression patterns of AmphiPax3/7 and other neural crest markers of amphioxus and tunicates suggest that cell populations that eventually gave rise to definitive vertebrate neural crest may have been present in ancestral invertebrate chordates. During later neurogenesis in amphioxus, AmphiPax3/7, like its vertebrate homologs, is expressed dorsally and dorsolaterally in the neural tube and may be involved in dorsoventral patterning. However, unlike its vertebrate homologs, AmphiPax3/7 is expressed only at the anterior end of the central nervous system instead of along much of the neuraxis; this amphioxus pattern may represent the loss of a primitive chordate character.

Ascidian Homologs of Mammalian Thyroid Transcription Factor-1 Gene Are Expressed in the Endostyle

Abstract The endostyle is a special organ in the pharynx of urochordates, cephalochordates and cyclostomes. During evolution of the primitive chordates, the endostyle was organized in their common ancestor(s) with a shift to internal feeding for extracting suspended food from the water. In addition, the endostyle has an iodine-concentrating activity and is therefore thought to be functionally homologous to the vertebrate thyroid gland. Human TITF1 and mouse TITF1 are members of the Nkx-2.1/TTF-1 gene subfamily, which encode an NK-2 type homeodomain transcription factor. The genes are expressed in the thyroid gland and are essential for thyroid-specific structural gene expression. In the present study, we isolated cDNA clones for ascidian homologs of TITF1 from Halocynthia roretzi and Ciona intestinalis, and examined whether the genes are expressed in the ascidian endostyle. Results clearly indicated that both the H. roretzi homolog Hrtitf1 and the C. intestinalis homolog Cititf1 are expressed specifically...

Tunicate tails, stolons, and the origin of the vertebrate trunk

ABSTRACTTunicates are primitive chordates that develop a transient ‘tail’ in the larval stage that is generally interpreted as a rudimentary version of the vertebrate trunk. Not all tunicates have tails, however. The groups that lack them, salps and pyrosomes, instead have a trunk‐like reproductive stolon located approximately where the tail would otherwise be. In salps, files of blastozooids are formed along the sides of the stolon. The tail and caudal trunk in more advanced chordates could have evolved from a stolon of this type, an idea referred to here as the ‘stolon hypothesis’. This means the vertebrate body could be a composite structure, since there is the potential for each somite to incorporate elements originally derived from a complete functional zooid. If indeed this has occurred, it should be reflected in some fashion in gene expression patterns in the vertebrate trunk. Selected morphological and molecular data are reviewed to show that they provide some circumstantial support for the stolon hypothesis. The case would be stronger if it could be demonstrated that salps and/or pyrosomes are ancestral to other tunicates. The molecular phylogenies so far available generally support the idea of a pelagic ancestor, but offer only limited guidance as to which of the surviving pelagic groups most closely resembles it. The principal testable prediction of the stolon hypothesis is that head structures (or their homologues) should be duplicated in series in the trunk in advanced chordates, and vice versa, i.e. trunk structures should occur in the head. The distribution of both rhabdomeric photoreceptors and nephridia in amphioxus conform with this prediction. Equally striking is the involvement of the Pax2 gene in the development of both the inner ear and nephric ducts in vertebrates. The stolon hypothesis would explain this as a consequence of the common origin of otic capsules and excretory ducts from atrial rudiments: from the paired rudiments of the parent oozooid in the case of the otic capsule (these express Pax2 according to recent ascidian data), and from tubular rudiments in the stolon in the case of the excretory ducts.

Tunicate tails, stolons, and the origin of the vertebrate trunk.

Tunicates are primitive chordates that develop a transient 'tail' in the larval stage that is generally interpreted as a rudimentary version of the vertebrate trunk. Not all tunicates have tails, however. The groups that lack them, salps and pyrosomes, instead have a trunk-like reproductive stolon located approximately where the tail would otherwise be. In salps, files of blastozooids are formed along the sides of the stolon. The tail and caudal trunk in more advanced chordates could have evolved from a stolon of this type, an idea referred to here as the 'stolon hypothesis'. This means the vertebrate body could be a composite structure, since there is the potential for each somite to incorporate elements originally derived from a complete functional zooid. If indeed this has occurred, it should be reflected in some fashion in gene expression patterns in the vertebrate trunk. Selected morphological and molecular data are reviewed to show that they provide some circumstantial support for the stolon hypothesis. The case would be stronger if it could be demonstrated that salps and/or pyrosomes are ancestral to other tunicates. The molecular phylogenies so far available generally support the idea of a pelagic ancestor, but offer only limited guidance as to which of the surviving pelagic groups most closely resembles it. The principal testable prediction of the stolon hypothesis is that head structures (or their homologues) should be duplicated in series in the trunk in advanced chordates, and vice versa, i.e. trunk structures should occur in the head. The distribution of both rhabdomeric photoreceptors and nephridia in amphioxus conform with this prediction. Equally striking is the involvement of the Pax2 gene in the development of both the inner ear and nephric ducts in vertebrates. The stolon hypothesis would explain this as a consequence of the common origin of otic capsules and excretory ducts from atrial rudiments: from the paired rudiments of the parent oozooid in the case of the otic capsule (these express Pax2 according to recent ascidian data), and from tubular rudiments in the stolon in the case of the excretory ducts.

Organic membranous skeleton of the Precambrian metazoans from Namibia

Unlike the celebrated Ediacara fossils, those from the roughly coeval localities of the Kuibis Quarzite of Namibia are preserved not as imprints on the sandstone bedding plane, but three-dimensionally, within the rock matrix. The pattern of deformation and the presence of sand in lower parts of the bodies of Ernietta , the most common and typical of those organisms, indicate that their three-dimensional preservation is a result of a density-controlled sinking of sand-filled organic skeletons within hydrated mud layers. Specimens of Ernietta have preserved various stages of migration across the mud beds. Their wall material, as documented by the mode of deformation, was not only flexible, but also elastic, which makes it unlike chitin. The walls thus seem to be proteinaceous, built probably of a collagenous fabric. The Ernietta skeleton was built of series of parallel chambers, which excludes the possibility that these were external body covers. The chambers apparently represent walls of hydraulic skeleton units, resembling the basement membrane of chaetognaths or the notochord sheath of primitive chordates. Such chambers are widespread among the earliest fossil animals represented by fossils preserved in sandstone. The rise and fall of the Ediacaran faunas thus seem to be partially preservational artifacts. The range of its occurrence is a result of two successive evolutionary events: the origin of an internal hydraulic skeleton enclosed by a strong basement membrane, and the appearance of decomposers with abilities to disintegrate such collagenous sheaths.

Retinoic acid affects patterning along the anterior-posterior axis of the ascidian embryo.

Because retinoic acid (RA) is known to affect anterior posterior patterning in vertebrate embryos, it was questioned whether it shows similar effects in a more primitive chordate, the ascidian Halocynthia roretzi. Ascidian embryos treated with RA exhibited truncated phenotypes in a dose-dependent manner similar to the anterior truncations seen in vertebrate embryos. The most severely affected larvae possessed a round trunk without the papillae characteristic of the anterior terminal epidermis. Retinoic acid also altered the expression of HrHox-1 and Hroth in a dose-dependent manner. Expression of HrHox-1 increased, whereas expression of Hroth decreased with increasing levels of RA. In treated embryos, HrHox-1 was first expressed pan-ectodermally, then degraded in all but specific regions of the embryo. By contrast, initiation of Hroth expression was not affected, but epidermal expression was lost while expression in the neural tube narrowed toward the anterior in tail-bud embryos. These alterations in the expression of homeobox genes appear to correlate closely to the morphological defects elicited by RA treatment, suggesting broad conservation of developmental patterning mechanisms within the Phylum Chordata.

Novel gene containing multiple epidermal growth factor-like motifs transiently expressed in the papillae of the ascidian tadpole larvae.

We have investigated molecular mechanisms of the embryonic development of an ascidian, a primitive chordate which shares features of both invertebrates and vertebrates, with a view to identifying genes involved in development and metamorphosis. We isolated 12 partial cDNA sequences which were expressed in a stage-specific manner using differential display. We report here the isolation of a full-length cDNA sequence for one of these genes which was specifically expressed during the tailbud and larval stages of ascidian development. This cDNA, 1213 bp in length, is predicted to encode a protein of 337 amino acids containing four epidermal growth factor (EGF)-like repeats and three novel cysteine-rich repeats. Characterization of its spatial expression pattern by in situ hybridisation in late tailbud and larval embryos demonstrated strong expression localised throughout the papillae and anteriormost trunk and weaker expression in the epidermis of the remainder of the embryo. As recent evidence indicates that the signal for metamorphosis originates in the anterior trunk region, these results suggest that this gene may have a role in signalling the initiation of metamorphosis.

Novel gene containing multiple epidermal growth factor–like motifs transiently expressed in the papillae of the ascidian tadpole larvae

We have investigated molecular mechanisms of the embryonic development of an ascidian, a primitive chordate which shares features of both invertebrates and vertebrates, with a view to identifying genes involved in development and metamorphosis. We isolated 12 partial cDNA sequences which were expressed in a stage-specific manner using differential display. We report here the isolation of a full-length cDNA sequence for one of these genes which was specifically expressed during the tailbud and larval stages of ascidian development. This cDNA, 1213 bp in length, is predicted to encode a protein of 337 amino acids containing four epidermal growth factor (EGF)–like repeats and three novel cysteine-rich repeats. Characterization of its spatial expression pattern by in situ hybridisation in late tailbud and larval embryos demonstrated strong expression localised throughout the papillae and anteriormost trunk and weaker expression in the epidermis of the remainder of the embryo. As recent evidence indicates that the signal for metamorphosis originates in the anterior trunk region, these results suggest that this gene may have a role in signalling the initiation of metamorphosis. Dev. Dyn. 1997;210:264–273. © 1997 Wiley-Liss, Inc.

Sulphated polyanions in cytoplasm and nuclei of epithelial cells of Branchiostoma demonstrated by the cationic dye Cupromeronic Blue

The intracellular occurrence and distribution of sulphated polyanions, interpreted to represent mucins, were studied in secretory epithelial cells in the primitive chordates Branchiostoma lanceolatum and B. floridae at the electron microscopical level by using Cupromeronic Blue (CMB). CMB-precipitates were mainly found within two potential types of mucin vesicles (apical and basal) and Golgi cisterns. The mucin vesicles form a distinct population of secretory granules different from another nonmucin granule population. Within the epidermal cells the staining intensity of the Golgi cisterns with CMB increased from the cis to the trans compartment. The pharyngeal mucous cells showed staining only in the trans Golgi compartment. These findings indicate, that CMB can be used for intracellular localization of mucins and that sulphation of the mucins in the investigated cells may occur within different compartments of the Golgi complex. Apparently the mucin is secreted apically but only in the epidermis it forms a dense layer covering the apical microvilli. In the Branchiostoma epidermal cells a layer of specialized basal vesicles occurred, containing unusually large and branched CMB-precipitates which possibly serve mechanical functions. In the nuclei CMB-precipitates were regularly demonstrated in the euchromatin of the cell types studied.

Expression of pharyngeal gill-specific genes in the ascidian Halocynthia roretzi

The most primitive chordates may have arisen with a shift to internal feeding through the use of the pharyngeal gill slits and endostyle for extracting suspended food from the water. Therefore, the pharyngeal gill and endostyle, in addition to notochord and nerve cord, are structures key to an understanding of the molecular developmental mechanisms underlying the origin and evolution of chordates. In this and a following study, isolation of cDNA clones for genes that are specifically expressed in the pharyngeal gill or endostyle in the ascidian Halocynthia roretzi was attempted. Differential screening of a pharyngeal gill cDNA library and an endostyle cDNA library with total pharyngeal-gill cDNA probes yielded cDNA clones for two pharyngeal gill-specific genes, HrPhG1 and HrPhG2. Northern blot analysis showed a 3.0-kb transcript of HrPhG1 and a 2.0-kb transcript of HrPhG2. Predicted amino acid sequences of the gene products suggested that both genes encode secretory proteins with no significant match to known proteins. In adults, both HrPhG1 and HrPhG2 genes were only expressed in the pharyngeal gill and not in other tissues including the endostyle, body-wall muscle, gonad, gut and digestive gland. HrPhG1 and HrPhG2 transcripts were undetectable in embryos and larvae, and were first detected in juveniles 3 days after initiation of metamorphosis. In situ hybridization revealed that the expression of HrPhG1 and HrPhG2 was restricted to differentiating pharyngeal-wall epithelium, with intense signals in the area surrounding the stigma or gill slit. These genes may serve as probes for further analyses of molecular mechanisms underlying the occurrence of pharyngeal gill and formation of gill slits during chordate evolution.

Spatial expression of the amphioxus homologue of Brachyury (T) gene during early embryogenesis of Branchiostoma belcheri.

Amphioxus are primitive chordates that share several common anatomical features with vertebrates, such as a notochord and a dorsal nerve cord. The Brachyury (T) gene is required for notochord differentiation in vertebrates and an ascidian homologue of this gene is expressed in notochord cells at the time of developmental fate restriction. To gain insight into the molecular and developmental mechanisms underlying the evolution of chordates, we amplified a cDNA fragment for an amphioxus homologue (BbT1) of the mouse T gene, with which we examined the spatial expression of the BbT1 gene. In situ hybridization showed that BbT1 transcript was undetectable in eggs, as well as in early embryos at the cleavage and blastula stages. Distinct hybridization signals were first evident in the mesoderm of gastrulae but neither the ectoderm nor endoderm showed the signals. As development proceeded to the neurula stage, the signals became conspicuous in the primordial somite at the dorsolateral wall of the archenteron, while the primordial notochord at the dorsomedian wall of the archenteron emitted less intense hybridization signals. These results suggested that the T gene is conserved by amphioxus and that the BbT1 gene is expressed in the mesodermal region of the gastrula and neurula.

Proprotein convertases in amphioxus: predicted structure and expression of proteases SPC2 and SPC3.

SPC2 and SPC3 are two members of a family of subtilisin-related proteases which play essential roles in the processing of prohormones into their mature forms in the pancreatic B cell and many other neuroendocrine cells. To investigate the phylogenetic origins and evolutionary functions of SPC2 and SPC3 we have identified and cloned cDNAs encoding these enzymes from amphioxus (Branchiostoma californiensis), a primitive chordate. The amino acid sequence of preproSPC2 contains 689 aa and is 71% identical to human SPC2. In contrast, amphioxus prproSPC3 consists of 774 aa and exhibits 55% identity to human SPC3. These results suggest that the primary structure of SPC2 has been more highly conserved during evolution than that of SPC3. To further investigate the function(s) of SPC2 and SPC3 in amphioxus, we have determined the regional expression of these genes by using a reverse transcriptase-linked polymerase chain reaction (RT-PCR) assay. Whole amphioxus was dissected longitudinally into four equal-length segments and RNA was extracted. Using RT-PCR to simultaneously amplify SPC2 and SPC3 DNA fragments, we found that the cranial region (section 1) expressed equal amounts of SPC2 and SPC3 mRNAs, whereas in the caudal region (section 4) the SPC2-to-SPC3 ratio was 5:1. In the mid-body sections 2 and 3 the SPC2-to-SPC3 ratio was 1:5. By RT-PCR we also determined that amphioxus ILP, a homologue of mammalian insulin/insulin-like growth factor, was expressed predominately in section 3. These results suggest that the relative levels of SPC2 and SPC3 mRNAs are specifically regulated in various amphioxus tissues. Furthermore, the ubiquitous expression of these mRNAs in the organism indicates that they are involved in the processing of other precursor proteins in addition to proILP.

An Ascidian Homolog of the Mouse Brachyury (T) Gene is Expressed Exclusively in Notochord Cells at the Fate Restricted Stage: (Ascidians/T (Brachyury) gene/sequence conservation/notochord cells/transient expression).

Ascidians are primitive chordates in which a well-organized notochord is formed in the tail of the tadpole larva. The Brachyury (T) gene in the mouse is essential for formation of mesoderm and, in particular, of notochord. We report here the expression of an ascidian homolog (As-T) of the mouse T gene. A cDNA clone for the As-T gene contains a single open reading frame that encodes a polypeptide of 471 amino acids. Although the overall degree of amino acid identity was not very high (39.9%, ascidian/mouse), in the N-terminal half the extent of amino acid identity was 78.5% (ascidian/mouse). The expression of As-T was transient. A single band corresponding to a 2.2 kb mRNA was first detected at the 64-cell stage, and a distinct band was found at the gastrula stage, at which time accumulation of the transcripts was maximal. The transcript was barely detectable at the larval stage and was undetectable in adult tissues. The occurrence of As-T transcripts was restricted to notochord-lineage cells, and no other cell types expressed the As-T gene. In addition, the timing of As-T expression coincided with the time of restriction of developmental fate exclusively to notochord in the notochord-lineage cells. It is likely that the Brachyury gene was present when the ancestors of chordates with a notochord emerged and that the primary function of this gene is to specify notochord cells.

Decay of Branchiostoma : implications for soft‐tissue preservation in conodonts and other primitive chordates

Decay experiments on the cephalochordate Branchiostoma lanceolatum (‘amphioxus’) demonstrate that the most decay resistant structures are the notochord sheath and the cartilaginous rods which support the gill bars. However, even more labile soft parts, such as the muscles and skin may survive for at least 124 days under totally anoxic conditions. As the chevron-shaped muscles of the myomeres shrink and collapse, those on opposite sides of the trunk maybe displaced, resulting in pronounced offsetting. Only 1.42% of the initial dry weight of Branchiostoma is resistant to alkali and acid hydrolysis, compared to 46% in the polychaete Nereis virens. Branchiostoma is only likely to be fossilized as a result of decay inhibition and replication by early diagenetic minerals. The results of these experiments cast light on the interpretation of a number of primitive fossil chordates. There is no reason to infer extracellular decay-resistant cuticle in the Burgess Shale Pikaia. The axial lies preserved in the conodont animal specimens from the Carboniferous of Edinburgh, Scotland, represent the notochord. The displacement of the elements to one side of the head reflects the true position of the apparatus - the surrounding tissue has been lost through decay. The chevron-shaped structures in the Carboniferous chordate Conopiscius are the muscles of the myomeres, not external scales. The lines delineating the segments in the Silurian Jamoytius most likely represent the myosepta. There is some doubt about the nature of the only specimen interpreted as a fossil cephalochordate, Palaeobranchiostoma hamatotergum from the Permian of South Africa. □Taphonomy, decay, softparts, Cephalochordata, Branchiostoma, lancelet, Chordata, Pikaia, conodont, Conopiscius, Jamoytius, Palaeobranchiostoma.

Phylogenetic Relationships between Solitary and Colonial Ascidians, as Inferred from the Sequence of the Central Region of their Respective 18S rDNAs.

Ascidians (tunicates) are primitive chordates. In spite of their elevated phylogenetic position in the animal kingdom, ascidians have evolved a varied reproductive repertoire; some of them live as individuals (solitary ascidians), while others form colonies (colonial ascidians). Colonial ascidians propagate asexually by budding and strobilation, and they have an extensive capacity for regeneration. However, the orthodox taxonomic classification of ascidians categorizes them into two major groups (the orders Enterogona and Pleurogona), irrespective of their solitary or colonial life style. To examine whether the orthodox classification of ascidians is substantiated by molecular phylogeny, the complete nucleotide sequence of a region of about 1000 base pairs in the central part of their respective 18S rDNAs was determined, and the sequences were compared among five solitary and three colonial ascidians. The phylogenetic tree deduced from these results suggests that the three species of Enterogona and the five species of Pleurogona examined form discrete and separate groups irrespective of their potential to form colonies. Therefore, a solitary or colonial life style is likely to have developed independently after the divergence of the two major groups of ascidians.

At the orgin of chordates

It is classically known that the oldest representatives of vertebrates are Ordovician, heterostracan-like, dermal bone-bearing animals from Australia and North America. Another taxon, phylogenetically related to one of the Australian species, has recently been discovered in the Ordovician of Bolivia. However other discoveries of Lower Palaeozoic fossils shed a new light on the oldest representatives of primitive chordates (urochordates, cephalochordates). Most recent publications on this topic are reviewed. Anatolepis is rejected from chordate affinities. Euconodonts, more and more often cited as myxinoid (hagfish) relatives, are considered with doubt. A phylogenetic scheme is suggested, constrained by the stratigraphic record, and the minimum age of appearance of the main taxa is proposed: chordates in Early Cambrian times, vertebrates in mid-Ordovician times.

Evolutionary trends of neurofilament proteins in fish

1. 1. Neurofilament complement was studied in an early chordate ( Ciona intestinalis) and six fish species by immunoblot with antisera specific for each of the three mammalian NF subunits. 2. 2. The anti-NF-H and anti-NF-M antisera were characterized as strict for phosphorylated epitopes located in the carboxyterminal domain 3. 3. The NF-L subunit is absent in primitive chordates and appears first in fish; it can be identified on the basis of its apparent mol. wt, its reactivity with the anti-IFA antibody and with polyclonal antibodies raised to the NF-L subunit of mammals. 4. 4. Primitive chordate neurofilaments are constituted by a single polypeptide of ca 160,000 mol. wt exhibiting only M-type phosphorylation-dependent epitopes. 5. 5. Primitive fish ( Acipenser transmontanus, Salmo gairdneri, Scorpaena porcus, Serranus scriba) possess only a single high mol. wt NF subunit reacting with both anti-NF-H and anti-NF-M antiserum while more recent species ( Mugil saliens, Perca Fluviatilis) possess two high mol. wt NF subunits which are immunologically distinct as to their phosphorylation structures. 6. 6. The existence in some fish species of two high mol. wt NF polypeptides suggests that the process of gene duplication and diversification supposed to have given rise to the two high mol. wt NF subunits of mammals and birds has occurred repeatedly in vertebrate evolution, and may be regarded as a case of convergent evolution.