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The liver architecture of vertebrates can be classified into two types, the portal triad type (having periportal bile ducts) and the non-portal triad type (having non-periportal bile ducts). The former is detectable in the tetrapod liver whereas the lungfish liver has the latter. It remains to be revealed which type of hepatic architecture the coelacanth, which together with the lungfish belongs to the Sarcopterygii, possesses. The present study was undertaken to determine the histological characteristics of the coelacanth liver, and to compare with those of other vertebrates. The coelacanth liver had periportal bile ducts and ductules as detected in mammalian livers. The hepatic artery was found around large portal veins. Hagfish, shark, bichir, sturgeon, bowfin and frog livers had periportal bile ducts and bile ductules, whereas most intrahepatic bile ducts of the lungfish were independent of the distribution of the portal veins as seen in the Otocephala and Euteleostei. The lungfish liver developed duct and ductule structures in the parenchyma. These data indicate that the coelacanth liver had a mammalian-type hepatic architecture with a portal triad, and that the ancestors of tetrapods may have had a portal triad-type liver architecture.

The present paper is the first comparative study on the astroglia of several actinopterygian species at different phylogenetical positions, teleosts (16 species), and non-teleosts (3 species), based on the immunohistochemical staining of GFAP (glial fibrillary acidic protein), the characteristic cytoskeletal intermediary filament protein, and immunohistochemical marker of astroglia. The question was, how the astroglial architecture reflexes the high diversity of this largest vertebrate group. The actinopterygian telencephalon has a so-called ‘eversive’ development in contrast to the ‘evagination’ found in sarcopterygii (including tetrapods). Several brain parts either have no equivalents in tetrapod vertebrates (e.g., torus longitudinalis, lobus inferior, lobus nervi vagi), or have rather different shapes (e.g., the cerebellum). GFAP was visualized applying DAKO polyclonal anti-GFAP serum. The study was focused mainly on the telencephalon (eversion), tectum (visual orientation), and cerebellum (motor coordination) where the evolutionary changes were most expected, but the other areas were also investigated. The predominant astroglial elements were tanycytes (long, thin, fiber-like cells). In the teleost telencephala a ‘fan-shape’ re-arrangement of radial glia reflects the eversion. In bichir, starlet, and gar, in which the eversion is less pronounced, the ‘fan-shape’ re-arrangement did not form. In the tectum the radial glial processes were immunostained, but in Ostariophysi and Euteleostei it did not extend into their deep segments. In the cerebellum Bergmann-like glia was found in each group, including non-teleosts, except for Cyprinidae. The vagal lobe was uniquely enlarged and layered in Cyprininae, and had a corresponding layered astroglial system, which left almost free of GFAP the zones of sensory and motor neurons. In conclusion, despite the diversity and evolutionary alterations of Actinopterygii brains, the diversity of the astroglial architecture is moderate. In contrast to Chondrichthyes and Amniotes; in Actinopterygii true astrocytes (stellate-shaped extraependymal cells) did not appear during evolution, and the expansion of GFAP-free areas was limited.

The association between lateral‐line canals and skull bones in fishes has been the subject of several studies and raised a series of controversies, particularly with regard to the hypothesized role of lateral‐line organs (i.e. neuromasts) in osteogenesis and the consequences for hypotheses of homology of the bones associated with lateral‐line canals. Polypteridae, a group of freshwater fishes that occupies a key phylogenetic position as the most basal extant lineage of ray‐finned fishes (Actinopterygii), provides an interesting model for the study of the relationships between lateral‐line canals and skull bones. We describe the development of bones associated with lateral‐line canals in the Senegal Bichir, Polypterus senegalus, and use these data to re‐address previous hypotheses of homology of skull bones of polypterids. We demonstrate that the lateral‐line canals constitute a separate component of the dermatocranium that may interact with a membranodermal component, thereby forming compound bones in the adult. Differences in the interactions between these components determine the characteristics of the development of each independent bone in the skull of adult P. senegalus. Our results shed light on long‐standing controversies about the identity of skull bones such as the rostral, preopercle, and sphenotic in Polypteridae, and suggest the presence of an ancestral two‐component pattern of formation of bones associated with lateral‐line canals in bony fishes. These findings reveal the need to re‐address previous hypotheses of homology of bones associated with lateral‐line canals in different groups of bony fishes, especially fossil taxa.

Despite the wide variety of adaptive modifications in the oral and facial regions of vertebrates, their early oropharyngeal development is considered strictly uniform. It involves sequential formation of the mouth and pharyngeal pouches, with ectoderm outlining the outer surface and endoderm the inner surface, as a rule. At the extreme anterior domain of vertebrate embryos, the ectoderm and endoderm directly juxtapose and initial development of this earliest ecto-endoderm interface, the primary mouth, typically involves ectodermal stomodeal invagination that limits the anterior expansion of the foregut endoderm. Here we present evidence that in embryos of extant non-teleost fishes, oral (stomodeal) formation is preceded by the development of prominent pre-oral gut diverticula (POGD) between the forebrain and roof of the forming mouth. Micro-computed tomography (micro-CT) imaging of bichir, sturgeon and gar embryos revealed that foregut outpocketing at the pre-oral domain begins even before the sequential formation of pharyngeal pouches. The presence of foregut-derived cells in the front of the mouth was further confirmed by in vivo experiments that allowed specific tracing of the early endodermal lining. We show that POGD in sturgeons contribute to the orofacial surface of their larvae, comprising oral teeth, lips, and sensory barbels. To our knowledge, this is the first thorough evidence for endodermal origin of external craniofacial structures in any vertebrate. In bichir and gar embryos, POGD form prominent cranial adhesive organs that are characteristic of the ancient bauplan of free-living chordate larvae. POGD hence seem arguably to be ancestral for all ray-finned fishes, and their topology, pharyngeal-like morphogenesis and gene expression suggest that they are evolutionarily related to the foregut-derived diverticula of early chordate and hemichordate embryos. The formation of POGD might thus represent an ancestral developmental module with deep deuterostome origins.

Polypterus senegalus Cuvier, 1829 is one of the most basal living actinopterygian fish and a member of the Actinopterygii. We analyzed the spatial and temporal pattern of cranial muscle development of P. senegalus using whole-mount immunostaining and serial sectioning. We described the detailed structure of the external gill muscles which divided into dorsal and ventral parts after yolk exhaustion. The pattern of the division is similar to that of urodeles. We suggest that, the external gill muscles of P. senegalus are involved in spreading and folding of the external gill stem and the branches. The fibers of the external gill muscles appear postero-lateral to the auditory capsule. In addition, the facial nerve passes through the external gills. Therefore, the external gill muscles are probably derived from the m. constrictor hyoideus dorsalis. In contrast to previous studies, we described the mm. interhyoideus and hyohyoideus fibers as independent components in the yolk-sac larvae. The m. hyohyoideus fibers appear lateral to the edge of the ventral portion of the external gill muscles, which are probably derived from the m. constrictor hyoideus dorsalis. These findings suggest that the m. hyohyoidues is derived from the m. constrictor hyoideus dorsalis in P. senegalus. In other actinopterygians, the m. hyohyoideus is derived from the m. constrictor hyoideus ventralis; therefore, the homology of the m. hyohyoidues of P. senegalus and other actinopterygians remains unclear. J. Morphol. 278:450-463, 2017. © 2017 Wiley Periodicals, Inc.

In most vertebrates, pharyngeal arches form in a stereotypic anterior-to-posterior progression. To gain insight into the mechanisms underlying evolutionary changes in pharyngeal arch development, here we investigate embryos and larvae of bichirs. Bichirs represent the earliest diverged living group of ray-finned fishes, and possess intriguing traits otherwise typical for lobe-finned fishes such as ventral paired lungs and larval external gills. In bichir embryos, we find that the anteroposterior way of formation of cranial segments is modified by the unique acceleration of the entire hyoid arch segment, with earlier and orchestrated development of the endodermal, mesodermal, and neural crest tissues. This major heterochronic shift in the anteroposterior developmental sequence enables early appearance of the external gills that represent key breathing organs of bichir free-living embryos and early larvae. Bichirs thus stay as unique models for understanding developmental mechanisms facilitating increased breathing capacity.

Cladistians are a group of basal actinopterygian fishes that constitute a good model for studying primitive brain features, most likely present in the ancestral bony fishes. The analysis of the nitrergic neurons (with the enzyme nitric oxide synthase; NOS) has helped in understanding important aspects of brain organization in all vertebrates studied. We investigated the nitrergic system of two cladistian species by means of specific antibodies against NOS and NADPH-diaphorase (NADPH-d) histochemistry, which, with the exception of the primary olfactory and terminal nerve fibers, labeled only for NADPH-d, yielded identical results. Double immunohistochemistry was conducted for simultaneous detection of NOS with tyrosine hydroxylase, choline acetyltransferase, calbindin, calretinin, and serotonin, to establish accurately the localization of the nitrergic neurons and fibers and to assess possible interactions between these neuroactive substances. The pattern of distribution in both species showed only subtle differences in the density of labeled cells. Distinct groups of NOS-immunoreactive cells were observed in pallial and subpallial areas, paraventricular region, tuberal and retromammillary hypothalamic areas, posterior tubercle, prethalamic and thalamic areas, optic tectum, torus semicircularis, mesencephalic tegmentum, interpeduncular nucleus, superior and middle reticular nuclei, magnocellular vestibular nucleus, solitary tract nucleus, nucleus medianus magnocellularis, the spinal cord and amacrine cells in the retina. Large neurons in cranial nerve sensory ganglia were also labeled. The comparison of these results with those from other vertebrates, using a neuromeric analysis, reveals a conserved pattern of organization of the nitrergic system from this primitive fish group to amniotes, including mammals.

The coelacanth, Latimeria chalumnae Smith, 1939 [1] (Sarcopterygii: Actinistia), together with the closely related L. menadoensis Pouyaud et al ., 1999 [2], remains the only living representative of one of the most basally-branching primary radiations of lobe-finned fishes (Sarcopterygii). Even though extant species cannot be considered ‘primitive’ due to the inherent logic of phylogenetic theory, the coelacanth nonetheless is invaluable for understanding evolutionary transformations in basal sarcopterygians as it can help in the determination of character polarity. The appearance of one novelty during early vertebrate evolution that had major implications for the success of a huge number of species is the origin of lungs. The conventional interpretation is that lungs evolved in basal bony fishes (Osteichthyes or Osteognathostomata), were maintained in the lobe-finned fishes, and eventually were transformed into a swimbladder among the ray-finned fishes (Actinopterygii) (e.g. [3]). However, the currently available data do not rule out separate origins of lungs and swimbladders from a common ‘respiratory pharynx’, even though this would require a slightly less parsimonious course of evolution [4,5]. The coelacanth is a key species in addressing this question and for this reason the data recently provided by Cupello and colleagues [6] are a very welcome addition to the discussion. Here, I would like to add a few points pertinent to lung evolution that appear to be a consequence of these exciting data. One of the most interesting aspects of the coelacanth is that it apparently exhibits an unpaired structure of putative homology with lungs [6–8]. In the Polypteriformes (bichir and reed fish), the lungs are paired [5,9,10], as are those of the lungfishes (Dipnoi) [11], except the Australian lungfish, Neoceratodus forsteri (Krefft, 1870) …

It has been reported that in chondrichthyans the cardiac outflow tract is composed of the myocardial conus arteriosus, while in most teleosteans it consists of the nonmyocardial bulbus arteriosus. Classical studies already indicated that a conus and a bulbus coexist in several ancient actinopterygian and teleost groups. Recent work has shown that a cardiac outflow tract consisting of a conus and a bulbus is common to both cartilaginous and bony fishes. Nonetheless and despite their position at the base of the actinopterygian phylogenetic lineage, the anatomical arrangement of the cardiac outflow tract of the Polypteriformes remained uncertain. The present study of hearts from gray bichirs was intended to fill this gap. The cardiac outflow tract of the bichir consists of two main components, namely a very long conus arteriosus, furnished with valves, and a short, intrapericardial, arterial-like bulbus arteriosus, which differs from the ventral aorta because it is covered by epicardium, shows a slightly different spatial arrangement of the histological elements and is crossed by coronary arteries. Histomorphologically, the outflow tract consists of three longitudinal regions, distal, middle and proximal, an arrangement which has been suggested to be common to all vertebrates. The distal region corresponds to the bulbus, while the conus comprises the middle and proximal regions. The present findings reinforce the notion that the bulbus arteriosus of fish has played an essential role in vertebrate heart evolution as it is the precursor of the intrapericardial trunks of the aorta and pulmonary artery of birds and mammals.

Summary The occurrence of pigment cells in the heart is well documented in amphibians, birds and mammals. By contrast, information on heart pigmentation in fish is extremely sparse. The aim is to report the presence of pigment cells over the entire surface of the heart in the gray bichir, Polypterus senegalus. The sample consisted of 12 hearts, which, after gross anatomical examination, were studied using histochemical and immunohistochemical techniques for light microscopy, and transmission electron microscopy. The pigment cells were located in the subepicardium, showing a regular distribution pattern across the whole heart, except for the anterior end of the outflow tract, where the pigmentation was much more intense. The cells contained dark, ovoid-shaped organelles which was consistent with a melanosome cell identity. As in other vertebrates, the physiological role of the pigment cells in the heart of the gray bichir is unknown. The absence of such cells in hearts of other polypteriforms suggests that cells containing melanin are not essential for normal fish heart function. Basing on literature data concerning tetrapods, it can be inferred that the pigment cells of the heart of the gray bichir derive from the neural crest. If this were true, our findings would provide the first evidence for the presence of neural crest-derived cells in the subepicardium of adult hearts of early actinopterygians.

Most actinopterygians replace their teeth continuously throughout life. To address the question of where and how replacement teeth form in actinopterygians, it is advisable to investigate well-chosen representatives within the lineage. The African bichir, Polypterus senegalus, belongs to the earliest diverged group of the actinopterygian lineage with currently living representatives. Its well characterized dentition, together with its phylogenetic position, make this species an attractive model to answer following questions: (1) when and where does the replacement tooth form and how is it connected with the dental organ of the predecessor, and (2) is there any evidence for the presence of epithelial stem cells, hypothesized to play a role in replacement? Serial sections show that one tooth family can contain up to three members, which are all interconnected by dental epithelium. Replacement teeth develop without the presence of a successional dental lamina. We propose that this is the plesiomorphic condition for tooth replacement in actinopterygians. BrdU pulse-chase experiments reveal cells in the outer and middle dental epithelium, proliferating at the time of initiation of a new replacement tooth. It is tempting to assume that these cell layers provide a stem cell niche. The observed absence of label-retaining cells after long chase times (up to 8 weeks) is held against the light of divergent views on cell cycling properties of stem cells. At present, our data do not support, neither reject, the hypothesis on involvement of epithelial stem cells within the process of continuous tooth replacement. J. Exp. Zool. (Mol. Dev. Evol.) 322B: 281–293, 2014. © 2014 Wiley Periodicals, Inc.

Cladistians (Polypteriformes) are currently considered basal to other living ray-finned fishes (actinopterygians), and their brain organization is therefore critical to providing information about the primitive neural characters that existed in the earliest ray-finned fishes. The organization of the serotonergic system in the brain has been carefully analyzed in most vertebrate groups, and in the present study we provide the first detailed information on the distribution of serotonergic cell bodies and fibers in the central nervous system of representative species of the two extant genera of cladistians, i.e. Polypterus senegalus and Erpetoichthys calabaricus, by means of immunohistochemistry against serotonin (5-HT). Distinct groups of immunoreactive cells were detected in the preoptic area, the hypothalamic paraventricular organ, the pineal organ, the pretectal region, the long column of the raphe in the rhombencephalic midline, the spinal cord, and amacrine cells in the inner nuclear layer of the retina. Fiber labeling was widely distributed in all main brain subdivisions but was more abundant in distinct pallial and subpallial areas, the preoptic area, the thalamus, the optic tectum, the tori semicircularis and lateralis, the rhombencephalic reticular formation, the nucleus of the solitary tract, and the dorsal aspect of the spinal cord. Our analysis makes it possible to establish which serotonergic structures characterized the earliest ray-finned fishes, and a comparison of these results with those from other classes of vertebrates, including a segmental analysis to correlate cell populations, reveals that most characteristics, such as the presence of serotonergic cells in the preoptic area and the basal hypothalamus, are preserved in all anamniotes. However, this system seems to be reduced in amniotes, mainly mammals, although important features are shared, such as the presence of serotonergic cells in the pineal organ, the retina, and the raphe nuclei.

Polypteriform fishes are believed to be basal to other living ray-finned bony fishes, and they may be useful for providing information of the neural organization that existed in the brain of the earliest ray-finned fishes. The calcium-binding proteins calretinin (CR) and calbindin-D28k (CB) have been widely used to characterize neuronal populations in vertebrate brains. Here, the distribution of the immunoreactivity against CR and CB was investigated in the olfactory organ and brain of Polypterus senegalus and compared to the distribution of these molecules in other ray-finned fishes. In general, CB-immunoreactive (ir) neurons were less abundant than CR-ir cells. CR immunohistochemistry revealed segregation of CR-ir olfactory receptor neurons in the olfactory mucosa and their bulbar projections. Our results confirmed important differences between pallial regions in terms of CR immunoreactivity of cell populations and afferent fibers. In the habenula, these calcium-binding proteins revealed right-left asymmetry of habenular subpopulations and segregation of their interpeduncular projections. CR immunohistochemistry distinguished among some thalamic, pretectal, and posterior tubercle-derived populations. Abundant CR-ir populations were observed in the midbrain, including the tectum. CR immunoreactivity was also useful for characterizing a putative secondary gustatory/visceral nucleus in the isthmus, and for distinguishing territories in the primary viscerosensory column and octavolateral region. Comparison of the data obtained within a segmental neuromeric context indicates that some CB-ir and CR-ir populations in polypteriform fishes are shared with other ray-finned fishes, but other positive structures appear to have evolved following the separation between polypterids and other ray-finned fishes.

The cholinergic system in the brain has been widely studied in most vertebrate groups, but there is no information available about this neurotransmission system in the brains of holostean fishes, a primitive and poorly understood group of actinopterygian fishes. The present study provides the first detailed information on the distribution of cholinergic cell bodies and fibers in the central nervous system in two holostean species, the Florida gar, Lepisosteus platyrhincus, and the bowfin, Amia calva. Immmunohistochemistry against the enzyme choline acetyltransferase (ChAT) revealed distinct groups of ChAT-immunoreactive (ChAT-ir) cells in the habenula, isthmic nucleus, laterodorsal tegmental nucleus, octavolateral area, reticular formation, cranial nerve motor nuclei and the motor column of the spinal cord, all of which seem to be highly conserved among vertebrates. Some ChAT-ir cells were detected in the basal telencephalon that appear in actinopterygians for the first time in the evolution of this neurotransmission system, whereas the remarkable cholinergic population in the optic tectum is a peculiar characteristic, the presence of which varies throughout evolution, although it is present in all teleosts studied. Abundant cholinergic fibers were found in the pretectal region and optic tectum, where they probably modulate vision, and in the hypothalamus and the interpeduncular neuropil. Some interspecific differences were also observed, such as the presence of ChAT-ir cells in the supraoptoparaventricular band only in Lepisosteus and in in the nucleus subglomerulosus only in Amia. In addition, ChAT-ir fibers in the olfactory bulb were detected only in Amia. Comparison of these results with those from other classes of vertebrates, and a segmental analysis to correlate cell populations, reveal that the pattern of the cholinergic system in holosteans is very close to that in ancestral actinopterygian fishes, as recently described in the bichir (Cladistia), although an important evolutionary novelty in holosteans is the presence of cholinergic cells in the basal telencephalon.

As a member of the most basal clade of extant ray-finned fishes (actinopterygians) and of one of the most basal clades of osteichthyans (bony fishes + tetrapods), Polypterus can provide insights into the ancestral anatomy of both ray-finned and lobe-finned fishes, including those that gave rise to tetrapods. The pectoral fin of Polypterus has been well described but, surprisingly, neither the bones nor the muscles of the pelvic fin are well known. We stained and dissected the pelvic fin of Polypterus senegalus and Polypterus delhezi to offer a detailed description of its musculoskeletal anatomy. In addition to the previously described adductor and abductor muscles, we found preaxial and postaxial muscles similar to those in the pectoral fin of members of this genus. The presence of pre- and postaxial muscles in both the pectoral and pelvic fins of Polypterus, combined with recent descriptions of similar muscles in the lobe-finned fishes Latimeria and Neoceratodus, suggests that they were present in the most recent common ancestor of bony fishes. These results have crucial implications for the evolution of appendicular muscles in both fish and tetrapods.

The lung is an important organ for air breathing in tetrapods and originated well before the terrestrialization of vertebrates. Therefore, to better understand lung evolution, we investigated lung development in the extant basal actinopterygian fish Senegal bichir (Polypterus senegalus). First, we histologically confirmed that lung development in this species is very similar to that of tetrapods. We also found that the mesenchymal expression patterns of three genes that are known to play important roles in early lung development in tetrapods (Fgf10, Tbx4 and Tbx5) were quite similar to those of tetrapods. Moreover, we found a Tbx4 core lung mesenchyme-specific enhancer (C-LME) in the genomes of bichir and coelacanth (Latimeria chalumnae) and experimentally confirmed that these were functional in tetrapods. These findings provide the first molecular evidence that the developmental program for lung was already established in the common ancestor of actinopterygians and sarcopterygians.

Hatching gland cells (HGCs) originate from different germ layers between frogs and teleosts, although the hatching enzyme genes are orthologous. Teleostei HGCs differentiate in the mesoendodermal cells at the anterior end of the involved hypoblast layer (known as the polster) in late gastrula embryos. Conversely, frog HGCs differentiate in the epidermal cells at the neural plate border in early neurula embryos. To infer the transition in the developmental origin of HGCs, we studied two basal ray-finned fishes, bichir (Polypterus) and sturgeon. We observed expression patterns of their hatching enzyme (HE) and that of three transcription factors that are critical for HGC differentiation: KLF17 is common to both teleosts and frogs; whereas FoxA3 and Pax3 are specific to teleosts and frogs, respectively. We then inferred the transition in the developmental origin of HGCs. In sturgeon, the KLF17, FoxA3, and HE genes were expressed during the tailbud stage in the cell mass at the anterior region of the body axis, a region corresponding to the polster in teleost embryos. In contrast, the bichir was suggested to possess both teleost- and amphibian-type HGCs, i.e. the KLF17 and FoxA3 genes were expressed in the anterior cell mass corresponding to the polster, and the KLF17, Pax3 and HE genes were expressed in dorsal epidermal layer of the head. The change in developmental origin is thought to have occurred during the evolution of basal ray-finned fish, because bichir has two HGCs, while sturgeon only has the teleost-type.

Epimorphic regeneration of fins was studied in different ray-finned fishes (Actinopterygii), but species representing the phylogenetically basal lineages of the taxon have remained outside the attention of researchers. Information on the regenerative abilities of these groups is important both for understanding the evolutionary origins of the epimorphic regeneration phenomenon and for assessing the universality of regenerative potencies in Actinopterygii. Addressing this problem, we studied for the first time fin regeneration in two members of the archaic family Polypteridae: the ropefish (Erpetoichthys calabaricus) and the Senegal bichir (Polypterus senegalus). Along with the ability to regenerate the bony rays of fins, widespread among Actinopterygii, polypterids show the ability to effectively regenerate the endoskeleton and musculature of their fins. This unusual feature allows us to suggest polypterids as new model organisms for the study of the mechanisms of vertebrate limb regeneration.

Summary Polypterid fishes are considered the basal-most group of extant actinopterygians and thus may serve as a direct link to understand the evolution of the first bony fishes. Embryonic and larval specimens, however, are extremely scarce, making it difficult to study their developmental patterns and processes. During the past few years we collected many embryos and larvae of the Senegal bichir Polypterus senegalus and in this paper we describe some novel observations concerning the supposed ecto-endoderm border in the mouth and consecutive initiation of dental development. The mouth of the Senegal bichir does not develop via a classical oropharyngeal membrane but instead, opening the mouth occurs via a separation of the upper and lower jaws that are connected by epithelial bridges. These structures are bilaterally symmetrical and are found invariantly at places of the earliest tooth bud development. It is suggested that the epithelial bridges may represent the ecto-endoderm bordering zone, are both structurally and functionally homologous to the oropharyngeal membrane and consequently it is hypothesized that the epithelial bridges and the developmental factors producing them play a key role in initiation and early distribution of the particular dental domains.

Amphibian holoblastic cleavage in which all blastomeres contribute to any one of the three primary germ layers has been widely thought to be a developmental pattern in the stem lineage of vertebrates, and meroblastic cleavage to have evolved independently in each vertebrate lineage. In extant primitive vertebrates, agnathan lamprey and basal bony fishes also undergo holoblastic cleavage, and their vegetal blastomeres have been generally thought to contribute to embryonic endoderm. However, the present marker analyses in basal ray-finned fish bichir and agnathan lamprey embryos indicated that their mesoderm and endoderm develop in the equatorial marginal zone, and their vegetal cell mass is extraembryonic nutritive yolk cells, having non-cell autonomous meso-endoderm inducing activity. Eomesodermin (eomes), but not VegT, orthologs are expressed maternally in these animals, suggesting that VegT is a maternal factor for endoderm differentiation only in amphibian. The study raises the viewpoint that the lamprey/bichir type holoblastic development would have been ancestral to extant vertebrates and retained in their stem lineage; amphibian-type holoblastic development would have been acquired secondarily, accompanied by the exploitation of new molecular machinery such as maternal VegT.

Diplogyrodactylus martini n. g. et sp., a viviparous monogenean, is described from the gills of gray bichir Polypterus senegalus Cuvier, 1829, in Senegal. This new genus can be readily distinguished from the other described viviparous genera based on the morphology of the attachment apparatus. The haptor is equipped with a pair of centrally positioned hamuli, a simple ventral bar without membrane and 8 pairs of marginal hooks of 2 types, 5 pairs of hooks with well-defined large falculate sickles, and 3 pairs of smaller hooks with well-articulated sickles. The new genus is unique in having a teardrop-shaped muscular tubular male copulatory organ which lacks spines. A partial sequence of the internal transcribed spacer (ITS rDNA) was obtained from 4 specimens of D. martini. The relationship among D. martini and other genera of the Gyrodactylidae is discussed based on the similarities of the attachment apparatus.

There is general agreement that the polypteriform fishes, like Polypterus senegalus, constitute a unique lineage in the evolution of the vertebrates. However, the precise position of these fishes had been a point of controversy since the time of Darwin and Huxley. There is now consensus that the polypteriform fishes are members of superorder Actinopterygii. However, within the Actinopterygii, it is still debatable as to whether the polypteriform fishes are an early offshoot of the Actinopterygii or a more recent sister group to the sturgeon and other extant chondrostean fishes. In this study the sequence of proopiomelanocortin (POMC), the common precursor for the melanocortins and beta-endorphin, was used to evaluate the phylogenetic position of the polypteriform fishes relative to other bony fishes. 3(')RACE and 5(')RACE protocols were used to amplify overlapping regions of a POMC cDNA from the brain of P. senegalus. The full-length POMC cDNA had an open reading frame that encoded 259 amino acids. As seen in most gnathostomes, P. senegalus POMC has three melanocortin sequences (ACTH/alpha-MSH, gamma-MSH, and beta-MSH), and a beta-endorphin region. For phylogenetic analysis, the following POMC sequences were aligned at the amino acid level and analyzed using a maximum parsimony algorithm: P. senegalus, dogfish, sturgeon A, paddlefish A, sockeye salmon A, tilapia, and gar. The dogfish POMC sequence was used as the out-group. In this analysis the P. senegalus POMC sequence formed a clade with the chondrostean POMC sequences (sturgeon A and paddlefish A), and not with the neopterygian sequences (sockeye salmon A, tilapia, and gar). P. senegalus POMC is remarkably similar to sturgeon POMC A. In particular, in both precursors there is evidence for degeneration at the proteolytic cleavage site that precedes the gamma-MSH sequence. Based on the analysis of this nuclear gene it would appear that P. senegalus belongs to a branch of the chrondrostean lineage rather than representing a lineage of ray-finned fish that is ancestral to the chondrostean and neoptyergian ray-finned fishes. Alternatively, if the polypteriform fishes are in fact an early offshoot of the Actinopterygii (the traditional view), then the observations made for P. senegalus POMC relative to the chondrostean POMC sequences is the result of convergence.

The connections of the dorsal telencephalon (pallium) of the Senegal bichir (Polypterus senegalus) were investigated in order to test the hypothesis that the simple everted pallium of polypterid fishes is directly comparable to the evaginated pallium of most vertebrates. Neuroanatomical tracers (DiI or biotinylinated dextran amines) were injected into the three traditionally recognized divisions of the bichir pallium to determine their afferent and efferent connections. The connections were corroborated with injections into regions identified as projecting to, or receiving input from, one or more pallial divisions. The results suggest that the bichir pallium consists not of three, but only two primary divisions: a dorsomedial one and a dorsolateral one. This reinterpretation refutes the hypothesis of a direct one-to-one comparison with the tripartite pallium of most vertebrates. Homologues of the bichir dorsomedial and dorsolateral pallium are recognized in the lateral and medial pallium of anuran amphibians, respectively. The pallium of bichirs is compared to that of derived ray-finned fishes as a link between derived ray-finned fishes and other vertebrates. The available information on the connections of the pallium of teleosts suggests that only the olfactory recipient (pars posterior) of the pallium can be directly compared to bichirs and amphibians and that the remaining divisions of the pallium in teleosts are uniquely derived features of the teleost telencephalon.

Cartilaginous fishes (chondrichthyans) have traditionally been taken as an early offshoot among jawed vertebrates. To examine some crucial chondrichthyan relationships, we have sequenced the mitochondrial genomes of the holocephalan Chimaera monstrosa (ratfish) and the basal galeomorph species Heterodontus francisci (horn shark) and analysed them together with the corresponding data set of several other chondrichthyans, teleosts, the coelacanth, the African lungfish and the bichir. The rooting point of the tree was established using unequivocal outgroups, the sea lamprey , the sea lancelet or echinoderms. The phylogenetic analyses identified monophyletic Chondrichthyes in a terminal position in the piscine tree, lending no support to the traditionally accepted basal position of cartilaginous fishes among extant gnathostomes. The findings suggest that the cartilage characterizing extant chondrichthyans is a retention of an embryonic condition, thus representing a derived rather than a primitive phylogenetic and developmental stage. Similarly, the analyses suggest that the open gill slits of neoselachians (sharks and rays) constitute a derived state compared to the operculum (gill cover) characterizing bony fishes and holocephalans. The analyses did not support the so-called Squalea/Galea hypothesis which posits that batomorphs (sharks, rays) have arisen from recent selachians (sharks). Inconsistent with the common understanding of piscine and gnathostome evolution, the two taxa having lungs, the African lungfish and the bichir, had a basal position in the piscine tree. The findings put into question the phylogenetic validity of the taxonomic nomenclature attributed to various vertebrate, notably piscine, clades.

The distribution of serotonin-immunoreactive (5HT-IR) neurons was studied in the brain of a primitive bony fish, Polypterus senegalus. The diencephalon contained a prominent 5HT-IR cell group (G2) consisting mainly of liquor-contacting neurons in the hypothalamic periventricular wall including the paraventricular organ. Their ependymofugal processes arborized profusely and gave rise to dense networks of varicose fibers in the hypothalamus. Other groups of 5HT-IR cells were situated in the preoptic area including the periventricular preoptic nucleus (G1) and in the dorsal thalamus (G3). In the brain stem, 5HT-IR cells were found in the medial part, mainly in the superior raphe nucleus (G4). No 5HT-IR cells were demonstrated in other regions of the brain. The cell groups referred to as G1 and G4 in the present study seem to be new components comprising the serotonergic systems in the Polypterus brain.

Peptide tyrosine–tyrosine (PYY) has been isolated from the intestines of two species of reptile, the desert tortoise Gopherus agassizii (Testudines) and the Burmese python Python molurus (Squamata), from the primitive Actinopterygian fish, the bichir Polypterus senegalis (Polypteriformes) and from two agnathans, the Southern-hemisphere lamprey Geotria australis (Geotriidae) and the holarctic lamprey Lampetra fluviatilis (Petromyzontidae). The primary structure of bichir PYY is identical to the proposed ancestral sequence of gnathostome PYY (YPPKPENPGE10 | DAPPEELAKY20 | YSALR HYINL30 | ITRQRY). Tortoise and python PYY differ by six and seven residues, respectively, from the ancestral sequence consistent with the traditional view that the Testudines represent an earlier divergence from the primitive reptilian stock than the Squamates. The current views of agnathan phylogeny favor the hypothesis that the Southern-hemisphere lampreys and the holarctic lampreys arose from a common ancestral stock but their divergence is of a relatively ancient (pre-Tertiary) origin. The Geotria PYY-related peptide shows only two amino acid substitutions (Pro10→Gln and Leu22→Ser) compared with PYY from the holarctic lamprey Petromyzon marinus. This result was unexpected as Petromyzon PYY differs from Lampetra PYY deduced from the nucleotide sequence of a cDNA (Soderberg et al. J. Neurosci. Res. 1994;37:633–640) by 10 residues. However, a re-examination of an extract of Lampetra intestine revealed the presence of a PYY that differed in primary structure from Petromyzon PYY by only one amino acid residue (Pro10→Ser). This result suggests that the structure of PYY has been strongly conserved during the evolution of Agnatha and that at least two genes encoding PYY-related peptides are expressed in Lampetra tissues.

The distribution of neuropeptide Y (NPY)-like immunoreactivity in the brain of the bichir, Polypterus senegalus, was examined immunohistochemically. NPY-like immunoreactivity was distributed widely in the brain, with the highest density in the diencephalon. NPY-positive perikarya were found in various areas, including the terminal nerve, the pallial zones of the telencephalon, the periventricular preoptic nucleus, the thalamic nucleus, the ventral hypothalamus of the diencephalon, the tegmentum of the mesencephalon, and the area intermedioventralis of the rhombencephalon. In the hypothalamus, NPY-positive liquor-contacting neurons were frequently observed. Immunoreactive neuron-like cells also appeared in the distal lobe of the hypophysis. NPY fibers were densely distributed in the ventral telencephalon, the hypothalamus, and the ventrolateral area of the rhombencephalon. They were also demonstrated in the terminal nerve. In the hypophysis, NPY fibers were dense in the median eminence, but sparse in the neural lobe. Electron-microscopic double immunostaining of the terminal nerve revealed the coexistence of NPY-like antigen with gonadotropin-releasing hormone-like and molluscan cardioexcitatory tetrapeptide-like antigens in the same cytoplasmic granules. These results suggest that NPY or a related substance is involved in neuroregulation of various areas of the bichir brain, by mainly acting as a neurotransmitter or neuromodulator.

The organization of the roots, ganglia and peripheral distribution of the rami of the cranial nerves of larval and juvenile Senegal bichirs was examined with a wide range of techniques, including gross dissection and histological preparations. The profundal nerve of bichirs is completely separate from the trigeminal nerve and innervates the skin overlying the orbit and snout, and there is no evidence that a superficial ophthalmic ramus of the trigeminal nerve exists. Bichirs appear to retain all six pairs of lateral line nerves that characterized the earliest gnathostomes, but the sensory ganglia of the anterodorsal and otic lateral line nerves have fused, and the sensory ganglia of the anterodorsal and anteroventral lateral line nerves are partially fused, which appears to be a shared derived character of ray-finned fishes. The anteroventral lateral line nerve of bichirs retains mandibular and opercular rami, a primitive feature shared with other bony fishes, and the fibers of the opercular ramus innervate all postotic ampullary organs. Surprizingly, bichirs, unlike most nonteleost bony fishes, have lost the spiracular organ. The lateral ramus of the posterior lateral line nerve of bichirs is divided into a pars dorsalis and a pars ventralis, which appears to be a shared derived character of bony fishes. The glossopharyngeal and vagal nerves of bichirs exhibit lateral and medial sensory ganglia, and this appears to be a shared derived character of bony fishes. The posttrematic rami of the glossopharyngeal nerve and both the pre- and posttrematic rami of the vagal nerve of bichirs are divided, which appears to be a shared derived feature of bony fishes. The hypobranchial nerve of bichirs is unusual in that it is formed by only two spinal nerves, a pattern also seen in gars, but not other bony fishes, and apparently a uniquely derived character.

Polypterid bony fishes are believed to be basal to other living ray-finned fishes, and their brain organization is therefore critical in providing information as to primitive neural characters that existed in the earliest ray-finned fishes. The cholinergic system has been characterized in more advanced ray-finned fishes, but not in polypterids. In order to establish which cholinergic neural centers characterized the earliest ray-finned fishes, the distribution of choline acetyltransferase (ChAT) is described in Polypterus and compared with the distribution of this molecule in other ray-finned fishes. Cell groups immunoreactive for ChAT were observed in the hypothalamus, the habenula, the optic tectum, the isthmus, the cranial motor nuclei, and the spinal motor column. Cholinergic fibers were observed in both the telencephalic pallium and the subpallium, in the thalamus and pretectum, in the optic tectum and torus semicircularis, in the mesencephalic tegmentum, in the cerebellar crest, in the solitary nucleus, and in the dorsal column nuclei. Comparison of the data within a segmental neuromeric context indicates that the cholinergic system in polypterid fishes is generally similar to that in other ray-finned fishes, but cholinergic-positive neurons in the pallium and subpallium, and in the thalamus and cerebellum, of teleosts appear to have evolved following the separation of polypterids and other ray-finned fishes.

The chemical components of nerve membranes of primitive bony fishes (coelacanth (Latimeria chalumnae), lungfish (Lepidosiren paradoxa and Protopterus aetiopicus), bichir (Polypterus senegalus), and sturgeon (Acipenser ruthenus)) were analyzed to investigate their phylogenetic relations. Representative myelin components such as cerebroside and myelin proteins and enzyme exhibited characteristic profiles in the different species. Coelacanth was characterized by an abundance of hydroxy components of cerebroside fatty acids and by the occurrence of a 28 000 molecular weight band (possibly P0-like protein) and proteolipid protein (PLP). Lungfish possessed glycosylated PLP, but lacked PLP; bichir and sturgeon showed the same protein compositions, PLP and P0-like protein being major components. The activity of 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNP), a myelin marker enzyme, was detected in coelacanth myelin, but was almost negligible in the others. The ganglioside compositions were simple in coelacanth and lungfish compared with those in bichir and sturgeon, which were abundant in polysialo components. These findings suggest that coelacanth nerve membranes are the most developed in these species; thus, coelacanth may be more advanced phylogenetically than the others. Bichir and sturgeon were suggested to occupy unique phylogenetic positions, showing similar chemical compositions of nerve membranes which were distinct from those of coelacanth and lungfish.

Anatomical and functional studies of the autonomic innervation in the lung and the heart of the bichirs are lacking. The present review paper describes the presence of nerve fibers located in the muscle layers of the lung and its submucosa, the collection of unipolar neurons found in the submucosal and muscle layers of the glottis in a bichir species (Polypterus bichir bichir). Putative oxygen chemoreceptive, neuroepithelial cells (NECs) in the lung mucosa are also included. The latter share many immunohistochemical characteristics similar to those observed in the carotid body and neuroepithelial bodies of mammals. A packed collection of paraganglion cells is located within the trunk of the pulmonary vagus nerves. The paper also examines the occurrence of intracardiac neurons and nerve fibers in the heart of the above species. These studies show that various neurotransmitters may indicate different patterns of innervation in the lung and the heart of the bichirs. However, there is still much to be discovered about the lung and cardiovascular nervous control of these primitive fishes.

Anatomical and physiological studies of cardiovascular control are lacking in the ray-finned fish, the bichirs. The present immunohistochemical studies on the bichir (Polypterus bichir bichir) demonstrated the occurrence of intracardiac neurons and nerve fibers in the heart. Immunoreactivity to tyrosine hydroxylase (TH) and acetylcholinesterase (AchE) and various neuropeptides (substance P, galanin, vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP)), including neuronal nitric oxide synthase (nNOS), was found in the nerve cell bodies lying close to the Sinus venosus and the sino-atrial region. The main intracardiac localization of the nervous tissue is a network of nerve fibers, presumably corresponding to the postganglionic outflow giving rise to nerve terminals and the nerve cell bodies. In addition, the heart is innervated by extrinsic monoamine-containing nerve fibers supplying the Conus arteriosus and Sinus venosus, and substance P and galanin immunopositive fibers probably originating from cranial and spinal ganglia. The adrenergic innervation of the heart of the bichir is similar to that of the teleosts, but further studies are required on nervous control of the heart.

To assess evolutional changes in the expression pattern of Otx paralogues, expression analyses were undertaken in fugu, bichir, skate and lamprey. Together with those in model vertebrates, the comparison suggested that a gnathostome ancestor would have utilized all of Otx1, Otx2 and Otx5 paralogues in organizer and anterior mesendoderm for head development. In this animal, Otx1 and Otx2 would have also functioned in specification of the anterior neuroectoderm at presomite stage and subsequent development of forebrain/midbrain at somite stage, while Otx5 expression would have already been specialized in epiphysis and eyes. Otx1 and Otx2 functions in anterior neuroectoderm and brain of the gnathostome ancestor would have been differentially maintained by Otx1 in a basal actinopterygian and by Otx2 in a basal sarcopterygian. Otx5 expression in head organizer and anterior mesendoderm seems to have been lost in the teleost lineage after divergence of bichir, and also from the amniotes after divergence of amphibians as independent events. Otx1 expression was lost from the organizer in the tetrapod lineage. In contrast, in a teleost ancestor prior to whole genome duplication, Otx1 and Otx2 would have both been expressed in the dorsal margin of blastoderm, embryonic shield, anterior mesendoderm, anterior neuroectoderm and forebrain/midbrain, at respective stages of head development. Subsequent whole genome duplication and the following genome changes would have caused different Otx paralogue usages in each teleost lineage. Lampreys also have three Otx paralogues; their sequences are highly diverged from gnathostome cognates, but their expression pattern is well related to those of skate Otx cognates.

Polypterid fishes are considered the basalmost group of extant actinopterygians and may be a direct link to understanding the systematics and evolution of the first bony fishes. Several investigations have been conducted on one member genus, Polypterus; however, since the first specimens of its sister taxon Erpetoichthys calabaricus were described, remarkably little work has been done on the species. We review terminology critical to understanding cranial morphology in polypterids and present a new description of the skull of E. calabaricus as observed through classical methods of skeletal preparation, X-radiographic microfocus computed tomography, and 3D-digital reconstruction. Differences among E. calabaricus and at least three species of Polypterus (P. bichir, P. senegalus, and P. endlicheri), besides the gross variation in size, include an overall elongation of the skull roof observable in most elements of E. calabaricus with a shortening of most associated processes. In addition, several elements present in species of Polypterus are absent in E. calabaricus. As a result, Polypterus should not be used as a proxy for the family Polypteridae to the exclusion of E. calabaricus in phylogenetic studies, which examine early actinopterygians. Each should be treated separately, to resolve inter- and intrarelationships of Polypteridae.

Anatomical and functional studies of the autonomic innervation in the lung of dipnoan fishes and the bichirs are lacking. The present immunohistochemical studies demonstrated the presence of nerve fibers in the muscle layers of the lung of the bichir, Polypterus bichir bichir, and identified the immunoreactive elements of this innervation. Tyrosine hydroxylase, acetylcholinesterase, and peptide immunoreactivity was detected in the intramural nerve fibers. Extensive innervation was present in the submucosa where adenylatecyclase/activating polypeptide 38, substance P, P2X2, and 5-hydroxytryptamine (5-HT)–immunoreactive nerve fibers mainly supplied blood vessels. A collection of monopolar neurons located in the submucosal and the muscular layers of the glottis expressed a variety of various transmitters. These neurons may be homologous to ganglion cells in the branchial and pharyngeal rami of the vagus in fishes. Nerves containing 5-HT and P2X2 receptor immunoreactivity projected to the lung epithelium. Associated with neuroepithelial cells in mucociliated epithelium, were neuronal nitric oxide synthase–immunopositive axons. The physiological function of this innervation is not known. The present study shows that the pattern of autonomic innervation of the bichir lung may by similar in its elements to that in tetrapods. Anat Rec, 290:1166–1177, 2007. © 2007 Wiley-Liss, Inc.

Immunohistochemical localisation of neurotransmitters was used to determine the distribution of unipolar neurons and neuroepithelial cells (NECs) in the respiratory tract of the bichir, Polypterus bichir bichir. NECs were commonly encountered in the mucociliated epithelium of the lung. Unipolar neurons were located in the submucosal and muscle layers of the glottis. The results suggest the presence of tyrosine hydroxylase (TH) and nNOS immunoreactivities in NECs. In addition, ACh-E/nNOS and TH/nNOS nerve fibers were also found associated with these cells. Unipolar neuronal cells showed a chemical code including the presence of 5-HT, ACh-E, peptides and P2×2 receptors. The present findings indicate that nitric oxide (NO) is a primitive transmitter of neuroepithelial oxygen-sensitive chemoreceptor cells together with acetylcholine. The coexistence of ACh-E with other substances in the unipolar neurons, but not with NO, may be a property of vagal postganglionic neurons since the emergence of the cranial autonomic pathways in the earliest vertebrates. It would be interesting to know about the provenance of the nerves in contact with NECs, which appear to have a complex innervation pattern.

It has been suggested that the increase in the number of Hox genes may have been one of the key events in vertebrate evolution. Invertebrates have one Hox cluster, while mammals have four. Interestingly, the number of Hox gene clusters is greater in the teleost fishes, zebrafish and medaka, than in mouse and human. The greater number of Hox clusters in the teleosts suggests that Hox gene duplication events have occurred during the radiation of ray-finned fishes. The question is when the Hox gene duplication event(s) that lead to seven Hox clusters in the teleosts actually occurred. We have addressed this question by studying the Hox genes in the bichir, Polypterus palmas. A preliminary PCR-estimation of the number of Hox genes suggests that Polypterus has five different Hox9 cognate group genes, which may be an indication of more than four Hox clusters in the bichir.

SUMMARYThe fast-start escape response is the primary reflexive escape mechanism in a wide phylogenetic range of fishes. To add detail to previously reported novel muscle activity patterns during the escape response of the bichir, Polypterus, we analyzed escape kinematics and muscle activity patterns in Polypterus senegalus using high-speed video and electromyography (EMG). Five fish were filmed at 250 Hz while synchronously recording white muscle activity at five sites on both sides of the body simultaneously (10 sites in total). Body wave speed and center of mass velocity, acceleration and curvature were calculated from digitized outlines. Six EMG variables per channel were also measured to characterize the motor pattern. P. senegalus shows a wide range of activity patterns, from very strong responses, in which the head often touched the tail, to very weak responses. This variation in strength is significantly correlated with the stimulus and is mechanically driven by changes in stage 1 muscle activity duration. Besides these changes in duration, the stage 1 muscle activity is unusual because it has strong bilateral activity, although the observed contralateral activity is significantly weaker and shorter in duration than ipsilateral activity. Bilateral activity may stiffen the body, but it does so by a constant amount over the variation we observed; therefore, P. senegalus does not modulate fast-start wave speed by changing body stiffness. Escape responses almost always have stage 2 contralateral muscle activity, often only in the anterior third of the body. The magnitude of the stage 2 activity is the primary predictor of final escape velocity.

The telencephalon in ray-finned fish (actinopterygians) is everted, in contrast to the evaginated telencephalic hemispheres in all other vertebrates. In the more derived ray-finned fish, the teleosts, proliferation of neurons and their migration from the ependymal zone of the pallium renders comparisons between telencephalic cell groups of the teleosts and members of other vertebrate groups extremely difficult. The telencephalon of Polypterus (a primitive living ray-finned fish), although everted, is cytoarchitecturally much simpler than that of teleosts. We have thus applied immunohistochemical techniques to the study of the telencephalon of Polypterus to help clarify the evolution of the telencephalon in teleosts and facilitate comparisons between the telencephalon in ray-finned fish and other vertebrates. Antisera against the following neuroactive substances were used: 1) serotonin (5HT), 2) tyrosine hydroxylase (TH), 3) substance P (SP), 4) leucine-enkephalin (ENK), 5) neuropeptide Y (NPY), and 6) the neurotensin-related hexapeptide LANT6. Several features of the labeling patterns obtained suggested that the dorsal and ventral subdivisions of the area ventralis are homologous as a field to the basal ganglia and septum plus other basal telencephalic regions of land vertebrates, sharks and lungfish: 1) an abundance of SP +, NPY +, and ENK+ fibers; 2) an abundance of TH+ fibers, possibly of posterior tubercle/tegmental origin; 3) the presence of an SP + fiber bundle that appeared to descend from basal telencephalic levels and terminate in the posterior tubercle/tegmentum, which contain TH + (possibly dopaminergic) neurons; and 4) an abundance of 5HT+ fibers, presumably of posterior tubercle/tegmental origin. It was not possible, however, to recognize distinct pallidal and striatal subdivisions within the area ventralis of Polypterus. The olfactory pallium (Pl) was generally poor in most of the substances examined, except for the presence of LANT6+ fibers. The P3 pallid field was conspicuously rich in SP+ and ENK+ fibers throughout its extent, and the caudal and lateral parts of the P2 field were rich in SP+ fibers and ENK+ fibers. Since this is characteristic of the medial pallial and/or dorsomedial pallial walls of the telencephalon in lungfish, sharks, frogs, and reptiles, the P3 field and caudolateral part of the P2 field may be homologous to these portions of the telencephalon in other vertebrates. More rostromedial parts of P2 may correspond to those parts of the pallium in land vertebrates that are in receipt of specific sensory input from the thalamus, since low neuropeptide levels are characteristic of these regions. These results indicate that the dorsal part of the ventral area of the telencephalon in ray-finned fish may be homologous to the basal ganglia in other vertebrates. These results also suggest that the pallial portions of the telencephalon in Polypterus may include a region comparable to part of the isocortex or its homologues in amniotes. o 1992 Wiley-Liss, Inc.

INTRODUCTIONAxis and germ-layer formations are central issues in vertebrate embryology that can be examined in the zebrafish, Xenopus, chick, and mouse. An intriguing question is how the mechanisms that existed in an ancestral vertebrate have been modified during vertebrate evolution. A major stream of vertebrates (Osteichthyes) evolved in two monophyletic lineages: the Sarcopterygii and Actinopterygii. The zebrafish is a well-known and well-studied teleost fish highly derived in the actinopterygian lineage, ray-finned fishes. The early cleavage pattern of teleosts is meroblastic, in contrast to the holoblastic cleavage seen in amphibians, which diverged early in the sarcopterygian lineage. There are many differences in the molecular and cellular mechanisms of embryogenesis between teleosts and amphibians or actinopterygians and sarcopterygians, including the differences in bauplan (body plan). However, the lineage leading to the teleost fishes has undergone a whole-genome duplication before their radiation, which may have also caused changes in molecular usage independent of the teleost bauplan. Polypterus diverged from all other actinopterygians ~400 million years ago (Mya) during the Devonian period, soon after the divarication of an ancestral bony fish into Actinopterygii and Sarcopterygii. Polypterus is thus uniquely well suited for studies assessing the ancestral state or bauplan of Osteichthyes and Actinopterygii, as well as the divergence of embryogenetic processes in teleosts and amphibians.

Polypterus mokelembembe sp. n. is described from the central Congo River basin. It is distinguished from all other Polypterus species except P. retropinnis, P. teugelsi and P. palmas palmas by its high predorsal scale count (32–37 vs. 11–28). It differs from P. teugelsi by having 57– 60 lateral line scales (vs. 63–65), from P. palmas palmas by a single large black blotch on the pectoral-fin base (vs. numerous small spots on the posterior part of the base), and from P. retropinnis by having a wider first dorsal-fin spine (6.4–8.1 vs. 4.6–6.1 % of HL), a smaller internostril distance (11.6–13.7 vs 14.3–18.0 % of HL), and fewer pectoral-fin rays (23–29 vs. 30– 34). Inasmuch as the type series (comprising three specimens) of Polypterus retropinnis Vaillant, 1899 is of mixed composition, and includes two specimens of P. mokelembembe, a lectotype is designated for P. retropinnis and a rediagnosis for that species is provided.

The ampullary organs of the bichir were examined by light and electron microscopy. Unlike most other ampullary organs, they are exclusively found in the epidermis and are never sunk into the subepidermal connective tissue. The sensory epithelium consists of sensory cells and supporting cells surrounded by mantle cells. The luminal surface of the sensory cell is provided with a cilium surrounded by several microvilli. In the apical cytoplasm are found numerous mitochondria and microtubules. In the basal part of the cell synaptic sheets or synaptic bodies opposite to afferent nerve endings are frequent.

The central projections of the olfactory bulb were studied in Polypterus using the Nauta and Fink-Heimer techniques. Two major target areas were identified in the subpallium: the lateral subpallial nucleus and the dorsal and ventral entopeduncular nuclei. The connections are predominantly, if not exclusively, ipsilateral. In the pallium a massive ipsilateral projection to the superficial third of the medial pallium was demonstrated while the remainder of the pallium was found to be free of degeneration. Thus it appears that the pallium of Polypterus is not uniform throughout, as has been suggested in the literature. This contention is also supported by an analysis of the pallial cytoarchitecture. Because the pallium of Polypterus is everted, rather than inverted and evaginated, the topographically medial pallium is in topological correspondence with the lateral pallium of tetrapods. On the basis of this topological correspondence and the similarity of afference from the olfactory bulb, it is argued that the “medial” pallium of Polypterus is homologous to the pyriform pallium of tetrapods. The findings of this study are compared to those of similar studies in teleosts, which also have an everted pallium. An apparent conflict appears and suggestions for resolving it are offered.

Electron microscopic studies of the neurohypophysis and saccus vasculosus of the bichir (Polypterus ornatipinnis) reveal an apocrine release of secretory material by ependymal cells (e.g., crown cells). The secretory material appears to migrate along the microtubular apparatus and the ciliary filaments to the cell buds. It is postulated that the formation of the buds and their release is mediated by cilial action. Secretion buds are noted in the cerebrospinal fluid and vascular sinusoids. Bulbous projections of neurohypophyseal secretory tracts extend into the ependymal lumen which also contains elementary secretion granules. Specialized “liquorkontaktneurone” are interspersed with ependymal cells. An interrelation between the hypothalamus, neurohypophysis, saccus vasculosus, and the meta-adenohypophysis is postulated.

The anatomy and ultrastructure of the sacculus, lagena, and utriculus of the ear of Polypterus bichir and Scaphirhynchus platorynchus were studied using the scanning electron microscope. The otolithic organs each contain a single dense calcareous otolith in close contact with a sensory epithelium (macula). The maculae have sensory hair cells typical of those found in other vertebrates, surrounded by microvilli-covered supporting cells. The hair cells on each macula are divided into several groups, with all of the cells in each group morphologically polarized in the same direction. The cells of the utricular macula in both species are divided into opposing groups in a pattern similar to that found in other vertebrates. The saccular and lagenar maculae are located in a single large chamber in both species. In Scaphirhychus the two maculae are on the same plane, while in Polypterus they are at right angles to one another. The hair cells on the saccular maculae of both species are divided into two oppositely oriented groups. In Scaphirhynchus the cells on the posterior half of the macula are oriented dorsally on the dorsal half of the macula and ventrally on the ventral half. The anterior region of the macula is rotated and the cells of the dorsal and ventral groups are shifted so that they are oriented on the animal's horizon plane. A similar pattern is found in Polypterus, except that this macula is shaped like a "J" with the vertical portion of the J having horizontal cells and the bottom portion vertical cells. The lagenar maculae in both species have dorsally oriented cells on the anterior side of the macula and ventrally oriented cells on the posterior half of the macula. While these data are not sufficient for clarifying the taxonomic relationship between the two species studied, it is clear that the ears in these species have a number of significant differences from the teleost ear that could have functional and/or taxonomic significance.

THE anal fin of the female of both Polypterus and Calamoichthys is narrow and pointed, whereas that of the male is wider and rounded, and can be erected into a cup-shaped organ. This diiference was described for P. bichir by Leydig1, who suggested that internal fertilization occurred, and subsequently Harrington2 stated that the anal fin of the male “is undoubtedly a copulatory organ”. Budgett3, noting sexual dimorphism of the anal fins of P. senegalus and P. bichir (= P. lapradei), wrote that “it remains to be seen to what use the male Polypterus puts this modified anal fin”, while he dismissed Leydig's suggestion that fertilization is internal. Traquair4 described sexual dimorphism of the anal fin for C. calabaricus but did not comment on the possible function of the anal fin of the male. Recent work by Arnoult5,6, who described the spawning behaviour of P. senegalus in aquaria, shows that the anal fin of the male undoubtedly forms a copulatory chamber. During spawning the male swims alongside the female with its anal fin folded up like a gutter towards the vent of the female, fertilizing the eggs as they are laid.