Relationship among coelacanths,lungfishes, and tetrapods: A phylogenetic analysis based on mitochondrial cytochrome oxidase I gene sequences

To clarify the relationship among coelacanths, lungfishes, and tetrapods, the amino acid sequences deduced from the nucleotide sequences of mitochondrial cytochrome oxidase subunit I (COI) genes were compared. The phylogenetic tree of these animals, including the coelacanth Latimeria chalumnae and the lungfish Lepidosiren paradoxa, was inferred by several methods. These analyses consistently indicate a coelacanth/lungfish clade, to which little attention has been paid by previous authors with the exception of some morphologists. Overall evidence of other mitochondrial genes reported previously and the results of this study equally support the coelacanth/lungfish and lungfish/tetrapod clades, ruling out the coelacanth/tetrapod clade.Correspondence to: K. Watanabe 0592     

Searching for the closest living relative(s) of tetrapods through evolutionary analyses of mitochondrial and nuclear data

The phylogenetic relationships of the African lungfish (Protopterus dolloi) and the coelacanth (Latimeria chalumnae) with respect to tetrapods were analyzed using complete mitochondrial genome DNA sequences. A lungfish + coelancanth clade was favored by maximum parsimony (although this result is dependent on which transition:transversion weights are applied), and a lungfish + tetrapod clade was supported by neighbor-joining and maximum-likelihood analyses. These two hypotheses received the strongest statistical and bootstrap support to the exclusion of the third alternative, the coelacanth + tetrapod sister group relationship. All mitochondrial protein coding genes combined favor a lungfish + tetrapod grouping. We can confidently reject the hypothesis that the coelacanth is the closest living relative of tetrapods. When the complete mitochondrial sequence data were combined with nuclear 28S rRNA gene data, a lungfish + coelacanth clade was supported by maximum parsimony and maximum likelihood, but a lungfish + tetrapod clade was favored by neighbor-joining. The seeming conflicting results based on different data sets and phylogenetic methods were typically not statistically strongly supported based on Kishino-Hasegawa and Templeton tests, although they were often supported by strong bootstrap values. Differences in rate of evolution of the different mitochondrial genes (slowly evolving genes such as the cytochrome oxidase and tRNA genes favored a lungfish + coelacanth clade, whereas genes of relatively faster substitution rate, such as several NADH dehydrogenase genes, supported a lungfish + tetrapod grouping), as well as the rapid radiation of the lineages back in the Devonian, rather than base compositional biases among taxa seem to be directly responsible for the remaining uncertainty in accepting one of the two alternate hypotheses.     

Sonic hedgehog antagonists reduce size and alter patterning of the frog inner ear

Sonic hedgehog (Shh) signaling plays a major role in vertebrate development, from regulation of proliferation to the patterning of various organs. In amniotes, Shh affects dorsoventral patterning in the inner ear but affects anteroposterior patterning in teleost ears. It remains unknown how altered function of Shh relates to morphogenetic changes that coincide with the evolution of limbs and novel auditory organs in the ear. In this study, we used the tetrapod, Xenopus laevis , to test how increasing concentrations of the Shh signal pathway antagonist, Vismodegib, affects ear development. Vismodegib treatment dose dependently alters the development of the ear, hypaxial muscle, and indirectly the Mauthner cell through its interaction with the inner ear afferents. Together, these phenotypes have an effect on escape response. The altered Mauthner cell likely contributes to the increased time to respond to a stimulus. In addition, the increased hypaxial muscle in the trunk likely contributes to the subtle change in animal C‐start flexion angle. In the ear, Vismodegib treatment results in decreasing segregation between the gravistatic sensory epithelia as the concentration of Vismodegib increases. Furthermore, at higher doses, there is a loss of the horizontal canal but no enantiomorphic transformation, as in bony fish lacking Shh. Like in amniotes, Shh signaling in frogs affects dorsoventral patterning in the ear, suggesting that auditory sensory evolution in sarcopterygians/tetrapods evolved with a shift of Shh function in axis specification. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1385–1400, 2017     

The Complete DNA Sequence of the Mitochondrial Genome of a ``living Fossil,'''' the Coelacanth (Latimeria Chalumnae)

The complete nucleotide sequence of the 16,407-bp mitochondrial genome of the coelacanth (Latimeria chalumnae) was determined. The coelacanth mitochondrial genome order is identical to the consensus vertebrate gene order which is also found in all ray-finned fishes, the lungfish, and most tetrapods. Base composition and codon usage also conform to typical vertebrate patterns. The entire mitochondrial genome was PCR-amplified with 24 sets of primers that are expected to amplify homologous regions in other related vertebrate species. Analyses of the control region of the coelacanth mitochondrial genome revealed the existence of four 22-bp tandem repeats close to its 3' end. The phylogenetic analyses of a large data set combining genes coding for rRNAs, tRNAs, and proteins (16,140 characters) confirmed the phylogenetic position of the coelacanth as a lobe-finned fish; it is more closely related to tetrapods than to ray-finned fishes. However, different phylogenetic methods applied to this largest available molecular data set were unable to resolve unambiguously the relationship of the coelacanth to the two other groups of extant lobe-finned fishes, the lungfishes and the tetrapods. Maximum parsimony favored a lungfish/coelacanth or a lungfish/tetrapod sistergroup relationship depending on which transversion:transition weighting is assumed. Neighbor-joining and maximum likelihood supported a lungfish/tetrapod sistergroup relationship.     

The developing lamprey ear closely resembles the zebrafish otic vesicle: otx1 expression can account for all major patterning differences

The inner ear of adult agnathan vertebrates is relatively symmetric about the anteroposterior axis, with only two semicircular canals and a single sensory macula. This contrasts with the highly asymmetric gnathostome arrangement of three canals and several separate maculae. Symmetric ears can be obtained experimentally in gnathostomes in several ways, including by manipulation of zebrafish Hedgehog signalling, and it has been suggested that these phenotypes might represent an atavistic condition. We have found, however, that the symmetry of the adult lamprey inner ear is not reflected in its early development; the lamprey otic vesicle is highly asymmetric about the anteroposterior axis, both morphologically and molecularly, and bears a striking resemblance to the zebrafish otic vesicle. The single sensory macula originates as two foci of hair cells, and later shows regions of homology to the zebrafish utricular and saccular maculae. It is likely, therefore, that the last common ancestor of lampreys and gnathostomes already had well-defined otic anteroposterior asymmetries. Both lamprey and zebrafish otic vesicles express a target of Hedgehog signalling, patched, indicating that both are responsive to Hedgehog signalling. One significant distinction between agnathans and gnathostomes, however, is the acquisition of otic Otx1 expression in the gnathostome lineage. We show that Otx1 knockdown in zebrafish, as in Otx1(-/-) mice, gives rise to lamprey-like inner ears. The role of Otx1 in the gnathostome ear is therefore highly conserved; otic Otx1 expression is likely to account not only for the gain of a third semicircular canal and crista in gnathostomes, but also for the separation of the zones of the single macula into distinct regions.     

Fgf9 signaling regulates inner ear morphogenesis through epithelial-mesenchymal interactions

The mammalian inner ear comprises the cochleovestibular labyrinth, derived from the ectodermal otic placode, and the encasing bony labyrinth of the temporal bone. Epithelial-mesenchymal interactions are thought to control inner ear development, but the modes and the molecules involved are largely unresolved. We show here that, during the precartilage and cartilage stages, Fgf9 is expressed in specific nonsensory domains of the otic epithelium and its receptors, Fgfr1(IIIc) and Fgfr2(IIIc), widely in the surrounding mesenchyme. To address the role of Fgf9 signaling, we analyzed the inner ears of mice homozygous for Fgf9 null alleles. Fgf9 inactivation leads to a hypoplastic vestibular component of the otic capsule and to the absence of the epithelial semicircular ducts. Reduced proliferation of the prechondrogenic mesenchyme was found to underlie capsular hypoplasticity. Semicircular duct development is blocked at the initial stages, since fusion plates do not form. Our results show that the mesenchyme directs fusion plate formation and they give direct evidence for the existence of reciprocal epithelial-mesenchymal interactions in the developing inner ear. In addition to the vestibule, in the cochlea, Fgf9 mutation caused defects in the interactions between the Reissner's membrane and the mesenchymal cells, leading to a malformed scala vestibuli. Together, these data show that Fgf9 signaling is required for inner ear morphogenesis.     

Dysmorphogenesis of the inner ear: disruption of extracellular matrix (ECM) formation by an L-proline analog in otic explants

L-azetidine-2-carboxylic acid (LACA), a l-proline analog, disrupts collagen secretion by cells and prevents normal morphogenesis of in vitro developing organ rudiments. Otic explants derived from 10.5-through 14-day-old mouse embryos were continuously exposed to LACA in the nutrient medium at concentrations of 75, 150, and 300 micrograms/ml. LACA disrupted normal in vitro otic morphogenesis in inner ears explanted from embryos of 10.5 through 13 days' gestation. Development of 14-day-old otic explants were not affected by LACA at the concentrations tested. There was a direct correlation between the embryonic age of the explant when exposed to LACA, and the severity of otic dysmorphogenesis. The younger explants (10.5-to 12-day-old) developed abnormalities of both vestibular and auditory structures, but with increasing embryonic age of the explants (12-to 13.5-day-old) abnormalities were confined more to the auditory portion of the inner ear. Disruption of collagen secretion of connective tissue cells of the otic explants are a major teratogenic action of LACA on inner ear development. Disrupted collagen secretion alters otic extracellular matrix production, which in turn affects the tissue interactions that regulate the progressive expression of otic morphogenesis and differentiation.     

Ultrastructure of the collagen fibrils in the coelacanth

The collagen fibrils in the rectal gland capsule of the coelacanth Latimeria chalumnae are c . 127 nm in diameter and resemble those fibrils that play an active mechanical role in trout skin. The fibrils having periodicity of about 54 nm and seven intrabands per period suggest that the collagen molecules in Latimeria are shorter, a feature they presumably share with lower tetrapods.     

Presence of Proteolipid Protein in Coelacanth Brain Myelin Demonstrates Tetrapod Affinities and Questions a Chondrichthyan Association

The protein and glycoprotein compositions of CNS myelin from the living coelacanth (Latimeria chalumnae) were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An unglycosylated component of 25 kilodaltons showed substantially stronger immunoblot reactivity with antibodies against mammalian proteolipid protein (PLP) than lungfish glycosylated PLP. DM-20 (intermediate protein) was not detectable in either fish. The presence of unglycosylated PLP in CNS myelin of the actinistian coelacanth contradicts an association with cartilaginous fishes but supports tetrapod affinities closer than those of lungfish.     

Role of the opercularis muscle in seismic sensitivity in the bullfrog Rana catesbeiana

The inner ear of anuran amphibians appears to be exceptionally sensitive to substrate vibration. The opercularis system, consisting of an opercularis muscle running from the shoulder girdle to a movable, cartilaginous operculum lying next to the inner ear, has been hypothesized to be involved in driving these seismic responses. Removal of the opercularis muscle of adult bullfrogs, Rana catesbeiana, caused clear decreases in microphonic responses of the inner ear to vibrations from 20-250 Hz and 0.05-5.0 cm/sec2 accelerations. Degree of decrease in responsiveness was variable between individuals and between different frequencies of stimulation, ranging up to 90% reduction at certain frequencies and in certain specimens. Decreases were most marked at lower frequencies below about 50 Hz. Additional removal of the levator scapulae superior muscle, which runs alongside the opercularis muscle from the shoulder girdle to ventrolateral portions of the otic capsule, also tended to depress responses, although this effect was substantially less (generally less than 10%) and also less consistent. As the opercularis muscle appears to be derived from the levator scapulae musculature, it is speculated that primitively seismic sensitivity was enhanced by a muscular connection that could transmit motion from the forelimb to the otic region, responsiveness being further enhanced by the subsequent evolution of the specialized opercularis system.     

Gene Structure and Amino Acid Sequence of Latimeria chalumnae (Coelacanth) Myelin DM20: Phylogenetic Relation of the Fish

The structure of Latimeria chalumnae (coelacanth) proteolipid protein/DM20 gene excluding exon 1 was determined, and the amino acid sequence of Latimeria DM20 corresponding to exons 2–7 was deduced. The nucleotide sequence of exon 3 suggests that only DM20 isoform is expressed in Latimeria. The structure of proteolipid protein/DM20 gene is well preserved among human, dog, mouse, and Latimeria. Southern blot analysis indicates that Latimeria DM20 gene is a single-copy gene. When the amino acid sequences of DM20 were compared among various species, Latimeria was more similar to tetrapods than other fishes including lungfish, confirming the previous finding by immunoreactivity (Waehneldt and Malotka 1989 J. Neurochem. 52:1941–1943). However, when phylogenetic trees were constructed from the DM20 sequences, lungfish was clearly the closest to tetrapods. Latimeria was situated outside of lungfish by the maximum likelihood method. The apparent similarity of Latimeria DM20 to tetrapod proteolipid protein/DM20 is explained by the slow amino acid substitution rate of Latimeria DM20.     

The role of Six1 in mammalian auditory system development

The homeobox Six genes, homologues to Drosophila sine oculis (so) gene, are expressed in multiple organs during mammalian development. However, their roles during auditory system development have not been studied. We report that Six1 is required for mouse auditory system development. During inner ear development, Six1 expression was first detected in the ventral region of the otic pit and later is restricted to the middle and ventral otic vesicle within which, respectively, the vestibular and auditory epithelia form. By contrast, Six1 expression is excluded from the dorsal otic vesicle within which the semicircular canals form. Six1 is also expressed in the vestibuloacoustic ganglion. At E15.5, Six1 is expressed in all sensory epithelia of the inner ear. Using recently generated Six1 mutant mice, we found that all Six1(+/-) mice showed some degree of hearing loss because of a failure of sound transmission in the middle ear. By contrast, Six1(-/-) mice displayed malformations of the auditory system involving the outer, middle and inner ears. The inner ear development in Six1(-/-) embryos arrested at the otic vesicle stage and all components of the inner ear failed to form due to increased cell death and reduced cell proliferation in the otic epithelium. Because we previously reported that Six1 expression in the otic vesicle is Eya1 dependent, we first clarified that Eya1 expression was unaffected in Six1(-/-) otic vesicle, further demonstrating that the Drosophila Eya-Six regulatory cassette is evolutionarily conserved during mammalian inner ear development. We also analyzed several other otic markers and found that the expression of Pax2 and Pax8 was unaffected in Six1(-/-) otic vesicle. By contrast, Six1 is required for the activation of Fgf3 expression and the maintenance of Fgf10 and Bmp4 expression in the otic vesicle. Furthermore, loss of Six1 function alters the expression pattern of Nkx5.1 and Gata3, indicating that Six1 is required for regional specification of the otic vesicle. Finally, our data suggest that the interaction between Eya1 and Six1 is crucial for the morphogenesis of the cochlea and the posterior ampulla during inner ear development. These analyses establish a role for Six1 in early growth and patterning of the otic vesicle.     

The amphibian octavo-lateralis system and its regressive and progressive evolution

The phylogenetic and ontogenetic changes in the octavolateralis system of sarcopterygian fish and tetrapods, presumed to be important for the formation of an amphibian auditory system, are reviewed. The lateral line system shows rudimentation of lines and loss of ampullary electroreceptors in many amphibians; in some amphibians it never develops. The metamorphic changes of the lateral-line system show different patterns in the different amphibian lineages with metamorphic retention in most urodeles and metamorphic loss in most anurans. The multitude of both ontogenetic and phylogenetic changes of the lateral line system among amphibians do exclude any prediction as to how this system might have changed in ancestral amniotes. The most important auditory epithelium of the tetrapod inner ear, the basilar papilla, seems to be primitively present in all tetrapods and Latimeria. In two amphibian lineages there is a trend towards rudimentation and loss of the basilar papilla. Only in the third order, the anurans, a tympanic ear develops and the inner ear shows a progressive evolution of the auditory epithelia. Together with the known differences in the periotic labyrinth of amphibians and amniotes, this scenario suggests a parallel evolution of the amniotic and anuran auditory periphery. All mechanoreceptive hair cells of the lateral line system and the inner ear appear to receive a common and bilateral efferent innervation. Among amphibians this pattern is represented only in some urodeles, whereas anurans show a derived pattern with loss of a bilateral component and presumably also of a common neuromast/inner ear component. Changes in the rhombencephalic nuclei which receive octavo-lateralis afferent fibers show a trend towards development of auditory nuclei only in the anuran lineage. The phylogenetic appearance of an auditory nucleus in this lineage coincides with the complete absence of formation of ampullary electroreceptors. In contrast, the earlier claim of a correlation between a metamorphic loss of the lateral line system and the formation of an auditory nucleus is not supported by more recent data: an auditory nucleus develops in anurans already prior to metamorphosis and is present in all anurans even when they retain the neuromast system. In anurans with a metamorphic loss of the neuromasts, the second order neurons degenerate as well. This independence of the auditory and the second order lateral line nuclei is further substantiated by their separate projection to other brain areas, like the torus semicircularis of the midbrain, and their functional properties.(ABSTRACT TRUNCATED AT 400 WORDS)     

Role of hindbrain in inner ear morphogenesis: analysis of Noggin knockout mice

Signaling from rhombomeres 5 and 6 of the hindbrain is thought to be important for inner ear patterning. In Noggin −/− embryos, the gross anatomy of the inner ear is distorted and malformed, with cochlear duct outgrowth and coiling most affected. We attributed these defects to a caudal shift of the rhombomeres caused by the shortened body axis and the kink in the neural tube. To test the hypothesis that a caudal shift of the rhombomeres affects inner ear development, we surgically generated chicken embryos in which rhombomeres 5 and 6 were similarly shifted relative to the position of the inner ears, as in Noggin mutants. All chicken embryos with shifted rhombomeres showed defects in cochlear duct formation indicating that signaling from rhombomeres 5 and 6 is important for cochlear duct patterning in both chicken and mice. In addition, the size of the otic capsule is increased in Noggin −/− mutants, which most likely is due to unopposed BMP signaling for chondrogenesis in the peri-otic mesenchyme.     

Extremely slow rate of evolution in the HOX cluster revealed by comparison between Tanzanian and Indonesian coelacanths

Coelacanths are known as "living fossils" because their morphology has changed very little from that in the fossil record. To elucidate why coelacanths have evolved so slowly is thus of primary importance in evolutionary biology. In the present study, we determined the entire sequence of the HOX cluster of the Tanzanian coelacanth (Latimeria chalumnae) and compared it with that of the Indonesian coelacanth (L. menadoensis), which was available in the literature. The most intriguing result was the extremely small genetic divergence between the two coelacanths. The synonymous divergence of the HOX coding region between the two coelacanths was estimated to be 0.07%, which is ~11-fold smaller than that of human-chimp. When we applied the estimated divergence time of the two coelacanths of 6 million years ago (MYA) and 30 MYA, which were proposed in independent mitochondrial DNA analyses, the synonymous substitution rate of the coelacanth HOX cluster was estimated to be ~11-fold and 56-fold smaller than that of human-chimp, respectively. Thus, the present study implies that the reduction of the nucleotide substitution rate in coelacanth HOX genes may account for the conservation of coelacanth morphology during evolution.     

The Second Recorded Living Coelacanth from North Sulawesi

A detailed account of the second coelacanth specimen known to have been captured in Indonesian waters is provided. The account is written in the official format of the Coelacanth Conservation Council's (CCC) inventory of known specimens of the living coelacanth Latimeria chalumnae, and the specimen is herein numbered CCC no. 175. Morphological measurements and fin ray counts are recorded. A comparison of these measurements and meristic data with those recorded for Comoran specimens of Latimeria chalumnae shows that the present specimen has a similar allometry to L. chalumnae. Ongoing genetic analyses should reveal the depth of divergence between the two known populations of living coelacanths and help determine if this specimen is conspecific with its Comoran counterparts.     

Hearing in the African lungfish (Protopterus annectens): pre-adaptation to pressure hearing in tetrapods?

Lungfishes are the closest living relatives of the tetrapods, and the ear of recent lungfishes resembles the tetrapod ear more than the ear of ray-finned fishes and is therefore of interest for understanding the evolution of hearing in the early tetrapods. The water-to-land transition resulted in major changes in the tetrapod ear associated with the detection of air-borne sound pressure, as evidenced by the late and independent origins of tympanic ears in all of the major tetrapod groups. To investigate lungfish pressure and vibration detection, we measured the sensitivity and frequency responses of five West African lungfish (Protopterus annectens) using brainstem potentials evoked by calibrated sound and vibration stimuli in air and water. We find that the lungfish ear has good low-frequency vibration sensitivity, like recent amphibians, but poor sensitivity to air-borne sound. The skull shows measurable vibrations above 100 Hz when stimulated by air-borne sound, but the ear is apparently insensitive at these frequencies, suggesting that the lungfish ear is neither adapted nor pre-adapted for aerial hearing. Thus, if the lungfish ear is a model of the ear of early tetrapods, their auditory sensitivity was limited to very low frequencies on land, mostly mediated by substrate-borne vibrations.