A molecular phylogeny of two extinct sloths

Xenarthra (Edentata) is an extremely diverse mammalian order whose modern representatives are the armadillos, anteaters, and sloths. The phylogeny of these groups is poorly resolved. This is particularly true for the sloths (phyllophagans), originally a large and diverse group now reduced to two genera in two different families. Both morphological analyses and molecular analyses of rDNA genes of living and extinct sloths have been used with limited success to elucidate their phylogeny. In an attempt to clarify relationships among the sloths, DNA was extracted and mitochondrial cytochrome b gene sequences were determined from representatives of two extinct groups of sloths (Mylodontidae and Megatheriidae), their two living relatives (two-toed sloths [Megalonychidae], three-toed sloths [Bradypodidae]), anteaters and armadillos. A consistent feature of the latter two species was the nuclear copies of cytochrome b gene sequences. Several methods of phylogenetic reconstruction were applied to the sequences determined, and the results were compared with 12S rDNA sequences obtained in previous studies. The cytochrome b gene exhibited a phylogenetic resolving power similar to that of the 12S rDNA sequences. When both data sets were combined, they tended to support the grouping of two-toed sloths with mylodontids and three-toed sloths with megatheriids. The results strengthen the view that the two families of living sloths adapted independently to an arboreal life-style.     

The evolution of armadillos, anteaters and sloths depicted by nuclear and mitochondrial phylogenies: implications for the status of the enigmatic fossil Eurotamandua

The mammalian order Xenarthra (armadillos, anteaters and sloths) is one of the four major clades of placentals, but it remains poorly studied from the molecular phylogenetics perspective. We present here a study encompassing most of the order's diversity in order to establish xenarthrans' intra-ordinal relationships, discuss the evolution of their morphological characters, search for their extant sister group and specify the timing of their radiation with special emphasis on the status of the controversial fossil Eurotamandua. Sequences of three genes (nuclear exon 28 of the Von Willebrand factor and mitochondrial 12S and 16S rRNAs) are compared for eight of the 13 living genera. Phylogenetic analyses confirm the order's monophyly and that of its three major lineages: armadillos (Cingulata), anteaters (Vermilingua) and sloths ('Tardigrada', renamed in 'Folivora'), and our results strongly support the grouping of hairy xenarthrans (anteaters and sloths) into Pilosa. Within placentals, Afrotheria might be the first lineage to branch off, followed by Xenarthra. The morphological adaptative convergence between New World xenarthrans and Old World pangolins is confirmed. Molecular datings place the early emergence of armadillos around the Cretaceous/Tertiary boundary, followed by the divergence between anteaters and sloths in the Early Eocene era. These Tertiary dates contradict the concept of a very ancient origin of modern xenarthran lineages. They also question the placement of the purported fossil anteater (Eurotamandua) from the Middle Eocene period of Europe with the Vermilingua and instead suggest the independent and convergent evolution of this enigmatic taxon.     

A syndrome of mutualism reinforces the lifestyle of a sloth

Arboreal herbivory is rare among mammals. The few species with this lifestyle possess unique adaptions to overcome size-related constraints on nutritional energetics. Sloths are folivores that spend most of their time resting or eating in the forest canopy. A three-toed sloth will, however, descend its tree weekly to defecate, which is risky, energetically costly and, until now, inexplicable. We hypothesized that this behaviour sustains an ecosystem in the fur of sloths, which confers cryptic nutritional benefits to sloths. We found that the more specialized three-toed sloths harboured more phoretic moths, greater concentrations of inorganic nitrogen and higher algal biomass than the generalist two-toed sloths. Moth density was positively related to inorganic nitrogen concentration and algal biomass in the fur. We discovered that sloths consumed algae from their fur, which was highly digestible and lipid-rich. By descending a tree to defecate, sloths transport moths to their oviposition sites in sloth dung, which facilitates moth colonization of sloth fur. Moths are portals for nutrients, increasing nitrogen levels in sloth fur, which fuels algal growth. Sloths consume these algae-gardens, presumably to augment their limited diet. These linked mutualisms between moths, sloths and algae appear to aid the sloth in overcoming a highly constrained lifestyle.     

An Overview of the Presence of Osteoderms in Sloths: Implications for Osteoderms as a Plesiomorphic Character of the Xenarthra

The presence of osteoderms in the skin of some extinct sloths and in cingulates (armadillos, pampatheres, and glyptodonts) has often been considered a pleisomorphic character of the Xenarthra. While osteoderms are known from the earliest cingulates, they are absent in most sloths including the two extant taxa and only appear late in their fossil record. Osteoderms are currently only reported from five genera of mylodonts and two megatheres, out of the over 100 currently recognized genera of sloths. Consequently, rather than a plesiomorphic character of the Xenarthra, which has been secondarily lost in sloths, it is more likely that osteoderms in sloths are the result of parallel evolution to the cingulates that independently evolved in one, possibly two different sloth clades.     

The evolution of hyoid apparatus in Xenarthra (Mammalia: Eutheria)

The hyoid apparatus reflects aspects of the form and function of feeding in living and extinct organisms and, despite the availability of information about this structure for Xenarthra, it remains little explored from an evolutionary perspective. Here we compare the morphology of the hyoid apparatus in xenarthrans, describing its general morphology and variation in each major clade and score these variations as phylogenetic characters, which were submitted to ancestral states reconstructions. The general hyoid morphology of Xenarthra consists of a v-bone (basihyal fused with the thyrohyals) and three paired bones (stylohyal, epihyal and ceratohyal), which are unfused in the majority of taxa. The clade-specific morphology observed here, allowed us to obtain additional synapomorphies for all major clades of Xenarthra (Cingulata, Pilosa, Folivora and Vermilingua), for Glyptodontididae, and for Nothrotheriidae. The fusion of hyoid elements are convergentelly achieved among the diphyletic extant tree sloths, some extinct ground sloths and glyptodontids. Despite the heavy influence of adaptive evolution related to feeding habits, the morphology of the hyoid apparatus proved to be a valuable source of phylogenetic information.     

Orientation of the lateral semicircular canal in Xenarthra and its links with head posture and phylogeny

The orientation of the semicircular canals of the inner ear in the skull of vertebrates is one of the determinants of the capacity of this system to detect a given rotational movement of the head. Past functional studies on the spatial orientation of the semicircular canals essentially focused on the lateral semicircular canal (LSC), which is supposedly held close to horizontal during rest and/or alert behaviors. However, they generally investigated this feature in only a few and distantly related taxa. Based on 3D‐models reconstructed from µCT‐scans of skulls, we examined the diversity of orientations of the LSC within one of the four major clades of placental mammals, that is, the superorder Xenarthra, with a data set that includes almost all extant genera and two extinct taxa. We observed a wide diversity of LSC orientations relative to the basicranium at both intraspecific and interspecific scales. The estimated phylogenetic imprint on the orientation of the LSC was significant but rather low within the superorder, though some phylogenetic conservatism was detected for armadillos that were characterized by a strongly tilted LSC. A convergence between extant suspensory sloths was also detected, both genera showing a weakly tilted LSC. Our preliminary analysis of usual head posture in extant xenarthrans based on photographs of living animals further revealed that the LSC orientation in armadillos is congruent with a strongly nose‐down head posture. It also portrayed a more complex situation for sloths and anteaters. Finally, we also demonstrate that the conformation of the cranial vault and nuchal crests as well as the orientation of the posterior part of the petrosal may covary with the LSC orientation in Xenarthra. Possible inferences for the head postures of extinct xenarthrans such as giant ground sloths are discussed in the light of these results.     

A PCR-RFLP assay for gender assignment in the three-toed sloths (Bradypus, Pilosa, Bradypodidae)

The three-toed sloths (Bradypus) are slow-moving arboreal neotropical mammals. Understanding demographic variables (such as sex ratio) of populations is a key for conservation purposes. Nevertheless, gender assignment of Bradypus is particularly challenging because of the lack of sexual dimorphism in infants and in adults, particularly B. torquatus, the most endangered of the three-toed sloths, in which sex is attributed by visual observation of the reproductively active males. Here, we standardized a method for sexing Bradypus individuals using PCR-RFLP of sex-linked genes ZFX/ZFY. This assay was validated with known-gender animals and proved accurate to assign gender on three Bradypus species.     

Bony labyrinth morphometry indicates locomotor adaptations in the squirrel-related clade (Rodentia,Mammalia)

The semicircular canals (SCs) of the inner ear detect angular acceleration and are located in the bony labyrinth of the petrosal bone. Based on high-resolution computed tomography, we created a size-independent database of the bony labyrinth of 50 mammalian species especially rodents of the squirrel-related clade comprising taxa with fossorial, arboreal and gliding adaptations. Our sampling also includes gliding marsupials, actively flying bats, the arboreal tree shrew and subterranean species. The morphometric anatomy of the SCs was correlated to the locomotion mode. Even if the phylogenetic signal cannot entirely be excluded, the main significance for functional morphological studies has been found in the diameter of the SCs, whereas the radius of curvature is of minor interest. Additionally, we found clear differences in the bias angle of the canals between subterranean and gliding taxa, but also between sciurids and glirids. The sensitivity of the inner ear correlates with the locomotion mode, with a higher sensitivity of the SCs in fossorial species than in flying taxa. We conclude that the inner ear of flying and gliding mammals is less sensitive due to the large information flow into this sense organ during locomotion.     

Conservation of placentation during the tertiary radiation of mammals in South America

The eutherian placenta is considered to possess great plasticity, but it is not clear how this variation reflects adaptation to different ecological niches. Because South America was isolated for most of the Tertiary, it represents a natural laboratory to examine this question. We here describe placentation in three South American groups: Xenarthra have been part of the fauna from at least the mid‐Paleocene whereas caviomorph rodents and Neotropical primates are each derived from a single founder that reached South America in the Eocene and Oligocene, respectively. The common ancestor of Xenarthra had a villous, haemochorial placenta, from which the labyrinthine, endotheliochorial placenta of sloths later evolved. Placentation in Caviomorpha follows an extraordinary stable pattern, characterized by a haemomonochorial, labyrinthine and highly lobed structure with specialized growing areas. This pattern was present before arrival of these rodents in South America and enabled a successful radiation especially during the spread of grasslands. Neotropical primates have haemochorial, trabecular placentas with a specialized maternal blood supply; a pattern that contrasts with that of Old World monkeys and may have been present in the founder generation on arrival in South America. In conclusion, there is a dichotomy within Xenarthra but otherwise the ancient South American mammals do not show much variation in principal placental characters. Thus, the successful radiation of these three groups, and their adaptation to diverse ecological niches, did not require substantial alterations in placentation. J. Morphol., 2013. © 2013 Wiley Periodicals, Inc.     

Immobilization of free-ranging Hoffmann's two-toed and brown-throated three-toed sloths using ketamine and medetomidine: a comparison of physiologic parameters

Free-ranging Hoffmann's two-toed sloths (Choloepus hoffmanni; n=26) and brown-throated three-toed sloths (Bradypus variegatus; n=15) were manually captured and immobilized with 2.5 mg/kg ketamine + 0.02 mg/kg medetomidine administered intramuscularly. Physical examinations were conducted on each sloth 10 min after initial injection, and blood, fecal, and ectoparasite samples were collected. Heart rate, respiratory rate, body temperature, indirect systolic blood pressure, and indirect peripheral oxygen saturation were monitored every 5 min for the duration of anesthesia. After 45 min, atipamazole (0.1 mg/kg) was administered intramuscularly, as an antagonist to medetomidine, in order to facilitate recovery. All recoveries were smooth, rapid, and uneventful. Physiologic parameters were compared across time, gender, and species. All sloths, regardless of species and gender, demonstrated a time-dependent decrease in heart rate and blood pressure, and an increase in respiratory rate, during the course of anesthesia. Peripheral oxygen saturation was similar for all sloths over time. There were significant species differences for heart rate (Choloepus > Bradypus), respiratory rate (Choloepus > Bradypus), and systolic blood pressure (Bradypus > Choloepus), while there were significant gender differences for body temperature (males > females) and blood pressure (males > females). Results of this study suggest that the ketamine-medetomidine mixture, as described above, is a safe and effective anesthetic combination in free-ranging two- and three-toed sloths, although peripheral blood pressure should be monitored during anesthesia.     

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish (Danio rerio)

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the cellular and molecular features of the epithelia, which are mainly composed of two types of cells: hair cells (HCs) and supporting cells (SCs). Here we choose to study the inner ear sensory epithelia in adult zebrafish not only because the epithelial structures are highly conserved in all vertebrates studied, but also because the adult zebrafish is able to regenerate HCs, an ability that mammals lose shortly after birth. We use the inner ear of adult zebrafish as a model system to study the mechanisms of inner ear HC regeneration in adult vertebrates that could be helpful for clinical therapy of hearing/balance deficits in human as a result of HC loss.Here we demonstrate how to do gross and fine dissections of inner ear sensory epithelia in adult zebrafish. The gross dissection removes the tissues surrounding the inner ear and is helpful for preparing tissue sections, which allows us to examine the detailed structure of the sensory epithelia. The fine dissection cleans up the non-sensory-epithelial tissues of each individual epithelium and enables us to examine the heterogeneity of the whole epithelium easily in whole-mount epithelial samples.Open in a separate windowClick here to view.^{(51M, flv)}     

A comparison of the external morphology of the membranous inner ear in elasmobranchs

Studies on the elasmobranch inner ear have focused predominantly on a small group of sharks, particularly, carcharhinids. As a result, subsequent studies in other species have subdivided species into two main groups: those typical and those atypical of carcharhinid sharks. This study proposes a different set of inner‐ear morphology groupings to those previously suggested. The inner ears from 17 species of elasmobranchs (representing both sharks and rays) are examined in this study and based on morphometric data some groups include both rays and sharks. Four groups are now proposed based predominantly on the shape and dimensions of the membranous otoconial organs, and characteristics of the semicircular canals. Evident morphological differences between the ear types belonging to the new groups include the membranes of the semicircular canals being bound to the otoconial organs in some species, while only being connected via the canal ducts in others, as well as clear variation present in saccular organ size. Previous studies examining variation in the inner ear have attributed differences to either phylogeny or functional significance. Results from this study suggest that neither phylogeny nor feeding strategy solely accounts for the morphological diversity present in the external morphology of the elasmobranch inner ear. J. Morphol., 2010. © 2010 Wiley‐Liss, Inc.     

Molecular Decay of the Tooth Gene Enamelin (ENAM) Mirrors the Loss of Enamel in the Fossil Record of Placental Mammals

Vestigial structures occur at both the anatomical and molecular levels, but studies documenting the co-occurrence of morphological degeneration in the fossil record and molecular decay in the genome are rare. Here, we use morphology, the fossil record, and phylogenetics to predict the occurrence of “molecular fossils” of the enamelin (ENAM) gene in four different orders of placental mammals (Tubulidentata, Pholidota, Cetacea, Xenarthra) with toothless and/or enamelless taxa. Our results support the “molecular fossil” hypothesis and demonstrate the occurrence of frameshift mutations and/or stop codons in all toothless and enamelless taxa. We then use a novel method based on selection intensity estimates for codons (ω) to calculate the timing of iterated enamel loss in the fossil record of aardvarks and pangolins, and further show that the molecular evolutionary history of ENAM predicts the occurrence of enamel in basal representatives of Xenarthra (sloths, anteaters, armadillos) even though frameshift mutations are ubiquitous in ENAM sequences of living xenarthrans. The molecular decay of ENAM parallels the morphological degeneration of enamel in the fossil record of placental mammals and provides manifest evidence for the predictive power of Darwin''s theory.     

Temporal and spatial resource use by female three-toed sloths and their young in an agricultural landscape in Costa Rica

The information on ecological behavior of wild sloths is very scarce. In this study we determined the home ranges and resources used by three adult female three-toed sloths (Bradypus variegatus) and their four young in an agricultural matrix of cacao (Theobroma cacao), pasture, riparian forests and living fencerows in Costa Rica. Births occurred during November-December and the young became independent at five to seven months of age. Initially, mothers remained fixed in one or a few trees, but expanded their use of resources as young sloths became independent from them. Mothers initially guided the young to preferred food and cover resources, but they gradually left their young in small nucleus areas and colonized new areas for themselves. Home range sizes for young sloths (up to seven months of age) varied between 0.04-0.6 hectares, while home range sizes for mothers varied from 0.04-25.0 hectares. During the maternal care period, 22 tree species were used, with the most common being Cecropia obtusifolia (30.9%), Coussapoa villosa (25.6%), Nectandra salicifolia (12.1%), Pterocarpus officinalis (5.8%) and Samanea saman (5.4%). However, young sloths used only 20 tree species, with the most common being C. villosa (18.4%), S. saman (18.5%) and N. salicifolia (16.7%). The cacao agroforest was used only by mother sloths and never by their young following separation. However, in the riparian forest, both mother sloths and young used the tree species. A total of 28 tree species were used by the mother sloth; including the food species: C. obtusifolia, C. villosa, N. salicifolia and P. officinalis. However, the young used 18 trees species in this habitat with N. salicifolia and S. saman most commonly used, although they rested and fed during the day in C. obtusifolia, C. villosa and O. sinnuata. The cacao agroforest with adjacent riparian forests and fencerows provides an important habitat type that links the smaller secondary forests and other patches.     

Molecular phylogeny of living xenarthrans and the impact of character and taxon sampling on the placental tree rooting

Extant xenarthrans (armadillos, anteaters and sloths) are among the most derived placental mammals ever evolved. South America was the cradle of their evolutionary history. During the Tertiary, xenarthrans experienced an extraordinary radiation, whereas South America remained isolated from other continents. The 13 living genera are relics of this earlier diversification and represent one of the four major clades of placental mammals. Sequences of the three independent protein-coding nuclear markers alpha2B adrenergic receptor (ADRA2B), breast cancer susceptibility (BRCA1), and von Willebrand Factor (VWF) were determined for 12 of the 13 living xenarthran genera. Comparative evolutionary dynamics of these nuclear exons using a likelihood framework revealed contrasting patterns of molecular evolution. All codon positions of BRCA1 were shown to evolve in a strikingly similar manner, and third codon positions appeared less saturated within placentals than those of ADRA2B and VWF. Maximum likelihood and Bayesian phylogenetic analyses of a 47 placental taxa data set rooted by three marsupial outgroups resolved the phylogeny of Xenarthra with some evidence for two radiation events in armadillos and provided a strongly supported picture of placental interordinal relationships. This topology was fully compatible with recent studies, dividing placentals into the Southern Hemisphere clades Afrotheria and Xenarthra and a monophyletic Northern Hemisphere clade (Boreoeutheria) composed of Laurasiatheria and Euarchontoglires. Partitioned likelihood statistical tests of the position of the root, under different character partition schemes, identified three almost equally likely hypotheses for early placental divergences: a basal Afrotheria, an Afrotheria + Xenarthra clade, or a basal Xenarthra (Epitheria hypothesis). We took advantage of the extensive sampling realized within Xenarthra to assess its impact on the location of the root on the placental tree. By resampling taxa within Xenarthra, the conservative Shimodaira-Hasegawa likelihood-based test of alternative topologies was shown to be sensitive to both character and taxon sampling.     

Recent Advances on Variability, Morpho-Functional Adaptations, Dental Terminology, and Evolution of Sloths

The occasion of the Xenarthra Symposium during the ICVM 9 meeting allowed us to reflect on the considerable advances in the knowledge of sloths made by the “X-community” over the past two decades, particularly in such aspects as locomotion, mastication, diet, dental terminology, intraspecific variation, sexual dimorphism, and phylogenetic relationships. These advancements have largely been made possible by the application of cladistic methodology (including DNA analyses) and the discovery of peculiar forms such as Diabolotherium, Thalassocnus, and Pseudoglyptodon in traditionally neglected areas such as the Chilean Andes and the Peruvian Pacific desert coast. Modern tree sloths exhibit an upside-down posture and suspensory locomotion, but the habits of fossil sloths are considerably more diverse and include locomotory modes such as inferred bipedality, quadrupedality, arboreality or semiarboreality, climbing, and an aquatic or semi-aquatic lifestyle in saltwater. Modern tree sloths are generalist browsers, but fossil sloths had browsing, grazing, or mixed feeding dietary habits. Discovery of two important sloth faunas in Brazil (Jacobina) and southern North America (Daytona Beach and Rancho La Brea) have permitted evaluation of the ontogenetic variation in Eremotherium laurillardi and the existence of possible sexual dimorphism in this sloth and in Paramylodon harlani. A new dental terminology applicable to a majority of clades has been developed, facilitating comparisons among taxa. An analysis wherein functional traits were plotted onto a phylogeny of sloths was used to determine patterns of evolutionary change across the clade. These analyses suggest that megatherioid sloths were primitively semiarboreal or possessed climbing adaptations, a feature retained in some members of the family Megalonychidae. Pedolateral stance in the hindfoot is shown to be convergently acquired in Mylodontidae and Megatheria (Nothrotheriidae + Megatheriidae), this feature serving as a synapomorphy of the latter clade. Digging adaptations can only be securely ascribed to scelidotheriine and mylodontine sloths, and the latter are also the only group of grazing sloths, the remainder being general browsers.     

Comparative anatomy and histology of xenarthran osteoderms

Reconstruction of soft tissues in fossil vertebrates is an enduring challenge for paleontologists. Because inferences must be based on evidence from hard tissues (typically bones or teeth), even the most complete fossils provide only limited information about certain organ systems. Osteoderms ("dermal armor") are integumentary bones with high fossilization potential that hold information about the anatomy of the skin in many extant and fossil amniotes. Their importance for functional morphology and phylogenetic research has recently been recognized, but studies have focused largely upon reptiles, in which osteoderms are most common. Among mammals, osteoderms occur only in members of the clade Xenarthra, which includes armadillos and their extinct relatives: glyptodonts, pampatheres, and, more distantly, ground sloths. Here, I present new information on the comparative morphology and histology of osteoderms and their associated soft tissues in 11 extant and fossil xenarthrans. Extinct mylodontid sloths possessed simple, isolated ossicles, the presence of which is likely plesiomorphic for Xenarthra. More highly derived osteoderms of glyptodonts, pampatheres, and armadillos feature complex articulations and surface ornamentation. Osteoderms of modern armadillos are physically associated with a variety of soft tissues, including nerve, muscle, gland, and connective tissue. In some cases, similar osteological features may be caused by two or more different tissue types, rendering soft-tissue inferences for fossil osteoderms equivocal. Certain osteological structures, however, are consistently associated with specific soft-tissue complexes and therefore represent a relatively robust foundation upon which to base soft-tissue reconstructions of extinct xenarthrans.     

Bony labyrinth morphometry reveals hidden diversity in lungless salamanders (Family Plethodontidae): Structural correlates of ecology,development, and vision in the inner ear

Lungless salamanders (Family Plethodontidae) form a highly speciose group that has undergone spectacular adaptive radiation to colonize a multitude of habitats. Substantial morphological variation in the otic region coupled with great ecological diversity within this clade make plethodontids an excellent model for exploring the ecomorphology of the amphibian ear. We examined the influence of habitat, development, and vision on inner ear morphology in 52 plethodontid species. We collected traditional and 3D geometric morphometric measurements to characterize variation in size and shape of the otic endocast and peripheral structures of the salamander ear. Phylogenetic comparative analyses demonstrate structural convergence in the inner ear across ecologically similar species. Species that dwell in spatially complex microhabitats exhibit robust, highly curved semicircular canals suggesting enhanced vestibular sense, whereas species with reduced visual systems demonstrate reduced canal curvature indicative of relaxed selection on the vestibulo‐ocular reflex. Cave specialists show parallel enlargement of auditory‐associated structures. The morphological correlates of ecology among diverse species reveal underlying evidence of habitat specialization in the inner ear and suggest that there exists physiological variation in the function of the salamander ear even in the apparent absence of selective pressures on the auditory system to support acoustic behavior.     

Role of diffusible and transcription factors in inner ear development: implications in regeneration

Organogenesis involves a dynamic balance of the mechanisms regulating cell division, differentiation and death. The development of the chicken embryo inner ear offers a well-characterised model at the morphological level to study which signals are implicated in the modulation of cellular activation and commitment. The early developmental decisions that control the origin of the inner ear elements are just beginning to be identified by complementary in vivo and in vitro studies. Insulin-like growth factor-I (IGF-I) and nerve growth factor (NGF) are among the best characterised diffusible factors acting during inner ear development. Although the cellular actions of these factors are beginning to be understood, the signalling pathways triggered by them still remain largely unknown. In this context, viral vehicles can be used to deliver genes and then analyse their functional roles during inner ear development. A model is proposed where the actions of IGF-I and NGF contribute to the combinatorial expression of Jun and Fos family members in particular domains of the otic vesicle. Some of these mechanisms may be also implicated in otic regeneration.