Most anurans possess a tympanic center ear (TME) the transmits sound tide to the within ear; however, numerous species lack part or all TME components. To recognize the development of this structures, we took on a an extensive assessment of their occurrence across anurans and performed genealogical character state reconstructions. Our evaluation indicates that the TME was fully lost at the very least 38 independent time in Anura. The inferred evolutionary history of the TME is exceptionally facility in true toads (Bufonidae), whereby it was lost in the most recent common ancestor, preceding a radiation the >150 earless species. Complying with that early stage loss, live independence regains of part or all TME structures were inferred within two minor clades and also in a radiation of >400 species. The reappearance that the TME in the latter clade was complied with by at least 10 accident of the whole TME. The countless losses and gains the the TME in anurans is unparalleled among tetrapods. Our results show that anurans and also especially bufonid toads, are wonderful model to research the behavioural correlates of earlessness, extratympanic sound pathways and also the genetic and also developmental mechanisms the underlie the morphogenesis of TME structures.

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The role of audition in frogs and toads (Anura) is mostly the late of airborne sounds, consisting of those connected in society communication1. Thus, hear in anurans is thought to it is in a vital trait because that survival and also reproduction. In many anurans, late of airborne sound is allowed by a tympanic middle ear (TME) created minimally of a tympanic membrane, middle ear cavity and middle ear bone (=columella, columella auris, stapes, plectrum) the conducts sound waves from the atmosphere to the inner ear wherein they are transduced into electric signals via hair cells1,2,3,4.

Among other tetrapods, a TME is absent in caecilians and salamanders1,5 but present in amniotes. Nevertheless, although the TME is primitively present in all extant amniotes, that is not homologous throughout amniote lineages, having developed independently at least 5 times in turtles, lepidosaurs, archosaurs, an extinct lineage of parareptiles and the synapsid ancestor of mammals6,7,8,9,10. TME casualty are very rare in amniotes. All mammals, turtles and archosaurs possess a finish TME and even the amphisbaenians, snakes and also lizards that have lost the tympanic membrane and also middle ear cavity maintain a columella11, the single exceptions gift the pygopod lizard Aprasia repens12 and also possibly the snakes Atractaspis and Xenocalamus13. In contrast, lose is widespread among anurans, with at the very least a couple of species that several families lacking the entire TME, a problem referred to together “earlessness”14.

Earlessness is especially typical in the true toad family Bufonidae, in which the TME is completely lacking in more than 200 species. Bufonidae is among the many diversified groups of amphibians, comprising much more than 580 varieties in 51 genera naturally distributed over countless ecoregions the the Americas, Africa and Eurasia15. Numerous authors have detailed the reduction and also loss that TME structures (e.g.,16,17,18,19,20) or morphological sports in center ear structure (e.g.,1,21,22,23) in bufonids, yet the phylogenetic circulation of earlessness has never been studied either in ~ Bufonidae or throughout Anura.

As such, the objectives of this study space to explore the order of gains and also losses of the TME evolution across all anurans and also evaluate in a phylogenetic structure the fads of diversification of TME in Bufonidae.

Tympanic middle ear morphology and also data collection

Although many variations in the framework of the anuran auditory device exist, a generalised model deserve to be described1,2,3: the lateral-most section of this device is composed of a highly differentiated disc of thin, non-glandular skin, termed the tympanic membrane. The in salt of the tympanic membrane is attached to a cartilaginous tympanic ring, the tympanic annulus. The middle ear cavity is a diverticulum of the pharynx that opens up ventrally come the buccal cavity via the Eustachian tubes. The columella contact the tympanic membrane laterally and the otic capsule medially and also is split into three portions1,24,25: (1) the pars externa plectri or extracolumella, a cartilaginous framework that contacts the tympanic membrane and also often gift a slim, flattened strip of cartilage dubbed the ascending procedure or pars ascendens plectri, that extends anterodorsally to call the crista parotica that the prootic, (2) the pars media plectri or columellar shaft, an ossified, rod-shaped part with a dilated medial end and also (3) the pars interna plectri, a mostly cartilaginous framework that is constant with the pars media and also extends posteriorly to lie medial come the operculum. The broadened medial end of the pars media and also the whole pars interna constitute the stapedial footplate, which fills the rostral section of the oval window of the otic capsule1,26. The footplate is connected to the suprascapula via the columellar muscle in some species1, although the individuality of this muscle has actually been wondered about in at least some cases27,28. The otic operculum (operculum fenestrae ovalis), discovered only in caudates and anurans5, is one ovoid aspect that is normally cartilaginous or occasionally partially calcified4,25, that contacts the stapedial footplate and covers the caudal part of the oval window. The operculum is present in all anurans. The opercularis muscle inserts top top the operculum and also originates top top the suprascapular cartilage the the pectoral girdle1,26.

In all observed anuran types (also see29), the presence/absence of TME structures complies with a continual pattern. Absence of a medial framework is accompanied by the absence of the an ext lateral structures, such that absence of the columella entails lack of the tympanic annulus and also tympanic membrane and lack of the tympanic annulus entails absence of the tympanic membrane however not the columella. Similarly, existence of a lateral structure is accompanied by the visibility of the more medial structures, such that existence of the tympanic membrane requires presence that the tympanic annulus and also columella and presence the the tympanic annulus requires presence that the columella yet not the tympanic membrane. Consequently, us made the following presumptions when scoring the presence/absence the the tympanic structures (Fig. 1): (1) absence of the tympanic membrane and tympanic annulus once the columella is absent; (2) absence of the tympanic membrane when the tympanic annulus is absent; (3) presence of the tympanic annulus and also columella once the tympanic membrane is present; and also (4) visibility of the columella once the tympanic annulus is present.


Schematic depiction of tympanic center ear structures in anurans, reflecting the presumptions followed here for coding lack and visibility of the various elements.

TM, tympanic membrane; TA, tympanic annulus; CO, columella

In total, we scored the condition of the TME for 556 species and 51 genera the Bufonidae, representing >94% of every described varieties in the family. Among the sampled species, 239 were consisted of in Pyron’s30 phylogenetic analysis. We also scored the conditions of these structures for 1860 the the 2538 non-bufonid anuran species (representing 53 families; see15) consisted of by Pyron30, as well as 147 non-bufonid anuran varieties not included in this analysis. The only frog household not sampled by Pyron30 is the recently explained Odontobatrachidae31. Although our outgroup sampling is no exhaustive, we contained data because that the vast majority of genera of every the households sampled by Pyron30. Details on product examined, considerations around character coding and also character states scored because that each change series and literature sources are listed as Supplementary product (section S1 the the Supplementary Information).

Ancestral state reconstructions

We employed the many recent and also densely sampled phylogenetic hypothesis available for Anura, the of Pyron30, for ancestral state reconstruction and we discuss bufonid varieties not contained in Pyron’s30 study however present in various other analyses (e.g., well-supported outcomes of32,33,34). We concentrated original data collection mostly on Bufonidae and also relied an ext extensively on literary works accounts because that non-bufonids, some of which to be unclear or ambiguous about the event of specific structures. In particular, taxonomic accounts regularly use imprecise ax to describe the external morphology the the otic region35. Consequently, us analysed the phylogenetic circulation of every of the TME frameworks in Bufonidae but only the columella in analyses that Anura.

To check the homology of the center ear structures individually and also explain your variation amongst anurans, most parsimonious ancestral state reconstructions36 on Pyron’s30 phylogenetic theory were performed making use of Mesquite v3.0337. We further explored different evolutionary scenarios for the finish loss of every TME structures within Bufonidae v maximum likelihood genealogical reconstructions using the package APE38 and also stochastic character mapping39 utilizing phytools40 in R41. We compared Akaike info Criterion (AIC) worths for a maximum likelihood design in which change rates were enabled to vary (ARD) throughout the tree and also a best likelihood model in which shift rates between states were same (ER). We provided the most supported shift rate from our maximum likelihood analyses (ER) to estimate the variety of gains and losses throughout Bufonidae making use of stochastic character mapping. Stochastic character mapping allowed us to check out the probability that ear transitions under miscellaneous evolutionary scenarios, providing us a much better understanding that the likelihood of regains transparent this family. We considered three scenarios: (1) equal shift rates, no restrictions; (2) equal change rates and also restricting the ancestor to being eared; and also (3) a Dollo’s version (no regains possible). We ran 10,000 simulations every scenario and counted a state change whenever nodes switched from better than 50% assistance for one character state to better than 50% assistance for the various other character state.

Tympanic center ear development in Anura

The event of a columella is plesiomorphic in Anura, although the sister clade of all other anurans (Ascaphidae + Leiopelmatidae) lacks this structure (see Discussion). Offered that the tympanic membrane and tympanic annulus space not fossilizable structures, their incident in fossil material cannot be assessed, making it impossible to identify if the visibility of those structures is likewise plesiomorphic in Anura.

The TME is fully absent in at the very least some varieties of no fewer 보다 20 anuran families: Ascaphidae, Alsodidae, Batrachylidae, Bombinatoridae, Brachycephalidae, Brevicepitidae, Bufonidae, Calyptocephalellidae, Craugastoridae, Dicroglossidae, Hemisotidae, Leiopelmatidae, Leptodactylidae, Megophryidae, Microhylidae, Myobatrachidae, Nasikabatrachidae, Rhinophrynidae, Sooglossidae and also Telmatobiidae (section S1 that the Supplementary Information).

Ancestral personality state reconstructions and also detailed description of the occurrence of the columella in anuran family members other 보다 Bufonidae are listed in ar S2 the the Supplementary Information. Ancestral state restoration using Pyron’s30 phylogenetic hypothesis mirrors that the complete loss that the TME, as confirmed by absence of the columella, emerged independently at the very least 25 times exterior Bufonidae, add to two additional losses once taxa not contained in Pyron’s30 study however present in various other phylogenetic analyses room considered.

Tympanic center ear evolution in Bufonidae

Based on the phylogenetic theory of Pyron30 and also the results of both the parsimony and probabilistic genealogical state reconstructions (Fig. 2 and section S3 that the Supplementary Information), the tympanic membrane, tympanic annulus and columella were shed in the many recent usual ancestor of bufonids, regained subsequently and then repetitively re-lost again. Listed below we summarize the results of the genealogical reconstructions the the tympanic membrane, tympanic annulus and columella (the numbers of regains and also re-losses that TME frameworks in Bufonidae different somewhat once taxa not sampled by Pyron30 space considered; see sections S1 and also S4 the the Supplementary Information and also Discussion, below).


Partial phylogenetic tree the Pyron30 mirroring parsimony ancestral state reconstructions for the columella in Bufonidae.

The lack of the columella is a synapomorphy that Bufonidae (see ar S2 that the Supplementary Information), v independent regains in a subclade of Atelopus, Frostius (not contained in this analysis, but see text) and the sisters clade that Nannophryne, adhered to by 10 elevation losses.

Parsimony genealogical state reconstruction

The tympanic membrane was shed in the most recent usual ancestor that Bufonidae and also reappeared in the sisters clade of Nannophryne. Succeeding independent losses developed at the very least 25 times (see sections S3 and S4 that the Supplementary Information). The absence of the tympanic annulus is the inferred ancestral condition in bufonids, with two elevation regains: (a) within Atelopus in a clade composed of A. Flavescens, A. Franciscus, A. Pulcher and A. Spumarius and also (b) in the sisters clade the Nannophryne. The obtain of the tympanic annulus in the latter clade was followed by 10 independent casualty (see sections S3 and S4 the the Supplementary Information). Finally, the phylogenetic circulation of the gains and also losses that the columella is identical to the of the tympanic annulus because that the taxa had in the hypothesis of Pyron30 (see Fig. 2 and section S4 that the Supplementary Information). However, if bufonid species not consisted of by Pyron30 are also considered, the columella sometimes occurs there is no a tympanic annulus (see Discussion and also section S1 that the Supplementary Information).

Stochastic mapping of finish tympanic center ear ns under miscellaneous constraints

Our stochastic character mapping estimated similar patterns of TME loss and also regain in ~ Bufonidae under miscellaneous evolutionary scenarios (see ar S3 the the Supplementary Information). As soon as we ran an equal rates version of advancement we discovered results similar to the parsimony reconstructions that the tympanic annulus and also columella with strong support because that an ancestor doing not have these structures, 2 regains and 10 losses within the tree. When we assumed the ancestor had these structures and also a version of equal change rates, we discovered support for 12 losses and also still two regains. Once restricting regains from arising (Dollo’s model), we discovered a complete of 17 losses across bufonids.

The lack of the columella in Ascaphidae and Leiopelmatidae, i m sorry together type the sister clade of all other extant anurans (=Lalagobatrachia), has generated much discussion about the plesiomorphic condition in Anura29,42,43,44. However, due to the fact that most proanurans and also stem anuran fossils have a columella (i.e. Mesophryne beipiaoensis, Notobatrachus spp., Prosalirus bitis, Triadobatrachus massinoti, Yizhoubatrachus macilentus45,46,47,48,49), the lack of this framework in Ascaphidae + Leiopelmatidae appears to it is in synapomorphic. A columella can not be determined in the stem anuran Vieraella herbstii; however, the state of conservation of the specimen is poor50, leading part authors to think about the event of a columella to be unknown (e.g.,47) and also others to take into consideration it come be lacking (e.g.,43). Regardless, offered the phylogenetic position of Vieraella herbstii46,47,50, this debate has no bearing on ours inferences that the evolutionary history of this structure in anurans.

Although accessible evidence plainly indicates the plesiomorphic visibility of the columella in Anura, that is unknown if the ancestral anuran additionally possessed a tympanic membrane and also annulus. The tympanic membrane and tympanic annulus room not fossilizable structures, for this reason their an accurate phylogenetic beginning is unknown. As such, 2 scenarios space compatible with existing evidence: the tympanic annulus and membrane might have been present in the most recent usual ancestor that Anura and lost through the columella in Ascaphidae + Leiopelmatidae, or they could have arisen in Lalagobatrachia.

Among non-bufonid anurans, the TME was completely lost at the very least 27 time (see above). All these casualty involve small clades scattered across the significant lineages the Anura, implying several putative synapomorphies (e.g., Atelognathus + Chaltenobatrachus, Brachycephalus, Nasikabatrachidae + Sooglossidae, Pseudophryne, Telmatobufo) or autapomorphies (e.g., Balebreviceps hilmani, Melanobatrachus indicus, Rhinophrynus dorsalis). For various other anuran clades (e.g., Alsodes, Microhyla, Nanorana, Scutiger, Telmatobius), denser taxon sampling is crucial to achieve adequate proof to know the advancement of the TME (see section S2 the the Supplementary information for a more exhaustive discussion around the development of this framework in these and other non-bufonid species).

Although TME frameworks were lost repeatedly in Anura, TME advancement in Bufonidae is especially complex. Every one of the households that are very closely related to Bufonidae30 (also see51,52) have a complete TME, make the absence of these frameworks a synapomorphy that Bufonidae. As such, the lack of a TME is plesiomorphic in the earliest diverging lineages (i.e., Amazophrynella, most types of Atelopus, Dendrophryniscus, Oreophrynella, Osornophryne, Melanophryniscus and also Nannophryne).

Independent that the methodological approach, accessible evidence indicates that the finish TME was regained within Bufonidae in Frostius and also the sister clade that Nannophryne, whereas the tympanic annulus and also columella to be regained in a subclade the Atelopus. Thus, these structures are not homologous through the indistinguishable structures found in the TME of other anurans, although that seems most likely that the underlying genetic basis for their development is homologous (i.e., deep homology53, view below). Subsequent losses developed several time in different clades, denote a facility evolutionary history of the TME in Bufonidae (see Fig. 2 and section S3 of the Supplementary Information).

The facility evolutionary background of the TME in Bufonidae is even much more unusual when contrasted to other tetrapods. As noted above, although extant caecilians and also salamanders execute not possess a tympanic membrane or middle ear cavity, it has been hypothesized the a TME can have been current plesiomorphically and that the lateral aspects might have been lost independently5,54. Regardless, return the remaining middle ear structures underwent considerable modification, the columella was shed only once in each group, having actually been greatly decreased or lost in salamandrid salamanders1,55 and lost in adult scolecomorphid caecilians (present together a cartilaginous facet in fetal and juvenile Scolecomorphus kirkii56).

Among amniotes, TME ns is very rare. There room no recorded losses amongst turtles (e.g.,57), archosaurs (e.g.,58,59,60), or mammals (e.g.,61), regardless of the remarkable center ear transformations in fossorial and also marine mammals62,63. Amongst lepidosaurs, plenty of lineages have lost the lateral-most contents of the TME, consisting of Serpentes, Amphisbaenia, Agamidae, Diploglossidae, Gymnophthalmidae, Lanthonotidae, Phrynosomatidae and also Scincidae11,60,64,65,66, yet the columella appears to be existing in all but the pygopod lizard Aprasia repens12 and, possibly, the lamprophid snake Atractaspis and also Xenocalamus13.

With couple of exceptions, the advance of TME frameworks in anurans adheres to a regular sequence that could explain the consistent pattern the co-occurrence of middle ear structures and also provides clues about the mechanisms connected in your loss and gain. First, the medial end of the pars media plectri develops as a chondrification in ~ the connective tissue membrane spanning the fenestra ovalis nearby to the currently formed operculum27,67,68,69,70. Next, the pars interna plectri begins to chondrify and, through the incipient pars media, form the future stapedial footplate. Subsequently, a socket-like structure begins to be defined, articulating with the anterior sheet of the operculum. The lateral-most section of the stapedial footplate elongates to finish the formation of the tower of the pars media plectri, i m sorry extends laterally towards the outside of the head. Meanwhile, the tympanic annulus and pars externa plectri build as cartilaginous condensations associated with the posterior margin of the palatoquadrate. Together the palatoquadrate ferris wheel posteriorly throughout metamorphosis, so too execute the tympanic annulus and pars externa plectri. Together the ontogenetic succession of advance of these frameworks progresses, they are positioned in the exact same medial-lateral plane. In ~ this point, the partes media and externa plectri connect synchondrotically to every other and the tympanic annulus induces the differentiation that the tympanic membrane27,70.

The assignment of losses and gains show up to be concerned the family member timing that the breakthrough of structures (heterochronies) and also tissue differentiation phenomena. Because that example, Helff69 prove the inductive results of the tympanic annulus top top the tegument to create the differentiation that the tympanic membrane, which explains why the tympanic membrane never ever occurs in the lack of a tympanic annulus. Similarly, Hetherington27, Smirnov71 and also Fabrezi and Goldberg68 emphasized the fairly late advance of TME structures. Hetherington27 and also Smirnov71 additionally observed that several species undergo post-metamorphic breakthrough of previously lacking or undeveloped frameworks (e.g., Sclerophrys regularis, Pseudacris crucifer, Bombina orientalis). Meanwhile, Smirnov72 mentioned that developmental heterochronies (progenesis, neoteny and also post-displacement) seem to play a major role in the post-metamorphic advancement of these structures. All these events occur in certain sequences and their disruption in certain stages might produce the observed trends of accident in the succeeding stages of development of the TME. Therefore, research into the genetic basis because that the lack of induction that lateral elements promises to be a fruitful line of investigation.

Additionally, genetic mechanisms that directly regulate the expression of this ear structures can be involved. Knowledge of the origin of the materials of the vertebrate auditory mechanism is incipient generally and for anurans particularly. However, current studies that Xenopus laevis assistance a design in i beg your pardon the cartilaginous elements of the TME are derived from three neural crest cell streams (see73): (1) the mandibular stream develops the tympanic annulus, (2) the hyoid stream offers rise come the partes media and also externa plectri and (3) the branchial stream creates the pars interna plectri. The regular patterns that co-occurrences observed in anurans indicate that a direct function of regulatory gene and/or warrior factors could be connected in the tissular differentiation that the tympanic membrane as result of inductive phenomena native the tympanic annulus. Also, the is likely that the advance of the tympanic annulus and pars externa plectri (in the spare part of the palatoquadrate) and the partes interna and also media plectri (in the otic capsule) outcomes from the initiation of a typical developmental module, as in the morphogenesis of numerous other structures74,75. Unfortunately, details on the developmental regulate genes that result in the development of facets in the amphibian center ear is unavailable. However, some genetic pathways connected in this differentiation process have been figured out in other vertebrates and also could be examined in frogs76.

The lateral–medial dependency in between the visibility and lack of tympanic center ear frameworks appears also to be related to functional constraints: a tympanic membrane there is no a tympanic annulus or columella would have no acoustic function, as would certainly a tympanic annulus there is no a columella. In contrast, the tympanic annulus maintain its acoustic duty in the absence of a tympanic membrane and the columella continues to be acoustically functional also in the absence of both structures, as evidenced by the middle ears of salamanders1. This asymmetric functional dependency shows up to have permitted these three frameworks to evolve sequentially fairly than together a single change series (i.e., presence or absence of every the 3 structures), v losses and also gains of every element arising sequentially in a lateral–medial dependency, throughout the bufonid tree.

The losses, regains and re-losses the TME frameworks in Bufonidae do true toads an excellent model to examine the behavioral correlates that TME morphology. Previous studies have actually hypothesized a relationship between earlessness and aquatic or fossorial habitats and also lack that acoustic interaction or manufacturing of low-frequency calls77. Additionally, based on the minimal evidence presently obtainable (see ar S5 the the Supplementary Information), the ns of TME structures in Bufonidae appears to be coincident through the beginning of a scramble vain mating system in i m sorry males in thick aggregations attempt amplexus indiscriminately and also struggle because that possession of females78. In this mating system, acoustic ar defence is missing and dependence on hearing because that mate choice is greatly reduced or eliminated, together is the efficiency of prezygotic isolating barriers like advertisement calls78, which presumably outcomes in the organic interspecific hybridization it was observed in numerous bufonid varieties (see79 and also references therein).

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Nevertheless, although most of the types of early on diverging clades the Bufonidae for which the mating device is well-known exhibit scramble vain (see section S5 that the Supplementary Information), the reproductive action of most species is unknown, making the personality state restoration of this behaviour in ~ the source node that Bufonidae ambiguous. Similarly, both in ~ Bufonidae and throughout Anura, numerous of the groups that absence a TME rental high frequency (>1 kHz) advertising calls during reproductive interaction (e.g., Atelopus80, Bombina81, Brachycephalus82, Melanophryniscus83, Osornophryne84, Rhinophrynus85 and also Sechellophryne + Sooglossus86). Indeed, in spite of the absence of a TME and the event of a scramble competition adjustment strategy, interspecific acoustic diversity is kept in most genera (e.g.,80,87,88,89) arguing that acoustic signal play a tho unclear function in interaction and/or friend choice. The maintenance of call diversity and also widespread production of advertising calls might be explained by extratympanic hear pathways in earless frogs. Many extratympanic pathways, including a lung pathway (e.g., Atelopus22,23, Bombina81, Nectophrynoides asperginis21), one opercularis pathway (reviewed by3,25,90,91) and bone conduction magnified by resonation that the dental cavity (Sechellophryne92) have been presented effective or hypothesized so far in a few earless species. Provided that in at least some anurans airborne sounds room transferred via both tympanic and also extratympanic pathways (reviewed by3,25), anurans might experience tranquil selective pressure on the TME if TME plasticity does not greatly influence acoustic acuity. If the generality of alternate sound deliver pathways for aerial sounds is corroborated across anuran diversity, then the pre-existence of alternative pathways for airborne sound transmission could explain the high price of TME ns in anurans. Nevertheless, at this time proposed sound localization pathways in anurans every require center ear coupling93, leaving an alternative mechanism by which earless types localize audible sound unknown.