The evolution of hearing in cetaceans is a matter of current interest given that odontocetes (toothed whales) are sensitive to high frequency sounds and mysticetes (baleen whales) are sensitive to low and potentially infrasonic noises. Earlier diverging stem cetaceans (archaeocetes) were hypothesized to have had either low or high frequency sensitivity. Through CT scanning, the morphology of the bony labyrinth of the basilosaurid archaeocete Zygorhiza kochii is described and compared to novel information from the inner ears of mysticetes, which are less known than the inner ears of odontocetes. Further comparisons are made with published information for other cetaceans. The anatomy of the cochlea of Zygorhiza is in line with mysticetes and supports the hypothesis that Zygorhiza was sensitive to low frequency noises. Morphological features that support the low frequency hypothesis and are shared by Zygorhiza and mysticetes include a long cochlear canal with a high number of turns, steeply graded curvature of the cochlear spiral in which the apical turn is coiled tighter than the basal turn, thin walls separating successive turns that overlap in vestibular view, and reduction of the secondary bony lamina. Additional morphology of the vestibular system indicates that Zygorhiza was more sensitive to head rotations than extant mysticetes are, which likely indicates higher agility in the ancestral taxon.

Introduction The evolution of whales from terrestrial to fully aquatic lifestyles is one of the most significant and famous transitions in vertebrate history. Those animals were faced with physiological challenges, especially when considering the special senses that had been evolving for hundreds of millions of years to function on land. The sense of hearing in stem cetaceans is of particular interest, given the physiological differences in the extant biota – the toothed whales (Odontoceti) are sensitive to high frequency and ultrasonic sound vibrations (Hall & Johnson, 1972; Ridgway et al. 1981; Brill et al. 2001; Hemilä et al. 2001; Ketten, 2004; Au et al. 2007; Nachtigall et al. 2007; Popov et al. 2007), whereas baleen whales (Mysticeti) are likely sensitive to lower frequency and potentially infrasonic noises based on behavioral models (Houser et al. 2001; Erbe, 2002; Parks et al. 2007). There is a great deal of interest in studying the evolution of hearing in cetaceans and two general hypotheses have been proposed for the attainment of the different auditory capabilities between the extant clades of whales. The first hypothesis is that low frequency sensitivity is ancestral for Neoceti (Odontoceti plus Mysticeti) and perhaps ancestral for Cetacea as a whole. Under that hypothesis, low frequency sensitivity is retained in mysticetes from their archaeocete ancestor with subsequent development of high frequency sensitivity in odontocetes. The low frequency hypothesis is supported by structures of the middle and inner ear (e.g. Fleischer, 1976; Thewissen et al. 1996; Nummela et al. 2004, 2007; Uhen, 2004). A second hypothesis is that high frequency sound reception is ancestral for Neoceti and retained in odontocetes with subsequent development of low frequency sensitivity in mysticetes. This intriguing hypothesis is supported by some data from the cochlea (Ketten, 1992b) as well as mandibular anatomy and asymmetry in archaeocete and odontocete skulls (e.g. Bianucci & Gingerich, 2011; Fahlke et al. 2011). Crucial to the testing of these hypotheses is the auditory capability of archaeocetes and other stem cetaceans. Numerous attempts have been made to reconstruct the hearing physiology of archaeocetes, primarily through comparative anatomy of the middle ear region as preserved on and around the petrosal bone and tympanic bulla (Lancaster, 1990; Nummela et al. 1999, 2004, 2007), the hypothesized sound transmission pathway through the lower jaw (e.g. Roth, 1978; Thewissen et al. 1996; Nummela et al. 2007; Steeman, 2009), cranial endocasts in the region of cranial nerves VII (facial) and VIII (vestibulocochlear) (Edinger, 1955), and cranial asymmetry as related to directional hearing (Fahlke et al. 2011). However, few studies have examined the functional unit of the auditory portion of the inner ear in early whales as contained within fluid‐filled chambers that contribute to the cochlea in mammals (exceptions include Fleischer, 1976; Uhen, 2004). The functional unit of the cochlea is the spiral organ of hearing (organ of Corti), which extends the length of the cochlea upon the basilar membrane. Several quantifiable features of the basilar membrane are thought to be correlated with hearing physiology in mammals, including the length, thickness, and width of the basilar membrane (e.g. Békésy, 1970; Wever et al. 1971b; Fleischer, 1976; Echteler et al. 1994; Ketten, 1994, 1997; Wartzok & Ketten, 1999). The morphology of the basilar membrane is difficult to study in extinct animals because the soft‐tissue structure is not preserved in the fossil record. However, the bony supports for the membrane, which are the primary and secondary bony laminae, are readily preserved in fossils as spiral ridges along the axial (inner) and radial (outer) walls of the cochlear canal, respectively (e.g. Fleischer, 1976; Court, 1992; Meng & Fox, 1995; Geisler & Luo, 1996; Ruf et al. 2009; Macrini et al. 2010; Ekdale & Rowe, 2011). Thus, many of those features thought to relate to hearing can be measured from a fossil, which offers the opportunity for documenting important transformations in the anatomy of the auditory system of extinct and extant cetaceans. Other features that are not directly related to basilar membrane morphology include the number of cochlear turns and gross anatomy of the cochlear spiral (e.g. West, 1985; Ketten & Wartzok, 1990; Manoussaki et al. 2006, 2008). In this paper, the anatomy of the bony labyrinth of the inner ear, including the cochlea and semicircular canals, is described for the extinct archaeocete Zygorhiza kochii. Zygorhiza is a basilosaurid archaeocete (Uhen, 2004) and commonly is used in phylogenetic analyses of cetaceans given its close relationship with Neoceti (e.g. Geisler & Sanders, 2003; Deméré et al. 2008; Ekdale et al. 2011; Geisler et al. 2011; Marx, 2011; Bisconti, 2012; Fordyce & Marx, 2013). The descriptions are supplemented with novel observations of the inner ears of extinct and extant mysticetes, for which little information is known compared with odontocetes, as well as comparisons with published information for other cetaceans. Imaging of the internal cavities of the bony labyrinths was accomplished through high resolution X‐ray computed tomography (CT) coupled with digital segmentation of inner ear cavities. Through detailed description of structures assumed to be associated with hearing sensitivity in the cochlea of Zygorhiza as compared with the ear regions of Neoceti, a more accurate interpretation of early cetacean physiology can be reconstructed.

Discussion One extreme difficulty in reconstructing the auditory physiology of extinct cetaceans is the lack of published audiograms for any extant mysticete. Thus, any comparisons using the morphology of baleen whales can only be made in a relative sense. Among extant cetaceans, odontocetes have been divided into two categories based on the ultrasonic frequencies of their vocalizations, which appear to correspond to auditory sensitivities, and morphological differences in cochlear shape (‘type I’ and ‘type II’ of Ketten, 1984; Ketten & Wartzok, 1990; Table 1). In general, each of those measurements is greater for the ‘type II’ cochlea of odontocetes than ‘type I’ (Ketten & Wartzok, 1990). The peak vocal frequencies of mysticetes are much lower than those of odontocetes (Edds, 1982; Ljungblad et al. 1982; Cummings & Holliday, 1987; Edds et al. 1993; Wartzok & Ketten, 1999; Erbe, 2002; Stimpert et al. 2007; Buchan et al. 2010), and all mysticete ears were binned within a single category (‘type M’; Ketten, 2000). There have been a few attempts to compare cochlear anatomy among archaeocetes, odontocetes, and mysticetes in order to reconstruct the auditory physiology of Zygorhiza and allied taxa (e.g. Fleischer, 1976; Ketten, 1992b; Uhen, 2004). The bony laminae were poorly preserved in a specimen of Zygorhiza kochii examined by Fleischer (1976), although he argued that the overall structure of the Zygorhiza cochlea was significantly different from either extant Odontoceti or Mysticeti in terms of basal ratio. However, there is little difference among most cetaceans examined here and elsewhere (Table 1). Ketten (1992b) reexamined Fleischer's data for Zygorhiza and supplemented those analyses with additional archaeocete data compiled from Kellogg (1936). Ketten concurred with Fleischer that the basilosaurid cochlea is unlike either odontocetes or mysticetes and she classified Zygorhiza as ‘types I’, ‘II’, and ‘M’. Ultimately, she hypothesized that Eocene archaeocetes likely were sensitive to high frequencies with subsequent evolution of low frequency sensitivity in mysticetes, based on the incomplete cochlea initially described by Fleischer (1976). Uhen (2004) studied the cochlea of the closely related basilosaurid Dorudon atrox, which was better preserved than the Zygorhiza cochlea examined by Fleischer (1976). Uhen concluded that Dorudon was much more comparable to extant Mysticeti than extant and extinct Odontoceti, particularly in terms of the laminar gap, and he hypothesized that basilosaurids likely were sensitive to similar lower frequency sound waves as has been hypothesized for extant Mysticeti (Uhen, 2004). Although some previous studies were unable to unite the cochlea of Zygorhiza with odontocetes or mysticetes (Fleischer, 1976; Ketten, 1992b), the dimensions of the cochlea can be compared. There are published data available for several odontocete species Ketten and Wartzok, 1990) that can be combined with the novel measurements of mysticetes produced in the present study. In terms of number of turns, the range of both types ‘II’ and ‘M’ overlap (1.7–2.5 and 2.0–2.7, respectively) and Zygorhiza (2.5) falls within that range (Table 1). Note that the number of turns calculated for the Tursiops truncatus specimen examined by Ekdale (2013) is nearly one half turn less than the value reported by previous authors (e.g. Ketten & Wartzok, 1990; Ketten, 1992b, 2000). In terms of axial pitch and absolute axial height, Zygorhiza falls between the ranges of odontocetes and mysticetes (Table 1). The basal and slope ratios of Zygorhiza fall within the ranges of mysticetes examined here and are separate from values calculated for ‘type I’ odontocetes (Table 1). Taking those observations together, it is unlikely that the cochlea of Zygorhiza is of ‘type I’, and although it may be classified as ‘type II’, there is more evidence for a ‘type M’ or mysticete‐like classification. Because the anatomy of the cetacean cochlea is related to auditory function and auditory thresholds initially defined the ‘types’, Zygorhiza likely would have been sensitive to similar low frequencies as are extant mysticetes. Most previous attempts to reconstruct the hearing physiology of extinct mammals have focused on the secondary bony lamina, and the laminar gap in particular (e.g. Fleischer, 1976; Geisler & Luo, 1996; Ekdale & Rowe, 2011). In life, the laminar gap and the robusticity of each bony spiral lamina affect the rigidity of the basilar membrane in such a way that a narrow gap and robust laminae indicate a stiffer membrane (Wever et al. 1971b; Pye, 1979). Stiffness and thickness of the membrane in turn are related to frequency sensitivity, in that a mammal with a stiffer basilar membrane will be sensitive to higher frequency vibrations than a mammal with a more flexible membrane (Békésy, 1970; Echteler et al. 1994; Ketten, 1994, 1997; Wartzok & Ketten, 1999). Following a transitive property, one mammal with a narrower laminar gap than another mammal will have a stiffer basilar membrane, and in turn will be more sensitive to higher frequencies. Even in the absence of neurophysiological data, the laminar gap can be used to interpret the relative hearing differences among mammals. However, the primary and secondary bony laminae are extremely thin and delicate, and their distal edges towards the center of the cochlear lumen often are missing or are not resolved in CT images of even the best preserved recent specimens (Fig. 4). The delicate nature of the bony laminae decreases the accuracy of basilar membrane width such that laminar gap estimates may be off by more than 100% when estimating actual membrane width at the basal end of the mysticete cochlea (Ketten, 2000). For odontocetes, estimates near the apex can be off by approximately 25%. Although the distal portions of the laminae are delicate, the bases are more robust and are more readily preserved, even in fossils. The proportion of the cochlear canal in which the secondary bony lamina is present is an indicator of relative high vs. low frequency sensitivity. Indeed, the secondary bony lamina is present for nearly 90% of the cochlea in Tursiops, but is restricted to the first three quarters in all mysticetes (Table S2). Among mysticetes, the secondary bony lamina is reduced to the greatest degree in extant balaenids, where it is restricted to the first third of the basal turn (Table S2). The secondary bony lamina extends for only half the cochlear canal in Zygorhiza, which suggests comparable development of the secondary bony laminae between the archaeocete and most mysticetes. Although gracile and short (less extensive) with respect to odontocetes, the secondary bony lamina is present in mysticetes. However, it has been hypothesized that the secondary bony laminae in mysticetes ‘are not functional equivalents’ of the secondary bony lamina in odontocetes and that ‘presence of the [secondary bony] lamina in mysticetes is a residual ancestral condition rather than a derived structure related to mysticete frequency ranges’ (Ketten, 1992a: p. 736). A secondary bony lamina is not visible in the endocasts of the early cetaceans Ichthyolestes and Indocetus that are figured by Spoor et al. (2002), although it is unclear whether the structure is in fact absent in those taxa. Both Ichthyolestes and Indocetus are archaeocetes, but they represent very different stages in the evolution of whales and degree of secondarily aquatic adaptation than Zygorhiza, which might account for morphological differences. The secondary bony lamina is described as absent for the terrestrial artiodactyl Sus (Ekdale, 2013), although the structure is described and figured for the early artiodactyl Diacodexis (Orliac et al. 2012), extinct notoungulates (Macrini et al. 2010, 2013), and the extant mouse deer Moschiola (Orliac et al. 2012). We agree that the secondary bony lamina is an ancestral retention in mysticetes, especially given the similarities in structure between mysticetes and Zygorhiza, but given that the bony lamina is attached to the basilar membrane, its very presence would affect the function of the membrane. A combination of basilar membrane length and number of cochlear turns is hypothesized to be useful in predicting high and low frequency thresholds in terrestrial mammals (West, 1985). In particular, West argued that the low frequency limit of hearing in terrestrial mammals could be predicted by the product of basilar membrane length multiplied by number of turns. Manoussaki et al. (2008) recovered a similar correlation, thereby corroborating West's hypothesis. The product of length times turns calculated for Zygorhiza falls between extant odontocetes and mysticetes (Table S2), but the metric is not entirely independent of body mass (length of the cochlear canal scales to body mass; Ekdale, 2013). Furthermore, West's calculations were based on audiograms measured in air. Pinnipeds have different auditory thresholds in water and air (e.g. Kastak & Schusterman, 1998, 1999), and subsequent investigations of West's correlations indicate that the dimensions may not hold for marine mammals, at least where the low frequency limit is concerned (Manoussaki et al. 2008). Therefore, this product may not be appropriate for the interpretation of auditory physiologies of extinct marine mammals, although future research is needed in this regard. The graded curvature of the cochlea appears to be more important for reconstructing auditory capabilities of extinct mammals (Manoussaki et al. 2006, 2008). Manoussaki et al. (2008) examined a broad range of placental mammals and concluded that taxa with wider basal turns relative to apical turns had a downward shift in low frequency thresholds. As might be expected, the grade calculated for most mysticetes is greater than that calculated for Tursiops (Table S2). In fact, the taxa examined by Manoussaki et al. (2008) that have grades approaching mysticetes are the cow and the elephant, both of which are known to be sensitive to very low frequency noises (Heffner & Heffner, 1982, 1983; Payne et al. 1986; Poole et al. 1988). Using the equation provided by Manoussaki et al. (2008), the low frequency threshold for Tursiops was estimated to be 187 Hz (in water at 120 dB re 1 μPa; range in the literature from 150 to 200 Hz; Ketten, 2000; Manoussaki et al. 2008) and extinct and extant mysticetes ranged from 10 Hz (Eubalaena) to 107 Hz (Balaena) (Table S2). Balaena is an outlier – the next highest threshold was estimated for the extinct eschrichtiid (60 Hz). A low frequency threshold for Zygorhiza was estimated at 8 Hz, which not only is below the range of normal human hearing (20 Hz to 20 kHz) but is lower than that estimated for any mysticete considered here. Although these results support the hypothesis that mysticetes are sensitive to low frequency noises, the specific frequency values that are estimated here should be considered tentative until audiograms of mysticetes are obtained and tested against the equation of Manoussaki et al. (2008), which was based primarily on hearing of terrestrial mammals in air. Related to the ‘tightness’ of coiling of the cochlea is the separation between basal and apical turns. Moving through the cochlea from the base to the apex, the walls separating the turns become thinner in all of the cetaceans examined here (Table S3; Fig. 5). The thickness of the wall at one‐quarter turn of the basal whorl relative to the basal diameter is nearly twice as large in Tursiops than in any mysticete, but Zygorhiza falls in between. However, when the cochlea is observed in cross‐section, it can be seen that the walls are really quite thin in apical regions of Zygorhiza (w in Fig. 3, slice 80, and Fig. 4A), and the walls of successive turns overlap as observed in mysticetes (Fig. 4), indicating more similar hearing lifestyles in those taxa. When compared with extant cetaceans, the cochlea of Zygorhiza is longer and coils to a greater degree, the wall separating the basal and apical whorls is relatively thinner, subsequent turns overlap, the secondary bony lamina is less robust and extends to a lesser degree within the cochlear canal, and the apical turn is coiled tighter (has a smaller relative radius) than the basal turn. All of these features point to physiology in line with extant mysticetes, which is a presumed sensitivity to low frequencies. If archaeocetes such as Zygorhiza were sensitive to low frequencies, then low frequency sensitivity would be the ancestral condition for crown Cetacea that is retained by extant mysticetes (Fig. 7). A subsequent specialization for high frequency sensitivity was developed in odontocetes. Figure 7 Open in figure viewer PowerPoint Generalized cladogram of Cetacea with mapped characters associated with auditory physiology. Thick line indicates low frequency sensitivity and thin line indicates high frequency sensitivity. Physiological significance of the characters is provided in the text. Although structure of the cochlea supports low frequency sensitivity in Zygorhiza, high frequency sensitivity in archaeocetes was hypothesized based on cranial asymmetry and mandibular architecture (Fahlke et al. 2011). The mandibular features that have been used to argue high frequency hearing sensitivity in archaeocetes include a possible pan bone and enlarged mandibular canal for a fat pad (Bianucci & Gingerich, 2011; Fahlke et al. 2011). However, recent evidence has shown that the acoustic fats and pan bone may not serve as the primary ‘acoustic window’ for reception of high frequency sounds as originally thought (e.g. Norris, 1968), but rather the primary sound transmission pathway of sound to the ear is through the gular region and into the fat pads leading to the ear complex (Cranford et al. 2008). In this sense, the enlarged mandibular foramen would serve as an ‘open door’ (Cranford et al. 2010) that likely facilitates hearing underwater in general for early whales, rather than hearing at specific frequencies. Furthermore, an enlarged mandibular foramen would allow access for sound waves with larger wavelengths (indicating lower frequencies) and higher amplitudes (Barroso et al. 2012). This should not imply that the animals were detecting low frequency vibrations, but that those vibrations could be transported to the ear. Likewise, cranial asymmetry in archaeocetes, as argued by Fahlke et al. (2011), likely is an adaptation for directional hearing underwater rather than evidence of biosonar or a particular bandwidth of hearing sensitivity, but more research is needed in this area. When considering the vestibular system and its relationship to agility and locomotion, cetaceans have been excluded from most correlations on account of the reduction of the vestibular system (e.g. Spoor et al. 2002) and cetacean estimates may not be comparable to terrestrial mammals (Spoor et al. 2007). However, the deviation of ipsilateral (same side) semicircular canal pairs from orthogonality (Malinzak et al. 2012; Berlin et al. 2013) is independent of body mass (unlike correlations based on semicircular canal arc radius; Spoor et al. 2007; Silcox et al. 2009) and it is perhaps more appropriate for extinct and extant cetaceans. In general, the closer two semicircular canal planes are to orthogonal (90°), the more sensitive the canals are to head rotations (Malinzak et al. 2012; Berlin et al. 2013). The average deviation from orthogonality for the three semicircular canal pairs is less for Zygorhiza than for any other cetacean examined, including Tursiops (Table S4), indicating high rotational sensitivity. Interestingly, the average deviation is low for extant Balaena, too. These results support a hypothesis that the deviation from orthogonality of the semicircular canals in mysticetes is likely a result of their shift from raptorial, single‐prey predation as hypothesized for basilosaurids (Uhen, 2004; Fitzgerald, 2010) to bulk filter feeding in later diverging mysticetes. One might argue that bulk filter feeding would not require semicircular canals as sensitive as those needed for active pursuit predators. However, the agility of mysticetes has not been investigated across the clade and several mysticete species exhibit elaborate movements during breaching and feeding (e.g. lunging in balaenopterids). Future investigations into the sensitivity of the semicircular canals to rotations of the head should assess intraspecific variation in canal shape and orientation. For example, a greater degree of variation in the orientations and morphology of the semicircular canals was observed in slow moving xenarthrans compared with faster moving species (Billet et al. 2012). We predict that if there is a reduction in the sensitivity of the semicircular canals that is associated with bulk filter feeding, and in turn agility, then a greater degree of variation in the deviation of the canal planes from orthogonality would be observed in mysticetes than in earlier diverging raptorial cetaceans. In summary, the morphology of the inner ear of Zygorhiza supports the hypothesis that low frequency sensitivity was ancestral for cetaceans and was retained in mysticetes with subsequent high frequency sensitivity in odontocetes. Given the potentially harmful effects of anthropogenic noise on cetaceans and other marine mammals, an accurate interpretation of auditory physiologies of living whales is of paramount importance. Thus, additional information from extinct mysticetes and odontocetes will uncover further patterns of the sensory evolution in cetaceans, which will lead to a better understanding of the physiologies of the living biota.

Acknowledgements This project was funded by the National Science Foundation, DEB‐0743869, DEB‐0743861, and DEB‐1146371. We thank M. Colbert and J. Maisano at UTCT for generating the CT data used in this study. We also thank A. Berta and T. Deméré for helpful discussions during the development of this project. Three anonymous reviewers provided helpful comments during the review process of the manuscript.

Author contributions Both authors contributed to the design of the project, acquisition of data and data analysis. E.G.E. drafted the original manuscript and both E.G.E. and R.A.R. critically revised and approved the completed manuscript prior to submission.

Supporting Information Filename Description joa12253-sup-0001-TableS1.pdfPDF document, 49.6 KB Table S1. Parameters used during novel CT scanning of cetaceans considered here. Daggers (†) indicate extinct taxa. Parameter abbreviations: Interslice – interslice spacing in μm; Interpixel – interpixel spacing in μm, calculated as field of reconstruction/image resolution; Reconstruction – field of reconstruction in mm; Resolution – image resolution of individual CT slices in pixels; Spec No – specimen numbers. Institutional abbreviations: HSU VM – Humboldt State University, Arcata, CA; LACM – Natural History Museum Los Angeles County, Los Angeles, CA; SDNHM/SDSNH – San Diego Natural History Museum, San Diego, CA; USNM – United States National Museum of Natural History, Washington, DC. joa12253-sup-0002-TableS2.pdfPDF document, 90.3 KB Table S2. Dimensions and shape ratios of the bony labyrinths of cetaceans. Data from the bony labyrinths of Balaenopteridae (NC) (from Pliocene of North Carolina) and Tursiops truncatus were taken from Ekdale ( 2013 3); Cw – width of basal turn of cochlea (mm); LF – estimated low frequency limit (Hz) based on graded curvature (following Manoussaki et al. 2008 3); ρ – graded curvature of cochlea, calculated as Crb/Cra). joa12253-sup-0003-TableS3.pdfPDF document, 110.8 KB Table S3. Dimensions of the internal structures of the cochlear canal at each quarter of every turn. First measurement location (0/4) is immediately behind the center of the fenestra cochleae. Values expressed in mm. Dashes (–) indicate that the structure is not present, and ‘NP’ indicates that the structure is not preserved. Bony labyrinths of Balaenopteridae (NC) (from Pliocene of North Carolina) and Tursiops truncatus are described further by Ekdale ( 2013 joa12253-sup-0004-TableS4.pdfPDF document, 77.9 KB Table S4. Dimensions and orientations of anterior (A), lateral (L), and posterior semicircular canals (P). Canal arc radii and lengths expressed in mm, and 90 var refers to average deviation of canal pair angles (90 A‐L , 90 A‐P , 90 L‐P ) and expressed in degrees. Data from the bony labyrinths of Balaenopteridae (NC) (from Pliocene of North Carolina) and Tursiops truncatus were taken from Ekdale ( 2013 Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.