bioRxiv preprint doi: https://doi.org/10.1101/490367; this version posted December 7, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Petrosal morphology and cochlear function in Mesozoic stem therians Tony Harper1, Guillermo Rougier2* 5 1Tony Harper, Center for Functional Anatomy and Evolution, Johns Hopkins University, 1830 E. Monument St., Baltimore, MD 21205, USA. ORCID: 0000-0002-9561-1562 2Department of Anatomical Sciences and Neurobiology, University of Louisville, 511 S. Floyd 10 St., Louisville, KY 40202, USA. ORCID: 0000-0003-3051-1373 *Corresponding Author: Guillermo W. Rougier [email protected] 15 502-852-4541 Short Title: Stem therian petrosals 20 1 bioRxiv preprint doi: https://doi.org/10.1101/490367; this version posted December 7, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Abstract Here we describe the bony anatomy of the inner ear and surrounding structures seen in three of the most plesiomorphic crown mammalian petrosal specimens in the fossil record. Our 25 study sample includes the stem therian taxa Priacodon fruitaensis from the Upper Jurassic of North America, and two isolated petrosal specimens colloquially known as the Höövör petrosals, recovered from Aptian-Albian sediments in Mongolia. The second Höövör petrosal is here described at length for the first time. All three of these stem therian petrosals and a comparative sample of extant mammalian taxa have been imaged using micro-CT, allowing 30 for detailed anatomical descriptions of osteological correlates of functionally significant neurovascular features, especially along the abneural wall of the cochlear canal. The high resolution imaging provided here clarifies several hypotheses regarding the mosaic evolution of features of the cochlear endocast in early mammals. In particular, these images demonstrate that the membranous cochlear duct adhered to the bony cochlear canal 35 abneurally to a secondary bony lamina before the appearance of an opposing primary bony lamina or tractus foraminosus. Additionally, while corroborating the general trend of reduction of venous sinuses and plexuses within the pars cochlearis seen in crownward mammaliaformes generally, the Höövör petrosals show the localized enlargement of a portion of the intrapetrosal venous plexus. This new excavation is for the vein of cochlear aqueduct, a 40 structure that is solely or predominantly responsible for the venous drainage of the cochlear apparatus in extant therians. However, given that these stem therian inner ears appear to have very limited high-frequency capabilities, the development of these modern vascular features the cochlear endocast suggest that neither the initiation or enlargement of the stria 2 bioRxiv preprint doi: https://doi.org/10.1101/490367; this version posted December 7, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. vascularis (a unique mammalian organ) is originally associated with the capacity for high- 45 frequency hearing or precise sound-source localization. Introduction 50 Therian mammals today display an impressive variety of auditory features facilitating the adept detection of airborne sound, making this group arguably the most acoustically sophisticated and diverse living or extinct clade of terrestrial vertebrates. The widespread capacity for high-acuity hearing (in terms of sensitivity, specificity, and highest detectable frequency) among the majority of extant therian mammals has reinforced the assumption 55 among neontologists that the Mesozoic members of the therian stem linage were stereotypically nocturnal forms that leveraged their auditory faculties to locate small prey and escape gigantic predators (i.e. [1]). However, this supposition has not been supported by the known fossil record of stem therians, with prior descriptions [2-7] demonstrating that these forms lacked the level of petrosal organization characterizing plesiomorphic marsupials [8], 60 afrotheres [9], eulipotyphlans, and primatomorphs [10]. Additionally, these reports have relied heavily on the description of the external morphology of the ear region, and for the most part remained silent or equivocal regarding the performance and/or physiological implications of the bony anatomy seen in fossil specimens. Conversely, several biomechanical/experimental studies on auditory anatomy and 65 physiology across extant tetrapods [11-15] highlight the unique nature of the therian cochlear apparatus (with its well-ordered acoustic hair-cell populations arrayed along the organ of 3 bioRxiv preprint doi: https://doi.org/10.1101/490367; this version posted December 7, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Corti, high endolymphatic potential, and absence of the lagenar macula), as well as the plesiomorphic nature of the monotreme cochlea with respect to many modern and fossil therians [16,17]. The complete loss of the lagenar macula in particular has been posited as a 70 therian-lineage evolutionary breakthrough that allowed for the later elongation and sophistication of the cochlear apparatus for non-linear amplification of high-frequency stimuli [18]. Conversely, the retention of a functional lagenar macula, along with its accompanying otoconial and neurovascular structures, in the monotreme and sauropsid lineagesis hard to conciliate with the soft tissue adaptations seen in modern therians. These “therian” features 75 include: 1) the exclusive reliance on the stria vascularis as the major endolymph producing organ, 2) the well-developed electromotility of prestins and other molecular components of the “cochlear amplifier”, and 3) the radical elongation and geometrical reorganization of the cochlear sensory epithelium. The distribution of these characteristics in extant animals, within the wider framework of cynodont evolution, points to a fundamental transformation of the 80 cochlear apparatus somewhere within the phylogenetic vicinity of the therian stem [18]. The lack of anatomical depiction within the Mesozoic fossil record is understandable given the general lack of high-fidelity bony correlates for key soft tissue structures such as the cochlear duct, lagenar macula, and stria vascularis. Additionally, prior studies using high- resolution imaging of the internal anatomy in pertinent fossils have not commented on or 85 depicted the possible existence of several physiologically significant bony structures that are not familiar to most paleontologists. This report uses high-resolution micro-CT information to update and qualify the descriptions of petrosal anatomy provided in the representative sample of stem therians focused on by [2] and [3], two large-scale studies characterizing the fossil record of early mammalian petrosal evolution (Fig 1). These images and reconstructions are 90 framed within a broader comparative and functional setting of modern mammalian auditory 4 bioRxiv preprint doi: https://doi.org/10.1101/490367; this version posted December 7, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. physiology, and morphology with the hope of bridging both fields which, due to the steep learning curves involved, have had only limited cross-referencing. These new high-resolution images allow the first descriptions of the labyrinthine anatomy in some of the most morphologically plesiomorphic petrosal regions known in the crown mammalian fossil record. 95 The three specimens focused on here include the relatively well-known triconodontid Priacodon fruitaensis and the two isolated petrosal specimens known as the Höövör petrosals. The second Höövör petrosal (Fig 1 a, b) has not been described or figured, and has only been cursorily referred to in previous publications (i.e. [2]). We regard this second petrosal as distinct from that of the previously described petrosal in [2], and therefore provide 100 here a complete description and assessment of this specimen. Fig 1. Stem therian petrosal specimens described in this report. a,b Höövör Petrosal 2 (PSS-MAE-119); c,d, Höövör Petrosal 1 (PSS-MAE-104); e,f left petrosal of Priacodon fruitaensis (LACM 120451). a,c,e in ventral view; b,d,f in dorsomedial view. Scale bar is 1 105 mm. The novel information presented here does little to overturn previous taxonomic attributions of these stem therians especially the first and second Höövör petrosals, one or both of which are tentatively referred to taxa within Gobiconodontidae and/or Tinodontidae. 110 However, the new anatomical representation provided by the images used here demonstrates the presence of a combination of plesiomorphic and derived character states of the cochlear canal that support their ancestry