Auditory region circulation in Lagomorpha: the internal carotid artery pattern revisited

The internal carotid artery (ICA) is one of the major vessels in the cranial circulation. Characters concerning the ICA, such as its course in the auditory region, have been employed frequently in phylogenetic analyses of mammals, including extinct taxa. In lagomorphs, however, our knowledge on vascular features of the auditory region has been based predominantly on living species, mostly on the European rabbit. We present the first survey on 11 out of 12 extant genera and key fossil taxa such as stem lagomorphs and early crown representatives (Archaeolagus and Prolagus). The ICA pattern shows a modified transpromontorial course in stem taxa (Litolagus, Megalagus and Palaeolagus) and Archaeolagus, which we propose as the ancestral character state for Lagomorpha, similar to that for the earliest rodents, plesiadapids and scandentians. The ICA pattern in leporids is perbullar, but shows structural similarities to stem taxa, whereas the extrabullar ICA course in Ochotona is apparently a highly derived condition. Prolagus shows a mixed character state between leporids and Ochotona in its ICA route. The persistence of the transpromontorial ICA course and similarities in the carotid canal structure among stem taxa and crown leporids support morphological conservatism in Lagomorpha, in contrast to their sister clade Rodentia. This article is part of the theme issue ‘The mammalian skull: development, structure and function’.


Introduction
The paired carotid artery is the most important blood vessel supplying the head. It arises from the aortic arch as the common carotid artery, which branches into the external carotid artery (ECA), supplying most of the face and neck, and the internal carotid artery (ICA), which passing through the auditory region supplies the brain, orbit and nasal cavity through its offshoots: the anterior, middle and posterior cerebral arteries, ophthalmic artery, and ethmoidal artery, respectively [1][2][3][4][5]. However, the ICA itself does not contribute substantially to the blood supply of the ear region.
Characters concerning the organization and course of the internal carotid artery have been employed frequently in phylogenetic analyses of mammals, including extinct taxa [6][7][8][9][10][11], because the ICA foramina, canals or grooves are often preserved in skulls of fossil specimens.
The ancestral course of the ICA for Mammalia is accepted as medial to the promontorium of the petrosal [7,12], whereas for the Eutheria as lateral to the promontorium and in the tympanic cavity (see e.g. [12][13][14]). Interestingly, the reconstructed position of the ICA in the Late Cretaceous stem placentals Asioryctes, Kennalestes and Zalambdalestes is medial [10,12]. As currently understood, Matthew's [15] hypothesis suggesting the presence of two ICA branches (medial and lateral as postulated for several early eutherian taxa) is not valid, as the ICA at the auditory capsule's level originates from a single embryonic dorsal aorta independent from its later course [16].
In Eutheria, three main types of the ICA arrangement are observed, in its relation to the auditory region: (1) transpromontorial, where the ICA goes through the tympanic cavity and crosses the ventral surface of the promontorium of the petrosal; (2) perbullar, with the ICA contained within a canal in the medial wall of the auditory bulla; and (3) extrabullar, with the ICA passing the ear region externally, medial to the auditory bulla and the tympanic cavity [7]. The transpromontorial route of the ICA is recognized as an ancestral character state for Eutheria [14], whereas the perbullar and extrabullar courses of the ICA are considered derived conditions [7,10,11].
Thus far, our knowledge on vascular features of the auditory region in lagomorphs (figure 1), such as the ICA organization, has been based almost exclusively on living species and mostly on the European rabbit (Oryctolagus cuniculus; see [1,3,17,18]). Bugge [17,18] provided a detailed description of the cephalic arterial system, i.e. internal and external carotid arteries and stapedial artery including anastomoses, of extant Lagomorpha (three genera). While the internal carotid artery is relatively well developed in O. cuniculus, European hare (Lepus europaeus) and Afghan pika (Ochotona rufescens), the stapedial artery is substantially reduced (figure 1a). Its tympanic ( proximal) part is obliterated and mostly lost, whereas the distal portion and its branches are supplied by the ECA via anastomoses (see [17;18, fig. 15D]). The two living lagomorph families, Ochotonidae and Leporidae, exhibit different character states of the ICA: the former, the extrabullar, while the latter, the perbullar condition [7,18].
The skull anatomy of several fossil lagomorph species has been described in detail (e.g. [19][20][21][22]), but only Wolniewicz & Fostowicz-Frelik [23] devoted more attention to the ICArelated features. Wu [24] marks the external opening of the carotid canal 'external carotid foramen' sensu [24] in the fossil ochotonid Alloptox, similar to the opening in Prolagus sardus shown in Dawson [21]; Meng et al. [25] describe the tympanic-petrosal complex in another fossil pika, tentatively assigned to Sinolagomys, in which the spongy bulla shows an external opening for the ICA. Other contributions concerning the auditory region in lagomorphs are scarce and focused mostly on the organs of hearing (see [26][27][28]). Thus, our knowledge on the cranial circulation in the ear region of extinct (and most extant) lagomorphs is extremely limited.
Here we provide the first comprehensive and µCT databased morphological survey of the ICA-related structures traceable in the skull of Lagomorpha. Our study includes all but one of the extant lagomorph genera and a few fossil taxa for which the course of the internal carotid artery through the ear region could be ascertained from osteological features. This paper furthers the view of lagomorphs as an emergent robust system (see [29]) in which we can study macro-and micro-scale patterns of morphological change.

Material and methods
The auditory region of seven fossil and 29 extant lagomorph specimens has been studied, using the µCT scans and virtual three-dimensional reconstructions performed in Avizo 9.0.1 (Thermo Fisher Scientific 1995-2019) and VG Studio MAX2.2 (Volume Graphics, Heidelberg, Germany) software.

Stem Lagomorpha
In Megalagus turgidus, the posterior carotid foramen (PCF, the external opening into the carotid canal) enters the bulla in its posteromedial corner, directly anteromedial to the jugular foramen (figure 2a-c). The entrance is completely formed by the ectotympanic. In its course, the short proximal (or posterior) carotid canal is formed by the ectotympanic and petrosal and continues as a distinct and deep sulcus crossing the medioventral surface of the promontorium in a lateral curve. Anteriorly, it leaves the tympanic cavity in the medial direction; this area is not well preserved in the specimen.  In Palaeolagus haydeni and Palaeolagus burkei, the ICA enters the basicranium via the PCF, which lies between the ectotympanic and petrosal right in front of the jugular foramen (figure 3). The foramen opens into a relatively short proximal carotid canal that runs anterodorsally between these two bones and opens into the tympanic cavity. The ICA continues in a shallow sulcus on the medial side of the promontorium (figure 1b) and then becomes enclosed again, between the bulla and petrosal bone into the distal (anterior) carotid canal. The ICA then bends medially and enters the brain cavity next to the sella turcica. The anterior course of the ICA from the distal carotid canal into the brain cavity could not be fully traced in P. burkei owing to poor preservation in that area. The promontorium of both specimens is smooth and no additional sulci were detected.
In Litolagus molidens (figure 2d-f ), the PCF leads into a short proximal carotid canal between the ectotympanic and petrosal. The posterior rim of the foramen is embraced by a small lamella of the bulla. The course of the ICA across the promontorium cannot be traced completely. Posteriorly, no sulcus is present, though it might be obscured by scan artefacts. Anteriorly, the sulcus for the ICA continues into a distal carotid canal formed by the petrosal and ectotympanic.
The whole course of the ICA through the ear region in stem Lagomorpha can be therefore divided into three distinct sections: (1) the posterior carotid foramen leading into a short proximal carotid canal (housing the posterior ICA section) between the ectotympanic and petrosal, (2) the middle (promontorial) section of the ICA, which runs in a sulcus at the ventromedial side of the promontorium, and (3) a long anterior section of the ICA, which is completely enclosed by the surrounding bones (especially the petrosal and ectotympanic) forming a distal (or anterior) carotid canal, before it enters the cranial cavity. Megalagus and Litolagus depart in certain respects from this scheme; in both species the posterior carotid foramen is totally or at least mostly located in the tympanic bulla, and in Litolagus the posterior part of the ICA promontorial section is not represented by a sulcus. In all cases, there is no evidence of the stapedial artery as a branch of the ICA, a known fact for the extant taxa [18]. However, it should be noted that the absence of specific osteological characters (e.g. sulci) may not, strictly speaking, attest to the absence of the respective blood vessels (see [8]).

Crown Lagomorpha
The early archaeolagine leporid Archaeolagus ennisianus shows a huge PCF located in front of the jugular foramen between the ectotympanic and petrosal (figure 4). The carotid canal and the course of the ICA across the promontorium resemble the pattern observed in the stem lagomorphs under study, i.e. there is a shallow sulcus formed in the tympanic surface of the petrosal. The skull of A. ennisianus is only partially preserved; thus, the anteriormost course of the ICA into the cranial cavity is not fully recognizable.
All studied extant leporid specimens have a similar pattern of the osteological features related to the perbullar course of the ICA in the ear region. The auditory bulla shows a distinct PCF (for the ICA entrance) that is entirely formed by the ectotympanic and located posteromedially to medially in the bulla (figure 5), in the vicinity of the jugular foramen. The PCF opens into a wide carotid canal for the ICA. The proximal (posterior) part of the canal is made up by the ectotympanic only (apart from Nesolagus timminsi and Pentalagus furnessi), whereas the distal (anterior) part runs anterodorsally between the ectotympanic and the petrosal. Along its course, the royalsocietypublishing.org/journal/rstb Phil. Trans. R. Soc. B 378: 20220088 carotid canal does not enter the tympanic cavity, in contrast to the fossil taxa under study. However, before it bends medially toward the brain cavity (area of the sella turcica), its lateral wall can be incomplete and, thus, the canal lumen becomes confluent with the anteriormost part of the tympanic cavity in the area of the foramen lacerum medium. Most studied specimens have no distinct sulci on the promontorium for e.g. branches of the tympanic plexus; however, some species show certain specific characters due to the exact position of the PCF and the course of the carotid canal, which can vary even within a genus. In Caprolagus hispidus, Lepus spp., Pronolagus spp. and Sylvilagus spp., the carotid canal is situated anterior to the bony labyrinth (and the promontorium) and thus shows a steeper course. Pronolagus cf. saundersiae has a smaller foramen (possibly for the internal carotid nerves) directly in front of a large foramen; we interpret the latter as the PCF. Both canals are confluent inside the ectotympanic. In Oryctolagus cuniculus and C. hispidus, a very small canal branches off the proximal part of the carotid canal and runs anterodorsally between the ectotympanic and petrosal; more anteriorly, it enters the tympanic cavity and continues into a shallow sulcus on the promontorium that runs anteriorly and medially to the fenestra vestibuli ( figure 5). This canal and sulcus may house a branch of the internal carotid nerves  royalsocietypublishing.org/journal/rstb Phil. Trans. R. Soc. B 378: 20220088 (internal carotid plexus). Krause [1] described a distinct tympanic sulcus on the promontorium of O. cuniculus that houses the tympanic nerve (branch of CN IX); however, our structure seems to be located more anteriorly and thus does not refer to the tympanic nerve as it is clearly associated with the carotid canal. Lepus townsendii, Pronolagus rupestris and Sylvilagus nuttallii also show the small additional canal but no sulcus on the promontorium. In Lepus sinensis and Sylvilagus brasiliensis, a tiny separate canal is present posterior to the carotid canal that shows the same course (including a shallow sulcus on the promontorium) and corresponds to the small canal observed in Oryctolagus and Caprolagus. Sylvilagus floridanus has two small canals branching off from the carotid canal; the anterior one leads into a sulcus on the anterior tip of the promontorium.
Pentalagus furnessi and Nesolagus spp. have very small auditory bullae compared with those of most other leporids; they also differ in the PCF location and morphology. In Pentalagus, the PCF lies just at the posteromedial rim of the ectotympanic. The foramen is not fully formed by the ectotympanic but in part also by the petrosal. The canal is quite short and is entirely made up by the ectotympanic and petrosal. Nesolagus netscheri and N. timminsi show two foramina in the region of interest. A large foramen, most probably the PCF, enters the auditory region right in front of the jugular foramen. In N. netscheri, this foramen lies within the ectotympanic; in N. timminsi, contrary to other leporids, it is clearly situated between the auditory bulla and petrosal. In both species, the foramen opens into a short carotid canal that runs entirely between the two bones (ectotympanic and petrosal), similar to Pentalagus. However, a smaller canal that is confluent with the former is totally enclosed by the ectotympanic and its entrance is situated anteromedially to the large foramen. The promontorium in both Nesolagus species shows no sulci connected to the carotid canal.
In extant ochotonids, the ICA enters the cranial cavity (figure 6a) via the foramen lacerum medium (carotid notch of the piriform fenestra according to [32]). According to Bugge [18], the ICA runs below the medial tympanic bulla in an anterior direction before entering the basicranium. Absence of a distinct intraosseous course of the ICA in the auditory region, and thus an extrabullar course of the ICA, is confirmed in all the pika species in our study.
Dawson [21] described the external cranial anatomy of the fossil ochotonid Prolagus sardus and observed the PCF located anteromedially in the bulla (see her fig. 4). This is confirmed by our studied specimen of P. sardus (AMNH 116812). A short carotid canal is traceable in the anterior auditory region that runs from ventral to dorsal into the cranial cavity ( figure 6b-d). The PCF is formed by the ectotympanic and opens into the anteromedial part of the auditory bulla. Anteriorly, the carotid canal is formed by the ectotympanic and petrosal. The canal does not enter the tympanic cavity. Unfortunately, our specimen shows an incomplete promontorium and posterior auditory bulla; thus, no further information on additional small canals and sulci is available.

Discussion
In a broader phylogenetic scheme of the Euarchontoglires clade, lagomorphs noticeably differ in the course of the ICA from rodents, their sister clade within the Glires cohort [8].
Rodents show a great diversity of the ICA course patterns and of related structures (see [18,33]); they display all three types of the ICA route-transpromontorial, perbullar and extrabullar [12]-early in their evolution. Some most basal taxa, such as Cocomys, Sciuravus and Paramys show a transpromontorial course of the ICA [8], although Reithroparamys, a more specialized ischyromyid rodent already displays complete lack of the ICA (see [8,34]). The ancestral state for Glires was presumably the transpromontorial course of the ICA as this character state is shared by stem Lagomorpha royalsocietypublishing.org/journal/rstb Phil. Trans. R. Soc. B 378: 20220088 with the primatomorph Plesiadapis ( [35]; see [36] for phylogenetic position) and above-mentioned earliest rodents (ctenodactyloids, ischyromyids and sciuravids). The exact spatial relationships between the promontorium and ICA course within the tympanic cavity are, however, not the same in stem Lagomorpha as in primatomorphs and other Euarchontoglires, which have the transpromontorial ICA course. Furthermore, the ICA (especially its promontorial part) in stem lagomorphs is positioned differently from the reconstructed ICA morphology in early eutherians (see [7,13,35]). In lagomorphs, the sulcus for the ICA on the promontorium is more medial (especially in Palaeolagus and Litolagus) than in other Euarchontoglires displaying this character state.
The transpromontorial course of the ICA is observed in some Euarchontoglires groups, such as treeshrews (Ptilocercus and Tupaiidae; see [37]), Strepsirhini, Paromomyidae and Plagiomenidae (see [12]), but in all these groups the course of the ICA runs mostly in the middle of the promontorium.
In Dermoptera the ICA is lost, although the presence of the internal carotid nerves indicates a previous transpromontorial course in a similar position (see fig. 6 in [35]). Therefore, the ICA course in stem Lagomorpha cannot be interpreted strictly as ancestral compared with the totality of Euarchontoglires. Its position is already derived, although, being still transpromontorial, it should be regarded as a modified character state in relation to the primitive course known for other Euarchontoglires. A somewhat similar situation occurs in Tupaia, in which a bony canal encloses the ICA, but still within the tympanic cavity, and thus it is a derived state compared with an open transpromontorial sulcus [8,35].
Nevertheless, the transpromontorial course of the ICA and the presence of a sulcus in the Oligocene stem lagomorphs and especially early Miocene Archaeolagus are yet further confirmation of the notable morphological conservatism observed in Lagomorpha (see [18,23,30,38]).
The only other derived feature in stem Lagomorpha seems to be a lack of the stapedial artery originally existing  in their placental ancestors [8]. However, the stapedial foramen, normally passed by the stapedial artery, is retained as a plesiomorphic mammalian character in Lagomorpha (see [39]). The uniform pattern of the ICA route in stem lagomorphs stands in opposition to the rapid evolutionary changes observed in rodents and even advanced eurymylid Glires, such as Rhombomylus, which has already lost the PCF and a whole ICA, whereas in the closely related Matutinia the ICA presumably showed an extratympanic bifurcation of the ICA into a branch representing the anterior continuation of this vessel and the stapedial artery, with separate entrances [8,40].
There is a disparity in the ICA course and the carotid canal structure among the stem and crown Lagomorpha (figure 7) as well as within the crown groups themselves (table 1 for details). In general, all studied stem fossil taxa and Archaeolagus allowed the reconstruction of a more or less medial transpromontorial course of the ICA. The extrabullar ICA course of the extant Ochotona is apparently highly derived, whereas the ICA course in extant Leporidae shows principally a similar pattern to that observed in stem lagomorphs (and Archaeolagus). However, both groups differ in the formation of the PCF, lack of the promontorial sulcus and total enclosure of the carotid canal, especially by the bullar wall.
Among extant leporids, Nesolagus and Pentalagus are exceptions having the posterior part of the carotid canal and PCF formed also by both the ectotympanic and petrosal (figure 7), in this respect slightly resembling the ICA structure in stem lagomorphs (disregarding the absence of a transpromontorial sulcus). It is hard to discern whether such similarity is a result of a preserved ancestral structure, or a secondary derived character state, and its adaptive meaning is also unclear. The other feature of the ear structure that both extant genera (Nesolagus and Pentalagus) have in common with Megalagus is a relatively small auditory bulla. On the other hand, Litolagus and Palaeolagus, which also have the carotid canal built by both the ectotympanic and petrosal, display large auditory bullae. The size of the bullae in lagomorphs is regarded as an adaptive character related to the habitat type [22]; the species living in open landscape habitats or mixed forest/grassland environments have larger bullae than typical forest/jungle inhabitants (such as Nesolagus and Pentalagus). In rodents, bullar hypertrophy is frequently associated with increased aridity and xeric habitats (see [44]).
As there is no direct functional relation between the carotid canal structure and the size of the auditory bullae, we can assume that the ICA-related structures such as the PCF and presence/absence of the canal, and its composition, can be perceived as features of phylogenetic meaning in Lagomorpha. Although our study does not cover many Archaeolaginae and early Leporinae, it captures the ICA route and structure in most crucial points of the lagomorph evolution: stem taxa, early crown members and all two/three crown groups (depending on whether or not Prolagus is placed within Ochotonidae).
The character polarization is clear, and it follows the character states observed by Wible [12] for Placentalia: the transpromontorial course of the ICA with a well-displayed sulcus is an ancestral state for Lagomorpha, whereas the perbullar and extrabullar states should be considered as derived.
Furthermore, the PCF formed in parts by the ectotympanic and petrosal represents another ancestral character state for Lagomorpha, whereas the PCF formed solely by the ectotympanic can be considered derived. We can hypothesize that the carotid canal formed equally by the ectotympanic and petrosal (as in Nesolagus and Pentalagus) is less advanced than that formed prevailingly by the ectotympanic (the rest of In Nesolagus spp. and P. furnessi, a short carotid canal is completely formed by the ectotympanic and petrosal. royalsocietypublishing.org/journal/rstb Phil. Trans. R. Soc. B 378: 20220088 the studied leporids). Following this hypothesis, it can be assumed that Nesolagus and Pentalagus are relatively 'basal' leporid taxa, which at least partly agrees with the recent molecular-based phylogenies of extant Lagomorpha (see e.g. [43]). Nevertheless, we cannot rule out that this is a secondary character because evolutionary parallelisms are frequent in lagomorph evolution [38,42]. The question remains unanswered until a more exhaustive study of the Neogene leporids is conducted. However, the persistence of the 'stem lagomorph' pattern of the ICA-related structures in Archaeolagus shows the evolutionary stability of this structure. The developmental aspect supports our conclusions as the pattern observed in the stem lagomorphs resembles the one described in prenatal ontogeny of Oryctolagus cuniculus [45].
In the European rabbit, the proximal part of the ICA becomes enclosed by the growing ectotympanic, and the distal part runs in a sulcus on the anterior promontorium but becomes enclosed by the ectotympanic and petrosal later.
In terms of the general ICA pattern found in Leporidae, it can be treated as an advanced variant of the basic stem lagomorph structure, especially when the fully extrabullar ICA course of modern ochotonids is considered ( figure 7). In this respect, the pattern displayed by Prolagus sardus seems halfway between the typical leporid and ochotonid structures, as it is extrabullar in its posterior part but then becomes perbullar and the canal structure is similar to that of leporids ( figure 7). Furthermore, the PCF is shifted far anteriorly and thus resembles a transitional stage. The Prolagus lineage is old, originating around the end of the Oligocene [46]. Whether Prolagus indeed shares a direct common ancestor with Ochotona is another matter, but the ICA structure and canal pattern show some similarity to the leporid structure, which suggests an independent evolutionary history of the Prolagus lineage from the rest of Ochotonidae. Until more fossil ochotonids are studied, it is hard to determine if the Prolagus pattern is ancestral for the entire ochotonid lineage and the fully extrabullar condition appeared later in the Ochotona lineage, or Prolagus is an independent group of the crown Lagomorpha.
Data accessibility. Virtual reconstructions of the auditory region of six fossil and two extant lagomorphs are available from the Dryad Digital Repository: doi:10.5061/dryad.prr4xgxrd [31]. Detailed data for all specimens, including µ-CT scan parameters, are provided in electronic supplementary material, table S1.