Pentalagus furnessi (Amami rabbit) is a medium-sized leporid and the only living species of the genus. Today, Pentalagus furnessi is restricted to Amami Oshima and Tokunoshima (Ryukyu Islands) in southern Japan and probably arrived at the latter during the early Middle Pleistocene. The species is known from only four fragmented extant populations, which in concert with habitat loss and introduction of dogs, cats and Herpestes javanicus accounts for the classification “Endangered” by the IUCN. The Amami rabbit differs from all other living Leporidae in having very short ears, small eyes, tail and hindfeet; the claws are significantly longer and used for digging burrows as well as climbing steep slopes; vocalization is comparable to Ochotona (Otsuka et al. 1980; Tomida and Otsuka 1993; Yamada et al. 2002; Yamada and Cervantes 2005; Schai-Braun and Hackländer 2016). Although Pentalagus furnessi is considered to be one of the most primitive extant lagomorphs it is well nested within Leporidae based on morphological and especially molecular data (e.g., Yamada et al. 2002; Ge et al. 2013; Schai-Braun and Hackländer 2016; Cano-Sánchez et al. 2022).

As an insular species, Pentalagus furnessi is enigmatic in several respects but still little is known about its cranial anatomy. While some external cranial and dental studies of Pentalagus furnessi exist (e.g., Otsuka et al. 1980; Tomida and Otsuka 1993; Ge et al. 2015), no anatomical descriptions of intracranial structures are available to date.

The mammalian cranium holds important structural complexes for understanding evolution, development and function of the sensory systems, dietary and locomotory adaptations as well as phylogenetic information (e.g., Maier 1993; Wible and Hopson 1993; Spoor et al. 2007; Usui and Tokita 2018; Martin and Koenigswald 2020). In their comprehensive review on the biology, systematics, and evolution of Lagomorpha, Kraatz et al. (2021) propose this order as an excellent model system to study a broad array of functional systems and evolutionary transformations. In this regard it is important to fill gaps of knowledge in lagomorph cranial anatomy.

The present study provides the first anatomical description of intracranial structures in Pentalagus furnessi focusing on nasal structures. The turbinal skeleton supports the respiratory and olfactory epithelia and provides phylogenetic as well as morphofunctional information as already demonstrated in several other mammalian orders (e.g., Maier 2000; Smith and Rossie 2008; Van Valkenburgh et al. 2011, 2014; Smith et al. 2012; Ruf 2014, 2020; Ruf et al. 2015, 2021; Martinez et al. 2018, 2020; Wagner and Ruf 2019, 2021). Systematically relevant turbinal characters were described for all extant lagomorph genera except for Pentalagus, Nesolagus, Bunolagus, and Brachylagus (Ruf 2014). Thus, the anatomical description of the turbinal skeleton of Pentalagus furnessi will help complete this data set for further phylogenetic and morphofunctional studies.

This study is based on two adult macerated skulls of Pentalagus furnessi that are housed in the National Museum of Nature and Science, Tokyo, Japan. Specimen M 12939 was scanned with the µCT scanner (Fraunhofer/ProConXray/Feinfocus) housed at the Senckenberg Forschungsinstitut und Naturmuseum Frankfurt, Frankfurt am Main, Germany. Scan parameters are 90 kV, 89 µA, 1500 ms exposure time, 1600 projections, and 0.0252 mm resolution (isotropic voxel size). Specimen M 12940 was scanned at Fraunhofer IIS, Deggendorf, Germany, with a TomoScope HV 500 by Werth Messtechnik GmbH Gießen; scan parameters are 150 kV, 300µA, 666ms exposure time, 1200 projections, and 0.0499 mm resolution (isotropic voxel size). The µCT data were processed with VGStudio MAX 2.2 (Volume Graphics, Heidelberg, Germany) and the virtual 3D model of the turbinal skeleton of M 12940 was manually segmented in Avizo 9.01 (Thermo Fisher Scientific FEI).

Anatomical terminology follows Ruf (2014) and Maier and Ruf (2014), although some other terminologies exists (see Smith and Bonar 2022 and table 1 for further synonyms of turbinal structures therein). Due to the lack of early ontogenetic stages of Pentalagus furnessi identification and homology of interturbinals are based on topography, medial extension compared to fronto- and ethmoturbinals as well as developmental patterns of these structures observed in other Lagomorpha and further mammalian species (e.g., Paulli 1900; Voit 1909; Frick and Heckmann 1955; Ruf 2014, 2020; Wagner and Ruf 2019, 2021). For comparative illustration of selected characters, µCT images of Romerolagus diazi (10977, Naturhistorisches Museum Basel, Switzerland), Poelagus marjorita (28732, Staatliches Museum für Naturkunde Stuttgart, Germany), Lepus europaeus (M6335, Institut für Geowissenschaften, Universität Bonn, Germany) and Ochotona alpina (100485, Museum für Naturkunde, Berlin, Germany) were used. For details on scan parameters see Ruf (2014).

Lagomorph phylogeny follows Robinson and Matthee (2005), Ge et al. (2013), and Cano-Sánchez et al. (2022) and is used for grundplan reconstruction in the sense of Hennig (1984) in order to elucidate apomorphic and plesiomorphic characters of the last common ancestor of a respective taxon.

Both studied Pentalagus furnessi specimens are very similar in their turbinal morphology. Thus, the anatomical description mainly refers to specimen M 12940, and specimen M 12939 is mentioned for intraspecific differences.

The nasal cavity of Pentalagus furnessi is large and extends below the endocranial cavity, where the olfactory bulbs are located (Fig. 1). The pars anterior of the nasal cavity houses the maxilloturbinal and the nasoturbinal. The maxilloturbinal has a great dorsoventral extension and is situated ventrally to the nasoturbinal. Anteriorly the maxilloturbinal extends far into the nasal aperture and posteriorly it ends below ethmoturbinal I (Figs 1A, 2A, B). In cross-section the maxilloturbinal is highly dendritic, i.e., it shows many lamellar ridges and additional outgrowths (epiturbinals). Its ventralmost lamella forms the medial wall of the nasolacrimal canal. The nasoturbinal arises from the nasal ridge of the nasal bone and forms a straight lamella that shows a sickle-shaped posterior border (Figs 2A, B, 3A). Here, the nasoturbinal is partly in close contact with the lamina semicircularis and thus, both structures form a morphological unit (Fig. 2A, B). In this area both structures are excavated to a recess that continues posteriorly into the pars lateralis of the nasal cavity (Fig. 3B).

The lamina semicircularis is sickle-shaped reflecting the posterior border of the nasoturbinal and forms the anteromedial wall of the pars lateralis as well as of the hiatus semilunaris (Fig. 2B, C). Its long dorsal lamella becomes laterally rolled up and merges with the lamina cribrosa; its ventral part is much shorter and forms a straight and distally slightly thickened lamella in the upper part of the maxillary sinus (processus uncinatus) (Fig. 4). The part that forms the anterior border of the hiatus semilunaris is not significantly rolled up (Fig. 2B, C).

The pars lateralis of the nasal cavity comprises the frontoturbinal recess (dorsally) and the large maxillary sinus (ventrally) that are separated by the lamina horizontalis but confluent anteriorly (Figs 3C, 4). The frontoturbinal recess houses several turbinals. Both studied specimens have two frontoturbinals and one interturbinal in between, all of which end on the lamina cribrosa (Fig. 2). The frontoturbinals are double scrolls. Frontoturbinal 1 arises anteriorly from the ossified nasal capsule and then moves onto the frontal bone while frontoturbinal 2 arises from the lamina horizontalis (Fig. 3C). The interturbinal between the frontoturbinals is simpler in shape (scrolled dorsally), smaller and shorter than the frontoturbinals and located in the posterior part of the frontoturbinal recess (Figs 2B, C, 3C). It arises from the ossified lateral wall of the nasal capsule (paries nasi) and only its posterior end is attached to the maxillary bone and the most lateral rim of the lamina cribrosa. Specimen M12939 shows some variation in that the left interturbinal forms a short straight lamella; the right frontoturbinal 2 is only a single scroll maybe due to constraints in space caused by the single-scrolled right interturbinal (Fig. 4B). In addition, both specimens show an additional small and short interturbinal between frontoturbinal 2 and ethmoturbinal I located in the anterior frontoturbinal recess (Fig. 2B, D). In specimen M 12940 this interturbinal is a straight but low lamella that diverges together with frontoturbinal 2 from the lamina horizontalis, runs posteromedially and merges with ethmoturbinal I (Fig. 4A). Specimen M 12939 shows a somewhat different pattern. Here, on the right side this additional interturbinal is a very short and low lamella arising from the lamina horizontalis, with no contact to frontoturbinal 2 or ethmoturbinal I. On the left side the additional interturbinal is just a very low ridge (Fig. 4B).

The pars posterior of the nasal cavity comprises the ethmoturbinal recess and houses three ethmoturbinals and one interturbinal between ethmoturbinal I and II (Fig. 2); all of these turbinals end on the ventral surface of the lamina cribrosa. Ethmoturbinal I is by far the largest olfactory turbinal. Anteriorly, it is separated into an anterior lamella, whose anterior process projects into the pars anterior of the nasal cavity, and a posterior lamella (Fig. 3B, C). Both lamellae are straight at their front end and arise from the lamina horizontalis. More posteriorly they are attached with a common root to the lamina horizontalis, that partly forms the lateral sidewall of the nasal cavity, and at the very end to the frontal bone. The posterior end of ethmoturbinal I and the lamina cribrosa together form a deep funnel that transmits the olfactory nerves (cranial nerve I) of that area. During their course both lamellae of ethmoturbinal I become single and double scrolls. Interestingly, both studied specimens show different scroll patterns. In M 12940 both lamellae are rolled up inversely, i.e., the anterior lamella ventromedially and the posterior lamella dorsolaterally (Fig. 4A). The anterior lamella becomes a double scroll that shifts to a single scroll (rolled up dorsolaterally), while the posterior lamella remains a single scroll (Fig. 3C); in addition, the posterior lamella of the left ethmoturbinal I has a short lateral epiturbinal. In contrast both lamellae of ethmoturbinal I in M 12393 are rolled up ventromedially in their anterior parts and then both become double scrolls (Fig. 4B); in the posterior lamella the double scroll portion is quite short as this is the transition from a ventromedially to dorsolaterally rolled up single scroll.

The interturbinal is relatively short and situated in the posterior part of the ethmoturbinal recess where it arises entirely from the lamina horizontalis (Figs 2B, D, 3C). The interturbinal is ventrally rolled up (single scroll) but forms a double scroll for a short distance.

Ethmoturbinal II is the second largest ethmoturbinal (Fig. 2A, B, D). Anteriorly it arises from the lamina terminalis and then it runs posterolaterally onto the maxillary bone (Figs 3C, 4B). The posterior end is an almost straight lamella hanging down from the lamina cribrosa (Fig. 3D). Ethmoturbinal II of M 12940 is mostly a single scroll, whose orientation of rolling up changes from ventrally to laterally. On the right side a short lateral epiturbinal is evident in the area where the orientation of rolling up changes. In specimen M 12939, the anterior part of ethmoturbinal II is almost straight and thus this turbinal is a single scroll (rolled up dorsolaterally). However, the left ethmoturbinal II shows a ventral epiturbinal that forms a short double scroll.

Ethmoturbinal III is the most posterior olfactory turbinal and its size is comparable to the interturbinal inside the ethmoturbinal recess (Fig. 2A, B, D). The anterior part of ethmoturbinal III is attached to the maxillary bone but the rest arises from the partly ossified lateral wall of the posterior nasal cavity (paries nasi) (Fig. 3D). Like ethmoturbinal II ethmoturbinal III shows a changing orientation of its single scroll, but in contrast to the former from dorsolaterally to ventrolaterally; in the transition area a short double scroll is present.

Generally, the turbinal skeleton of Pentalagus furnessi resembles the pattern and characters observed in other Leporidae (Ruf 2014). Pentalagus furnessi resembles the lagomorph grundplan in showing a dendritic maxilloturbinal and an excavated nasoturbinal that contacts the lamina semicircularis, and the leporid grundplan in having an interturbinal between frontoturbinal 1 and 2 (Ruf 2014) (Fig. 5). All these characters are also present in the stem-lagomorph Palaeolagus haydeni (Ruf et al. 2021). A nasoturbinal that contacts or is continuous with the lamina semicircularis has also been observed in Potamogale velox and in marsupials (Macrini 2012; Ruf et al. 2020) but is not present in Sciurus vulgaris, muroid rodents and Scandentia (Ruf 2014; Ruf et al. 2015). Concerning the turbinal pattern in the pars posterior of the nasal cavity (three ethmoturbinals, one interturbinal between ethmoturbinal I and II, ethmoturbinal I with two major lamellae) Pentalagus furnessi represents a common mammalian pattern. This has been also observed in Palaeolagus haydeni, many leporids (Poelagus marjorita, Pronolagus cf. saundersiae, Oryctolagus cuniculus, Sylvilagus spp., Lepus spp.), many rodents (e.g., Sciurus vulgaris, Mus musculus, Mesocricetus auratus), and Scandentia (Ruf 2014, 2020; Ruf et al. 2015, 2021).

In addition, Pentalagus furnessi shows some apomorphic characters in the turbinal skeleton (Fig. 5). A derived feature that unites Lagomorpha and Palaeolagus haydeni is a significantly rolled up posterior margin of the lamina semicircularis (Ruf 2014; Ruf et al. 2021), which is not the case in Pentalagus furnessi (Fig. 6A). The latter is derived from the leporid grundplan in that the ventral lamella of the lamina semicircularis is straight. Due to the thickened free margin it differs from the straight lamina semicircularis in Ochotonidae (Ruf 2014). A further apomorphic character of Pentalagus furnessi is the reduced (proportionally short and single-scrolled) interturbinal between frontoturbinal 1 and 2 (Fig. 6B). It should be mentioned that reduction and especially loss of this interturbinal evolved several times independently within Lagomorpha, e.g., in Ochotonidae, Romerolagus diazi, and Pronolagus spp. (Ruf 2014).

The two studied specimens of Pentalagus furnessi show some variation in the detailed morphology of certain olfactory turbinals including left-right asymmetry, e.g., the additional interturbinal between frontoturbinal 2 and ethmoturbinal I. Intraspecific variation of smaller interturbinals has already been described in Oryctolagus cuniculus, some Sylvilagus and Lepus species, and in Canis lupus familiaris (Ruf 2014; Wagner and Ruf 2019, 2021). The general morphology and number of the major olfactory turbinals (frontoturbinals, ethmoturbinals, interturbinal between ethmoturbinal I and II, lagomorph interturbinal between frontoturbinals) used for comparative and systematic purposes is normally not affected. However, there are very few observations on variation in these major turbinals: Myocastor coypus is generally lacking frontoturbinals but some studied specimens have one (Martinez et al. 2020; pers. obs. of QM and IR), Notoryctes typhlops shows some intraindividual variation in having two frontoturbinals on the left side and three ones on the right side (Macrini 2012) and a fetal stage of Daubentonia madagascariensis has four ethmoturbinals in contrast to an adult stage with only three ethmoturbinals (Maier and Ruf 2014). As the virtual reconstruction of the turbinal skeleton is quite time consuming most studies rely only on a single specimen per species and thus intraspecific variation has not been detected very often. Intraspecific variation can only be elucidated by larger sample sizes in future studies.

The sister-group relationship of Pentalagus furnessi is still debated and several hypotheses exist. Possible candidates are Pronolagus spp. (eastern and southern Africa), Bunolagus monticularis (South Africa), Poelagus marjorita (The Democratic Republic of the Congo, South Sudan, Sudan, Uganda) and Caprolagus hispidus (Himalayan region of India, Bhutan, Nepal) although morphological similarities with the former may be due to convergent evolution (Dawson 1981; Yamada et al. 2002; Stoner et al. 2003; Matthee et al. 2004; Robinson and Matthee 2005; Ge et al. 2013; Cano-Sánchez et al. 2022). The turbinal skeleton does not elucidate the sister-group relationship of Pentalagus furnessi as much as expected because a puzzling pattern becomes evident. Among the studied Pronolagus specimens only Pronolagus cf. saundersiae shows the same turbinal count as Pentalagus furnessis (except for the additional small interturbinal in the frontoturbinal recess) as well as a very reduced interturbinal between the frontoturbinals. However, ethmoturbinal I of Pronolagus cf. saundersiae is very different from that in Pentalagus furnessi in being slender with a short posterior lamella. Pronolagus rupestris and Pronolagus randensis lack certain turbinals and thus are highly derived from the lagomorph grundplan (Ruf 2014). However, as long as the turbinal skeleton of Pronolagus crassicaudatus remains undescribed the polarization within that genus remains ambiguous and thus, comparison with the Amami rabbit is limited. Pentalagus furnessi and Poelagus marjorita show the same number of major olfactory turbinals but they differ significantly in their proportions. While the turbinals of the ethmoturbinal recess of Poelagus marjorita are more slender, the interturbinal between the frontoturbinals is proportionally longer than in Pentalagus furnessi. However, all turbinals of the frontoturbinal recess of Poelagus marjorita are reduced in that they form single scrolls but in contrast to Pentalagus furnessi the interturbinal is scrolled ventrally (Fig. 6B); this indicates a convergently evolved pattern (Fig. 5). Furthermore, in Poelagus marjorita the lamina semicircularis is proportionally much larger and resembles the leporid grundplan; the ventral lamella of Poelagus marjorita differs from other lagomorphs in forming a large cavity (Fig. 6A). In contrast, the maxilloturbinal of Poelagus marjorita appears to be shorter and lower with less ridges, although this could be an artefact due to poor preservation (Ruf 2014). However, one should bear in mind that differences in turbinal number, complexity, proportion and relative size may be related to ecology and reflect potential adaptations (e.g., Martinez et al. 2018, 2020). In terms of overall proportions and pattern of the turbinal skeleton Pentalagus furnessi also compares to Caprolagus hispidus, although the latter has four ethmoturbinals and a larger interturbinal between the frontoturbinals. Furthermore, Pronolagus cf. saundersiae, Poelagus marjorita and Caprolagus hispidus, are all lacking further small interturbinals (Ruf 2014). Unfortunately, the turbinal skeleton of Bunolagus monticularis has not been studied to date.

The first anatomical description of the turbinal skeleton in Pentalagus furnessi sheds new light on intracranial diversity and cranial variation in Leporidae. The Amami rabbit shows a combination of typical leporid and autapomorphic characters. The latter comprise the relatively short and single-scrolled interturbinal between frontoturbinal 1 and 2 and a straight ventral lamella of the lamina semicircularis. Pentalagus furnessi differs from the turbinal skeleton of possible sister-group species in several respects. This underpins the great variation observed in the lagomorph skull that contradicts the generally assumed conservative morphology of Lagomorpha (e.g., Fostowicz-Frelik and Meng 2013; Kraatz et al. 2021). For instance, variation in skull shape changes might reflect some loss of phylogenetic signal at the generic level and a high degree of convergent adaptations due to extensive extinctions especially in Ochotonidae and the radiation of modern Leporidae at the end and after the Miocene (Ge et al. 2013, 2015). However, the present study clearly shows the need for more detailed comparative morphological studies of cranial characters ideally covering all living lagomorph genera and species.

Pentalagus furnessi has a heavier and thicker skull than many other leporid species (Ge et al. 2015), which refers to its smaller eyes and ear regions and implies some primitive pattern; compared to Lepus europaeus and Oryctolagus cuniculus the rostrum of Pentalagus furnessi appears to be proportionally smaller and less robust (Otsuka et al. 1980: figs. 2, 3), which indicates a smaller nasal cavity. Future morphometric studies on surface area and complexity of the respiratory and olfactory turbinal skeleton (according to Martinez et al. 2018 and Wagner and Ruf 2019) may help to elucidate morphofunctional adaptations related to environment, climate, foraging and social behavior in the Amami rabbit as well as in other living lagomorph species.