Systematic distribution and evolution of petrosal characters
The majority of the petrosal characters seen in the holotype of Henkelotherium are also present in Vincelestes, albeit in slightly modified conditions. In fact, Henkelotherium more closely resembles the morphological condition observed in prototribosphenidans in general than in other more basal Mesozoic mammalian clades. Henkelotherium shows a more bulbous and oval-shaped promontorium similar to Vincelestes (Rougier et al. 1992) and the more derived metatherians and eutherians. This contrasts with the cylindrical (elongate, narrow and not inflated) promontorium in the Early Cretaceous spalacotheroid Zhangheotherium (Hu et al. 1997) and in triconodontids such as the Late Jurassic Priacodon (Rougier et al. 1996), the Early Cretaceous Cloverly Formation tricondontid (Crompton & Luo, 1993) and the British Early Cretaceous Trioracodon (Wible & Hopson, 1993). Henkelotherium has a distinct sulcus on the promontorium for the internal carotid artery, a feature also present in more derived Vincelestes and most basal eutherians (Rougier et al. 1992; McKenna et al. 2000; Wible et al. 2001, 2007, in press; Ekdale et al. 2004). This differs from eutriconodonts, most multituberculates, Zhangheotherium and metatherians, which lack the promontorial sulcus for the internal carotid artery (Miao, 1988; Wible, 1990; Wible & Hopson 1993, 1995; Rougier et al. 1996; Wible & Rougier, 2000; Ladevèze, 2004, 2007; Ladevèze et al. 2008; Horovitz et al. 2008).
The lateral trough is broad in cynodonts, tritylodontids, tritheledontids, brasilitheriids and a wide range of pre-mammalian mammaliaforms (Wible & Hopson, 1993, 1995; Luo et al. 1995, 2001; Rougier et al. 1996; Bonaparte et al. 2005). In eutriconodonts, the lateral trough is reduced in size and becomes narrow. The narrow lateral trough is partially contacted by the lateral flange in multituberculates (Wible & Hopson, 1995; Rougier et al. 1996; Wible & Rougier, 2000). In Vincelestes, this trough is further reduced (Rougier et al. 1992) and the preserved part of the lateral trough in Henkelotherium is similar to that of Vincelestes.
Although the cavum supracochleare floor is fractured and broken in Henkelotherium, the cavum is reconstructed tentatively as having a complete bony floor for the geniculate ganglion of the facial nerve, a derived feature of eutriconodonts, multituberculates, Vincelestes and most therians (Rougier et al. 1996, 1998). However, because the cavum epiptericum region is completely missing and the cavum supracochleare floor is broken in Henkelotherium, it is not clear if it would resemble the triconodontid and multituberculate condition in which the cava supracochleare and epiptericum are completely confluent with each other by the broadly open semilunar notch. However, it cannot be excluded that Henkelotherium resembles Vincelestes and derived therians such as Prokennalestes, in which the cavum supracochleare is enclosed by the petrosal but is interconnected with the cavum epiptericum through the fenestra semilunaris (Rougier et al. 1992; Wible et al. 2001).
The post-promontorial tympanic sinus and the caudal tympanic process in Henkelotherium (Fig. 3A, pps, ctpp) are also present in Zhangheotherium, Vincelestes and therians (Rougier et al. 1992; Hu et al. 1997; Wible & Rougier, 2000; Wible et al. 2001; Ekdale et al. 2004; Ladevèze, 2004, 2007). These are synapomorphies of trechnotherians (Zhangheotherium through Henkelotherium to therians) but are absent in most (but not all) multituberculates, eutriconodonts, the monotreme Ornithorhynchus and mammaliaforms such as Sinoconodon, Morganucodon and Haldanodon. Of other preserved characters on the petrosal of Henkelotherium, the presence of the prootic canal and the relatively shallow internal acoustic meatus are primitive features of mammaliaforms (Kermack et al. 1981; Lillegraven & Krusat, 1991; Wible & Hopson, 1993; Luo et al. 1995).
In Sinoconodon, Morganucodon, Priacodon, multituberculates and the monotreme Ornithorhynchus, there is an open groove (for the perilymphatic duct) on the petrosal from the jugular notch that enters the inner ear through an opening on the back of the promontorium, the perilymphatic foramen; a fenestra cochleae (round window) is lacking (Lillegraven & Hahn, 1993; Zeller, 1993; Luo, 1994; Rougier et al. 1996; Hurum, 1998). During phylogeny with the enlargement of the pars cochlearis and isolation of the inner ear from the middle ear cavity, a processus recessus has grown to enclose the perilymphatic duct and separates the fenestra cochleae from the perilymphatic foramen in therians (Zeller, 1985). Among living mammals, an enclosed perilymphatic canal, the cochlear canaliculus, occurs in the vast majority of therians and most probably convergently in the adult monotreme Tachyglossus (De Beer, 1937; Kuhn, 1971; Zeller, 1993). Among fossils, the cochlear canaliculus has been described only in Vincelestes and therians (Wible, 1990; Rougier et al. 1992; Wible et al. 2001; Ekdale et al. 2004; Ladevèze, 2004, 2007; Ladevèze et al. 2008; Schmelzle et al. 2007), and in an isolated petrosal that is referable to the ‘symmetrodont’Gobiotheriodon (Wible et al. 1995). Because the cochlear canaliculus is also present in more derived taxa than ‘symmetrodonts’ including Henkelotherium, it probably represents an apomorphic trechnotherian character. The formation of the perilymphatic canal occurred earlier in phylogenetic evolution of therian mammals than the development of a cochlear canal coiled through at least 270°.
Implication for evolution of hearing in Mesozoic mammals
Inner ear structures underwent fundamental changes during the evolution from non-mammalian cynodonts to modern mammals (Allin & Hopson, 1992; Luo, 2001; Kielan-Jaworowska et al. 2004). The elongation of the bony cochlear canal is correlated with the development of a ventral eminence of the pars cochlearis, leading to the fully developed promontorium, as described for Sinoconodon, Morganucodon, Haldanodon, multituberculates and Zhangheotherium (Kermack & Musset, 1983; Graybeal et al. 1989; Lillegraven & Krusat, 1991; Luo & Ketten, 1991; Luo et al. 1995; Meng & Wyss, 1995; Hurum, 1998). Both the elongate cochlear canal and promontorium are regarded as apomorphic features of mammaliaforms (Lucas & Luo, 1993; Wible & Hopson, 1993; Luo, 1994; Rougier et al. 1996). In some pre-mammalian mammaliamorphs, such as the tritylodontid Yunnanodon, the cochlear canal has already formed (Luo, 2001). In most basal mammaliaforms, the cochlear canal is longer relative to skull length (Table 1 in Luo et al. 1995) than in pre-mammaliaform cynodonts. A straight, elongate cochlear canal is also present in eutriconodonts, such as Jeholodens, and in the spalacotheroid Zhangheotherium (Hu et al. 1997; Ji et al. 1999; Luo, unpublished data). In multituberculates, the cochlear canal can be straight (Luo & Ketten, 1991; Lillegraven & Hahn, 1993) or slightly curved (Meng & Wyss, 1995; Fox & Meng, 1997; Hurum, 1998).
Monotremes and therians have membranous cochlear ducts that are coiled, which distinguishes them from all living non-mammalian amniotes. In monotremes, the membranous cochlear duct is coiled (Ornithorhynchus anatinus) or distally hooked (Tachyglossus aculeatus) (Alexander, 1904; Fleischer, 1973; Zeller, 1989) but the bony cochlear canal is less curved than the membranous cochlear duct (Fig. 6 in Fox & Meng, 1997). There is not a close correlation between coiling of the membranous cochlear duct and coiling of the ossified cochlear canal. The coiled basilar membrane in monotremes is not supported by any bony lamina(e) inside the bony labyrinth. This observation has been made in embryonic sections (Zeller, 1989, 1993), in naturally broken adult petrosals with desiccated membranous labyrinth tissues inside (Luo & Ketten, 1991) and in a scanning electron microscope study of the broken pars cochlearis of adults (Fig. 7 in Fox & Meng, 1997). Although Fleischer (1973, 177, Abb. 78) speculated that Ornithorhynchus may have a short primary bony lamina, this has not been confirmed by the more recent studies that show no such structure in serial sections of late-stage embryos (Zeller, 1989) or in the exposed interior of the cochlear canal through dissection of the pars cochlearis (Fig. 7 in Fox & Meng, 1997).
In contrast, in extant marsupials and placentals, the soft-tissue cochlear duct of the membranous labyrinth and the cochlear canal of the bony labyrinth are coiled together. The coiled basilar membrane within the cochlea is supported by the primary bony lamina. The latest reviews of these characters present the consensus that the coiling of the membranous cochlear duct is not homologous between monotremes and extant therians, as indicated by these fundamental structural differences (e.g. Lewis et al. 1985; Zeller, 1989; Luo & Ketten, 1991; Hu et al. 1997; Fox & Meng, 1997; Luo et al. 2002). This view is also supported by recent phylogenies (e.g. Luo & Wible, 2005; Rougier et al. 2007; Luo, 2007; Luo et al. 2007). The primary bony lamina (or a bony base for the basilar membrane lamina) of Henkelotherium, as identified in this study (Figs 2E,F, 5B,D, 6B), is ventral to the entry point of the cochlear nerve, whereas the putative primary lamina, as identified for Ornithorhynchus by Fleischer (1973, Abb. 78), is dorsal to the entry point of the cochlear nerve. This is consistent with the broad difference in the bony supporting structure (or the lack thereof) for the membranous labyrinth cochlear duct between monotremes on the one hand and the cladotherians on the other.
Previously, Early Cretaceous Vincelestes was the most primitive-known stem taxon to extant therians to have a bony cochlear canal coiled through 270°. Vincelestes also has the secondary bony lamina on a naturally exposed cochlear canal endocast (Fig. 34 in Rougier, 1993), although it is unknown if Vincelestes has the primary bony lamina as the relevant part of the inner ear is not exposed or studied by CT scans. Late Cretaceous stem metatherians and eutherians are the most primitive-known therians to have the primary and secondary bony laminae in the cochlear canal (Meng & Fox, 1995). This CT study of the cochlear structure of Henkelotherium reveals a coiled cochlear canal with primary and secondary bony laminae for the basilar membrane. Therefore, a cochlear canal curved through 270° with primary and secondary bony laminae is part of the apomorphic groundplan for the inner ear of the Cladotheria, the clade defined by the common ancestor of dryolestoids (including Henkelotherium) and therians. This is a more ancient origin for cochlear coiling with bony laminae for the basilar membrane in therian evolution in a more inclusive clade than Protribosphenida (Vincelestes and therians).
The functional significance of the primary and secondary bony laminae for the basilar membrane has been studied in placental mammals. The distribution, size and structural form of the primary and secondary bony laminae and their relationship to the basilar membrane in the Organ of Corti for hearing are known in cetaceans and bats (Yamada & Yoshizaki, 1959; Pye, 1970; Wever et al. 1971; Fleischer, 1976a,b; Ramprashad et al. 1979; Ketten & Wartzok, 1990; Geisler & Luo, 1996; Luo & Marsh, 1996). A narrow width of the basilar membrane and a more rigid bony support for the basilar membrane by well-developed primary and secondary bony laminae in the basal part of the cochlear canal have been shown to be correlated with highly sensitive hearing in ultra-high-frequency sound for microchiropteran bats and odontocete whales (Wever et al. 1971; Fleischer, 1973, 1976b; Ramprashad et al. 1979). This provides a basis for understanding the functional implications of the newly discovered structures in Henkelotherium.
The secondary bony lamina is a derived feature because it is known in many (but not all) placental taxa and has a more restricted systematic distribution than the primary bony lamina. The latter structure is universally present in all extant therians and is now by the present study extended to be synapomorphic for the clade of extant therians plus Henkelotherium. In those living placentals that have the secondary bony lamina, the lamina is on the radial wall of the cochlear canal to reinforce the radial edge of the basilar membrane (Pye, 1970; Fleischer 1976b; Ramprashad et al. 1979; Ketten, 1992). With the exception of some primates, all of chiropterans and cetaceans, this structure is confirmed to be absent in many extant placentals, such as humans, manatees, camels and pigs (Bast & Anson, 1949; Fleischer, 1973; personal observation by authors on several taxa). Contrary to an earlier (and anecdotal) reference to this structure in some rodents (Reysenbach de Haan, 1957), most rodents that have been examined by histological sections lack this feature (Pye, 1977, pl. 1, 1979, pl. I). A weakly developed and short secondary lamina is reported by Fleischer (1973) for a range of placental mammals (including pangolins and xenarthrans) but not confirmed for some of these taxa by other studies using histological sections (Pye 1979, pls. II and III). Information on the secondary bony lamina is more limited on living marsupials than on living placentals. It is not seen in the histological sections of some didelphids (Pye, 1979) but appears to be present on the virtual endocast of the inner ear from CT scans of the didelphid Caluromys (Sánchez-Villagra & Schmelzle, 2007). It seems to be absent from the inner ear virtual endocasts of the fossil marsupials Necrolestes (Ladevèze et al. 2008) and Herpetotherium (Horovitz et al. 2008). In light of its scattered distribution, the secondary bony lamina is homoplastic among the diverse groups of living therians, possibly down to low taxonomic levels. It cannot be ruled out that this character can be variable among individuals of the same taxa and that some of the conflicting reports are due to sampling with different techniques. When present, the secondary bony lamina has its greatest width at the basal-most part in the basal cochlear turn and becomes diminishingly narrower toward the apex of the cochlea. Consequently, the width or other size-related features can vary with different locations along the basal cochlear canal turns. A full-scale assessment of the variability of this feature is beyond the scope of the present study.
Our CT scanning clearly demonstrates the presence of the secondary bony lamina in Henkelotherium. This is consistent with an earlier observation of this structure in Vincelestes (Rougier, 1993) and in the broken cochlear canal of some Cretaceous metatherian and eutherian petrosals, which appears to be representative of the character conditions (groundplan) of metatherians and eutherians as a whole (Meng & Fox, 1995; Fox & Meng, 1997). The consistent distribution of secondary bony lamina in the phylogenetically and successively nested taxa from Henkelotherium and Vincelestes to the representative metatherians and eutherians is in contrast to their conspicuous absence in many extant marsupials and placentals. For the systematic distribution of this feature in the above-mentioned clades, there can be two alternative hypotheses of the phylogenetic evolution of this character. (i) Because the secondary bony lamina is present in the basal members of the cladotherian clade for which this character has been investigated, it is parsimonious to hypothesize that the secondary lamina is an apomorphy of the cladotherian clade (Henkelotherium, Vincelestes through therians) and that its absence in the majority of extant therian mammals would present a secondary loss within this clade. (ii) Alternatively, the absence of the secondary bony lamina would be the ancestral condition of many extant therian mammals, shared by the pre-cladotherian ‘symmetrodonts.’ The secondary lamina is homoplastic and evolved independently in the basal cladotherians Henkelotherium, Vincelestes, some stem metatherians and some stem eutherians, and separately in chiropterans and in cetaceans within the placental group. The latter hypothesis is consistent with the fact that chiropterans with this lamina are nested within laurasiatherians, many of which lacked this feature ancestrally, and that cetaceans are nested within cetartiodactyls, many of which also lacked this feature ancestrally. Unless it is demonstrated by future CT scanning or histological studies of cochlear structure that the secondary lamina is present in more taxa of the basal-most eutherians and metatherians, we prefer the former hypothesis.
Regardless of the uncertainty in interpreting the pattern of phylogenetic distribution of the secondary bony lamina among the basal-most therians, there is clear insight into the hearing of Henkelotherium from the newly observed secondary bony lamina (in addition to the primary bony lamina). The presence of these structures suggests that the basilar membrane, whose dimension and structural support determine the sensitivity of hearing frequencies, had a more rigid structural support in Henkelotherium, at least more so than in the pre-cladotherian Mesozoic mammals lacking these structures. Presence of the secondary lamina would narrow the width of the basilar membrane and would provide more rigid structural support for the basilar membrane. Both characters are correlated with more sensitive hearing of high-frequency sounds [reviewed by Fleischer (1976b) and Ketten (1992)]. This can be hypothesized to indicate that Henkelotherium had more acute hearing for high-frequency sounds than other contemporary fossil mammals without such structures, everything else being equal for the relevant soft-tissue structures. The fossil evidence of the primary and secondary bony laminae in Henkelotherium has pushed back the first appearance of this improved capacity for high-frequency hearing from the Early Cretaceous to the Late Jurassic. Henkelotherium represents a transitional stage in the earliest evolution of the therian cochlear structure, according to our assumption that the presence of this secondary bony lamina is a synapomorphy of cladotherians.
Even though the pars vestibularis of the bony labyrinth of Henkelotherium is partly preserved, the morphology of the semicircular canals resembles the typical mammalian condition. As is the case in Henkelotherium, in the Oligocene metatherian Herpetotherium (Horovitz et al. 2008), several plesiomorphic extant marsupials (Isoodon, Monodelphis, Dasyurus and Caluromys) (Sánchez-Villagra & Schmelzle, 2007; Schmelzle et al. 2007) as well as early eutherians (Meng & Fox, 1995), certain extant carnivorans (Hyrtl, 1845; Gray, 1908) and rodents (personal observations), the posterior and lateral semicircular canals produce a secondary crus commune. Because the distribution of the secondary crus commune has not been mapped in other Mesozoic mammals, currently it is difficult to provide a phylogenetic interpretation of this character.