2006 Zoological Society of Japan
ZOOLOGICAL SCIENCE 23: 427–434 (2006)
Early Skeletal Development in Talpa europaea, the Common European Mole Jan Prochel* Zoologisches Institut, Spezielle Zoologie, Universität Tübingen, Auf der Morgenstelle 28, 72076 Tübingen, Germany
An ontogenetic series of 22 cleared and double-stained prenatal specimens was used to study the sequence of ossification of selected postcranial skeletal elements of Talpa europaea. Results were compared with nine other therian mammals, with Alligator, Chelydra, and Lacerta as outgroups. Using the event-pairing method, shifts in the onset of ossification in T. europaea, Sus, and Homo were identified. In T. europaea, the ossification of the cervical vertebrae starts before the metatarsals. In Homo and Sus, the tarsals ossify before the pubic bone. These shifts in the sequence of ossification are unique among the mammals examined, whereas many other changes, characterising monophyletic groups and/or evolving convergently, were also identified. Particular attention was given to some peculiar calcified elements of the hand in T. europaea, which were identified as accessory ‘sesamoid bones’, and do not display a chondrified precursor. They start to calcify before all others of the hand and later fuse. They appear in all fingers and function as reinforcement for the distal phalanges, most likely as an adaptation for burrowing. The development of the sesamoid bones was examined using histological sections and macerated adults. Key words: Mammalia, Talpidae, Eulipotyphla, sesamoids, ontogeny, staining
INTRODUCTION Most of the recent treatments of skeletal development in mammals have concentrated on the studies of model organisms such as the mouse (Wanek et al., 1989) and Monodelphis domestica, the grey short-tailed opossum (Maunz and German, 1997), where comprehensive data are easy to obtain in the literature. The marsupial/placental dichotomy (Sánchez-Villagra, 2002) and some clades such as bats (Adams, 1992) and primates (Kivell, 2005) were also of interest for several authors, due to their relevance to understand the phylogeny of those clades. In recent years, some attempts have been made to detect phylogenetically relevant characters in the morphology of talpids, such as myology (Whidden, 2000) and the anatomy of the humerus (Sánchez-Villagra et al., 2004), hand (Sánchez-Villagra and Menke, 2005), or whole skeleton (Sánchez-Villagra et al., 2006). Molecular data have generated new hypotheses about the phylogeny of this group (Shinohara et al., 2004). In contrast to this new information on adult anatomy, there has been no attempt to analyse developmental sequences in this clade. Several authors have discussed the phylogenetic relevance of developmental sequences (e.g., Jeffery et al., 2002; Schulmeister and Wheeler, 2004), but few empirical studies of this kind exist in the literature. Therefore, I have added the highly specialised fossorial common European mole to a previous analysis by Sánchez-Villagra * Corresponding author. Phone: +49-7071-29-72613; Fax : +49-7071-29-5150; E-mail:
[email protected] doi:10.2108/zsj.23.427
(2002), in which he discussed the marsupial/placental dichotomy, using the event-pairing approach (Smith, 1997, 2001). This work is part of a series, intending to examine sequences of ossification in mammals, especially eulipotyphlans, using a comparative ontogenetic approach (Prochel et al., 2004). One of the first scientific works on the common European mole (Jacobs, 1816) discussed and illustrated much of the whole anatomy of Talpa europaea. Several later publications, that described the morphology of the forelimb of this species (Flower, 1888), dealt with carpal anatomy, including the very specialised praepollex (os falciforme), the ‘mole’s thumb’ (Sánchez-Villagra and Menke, 2005). The skeletal development of Talpa europaea was examined first by Sterba and Zeleny (1974), who discussed the prenatal development of the pelvic and later the thoracic limb (Sterba, 1976). None of these authors considered the development of several extra-calcified elements in the hand of T. europaea. Flower (1888) interpreted them as elements homologous to the distal phalanges. In this study, I attempt to contribute to the discussion on the phylogenetic relevance and interpretation of sequences of development (Jeffery et al., 2005). I also give insights into the structure and development of calcified elements in the forelimbs of Talpa europaea. The development of sesamoid bones is frequently discussed, and several definitions of them appear in the literature (Andersen, 1961; Bland and Ashhurst, 1997; Hall, 2005). As Vickaryous and Olson (in press) discussed, in most cases sesamoid bones are defined as ossified parts of a tendon, usually with secondary chondrified precursors.
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MATERIALS AND METHODS
RESULTS
An ontogenetic series of 22 cleared and double-stained prenatal specimens (size range 14–34 mm in crown-rump length (CRL); body-size around birth, 40–42 mm according to Mohr, 1933; see also Sterba, 1980) of Talpa europaea was used to document skeletal development. Supplementary Table 1 shows the specifications of the specimens examined (http://dx.doi.org/10.2108/zsj.23.427). The relative timing of ossification of 24 selected postcranial elements was recorded for three outgroups and ten therian mammals. Data for taxa other than T. europaea were taken from Sánchez-Villagra (2002) and literature cited therein, except data for Sus scrofa (Stöckli, 1922). All data were used to construct a matrix and were mapped onto a recent phylogeny of placental mammals (Springer et al., 2003) using MacClade® software, following the event-pairing method (Smith, 1997; Velhagen, 1997). This resulted in 276 eventpairs (characters) among 13 species. The onset of ossification was coded as follows: an element starts to ossify 0=before, 1=simultaneously with, or 2=after another skeletal element.
In comparison with the other species analysed, there are several notable differences in the sequence of ossifica-
Clearing and staining methods The clearing and double-staining method used was a modified version of the one introduced by Dingerkus and Uhler (1977). After skinning, all visible fat and the intestines were removed, and the brain was destroyed with a needle by piercing through one of the fissures of the head (which later helped to remove the bubble appearing in the head). Specimens were dehydrated in alcohol series up to 99% ethanol (about 2–4 hours in each solution). Cartilage was stained with a solution of 150 mg/l alcian blue 8 GX (Sigma Aldrich A-3157) in 80% ethanol and 20% acetic anhydride. The animals remained in this solution until staining of the cartilage was visible (4 to 24 hours). After washing in 99% ethanol and transfer back into water via ethanol series, the specimens were bleached and degreased in a solution of 1% potassium hydroxide (80%) and 3% hydrogen peroxide (20%) about one hour to one day. The animals were neutralised afterwards in a buffer solution of 4.2 g/l NaH2PO4 and 6.5 g/l Na2HPO4 in demineralised water (pH=7.00). After the animals were placed in the buffer solution, all bubbles were removed with an exsiccator. For digestion, the animals were incubated in a solution of 3 g/l trypsin (Merck 108367; 200 FIP-U/ g) in a mixture containing 66% phosphate buffer and 34% saturated aqueous solution of sodium borate, at 36 °C. In order to decelerate the autodigestion of the enzyme and to increase the speed of the clearing process, 0.22 g/l CaCl2 were added to the solution. Once the specimens were translucent and no tissue was visible, the animals were put into a solution of alizarin red (Sigma Aldrich A-5533) in 1% potassium hydroxide (10 mg/100 ml). This staining process required careful observation, because alizarin red can recolour the cartilage. The specimens were transferred to 99% glycerol following the method of Dingerkus und Uhler (1977). Study of hand sesamoids Histological sections of a neonate (CRL=45 mm, slice thickness 10 µm, stained with azan using the method of Domagk, 1933) were used to study the later development of skeletal elements in the hand. Two macerated adult Talpa europaea from the Zoologische Schausammlung Tübingen (SZ 7565, 6657) were used to document skeletal hand anatomy. The ultrastructure of selected postcranial bones of one mole (SZ 6657) was examined by scanning electron microscopy (SEM) in order to compare the texture of a replacement (radius) and a dermal bone (clavicle) with that of the praepollex. Additionally, one distal phalanx of the hand, the praehallux, and the extra-calcified elements were scanned in this way to get additional information about the texture of these elements.
Fig. 1. Examples of patterns of transformation resulting from a comparison of the sequence of ossification among selected postcranial elements. The three character states reflect the relative timing of one event relative to another: before (0), simultaneously (1), or after (2). (a) First cervical vertebra versus femur. (b) Scapula versus humerus. (c) First thoracic vertebra versus humerus. (d) First rib versus humerus.
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Fig. 2. All elements in the hand of two cleared and stained embryonic stages of Talpa europaea (crown-rump-length a: 26.9 mm, b: 34 mm) are chondrified, except for the ‘accessory elements’ (ae). (a) Two independent centres of calcification appear in fingers 2–4. (b) Disto-palmar to all distal phalanges, the elements start to calcify; in fingers 2–4, they are fused.
Fig. 3. (a) A horizontal histological section near the level of the palmar side of the distal phalanx (dp3) of the third finger shows parts of the later-calcified accessory element (ae). (b) Enlarged detail of the histological section showing the former tendon of the Musculus flexor digitorum profundus with calcified irregular particles (cp).
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tion. Fig. 1 shows examples of the changes that occur in the onset of ossification in Talpa europaea. This and the marsupial Didelphis are the only species considered in which the first cervical vertebra ossifies before the femur. In three of the rodents, except Cavia, as well as in the marsupial Sminthopsis (Fig. 1a), this event was unresolved. Talpa europaea is the only species in this study in which the scapula starts to ossify before the humerus. In Mus and the two marsupials, this event-pair was unresolved (Fig. 1b). Fig. 1c shows that T. europaea is also the only species showing signs of ossification in the first thoracic vertebra before ossification of the humerus. This again was unresolved in Didelphis and Mus. As seen in Fig. 1d, another character observed only in Talpa is the ossification of the first rib before ossification of the humerus, although this pair was unresolved in several species in this study (Chelydra, Didelphis, and all rodents exceptCavia). Characters that represent unequivocal synapomorphies or autapomorphies in other species are listed in Supplementary Table 2 (http://dx.doi.org/10.2108/zsj.23.427). Some skeletal elements that were not taken into account in the analysis show unusual development: in the hand, a pair of elements appears disto-palmar to the distal phalanx of the middle finger (Fig. 2a). In later stages, analogous elements appear in the second and fourth fingers, and still later in the first and then the fifth phalanx (Fig. 2b). These elements do not have any cartilaginous precursor, and they are the first elements to calcify in the hand. The chondrified precursor of the praepollex is the dominant element on the radial side of the hand. Also, in relative stage 11 (19.3 mm), a chondrified patella appears which does not ossify in the prenatal stages documented here. There is no sign of other sesamoids in the hand in these stages. The histological sections of the hand show a special tissue: in the tendon of the musculus flexor digitorum profundus are tiny calcified particles (Fig. 3a and b). In older stages, these particles increase and form extra-calcified elements. In adults, these elements become a groove for the distal phalanges and form tiny splits (Fig. 4).
Fig. 4. Photo of the right hand of an adult specimen (SZ 7565) shows all dorsal ‘sesamoids’ (se), and the ‘accessory elements’ (ae) forming splits for the distal phalanges.
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Fig. 5. (a) SEM of the distal process of the right radius. (b) Enlarged detail of this bone, showing the region of the epiphysis. (c) SEM of the left clavicle. (d) Enlarged detail of the clavicle.
Fig. 6. (a) SEM of the distal phalanx of a finger and the associated extra-calcified element in an adult specimen (SZ 6657). (b) Enlarged details of the distal phalanx (dp3). (c) Enlarged details of the accessory element. (d) Enlarged details of the proximal end of the accessory element.
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Fig. 7. (a) SEM of the praepollex of the right hand of Talpa europaea (SZ 6657). SEM of (b) the superficial and (c) the deeper layer of this bone.
Several sesamoid bones are visible in the macerated specimens (Fig. 4). They are located dorsal to the articulations of the metacarpals and the proximal and medial phalanges. In the SEM image, no difference is evident in the textures of the right radius and the dermal ossified left clavicle (Fig. 5). Fig. 6a shows the palmar view of the distal phalanx of the right hand and the associated extra-calcified element. The ultrastructural texture of the distal phalanx (Fig. 6b) is similar to the texture of the radius and the clavicle, whereas the texture of the extra-calcified element (Fig. 6c and d) is distinct. The surface of this element (Fig. 6c) and its proximal edge (Fig. 6d) are similar to the parallel collagen fibrils of the former tendon. The os falciforme, which has a chondrified precursor, shows two different textures (Fig. 7a). The texture of the superficial layer is nearly similar to that of the radius or clavicle (Fig. 7b), whereas the deeper parts of this bone resemble the surface of the extra-calcified element (Fig. 7c).
As seen in Fig. 8, the praehallux has a more spongy texture than the praepollex, whereas the distal phalanx shows two different textures. In the lateral part it is similar to the humerus and the clavicle, whereas in the proximal part it resembles the more fibrous structure of the extra-calcified elements. DISCUSSION Some shifts in the timing of ossifications are shared by some groups and seem to provide phylogenetic signal. On the other hand, these shifts present several problems when used for phylogenetic reconstruction, e.g., by treating dependant characters as independant and potentially leading to impossible ancestors (Schulmeister and Wheeler, 2004). Several unique changes in the sequence of ossification occur in the postcranial elements of Talpa europaea in comparison to other eutherian mammals. Most of these shifts appear in the cervical and thoracic region of the vertebral
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Fig. 8. (a) SEM of the praehallux of the left foot of Talpa europaea (SZ 6657). (b) Enlarged details of the praehallux. (c) Ultrastructure of the surface of this bone.
column. These elements allow the altricial mole (Sterba, 1975) to have a stabilised body axis and cervical region after birth. Later on in development, when the mole starts to crawl around and dig, the associated elements start to ossify. It has been shown for marsupials, that shifts in the prenatal onset of ossification can be explained by examining their behaviour directly after birth (Sánchez-Villagra, 2002). In marsupials, the distal phalanges start to ossify before all other elements of the hands and the feet, because the animals have to crawl to their mothers’ teats directly after being born (Maier, 1999; Sears, 2004). Jacobs (1816) discussed and illustrated all common carpal elements, as well as nearly all sesamoids. He did not mention the extra-calcified elements correlated with the distal phalanges and several elements in this study identified as sesamoids. Other authors (Galis et al., 2001) described some elements that show bifurcated distal ends, but interpreted them as distal phalanges. As a matter of fact, these skeletal elements are the first to calcify in the hand of Talpa europaea. They build up a groove for the distal phalanges, but do not fuse with them and therefore do not represent traction epiphyses, which are independent of the bones early on and later in development fuse to the bone they are associated with (Barnett and Lewis, 1958). Patterson (1977) discussed several modes of ossification of bones and the nomenclatural confusion surrounding them. Following his definitions, the accessory bones described here should be included in the group he called membrane bones. These are bones that develop directly, which means that they do not have cartilaginous precursors. He also included bones such
as sesamoids in this group of skeletal elements. In contrast to this definition, it is nowadays clear that many sesamoids preform in cartilage, for example the os falciforme in moles (Sánchez-Villagra and Menke, 2005). These elements probably enlarge the hand in the region of the distal phalanges and are likely an adaptation to the digging activities of the mole. Fig. 2b shows that these extra-calcified elements are formed by small, calcified particles incorporated in the fibrous structure of the tendon of the musculus flexor digitorum profundus, which seems to be similar to the occurrence of mineralisation of the gastrocnemicus tendon in turkeys (Landis and Silver, 2002). Later in development, these particles fuse and form a solid element in the hand. They show a different texture than the radius and the clavicle (Fig. 4), which are representatives of the standard replacement and dermal modes, respectively, of ossification in vertebrates (Starck, 1975). The function, structure, and nomenclature of the praepollex (os falciforme in moles) have often been discussed in the literature. In several mammal species, such as the giant panda (‘pseudo thumb’, e.g., Endo et al., 1996; see also Endo et al., 1999), it is hypothesised that the prepollex helps to grasp pieces of bamboo. In the mole, the prepollex broadens the shovel-like hands to support digging through the substrate (Yalden, 1966). In Talpa europaea, it has a cartilaginous precursor (Fig. 2; see also Sánchez-Villagra and Menke, 2005) that totally ossifies in adults. This is in contrast to the prepollex of the lesser panda, Ailurus fulgens, where the ulnar part of this element remains cartilaginous in
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adults (Endo et al., 2001). It has been discussed in the literature whether the praepollex should be named a sesamoid bone or a ‘true’ carpal, and therefore can be considered homologous among all therian mammals (Lessertisseur and Saban, 1967). Early development supports the hypothesis of prepollici as true carpals, because also in relatively basal marsupials such as Monodelphis domestica, there seems to be no difference in the mode by which the prepollex develops in comparison to the other carpals (Prochel and Sánchez-Villagra, 2003). However, in SEM ultrastructure, the praepollex looks more like the sesamoid patella (Clark and Stechschulte, 1998). The SEM of the superficial and deeper surface of the os falciforme shows two different textures. The structure of the superficial part of this bone is similar to that of the radius and the clavicle, whereas the more profound region shows similarities to the texture of the calcified tendon of the musculus flexor digitorum profundus. Clark and Stechschulte (1998) discussed a structure at the quadriceps insertion site of the patella in different mammals (e.g., the rabbit) that they called lamellar bone. They showed SEM images of ossified fibro-cartilage with structures they interpreted as chondrocytes and osteocytes. The texture of this insertion seems to be similar to that of the accessory elements in T. europaea. One fundamental difference between these structures is the existence of remains of chondrocytes in the ossified insertion, whereas no sign of chondrification appears at any time during the development of the accessory elements. Furthermore, in the patella the ossified tendons appear only close to the surface, whereas the elements discussed form an extra-calcified element. Therefore, these extra-calcified structures can be interpreted as ‘sesamoid bones’ sensu lato, or more exactly as calcified tendons (W. Maier, pers. comm.), following the classical, human-anatomical definition that bones are strictly built up by osteoblasts (Starck, 1975). Conclusions The relative timing in development of several postcranial elements in Talpa is convergent with the timing in the marsupials examined. This can be related to the importance of the forelimb for the locomotion of the moles. A sesamoid bone sensu strictu should be defined as a bone that ossifies within a tendon, has a chondrified precursor, and helps the attended tendon to transmit force. Therefore the accessory elements, disto-palmar to the distal phalanx in the specimens examined, should be called ‘membrane bones’ or just ‘calcified tendons’, rather than sesamoid bones. They show a mode of development that is different from all other skeletal elements in Talpa europaea: they appear without a visible chondrified precursor and are the first elements in the development of the hand of T. europaea to show calcification. The examination of other talpids, especially the anatomy of the ‘sesamoid bones’ of semi- and non-fossorial species, and non-talpid fossorial mammals, will be relevant to elucidating the ecological or phylogenetic significance of these elements. ACKNOWLEDGMENTS I thank M.R. Sánchez-Villagra for very useful suggestions and comments he made concerning my work, and for his support. For permitting access to the specimens used for clearing and staining,
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I thank M. R. Sánchez-Villagra and J. Narraway, formerly at the Netherlands Institute for Developmental Biology in Utrecht. I thank W. Maier for his support, the histological sections, and discussions; E. Weber and J. Rösinger from the Zoologische Schausammlung Tübingen for allocating the macerated adults; O. Betz for providing access to the SEM; K.-H. Hellmer for SEM work; S.C. Eger and N.K. Schnell for technical assistance; P. Ziegler for the translation of Jacobs (1816); and M.K. Vickaryous for providing me an unpublished version of his manuscript on sesamoid bones. I thank A. Goswami, M. Laumann, M. Nilsson, I. Ruf, and T. Schmelzle for discussions and/or comments, and two anonymous reviewers and M.R. Sánchez-Villagra for improving the manuscript. The Department of Systematic Zoology at the University of Tübingen (Prof. Maier) provided financial support for this project. The Synthesys program of the European Union funded a visit to the lab of M. R. Sánchez-Villagra at the Natural History Museum, London.
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