All 140 adult B. subaspera (male: n = 76 and female: n = 64) captured in the field possessed the pseudothumb that was separated from the first finger in the hand. Radiographs and whole-mount skeletal preparations of adult specimens (n = 5) revealed that well-developed prepollex was completely ossified, in addition to skeletal elements of each finger (b). The tip of the prepollex was very sharp, approaching the condition of a prepollical spine. A prepollex consists of two articulated bones: the distal one that constitutes main part of the prepollical spine and the proximal one that articulates with more proximal Element Y and carpal 1, which has a spherical surface (electronic supplementary material 2). Anatomical comparison of architectural patterns of the hand skeletal muscles showed no obvious differences among species. In B. subaspera, the distal part of the prepollex was accompanied by a large amount of connective tissue. By examining museum specimens, we confirmed that B. holsti also possessed the pseudothumb and prepollical spine (n = 2; electronic supplementary material 3).
Subsequently, skeletal anatomy in juveniles (stage 45) was compared between non-Babina frog species and B. subaspera. In both groups, the shaft of the radioulna, metacarpals and phalanges were already ossified, as indicated by Alizarin uptake (c,d). The prepollex was present in all species examined, but was still cartilaginous, stained by Alcian blue, as well as carpal elements. The size of the prepollex varied among species. In non-Babina species, the tip of the prepollex never extended beyond the distal end of the first metacarpal (c,e). By contrast, in B. subaspera, the prepollex was elongated and its tip extended to the level of the distal end of the first metacarpal (d,f). The most striking difference between the two groups was the direction in which the prepollex curved. In non-Babina species, the prepollex was bent in the direction of the first finger (c,e). On the other hand, in B. subaspera, the prepollex was curved in the opposite direction of the first finger, separated from it (d,f and electronic supplementary material 4).
In larvae (stage 37), the whole prepollex was encased in the sheath of the first finger in non-Babina ranids (G. rugosa, L. catesbeiana, R. japonica; a,b). By contrast, the future pseudothumb region was separated from the first finger by a notch of thickened epidermis in B. subaspera (c,d). Surprisingly, the domain where the future prepollex should be established had already swelled in the forelimb anlagen of B. subaspera at the earlier larval stage (stage 36), whereas no such bud-like structure was observed at the corresponding domain in non-Babina ranids (G. rugosa and L. catesbeiana) at the same ontogenetic stage (e,g). Histological sections revealed that the newly developed cartilaginous prepollex was anteriorly covered with a thin epidermal layer and a small number of mesenchymal cells in non-Babina ranids (f). On the other hand, the newly formed prepollex was overlaid by a large number of mesenchymal cells that occupies the inside of the bud-like structure in B. subaspera (h).
Figure 2. Comparison of early prepollex development between non-Babina ranids and Babina subaspera. (a) Ventral view of right forelimb anlage of Lithobates catesbeiana larva at stage 37. (b) Histological section showing the transverse plane of the first and second (more ...)