An overview of the osseous palmar sesamoid in Anura, with the particular case of some Rhinella species

Introduction

Material and Methods

Thirteen all adults specimens of eight species of Rhinella were dissected for this study belonging to institutional herpetological collections from Fundacion Miguel Lillo, CICyTTP-CONICET (DIAM) and L. Ponssa’s personal collection (Material S1). Since the material used was already deposited in collections, our study did not raise ethical concerns. Skeletal whole-mounts specimens were cleared and double-dyed with Alcian Blue and Alizarin Red S following Wassersug’s (1976) protocol. Some of the specimens(R. arenarum 8 sp.; R. dorbigny 8 sp.; R. margaritifera 3 sp.; R. diptycha 2 sp.) were not cleared and used for anatomical descriptions of muscles and tendons. Specifically, we considered the variation of the sesamoid shape and its relationship to the distal carpal, the flexor plate, and the related muscles (Rhinella species, comparative table in Material S2). These specimens were preserved in 70% ethanol and, at the time of observation, temporarily stained with iodine solution to better contrast the structures. Observations, illustrations, and photographs were made with Nikon SMZ1000 and Zeiss stereo microscopes and a Leica DM light microscope equipped with a digital camera and camera lucida. We observed and dissected both left and right carpus. A movement of the flexor plate, with the sesamoid embedded, was achieved by pulling the flexor digitorum communis muscle and the associated tendons in preserved manus (simulating the mechanism of contraction of the muscle) to corroborate the manus movement possibilities when the palmar sesamoid is present. This movement was compared to that of a preserved manus without sesamoid to control the effect of the osseous sesamoid presence.

We also review and describe the osseous palmar sesamoid of both manus of 170 anuran species from CT images downloaded from the Morphosource, and the University of California, representing almost all Anuran families. Since the CT scan images show only bony sesamoids, and it has been reported that fibrocartilaginous sesamoid can be usual in anurans (e.g., Abdala, Vera & Ponssa, 2017) our interpretations on sesamoid absences should be taken with caution. We include a description of the carpus of those specimens exhibiting palmar sesamoid as a way to provide the anatomical context of the palmar sesamoid in anurans. From all examined material, we selected three characters (character matrix in Material S3): (A) Osseous palmar sesamoid: 0 = Absent, 1 = Present; (B) Palmar surface of the distal carpals: 0 = concave, 1 = protruded, 2 = flat; (C) habitat use: 0 = terrestrial, 1 = aquatic, 2 = arboreal. Differences in the detail of the descriptions are due to the differences in the display possibilities of the specimens in the database.

We performed a standard ancestral state reconstruction by optimizing the selected characters. We follow the character changes, i.e., character evolution, through a phylogeny based on Jetz & Pyron (2018) and Feng et al. (2017) for anuran phylogeny, Pereyra et al. (2015) for Bufonidae, and Wiens et al. (2010) for Hylidae, integrating both morphosource and dissections data. We used parsimony as our methodological criterion for optimal mapping applied with Mesquite 3.70 (Maddison & Maddison, 2019). We consider that parsimony is an appropriate methodological option to deal with morphological characters (Goloboff, 2022), especially in this context of mapping characters on phylogenies, as the branch lengths are not considered (Pol, 2001). Through character mapping, we investigate the relationship between the palmar sesamoid, the carpal ventral surface, phylogeny, and the habitat use within the anuran groups.

Results

Anatomical descriptions of the bony palmar sesamoid, the associated muscles, and tendinous system showing the variations between four Rhinella species with different habitat use were done (Material S2; Figs. 1A, 1B, 1C, 1D, 1E and 1F). The data from the 170 specimens obtained from Morphsource are also recorded. The descriptions are detailed in Material S2 in a comparative manner. Below are the descriptions of the osseous palmar sesamoid and the distal carpal surface anatomy based on specimens’ CT images of Morphosource. Only those taxa with osseous palmar sesamoid are included in this list, from which characters A and B were surveyed. Character A, the presence/absence of the bony sesamoid, represents the variation found in the form and distribution of this sesamoid (Fig. 2). Character B, represents the shape of the distal carpals. These carpals are bones that could be independent at the base of each digit (2, 3, 4, and 5) or fused during the development of the anuran carpus as one bone, distal carpal 3+4, or 3+4+5, or 4+5 (Fabrezi & Alberch, 1996). The palmar surface of the distal carpal unique bone could be protruded, flat or concave due to two parallel crests located on the edges of the bone. Both characters were used to perform the ancestral state reconstruction of the palmar sesamoid presence and the distal carpal palmar surface.

Distal carpals—Sesamoid relationship (Figs. 2A, 2B, 2C, 2D, 2E; 3A, 3B, 3C)

The distal carpal 3+4+5 presents two parallel processes projected from the palmar surface. One is at the preaxial edge of the carpal (as in the case of that of hamalus in the hamate bone of the human manus), and the other on the postaxial edge of the distal carpal. These processes are not always evident, such as in Hadromophryne natalensis (Fig. 2D). More developed processes are seen in, e.g., Rhinella margaritifera (Fig. 1F), Truebella tothastes, Anaxyrus terrestris (Fig. 2C), showing a deep depression between them, which form a concavity. In all cases, the distal carpal is located beside the base of the digits III to V, dorsal to the palmar sesamoid, and the flexor plate. In most cases, the sesamoid embedded on the flexor plate, rests onto the distal carpal, see, e.g., Hemisus guineensis (Fig. 2A), or as in the case of Melanoprhyniscus stelzneri (Fig. 2E). In Rhinella, the development in the size of the palmar sesamoid varies from a small oblong bone to big oval with a proximal process (Figs. 3A, 3B and 3C). In R. margaritifera, the sesamoid is cylindrical (Fig. 1F). It fits into the deep depression, among the two parallel processes of the distal carpal, totally covered by the flexor plate and fused to it ventrally. The tendon that integrates the flexor plate varies in size even overpassing the size of the sesamoid.

Ancestral state reconstruction of the palmar sesamoid presence, morphology of the distal carpal palmar surface, and the habitat use (Figs. 4A, 4B, 4C)

The optimization of the bony palmar sesamoid in the anuran phylogeny (Feng et al., 2017; Jetz & Pyron, 2018) shows that its absence is a widespread character among anurans, being present in specific, mostly neobatrachia clades; we name this big clade as PS (with bony palmar sesamoid). Thus a phylogenetic association is shown by our data. Interestingly, the taxa within PS clade are strongly terrestrial, and those secondarily arboreal, such as Rhinella margaritifera and Frostrius, exhibit a modified shape of the palmar sesamoid. This clade presents some reversions to the ancestral state without palmar sesamoid within the mostly arboreal Hyloydea clade in the dendrobatid Minyobates minutus, in the odontophrynids Proceratophrys bioei and Macrogeniaglottus alipioi; in the hylodid Crossodactylus gaudichaudii and C. trachystomus. Other taxa exhibiting reversions are the cycloramphids Zachaenus parvulus and Cycloramphus asper; the miobatrachid Crinia signifera, and Hadromophryne, a Heleophrynidae, the sister clade of Hyloidea+Ranoidea. Among ranoids, the palmar sesamoid is exhibited in the hemisotid Hemisus guineensis; the microhylids Chiasmocleis crucis, Gastrophryne carolinensis, Syncope antenori, and Melanobatrachus indicus; the pyxicephalids Cacosternum boettgeri and C. namaquense. The only taxon with a palmar sesamoid among Archeobatrachia belongs to the Pelobatoidea clade, Leptobrachium hasseltii.

The shape of the distal carpal ventral surface shows no clear relationship with the presence/absence of the osseous palmar sesamoid. The presence of the parallel crests on the distal carpal, as edges of a concave surface, is independent of the palmar sesamoid presence (CT image of the skeleton of the ventral surface of Lymnodinastes dorsalis can be seen in Material S5).

Discussion

In this study we present a survey of the osseous palmar sesamoid and its relationships with the carpal surface within the anuran amphibian group, to test whether an association can be proposed between these anatomical structures, the phylogeny of the anuran groups, and the habitat use of these taxa by using an ancestral state reconstruction. We consider that if we find that the selected characters change rapidly in response to varying environments, i.e., habitat use, they should not be well correlated with phylogeny.

The osseous palmar sesamoid optimization in the anuran phylogeny showed that its presence is strongly related to specific clades and not as widespread as expected (Ponssa, Goldberg & Abdala, 2010). We can propose that the osseous palmar sesamoid arises early in Neobatrachian groups, being a parallelism or an independent acquisition in the Pelobatoidea Leptobranchium hasseltii. Our data indicate that the osseous palmar sesamoid is highly variable in shape and size, with some taxa such as Melanophryniscus stelzneri, presenting two overlapping palmar sesamoids (Fig. 4). Deforel et al. (2021) described a single, large, and ossified palmar sesamoid as present, in general, in the genus Melanophryniscus. However, Abdala et al. (2022) did not find this sesamoid in this Melanophryniscus species, suggesting that this variability should be further addressed.

The scattered presence of the osseous palmar sesamoid among anuran clades suggests that its genesis can be explained not only by phylogenetic inheritance but also by other factors (such as environmental ones), maybe related to the anuran habitat use. Interestingly, the terrestrial habitat use optimizes as ancestral to the anuran group, which allows us to suppose that the osseous palmar sesamoid absence may indicate a palmar sesamoid of another tissue composition. Bufonidae-Dendrobatidae-Leptodactylidae-Brachicephalidae (PS clade), clearly united by a common ancestor, present a bony palmar sesamoid and share a terrestrial habitat use, inhabiting the forest floor litter and lowland rainforests. Leptobrachium hasseltii, an archeobatrachian pelobatoid frog, also presents a bony palmar sesamoid, shows cryptic habits and also similar habitat use, does not belong to this clade, leaving some room for the discussion of non-phylogenetic factors. Tadpoles of these taxa usually live in quiet pools and ponds. Leptobrachium hasseltii would represent the ancestral mode of life of frogs (Herrel et al., 2016), and it is particularly special since its osseous palmar sesamoid is located between two crests formed by the protrusion edge of the distal carpals 5 and 3 +4 (Fig. 2B). The re-examination of the ancestral state unveils another captivating pattern. It seems that the absence of a crest results in a flat and smooth carpal surface without a palmar sesamoid. Nonetheless, the existence of crests does not hold a direct association with the existence or non-existence of a palmar sesamoid. This suggests that a different element, apart from the crests, is imperative for the connection of the palmar sesamoid to the carpus.

Among bufonids, almost all species examined have osseous palmar sesamoid and primary terrestrial habitat use, which would be the ancestral state according to Herrel et al. (2016). Functionally, the presence of such a structure in the middle of the manus could prevent the closure of the manus by limiting the digits from touching each other (Sustaita et al., 2013). Manzano, Abdala & Herrel (2008) and Manzano, Fontanarrosa & Abdala (2019) noted that tree frogs could grasp because they not only do not have a bony sesamoid embedded in the flexor plate, but also a reduced or absent flexor plate. Our results evidence the absence of the osseous palmar sesamoid in tree frogs (Manzano, Abdala & Herrel, 2008). These data are in accordance with studies in other groups of tetrapods such as lizards and marsupials (Moro & Abdala, 2006; Abdala et al., 2009; Abdala et al., 2009; Fontanarrosa & Abdala, 2014; Fontanarrosa & Abdala, 2016) supporting the pervasive link between the invasion of the narrow branches niche and the lack or reduction of the palmar sesamoid. The examination and comparison of dissected specimens revealed that a manus devoid of an ossified sesamoid exhibits an increased range of motion. This implies that, from a functional standpoint, the presence of this structure may hinder climbing capabilities. This is a fascinating conclusion, and we are inclined to accept it. However, exceptions do exist, such as in certain species of Bufonidae, such as Rhinella margaritifera and R. paraguas, which possess an osseous palmar sesamoid and exhibit scansorial behavior, enabling them to ascend shrubs and small trees (De Noronha et al., 2013; Vassallo et al., 2021; Ceballos-Castro, Cabra-García & Ospina-Sarria, 2023). Our data indicate that in such a case, the shape of the sesamoid is a crucial factor, functionally enabling the manus to grasp. The Rhinella margaritifera’ s palmar sesamoid has a convex surface that articulates with the deep concave depression of the carpal distal 3+4+5, like a hinge joint. This kind of articulation enables the movement of closing the manus around a thin object, such as a small branch, and the sesamoid, in this case, has a cylindrical shape. Curiously, the Bufonidae osseous palmar sesamoid, which can be big or small, has a particular drop shape, and lays superficial to the distal carpals, being usually flat or with a slight depression limited by two crests (Figs. 1 and 3). Our ancestral state reconstruction revealed that the evolution of the distal carpal depression and the sesamoid seem to be independent of each other, suggesting that their relationship is limited to a functional interaction in the manus of R. margaritifera. Thus, this association between the palmar sesamoid shape and the carpal surface can be proposed as an exaptation that led to a grasping manus.

Fontanarrosa, Fratani & Vera (2020) assigned the palmar sesamoid to the pectoral—forelimb module, formed by the coracoid elements and the forelimb, showing the high intermediacy of this sesamoid, comparable to that of the canonical bones. This high value of this network parameter suggests a functional significance, probably related to the requirements of the strongly terrestrial and burrowing mode of life. Additionally, the pectoral and forelimb region has the important function of absorbing the stress of the impact in the landing phase of the jump (Emerson, 1982; Fontanarrosa, Fratani & Vera, 2020). It has been proposed that in most frogs, the palmar sesamoid could be participating of the landing mechanism (Abdala et al., 2022) by regulating the curvature of the distal phalanges of the manus. Thus, the palmar sesamoid could be contributing to an indirect mechanical stress absorption during landing.

Conclusion

The optimization of sesamoids in the anuran phylogeny revealed that an osseous palmar sesamoid is strongly associated with specific clades, contrary to what was previously thought. The presence of a bony palmar sesamoid can be observed in the clade made up of Bufonidae, Dendrobatidae, Leptodactylidae, and Brachicephalidae, which we have named the PS clade, as well as in the archeobatrachian pelobatoid Leptobranchium. These species are all strongly terrestrial and burrowing, though there are exceptions. Bufonidae always has a bony palmar sesamoid, but its form and size vary depending on how the species uses its manus, such as in Rhinella margaritifera, which has a cylindrical one and the ability to grasp through closure of its manus. The scattered presence of the bony palmar sesamoid among different anuran clades raises the question of whether this sesamoid can exist with different tissue compositions in other groups.

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