María Laura Ponssa

Sesamoids in Anurans: New Data, Old Issues

Sesamoids are skeletal elements rarely considered in studies of the vertebrate skeleton. In this work, we integrate ontogenetic data of anuran sesamoids in two species (Leptodactylus latinasus and Pleurodema cf. guayapae), the related structures (tendons, muscles, and joints) in L. latinasus, and a survey of sesamoid distribution in 185 anuran taxa. Our main goals are: (1) to contribute to the knowledge of the comparative anatomy of sesamoids in tetrapods; (2) to provide additional developmental evidence to interpret the ontogenetic pattern of sesamoids in anurans, as a key to elucidate that of tetrapods in general; (3) to provide data about tendon development in relation to sesamoid development in anurans for the first time; and (4) to propose a pattern of anuran sesamoid distribution. The homologies of sesamoids across tetrapods are discussed here. Observations were made in cleared and stained skeletal whole‐mounts. Fifty‐four sesamoids were found in anurans, thirty‐seven of which occur in L. latinasus. The traditional point of view of embedded sesamoids always resulting from biomechanical stimuli of a previously existing tendon is not sustained by our data. Many sesamoids arise before the differentiation of a tendinous tissue. Our survey results in a data set where the two big anuran clades, Hyloides (12 families) and Ranoides (14 families), were represented. The matrix has 38% missing entries. Most of the surveyed sesamoids have multiple origins, with only three of them (about 19%) having one origin. Anat Rec 293:1646–1668, 2010.

Patterns of skull development in anurans: size and shape relationship during postmetamorphic cranial ontogeny in five species of the Leptodactylus fuscus Group (Anura: Leptodactylidae)

The effect of allometric ontogenetic changes on morphology has been examined primarily in larval stages of anurans. To our knowledge, such studies after metamorphosis are non-existent, and this information is important because the skull acquires its adult configuration in that period. Using geometric morphometrics, we studied postmetamorphic shape changes in the skull of five species of the Leptodactylus fuscus Group (Leptodactylus bufonius, Leptodactylus elenae, Leptodactylus fuscus, Leptodactylus latinasus, and Leptodactylus mystaceus), a group of small- to medium-sized frogs. Size change is an important factor in explaining shape change during postmetamorphic growth in four of these species; ontogenetic trajectories have in general parallel directions and similar rates of shape change. L. latinasus skulls tend to differ in size and shape from the others, and the allometric model, although significant, explains low percentages of shape change. The diverging slope of its ontogenetic trajectory indicates non-heterochronic, allometric repatterning change regarding the ontogenies of L. bufonius, L. elenae, and L. fuscus. Conversely, ontogenetic scaling appears as the main mechanism modeling shape change as regard to L. mystaceus; hence, we suggest that a process of progenesis determines the small, juvenile-like cranium of L. latinasus. The disparity analysis shows a broader morphological divergence in metamorph morphospace than in adults, suggesting that postmetamorphic stages can contribute with informative characters to phylogenetic analysis. Differences in shapes between metamorphs and adults indicate that many changes occur after metamorphosis, but whether these changes result from internal or ecological requirements at different stages remains unknown.

Life in the Slow Lane: The Effect of Reduced Mobility on Tadpole Limb Development

Movement is thought to be a primary agent eliciting basic responses in the vertebrate body, such as the proper development of the musculoskeletal system. Embryos do not passively await hatching or birth but rather begin active movement very early on in their development. Most studies dealing with embryonic responses to changes in mobility have been performed in chickens or mammals. Herein, we investigate for the first time whether the embryos of organisms that are free‐living during development demonstrate the same morphological responses to reduced mobility as embryos that undergo development in controlled environments such as in utero or in a shelled egg. We changed the viscosity of the environment in which free‐living anuran tadpoles grow by rearing them in an agar medium. We thus increased the viscosity of the growth medium resulting in a decrease in larval movement. We predicted that a substantial increase in viscosity of the medium in which the larvae were reared would have at least two consequences: (1) a reduction of tadpole mobility and (2) a delayed onset of skeletogenesis thus producing shorter long bones. Our predictions were upheld and tadpoles reared in an agar medium remain immobile longer and showed a delayed onset of skeletogenesis compared with controls. We propose that the developmental responses to the same stimulus are similar throughout tetrapods, regardless of their developmental context (i.e., intrauterine, within an egg, or free‐living). Anat Rec, 2012.

Network architecture associated with the highly specialized hindlimb of frogs

Network analyses have been increasingly used in the context of comparative vertebrate morphology. The structural units of the vertebrate body are treated as discrete elements (nodes) of a network, whose interactions at their physical contacts (links) determine the phenotypic modules. Here, we use the network approach to study the organization of the locomotor system underlying the hindlimb of frogs. Nodes correspond to fibrous knots, skeletal and muscular units. Edges encode the ligamentous and monoaxial tendinous connections in addition to joints. Our main hypotheses are that: (1) the higher centrality scores (measured as betweenness) are recorded for fibrous elements belonging to the connective system, (2) the organization of the musculoskeletal network belongs to a non-trivial modular architecture and (3) the modules in the hindlimb reflect functional and/or developmental constraints. We confirm all our hypotheses except for the first one, since bones overpass the fibrous knots in terms of centrality. Functionally, there is a correlation between the proximal-to-distal succession of modules and the progressive recruitment of elements involved with the motion of joints during jumping. From a developmental perspective, there is a correspondence between the order of the betweenness scores and the ontogenetic chronology of hindlimbs in tetrapods. Modular architecture seems to be a successful organization, providing of the building blocks on which evolution forges the many different functional specializations that organisms exploit.

Comparative Morphometrics in Leptodactyline Frogs (Anura, Leptodactylidae, Leptodactylinae): Does Burrowing Behavior Relate to Sexual Dimorphism?

Abstract Fossorial habits occur in many animal lineages and usually involve both morphological and physiological adaptations that may evolve independently. Burrowing behavior in some species of the anuran subfamily Leptodactylinae involves the specialized use of the hind limbs and/or head. The aim of this study was to identify the morphometric characters associated with burrowing behavior in species of this subfamily. We then hypothesized that, as this habit is usually associated with males, we would find sexual dimorphism in head and hind-limb morphology in the burrowing species but not in the nonburrowing species. We compared 500 specimens from 24 species using phylogenetic statistical analyses and phylogenetic mapping of sexually dimorphic characters. The results demonstrated the following: 1) There was no correlation between the measurements of the limbs, head, or tarsal tubercle and burrowing behavior in the analyzed species; 2) there was no sexual dimorphism related to burrowing behavior reflected in measurements of the head or hind limbs; and 3) sexual dimorphism in the morphometric characters always was derived. Modifications of the ridged snout and increased ossification in the nasal region of the males of the fossorial species appear to be sufficient adaptations for burrowing.

On the presence of the patella in frogs

The patella is one of the most studied sesamoids. Historically, the patella is described as a big sesamoid embedded in the tendon of the quadriceps femoris muscle. This sesamoid is studied from developmental, functional, clinical, and anatomical perspectives. The presence of a patella is reported in squamatans, birds, and mammals. Lissamphibians are identified as the major lineage that fail to develop a patella. However, this sesamoid is reported at least once in anurans, but without detailed anatomical discussions. Through anatomical and histological studies we examined the topography and tissue composition of two structures that we identify as the proximal and distal patellae in several anuran species. We explored the evolution of these sesamoids through ancestral state reconstruction, finding that they are ancestral for amphibians and possibly tetrapods as a whole. The presence of these patellae in anurans would roll back their origin to the last common ancestor of tetrapods. From a functional perspective, the overwhelming evidence of fibrocartilage as a clear response to compression suggests that the fibrocartilaginous patellae could also withstand the mechanical stress generated on the knee undergoing compression during limb extension. Anat Rec, 2017.

Phenotypical expression of reduced mobility during limb ontogeny in frogs: the knee-joint case

Movement is one of the most important epigenetic factors for normal development of the musculoskeletal system, particularly during genesis and joint development. Studies regarding alterations to embryonic mobility, performed on anurans, chickens and mammals, report important phenotypical similarities as a result of the reduction or absence of this stimulus. The precise stage of development at which the stimulus modification generates phenotypic modifications however, is yet to be determined. In this work we explore whether the developmental effects of abnormal mobility can appear at any time during development or whether they begin to express themselves in particular phases of tadpole ontogeny. We conducted five experiments that showed that morphological abnormalities are not visible until Stages 40–42. Morphology in earlier stages remains normal, probably due to the fact that the bones/muscles/tendons have not yet developed and therefore are not affected by immobilization. These results suggest the existence of a specific period of phenotypical expression in which normal limb movement is necessary for the correct development of the joint tissue framework.

Development and morphological variation of the axial and appendicular skeleton in hylidae (Lissamphibia, Anura)

The axial and appendicular skeleton, the associated musculature and tendons form a functional system related to specific modes of locomotion in anurans. Many transformations in the structures linked with the locomotor function of the adult occur during larval stages and metamorphosis. In this study, we present the larval ontogeny and adult morphology of the axial and appendicular skeletons of 14 species of frogs in the family Hylidae with different locomotor modes and habitat uses. Among Hylidae, a diversity of shapes, locomotory types occurs (e.g., walker, swimmer, jumper, hopper) and different habitat types occupied (shrubby, terrestrial, aquatic, arboreal). Many elements complete differentiation at the end of metamorphosis; others, such as sesamoids, still show an incomplete development at that stage. Sixty seven characters were scored and optimized in an available phylogeny. Nine characters of developmental timing and adult osteology are optimized as synapomorphies of specific groups. Some characters appear to be related to the locomotor type (e.g., the sacro‐urostyle region configuration is highly linked with the jumping mode; nonexpanded diapophyses would related to aquatic habitat use). Nevertheless, the functional interpretations are quite particular to this family. Monophyletic clades are also groups with shared locomotory modes or habitat uses. Hence, the hypothesis of common ancestry or adaptation can be evaluated, taking into account the analysis level of the phylogenetic context, so that, when a character is inherited via common ancestry, it necessarily means that functional constraints could also be inherited. Here, we outline the basis for further work on: postmetamorphic development as a fundamental period for the complete differentiation of structures related to a full locomotor functionality; the biomechanical performance in relationship to the variation in ligaments and sesamoids; the importance of analyzing these topics within the frame of heterochrony. J. Morphol. 277:786–813, 2016.

Frog tendon structure and its relationship with locomotor modes

Tendon collagen fibrils are the basic force‐transmitting units of the tendon. Yet, surprisingly little is known about the diversity in tendon anatomy and ultrastructure, and the possible relationships between this diversity and locomotor modes utilized. Our main objectives were to investigate: (a) the ultra‐structural anatomy of the tendons in the digits of frogs; (b) the diversity of collagen fibril diameters across frogs with different locomotor modes; (c) the relationship between morphology, as expressed by the morphology of collagen fibrils and tendons, and locomotor modes. To assess the relationship between morphology and the locomotor modes of the sampled taxa we performed a principal component analysis considering body length, fibrillar cross sectional area (CSA) and tendon CSA. A MANOVA showed that differences between species with different locomotor modes were significant with collagen fibril diameter being the discriminating factor. Overall, our data related the greatest collagen fibril diameter to the most demanding locomotor modes, conversely, the smallest collagen fibril CSA and the highest tendon CSA were observed in animals showing a hopping locomotion requiring likely little absorption of landing forces given the short jump distances.

Further Data on Sesamoid Identity from Two Anuran Species

Considering that the identification of equivalent entities is the basis for any comparative analysis, we compare the histology, histochemistry, shape and dimensions of epiphyses, carpal and sesamoids in two anuran frogs. Our goal was to explore the morphological correspondence among these three skeletal elements in order to clarify the sesamoid identity. We studied the skeletogenesis, contour geometric morphometry and dimensions of forelimb elements of juveniles of two anurans species Leptodactylus bufonius and Rhinella arenarum. Skeletogenesis in anurans present a common trait between carpals and sesamoids: both elements exhibit endochondral ossification. A difference between these elements is the presence of fibrocartilage in the development of sesamoids. The geometric morphometry does not allow us to establish a shape pattern that can be compared either between sesamoids and epiphyses or carpals. With regard to dimensions, our data indicate that bones categorization based on these aspects is ambiguous and therefore is useless to classify of skeletal bones. The data about tissue differentiation of sesamoids provide evidence that support the idea that these elements should be considered part of the typical endowment of the vertebrate skeleton. Anat Rec, 298:1376–1394, 2015.