Evolutionary and developmental considerations of the diet and gut morphology in ceratophryid tadpoles (Anura).

BACKGROUND: Before metamorphosis, almost all anuran tadpoles are omnivores. Larval carnivory occurs in some species and, it is associated with distinctive morphotypes. Obligatory carnivorous tadpoles exhibit structural changes in the gastrointestinal tract compared to larvae that are predominately omnivores. The most distinctive feature of the anuran family Ceratophyridae (three genera) overall is the enormous gape of adults. This feature increases their ability to capture extremely large and active prey. The larvae of Ceratophyrid genera are remarkably distinct from each other and carnivory has diversified in a manner unseen in other anurans. The larvae of one genus, Lepidobatrachus, has a massive gape like the adult. Herein, we report on larval developmental variation, diet, gross morphology of the gastrointestinal tract, and histology of the cranial segment of the gut before, during and after metamorphosis in larval series for the following ceratophryid species: Chacophrys pierottii, Ceratophrys cranwelli, Lepidobatrachus laevis and Lepidobatrachus llanensis. RESULTS: We described patterns of larval development with variation in growth with consequence to the final size at the end of metamorphosis. These patterns seem to be influenced by food quantity/quality, and most predominant by animal protein. Prey items found in pre and post-metamorphic Lepidobatrachus spp. are similar. Tadpoles of Ceratophrys and Chacophrys (and other anurans) share a short cranial segment of the gut with an internal glandular, mucous secreting epithelium, a double coiled intestine and the sequence of metamorphic changes (tract is empty, the stomach differentiates and the intestine shortens abruptly). In contrast, Lepidobatrachus tadpoles have a true stomach that acquires thickness and increased glandular complexity through development. As larvae they have a short intestine without double coils, and the absence of intestine shortening during metamorphosis. CONCLUSIONS: The larval development of the gastrointestinal tract of Lepidobatrachus is unique compared with that of other free-living anuran larvae. An abrupt metamorphic transformation is missing and most of the adult structural features start to differentiate gradually at the beginning of larval stages.

Histology and microscopic anatomy of the thyroid gland during the larval development of Pseudis platensis (Anura, Hylidae).

Several hormones regulate anuran larval development, most notably thyroid hormones (THs). In anurans, metamorphosis fails when the thyroid gland is absent or inactivated, resulting in giant tadpoles. Larval gigantism occurs naturally in neotropical frogs of the genus Pseudis as a result of a prolonged larval period. Its thyroid function is poorly investigated and the focus of this study. We describe qualitative and quantitative variations in larval development for field-captured specimens of Pseudis platensis and compare those to the development of two sympatric species, Phyllomedusa sauvagii and Pithecopus azureus, which have small tadpoles and a shorter larval period. We describe morphological changes in the thyroid glands of larval and adult specimens. In contrast to other species with similar ecological requirements, P. platensis exhibits distinct glandular activity. During premetamorphosis, there was little or no thyroid activity, a period in which the tadpole reached 70% of its maximum size. Development and degree of activity of the thyroid gland determine the duration of the early stages of the larval period. Thyroid gland histology in tadpoles appears to correlate with the TH activity, and in turn with the diversity in anuran life history transitions.

Differences in responsiveness and sensitivity to exogenous disruptors of the thyroid gland in three anuran species.

Anuran larval development comprises tissues/organs/systems that are: exclusively of larvae, able to be remodelled, and those of postmetamorphic stages. Also, the anuran larval development is characterized by inter-related parameters: time, size and shape forming part of growth and differentiation. The anuran metamorphosis starts when growth and differentiation achieve a threshold that differs among species since it is regulated by a number of external (environmental) and internal (hormonal) processes. Here we explore the consequences of exogenous disruptors on the thyroid gland (e.g., methimazole and thyroxine as T4) of three species by immersing premetamorphic tadpoles in predetermined concentrations of the disruptors for short periods (10 or 16 days). The species were Pleurodema borellii, Leptodactylus chaquensis, and Dermatonotus muelleri, which all breed in small temporary ponds during the summer, but differ in their ecomorphology. The experiments were conducted to evaluate the effects of these substances on larval development (based in Gosner larval stages), morphometric variation in body parameters (snout-vent and total length by larval stages), and thyroid gland histopathology at the end of the assays. In P. borelli and L. chaquensis, methimazole produces significant increment of size measurements (nonparametric Kruskal-Wallis, p < .05) during stages of digit differentiation and induced thyroid gland hypertrophy. In the three species, T4 exposure accelerated limb development and caused atrophy of thyroid gland. Prolonged T4 exposure in L. chaquensis and D. muelleri triggered metamorphic transformation in the gut and skull cartilages. Discussion about interspecific differences in responsiveness and sensitivity elucidates the importance of hormonal signals to morphological evolution.

Intraspecific developmental variation in the life cycle of the Andean Treefrog (Boana riojana): A temporal analysis.

Intraspecific variation during the anuran larval period has been analyzed mainly in relation to the timing of metamorphosis and body size at metamorphosis. However, other traits may vary as well. We examined two developmental series of Boana riojana from the same population in two consecutive years and describe intraspecific variation in larvae of this species. We discuss how variation, if present, may influence its life cycle. We found that both larval series differed in the larval period length, one twice as long as the other. This variation primarily depended on when breeding occurred, metamorphosis was achieved during late spring in both generations and at similar sizes, and only the rate of larval development during premetamorphosis varied extensively between years. This is consistent with thyroid gland activity because when it became active the developmental trajectory became more canalized. No variation of staging sequence occurred in relation to the different durations of the larval period. However, in the long-lasting series we found two different morphs. Also, integument, thyroid gland, skeleton, and testis differentiation events occurred at the same developing stages. In contrast, ovarian differentiation proceeded at the same absolute age in both series. Sexual dimorphism becomes evident within the year after metamorphosis. The intraspecific heterochrony that we describe for the larval development of B. riojana does not lead to phenotypic variation at the end of metamorphosis. We discuss the importance of analyzing growth and development independently. Each proceeds differently in time, but with an interdependence at some point, because size and shape do not vary at the end of metamorphosis.

The peripheral nerves of Lepidobatrachus tadpoles (Anura, Ceratophryidae).

The peripheral nervous system of anuran larvae has traditionally been assumed to be largely invariant. Here, we describe the organization of cranial, spinal, and lateral line nerves at different larval stages of Lepidobatrachus spp. based on whole mounts. This is the first detailed description of cranial, spinal, and lateral lines innervation at premetamorphic stages of anuran larvae with notes on temporal variation. We distinguish three sources of morphological variation with respect to other anuran larvae: (a) the loss or reduction of some exclusively larval elements (i.e., the absence of the middle lateral line nerve); (b) spatial changes in the lateral line system (i.e., the supralabial arrangement of component of the anteroventral lateral line nerve); and (c) temporal changes in the disappearance of most of the lateral line system and in the premetamorphic repatterning of the spatial relationships of mandibularis ramus of the trigeminal (V) and hyomandibularis ramus of facial (VII). The innervation of limbs is achieved during late larval stages. Furthermore, comparisons among selected anurans reveal differences in tadpole brain morphology. The spatial and temporal variation found in the peripheral nerves of Lepidobatrachus larvae testifies to previously unappreciated variation in anuran larval morphology.

Morphological Variation in Anuran Limbs: Constraints and Novelties.

Anurans have three primary types of locomotion: walking, jumping, and swimming. Additionally, they may dig, climb, grasp, etc. All adult anurans have four limbs, with four fingers on the hands and five toes on the feet. We summarized and updated knowledge on the interspecific variation within anuran limbs, then discuss how developmental constraints (e.g., in size) and novelties may have influenced anuran diversification through the locomotion. We analyze morphological variation from limb bud stages up to the final limb form resulting from certain skeletal organization and growth. We find limited morphometric variations in the skeleton of different developmental modules (i.e., skull, trunk, urostyle, limbs) indicate that the anuran body shape is largely constrained. We identify specializations of the stylopodium, zeugopodium, and proximal carpals/tarsals that have evolved to facilitiate saltatorial locomotion. We show that the anuran prepollex and prehallux are not vestigial digits and that they have come to serve specialized function. Medial rotation of the manus in anurans appears to have evolved to help distribute the force of impact upon landing at the end of a jump. Additional skeletal elements in anuran limbs are intercalary elements and sesamoids. The intercalary elements appear within neobatrachians and are integrated with digital pads in lineages capable of locomotion on smooth vertical surfaces. They have allowed arboreal anurans to occupy a wide range of arboreal habitats.

The distribution of the Bururi Long-fingered Frog (Cardioglossa cyaneospila, family Arthroleptidae), a poorly known Albertine Rift endemic.

The species diversity of the frog genus Cardioglossa (family Arthroleptidae) is concentrated in the Lower Guinean Forest Zone of Central Africa with most of the 19 species occurring in Cameroon and neighboring countries (Amiet 1972a,b; Blackburn 2008; Hirschfeld et al. 2015). These small leaf-litter frogs are typically found in primary or secondary forest, have shrill whistling calls, are characterized by a variety of color patterns, and lay terrestrial eggs that hatch and develop into elongate, stream-adapted tadpoles (Amiet 1972a,b, 1973; Rödel et al. 2001; Hirschfeld et al. 2012). One of the most poorly known species-the Bururi Long-fingered Frog Cardioglossa cyaneospila Laurent, 1950-is also among the most geographically peripheral to the rest of the species diversity. To date, it is known only from two locations in Burundi and four in eastern Democratic Republic of Congo, regions in which armed conflicts have long hampered scientific research. In this short contribution, we (1) document both new and long unpublished records of C. cyaneospila, associate these with known museum records, and extend its geographic range, (2) highlight fruitful areas for future field surveys based on predicting an environmental envelope for this species, and (3) summarize what little is known of its natural history.

Developmental changes and novelties in ceratophryid frogs.

The Neotropical frog genera Ceratophrys, Chacophrys and Lepidobatrachus form the monophyletic family Ceratophryidae. Although in- and out-group relationships are not fully resolved, the monophyly of the three genera is well supported by both morphological and molecular data. Much is known about the morphology of the ceratophryids, but there is little comparative information on how modification of a common ancestral developmental pathway played a role in shaping their particular body plans. Herein, we review morphological variation during ceratophryid ontogeny in order to explore the role of development in their evolution. The ceratophryids are collectively characterized by rapid larval development with respect to other anurans, yet the three genera differ in their postmetamorphic growth rates to sexual maturity. Derived traits in the group can be divided into many homoplastic features that evolved in parallel with those of anurans with fossorial/burrowing behaviors in semiarid environments, and apomorphies. Morphological novelties have evolved in their feeding mechanism, which makes them capable of feeding on exceptional large prey. Lepidobatrachus is unusual in having reduced the ecomorphological differences between its larvae and adults. As a result, both the larvae and the frog are similarly able to capture large prey underwater. Some unique features in Lepidobatrachus are differentiated in the tadpole and then exaggerated in the adult (e.g., the posterior displaced jaw articulation) in a manner unobserved in any other anurans.

The lateral line system in anuran tadpoles: neuromast morphology, arrangement, and innervation.

Anuran larvae have been classified into four morphological types which reflect intraordinal macroevolution. At present, complete characterizations of the lateral line system are only available for Xenopus laevis (Type I) and Discoglossus pictus (Type III). We analyzed the morphology, arrangement, and innervation of neuromasts related to the anterodorsal and anteroventral lateral line nerves in 10 anuran species representing Types I, II, and IV with the aim of interpreting the existing variation and discussing the evolution of the lateral line in anuran larvae. We found: (1) the presence of two orbital and three mandibular neuromast lines in all anuran larvae studied, (2) the ventral arrangement of mandibular neuromast lines appears to have evolved convergently in Larval Types I and II, and the lateroventral arrangement of mandibular lines of neuromasts appears to have evolved in Larval Types III and IV; (3) interspecific variation in the organization, size, and number of sensory cells per neuromast within the lines; and (4) the supralabial extension of the Angular line in Lepidobatrachus spp. and the tentacular location of the Oral neuromasts in X. laevis are concomitant with their particular morphologies. Based on the variation described we find that the lateral line system in anuran larvae seems to have been maintained without significant changes, with the exception of Lepidobatrachus spp. and Xenopus. These unique features added to other of Lepidobatrachus tadpoles are sufficient to propose a new Larval Type (V).

Ontogenetic and structural variation of mineralizations and ossifications in the integument within ceratophryid frogs (anura, Ceratophryidae).

Ceratophryidae represent a monophyletic group of terrestrial and aquatic frogs inhabiting lowlands of South America where they are more diverse in semiarid environments of the Chaco region. Adult morphology of ceratophryids presents some features associated to terrestrial and fossorial life such as hyper-ossified skulls, spade feet for digging, among others. For anurans, different mineralized structures have been described in the integument as calcium reservoirs and related to the terrestrial life and water balance (e.g., the calcified layer and dermal ossifications). We describe the ontogeny of the integument in the three genera of ceratophryids (Chacophrys, Ceratophrys, and Lepidobatrachus) that inhabit in semiarid environments. Data obtained demonstrated the early acquisition of metamorphic transformations in the integument layers in larvae of Ceratophrys cranwelli and Lepidobatrachus spp. and a continuous increment in the thickness of them up to old postmetamorphic stages. The integument of ceratophryids develops calcium deposits as the calcified layer during postmetamorphic stages. Furthermore, dorsal shields are also present in adult stages independently of terrestrial versus aquatic lifestyles. While the calcified layer seems to be a feature of a fully developed integument, in which their layers have acquired the adult thickness, dorsal shields develop at premetamorphic stages in L. llanensis and postmetamorphic individuals of C. cranwelli. In ceratophryids, similar to other studied taxa (e.g., Brachycephalus spp.) dorsal shields develop via an intramembranous ossification in which the calcified layer does not precede its differentiation. Within anurans, the occurrence of dorsal shields in the monophyletic ceratophryids suggested a distinctive evolutionary history in the lineage.

The ontogeny of Pseudis platensis (Anura, Hylidae): Heterochrony and the effects of larval development on postmetamorphic life.

Recent studies have described the giant tadpole, delayed metamorphic transformations, and absence of postmetamorphic growth of the skeleton of Pseudis Platensis. These features address questions about derived patterns of life cycles and the role of the heterochrony during the metamorphosis in anurans. Using anatomical methods, we provide new data on the development of reproductive, digestive and integument systems, and age inference obtained from ontogenetic series of Pseudis platensis. Our results indicate that at the end of metamorphosis, the adult skin is completely differentiated, including the calcified dermal layer; the testis has seminiferous tubules with spermatogonia, spermatocytes, and spermatids; ovarian sacs present previtellogenic ova; and the adult digestive tract is fully formed. The froglets differ from adults only in being unable to reproduce. The entire life cycle of P. platensis can occur in 4 years. In the first year, larval development, growth to adult size, and gonad differentiation are completed. Long larval development rather than size of the tadpoles seems to be involved in the absence of juvenile stages.

Hyoid skeleton, its related muscles, and morphological novelties in the frog Lepidobatrachus (anura, ceratophryidae).

Many traits of the skull of ceratophryines are related to the capture of large prey independently of aquatic or terrestrial feeding. Herein, detailed descriptions of the development of hyoid skeleton and the anatomy of muscles responsible for hyoid and tongue movements in Lepidobatrachus laevis and L. llanensis are provided and compared with those of other neobatrachians. The aquatic Lepidobatrachus has special features in its hyoid skeleton that integrates a set of derived features convergent with the conditions observed in non-neobatrachian anurans and morphological novelties (e.g., dorsal dermal hyoid ossification) that deviate from the generalized pattern found in most frogs. Further, reduction of fibers of muscles of buccal floor, reduction or loss of hyoid muscles (m. geniohyoideus rama lateralis, anterior pair of m. petrohyoideus posteriores), small tongue, and simplified tongue muscles are also morphological deviations from the pattern of terrestrial ceratophryines, and other aquatic ceratophryids (e.g., Telmatobius) that seem to be related to feeding underwater. The historical derived features shared with Chacophrys and Ceratophrys involved in megalophagy are conserved in Lepidobatrachus and morphological changes in the hyoglossal apparatus define a unique functional complex among anurans.

Heterochrony during skeletal development of Pseudis platensis (Anura, Hylidae) and the early offset of skeleton development and growth.

The aquatic frog Pseudis platensis has a giant tadpole, long developmental time, and dissociated metamorphic events that include later offset of larval somatic morphologies. Moreover, when the tadpole metamorphoses, the young frog is nearly the size of an adult, suggesting that this species has low rates of postmetamorphic growth. Herein, we study the development of the skeleton during larval development up to the end of metamorphosis, which is denoted by the complete lost of the tail in P. platensis. Our study revealed heterochronic differences in skeletal development compared with that of most anurans; these involve the complete differentiation of skull bones and the extensive ossification of the postcranial skeleton before completion of metamorphosis. The skull of metamorphosing P. platensis has an ossified sphenethmoid and a fully formed plectral apparatus, thus differing with regard to the pattern observed in most anurans in which both developmental events take place during the postmetamorphic life. Despite the fact that the iliosacral articulation and the urostyle are present at the end of metamorphosis as in most anurans, ossification/calcification of carpus, tarsus, and limb epihyses during metamorphosis of P. platensis suggests that the postcranial skeleton lacks postmetamorphic growth. This study also includes a discussion of the pattern of development of the plectral apparatus, which allows us to propose a new hypothesis regarding pars externa plectri homology.

Intercalary elements, treefrogs, and the early differentiation of a complex system in the Neobatrachia.

Intercalary elements are additional skeletal structures of digits of many anuran amphibians. Twelve terminal clades in the neobatrachian lineage of frogs have intercalary elements revealing it is a homoplastic character with five to seven gains and two to four losses along a consensus phylogeny of the Neobatrachia. We analyzed anatomical variation of intercalary elements, related structures (distal phalanges, tendons, and muscles), and articulations of digits of 45 anuran species, representing eight suprageneric terminal taxa. The intercalary elements are integrated in a complex system that is probably related to different types of movements, which are produced by a similar set of muscles and tendons with limited variation among the studied taxa. Species in the clades Hyloides and Ranoides show distinctive patterns of morphostructural features in their intercalary elements that are usually wedge-shaped and composed of hyaline cartilage in Ranoides, and biconcave and composed of embryonic cartilage in Hyloides. Features derived from the typical hyloid condition may only be interpreted in some Hylidae (Pseudis and Lysapsus) and Centrolenidae. In Ranoides, the described features of the intercalary elements are found in all taxa examined with the exception of Leptopelis, which have an intercalary element similar to the other Ranoides but formed by connective tissue. Several features are shared by all taxa having intercalary elements: (1) the intercalary elements differ from the phalanges by lacking terminal epiphyses, (2) they are present in hands and feet, and (3) they appear in all digits. This finding suggests that the genetic basis for presence of intercalary elements may be homologous in all these taxa and may have evolved only once early in neobatrachian history.

Developmental basis of limb homology in lizards.

Shubin and Alberch (Evol Biol 1986;20:319-387) proposed a scheme of tetrapod limb development based on cartilage morphogenesis that provides the arguments to interpret the homologies of skeletal elements and sets the basis to explain limb specialization through later developmental modification. Morphogenetic evidence emerged from the study of some reptiles, but the availability of data for lizards is limited. Here, the study of adult skeletal variation in 41 lizard taxa and ontogeny in species of Liolaemus and Tupinambis attempts to fill in this gap and provides supporting evidence for the Shubin-Alberch scheme. Six questions are explored. Is there an intermedium in the carpus? Are there two centralia in the carpus? Is there homology among proximal tarsalia of reptiles? Does digit V belong to the digital arch? Is the pisiform an element of the autopodium plan? And should the ossification processes be similar to cartilage morphogenesis? We found the following answers. Some taxa exhibit an ossified element that could represent an intermedium. There is one centrale in the carpus. Development of proximal tarsalia seems to be equivalent with that observed among reptiles. Digit V could arise from the digital arch. Pisiform does not arise as part of the limb plan. And different patterns of ossification occur following a single and conservative cartilaginous configuration. Lizard limb development shows an early pattern common to other reptiles with clear primary axis and digital arch. The pattern then becomes lizard-specific with specialization involving some reduction in prechondrogenic elements.