Amphibian Hormones, Calcium Physiology, Bone Weight, and Lung Use Call for a More Inclusive Approach to Understanding Ossification Sequence Evolution

Skeleton plays a huge role in understanding how vertebrate animals have diversified in phylogeny, ecology and behavior. Recent evo-devo research has used ossification sequences to compare skeletal development among major groups, to identify conserved and labile aspects of a sequence within a group, to derive ancestral and modal sequences, and to look for modularity based on embryonic origin and type of bone. However, questions remain about how to detect and order bone appearances, the adaptive significance of ossification sequences and their relationship to adult function, and the utility of categorizing bones by embryonic origin and type. Also, the singular focus on bone appearances and the omission of other tissues and behavioral, ecological and life history events limit the relevance of such analyses. Amphibians accentuate these concerns because of their highly specialized biphasic life histories and the exceptionally late timing, and high variability of their ossification sequences. Amphibians demonstrate a need for a whole-animal, whole-ontogeny approach that integrates the entire ossification process with physiology, behavior and ecology. I discuss evidence and hypotheses for how hormone mediation and calcium physiology might elicit non-adaptive variability in ossification sequence, and for adaptive strategies to partition larval habitats using bone to offset the buoyancy created by lung use. I also argue that understanding plasticity in ossification requires shifting focus away from embryonic development and adult function, and toward postembryonic mechanisms of regulating skeletal growth, especially ones that respond directly to midlife environments and behaviors.

What do ossification sequences tell us about the origin of extant amphibians?

The origin of extant amphibians has been studied using several sources of data and methods, including phylogenetic analyses of morphological data, molecular dating, stratigraphic data, and integration of ossification sequence data, but a consensus about their affinities with other Paleozoic tetrapods has failed to emerge. We have compiled five datasets to assess the relative support for six competing hypotheses about the origin of extant amphibians: a monophyletic origin among temnospondyls, a monophyletic origin among lepospondyls, a diphyletic origin among both temnospondyls and lepospondyls, a diphyletic origin among temnospondyls alone, and two variants of a triphyletic origin, in which anurans and urodeles come from different temnospondyl taxa while caecilians come from lepospondyls and are either closer to anurans and urodeles or to amniotes. Our datasets comprise ossification sequences of up to 107 terminal taxa and up to eight cranial bones, and up to 65 terminal taxa and up to seven appendicular bones, respectively. Among extinct taxa, only two or three temnospondyl can be analyzed simultaneously for cranial data, but this is not an insuperable problem because each of the six tested hypotheses implies a different position of temnospondyls and caecilians relative to other sampled taxa. For appendicular data, more extinct taxa can be analyzed, including some lepospondyls and the finned tetrapodomorph Eusthenopteron, in addition to temnospondyls. The data are analyzed through maximum likelihood, and the AICc (corrected Akaike Information Criterion) weights of the six hypotheses allow us to assess their relative support. By an unexpectedly large margin, our analyses of the cranial data support a monophyletic origin among lepospondyls; a monophyletic origin among temnospondyls, the current near-consensus, is a distant second. All other hypotheses are exceedingly unlikely according to our data. Surprisingly, analysis of the appendicular data supports triphyly of extant amphibians within a clade that unites lepospondyls and temnospondyls, contrary to all phylogenies based on molecular data and recent trees based on paleontological data, but this conclusion is not very robust.

Comparative description and ossification patterns ofDendropsophus labialis(Peters, 1863) andScinax ruber(Laurenti, 1758) (Anura: Hylidae)

Although comparative studies of anuran ontogeny have provided new data on heterochrony in the life cycles of frogs, most of them have not included ossification sequences. Using differential staining techniques, we observe and describe differences and similarities of cranial and postcranial development in two hylid species,Scinax ruber(Scinaxinae) andDendropsophus labialis(Hylinae), providing new data of ontogenetic studies in these Colombian species. We examined tadpoles raining from Gosner Stages 25 to 45. We found differences between species in the infrarostral and suprarostral cartilages, optic foramen, planum ethmoidale, and gill apparatus. In both species, the first elements to ossify were the atlas and transverse processes of the vertebral column and the parasphenoid. Both species exhibited suprascapular processes as described in other hylids. Although the hylids comprise a large group (over 700 species), postcranial ossification sequence is only known for 15 species. Therefore, the descriptions of the skeletal development and ossification sequences provided herein will be useful for future analyses of heterochrony in the group.

Comparative description and ossification patterns of Dendropsophus labialis (Peters, 1863) and Scinax ruber (Laurenti, 1758)(Anura: Hylidae)

Although comparative studies of anuran ontogeny have provided new data on heterochrony in the life cycles of frogs, most of them have not included Colombian species. Using different staining techniques, we describe the cranial and poscranial elements development in two hylid species, Scinax ruber and Dendropsophus labialis, providing new data for more comprehensive ontogenetic studies in Neotropical frogs. We examined specimens from Gosner stages 25 to 45. We found differences in the infrarostral and suprarostral cartilages, optic foramen, planum ethmoidale, and the gill apparatus. In the ossification sequence, one of the first elements to ossify were the transverse process of spinal column and atlas in both species, and the parasphenoid in the skull. New descriptions of skeletal development and ossification sequences of larval stages of these two species, especially data concerning the postcranium, contribute with useful information for analysis of sequential heterochrony, because although the hylids are widely known, there are few works (15 of 700 species) about ossification sequence that include the whole skeleton.

Comparative description and ossification patterns of Dendropsophus labialis (Peters, 1863) and Scinax ruber (Laurenti, 1758)(Anura: Hylidae)

Although comparative studies of anuran ontogeny have provided new data on heterochrony in the life cycles of frogs, most of them have not included Colombian species. Using different staining techniques, we describe the cranial and poscranial elements development in two hylid species, Scinax ruber and Dendropsophus labialis, providing new data for more comprehensive ontogenetic studies in Neotropical frogs. We examined specimens from Gosner stages 25 to 45. We found differences in the infrarostral and suprarostral cartilages, optic foramen, planum ethmoidale, and the gill apparatus. In the ossification sequence, one of the first elements to ossify were the transverse process of spinal column and atlas in both species, and the parasphenoid in the skull. New descriptions of skeletal development and ossification sequences of larval stages of these two species, especially data concerning the postcranium, contribute with useful information for analysis of sequential heterochrony, because although the hylids are widely known, there are few works (15 of 700 species) about ossification sequence that include the whole skeleton.

Ontogenetic sequence reconstruction and sequence polymorphism in extinct taxa: an example using early tetrapods (Tetrapoda: Lepospondyli)

Ontogenetic sequence reconstruction is challenging particularly for extinct taxa because of when, where, and how fossils preserve. Different methods of reconstruction exist, but the effects of preservational bias, the applicability of size-independent methods, and the prevalence of sequence polymorphism (intraspecific variation) remain unexplored for paleontological data. Here I compare five different methods of ontogenetic sequence reconstruction and their effects on the detection of sequence polymorphism, using a large collection of the extinct vertebrates Microbrachis pelikani and Hyloplesion longicostatum. The postcranial ossification sequences presented here for those taxa are the first examples known for extinct lepospondyls. Sequences were reconstructed according to skull length, trunk length, increasing number of ontogenetic events, majority-rule consensus, and Ontogenetic Sequence Analysis (OSA). Results generally were in agreement, demonstrating that paleontological data may be used to robustly reconstruct developmental patterns. When reconstructing sequences based on fossils, size-based methods and OSA are more objective and less dependent on preservational bias than other techniques. Apart from the other methods, OSA also allows for statistical analysis of observed and predicted polymorphism. However, OSA requires a large sample size to yield meaningful results, and size-based methods are justified in paleontological studies when sample size is limited by poor preservation. Different methods of reconstruction detected different patterns of sequence polymorphism, although across all methods the magnitude of sequence variation for M. pelikani and H. longicostatum (1.3−3.4%) was within the lower range of values reported for extant vertebrates. Compared with other extinct and extant tetrapods, all sequence reconstruction methods consistently showed that M. pelikani and H. longicostatum exhibit advanced ossification of the pubis and delayed ossification of the scapula. However, the postcranial ossification sequences of these two taxa largely are congruent with those of other tetrapods, suggesting an underlying conservative ancestral pattern that evolved early in tetrapod history.