Morphological variation in the vomer of aquatic and terrestrial spelerpini salamanders.

The vomer is an important tooth-bearing cranial bone in the lungless salamanders (Caudata: Plethodontidae) that serves different functional roles in aquatic versus terrestrial feeding. Vomerine tooth rows that run parallel with the maxillary teeth are thought to help grasp prey while expelling water from the mouth, while posterior extensions of the tooth row may help terrestrial taxa bring prey down the throat. We hypothesize that these two general morphological types will correlate with the habitat (aquatic vs. terrestrial) of adult salamanders. Alternatively, variation in form may be due to taxonomic effects, such that closely related species will have similar vomer morphology regardless of adult habitat. To test this hypothesis, we examined vomer shape on a set of species of the morphologically diverse tribe Spelerpini, in which two of the five genera (Eurycea and Gyrinophilus) include both aquatic and terrestrial species. Data were collected using micro computed tomography (micro-CT) scans from specimens from the Field Museum of Natural History and the Illinois Natural History Survey; additional data was obtained from public online repositories including Morphosource.org. Two-dimensional geometric morphometric analyses were performed to capture shape variation of both the vomer and the vomerine tooth row. We found clear separation between aquatic and terrestrial taxa, with most of the variation due to differences in the vomerine tooth row. Differences ascribed to habitat use likely correspond to feeding behavior, and the functional role of the vomer in prey processing warrants further investigation in this species-rich salamander family.

Triassic stem caecilian supports dissorophoid origin of living amphibians.

Living amphibians (Lissamphibia) include frogs and salamanders (Batrachia) and the limbless worm-like caecilians (Gymnophiona). The estimated Palaeozoic era gymnophionan-batrachian molecular divergence1 suggests a major gap in the record of crown lissamphibians prior to their earliest fossil occurrences in the Triassic period2-6. Recent studies find a monophyletic Batrachia within dissorophoid temnospondyls7-10, but the absence of pre-Jurassic period caecilian fossils11,12 has made their relationships to batrachians and affinities to Palaeozoic tetrapods controversial1,8,13,14. Here we report the geologically oldest stem caecilian-a crown lissamphibian from the Late Triassic epoch of Arizona, USA-extending the caecilian record by around 35 million years. These fossils illuminate the tempo and mode of early caecilian morphological and functional evolution, demonstrating a delayed acquisition of musculoskeletal features associated with fossoriality in living caecilians, including the dual jaw closure mechanism15,16, reduced orbits17 and the tentacular organ18. The provenance of these fossils suggests a Pangaean equatorial origin for caecilians, implying that living caecilian biogeography reflects conserved aspects of caecilian function and physiology19, in combination with vicariance patterns driven by plate tectonics20. These fossils reveal a combination of features that is unique to caecilians alongside features that are shared with batrachian and dissorophoid temnospondyls, providing new and compelling evidence supporting a single origin of living amphibians within dissorophoid temnospondyls.

Phalanx morphology in salamanders: A reflection of microhabitat use, life cycle or evolutionary constraints?

Morphological patterns are modeled by the interaction of functional, phylogenetic, ecological, and/or developmental constraints. In addition, the evolution of life cycle complexity can favor phenotypic diversity; however, the correlation between stages of development may constrain the evolution of some organs. Salamanders present microhabitat and life cycle diversity, providing an excellent framework for testing how these factors constrain phenotypic evolution. We reconstructed the morphological evolution of the terminal phalanx using a sample of 60 extinct and living species of salamanders. Using a geometric morphometric approach combined with comparative analyses, we further investigated the impact of phylogenetic, ecological, and/or life cycle factors on the shape of the terminal phalanx. We find that the phylogeny has some influence in determining the dorsal shape of the phalanges; whereas a relationship between microhabitat or life cycle and the dorsal and lateral shapes of the phalanx was not observed in the analyzed species. The allometric pattern found in the phalanx shape implies that small phalanges are more curved and with more truncated end than bigger phalanges. The evolutionary rate of phalanx shape was higher in the semiaquatic species, and the morphological disparity was significantly higher on biphasic groups. These results contradict the hypothesis that a complex life cycle constrains body shape. Finally, the phalanx shape of the salamander remains quite conserved from the Mesozoic. This configuration would allow them to occur in the different microhabitats occupied by the salamander lineages.

Middle Jurassic fossils document an early stage in salamander evolution.

Salamanders are an important group of living amphibians and model organisms for understanding locomotion, development, regeneration, feeding, and toxicity in tetrapods. However, their origin and early radiation remain poorly understood, with early fossil stem-salamanders so far represented by larval or incompletely known taxa. This poor record also limits understanding of the origin of Lissamphibia (i.e., frogs, salamanders, and caecilians). We report fossils from the Middle Jurassic of Scotland representing almost the entire skeleton of the enigmatic stem-salamander Marmorerpeton. We use computed tomography to visualize high-resolution three-dimensional anatomy, describing morphologies that were poorly characterized in early salamanders, including the braincase, scapulocoracoid, and lower jaw. We use these data in the context of a phylogenetic analysis intended to resolve the relationships of early and stem-salamanders, including representation of important outgroups alongside data from high-resolution imaging of extant species. Marmorerpeton is united with Karaurus, Kokartus, and others from the Middle Jurassic-Lower Cretaceous of Asia, providing evidence for an early radiation of robustly built neotenous stem-salamanders. These taxa display morphological specializations similar to the extant cryptobranchid "giant" salamanders. Our analysis also demonstrates stem-group affinities for a larger sample of Jurassic species than previously recognized, highlighting an unappreciated diversity of stem-salamanders and cautioning against the use of single species (e.g., Karaurus) as exemplars for stem-salamander anatomy. These phylogenetic findings, combined with knowledge of the near-complete skeletal anatomy of Mamorerpeton, advance our understanding of evolutionary changes on the salamander stem-lineage and provide important data on early salamanders and the origins of Batrachia and Lissamphibia.

Genome size drives morphological evolution in organ-specific ways.

Morphogenesis is an emergent property of biochemical and cellular interactions during development. Genome size and the correlated trait of cell size can influence these interactions through effects on developmental rate and tissue geometry, ultimately driving the evolution of morphology. We tested whether variation in genome and body size is related to morphological variation in the heart and liver using nine species of the salamander genus Plethodon (genome sizes 29-67 gigabases). Our results show that overall organ size is a function of body size, whereas tissue structure changes dramatically with evolutionary increases in genome size. In the heart, increased genome size is correlated with a reduction of myocardia in the ventricle, yielding proportionally less force-producing mass and greater intertrabecular space. In the liver, increased genome size is correlated with fewer and larger vascular structures, positioning hepatocytes farther from the circulatory vessels that transport key metabolites. Although these structural changes should have obvious impacts on organ function, their effects on organismal performance and fitness may be negligible because low metabolic rates in salamanders relax selective pressure on function of key metabolic organs. Overall, this study suggests large genome and cell size influence the developmental systems involved in heart and liver morphogenesis.

External nasal gland morphology of Eurycea bislineata (Amphibia, Urodela, Plethodontidae).

Plethodontid salamanders possess numerous courtship glands. Previous studies have shown that the glands are more prominent in male individuals than females, and often experience periods of atrophy and hypertrophy throughout the year that correlate to the nonmating and mating seasons, respectively. We sampled male and female Eurycea bislineata throughout the year to test the hypothesis that external nasal glands are courtship glands. External nasal glands are paired, branched tubular glands that extend from excretory ducts dorsal to the nares to terminal secretory units posterior to the eyes. We found that the glands hypertrophy and stain/react more intensely with histochemical procedures during the mating season. Hypertrophy of the glands is more pronounced in males, and seasonal variation in epithelial height of external nasal glands of males is significantly correlated to that of seasonal variation in mental gland epithelial height, a known courtship gland found in males, when compared throughout the year. This correlation was not as strong in females, confirming sexual dimorphism of external nasal glands in terms of seasonal variation. We found no ultrastructural differences between male and female external nasal glands. In all specimens, the glandular tubules were lined by a simple, columnar epithelium that was packed with secretory granules that often obscured other cytoplasmic contents.

Palatal morphology predicts the paleobiology of early salamanders.

Ecological preferences and life history strategies have enormous impacts on the evolution and phenotypic diversity of salamanders, but the yet established reliable ecological indicators from bony skeletons hinder investigations into the paleobiology of early salamanders. Here, we statistically demonstrate by using time-calibrated cladograms and geometric morphometric analysis on 71 specimens in 36 species, that both the shape of the palate and many non-shape covariates particularly associated with vomerine teeth are ecologically informative in early stem- and basal crown-group salamanders. Disparity patterns within the morphospace of the palate in ecological preferences, life history strategies, and taxonomic affiliations were analyzed in detail, and evolutionary rates and ancestral states of the palate were reconstructed. Our results show that the palate is heavily impacted by convergence constrained by feeding mechanisms and also exhibits clear stepwise evolutionary patterns with alternative phenotypic configurations to cope with similar functional demand. Salamanders are diversified ecologically before the Middle Jurassic and achieved all their present ecological preferences in the Early Cretaceous. Our results reveal that the last common ancestor of all salamanders share with other modern amphibians a unified biphasic ecological preference, and metamorphosis is significant in the expansion of ecomorphospace of the palate in early salamanders.

Salamander braincase morphology as revealed by micro-computed tomography.

Morphological data sets are misleading in salamander (Caudata) phylogeny due to the relative homoplasy of the dermal skull observed in paedomorphic forms, leading to the trend of excluding morphology when exploring questions of salamander phylogeny. Investigations in caecilians (Gymnophiona) have demonstrated that the inclusion of braincase morphology can rescue morphological phylogenetic analyses and produce topologies congruent with molecular data sets. We scanned 28 species (25 genera) of salamander, representing all 10 families, with high-resolution micro-computed tomography to investigate braincase variation. We describe the morphology of the braincase for all 10 families and distinguish between paedomorphic and metamorphic morphologies. Our results demonstrate a general uniformity amongst metamorphic species with variation largely restricted to the occipito-otic region. A greater range of variation is observed within paedomorphic forms than would be expected when considering the homoplasy of the dermal skull. Obligate paedomorphic forms demonstrate considerably more variation in the anterior braincase than do facultative paedomorphs, which we suggest is evidence of a greater complexity in the evolution and development of these forms than neoteny alone would produce. This raises the question of character independence within morphological data sets and warrants further investigation into the correlation of other characters before morphological data are omitted.

Osteological characteristics of the Setouchi salamander Hynobius setouchi (Urodela, Hynobiidae).

This study described the detailed osteological features of the Setouchi salamander Hynobius setouchi. H. setouchi, which is endemic to central Japan, was recently delineated from the wide range of H. nebulosus species based on molecular and external morphological characteristics. However, the osteological features of the species belonging to the genus Hynobius have not been examined, which has hindered the detailed understanding of morphological diversity in the genus. To address this problem, this study elucidated the osteological features of the salamander using micro-computed tomography scanning and whole-mount double staining methods. The articular, a part of the hypobranchial I, a part of coracoid, pubis, carpus, and tarsus were cartilaginous in small-sized individuals and were partially or completely ossified in large-sized individuals. The degree of ossification varied in the operculum, basibranchial II, and ascending process of the palatoquadrate. However, ossification was not associated with body size. The lacrimal, ossified manus, and ossified pes exhibited varying degrees of articulation or fusion irrespective of body size. Moreover, the cranium and tarsus of H. setouchi exhibited several unique characteristics. These osteological characteristics will aid in revising the taxonomy and phylogeny of the Hynobius species.

Genetic basis for an evolutionary shift from ancestral preaxial to postaxial limb polarity in non-urodele vertebrates.

In most tetrapod vertebrates, limb skeletal progenitors condense with postaxial dominance. Posterior elements (such as ulna and fibula) appear prior to their anterior counterparts (radius and tibia), followed by digit-appearance order with continuing postaxial polarity. The only exceptions are urodele amphibians (salamanders), whose limb elements develop with preaxial polarity and who are also notable for their unique ability to regenerate complete limbs as adults. The mechanistic basis for this preaxial dominance has remained an enigma and has even been proposed to relate to the acquisition of novel genes involved in regeneration. However, recent fossil evidence suggests that preaxial polarity represents an ancestral rather than derived state. Here, we report that 5'Hoxd (Hoxd11-d13) gene deletion in mouse is atavistic and uncovers an underlying preaxial polarity in mammalian limb formation. We demonstrate this shift from postaxial to preaxial dominance in mouse results from excess Gli3 repressor (Gli3R) activity due to the loss of 5'Hoxd-Gli3 antagonism and is associated with cell-cycle changes promoting precocious cell-cycle exit in the anterior limb bud. We further show that Gli3 knockdown in axolotl results in a shift to postaxial dominant limb skeleton formation, as well as expanded paddle-shaped limb-bud morphology and ensuing polydactyly. Evolutionary changes in Gli3R activity level, which also played a key role in the fin-to-limb transition, appear to be fundamental to the shift from preaxial to postaxial polarity in formation of the tetrapod limb skeleton.

A new species of Bolitoglossa (Caudata: Plethodontidae) of the Bolitoglossa franklini group from an isolated cloud forest in northern Guatemala.

We describe Bolitoglossa qeqom sp. nov. from an isolated cloud forest in Cerro Guachmalén, Alta Verapaz, Guatemala, based on multiple lines of evidence (morphological, molecular, and biogeographic data). This region comprises a mountain ridge without previous herpetological surveys. The new species is a large salamander with uniform purplish-black coloration and is distinguished by having relatively long legs with only one costal groove between adpressed limbs, numerous maxillary teeth, few vomerine teeth, only one phalange free of webbing in digit III of feet, and a relatively short tail. It is geographically closest to its sister clade of B. lincolni + B. franklini and the xeric Chixoy river canyon appears to be the major biogeographic barrier that isolated the new taxon. The cloud forest inhabited by this species has undergone severe habitat destruction in the region and land conservation actions are urgent.

Energy storage in salamanders' tails: the role of sex and ecology.

In vertebrates, the main tissue devoted to energy storage is the adipose tissue. In salamanders, energy reserves can also be stored in the adipose tissues of the tail. Therefore, we evaluated if energy storage in salamanders' tails is related to individual body condition, life cycle and environmental constraints. We calculated a scaled measure of tail width for 345 salamanders belonging to six Mediterranean taxa exhibiting wide phylogenetic, behavioural and ecological variation. We related this measure to the Scaled Mass Index (SMI), a body condition index which reliably predicts body fat. We found significant relationships between the SMI and scaled tail width in the terrestrial Spectacled salamander and Alpine salamanders, independently of sex. At the same time, we found that energy storage in the tail is maximum in Alpine Salamanders, which experience reduced activity periods and restricted access to resources. Conversely, we found a significant effect of sex in Imperial cave salamanders, where females store reserves in the tail to counterbalance resource investment in parental care, and in Corsican Brook Newts, where the reproductive function of males' tails may imply a greater tail width. Finally, in the biphasic Great Crested Newt, tail width was not related to SMI in both sexes.

Comparative osteology of the hynobiid complex Liua-Protohynobius-Pseudohynobius (Amphibia, Urodela): â . Cranial anatomy of Pseudohynobius.

Hynobiidae are a clade of salamanders that diverged early within the crown radiation and that retain a considerable number of features plesiomorphic for the group. Their evolutionary history is informed by a fossil record that extends to the Middle Jurassic Bathonian time. Our understanding of the evolution within the total group of Hynobiidae has benefited considerably from recent discoveries of stem hynobiids but is constrained by inadequate anatomical knowledge of some extant forms. Pseudohynobius is a derived hynobiid clade consisting of five to seven extant species living endemic to southwestern China. Although this clade has been recognized for over 37 years, osteological details of these extant hynobiids remain elusive, which undoubtedly has contributed to taxonomic controversies over the hynobiid complex Liua-Protohynobius-Pseudohynobius. Here we provide a bone-by-bone study of the cranium in the five extant species of Pseudohynobius (Ps. flavomaculatus, Ps. guizhouensis, Ps. jinfo, Ps. kuankuoshuiensis and Ps. shuichengensis) based on x-ray computer tomography data for 18 specimens. Our results indicate that the cranium in each of these species has a combination of differences in morphology, proportions and articulation patterns in both dermal and endochondral bones. Our study establishes a range of intraspecific differences that will serve as organizing hypotheses for future studies as more extensive collections of these species become available. Morphological features in the cranium for terrestrial ecological adaptation in Hynobiidae are summarized. Based on the results, we also discuss the evolution and development of several potential synapomorphies of Hynobiidae, including features of the orbitosphenoid and articular.

Miniaturization, Genome Size, and Biological Size in a Diverse Clade of Salamanders.

AbstractGenome size (C-value) can affect organismal traits across levels of biological organization from tissue complexity to metabolism. Neotropical salamanders show wide variation in genome and body sizes, including several clades with miniature species. Because miniaturization imposes strong constraints on morphology and development and because genome size is strongly correlated with cell size, we hypothesize that body size has played an important role in the evolution of genome size in bolitoglossine salamanders. If this hypothesis is correct, then genome size and body size should be correlated in this group. Using Feulgen image analysis densitometry, we estimated genome sizes for 60 species of Neotropical salamanders. We also estimated the "biological size" of species by comparing genome size and physical body sizes in a phylogenetic context. We found a significant correlation between C-value and physical body size using optimal regression with an Ornstein-Uhlenbeck model and report the smallest salamander genome found to date. Our index of biological size showed that some salamanders with large physical body size have smaller biological body size than some miniature species and that several clades demonstrate patterns of increased or decreased biological size compared with their physical size. Our results suggest a causal relationship between physical body size and genome size and show the importance of considering the impact of both on the biological size of organisms. Indeed, biological size may be a more appropriate measure than physical size when considering phenotypic consequences of genome size evolution in many groups.

A new species of the genus Batrachuperus (Urodela: Hynobiidae) from Southwestern China.

A new stream salamander species, Batrachuperus daochengensis sp. nov., from southwestern China, is described herein based on morphological and molecular evidence. Molecular phylogeny derived from the mitochondrial gene together with previous nuclear data revealed that B. daochengensis sp. nov. is sister to B. yenyuanensis. The new species differs from all other species of the genus by the following combination of characters: brown horny epidermis on tips of fingers and toes absent; tubercles on palms and soles absent; costal grooves 12; dorsal brown, mottled with blackish spots; fingers 2-3-4-1 in order of decreasing length; tips of longest digits of fore- and hindlimbs largely separated by one to two costal spaces when adpressed towards each other along sides of body. The new species is currently known in the central and southern Shaluli Mountains in southwestern China.

Intraspecific Variation in the External and Skeletal Morphology of Hynobius setouchi Matsui, Okawa, Tanabe et Misawa, 2019 (Amphibia: Urodela: Hynobiidae).

The hynobiid salamander genus Hynobius is a basal taxon in the urodeles that plays a key role in understanding the evolution of sexual dimorphism in urodeles. We examined the age (juveniles vs. adults) and sexual differences in the external and skeletal morphology of a Japanese lentic breeding salamander, Hynobius setouchi. We found that juveniles had larger heads than those of adults, and most characters were larger in adult males than in females, except for trunk size. This was also observed in skeletal morphology, although some tail characters differed only in the skeleton. We suggest that intra- and interspecific comparisons of the external and skeletal characters of salamanders should consider age, sexual, and seasonal differences.

A Triassic stem-salamander from Kyrgyzstan and the origin of salamanders.

The origin of extant amphibians remains largely obscure, with only a few early Mesozoic stem taxa known, as opposed to a much better fossil record from the mid-Jurassic on. In recent time, anurans have been traced back to Early Triassic forms and caecilians have been traced back to the Late Jurassic Eocaecilia, both of which exemplify the stepwise acquisition of apomorphies. Yet the most ancient stem-salamanders, known from mid-Jurassic rocks, shed little light on the origin of the clade. The gap between salamanders and other lissamphibians, as well as Paleozoic tetrapods, remains considerable. Here we report a new specimen of Triassurus sixtelae, a hitherto enigmatic tetrapod from the Middle/Late Triassic of Kyrgyzstan, which we identify as the geologically oldest stem-group salamander. This sheds light not only on the early evolution of the salamander body plan, but also on the origin of the group as a whole. The new, second specimen is derived from the same beds as the holotype, the Madygen Formation of southwestern Kyrgyzstan. It reveals a range of salamander characters in this taxon, pushing back the rock record of urodeles by at least 60 to 74 Ma (Carnian-Bathonian). In addition, this stem-salamander shares plesiomorphic characters with temnospondyls, especially branchiosaurids and amphibamiforms.

Evolution of a high-performance and functionally robust musculoskeletal system in salamanders.

The evolution of ballistic tongue projection in plethodontid salamanders-a high-performance and thermally robust musculoskeletal system-is ideal for examining how the components required for extreme performance in animal movement are assembled in evolution. Our comparative data on whole-organism performance measured across a range of temperatures and the musculoskeletal morphology of the tongue apparatus were examined in a phylogenetic framework and combined with data on muscle contractile physiology and neural control. Our analysis reveals that relatively minor evolutionary changes in morphology and neural control have transformed a muscle-powered system with modest performance and high thermal sensitivity into a spring-powered system with extreme performance and functional robustness in the face of evolutionarily conserved muscle contractile physiology. Furthermore, these changes have occurred in parallel in both major clades of this largest family of salamanders. We also find that high-performance tongue projection that exceeds available muscle power and thermal robustness of performance coevolve, both being emergent properties of the same elastic-recoil mechanism. Among the taxa examined, we find muscle-powered and fully fledged elastic systems with enormous performance differences, but no intermediate forms, suggesting that incipient elastic mechanisms do not persist in evolutionary time. A growing body of data from other elastic systems suggests that similar coevolution of traits may be found in other ectothermic animals with high performance, particularly those for which thermoregulation is challenging or ecologically costly.

A new small-sized stem salamander from the Middle Jurassic of Western Siberia, Russia.

Salamanders (Caudata) are one of the three modern groups of amphibians known from the Middle Jurassic. The early stages of evolution of these amphibians are still poorly known, especially for stem taxa of Jurassic age. A new small-sized stem salamander, Egoria malashichevi gen. et sp. nov., from the Middle Jurassic (Bathonian) Itat Formation of the Berezovsk Quarry locality in Western Siberia, Russia, is described on the basis of isolated vertebrae, including an atlas centrum and a fragmentary trunk vertebra centrum previously referred to an undescribed salamander taxon ("Berezovsk salamander A"). The new taxon is diagnosed by the following unique combination of vertebral characters: atlantal anterior cotyles with elliptical anterior outline, located at an angle of approximately 135-137 degrees to each other; wide posterior portion of the atlantal centrum; ossified portion of the intercotylar tubercle represented by dorsal and ventral lips; absence of a deep depression on the ventral surface of the atlantal centrum; absence of pronounced ventrolateral ridges on the atlas; absence of spinal nerve foramina; presence of a pitted texture on the ventral and lateral surfaces of the centra and lateral surfaces neural arch pedicels; presence of a short atlantal neural arch with its anterior border situated behind the level of the anterior cotyles; short trunk vertebrae; and upper transverse process (= diapophysis) larger than lower transverse process (= parapophysis) on the trunk vertebrae; notochordal canal opens in the upper half of the cotyle (= the lower portion of the centrum is more massive and less compact than the upper portion). The microanatomical organization of the atlas and trunk vertebrae is characterized by the presence of inner cancellous endochondral bone. The small body size (about 180-215 mm) of Egoria malashichevi gen. et sp. nov. indicates that that not all stem salamanders were large neotenic forms (up to 550-600 mm in Urupia and Marmorerpeton) and hints at a broader ecological role for stem salamanders.

Salamanders and other amphibians are aglow with biofluorescence.

Biofluorescence is the absorption of electromagnetic radiation (light) at one wavelength followed by its reemission at a lower energy and longer wavelength by a living organism. Previous studies have documented the widespread presence of biofluorescence in some animals, including cnidarians, arthropods, and cartilaginous and ray-finned fishes. Many studies on biofluorescence have focused on marine animals (cnidarians, cartilaginous and ray-finned fishes) but we know comparatively little about the presence of biofluorescence in tetrapods. We show for the first time that biofluorescence is widespread across Amphibia, with a focus on salamanders (Caudata), which are a diverse group with a primarily Holarctic distribution. We find that biofluorescence is not restricted to any particular family of salamanders, there is striking variation in their fluorescent patterning, and the primary wavelengths emitted in response to blue excitation light are within the spectrum of green light. Widespread biofluorescence across the amphibian radiation is a previously undocumented phenomenon that could have significant ramifications for the ecology and evolution of these diverse and declining vertebrates. Our results provide a roadmap for future studies on the characterization of molecular mechanisms of biofluorescence in amphibians, as well as directions for investigations into the potential impact of biofluorescence on the visual ecology and behavior of biofluorescent amphibians.