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The neural crest is a distinct population of embryonic cells found only in craniates. Several genes, including BMP-4and the zinc-finger transcription factor Slug, have been shown to mediate neural crest development in both chickens and mice. We examined the function of Slug in the frog Xenopus laevisby injecting antisense RNA (ASR) targeted to the 3' UTR of Xenopus Slug (XSlug). Injection of XSlugASR inhibits neural crest-cell migration and leads to reduction or loss of many neural crest derivatives, including pigment cells and cranial cartilages. Moreover, regions injected with XSlug ASR show significantly elevated levels of cell death, and expanded regions of the BMP-4antagonist, Chordin. In addition to its important role in neural crest migration, XSlug may mediate the cell-death-signaling pathway and other significant developmental pathways in Xenopus laevis. Supported by NSF.

Derivation of most if not all of the ossified skull from the embryonic neural crest is an accepted axiom of contemporary developmental biology. Yet, this 'fact' is based on direct empirical results involving just one species of vertebrate, the domestic chicken. Anuran amphibians (frogs) pose a particularly difficult challenge in this regard: bone typically forms many weeks after hatching, and tracing individual cells over this lengthy interval is technically difficult. We used RNA encoding for green fluorescent protein (GFP) to label premigratory neural crest in embryos of Xenopus laevis, and assessed cranial derivatives both before and after metamorphosis. Results confirm the neural crest contribution to larval cranial cartilages demonstrated previously by using conventional vital stains, lineage markers, and ablation techniques. They also provide evidence for the neural crest derivation of a major paired skull bone, the frontoparietal; other, later-forming bones are currently under study. This is the first direct empirical demonstration of neural crest contribution to the ossified skull in any amphibian. Supported by NSF.

In the chicken embryo, neural crest cells contribute to the connective tissue components of cranial muscle. Furthermore, a 'cryptic segmentation' exists, in which mesodermal and neural crest components of a given muscle are derived from the same visceral arch. Larval anurans have a highly apomorphic head. Unique features include, for example, second (hyoid) arch muscles that both originate and insert on cartilages from the first (mandibular) arch. This makes anurans an excellent testing ground for the hypothesis that cryptic segmentation is a conserved vertebrate trait that persists even in clades that have undergone drastic evolutionary changes in cranial morphology. Vital dye labelling and ablation of selected regions of migrating neural crest cells in Bombina orientalis indicate that neural crest contributes to connective tissue components at the attachment sites of several cranial muscles. Crest ablation severely disrupts cranial muscle patterning. Preliminary data indicate that cryptic segmentation is conserved in frogs. Supported by NSF (to J.H.) and by the Wenner-Gren Center Foundations and the Swedish Institute (to L.O.).

Slug is a Zn-finger transcription factor expressed in neural-crest cells. Slugs function has been studied in both mouse and chicken, with conflicting results, e.g. slug appears to be critical for neural crest migration in the chicken, but not in the mouse. To study the role of slug in amphibians, we injected fertilized eggs of Xenopus laevis with antisense slug RNA. This caused reduced expression of both slug and the related transcription factor snail, as assessed by in situ hybridization. In addition to gastrulation defects, we found a distinct inhibition of neural crest-cell migration and the reduction or loss of many neural crest derivatives. The effects of antisense slug RNA were not uniform, however. While rostral cartilages were often severely affected, and in some cases missing altogether, posterior cartilages always appeared quite normal. This suggests that the differentiation of these more caudal neural crest derivatives is less dependent upon slug/snail activity. These results are similar to those found in chicken and indicate that slug and snail are critical for neural crest development in Xenopus. Supported by NSF (T.F.C, J.H.), and NIH (M.W.K.).

The plethodontid salamander genus Thorius comprises a clade of miniaturized salamanders endemic to southern Mexico. Though the genus was erected (by E. D. Cope) more than 130 years ago, for more than 70 years following its initial description Thorius was thought to include just a single species. Nine named forms were added by 1970, and subsequent morphological and molecular studies have continued to reveal additional species. At present, 23 valid species are recognized, yet at least 11 undescribed species await formal description. These species coexist with and replace one another in complex geographic and elevational patterns, which include numerous instances of sympatry involving two, three, and possibly four species. Phylogenetic analyses suggest surprising trends in morphological evolution associated with miniaturization, including frequent homoplasy involving character loss and gain. While such cryptic diversity was not anticipated by early students of Thorius, it is proving to be a common theme in contemporary studies of amphibian systematics. The total number of valid, named species has been increasing at a rate of nearly 2% or more per year for the last two decades.

Most anuran amphibians exhibit a complex, biphasic life history consisting of a free-living aquatic larva (tadpole) and a terrestrial adult. Metamorphosis in anurans is characterized by extensive remodeling of larval tissues via growth or resorption and the de novo formation of many adult structures. This process is dependent on thyroid hormone, which initiates--via nuclear receptors--a cascade of gene expression in responding tissues. It has been hypothesized that changes in the thyroid hormone-regulated metamorphic program mediate the evolutionary diversification of larval and adult morphology. This could be accomplished via temporal changes in tissue sensitivity and responsiveness to thyroid hormone on a tissue or organ-specific level. We are interested in the role that thyroid hormone response plays in the diversification of a specific tissue, Meckel's cartilage, a prominent skeletal element in the larval lower jaw. We investigated the presumed ancestral pattern of thyroid hormone receptor (TRa and TRa) expression during tadpole development and metamorphosis, and compared this to patterns in other species with varied jaw morphologies associated with larval feeding adaptations. Additionally, we assessed whether patterns of cell death and proliferation correlate with thyroid hormone response. This study will contribute to our understanding of the biphasic developmental program of anuran amphibians and its role in the generation of anuran diversity.

Examines the role of molecular heterochrony in the pattern of fibronectin expression during gastrulation in amphibians. Evolutionary concept of heterochrony; Expression of extracellular matrix molecules on the blastocoel roof during gastrulation; Patterns of expression of fibronectin on the surface of blastocoel roofs; Analysis of the blastula and gastrula stages.

The impact of preserved museum specimens is transforming and increasing by three-dimensional (3D) imaging that creates high-fidelity online digital specimens. Through examples from the openVertebrate (oVert) Thematic Collections Network, we describe how we created a digitization community dedicated to the shared vision of making 3D data of specimens available and the impact of these data on a broad audience of scientists, students, teachers, artists, and more. High-fidelity digital 3D models allow people from multiple communities to simultaneously access and use scientific specimens. Based on our multiyear, multi-institution project, we identify significant technological and social hurdles that remain for fully realizing the potential impact of digital 3D specimens., (© The Author(s) 2023. Published by Oxford University Press on behalf of the American Institute of Biological Sciences.)

Southern Amazonia is one of the less-explored regions by anuran taxonomists. We describe a small new species of snouted treefrog, genus Scinax, from this region, from a fluvial archipelago in the Juruena River, state of Mato Grosso, Brazil. The description is based on external morphology of adults and tadpoles, advertisement call and molecular data. The species is phylogenetically related to other snouted treefrogs of the Scinax cruentomma species group and shows the most southeastern distribution in Amazonia among its close relatives. It is distinguished from congeners mainly by its larger adult body size and bilobate vocal sac that reaches the level of the pectoral fold, a reddish-brown horizontal stripe on the iris, dark melanophores or blotches on the vocal sac and the throat of females, and the uniformly brown posterior portion of the thigh. The advertisement call comprises one pulsed note emitted at regular intervals, with a duration of 189-227 ms, 30-35 pulses/note and a dominant frequency of 2,250-2,344 Hz. The type locality is suffering several environmental impacts, including illegal mining, overfishing, unsustainable agriculture, uncontrolled logging and degradation associated with the construction of new hydroelectric dams. Further study of the biology and regional distribution of the new species is required to propose mitigation measures needed for its conservation., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ferrão et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Frogs are an ecologically diverse and phylogenetically ancient group of anuran amphibians that include important vertebrate cell and developmental model systems, notably the genus Xenopus. Here we report a high-quality reference genome sequence for the western clawed frog, Xenopus tropicalis, along with draft chromosome-scale sequences of three distantly related emerging model frog species, Eleutherodactylus coqui, Engystomops pustulosus, and Hymenochirus boettgeri. Frog chromosomes have remained remarkably stable since the Mesozoic Era, with limited Robertsonian (i.e., arm-preserving) translocations and end-to-end fusions found among the smaller chromosomes. Conservation of synteny includes conservation of centromere locations, marked by centromeric tandem repeats associated with Cenp-a binding surrounded by pericentromeric LINE/L1 elements. This work explores the structure of chromosomes across frogs, using a dense meiotic linkage map for X. tropicalis and chromatin conformation capture (Hi-C) data for all species. Abundant satellite repeats occupy the unusually long (~20 megabase) terminal regions of each chromosome that coincide with high rates of recombination. Both embryonic and differentiated cells show reproducible associations of centromeric chromatin and of telomeres, reflecting a Rabl-like configuration. Our comparative analyses reveal 13 conserved ancestral anuran chromosomes from which contemporary frog genomes were constructed., (© 2024. The Author(s).)

Natural history museums are vital repositories of specimens, samples and data that inform about the natural world; this Formal Comment revisits a Perspective that advocated for the adoption of compassionate collection practices, querying whether it will ever be possible to completely do away with whole animal specimen collection., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Modularity (segmentation), homology and heterochrony were essential concepts embraced by Gavin de Beer in his studies of the development and evolution of the vertebrate skull. While his pioneering contributions have stood the test of time, our understanding of the biological processes that underlie each concept has evolved. We assess de Beer's initial training as an experimental embryologist; his switch to comparative and descriptive studies of skulls, jaws and middle ear ossicles; and his later research on the mammalian skull, including his approach to head segmentation. The role of cells of neural crest and mesodermal origin in skull development, and developmental, palaeontological and molecular evidence for the origin of middle ear ossicles in the evolutionary transition from reptiles to mammals are used to illustrate our current understanding of modularity, homology and heterochrony. This article is part of the theme issue 'The mammalian skull: development, structure and function'.

One or more members of four living amphibian clades have independently dispensed with pulmonary respiration and lack lungs, but little is known of the developmental basis of lung loss in any taxon. We use morphological, molecular, and experimental approaches to examine the Plethodontidae, a dominant family of salamanders, all of which are lungless as adults. We confirm an early anecdotal report that plethodontids complete early stages of lung morphogenesis: Transient embryonic lung primordia form but regress by apoptosis before hatching. Initiation of pulmonary development coincides with expression of the lung-specification gene Wnt2b in adjacent mesoderm, and the lung rudiment expresses pulmonary markers Nkx2.1 and Sox9 . Lung developmental-genetic pathways are at least partially conserved despite the absence of functional adult lungs for at least 25 and possibly exceeding 60 million years. Adult lung loss appears associated with altered expression of signaling molecules that mediate later stages of tracheal and pulmonary development.

Cryptic diversity is extremely common in widespread Amazonian anurans, but especially in nurse frogs of the genus Allobates . There is an urgent need to formally describe the many distinct but unnamed species, both to enable studies of their basic biology but especially to facilitate conservation of threatened environments in which many are found. Here, we describe through integrative taxonomy a new species of the Allobates tapajos species complex from the upper Madeira River, southwestern Amazonia. Species delimitation analyses based on molecular data are congruent and delimit five candidate species in addition to A. tapajos sensu stricto. The new species is recovered as sister to A. tapajos clade F, a candidate species from Teles-Pires River, southeastern Amazonia. The new species differs from nominal congeners in adult and larval morphology and in male advertisement call. Egg deposition sites differ between east and west banks of the upper Madeira River, but there is no evidence of corresponding morphologic or bioacoustic differentiation. The new species appears to be restricted to riparian forests; its known geographic range falls entirely within the influence zone of reservoirs of two large dams, which underscores the urgent need of a conservation assessment through long-term monitoring. This region harbors the richest assemblage of Allobates reported for Brazilian Amazonia, with six nominal species and four additional candidate species awaiting formal description., Competing Interests: The authors declare there are no competing interests., (©2022 Ferrão et al.)

Large diversifications of species are known to occur unevenly across space and evolutionary lineages, but the relative importance of their driving mechanisms, such as climate, ecological opportunity and key evolutionary innovations (KEI), remains poorly understood. Here, we explore the remarkable diversification of rhacophorid frogs, which represent six percent of global amphibian diversity, utilize four distinct reproductive modes, and span a climatically variable area across mainland Asia, associated continental islands, and Africa. Using a complete species-level phylogeny, we find near-constant diversification rates but a highly uneven distribution of species richness. Montane regions on islands and some mainland regions have higher phylogenetic diversity and unique assemblages of taxa; we identify these as cool-wet refugia. Starting from a centre of origin, rhacophorids reached these distant refugia by adapting to new climatic conditions ('niche evolution'-dominant), especially following the origin of KEIs such as terrestrial reproduction (in the Late Eocene) or by dispersal during periods of favourable climate ('niche conservatism'-dominant)., (© 2022. The Author(s).)

Competing Interests: The author declares no competing interest.

Nurse frogs (Aromobatidae: Allobates ) are probably the most extensively studied genus by taxonomists in Brazilian Amazonia. The southwestern portion of Amazonia is the most species-rich: as many as seven species may occur in sympatry at a single locality. In this study, we describe a new species of nurse frog from this region. The description integrates data from larval and adult morphology, advertisement calls and DNA sequences. Allobates velocicantus sp. nov. is distinguished from other Allobates mainly by the absence of hourglass-shaped dark marks on the dorsum and dark transverse bars on the thigh; a throat that is white centrally and yellow marginally; basal webbing on toes II and III; finger I longer than finger II; and an advertisement call composed of 66-138 pulsed notes with a note duration of 5-13 ms, inter-note intervals of 10-18 ms and a dominant frequency of 5,512-6,158 Hz. Tadpoles of the new species have 3-4 short, rounded papillae on the anterior labium, 16-23 papillae on the posterior labium, and a labial keratodont row formula 2(2)/3(1). This is the fifth species of Allobates described from the state of Acre, southwestern Brazilian Amazonia., Competing Interests: The authors declare that they have no competing interests., (© 2020 Souza et al.)

Recent studies have shown that species diversity of the South American frog genus Dendropsophus is significantly underestimated, especially in Amazonia. Herein, through integrative taxonomy a new species of Dendropsophus from the east bank of the upper Madeira River, Brazil is described. Based on molecular phylogenetic and morphological analyses, the new species is referred to the D. microcephalus species group, where it is differentiated from its congeners mainly by having a green bilobate vocal sac and an advertisement call comprising 1-4 monophasic notes emitted with a dominant frequency of 8,979-9,606 Hz. Based on intensive sampling conducted in the study area over the last ten years, the new species is restricted to the east bank of the upper Madeira River, although its geographic range is expected to include Bolivian forests close to the type locality., (Miquéias Ferrão, Jiří Moravec, James Hanken, Albertina Pimentel Lima.)

The origin and evolution of the vertebrate skull have been topics of intense study for more than two centuries. Whereas early theories of skull origin, such as the influential vertebral theory, have been largely refuted with respect to the anterior (pre-otic) region of the skull, the posterior (post-otic) region is known to be derived from the anteriormost paraxial segments, i.e. the somites. Here we review the morphology and development of the occiput in both living and extinct tetrapods, taking into account revised knowledge of skull development by augmenting historical accounts with recent data. When occipital composition is evaluated relative to its position along the neural axis, and specifically to the hypoglossal nerve complex, much of the apparent interspecific variation in the location of the skull-neck boundary stabilizes in a phylogenetically informative way. Based on this criterion, three distinct conditions are identified in (i) frogs, (ii) salamanders and caecilians, and (iii) amniotes. The position of the posteriormost occipital segment relative to the hypoglossal nerve is key to understanding the evolution of the posterior limit of the skull. By using cranial foramina as osteological proxies of the hypoglossal nerve, a survey of fossil taxa reveals the amniote condition to be present at the base of Tetrapoda. This result challenges traditional theories of cranial evolution, which posit translocation of the occiput to a more posterior location in amniotes relative to lissamphibians (frogs, salamanders, caecilians), and instead supports the largely overlooked hypothesis that the reduced occiput in lissamphibians is secondarily derived. Recent advances in our understanding of the genetic basis of axial patterning and its regulation in amniotes support the hypothesis that the lissamphibian occipital form may have arisen as the product of a homeotic shift in segment fate from an amniote-like condition., (© 2020 The Authors. Biological Reviews published by John Wiley & Sons Ltd on behalf of Cambridge Philosophical Society.)

The genus Chiropterotriton is endemic to Mexico with a geographical distribution along the Sierra Madre Oriental, the Trans Mexican Volcanic Belt and the Sierra de Juárez. The recent use of molecular tools has shown that Mexico's amphibian diversity is highly underestimated, including a large number of cryptic, unnamed species. Chiropterotriton has 18 described species including terrestrial, arboreal and cave-dwelling species. In previous molecular studies, the presence of multiple undescribed species was evident. We present a phylogenetic hypothesis based on mitochondrial data, which includes all described species and six undescribed taxa. Based on the morphological analyses and, when available, combined with molecular data, we describe five new species of the genus; Chiropterotriton casasi sp. nov., C. ceronorum sp. nov., C. melipona sp. nov., C. perotensis sp. nov. and C. totonacus sp. nov. In addition, we redescribe two others: Chiropterotriton chiropterus and C. orculus , and provide a comparable account of one additional sympatric congener. This increases the number of species in the genus to 23, which represent a considerable component of Mexican plethodontid richness., Competing Interests: Gabriela Parra Olea is an Academic Editor for PeerJ., (© 2020 Parra Olea et al.)

Developmental novelties often underlie the evolutionary origins of key metazoan features. The anuran urostyle, which evolved nearly 200 MYA, is one such structure. It forms as the tail regresses during metamorphosis, when locomotion changes from an axial-driven mode in larvae to a limb-driven one in adult frogs. The urostyle comprises of a coccyx and a hypochord. The coccyx forms by fusion of caudal vertebrae and has evolved repeatedly across vertebrates. However, the contribution of an ossifying hypochord to the coccyx in anurans is unique among vertebrates and remains a developmental enigma. Here, we focus on the developmental changes that lead to the anuran urostyle, with an emphasis on understanding the ossifying hypochord. We find that the coccyx and hypochord have two different developmental histories: First, the development of the coccyx initiates before metamorphic climax whereas the ossifying hypochord undergoes rapid ossification and hypertrophy; second, thyroid hormone directly affects hypochord formation and appears to have a secondary effect on the coccygeal portion of the urostyle. The embryonic hypochord is known to play a significant role in the positioning of the dorsal aorta (DA), but the reason for hypochordal ossification remains obscure. Our results suggest that the ossifying hypochord plays a role in remodeling the DA in the newly forming adult body by partially occluding the DA in the tail. We propose that the ossifying hypochord-induced loss of the tail during metamorphosis has enabled the evolution of the unique anuran bauplan ., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Amphibians form fingers without webbing by differential growth between digital and interdigital regions. Amniotes, however, employ interdigital cell death (ICD), an additional mechanism that contributes to a greater variation of limb shapes. Here, we investigate the role of environmental oxygen in the evolution of ICD in tetrapods. While cell death is restricted to the limb margin in amphibians with aquatic tadpoles, Eleutherodactylus coqui, a frog with terrestrial-direct-developing eggs, has cell death in the interdigital region. Chicken requires sufficient oxygen and reactive oxygen species to induce cell death, with the oxygen tension profile itself being distinct between the limbs of chicken and Xenopus laevis frogs. Notably, increasing blood vessel density in X. laevis limbs, as well as incubating tadpoles under high oxygen levels, induces ICD. We propose that the oxygen available to terrestrial eggs was an ecological feature crucial for the evolution of ICD, made possible by conserved autopod-patterning mechanisms., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Direct development is a reproductive mode in amphibians that has evolved independently from the ancestral biphasic life history in at least a dozen anuran lineages. Most direct-developing frogs, including the Puerto Rican coquí, Eleutherodactylus coqui , lack a free-living aquatic larva and instead hatch from terrestrial eggs as miniature adults. Their embryonic development includes the transient formation of many larval-specific features and the formation of adult-specific features that typically form postembryonically-during metamorphosis-in indirect-developing frogs. We found that pre-hatching developmental patterns of thyroid hormone receptors alpha ( thra ) and beta ( thrb ) and deiodinases type II ( dio2 ) and type III ( dio3 ) mRNAs in E. coqui limb and tail are conserved relative to those seen during metamorphosis in indirect-developing frogs. Additionally, thra, thrb , and dio2 mRNAs are expressed in the limb before formation of the embryonic thyroid gland. Liquid-chromatography mass-spectrometry revealed that maternally derived thyroid hormone is present throughout early embryogenesis, including stages of digit formation that occur prior to the increase in embryonically produced thyroid hormone. Eleutherodactylus coqui embryos take up much less 3,5,3'-triiodothyronine (T 3 ) from the environment compared with X. tropicalis tadpoles. However, E. coqui tissue explants mount robust and direct gene expression responses to exogenous T 3 similar to those seen in metamorphosing species. The presence of key components of the thyroid axis in the limb and the ability of limb tissue to respond to T 3 suggest that thyroid hormone-mediated limb development may begin prior to thyroid gland formation. Thyroid hormone-dependent limb development and tail resorption characteristic of metamorphosis in indirect-developing anurans are evolutionarily conserved, but they occur instead in ovo in E. coqui .

Pseudophilautus comprises an endemic diversification predominantly associated with the wet tropical regions of Sri Lanka that provides an opportunity to examine the effects of geography and historical climate change on diversification. Using a time-calibrated multi-gene phylogeny, we analyze the tempo of diversification in the context of past climate and geography to identify historical drivers of current patterns of diversity and distribution. Molecular dating suggests that the diversification was seeded by migration across a land-bridge connection from India during a period of climatic cooling and drying, the Oi-1 glacial maximum around the Eocene-Oligocene boundary. Lineage-through-time plots suggest a gradual and constant rate of diversification, beginning in the Oligocene and extending through the late Miocene and early Pliocene with a slight burst in the Pleistocene. There is no indication of an early-burst phase of diversification characteristic of many adaptive radiations, nor were there bursts of diversification associated with favorable climate shifts such as the intensification of monsoons. However, a late Miocene (8.8 MYA) back-migration to India occurred following the establishment of the monsoon. The back migration did not trigger a diversification in India similar to that manifest in Sri Lanka, likely due to occupation of available habitat, and consequent lack of ecological opportunity, by the earlier radiation of a sister lineage of frogs (Raorchestes) with similar ecology. Phylogenetic area reconstructions show a pattern of sister species distributed across adjacent mountain ranges or from different parts of large montane regions, highlighting the importance of isolation and allopatric speciation. Hence, local species communities are composed of species from disparate clades that, in most cases, have been assembled through migration rather than in situ speciation. Lowland lineages are derived from montane lineages. Thus, the hills of Sri Lanka acted as species pumps as well as refuges throughout the 31 million years of evolution, highlighting the importance of tropical montane regions for both the generation and maintenance of biodiversity., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Numerous physiological and morphological adaptations were achieved during the transition to lungless respiration that accompanied evolutionary lung loss in plethodontid salamanders, including those that enable efficient gas exchange across extrapulmonary tissue. However, the molecular basis of these adaptations is unknown. Here, we show that lungless salamanders express in the larval integument and the adult buccopharynx-principal sites of respiratory gas exchange in these species-a novel paralogue of the gene surfactant-associated protein C ( SFTPC ), which is a critical component of pulmonary surfactant expressed exclusively in the lung in other vertebrates. The paralogous gene appears to be found only in salamanders, but, similar to SFTPC , in lunged salamanders it is expressed only in the lung. This heterotopic gene expression, combined with predictions from structural modelling and respiratory tissue ultrastructure, suggests that lungless salamanders may produce pulmonary surfactant-like secretions outside the lungs and that the novel paralogue of SFTPC might facilitate extrapulmonary respiration in the absence of lungs. Heterotopic expression of the SFTPC paralogue may have contributed to the remarkable evolutionary radiation of lungless salamanders, which account for more than two thirds of urodele species alive today., (© 2018 The Authors.)

Direct-developing amphibians form limbs during early embryonic stages, as opposed to the later, often postembryonic limb formation of metamorphosing species. Limb patterning is dramatically altered in direct-developing frogs, but little attention has been given to direct-developing salamanders. We use expression patterns of two genes, sox9 and col2a1, to assess skeletal patterning during embryonic limb development in the direct-developing salamander Plethodon cinereus. Limb patterning in P. cinereus partially resembles that described in other urodele species, with early formation of digit II and a generally anterior-to-posterior formation of preaxial digits. Unlike other salamanders described to date, differentiation of preaxial zeugopodial cartilages (radius/tibia) is not accelerated in relation to the postaxial cartilages, and there is no early differentiation of autopodial elements in relation to more proximal cartilages. Instead, digit II forms in continuity with the ulnar/fibular arch. This amniote-like connectivity to the first digit that forms may be a consequence of the embryonic formation of limbs in this direct-developing species. Additionally, and contrary to recent models of amphibian digit identity, there is no evidence of vestigial digits. This is the first account of gene expression in a plethodontid salamander and only the second published account of embryonic limb patterning in a direct-developing salamander species., (© 2018 The Authors. Evolution and Development Published by Wiley Periodicals, Inc.)

Nearly two thirds of the approximately 700 species of living salamanders are lungless. These species respire entirely through the skin and buccopharyngeal mucosa. Lung loss dramatically impacts the configuration of the circulatory system but the effects of evolutionary lung loss on cardiac morphology have long been controversial. For example, there is presumably little need for an atrial septum in lungless salamanders due to the absence of pulmonary veins and the presence of a single source of mixed blood flowing into the heart, but whether lungless salamanders possess an atrial septum and whether the sinoatrial aperture is located in the left or right atrium are unresolved; authors have stated opposing claims since the late 1800s. Here, we use micro-computed tomography (μ-CT) imaging, gross dissection and histological reconstruction to compare cardiac morphology among lungless plethodontid salamanders (Plethodontidae), salamanders with lungs, and the convergently lungless species Onychodactylus japonicus (Hynobiidae). Plethodontid salamanders have partial atrial septa and incomplete separation of the atrium into left and right halves. Partial septation is also seen in O. japonicus. Hence, lungless salamanders from two lineages convergently evolved similar morphology of the atrial septum. The partial septum in lungless salamanders can make it appear that the sinoatrial aperture is in the left atrium, but this interpretation is incorrect. Outgroup comparisons demonstrate that the aperture is located in a posterodorsal extension of the right atrium into the left side of the heart. Independent evolutionary losses of the atrial septum may have a similar developmental basis. In mammals, the lungs induce formation of the atrial septum by secreting morphogens to neighboring mesenchyme. We hypothesize that the lungs induce atrial septum development in amphibians in a similar fashion to mammals, and that atrial septum reduction in lungless salamanders is a direct result of lunglessness., (© 2016 The Authors. Journal of Anatomy published by John Wiley & Sons Ltd on behalf of Anatomical Society.)

We describe three new species of minute salamanders, genus Thorius , from the Sierra Madre del Sur of Oaxaca, Mexico. Until now only a single species, T. minutissimus , has been reported from this region, although molecular data have long shown extensive genetic differentiation among geographically disjunct populations. Adult Thorius pinicola sp. nov., T. longicaudus sp. nov., and T. tlaxiacus sp. nov. are larger than T. minutissimus and possess elliptical rather than oval nostrils; T. pinicola and T. longicaudus also have longer tails. All three new species occur west of the range of T. minutissimus , which has the easternmost distribution of any member of the genus. The new species are distinguished from each other and from other named Thorius in Oaxaca by a combination of adult body size, external morphology and osteology, and by protein characters (allozymes) and differences in DNA sequences. In addition, we redescribe T. minutissimus and a related species, T. narisovalis , to further clarify the taxonomic status of Oaxacan populations and to facilitate future studies of the remaining genetically differentiated Thorius that cannot be satisfactorily assigned to any named species. Populations of all five species considered here appear to have declined dramatically over the last one or two decades and live specimens are difficult to find in nature. Thorius may be the most endangered genus of amphibians in the world. All species may go extinct before the end of this century., Competing Interests: The authors declare that they have no competing interests.

Bones of the cranial vault appear to be highly conserved among tetrapod vertebrates. Moreover, bones identified with the same name are assumed to be evolutionarily homologous. However, recent developmental studies reveal a key difference in the embryonic origin of cranial vault bones between representatives of two amniote lineages, mammals and birds, thereby challenging this view. In the mouse, the frontal is derived from cranial neural crest (CNC) but the parietal is derived from mesoderm, placing the CNC-mesoderm boundary at the suture between these bones. In the chicken, this boundary is located within the frontal. This difference and related data have led several recent authors to suggest that bones of the avian cranial vault are misidentified and should be renamed. To elucidate this apparent conflict, we fate-mapped CNC and mesoderm in axolotl to reveal the contributions of these two embryonic cell populations to the cranial vault in a urodele amphibian. The CNC-mesoderm boundary in axolotl is located between the frontal and parietal bones, as in the mouse but unlike the chicken. If, however, the avian frontal is regarded instead as a fused frontal and parietal (i.e. frontoparietal) and the parietal as a postparietal, then the cranial vault of birds becomes developmentally and topologically congruent with those of urodeles and mammals. This alternative hypothesis of cranial vault homology is also phylogenetically consistent with data from the tetrapod fossil record, where frontal, parietal and postparietal bones are present in stem lineages of all extant taxa, including birds. It further implies that a postparietal may be present in most non-avian archosaurs, but fused to the parietal or supraoccipital as in many extant mammals.

Vertebrate neck musculature spans the transition zone between head and trunk. The extent to which the cucullaris muscle is a cranial muscle allied with the gill levators of anamniotes or is instead a trunk muscle is an ongoing debate. Novel computed tomography datasets reveal broad conservation of the cucullaris in gnathostomes, including coelacanth and caecilian, two sarcopterygians previously thought to lack it. In chicken, lateral plate mesoderm (LPM) adjacent to occipital somites is a recently identified embryonic source of cervical musculature. We fate-map this mesoderm in the axolotl (Ambystoma mexicanum), which retains external gills, and demonstrate its contribution to posterior gill-levator muscles and the cucullaris. Accordingly, LPM adjacent to the occipital somites should be regarded as posterior cranial mesoderm. The axial position of the head-trunk border in axolotl is congruent between LPM and somitic mesoderm, unlike in chicken and possibly other amniotes.