Identity of the holotype and type locality of Rhabdophis leonardi (Wall, 1923) (Colubridae: Natricinae), with notes on the morphology and natural history of the species in southwestern China.

The original description of Natrix leonardi (currently Rhabdophis leonardi) by Frank Wall in 1923, based on a specimen from the "Upper Burma Hills," lacked important morphological details that have complicated the assignment of recently collected material. Furthermore, although the holotype was never lost, its location has been misreported in one important taxonomic reference, leading to further confusion. We report the correct repository of the holotype (Natural History Museum, London), together with its current catalog number. We also describe key features of that specimen that were omitted from the original description, and provide new details on the morphology of the species, including sexual dichromatism unusual for the genus, based upon specimens from southern Sichuan, China. Rhabdophis leonardi is distinguished from its congeners by the following characters: 15 or 17 DSR at midbody and 6 supralabials; distinct annulus around the neck, broad and red in males, and narrow and orange with a black border in females; dorsal ground color light green or olive; some lateral and dorsal scales possessing black edges, the frequency of black edges gradually increasing from anterior to posterior, forming irregular and ill-defined transverse black bands; eye with prominent green iris; black ventral spots with a red edge, most numerous at midbody but extending halfway down the length of the tail. In southwestern China, this species is frequently found at 1730-2230 m elevation. It has been documented to prey upon anuran amphibians, including toads. A recently published phylogenetic analysis showed this species to be deeply nested with the genus Rhabdophis, as a member of the R. nuchalis Group. That analysis also revealed the existence of two closely related but geographically distinct subclades in the molecular analysis, one of which may represent an unnamed taxon.

Cryptic diversity and phylogeography of the Rhabdophis nuchalis group (Squamata: Colubridae).

Previous studies, have found that the rapid uplift of the Tibetan plateau accelerated the diversification of species. However, there are few relevant biogeographic data for the Colubridae in this region. We conducted a comprehensive study of the Rhabdophis nuchalis Group, which presently contains four nominal species, R. nuchalis, R. pentasupralabialis, R. leonardi, and R. chiwen. Building upon previous studies with specimens we have recently examined, greater interspecific and intraspecific diversity has been revealed. Here we address three questions: (1) Do the intraspecific differences represent only geographic variation within lineages, or are there cryptic species? (2) What are the interspecific relationships among members of the R. nuchalis Group? (3) What has been the biogeographic history of this species group? To resolve these questions we used four mitochondrial gene sequences and one nuclear sequence to investigate the molecular phylogenetic and geographic relationships among populations. Our molecular analysis reveals cryptic species diversity within the R. nuchalis Group, and seven clades were identified in the analysis. Ancestral area estimation suggests that the R. nuchalis Group originated in the Hengduan Mountains approximately 6.24 Mya and expanded its range northward to the Qinling-Daba Mountains. The Sichuan Basin appears to have been a barrier to migration. Species divergence seems to have been related to the rapid uplift of the Qinghai-Tibet Plateau.

Physiology labs during a pandemic: What did we learn?

This article captures a collective reflection on the successes and challenges we experienced when teaching physiology laboratories online during the COVID-19 pandemic. Physiology instructors from six institutions discussed their own efforts to redesign meaningful physiology laboratories that could be taught remotely, as the nation scrambled to respond to the sudden shift out of the classroom. Despite the complexity of this task, clear themes emerged as our courses transitioned to an online format in spring 2020 and were solidified in the fall of 2020. This article reflects on the history, features, benefits, and challenges of current laboratory teaching when applying a scientific teaching approach to facilitate the redesign process. We believe online networks like ours can facilitate information sharing, promote innovations, and provide support for instructors. The insights we gained through this collaboration will influence our thinking about the future of the physiology lab, whether online or in person.

New Insights Into Dietary Toxin Metabolism: Diversity in the Ability of the Natricine Snake Rhabdophis tigrinus to Convert Toad-Derived Bufadienolides.

The Japanese natricine snake Rhabdophis tigrinus sequesters cardiotonic steroids, bufadienolides (BDs), from ingested toads in the nuchal glands as defensive toxins. A previous study showed that R. tigrinus in captivity converts dietary BDs when it sequesters them. However, it is unknown whether the dietary BDs are actually converted and the modified products accumulated under natural conditions. It is also unknown to what extent the BD profile of ingested toads is reflected in that of the snake. We collected 123 snakes from throughout Japan, analyzed their BD profiles by liquid chromatography/mass spectrometry, and identified 15 BDs from R. tigrinus by nuclear magnetic resonance analyses. We also compared their BD profiles using hierarchical cluster analysis (HCA). HCA exhibited two main clusters associated with their collection locations: eastern and western regions of the Japanese main islands. These results, coupled with previous findings on the BDs of Japanese toads, suggest that 1) R. tigrinus converts toad-derived BDs into other compounds under natural conditions; 2) there are both universal and regionally-specific conversions of dietary BDs by R. tigrinus; and 3) geographic variation in toad BD profiles is partially reflected in the variation of snake BD profiles.

Differences in morphology and in composition and release of parotoid gland secretion in introduced cane toads (Rhinella marina) from established populations in Florida, USA.

Cane toads are highly toxic bufonids invasive in several locations throughout the world. Although physiological changes and effects on native predators for Australian populations have been well documented, Florida populations have received little attention. Cane toads were collected from populations spanning the invaded range in Florida to assess relative toxicity, through measuring morphological changes to parotoid glands, likelihood of secretion, and the marinobufagenin (MBG) content of secretion. We found that residual body indices increased in individuals from higher latitude populations, and relative parotoid gland size increased with increasing toad size. There was no effect of latitude on the allometric relationship between gland size and toad size. We observed an increase in likelihood of secretion by cane toads in the field with increasing latitude. Individuals from southern and northern populations did not vary significantly in the quantity of MBG contained in their secretion. Laboratory-acclimated cane toads receiving injections of epinephrine were more likely to secrete poison with increasing dose, although there was no difference in likelihood of secretion between southern and northern populations. This suggests that differences between populations in the quantities of epinephrine released in the field, due to altered hypothalamic sensitivity upon disturbance, may be responsible for the latitudinal effects on poison secretion. Our results suggest that altered pressures from northward establishment in Florida have affected sympathetic sensitivity and defensive mechanisms of cane toads, potentially affecting risk to native predators.

Variation in Bufadienolide Composition of Parotoid Gland Secretion From Three Taxa of Japanese Toads.

Toads of the genus Bufo synthesize and accumulate bufadienolides (BDs) in their parotoid glands. BDs are cardiotonic steroids that play an important role in defense against the toads' predators. Three bufonid taxa occur in mainland Japan, Bufo japonicus formosus, B. j. japonicus, and B. torrenticola. The chemical structures of BDs isolated from B. j. formosus were studied several decades ago, but there is no further information on the toxic components of Japanese toads and their metabolism. In this study, we analyzed BDs of toads from throughout Japan and compared the BD profiles by liquid chromatography/mass spectrometry (LC/MS) and hierarchical cluster analysis (HCA). We observed BDs in three taxa of Japanese toads, and identified five of the most common BDs by nuclear magnetic resonance (NMR) analyses. Of the five BDs, only bufalin was detected in all individuals. HCA of individual BD profiles divided the three taxa into five primary clusters and several subclusters. This result indicates that BD profiles differ both among and within the taxa. The clustering pattern of BDs is generally concordant with a phylogenetic tree reconstructed from the mitochondrial cytochrome b gene of Japanese toads. Our results suggest that the BDs of Japanese toads have diversified not in response to specific selective pressures, but simply due to population structuring over evolutionary time.

Dramatic dietary shift maintains sequestered toxins in chemically defended snakes.

Unlike other snakes, most species of Rhabdophis possess glands in their dorsal skin, sometimes limited to the neck, known as nucho-dorsal and nuchal glands, respectively. Those glands contain powerful cardiotonic steroids known as bufadienolides, which can be deployed as a defense against predators. Bufadienolides otherwise occur only in toads (Bufonidae) and some fireflies (Lampyrinae), which are known or believed to synthesize the toxins. The ancestral diet of Rhabdophis consists of anuran amphibians, and we have shown previously that the bufadienolide toxins of frog-eating species are sequestered from toads consumed as prey. However, one derived clade, the Rhabdophis nuchalis Group, has shifted its primary diet from frogs to earthworms. Here we confirm that the worm-eating snakes possess bufadienolides in their nucho-dorsal glands, although the worms themselves lack such toxins. In addition, we show that the bufadienolides of R. nuchalis Group species are obtained primarily from fireflies. Although few snakes feed on insects, we document through feeding experiments, chemosensory preference tests, and gut contents that lampyrine firefly larvae are regularly consumed by these snakes. Furthermore, members of the R. nuchalis Group contain compounds that resemble the distinctive bufadienolides of fireflies, but not those of toads, in stereochemistry, glycosylation, acetylation, and molecular weight. Thus, the evolutionary shift in primary prey among members of the R. nuchalis Group has been accompanied by a dramatic shift in the source of the species' sequestered defensive toxins.

Evolution of nuchal glands, unusual defensive organs of Asian natricine snakes (Serpentes: Colubridae), inferred from a molecular phylogeny.

A large body of evidence indicates that evolutionary innovations of novel organs have facilitated the subsequent diversification of species. Investigation of the evolutionary history of such organs should provide important clues for understanding the basis for species diversification. An Asian natricine snake, Rhabdophis tigrinus, possesses a series of unusual organs, called nuchal glands, which contain cardiotonic steroid toxins known as bufadienolides. Rhabdophis tigrinus sequesters bufadienolides from its toad prey and stores them in the nuchal glands as a defensive mechanism. Among more than 3,500 species of snakes, only 17 Asian natricine species are known to possess nuchal glands or their homologues. These 17 species belong to three nominal genera, Balanophis, Macropisthodon, and Rhabdophis. In Macropisthodon and Rhabdophis, however, species without nuchal glands also exist. To infer the evolutionary history of the nuchal glands, we investigated the molecular phylogenetic relationships among Asian natricine species with and without nuchal glands, based on variations in partial sequences of Mt-CYB, Cmos, and RAG1 (total 2,767 bp). Results show that all species with nuchal glands belong to a single clade (NGC). Therefore, we infer that the common ancestor of this clade possessed nuchal glands with no independent origins of the glands within the members. Our results also imply that some species have secondarily lost the glands. Given the estimated divergence time of related species, the ancestor of the nuchal gland clade emerged 19.18 mya. Our study shows that nuchal glands are fruitful subjects for exploring the evolution of novel organs. In addition, our analysis indicates that reevaluation of the taxonomic status of the genera Balanophis and Macropisthodon is required. We propose to assign all species belonging to the NGC to the genus Rhabdophis, pending further study.

Snakes exhibit tissue-specific variation in cardiotonic steroid sensitivity of Na+/K+-ATPase.

Toads are among several groups of organisms chemically defended with lethal concentrations of cardiotonic steroids. As a result, most predators that prey on amphibians avoid toads. However, several species of snakes have gained resistance-conferring mutations of Na+/K+-ATPase, the molecular target of cardiotonic steroids, and can feed on toads readily. Despite recent advances in our understanding of this adaptation at the genetic level, we have lacked functional evidence for how mutations of Na+/K+-ATPase account for cardiotonic steroid resistance in snake tissues. To address this issue, it is necessary to determine how the Na+/K+-ATPases of snakes react to the toxins. Some tissues might have Na+/K+-ATPases that are more susceptible than others and can thus provide clues about how the toxins influence organismal function. Here we provide a mechanistic link between observed Na+/K+-ATPase substitutions and observed resistance using actual snake Na+/K+-ATPases. We used an in vitro approach to determine the tissue-specific levels of sensitivity to cardiotonic steroids in select resistant and non-resistant snakes. We compared the sensitivities of select tissues within and between species. Our results suggest that resistant snakes contain highly resistant Na+/K+-ATPases in their heart and kidney, both of which rely heavily on the enzymes to function, whereas tissues that do not rely as heavily on Na+/K+-ATPases or might be protected from cardiotonic steroids by other means (liver, gut, and brain) contain non-resistant forms of the enzyme. This study reveals functional evidence that tissue-level target-site insensitivity to cardiotonic steroids varies not only among species but also across tissues within resistant taxa.

Toad toxin-resistant snake (Thamnophis elegans) expresses high levels of mutant Na+/K+-ATPase mRNA in cardiac muscle.

Toads are chemically defended by bufadienolides, which are lethal to most predators. These toxins exert their lethal effects by binding to and disabling the Na+/K+-ATPases of cell membranes. Many species of snakes exhibit resistance to the effects of bufadienolides due to target-site insensitivity of the Na+/K+-ATPase. Mutations that confer resistance have previously been identified in ATP1a3, the gene that codes for the Na+/K+-ATPase α-3 paralog. We have found that this mutant gene is expressed at a significantly elevated level in heart tissue compared to gut, kidney, and liver of the bufadienolide-resistant snake, Thamnophis elegans. Furthermore, we found that exposure to bufadienolides elicits a significant increase in the expression levels of ATP1a3 in the heart, but not in the kidneys, liver, or gut 1h after exposure. We suggest that upregulation of ATP1a3 in the heart plays an important role in the physiological processes involved in tolerance of bufadienolides among genetically resistant snakes.

Corticosteroid responses of snakes to toxins from toads (bufadienolides) and plants (cardenolides) reflect differences in dietary specializations.

Toads are chemically defended by cardiotonic steroids known as bufadienolides. Resistance to the acute effects of bufadienolides in snakes that prey on toads is conferred by target-site insensitivity of the toxin's target enzyme, the Na+/K+-ATPase. Previous studies have focused largely on the molecular mechanisms of resistance but have not investigated the physiological mechanisms or consequences of exposure to the toxins. Adrenal enlargement in snakes often is associated with specialization on a diet of toads. These endocrine glands are partly composed of interrenal tissue, which produces the corticosteroids corticosterone and aldosterone. Corticosterone is the main hormone released in response to stress in reptiles, and aldosterone plays an important role in maintaining ion balance through upregulation of Na+/K+-ATPase. We tested the endocrine response of select species of snakes to acute cardiotonic steroid exposure by measuring circulating aldosterone and corticosterone concentrations. We found that Rhabdophis tigrinus, which specializes on a diet of toads, responds with lower corticosterone and higher aldosterone compared to other species that exhibit target-site resistance to the toxins but do not specialize on toads. We also found differences between sexes in R. tigrinus, with males generally responding with higher corticosterone and aldosterone than females. This study provides evidence of physiological adaptations, beyond target-site resistance, associated with tolerance of bufadienolides in a specialized toad-eating snake.

Toxin-resistant isoforms of Na+/K+-ATPase in snakes do not closely track dietary specialization on toads.

Toads are chemically defended by bufadienolides, a class of cardiotonic steroids that exert toxic effects by binding to and disabling the Na+/K+-ATPases of cell membranes. Some predators, including a number of snakes, have evolved resistance to the toxic effects of bufadienolides and prey regularly on toads. Resistance in snakes to the acute effects of these toxins is conferred by at least two amino acid substitutions in the cardiotonic steroid binding pocket of the Na+/K+-ATPase. We surveyed 100 species of snakes from a broad phylogenetic range for the presence or absence of resistance-conferring mutations. We found that such mutations occur in a much wider range of taxa than previously believed. Although all sequenced species known to consume toads exhibited the resistance mutations, many of the species possessing the mutations do not feed on toads, much less specialize on that food source. This suggests that either there is little performance cost associated with these mutations or they provide an unknown benefit. Furthermore, the distribution of the mutation among major clades of advanced snakes suggests that the origin of the mutation reflects evolutionary retention more than dietary constraint.

Mutations to the cardiotonic steroid binding site of Na(+)/K(+)-ATPase are associated with high level of resistance to gamabufotalin in a natricine snake.

Although toads are defended by bufadienolide toxins, some snakes have evolved resistance to bufadienolides and feed heavily on toads. We compared resistance in Nerodia rhombifer, which possesses mutations that confer target-site resistance, to Pituophis catenifer, which lacks those mutations. Even at the highest dosage tested, Nerodia showed no effects, whereas the lowest dose was lethal to Pituophis. Our results demonstrate a striking level of resistance to bufadienolides in a species possessing the mutations for resistance.

Sequestered defensive toxins in tetrapod vertebrates: principles, patterns, and prospects for future studies.

Chemical defenses are widespread among animals, and the compounds involved may be either synthesized from nontoxic precursors or sequestered from an environmental source. Defensive sequestration has been studied extensively among invertebrates, but relatively few examples have been documented among vertebrates. Nonetheless, the number of described cases of defensive sequestration in tetrapod vertebrates has increased recently and includes diverse lineages of amphibians and reptiles (including birds). The best-known examples involve poison frogs, but other examples include natricine snakes that sequester toxins from amphibians and two genera of insectivorous birds. Commonalities among these diverse taxa include the combination of consuming toxic prey and exhibiting some form of passive defense, such as aposematism, mimicry, or presumptive death-feigning. Some species exhibit passive sequestration, in which dietary toxins simply require an extended period of time to clear from the tissues, whereas other taxa exhibit morphological or physiological specializations that enhance the uptake, storage, and/or delivery of exogenous toxins. It remains uncertain whether any sequestered toxins of tetrapods bioaccumulate across multiple trophic levels, but multitrophic accumulation seems especially likely in cases involving consumption of phytophagous or mycophagous invertebrates and perhaps consumption of poison frogs by snakes. We predict that additional examples of defensive toxin sequestration in amphibians and reptiles will be revealed by collaborations between field biologists and natural product chemists. Candidates for future investigation include specialized predators on mites, social insects, slugs, and toxic amphibians. Comprehensive studies of the ecological, evolutionary, behavioral, and regulatory aspects of sequestration will require teams of ecologists, systematists, ethologists, physiologists, molecular biologists, and chemists. The widespread occurrence of sequestered defenses has important implications for the ecology, evolution, and conservation of amphibians and reptiles.

Tail morphology in the Western Diamond-backed rattlesnake, Crotalus atrox.

The shaker muscles in the tails of rattlesnakes are used to shake the rattle at very high frequencies. These muscles are physiologically specialized for sustaining high-frequency contractions. The tail skeleton is modified to support the enlarged shaker muscles, and the muscles have major anatomical modifications when compared with the trunk muscles and with the tail muscles of colubrid snakes. The shaker muscles have been known for many years to consist of three large groups of muscles on each side of the tail. However, the identities of these muscles and their serial homologies with the trunk muscles were not previously known. In this study, we used dissection and magnetic resonance imaging of the tail in the Western Diamond-backed Rattlesnake, Crotalus atrox, to determine that the three largest muscles that shake the rattle are the M. longissimus dorsi, the M. iliocostalis, and the M. supracostalis lateralis. The architecture of these muscles differs from their serial homologs in the trunk. In addition, the rattlesnake tail also contains three small muscles. The M. semispinalis-spinalis occurs in the tail, where it is a thin, nonvibratory, postural muscle that extends laterally along the neural spines. An additional muscle, which derives from fusion of the M. interarticularis inferior and M. levator costae, shares segmental insertions with the M. longissimus dorsi and M. iliocostalis. Several small, deep ventral muscles probably represent the Mm. costovertebrocostalis, intercostalis series, and transversohypapophyseus. The architectural rearrangements in the tail skeleton and shaker muscles, compared with the trunk muscles, probably relate to their roles in stabilizing the muscular part of the tail and to shaking the rattle at the tip of the tail. Based on comparisons with the tail muscles of a colubrid snake described in the literature, the derived tail muscle anatomy in rattlesnakes evolved either in the pitvipers or within the rattlesnakes. J. Morphol., 2008. (c) 2008 Wiley-Liss, Inc.

Dietary sequestration of defensive steroids in nuchal glands of the Asian snake Rhabdophis tigrinus.

The Asian snake Rhabdophis tigrinus possesses specialized defensive glands on its neck that contain steroidal toxins known as bufadienolides. We hypothesized that R. tigrinus does not synthesize these defensive steroids but instead sequesters the toxins from toads it consumes as prey. To test this hypothesis, we conducted chemical analyses on the glandular fluid from snakes collected in toad-free and toad-present localities. We also performed feeding experiments in which hatchling R. tigrinus were reared on controlled diets that either included or lacked toads. We demonstrate that the cardiotonic steroids in the nuchal glands of R. tigrinus are obtained from dietary toads. We further show that mothers containing high levels of bufadienolides can provision their offspring with toxins. Hatchlings had bufadienolides in their nuchal glands only if they were fed toads or were born to a dam with high concentrations of these compounds. Because geographic patterns in the availability of toxic prey are reflected in the chemical composition of the glandular fluid, snakes in toad-free regions are left undefended by steroidal toxins. Our findings confirm that the sequestration of dietary toxins underlies geographic variation in antipredatory behavior in this species and provide a unique example of sequestered defensive compounds in a specialized vertebrate structure.

Mechanical properties of the integument of the common gartersnake, Thamnophis sirtalis (Serpentes: Colubridae).

The evolution of the ophidian feeding mechanism has involved substantial morphological restructuring associated with the ability to ingest relatively large prey. Previous studies examining the morphological consequences of macrophagy have concentrated on modifications of the skull and cephalic musculature. Although it is evident that macrophagy requires highly compliant skin, the mechanical properties of the ophidian integument have received limited attention, particularly in the context of feeding. We examined mechanical properties of skin along the body axis in Thamnophis sirtalis (Colubridae). Data were collected from tensile tests and were analyzed using a multivariate analysis of variance (MANOVA) and post-hoc multiple comparison tests. Significant differences in mechanical properties were detected among regions of the body. In general, prepyloric skin is more compliant than postpyloric skin, consistent with the demands of macrophagy.

Vasculature of the parotoid glands of four species of toads (bufonidae: bufo).

The parotoid glands of toads (Bufonidae) consist of large aggregations of granular glands located between the otic region of the skull and the scapular region. To determine the circulatory pattern of these glands, we perfused the vascular systems of Bufo alvarius, B. marinus, B. terrestris, and B. valliceps with either India ink or Microfil, a fine latex. The perfused glands were studied by gross dissection, microscopic examination, and histology. The vascular patterns of the parotoid glands were compared to the arrangement of vessels in the dorsal skin of Rana sphenocephala (Ranidae), a frog that lacks parotoid glands. The parotoid glands of the four species of toads are supplied with blood by the lateral and dorsal cutaneous arteries and are drained by one or more branches of the internal jugular vein. The dorsal cutaneous artery supplies most of the blood to the parotoid glands in B. terrestris and B. valliceps. In B. alvarius and B. marinus, both the lateral and dorsal cutaneous arteries serve major roles in the blood supply of the glands. These patterns of blood flow have not been described previously for parotoid glands and conflict with earlier accounts for B. alvarius and B. marinus. The arteries and veins associated with the parotoid glands of toads are present in R. sphenocephala, but are arranged differently. In R. sphenocephala, the lateral cutaneous artery supplies the dorsal and lateral skin posterior to the shoulder region, whereas the dorsal cutaneous artery supplies the skin of the shoulder region. In toads, both the lateral and dorsal cutaneous arteries supply the skin of the shoulder region and ramify into subcutaneous capillaries that surround the secretory units of the parotoid glands. Extensive vasculature presumably is important for delivering cholesterol and other precursor molecules to the parotoid glands, where those compounds are converted into toxins.