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Buergeria japonica (Rhacophoridae) thrives in diverse habitats, from montane forests to the mouths of small rivers opening onto sandy beaches. The osmoregulatory system of B. japonica may differ between populations living in these contrasting environments. In this study, we compared the activity of cutaneous Na+/K+-ATPase in two populations of B. japonica from different habitats (montane vs. coastal). Our results reveal a significant difference between the Na+/K+-ATPase activity of the two populations of B. japonica, with the coastal population having higher Na+/K+-ATPase activity. Because the Na+/K+-ATPase plays a crucial role in osmoregulation in high salinity, these results are consistent with the higher salinity tolerance of the coastal population. It is not known whether the difference in Na+/K+-ATPase activity between populations is a result of local adaptation or phenotypic plasticity. More detailed work will be needed to disentangle the patterns of differentiation in physiological responses to salt in these amphibians.

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.

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.

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.

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.

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.

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.

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.

During a 17-yr telemetry study, we examined the diet and ambush behavior of a population of Crotalus horridus in southeastern Virginia. Forty dietary items were identified from 37 fecal samples. We documented 722 instances of snakes in an ambush posture, 61% of which were in a vertical-tree posture, as if hunting arboreal prey at the base of a tree. The most common prey items were Eastern Gray Squirrels (Sciurus carolinensis), which accounted for 45% of all dietary items and represented an estimated 78% of total biomass consumed by C. horridus. Prey was not consumed in proportion to availability, based on small mammal surveys. Our analysis provides indirect evidence that the vertical-tree foraging behavior is adopted to target arboreal Eastern Gray Squirrels. Further, we provide support for the hypothesis that C. horridus alters ambush behavior to forage selectively for specific prey types.

We report the presence of nucho-dorsal glands in Rhabdophis guangdongensis. This recently described species is the 18th member of the genus known to possess integumentary defensive glands. Nine to ten pairs of nuchal glands, separated by a diastema from 116–118 pairs of dorsal glands were observed in two specimens. Because the species was previously known only from the female holotype, we also report data on the size and scalation of five additional specimens and on the morphology of the hemipenis.

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.

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.

The distribution and genetic structure of wildlife populations may be impacted by landscape features and anthropogenic factors. The role of these factors in the genetic relationships among Timber Rattlesnakes (Crotalus horridus) from 21 locations in Pennsylvania, New Jersey, and Virginia was investigated using microsatellite genetic markers. Evidence for null alleles was found in 8 of the 21 populations studied, with frequencies ranging from 0.018 to 0.185. However, the presence of null alleles appeared to have no effect on the interpretation of the patterns of genetic relationships among the populations studied. The analyses showed that the overall extent of genetic differentiation was consistent with isolation by distance; however, a substantial amount of among-groups variation could not be accounted for by geographic distance alone. Bayesian analysis (STRUCTURE), genetic distance analysis, and factorial correspondence analysis (FCA) all indicated that the sampled populations represented three genetic g...

Despite large-scale population decline and geographic range contraction, Timber Rattlesnakes (Crotalus horridus) continue to occupy sites across much of eastern North America, including a ...

Toads are defended chemically by bufadienolides, a class of cardiotonic steroids lethal to most predators. Bufadienolides bind to Na+,K+-ATPases, inhibiting the ability of those cellular pumps to transport ions. In cardiocytes, this inhibition causes arrhythmia and increased contractile strength, which, if prolonged, lead to death. However, several snakes are resistant to bufadienolides and consume toads with no apparent ill effects. Adrenal glands produce hormones that function in the maintenance of Na+,K+-ATPases, and may therefore play an important role in countering the negative effects of bufadienolides. We hypothesized that bufophagous (toad-eating) snakes have enlarged adrenal glands that contribute to the snakes' resistance to bufadienolides, and that sexual dimorphism in adrenal gland size is a general characteristic of bufophagous snakes. We compared phylogenetically independent pairs of taxa to investigate differences in adrenal morphology between bufophagous and nonbufophagous species. We also compared adrenal masses between males and females of bufophagous and nonbufophagous species to test whether sexual dimorphism in adrenal size reported for one species of bufophagous snakes represents a more widespread phenomenon. Our results demonstrate for the first time that the allometric relationship between adrenal mass and body size is significantly different between several bufophagous species and related nonbufophagous species; adrenal size differs between males and females in those bufophagous species, with males having larger adrenal glands, but no such dimorphism exists in related nonbufophagous species. These results demonstrate that parallel morphological responses have occurred repeatedly in bufophagous snakes and suggest that the adrenal glands play a role in mediating the negative effects of bufadienolide ingestion in bufophagous snakes.

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

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.

Species that sequester toxins from prey for their own defense against predators may exhibit population-level variation in their chemical arsenal that reflects the availability of chemically defended prey in their habitat. Rhabdophis tigrinus is an Asian snake that possesses defensive glands in the skin of its neck (‘nuchal glands’), which typically contain toxic bufadienolide steroids that the snakes sequester from consumed toads. In this study, we compared the chemistry of the nuchal gland fluid of R. tigrinus from toad-rich and toad-free islands in Japan and determined the effect of diet on the nuchal gland constituents. Our findings demonstrate that captive-hatched juveniles from toad-rich Ishima Island that had not been fed toads possess defensive bufadienolides in their nuchal glands, presumably due to maternal provisioning of these sequestered compounds. Wild-caught juveniles from Ishima possess large quantities of bufadienolides, which could result from a combination of maternal provisioning and sequestration of these defensive compounds from consumed toads. Interestingly, juvenile females from Ishima possess larger quantities of bufadienolides than do juvenile males, whereas a small sample of field-collected snakes suggests that adult males contain larger quantities of bufadienolides than do adult females. Captive-born hatchlings from Kinkasan Island lack bufadienolides in their nuchal glands, reflecting the absence of toads on that island, but they can sequester bufadienolides by feeding on toads (Bufo japonicus) in captivity. The presence of large quantities of bufadienolides in the nuchal glands of R. tigrinus from Ishima may reduce the risk of predation by providing an effective chemical defense, whereas snakes on Kinkasan may experience increased predation due to the lack of defensive compounds in their nuchal glands.

We describe the diet composition of six species of dipsadines (Sibon longifrenis, S. argus, S. annulatus, S. nebulatus, Dipsas nicholsi, Dipsas sp.) from two montane forest sites in central Panama. Between 2005 and 2007 we captured 473 individual snakes, of which 305 contained no prey and two contained gut contents. Of the 166 fecal samples collected, 41 had no identifiable prey and 125 contained identifiable prey. We analyzed fecal samples and found that dipsadines at our study sites feed on mollusks, annelids, and amphibian eggs. None of the six species examined feed exclusively on mollusks, although most do feed on a variety of soft-bodied prey. Dipsas feeds extensively on earthworms, S. annulatus feeds on mollusks and earthworms, and S. longifrenis and S. argus feed heavily on amphibian eggs when available.

Of the various chemical defensive adaptations of vertebrates, nuchal glands are among the most unusual. First described in a Japanese natricine snake, Rhabdophis tigrinus, in 1935, these organs are embedded under the skin of the neck region as a series of paired glands that have neither lumina nor ducts. The major chemical components of the glandular fluid are bufadienolides, which are cardiotonic steroids also found in the skin secretion of toads. Here we review early studies of nuchal glands and briefly introduce our recent findings on the sequestration of bufadienolides from consumed toads and the maternal provisioning of those sequestered compounds. We summarize behavioral studies associated with the antipredator function of the nuchal glands, which have been conducted during our more than decade-long collaboration. Results of preliminary analyses on the possible costs of toad-eating and on the ultrastructure of the nuchal glands are also presented. Finally, we discuss the evolutionary origin of the nuchal glands and suggest future directions designed to understand the biological importance of these novel vertebrate organs, which have evolved in a limited number of snake species.

Rhabdophis tigrinus is an Asian natricine snake that possesses unusual defensive glands on the dorsal surface of its neck. These nuchal glands typically contain cardiotonic steroidal toxins known as bufadienolides, which are also abundant in the skin of toads. Feeding experiments demonstrated that toads consumed as prey are the ultimate sources of the bufadienolides in nuchal glands of R. tigrinus. Indeed, snakes on a toad-free Japanese island (Kinkasan, Miyagi Prefecture) lack these compounds in their nuchal glands, confirming that these snakes are unable to synthesize defensive bufadienolides. However, when snakes from Kinkasan are fed toads in the laboratory, they accumulate bufadienolides in their nuchal glands, indicating that they have not lost the ability to sequester defensive compounds from prey. In contrast, R. tigrinus from a toad-rich island (Ishima, Tokushima Prefecture) possess large quantities of bufadienolides, reflecting the abundance of toads from which these compounds can be sequestered. Feeding experiments involving gravid R. tigrinus demonstrated that bufadienolides can be provisioned to offspring so that hatchlings are chemically defended before their first toad meal. Maternal provisioning of bufadienolides can take place through two routes: by deposition in yolk and by diffusion in utero, even late in gestation. We applied bufadienolides to the surface of eggs from Kinkasan and found that the embryos are able to take up these compounds into their nuchal glands, demonstrating the feasibility of uptake across the eggshell. Female R. tigrinus provision bufadienolides to their offspring in direct proportion to their own level of chemical defense. By feeding toad-derived bufotoxins to R. tigrinus hatchlings, we determined that the sequestration of these compounds involves at least three types of modification: hydrolytic cleavage of suberylarginine side chains, hydroxylation, and epimerization.

Rhabdophis tigrinus obtains defensive steroids (bufadienolides) from its diet and sequesters those compounds in specialized structures on its neck known as nuchal glands. Hatchling snakes lacking these steroids must acquire them from toads consumed as prey. Here we show that females provision bufadienolides to their offspring in amounts correlated to the quantity in their own nuchal glands; thus, chemically protected mothers produce defended offspring. Bufadienolides can be provisioned to embryos via deposition in yolk and by transfer across the egg membranes within the oviducts. Maternally provisioned bufadienolides persist in the nuchal glands of juvenile snakes from the time of hatching in late summer until the following spring, when toads of ingestible size become abundant. Therefore, maternal provisioning may provide chemical protection from predators for young R. tigrinus in the absence of dietary sources of bufadienolides.

SUMMARY 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.

Ambystoma mabeei is listed as threatened in Virginia due to its rarity and susceptibility to urbanization and poor forestry practices. The purposes of this study were to determine the composition of the diet of larval A. mabeei and to compare larval diet in different geographic regions to gain insights into the ecology of Virginia populations. Ninety-eight percent (N = 75) of all A. mabeei larvae dissected contained food items. Virginia samples harbored higher loads of gastric parasites (Nematodes) than individuals from other locations. Further- more, prey species found in the stomachs of Virginia specimens were different than those of conspecifics from more southern locations. Higher loads of gastric parasites in Virginia animals may be related to their diet, which in turn is related to the forest cover over natural ponds. Habitat disturbance and parasite preva- lence may have major implications both for larval survival and for the long-term persistence of A. mabeei populations in this portion of their range.

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.

The Timber Rattlesnake (Crotalus horridus) is beset by a variety of conservation problems, including habitat loss and persecution. Effective management plans require an understanding of rangewide population structure and intraspecific evolutionary subdivisions. Northern and southern populations have been recognized as distinct subspecies, but this classification remains controversial. A proposed alternative arrangement recognizes southern, northern, and western morphotypes. To resolve intraspecific partitions, we examined a 319 base-pair (bp) fragment of mtDNA cytochrome b in 123 specimens of C. horridus. Neighbor-joining and parsimony analyses reveal a shallow gene genealogy (dmax = 0.024) and sharing of haplotypes among putative subspecies. Analysis of molecular variance demonstrates that traditional subspecific divisions explain only 3.5% of variation, whereas the alternative geographic classification (southern, northern, and western regions) explains 18.6% of genetic variation. The superior p...

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.

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.

By the early 1990s John W. Daly, of the National Institutes of Health, had been collecting and cataloguing toxins from the skins of frogs for about 30 years, on the assumption that the enormous variety of alkaloid toxins (by then numbering in the hundreds) were synthesized by the frogs themselves (Daly 1998). However, he had begun to detect patterns that suggested the toxins might have a dietary origin, including temporal variation in toxin profiles within a population, similarity of toxins among frogs of four distantly related families (and even in a bird), the absence of alkaloids in captive-bred frogs, and the discovery of some identical compounds in a variety of small arthropods (Saporito et al. 2009). In a series of experiments in the mid1990s, Daly and his collaborators demonstrated that captive-reared and nontoxic frogs of the genus Dendrobates could acquire toxins from prey that had been dusted with alkaloids in the lab (Daly et al. 1994b). Furthermore, captive-reared frogs would, if housed with access to wild arthropods as prey, acquire alkaloid toxins (Daly et al. John W. Daly in the field in Colombia, 1970 (left from Myers 2009, courtesy of Charles W. Myers and Robert W. Hansen) and Panama, 2003 (right courtesy of Maureen A. Donnelly)

The timber rattlesnake (Crotalus horridus) occurs in discontinuous populations across the eastern and central United States. The species exhibits high levels of polymorphism in morphological traits, especially in coloration and pattern. Previous studies recognized either distinct northern and southern subspecies or three regional morphs (northern, southern, and western), but the conflicting data sets and limited geographic sampling of previous studies have left the relationships among those regional variants unclear. In this study, univariate and multivariate statistics, analyzed in conjunction with a geographic information system, were used to examine geographic variation in 36 morphological characters recorded from 2420 specimens of C. horridus across its range.Univariate analyses detected substantial geographic variation in all meristic characters. Scutellation exhibited a general north–south pattern of variation, and most scale counts averaged higher in southern regions. Pattern characters differentiated the northeastern, central-eastern, and north-central regions from the southern and western regions. Coloration displayed a pattern of strong clinal variation among three broad areas consisting of the combined northeastern, central-eastern, and southern Appalachian regions, the northwestern regions, and the southern regions. Morphometric characters exhibited a general north–south pattern of geographic variation, with larger head and body sizes in southern regions. Sexual dimorphism was strong in ventral and subcaudal scales, and weak to moderate in band length and band spacing.Principal component analysis indicated that band length was the most important variable for characterizing geographic variation. The northeastern regions remained moderately distinct in all multivariate cluster analyses. The northwestern regions appeared very distinct in most cluster analyses for females. However, the clusters in all models showed extensive geographic overlap. Maps of the clusters revealed two north–south patterns of clinal variation across the northeastern, northwestern, and southern regions. The discordant patterns of variation among individual characters, overlapping patterns of coloration, and extensive overlap among the multivariate clusters collectively indicate that the putative zones of intergradation among recognized subpopulations of C. horridus are much broader than previously thought. Furthermore, because the general patterns of geographic variation are strongly clinal, our results support the conclusion that C. horridus is a single widespread species with variation too extensive and complex to be reflected by formal subspecific designations.

Iguanognathus is a monotypic genus known from only one specimen and notable for the unusual morphology of its teeth. Here, we study the tooth morphology of Iguanognathus werneri in detail, using scanning electron microscopy. The teeth of Iguanognathus are found to be hinged. Hinged teeth in other snakes are often correlated with dietary specialization, and an easily broken tail. Examination of Iguanognthus reveals an arthropod as gut contents and a broken tail. GUANOGNATHUS (Colubridae, sensu lato) is a monotypic genus, known from only one specimen. This snake has a very distinctive tooth morphology, but beyond that little is known about its morphology or natural history.

We quantified embryonic growth in 10 species of the viviparous thamnophiine snakes and three oviparous colubrid snakes. We fit data on mass, total length, and snout-vent length to linear and exponential growth models; all three size measures (particularly the lengths) fit the linear model better. For all three measures, there were significant differences among species in the absolute growth rates obtained from the linear model; these correlated with differences in neonate size. For the relative growth rates obtained from the exponential model, significant differences among species were seen only for mass; these were only moderately correlated with neonate size. Gestation periods, estimated from absolute growth rates and neonate size, generally increased with size. Comparisons with outgroup species suggest that having large neonates is the ancestral condition for the thamnophiines and that small neonate size evolved at least twice within the clade. Size reduction was achieved

Six genera of snakes, representing at least three lineages, possess teeth that fold backward against the jaws rather than being firmly ankylosed. This condition, effected by a connective tissue hinge at the base of each tooth, is associated with suites of cephalic modifications that enable the snakes to grasp and to swallow hard-bodied prey.

Patterns of morphological variation among fossorial, piscivorous, and durophagic snakes reveal several principles underlying character co-occurrence. Homoplasy is widespread among all three groups. Alternative adaptive responses to specialized habits include modifications for cranial reinforcement in fossorial and cryptozoic snakes and columellar adjustments to accommodate large excursion of the quadrates in piscivores.Among durophagic species alternatives include dental attachment in both scincivores and crustacean-eaters. Although it may be difficult to distinguish primary adaptive responses from secondary ones, specialization frequently elicits pervasive modifications across manymorphological systems. Some are associated with the foraging microhabitat, such as valvularity in piscivores and arboreal adaptations in cochleophages. Others, such as mimicry in Dasypeltis and Scaphiodontophis , are defensive. Still others reflect special aspects of prey processing, such as hypertrophy of the gastric smooth muscle and presumptive salt glandin Fordoma , a cancrivore. The extent to which coordinated morphological responses necessarily accompany specialization is important in formulating hypotheses of macroevolutionary mechanisms.

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.

The recent proposal that front-fanged venom delivery systems have been derived independently from the rear-fanged condition at least five times in the history of snakes (Savitsky, 1978) suggests the need for a reassessment of the role of venom delivery in ophidian history. Despite the fact that serous oral glands have long been known in aglyphous (fangless) and opisthoglyphous (rear-fanged) members of the family Colubridae (see Taub, 1967, p. 1618 for a historical resume), venom delivery has been ascribed a major role only in the evolution of front-fanged snakes. The latter group embraces two polyphyletic assemblages, the solenoglyphs (such as vipers, pit vipers, and mole vipers) with relatively kinetic fang-bearing maxillae, and the proteroglyphs (such as cobras, coral snakes, and seasnakes), which generally possess less mobile maxillae. Both groups possess specialized glands for the secretion and storage of venom and gland-deforming musculature to aid in delivering the venom. My premise is that envenomation has been a key adaptation in determining the success of all of the higher snakes. Furthermore, although envenomation is not the first innovative feeding strategy in ophidian history, its special role in the adaptive radiation of advanced snakes derives from a complex morphological response to changing environmental conditions that embraced both the locomotor and feeding systems of snakes. The current fluid nature of ophidian

Sceloporus adleri, a new species founded on 41 specimens, is related to the geographically adjacent species S. formosus, and is presumably limited to the Sierra Madre del Sur, Guerrero, Mexico. This Sierra appears to possess sufficient biotic endemism to justify recognition as a distinctive biotic province. Among specimens secured in Guerrero, Mexico, by Kraig Adler and his group during the winter of 1969, are 12 of a remarkably colorful species of Sceloporus cryptically related to S. formosus Wiegmann. Several other specimens of the same species had been secured by Adler earlier in the same area, and an additional series was secured for us in 1972 by David M. Dennis. We name this population Sceloporus adleri sp. nov. Holotype.-University of Michigan Museum of Zoology (UMMZ) 131689, a male, taken at Asoleadero, Guerrero, Mexico, 2520 m, on 17 December 1969, by Kraig Adler, David M. Dennis and David H. Snyder. Field no. MN 4466. Paratypes.-Forty, all from Guerrero, including UMMZ 126282 (5, field nos. IJ 1522-6), Cerro Teotepec (ca. 125 km by road W Xochipala), 3300-3400 m., 10 August 1964, Theodore J. Cohn and Jean Cohn; UMMZ 130134 (6, field nos. GH 9264-7, 9270-1), between Puerto Chico and Asoleadero, 2550-2600 m, 13-14 June 1964, Kraig Adler, Guy G. Musser and James H. Brown; Colorado University Museum (CUM) 50651-3, 5 km E Escalerillo, 2460 m, 20 December 1972, David M. Dennis, David H. Snyder, Mike Silvey, Don F. Harker; Field Museum of Natural History 191335-6, U.S. National Museum (USNM) 193236, University of Illinois Museum of Natural History 93532, all topoparatypes, 16 December 1969; Kansas University Museum of Natural History (KUMNH) 143612, CUM 46010, Museum of Comparative Zoology (MCZ) 129779, Brigham Young Univ. Mus. 40188, American Museum of Natural History (AMNH) 108211, California Academy of Science 133089, all topoparatypes, 17 December 1969; British Museum (Natural History) 1971.1856, ca. 3km (by road) E Puerto Gallo, 2740 m, 19 December 1969; CUM 50636-50, 2415 m; all topoparatypes, 19 December 1972, David M. Dennis, David H. Snyder, Mike Silvey, Don F. Harker. Twenty (USNM, KUMNH, CUM 46010, 50639-42, 50644, 50646-7, 50649, 50652, MCZ, AMNH, UMMZ field nos. 1522-3, 1526, 9264-5, 9270) are males; all others (20) are females. Collectors are Kraig Adler, David M. Dennis and David H. Snyder, unless otherwise noted. Definition.-A Sceloporus of the formosus group, distinguished from other members by the following combination of characters: mean dorsal scales 38.4; mean ventral scales 49.4; mean scales around midbody 43.7; mean femoral pores 13.3; mean scales between femoral pore series 12.8; median frontonasal in contact with lateral frontonasals; supraoculars generally not distinctly wider than long and generally not in contact with median head scales; scales on posterior surface of thigh relatively small; an incomplete, posteriorly light-bordered black nuchal collar; a pair of dorsolateral light stripes (obscure in large males); tan to dark brown dorsally in *Present address: Museum of Natural History, University of Kansas, Lawrence, Kansas 66045, USA.

The fringe-toed sand lizard Uma exsul Schmidt and Bogert is the most primitive member of its genus, and has been reported by several authors from sandy areas of heavy plant growth in the dry Lagunas de Mayran and Viesca in southwestern Coahuila, Mexico. Such sandy areas fall clearly into Norris' (1958) category of "sand hummocks or accretion dunes". The former term appears to be more applicable in this case, for there is a strong possibility, especially in light of the observations here reported, that the dunes in this region are the result of subsequent plant growth on sand dunes formed by other means than accretion around vegetation. Although U. exsul has been recorded from numerous such situations of small sand hills topped with shrubs, no record is known to us of the existence of this primitive form on "live" dunes. Four series of Uma exsul were collected by Martha L. Commins, Alan H. Savitzky, and Robert F. Zogg during August 1971, at 3 locations: sand dunes N Bilbao (a village 7.4 mi NW Viesca, along Coah. 15), elevation approximately 3,415 ft; sand dunes 4.1 mi NW Viesca, along Coah. 15, and 2.2 mi N Coah. 15, along a gravel road, elevation approximately 3,530 ft; and sand dunes 5.8 mi NW Viesca, along Coah. 15, elevation approximately 3,500 ft. The dunes are located in the Laguna de Viesca, in a part of the dry lake situated south of the Sie rr as, west of the Sierra de Parras and smaller ranges, and north of the Sierra deSierra de Santiago. The Sierra de las Burras separates the Laguna de Viesca from the more northern Laguna de Mayran, where the type locality of Uma exsul is located. The Laguna de Viesca extends westward to the vicinity of Torreon, and includes the area about Matamoros, where numerous specimens of Uma exsul have been collected by various workers. The Bilbao and Viesca localities extend the known range of the species southeast approximately 11 air mi. The dunes in the vicinity of Bilbao and Viesca were of the type considered by Norris (1958) as "barchan dunes", crescent-shaped dunes which are formed by unidirectional wind and which are in a state of unidirectional motion as a result of such wind. The orientation of the Bilbao-Viesca dunes indicates a wind blowing from the east, forming dune crests which in places rise to a height of approximately 5 m. The dunes lying on the north edge of the village of Bilbao are the largest and least overgrown of those visited. Most of the Uma exsul were seen in the basins of the dunes and especially along the edges of the dunes, in the open or under scattered vegetation. Some were also seen on the dunes proper (between basins), and a few were observed off the main dune area on a small strip of less sandy flatlands by the south edge of the dunes. Specimens of Sceloporus magister bimaculatus, Cnemidophorus tigris variolosus, and Coleonyx brevis were also collected in this area, and Uta stansburiana steinegeri observed. This association of Uma exsul with the latter 3 species was also observed by Williams, Smith, and Chrapliwy (1960); and with Chemidophorus tigris variolosus by Zweifel (1959). When approached on the interior of the duhes, the Uma exsul would usually run and hide in tumbleweed found in the basins or in cracks sometimes formed by the sand accumulated under the tumbleweed. On the edges of the dunes, Uma would generally take refuge in vegetation. "Sand swimming" was also occasionally observed, as seen under laboratory conditions (Carpenter, 1967). Sometimes they would lie motionless and flat against the sand, blending

Tantilla briggsi is described on the basis of a single specimen from 12 de Julio, 3 leagues W Donaji (formerly Tolosa), on the Caribbean drainage of the trans-lsthmus railway, Oaxaca.

Dowling, Herndon G. 1957. Amphibians and reptiles in Arkansas. Occ. Pap. Univ. Ark. Mus., No. 3: 1-51. Hardy, Laurence M. and Charles J. Cole. 1968. Morphological variation in a population of the snake, Tantilla gracilis Baird and Girard. Univ. Kans. Mus. Nat. Hist. Pubs., 17(15): 613-629. Kirn, Albert J., W. Leslie Burger, and Hobart M. Smith. 1949. The subspecies of Tantilla gracilis. Amer. Midland Naturalist, 42(1): 238-251. Taylor, Edward H. 1936. Notes and comments on certain American and Mexican snakes of the genus Tantilla, with descriptions of new species. Trans. Kans. Acad. Sci., 39: 335-348.