Fluorescent dUTP helps characterize 10 novel tetranucleotide microsatellites from an enriched salamander (Ambystoma texanum) genomic library

Ten tetranucleotide microsatellite loci were isolated and characterized from Indiana (USA) populations of the smallmouth salamander, Ambystoma texanum. As opposed to individually labelling primers that were not yet known to be polymorphic, we used fluorescent dUTP to assess genetic variability and found it to be very effective. Allelic diversity ranged from two to 18 alleles per locus and observed heterozygosity ranged from 0.17 to 0.91 among roughly 25 individuals. One of the 10 markers also amplified and was polymorphic in the tiger salamander (A. tigrinum).     

Polymorphic microsatellite loci for tiger salamanders,Ambystoma tigrinum

Microsatellites have been developed for few amphibian species. However, developing genetic markers for population genetic studies in amphibians is critical because amphibians are declining globally. The tiger salamander, Ambystoma tigrinum, is widespread throughout the United States and includes the endangered subspecies, A. t. stebbinsi. We present primers and amplification conditions for 10 polymorphic microsatellite loci that have produced successful results in three subspecies of A. tigrinum. Number of alleles per locus ranged from one to 11 and heterozygosity ranged from 0 to 0.815 depending on the subspecies and locus analysed. These markers should prove useful for future studies of genetic diversity and population subdivision.     

Mortality Rates Differ Among Amphibian Populations Exposed to Three Strains of a Lethal Ranavirus

Infectious diseases are a growing threat to biodiversity, in many cases because of synergistic effects with habitat loss, environmental contamination, and climate change. Emergence of pathogens as new threats to host populations can also arise when novel combinations of hosts and pathogens are unintentionally brought together, for example, via commercial trade or wildlife relocations and reintroductions. Chytrid fungus (Batrachochytrium dendrobatidis) and amphibian ranaviruses (family Iridoviridae) are pathogens implicated in global amphibian declines. The emergence of disease associated with these pathogens appears to be at least partly related to recent translocations over large geographic distances. We experimentally examined the outcomes of novel combinations of host populations and pathogen strains using the amphibian ranavirus Ambystoma tigrinum virus (ATV) and barred tiger salamanders (Ambystoma mavortium, formerly considered part of the Ambystoma tigrinum complex). One salamander population was highly resistant to lethal infections by all ATV strains, including its own strain, and mortality rates differed among ATV strains according to salamander population. Mortality rates in novel pairings of salamander population and ATV strain were not predictable based on knowledge of mortality rates when salamander populations were exposed to their own ATV strain. The underlying cause(s) for the differences in mortality rates are unknown, but local selection pressures on salamanders, viruses, or both, across the range of this widespread host–pathogen system are a plausible hypothesis. Our study highlights the need to minimize translocations of amphibian ranaviruses, even among conspecifc host populations, and the importance of considering intraspecific variation in endeavors to manage wildlife diseases.     

The origin of tiger salamander (Ambystoma tigrinum) populations in California, Oregon, and Nevada: introductions or relicts?

Whether intentionally or accidentally introduced, exotic species have the capacity to dramatically disrupt native communities. In central California, tiger salamanders (Ambystoma tigrinum) have been introduced as a by-product of the sport fishing bait industry. Some of these introductions are relatively well known and have resulted in the formation of hybrids with the imperiled native California tiger salamander (A. californiense). Other populations of A. tigrinum, particularly in the northern and eastern parts of the state, remain poorly characterized and are present in regions where relictual amphibian populations of other species have persisted, suggesting that these might be relictual, native A. tigrinum. We used genetic sequence data to determine the provenance of all known extralimital A. tigrinum populations in California and adjacent Oregon and Nevada through comparison with reference samples from the native range of A. tigrinum. Our results suggest that A. tigrinum have been introduced in Northern California, Southern California and the Sierra Nevada, originating from multiple sources across the Great Plains of the US. Furthermore, two populations near the California-Oregon border are most closely related to A. tigrinum populations from Washington and Oregon and may represent native tiger salamander lineages.     

Olfactory responses of aquatic and terrestrial tiger salamanders to airborne and waterborne stimuli

Summary Electro-olfactograms (EOGs) were used to assess olfactory responding by aquatic larval and terrestrial adult tiger salamanders (Ambystoma tigrinum) to airborne volatile compounds, and volatile and non-volatile compounds in aqueous solution. Both forms of salamander showed saturation effects to presentations of airborne stimuli (Fig. 2). Saturation was not observed, however, to stimulus presentations in aqueous solution (Figs. 2, 3). When threshold values and concentration-response curve parameters were compared, non-volatile amino acids in solution were more potent stimuli for larvae while airborne volatiles were more potent stimuli for adults (Tables 1, 2). We infer that metamorphosis in the tiger salamander is accompanied by changes in olfactory response characteristics, due possibly to changes in receptor population, changes in perireceptor properties (e.g. mucus) or to changes in stimulus access.Abbreviations EOG electro-olfactogram - PPM (ppm) parts per million     

Banding differences between tiger salamander and axolotl chromosomes

The Hoechst 33258 - Giemsa banding patterns were compared on axolotl (Ambystoma mexicanum Shaw) and axolotl - tiger salamander (Ambystoma tigrinum Green) species hybrid prophase chromosomes. Approximately 369 bands per haploid chromosome set were seen in the axolotl and about 344 bands in the tiger salamander. In the haploid set of 14 chromosomes, chromosome 3 has a constant short or q-arm terminal constriction at the location of the nucleolar organizer. Chromosomes 14 Z and W carry the sex determinants, the female being the heterogametic sex (ZW). The banding patterns of chromosomes 1, 6, 11, and 14 Z of the two species are apparently indistinguishable by our banding method. In the axolotl, chromosome 9 has a small long or p-arm terminal deletion. In the tiger salamander, the remaining 10 chromosomes have terminal or internal deletions. No translocations or inversions seem to have occurred since the gene pool separation of the two closely related species.     

Experimental morphology of the feeding mechanism in salamanders

The subarcualis rectus I muscle (SAR) in the feeding mechanism of four tiger salamanders (Ambystoma tigrinum) was removed early in ontogeny and these individuals were allowed to complete metamorphosis. This procedure resulted in postmetamorphic tiger salamanders which differed from control individuals in the size (and thus force generating capacity) of the SAR muscle. The experimental manipulation of muscle ontogeny allowed a test of previous hypotheses of SAR function in postmetamorphic individuals. Multivariate analysis of variance for kinematic variables measured from high-speed video records of feeding revealed that experimentally modified tiger salamanders did not protract the hyobranchial apparatus or project the tongue from the mouth during feeding. Removal of the SAR muscle resulted in significantly reduced hyobranchial elevation in the buccal cavity and reduced maximum tongue projection distance.     

Geographically variable selection in Ambystoma tigrinum virus (Iridoviridae) throughout the western USA

We investigated spatially variable selection in Ambystoma tigrinum virus (ATV) which causes frequent and geographically widespread epizootics of the tiger salamander, Ambystoma tigrinum. To test for evidence of selection, we sequenced several coding and noncoding regions from virus strains isolated from epizootics throughout western North America. Three of the sequenced regions contained homologues for genes putatively involved in host immune evasion and virulence: eIF‐2α, caspase activation and recruitment domain (CARD) and β‐OH‐steroid oxidoreductase. Selection analysis showed evidence of very strong purifying selection on eIF‐2α, purifying selection within certain viral clades on CARD and positive selection on β‐OH‐steroid oxidoreductase within certain clades. Analysis using multidivtime and Tajima’s relative rate tests indicate accelerated rates of evolution within clades associated with anthropogenic movement. These clades also demonstrate greater spatial variability in selection, suggesting a lack of local adaptation (i.e. locally adapted populations should exhibit little to no selection because of absent or reduced variation in fitness once a fitness optimum is reached). Increased transfer of non‐native viral strains to naïve salamander populations, in conjunction with local maladaptation as a result of local selection pressures, may explain the spread and emergence of ATV epizootics in A. tigrinum in western North America.     

Evidence for emergence of an amphibian iridoviral disease because of human-enhanced spread

Our understanding of origins and spread of emerging infectious diseases has increased dramatically because of recent applications of phylogenetic theory. Iridoviruses are emerging pathogens that cause global amphibian epizootics, including tiger salamander (Ambystoma tigrinum) die-offs throughout western North America. To explain phylogeographical relationships and potential causes for emergence of western North American salamander iridovirus strains, we sequenced major capsid protein and DNA methyltransferase genes, as well as two noncoding regions from 18 geographically widespread isolates. Phylogenetic analyses of sequence data from the capsid protein gene showed shallow genetic divergence (< 1%) among salamander iridovirus strains and monophyly relative to available fish, reptile, and other amphibian iridovirus strains from the genus Ranavirus, suggesting a single introduction and radiation. Analysis of capsid protein sequences also provided support for a closer relationship of tiger salamander virus strains to those isolated from sport fish (e.g. rainbow trout) than other amphibian isolates. Despite monophyly based on capsid protein sequences, there was low genetic divergence among all strains (< 1.1%) based on a supergene analysis of the capsid protein and the two noncoding regions. These analyses also showed polyphyly of strains from Arizona and Colorado, suggesting recent spread. Nested clade analyses indicated both range expansion and long-distance colonization in clades containing virus strains isolated from bait salamanders and the Indiana University axolotl (Ambystoma mexicanum) colony. Human enhancement of viral movement is a mechanism consistent with these results. These findings suggest North American salamander ranaviruses cause emerging disease, as evidenced by apparent recent spread over a broad geographical area.     

Amphibian survival,growth and development in response to mineral nitrogen exposure and predator cues in the field: an experimental approach

Mineral nitrogen (N) has been suggested as a potential factor causing declines in amphibian populations, especially in agricultural landscapes; however, there is a question as to whether it remains in the water column long enough to be toxic. We explored the hypothesis that mineral N can cause both lethal and sublethal toxic effects in amphibian embryos and larvae in a manipulative field experiment. We sampled 12 ponds, fertilizing half with ammonium nitrate fertilizer early in the spring, and measured hatching, survival, development, growth, and the incidence of deformities in native populations of wood frog (Rana sylvatica) and eastern tiger salamander (Ambystoma tigrinum tigrinum) embryos and larvae held in in situ enclosures. We found that higher ammonium concentrations negatively affect R. sylvatica more strongly than A. tigrinum. R. sylvatica tended to have lower survival as embryos and young tadpoles, slowed embryonic development, and an increased proportion of hatchlings with deformities at experimentally elevated ammonium. A. tigrinum did not experience significantly reduced survival, but their larval development was slowed in response to elevated ammonium and the abundance of large invertebrate predators. Variable species susceptibility, such as that shown by R sylvatica and A. tigrinum, could have large indirect effects on aquatic community structure through modification of competitive or predator-prey relationships. Ammonium and nitrate + nitrite concentrations were not correlated with other measures that might have affected amphibians, such as pH, pond area, depth, or vegetation. Our results highlight the potential importance of elevated ammonium on the growth, development and survival of amphibians, especially those that breed in surface waters receiving anthropogenic N inputs. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Adrenoceptor types in the respiratory vasculature of the salamander gill

Summary Adrenoreceptor types were determined in the branchial respiratory vasculature of the neotenic tiger salamander,Ambystoma tigrinum. Phenoxybenzamine antagonized increases in branchial vascular resistance caused by both epinephrine and norepinephrine. Propranolol antagonized both epinephrine-and norepinephrine-induced dilation of this vascular bed. Isoproterenol produced solely vasodilation, phenylephrine had no effect, and methoxamine caused constriction only at a very high dose. It is concluded that alpha-adrenoceptors mediate the catecholamine-induced vasoconstriction, and beta-adrenoceptors the catecholamine-induced vasodilation in the respiratory circulation of the gill.     

Shaping intraspecific variation: Development,ecology and the evolution of morphology and life history variation in tiger salamanders

The tiger salamander,Ambystoma tigrinum, is a geographically widespread, morphologically variable, polytipic species. It is among the most variable species of salamanders in morphology and life history with two larval morphs (typical and cannibal) and three adult morphs (metamorphosed, typical branchiate, cannibal branchiate) that vary in frequency between subspecies and between populations within subspecies. We report morphometric evidence suggesting that branchiate cannibals arose through intraspecific change in the onset or timing of development resulting in the wider head and hypertrophied tooth-bearing skull bones characteristic of this phenotype. We also quantified bilateral symmetry of gill raker counts and abnormalities, then evaluated fluctuating asymmetry as a measure of the developmental stability of each morph. There was a significant interaction between fluctuating asymmetry of developmental abnormalities in cannibals and typicals and the locality where they were collected, suggesting that relative stability of each phenotype could vary among populations. While altered timing of developmental events appears to have a role in the evolution and maintenance of morphs, novel phenotypes persist only under favorable ecological conditions. Predictability of the aquatic habitat, genetic variation, kinship, body size, intraspecific competition and predation all affect expression and survival of the morphs inA. tigrinum. This taxon provides an excellent model for understanding the diversity and complexity of developmental and ecological variables controlling the evolution and maintenance of novel phenotypes.     

Thyroid hormone responsive QTL and the evolution of paedomorphic salamanders

The transformation of ancestral phenotypes into novel traits is poorly understood for many examples of evolutionary novelty. Ancestrally, salamanders have a biphasic life cycle with an aquatic larval stage, a brief and pronounced metamorphosis, followed by a terrestrial adult stage. Repeatedly during evolution, metamorphic timing has been delayed to exploit growth-permissive environments, resulting in paedomorphic salamanders that retain larval traits as adults. We used thyroid hormone (TH) to rescue metamorphic phenotypes in paedomorphic salamanders and then identified quantitative trait loci (QTL) for life history traits that are associated with amphibian life cycle evolution: metamorphic timing and adult body size. We demonstrate that paedomorphic tiger salamanders (Ambystoma tigrinum complex) carry alleles at three moderate effect QTL (met1–3) that vary in responsiveness to TH and additively affect metamorphic timing. Salamanders that delay metamorphosis attain significantly larger body sizes as adults and met2 explains a significant portion of this variation. Thus, substitution of alleles at TH-responsive loci suggests an adaptive pleiotropic basis for two key life-history traits in amphibians: body size and metamorphic timing. Our study demonstrates a likely pathway for the evolution of novel paedomorphic species from metamorphic ancestors via selection of TH-response alleles that delay metamorphic timing and increase adult body size.     

Biochemical plasticity in the Arizona tiger salamander (Ambystoma tigrinum nebulosum).

The Arizona tiger salamander, Ambystoma tigrinum nebulosum, is a developmentally polymorphic species. Some individuals become sexually mature while retaining some larval traits (paedomorphs), while other individuals mature as metamorphosed salamanders. In this study, relative enzyme activities of the products of two duplicate loci in each of three enzyme systems (aconitase, malate dehydrogenase, and isocitrate dehydrogenase) were measured in paedomorphs and in paedomorphs forced to metamorphose by treatment with thyroxine. We found that thyroxine and laboratory conditions affect enzyme activities of four of the six enzymes examined and that activities of products of duplicate loci are altered to different degrees.     

Predator size and phenology shape prey survival in temporary ponds

Theoretical efforts suggest that the relative sizes of predators and their prey can shape community dynamics, the structure of food webs, and the evolution of life histories. However, much of this work has assumed static predator and prey body sizes. The timing of recruitment and the growth patterns of both predator and prey have the potential to modify the strength of predator–prey interactions. In this study, I examined how predator size dynamics in 40 temporary ponds over a 3-year period affected the survival of spotted salamander (Ambystoma maculatum) larvae. Across communities, gape-limited predator richness, but not size, was correlated with habitat duration (pond permanence). Within communities, mean gape-limited predator size diminished as the growing season progressed. This size reduction occurred because prey individuals grew into a body size refuge and because the largest of the predators left ponds by mid-season. Elevated gape-limited predation risk across time and space was predicted by the occurrence of two large predatory salamanders: marbled salamander larvae (Ambystoma opacum) and red-spotted newt adults (Notophthalmus viridescens). The presence of the largest gape-limited predator, A. opacum, predicted A. maculatum larval survival in the field. The distribution of large predatory salamanders among ponds and across time is expected to lead to differing community dynamics and to generate divergent natural selection on early growth and body size in A. maculatum. In general, a dynamic perspective on predator size often will be necessary to understand the ecology and evolution of species interactions. This will be especially true in frequently disturbed or seasonal habitats where phenology and ontogeny interact to determine body size asymmetries. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Sublethal Effects of Nitrite on Eastern Tiger Salamander (<Emphasis Type="Italic">Ambystoma tigrinum tigrinum</Emphasis>) and Wood Frog (<Emphasis Type="Italic">Rana sylvatica</Emphasis>) Embryos and Larvae: Implications for Field Populations

Ephemeral pools, which can have high animal biomass and low dissolved oxygen, may be prone to nitrite accumulation. As such, it is important to understand how exposure to nitrite might affect development and growth of amphibians that breed in these ephemeral pools. Wood frog (Rana sylvatica) and eastern tiger salamander (Ambystoma tigrinum tigrinum) embryos and tadpoles and young larvae were exposed to elevated concentrations of nitrite derived from sodium nitrite: 0, 0.3, 0.6, 1.2, 2.1, 4.6, and 6.1 mg l⁻¹ NO₂–N. Increasing nitrite exposure slowed embryonic and larval development in both the eastern tiger salamander and the wood frog, reduced growth in tiger salamander embryos and larvae, and delayed metamorphosis in the wood frog. At concentrations less than 2 mg l⁻¹ NO₂–N nitrite delayed hatching, and at concentrations above 2 mg l⁻¹ time to hatching decreased causing more individuals to hatch at less developed stages. Nitrite also increased asynchrony in tiger salamander hatching. The sublethal effects of nitrite on amphibian development, growth and hatching could have serious repercussions on amphibian fitness in ephemeral environments. Potential increases in mortality on field populations caused by sublethal effects of nitrite are discussed.     

Distribution and morphology of motoneurons innervating different muscle fiber types in an amphibian muscle complex

The distribution and morphology of motoneurons innervating specific types of muscle fibers in the levator scapulae superior (LSS) muscle complex of the bullfrog (Rana catesbeiana) and tiger salamander (Ambystoma tigrinum) were studied by retrograde labelling with cholera toxin-conjugated horseradish peroxidase (CT-HRP). The LSS muscle complex in both of these amphibians has a segregated pattern of muscle-fiber types (tonic; fast oxidative-glycolytic twitch [FOG]; fast glycolytic twitch [FG]) along an anteroposterior axis. The entire motor pool was labelled by injection of CT-HRP into the whole LSS muscle complex. The motoneurons innervating specific fiber types were labelled by injection of CT-HRP into certain muscle regions. The organization of the motoneuron pool of the LSS complex of both species was arranged in two columns—one ventrolateral and one medial. In bullfrogs, the ventrolateral column contains motoneurons innervating FG and tonic fiber types and the medial column contains motoneurons innervating FOG fiber types. In tiger salamanders, the ventrolateral column contains motoneurons innervating FG fiber types and the medial column contains motoneurons innervating FOG and tonic fiber types. The different motoneuron types also have different soma sizes and patterns of dendritic arborization. In both species, FG motoneurons are the largest, whereas FOG motoneurons are intermediate in size and tonic motoneurons are the smallest. In bullfrogs, the main dendrites of FG motoneurons extend into the dorsolateral and the ventrolateral gray matter of the spinal cord, whereas the dendrites of FOG motoneurons extend into the ventral and medial cord. In the tiger salamander, dendrites of FG motoneurons extend into the ventrolateral spinal cord and dendrites of the FOG motoneurons extend more generally into the ventral cord. Dendrites of tonic motoneurons in both amphibians were small and short, and difficult to observe. These results establish that motoneurons innervating different types of muscle fibers in the LSS muscle complex are segregated spatially and display consistent morphological differences. © 1993 Wiley-Liss, Inc.