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.     

Instrumentally conditioned avoidance by tiger salamanders (Ambystoma tigrinum) to reagent grade odorants

Tiger salamanders (Ambystoma tigrinum) were given pairings ofair dilutions of either n-amyl acetate or d-camphor and intenselight in a stainless steel and glass chamber using a simpleflow dilution olfactometer. Avoidance responses were conditionedto presentations of amyl acetate but avoidance responses didnot condition to presentations of camphor     

Molecular characterization of major histocompatibility complex class II alleles in wild tiger salamanders (Ambystoma tigrinum)

Major histocompatibility complex (MHC) class II genes are usually among the most polymorphic in vertebrate genomes because of their critical role (antigen presentation) in immune response. Prior to this study, the MHC was poorly characterized in tiger salamanders (Ambystoma tigrinum), but the congeneric axolotl (Ambystoma mexicanum) is thought to have an unusual MHC. Most notably, axolotl class II genes lack allelic variation and possess a splice variant without a full peptide binding region (PBR). The axolotl is considered immunodeficient, but it is unclear how or to what extent MHC genetics and immunodeficiency are interrelated. To study the evolution of MHC genes in urodele amphibians, we describe for the first time an expressed polymorphic class II gene in wild tiger salamanders. We sequenced the PBR of a class II gene from wild A. tigrinum (n=33) and identified nine distinct alleles. Observed heterozygosity was 73%, and there were a total of 46 polymorphic sites, most of which correspond to amino acid positions that bind peptides. Patterns of nucleotide substitutions exhibit the signature of diversifying selection, but no recombination was detected. Not surprisingly, transspecies evolution of tiger salamander and axolotl class II alleles was apparent. We have no direct data on the immunodeficiency of tiger salamanders, but the levels of polymorphism in our study population should suffice to bind a variety of foreign peptides (unlike axolotls). Our tiger salamander data suggest that the monomorphism and immunodeficiencies associated with axolotl class II genes is a relict of their unique historical demography, not their phylogenetic legacy. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Metamorphosis of cranial design in tiger salamanders (Ambystoma tigrinum): A morphometric analysis of ontogenetic change

While ontogenetic analyses of skull development have contributed to our understanding of phylogenetic patterns in vertebrates, there are few studies of taxa that undergo a relatively discrete and rapid change in morphology during development (metamorphosis). Morphological changes occurring in the head at metamorphosis in tiger salamanders (Ambystoma tigrinum) were quantified by a morphometric analysis of cranial osteology and myology to document patterns of change during metamorphosis. We employed a cross-sectional analysis using a sample of larvae just prior to metamorphosis and a sample of transformed individuals just after metamorphosis, as well as larvae undergoing metamorphosis. There were no differences in external size of the head among the larval and transformed samples. The hyobranchial apparatus showed many dramatic changes at metamorphosis, including shortening of ceratobranchial 1 and the basibranchial. The subarcualis rectus muscle increased greatly in length at metamorphosis, as did hypobranchial length and internasal distance. A truss analysis of dorsal skull shape showed that at metamorphosis the snout becomes wider, the maxillary and squamosal triangles rotate posteromedially, and the neurocranium shortens (while maintaining its width), resulting in an overall decrease in skull length at metamorphosis. These morphometric differences are interpreted in light of recent data on the functional morphology of feeding in salamanders. Morphological reorganization of the hyobranchial apparatus and shape changes in the skull are related to the acquisition of a novel terrestrial feeding mode (tongue projection) at metamorphosis. Metamorphic changes (both internal and external) that can be used to judge metamorphic condition are discussed.     

Molecular evidence for historical and recent population size reductions of tiger salamanders (Ambystoma tigrinum) in Yellowstone National Park

Population declines caused by natural and anthropogenic factors can quickly erode genetic diversity in natural populations. In this study, we examined genetic variation within 10 tiger salamander populations across northern Yellowstone National Park in Wyoming and Montana, USA using eight microsatellite loci. We tested for the genetic signature of population decline using heterozygosity excess, shifts in allele frequencies, and low ratios of allelic number to allelic size range (M-ratios). We found different results among the three tests. All 10 populations had low M-ratios, five had shifts in allele frequencies and only two had significant heterozygosity excesses. These results support theoretical expectations of different temporal signatures among bottleneck tests and suggest that both historical fish stocking, recent, sustained drought, and possibly an emerging amphibian disease have contributed to declines in effective population size.     

Effect of microhabitat on fitness components of larval tiger salamanders,Ambystoma tigrinum nebulosum

Summary Survivorship and growth of larval tiger salamanders, Ambystoma tigrinum nebulosum, in the White Mountains of east-central Arizona were compared in six microhabitats using field enclosures during summers, 1983–85. Microhabitats were vegetated and nonvegetated shallows, surface, middle, and bottom horizontal layers of limnetic areas, and the vertical limnetic water column. Initial enclosure densities (0.025 larvae per 1) were identical among microhabitats. Three enclosures were placed in each microhabitat in two ponds. Larval survivorship and growth were usually greatest in vegetated shallows in lowest in middle and bottom limnetic enclosures, despite several population dieoffs. Lower fitness, as reflected in survivorship and growth, in these latter enclosures was correlated with lower food levels, temperatures, and oxygen concentrations in deeper limnetic areas. Relative fitness varied greatly between years while food levels, temperatures, and oxygen concentrations within microhabitats remained relatively constant indicating additional factors affected fitness. Disparities in fitness between microhabitats apparently affect habitat use patterns of larvae.     

Metamorphosis of the feeding mechanism in tiger salamanders (Ambystoma tigrinum): the ontogeny of cranial muscle mass

Most previous research on metamorphosis of the musculoskeletal system in vertebrates has focused on the transformation of the skeleton. In this paper we focus on the transformation of the muscles of the head during metamorphosis in tiger salamanders ( Ambystoma tigrinum ) in order (1) to provide new data on changes in myology during ontogeny, and (2) to aid in interpreting previous data on the metamorphosis of function in the head of salamanders.
The physiological cross-sectional area of nine head muscles was calculated by measuring fibre angles, fibre lengths, and muscle mass in two samples of tiger salamanders obtained just before and just after metamorphosis. The major mouth-opening muscles (rectus cervicis and depressor mandibulae) exhibit a significant decrease in estimated maximum tetanic tension (MTT) across metamorphosis of about 36%. The jaw-closing muscles (adductor mandibulae internus and externus) and the head-lifting muscles (epaxials) also decrease in MTT but not significantly. The muscles associated with tongue projection during feeding on land (the subarcualis rectus I, geniohyoideus, interhyoideus and intermandibularis) all show a slight increase in MTT at metamorphosis.
Metamorphic transformation of feeding behaviour in Ambystoma tigrinum involves changes in performance, the design of skeletal elements, changes in muscle force-generating capability, and changes in hydrodynamic design from unidirectional flow in larvae to bidirectional flow during aquatic feeding after metamorphosis. Although muscle activity patterns during aquatic feeding do not change across metamorphosis, tongue-based terrestrial feeding involves a suite of novel muscle activity patterns, morphological characters acquired at metamorphosis, and a metamorphic increase in the masses of muscles important in tongue projection.     

Pigment pattern formation in larval ambystomatid salamanders: Ambystoma tigrinum tigrinum

We have begun a comparative study of pigment patterns and their mechanisms of formation in ambystomatid salamanders in an attempt to elucidate the evolution of these traits in this family. In Ambystoma t. tigrinum, the migration of the prospective pigment cells was followed by using scanning electron microscopy and light microscopy combined with markers (dopa incubation for detecting melanophores, ammonia-induced pterin fluorescence for detecting xanthophores). The pigment pattern resulting from the cell migration shares features both with the alternating vertical xanthophore and melanophore bars of A. mexicanum and the horizontal stripes of certain salamandrids and ambystomatids. The pigment pattern of A. t. tigrinum is interpreted here as an intermediate evolutionary step between a primitive horizontal stripe pattern and a derived vertical bar pattern. The initiation of pigment pattern formation resembles the situation in A. mexicanum, probably reflecting the close phylogenetic relationship between the two taxa.     

Landscape genetics of the blotched tiger salamander (Ambystoma tigrinum melanostictum)

The field of landscape genetics has great potential to identify habitat features that influence population genetic structure. To identify landscape correlates of genetic differentiation in a quantitative fashion, we developed a novel approach using geographical information systems analysis. We present data on blotched tiger salamanders (Ambystoma tigrinum melanostictum) from 10 sites across the northern range of Yellowstone National Park in Montana and Wyoming, USA. We used eight microsatellite loci to analyse population genetic structure. We tested whether landscape variables, including topographical distance, elevation, wetland likelihood, cover type and number of river and stream crossings, were correlated with genetic subdivision (F(ST)). We then compared five hypothetical dispersal routes with a straight-line distance model using two approaches: (i) partial Mantel tests using Akaike's information criterion scores to evaluate model robustness and (ii) the BIOENV procedure, which uses a Spearman rank correlation to determine the combination of environmental variables that best fits the genetic data. Overall, gene flow appears highly restricted among sites, with a global F(ST) of 0.24. While there is a significant isolation-by-distance pattern, incorporating landscape variables substantially improved the fit of the model (from an r2 of 0.3 to 0.8) explaining genetic differentiation. It appears that gene flow follows a straight-line topographic route, with river crossings and open shrub habitat correlated with lower F(ST) and thus, decreased differentiation, while distance and elevation difference appear to increase differentiation. This study demonstrates a general approach that can be used to determine the influence of landscape variables on population genetic structure.     

Morphology and escape performance of tiger salamander larvae (Ambystoma tigrinum mavortium)

The ability of an individual to escape predators is an important component of fitness. Several adaptive explanations of body shape variation in amphibians hypothesize relationships between swimming performance and morphology, but these ideas have rarely been tested. Here we investigate bivariate and multivariate relationships between natural variation in morphology and performance. We used high-speed video to examine fast-starts associated with escape responses in small tiger salamander larvae (Ambystoma tigrinum). Our results indicate that performance is influenced by interactions among aspects of morphology, physiology, and behavior. Relationships between morphometric variables and velocity could be detected with multivariate, but not bivariate statistical analyses. In particular, relationships between morphology and velocity depend on tail beat frequency (potentially a measure of effort or vigor). Relationships between morphology and acceleration were detected with bivariate analyses, but multivariate analysis suggests that acceleration performance, too, depends on interactions between morphology and tail beat frequency. We found a positive relationship between tail area and propulsive performance, which supports adaptive interpretations of variation in larval tail shape within and between amphibian species.     

Allopurinol-induced melanism in the tiger salamander (Ambystoma tigrinum nebulosum).

The enzyme, xanthine dehydrogenase (XDH), has been examined in Ambystoma tigrinum nebulosum with respect to its role in pigmentation. It now seems probable that the melanoid gene (m) either codes directly for XDH or is somehow intimately connected with the normal function of this enzyme. Inhibition of XDH using the drug, allopurinol, results in animals which appear to be phenocopies of melanoid mutants as described for the Mexican axolotl (Ambystoma mexicanum). The effects of allopurinol in terms of specific pigmentary alterations were examined, and a new method for analyzing heterogeneous extracts of skin pigments (e.g., purines and pteridines) is presented. The significance of the link between XDH and melanism is discussed with emphasis on possible mechanisms of pigment induction and general applicability to biological systems.     

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.     

Influence of temperature on Ranavirus infection in larval salamanders Ambystoma tigrinum

Temperature strongly influenced percent mortality and time to death of salamanders exposed to the Ambystoma tigrinum virus (iridovirus) (ATV). Most salamanders survived when exposed at 26 degrees C, whereas all died at 18 degrees C and nearly all died at 10 degrees C. Some asymptomatic salamanders that survived 60 d at 10 or 26 degrees C were found to be carrying virus. Polymerase chain reaction (PCR) confirmed the presence of virus in ATV-exposed salamanders but was found to be less sensitive than cell culture in detecting ATV at low concentrations. PCR products were 100% identical to ATV in the major capsid protein sequence. Virus titer was higher in salamanders held at 10 degrees C than at 18 degrees C but little virus, if any, was present in the small number of salamanders that died at 26 degrees C. These results may help explain periodic viral epizootics in field populations of A. tigrinum where water temperatures fluctuate widely.     

Insights into the mating habits of the tiger salamander (Ambystoma tigrinum tigrinum) as revealed by genetic parentage analyses

Among urodeles, ambystomatid salamanders are particularly amenable to genetic parentage analyses because they are explosive aggregate breeders that typically have large progeny arrays. Such analyses can lead to direct inferences about otherwise cryptic aspects of salamander natural history, including the rate of multiple mating, individual reproductive success, and the spatial distribution of clutches. In 2002, we collected eastern tiger salamander (Ambystoma tigrinum tigrinum) egg masses (> 1000 embryos) from a approximately 80 m linear transect in Indiana, USA. Embryos were genotyped at four variable microsatellite loci and the resulting progeny array data were used to reconstruct multilocus genotypes of the parental dams and sires for each egg mass. UPGMA analysis of genetic distances among embryos resolved four instances of egg mass admixture, where two or more females had oviposited at exactly the same site resulting in the mixing of independent cohorts. In total, 41 discrete egg masses were available for parentage analyses. Twenty-three egg masses (56%) consisted exclusively of full-siblings (i.e. were singly sired) and 18 (44%) were multiply sired (mean 2.6 males/clutch). Parentage could be genetically assigned to one of 17 distinct parent pairs involving at least 15 females and 14 different males. Reproductive skew was evident among males who sired multiply sired clutches. Additional evidence of the effects of sexual selection on male reproductive success was apparent via significant positive correlations between male mating and reproductive success. Females frequently partitioned their clutches into multiple discrete egg masses that were separated from one another by as many as 43 m. Collectively, these data provide the first direct evidence for polygynandry in a wild population of tiger salamanders.     

Behavioral determinations of thresholds for n-butyl acetate and nbutyl alcohol in the tiger salamander (Ambystoma tigrinum)

Two groups of tiger salamanders (Ambystoma tigrinum) were conditionedto respond to odorant-air mixtures of n-butyl acetate (8.9 x10^–5M) or n-butyl alcohol (6.7 x l0^–5M). They werethen given tests with various concentrations of the trainingodorants presented using a temporal forced-choice method ofascending limits. Results showed that reliable responses toodorant-air presentations were obtained with concentrationsof n-butyl acetate above 2.4 x l0^–7M and with concentrationsof n-butyl alcohol above 8.5 x 10^–8M. These results arein substantial agreement with previous dectrophysiological findings.