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.     

Effect of fasting and thermal acclimation on metabolism of juvenile axolotls (Ambystoma mexicanum)

Juvenile axolotls were acclimated to 15○C or 25○C and either fed or fasted at both temperatures, to study the interaction of thermal acclimation and nutritional state on metabolism. Fasting but not thermal acclimation significantly increased oxygen consumption at 15○C. Fasting also increased the specific activities of two oxidative metabolic enzymes – citrate synthase and cytochrome oxidase – but not that of the glycolytic enzyme lactic dehydrogenase. The specific activity of cytochrome oxidase was further stimulated by cold acclimation. Triglycerides and fatty acids were severely depleted in fasted animals, but thermal acclimation had no significant effect on lipid stores. This study illustrates: (1) the differential nature of various metabolic responses to fasting; and (2) the confounding interaction of the nutritional state on thermal acclimation experiments in an ectotherm.     

THE POLYTYPIC SPECIES REVISITED: GENETIC DIFFERENTIATION AND MOLECULAR PHYLOGENETICS OF THE TIGER SALAMANDER AMBYSTOMA TIGRINUM (AMPHIBIA: CAUDATA) COMPLEX

We present a phylogenetic analysis of the Ambystoma tigrinum complex, based on approximately 840 base pairs of mitochondrial-DNA sequence from the rapidly evolving D-loop and an adjacent intron. Our samples include populations of the continentally distributed species, A. tigrinum, plus all described species of Mexican ambystomatids. Sequence divergence is low, ranging from 0–8.5%, and most phylogenetic groupings are weakly supported statistically. We identified eight reasonably well-defined clades from the United States and Mexico, with the geographically isolated A. californiense from California as the probable sister group to the remaining taxa. Our sequence data are not capable of resolving the relationships among these clades, although the pattern of transitional-site evolution suggests that these eight lineages diverged during a period of rapid cladogenesis. We roughly calibrate a molecular clock and identify a few lineages that significantly deviate from the slow, baseline rate of 0.5–0.75% per million years. Our data also suggest that species boundaries for several U.S. and Mexican species need to be altered and that the concept of a continentally distributed, polytypic tiger salamander is not valid.     

GYNOGENETIC REPRODUCTION IN HYBRID MOLE SALAMANDERS (GENUS AMBYSTOMA)

Ambystoma platineum, a unisexual clonal triploid taxon of mole salamander, originated by hybridization between the Mendelian species A. jeffersonianum and A. laterale. Studies of lampbrush chromosomes indicated that A. platineum reproduces gynogenetically, that is, sperm from a sexual host species is required to activate egg development but makes no genetic contribution to the developing embryo. Nevertheless, electrophoretic diversity in populations of some hybrid Ambystoma suggested continual in situ recreation of unisexual hybrids and bidirectional gene exchange between the parental species and the hybrids. A. platineum usually lives with, and is sexually dependent on, one of its parental species, A. jeffersonianum. In central Indiana, however, A. platineum populations have shifted their host dependency to A. texanum. Such A. texanum-dependent populations of A. platineum provide an almost ideal system for studying reproductive mode in A. platineum, because both replacement of a jeffersonianum or laterale genome of A. platineum by a texanum genome, and movement of genes from A. platineum to the host species, A. texanum, would be readily detected by electrophoretic markers. Our samples of A. texanum provided no evidence for the transfer of jeffersonianum or laterale genes into A. texanum. Similarly, among 32 A. platineum sampled from six localities in east-central Illinois and central Indiana, we find no texanum alleles, and thus no evidence for genome replacement. The one diploid hybrid individual contained only a jeffersonianum and a laterale genome; because of the absence of either parental species from these populations, this hybrid could only have come from a diploid ovum produced by A. platineum. Both morphometric and electrophoretic results for the two tetraploid individuals indicate that they resulted from fertilization of triploid oocytes of A. platineum by sperm of A. texanum. Because genome replacement in A. texanum-dependent populations of A. platineum is irreversible, the persistence of A. platineum in A. texanum-dependent populations demonstrates conclusively that the major mode of reproduction in A. platineum populations is clonal: A. platineum produces mainly triploid eggs that develop gynogenetically.     

GENE FLOW AND INEFFECTIVE ANTIPREDATOR BEHAVIOR IN A STREAM-BREEDING SALAMANDER

Predators often feed on prey that show ineffective antipredator behavior. Gene flow among populations may constrain evolution of effective antipredator ability in larvae of the streamside salamander, Ambystoma barbouri, a species that occupies distinctly different habitats with conflicting selection pressures. Some streams are ephemeral, where larvae should be active to feed and reach metamorphosis before stream drying. In contrast, other streams are more permanent and contain pools with predatory fish, where larvae should remain inactive to avoid fish predation. Feeding rates and predator escape behavior were assayed for laboratory-reared larvae from 15 populations. Larval survival was also compared among populations in artificial streams with natural predators. Five populations represented streams subjected to fish predation along a gradient of genetic and geographic isolation from populations without fish; the remaining 10 populations were ephemeral and without fish. Individuals from populations with fish had significantly stronger behavioral responses to fish (i.e., decreased feeding rate associated with the presence of fish and increased escape response) than individuals from fishless populations. Larvae from populations containing fish that were more isolated from fishless populations showed stronger antipredator responses than less isolated populations. Further, larvae from more isolated populations survived longer in the predation experiment, indicating that the behaviors measured were related with survival. These results suggest that gene flow between populations with conflicting selection pressures limits local adaptation in some salamander populations with fish. While previous studies have typically focused on the role of gene flow in pairs of populations, the results of this study suggest that gene flow is acting to swamp local adaptation across several populations.     

Environment-dependent admixture dynamics in a tiger salamander hybrid zone

After an estimated five million years of independent evolution, the barred tiger salamander (Ambystoma tigrinum mavortium) was introduced by bait dealers into the native range of the California tiger salamander (A. californiense). Hybridization and backcrossing have been occurring in central California for 50-60 years, or an estimated 15-30 generations. We studied genetic and ecological factors influencing admixture of these two divergent gene pools by analyzing frequencies of hybrid genotypes in three kinds of breeding habitats: natural vernal pools, ephemeral man-made cattle ponds, and perennial man-made ponds. Perennial ponds tended to have higher frequencies of nonnative alleles than either type of seasonal pond, even in cases where perennial and seasonal ponds are within a few hundred meters. Thus, the hybrid zone has a mosaic structure that depends on pond hydrology or ecology. The presence of some broadly acting constraints on admixture is suggested by linkage disequilibria between physically unlinked molecular markers within ponds. In addition, we found several marker-specific deviations from Hardy-Weinberg equilibrium. One marker showed a consistent deficit of heterozygotes across pond types. Another showed heterozygote deficits only in vernal pools. A third was more likely to have heterozygote excess in ephemeral cattle ponds. These patterns indicate that admixture is influenced by complex genotype-by-environment interactions.     

Interaction of an insecticide with larval density in pond-breeding salamanders (Ambystoma)

1. Amphibian populations residing in or near agricultural areas are often susceptible to pesticide contamination. Recent evidence suggests that the effects of pesticides on amphibians often exceed those estimated in laboratory toxicity tests because other environmental factors (e.g. predators, resource abundance) can influence pesticide toxicity. 2. To examine the effects of an insecticide (carbaryl) on two species of Ambystoma salamanders experiencing the natural stress of competition, we manipulated chemical concentration (control, 3.5 and 7.0 mg L^?1) and larval density (low and high). We determined the effect of treatments on snout‐vent length (SVL), growth rate, lipid reserves, time to metamorphosis, per cent survival and per cent metamorphosis. 3. Carbaryl negatively affected all response variables of Ambystoma maculatum significantly, and significantly reduced survival and metamorphosis of A. opacum. Increased density significantly influenced SVL, lipid reserves, growth rate and metamorphosis of A. maculatum. 4. The effects of carbaryl and increased density on per cent metamorphosis were nearly additive, but were generally less than additive on other variables. 5. The negative effects of chemical contamination on salamanders were likely because of pesticide‐induced reductions of food resources, as zooplankton abundance decreased by as much as 97% following carbaryl application. 6. Our study demonstrates the importance of the interactive effects that chemical contamination and natural environmental factors have on salamanders.