Distribution of tiger salamanders in northern Sonora, Mexico: comparison of sampling methods and possible implications for an endangered subspecies

Many aquatic species in the arid USA-Mexico borderlands region are imperiled, but limited information on distributions and threats often hinders management. To provide information on the distribution of the Western Tiger Salamander (Ambystoma mavortium), including the USA-federally endangered Sonoran Tiger Salamander (Ambystoma mavortium stebbinsi), we used traditional (seines, dip-nets) and modern (environmental DNA [eDNA]) methods to sample 91 waterbodies in northern Sonora, Mexico, during 2015-2018. The endemic Sonoran Tiger Salamander is threatened by introgressive hybridization and potential replacement by another sub-species of the Western Tiger Salamander, the non-native Barred Tiger Salamander (A. m. mavortium). Based on occupancy models that accounted for imperfect detection, eDNA sampling provided a similar detection probability (0.82 [95% CI: 0.56-0.94]) as seining (0.83 [0.46-0.96]) and much higher detection than dip-netting (0.09 [0.02-0.23]). Volume of water filtered had little effect on detection, possibly because turbid sites had greater densities of salamanders. Salamanders were estimated to occur at 51 sites in 3 river drainages in Sonora. These results indicate tiger salamanders are much more widespread in northern Sonora than previously documented, perhaps aided by changes in land and water management practices. However, because the two subspecies of salamanders cannot be reliably distinguished based on morphology or eDNA methods that are based on mitochondrial DNA, we are uncertain if we detected only native genotypes or if we documented recent invasion of the area by the non-native sub-species. Thus, there is an urgent need for methods to reliably distinguish the subspecies so managers can identify appropriate interventions.

Geography is more important than life history in the recent diversification of the tiger salamander complex

The North American tiger salamander species complex, including its best-known species, the Mexican axolotl, has long been a source of biological fascination. The complex exhibits a wide range of variation in developmental life history strategies, including populations and individuals that undergo metamorphosis; those able to forego metamorphosis and retain a larval, aquatic lifestyle (i.e., paedomorphosis); and those that do both. The evolution of a paedomorphic life history state is thought to lead to increased population genetic differentiation and ultimately reproductive isolation and speciation, but the degree to which it has shaped population- and species-level divergence is poorly understood. Using a large multilocus dataset from hundreds of samples across North America, we identified genetic clusters across the geographic range of the tiger salamander complex. These clusters often contain a mixture of paedomorphic and metamorphic taxa, indicating that geographic isolation has played a larger role in lineage divergence than paedomorphosis in this system. This conclusion is bolstered by geography-informed analyses indicating no effect of life history strategy on population genetic differentiation and by model-based population genetic analyses demonstrating gene flow between adjacent metamorphic and paedomorphic populations. This fine-scale genetic perspective on life history variation establishes a framework for understanding how plasticity, local adaptation, and gene flow contribute to lineage divergence. Many members of the tiger salamander complex are endangered, and the Mexican axolotl is an important model system in regenerative and biomedical research. Our results chart a course for more informed use of these taxa in experimental, ecological, and conservation research.

Short-term storage of tiger salamander (Ambystoma tigrinum) spermatozoa: The effect of collection type, temperature and time

The aims of this project were to characterize tiger salamander (Ambystoma tigrinum) spermatozoa motility over time, when excreted as either milt or spermic urine prior to packaging into a spermatophore, and to determine the effect of temperature on sperm motility. A split-plot design was utilized to assess the motility of the two pre-spermatophore sample types at two temperatures, 0°C and 20°C (n = 10 for each treatment). Spermiation was induced through exogenous hormone treatment of luteinizing hormone releasing hormone analog in order to collect both milt and spermic urine, which were evaluated for motility, divided into two separate aliquots, and subsequently stored in either an ice-bath (0°C) or on the benchtop (20°C). The decay rate of sperm motility was assessed by reevaluating subsamples at 0.5, 1, 2, 3, 5, 7, and 24 hours following the initial assessment. Results showed that sperm stored at 0°C had significantly higher progressive, non-progressive, and total motility for both sperm collection types over time. An interaction was found between collection type and time, with milt exhibiting lower initial motility that was more sustainable over time, compared to spermic urine. For both milt and spermic urine, motility decreased rapidly with storage duration, indicating samples should be used as soon as possible to maximize motility for in-vitro fertilization and cryopreservation. This is the first study to describe the differences in sperm motility between milt and spermic urine from an internally fertilizing caudate and demonstrates the benefits of near freezing temperatures on sperm longevity.

ATP-mediated Increase in H+ Flux from Retinal Müller Cells: a Role for Na+/H+ Exchange

Small alterations in extracellular H+ can profoundly alter neurotransmitter release by neurons. We examined mechanisms by which extracellular ATP induces an extracellular H+ flux from Müller glial cells, which surround synaptic connections throughout the vertebrate retina. Müller glia were isolated from tiger salamander retinae and H+ fluxes examined using self-referencing H+-selective microelectrodes. Experiments were performed in 1 mM HEPES with no bicarbonate present. Replacement of extracellular sodium by choline decreased H+ efflux induced by 10 µM ATP by 75%. ATP-induced H+ efflux was also reduced by Na+/H+ exchange inhibitors. Amiloride reduced H+ efflux initiated by 10 µM ATP by 60%, while 10 µM cariporide decreased by 37%, and 25 µM zoniporide reduced H+ flux by 32%. ATP-induced H+ fluxes were not significantly altered by the K+/H+ pump blockers SCH28080 or TAK438, and replacement of all extracellular chloride with gluconate was without effect on H+ fluxes. Recordings of ATP-induced H+ efflux from cells simultaneously whole-cell voltage-clamped revealed no effect of membrane potential from -70 mV to 0 mV. Restoration of extracellular potassium after cells were bathed in 0 mM potassium produced a transient alteration in ATP-dependent H+ efflux. The transient response to extracellular potassium occurred only when extracellular sodium was present and was abolished by 1 mM ouabain, suggesting alterations in sodium gradients mediated by Na+/K ATPase activity. Our data indicate that the majority of H+ efflux elicited by extracellular ATP from isolated Müller cells is mediated by Na+/H+ exchange.

Geography is more important than life history in the recent diversification of the tiger salamander complex

The North American tiger salamander species complex, including its flagship species the axolotl, has long been a source of biological fascination. The complex exhibits a wide range of variation in developmental life history strategies, including populations and individuals that undergo metamorphosis and those able to forego metamorphosis and retain a larval, aquatic lifestyle (i.e., paedomorphosis). Such disparate life history strategies are assumed to cause populations to become reproductively isolated, but the degree to which they have actually shaped population- and species-level boundaries is poorly understood. Using a large multi-locus dataset from hundreds of samples across North America, we identified genetic clusters with clear signs of admixture across the geographic range of the tiger salamander complex. Population clusters often contain a mixture of paedomorphic and metamorphic taxa, and we conclude that geography has played a large role in driving lineage divergence relative to obligate paedomorphosis in this system. This conclusion is bolstered by model-based analyses demonstrating gene flow between metamorphic and paedomorphic populations. Even the axolotl, a paedomorphic species with an isolated native range, apparently has a history of gene flow with its neighboring populations. This fine-scale genetic perspective on life-history variation establishes a framework for understanding how plasticity, local adaptation, and gene flow contribute to lineage divergence. The axolotl is currently used as the vertebrate model system in regenerative biology, and our findings chart a course for more informed use of these and other tiger salamander species in experimental and field research, including conservation priorities.Significance StatementPopulation structure and speciation are shaped by a variety of biotic and abiotic factors. In the tiger salamander complex, one factor that may influence diversification is life history: some taxa are obligately paedomorphic–a condition where adults maintain an aquatic, larval phenotype–while others are facultatively paedomorphic or entirely metamorphic. Using a large multi-locus dataset, we found evidence of gene flow and/or panmixia between obligately and facultatively paedomorphic taxa, suggesting that an obligately paedomorphic life history is not a strong driver of speciation in the tiger salamander complex. We also recovered a history of gene flow between the critically endangered axolotl and its neighboring populations, providing important information for its conservation and captive management.