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When invasive and endangered native taxa hybridize, the resulting admixture introduces novel conservation challenges. Across a large region of central California, a hybrid swarm consisting of admixed endangered California tiger salamanders (CTS) (Ambystoma californiense) and introduced barred tiger salamanders (BTS) (Ambystoma mavortium) has replaced native populations, threatening the genetic integrity of CTS and the vernal pool systems they inhabit. We employed a large-scale, genomically informed field experiment to test whether shortening breeding pond hydroperiod would favor native CTS genotypes. We constructed 14 large, seminatural ponds to evaluate the effect of hydroperiod duration on larval survival and mass at metamorphosis. We tracked changes in non-native allele frequencies with a 5237-gene exon capture array and employed a combination of custom Bayesian and generalized linear models to quantify the effect of pond duration on salamander fitness. Earlier work on this system showed hybrid superiority under many conditions and suggested that hybrids are favored in human-modified ponds with artificially long hydroperiods. Consistent with these earlier studies, we found overwhelming evidence for hybrid superiority. Very short hydroperiods substantially reduced the mass (1.1-1.5 fold) and survival probability (10-13 fold) of both native and hybrid larvae, confirming that hydroperiod likely exerts a strong selective pressure in the wild. We identified 86 genes, representing 1.8% of 4723 screened loci, that significantly responded to this hydroperiod-driven selection. In contrast to earlier work, under our more natural experimental conditions, native CTS survival and size at metamorphosis were always less than hybrids, suggesting that hydroperiod management alone will not shift selection to favor native larval genotypes. However, shortening pond hydroperiod may limit productivity of hybrid ponds, complementing other strategies to remove hybrids while maintaining vernal pool ecosystems. This study confirms and expands on previous work that highlights the importance of hydroperiod management to control invasive aquatic species., (© 2023 The Authors. Conservation Biology published by Wiley Periodicals LLC on behalf of Society for Conservation Biology.)

Proteins encoded by antigen-processing genes (APGs) provide major histocompatibility complex (MHC) class I (MHC-I) with antigenic peptides. In mammals, polymorphic multigenic MHC-I family is served by monomorphic APGs, whereas in certain nonmammalian species both MHC-I and APGs are polymorphic and coevolve within stable haplotypes. Coevolution was suggested as an ancestral gnathostome feature, presumably enabling only a single highly expressed classical MHC-I gene. In this view coevolution, while optimizing some aspects of adaptive immunity, would also limit its flexibility by preventing the expansion of classical MHC-I into a multigene family. However, some nonmammalian taxa, such as salamanders, have multiple highly expressed MHC-I genes, suggesting either that coevolution is relaxed or that it does not prevent the establishment of multigene MHC-I. To distinguish between these two alternatives, we use salamanders (30 species from 16 genera representing six families) to test, within a comparative framework, a major prediction of the coevolution hypothesis: the positive correlation between MHC-I and APG diversity. We found that MHC-I diversity explained both within-individual and species-wide diversity of two APGs, TAP1 and TAP2, supporting their coevolution with MHC-I, whereas no consistent effect was detected for the other three APGs (PSMB8, PSMB9, and TAPBP). Our results imply that although coevolution occurs in salamanders, it does not preclude the expansion of the MHC-I gene family. Contrary to the previous suggestions, nonmammalian vertebrates thus may be able to accommodate diverse selection pressures with flexibility granted by rapid expansion or contraction of the MHC-I family, while retaining the benefits of coevolution between MHC-I and TAPs., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Hybridization between native and non-native species is an ongoing global conservation threat. Hybrids that exhibit traits and tolerances that surpass parental values are of particular concern, given their potential to outperform native species. Effective management of hybrid populations requires an understanding of both physiological performance and the underlying mechanisms that drive transgressive hybrid traits. Here, we explore several aspects of the hybridization between the endangered California tiger salamander (Ambystoma californiense; CTS) and the introduced barred tiger salamander (Ambystoma mavortium; BTS). We assayed critical thermal maximum (CTMax) to compare the ability of CTS, BTS and F1 hybrids to tolerate acute thermal stress, and found that hybrids exhibit a wide range of CTMax values, with 33% (4/12) able to tolerate temperatures greater than either parent. We then quantified the genomic response, measured at the RNA transcript level, of each salamander, to explore the mechanisms underlying thermal tolerance strategies. We found that CTS and BTS have strikingly different values and tissue-specific patterns of overall gene expression, with hybrids expressing intermediate values. F1 hybrids display abundant and variable degrees of allele-specific expression (ASE), likely arising from extensive compensatory evolution in gene regulatory mechanisms between CTS and BTS. We found evidence that the proportion of genes with allelic imbalance in individual hybrids correlates with their CTMax, suggesting a link between ASE and expanded thermal tolerance that may contribute to the success of hybrid salamanders in California. Future climate change may further complicate management of CTS if hybrid salamanders are better equipped to deal with rising temperatures., (© 2020 John Wiley & Sons Ltd.)

The use of taxon substitutes for extinct or endangered species is a controversial conservation measure. We use the example of the endangered California tiger salamander (Ambystoma californiense; CTS), which is being replaced by hybrids with the invasive barred tiger salamander (Ambystoma mavortium), to illustrate a strategy for evaluating taxon substitutes based on their position in a multivariate community space. Approximately one-quarter of CTS's range is currently occupied by "full hybrids" with 70% nonnative genes, while another one-quarter is occupied by "superinvasives" where a specific set of 3/68 genes comprising 4% of the surveyed genome is nonnative. Based on previous surveys of natural CTS breeding ponds, we stocked experimental mesocosms with field-verified, realistic densities of tiger salamander larvae and their prey, and used these mesocosms to evaluate ecological equivalency between pure CTS, full hybrids, and superinvasives in experimental pond communities. We also included a fourth treatment with no salamanders present to evaluate the community effects of eliminating Ambystoma larvae altogether. We found that pure CTS and superinvasive larvae were ecologically equivalent, because their positions in the multivariate community space were statistically indistinguishable and they did not differ significantly along any univariate community axes. Full hybrids were ecologically similar, but not equivalent, to the other two genotypes, and the no-Ambystoma treatment was by far the most divergent. We conclude that, at least for the larval stage, superinvasives are adequate taxon substitutes for pure CTS and should probably be afforded protection under the Endangered Species Act. The proper conservation status for full hybrids remains debatable.

Biological invasions and habitat alteration are often detrimental to native species, but their interactions are difficult to predict. Interbreeding between native and introduced species generates novel genotypes and phenotypes, and human land use alters habitat structure and chemistry. Both invasions and habitat alteration create new biological challenges and opportunities. In the intensively farmed Salinas Valley, California (U.S.A.), threatened California tiger salamanders (Ambystoma californiense) have been replaced by hybrids between California tiger salamander and introduced barred tiger salamanders (Ambystoma tigrinum mavortium). We conducted an enclosure experiment to examine the effects habitat modification and relative frequency of hybrid and native California tiger salamanders have on recruitment of salamanders and their prey, Pacific chorus frogs (Pseudacris regilla). We tested whether recruitment differed among genetic classes of tiger salamanders (hybrid or native) and pond hydroperiod (seasonal or perennial). Roughly 6 weeks into the experiment, 70% (of 378 total) of salamander larvae died in 4 out of 6 ponds. Native salamanders survived (n = 12) in these ponds only if they had metamorphosed prior to the die-offs. During die-offs, all larvae of native salamanders died, whereas 56% of hybrid larvae died. We necropsied native and hybrid salamanders, tested water quality, and queried the California Department of Pesticide Regulation database to investigate possible causes of the die-offs. Salamander die-offs, changes in the abundance of other community members (invertebrates, algae, and cyanobacteria), shifts in salamander sex ratio, and patterns of pesticide application in adjacent fields suggest that pesticide use may have contributed to die-offs. That all survivors were hybrids suggests that environmental stress may promote rapid displacement of native genotypes., (© 2012 Society for Conservation Biology.)

Quantifying the influence of the landscape on the genetic structure of natural populations remains an important empirical challenge, particularly for poorly studied, ecologically cryptic species. We conducted an extensive microsatellite analysis to examine the population genetics of the southern long-toed salamander (Ambystoma macrodactylum sigillatum) in a naturally complex landscape. Using spatially explicit modelling, we investigated the influence of the Sierra Nevada topography on potential dispersal corridors between sampled populations. Our results indicate very high-genetic divergence among populations, high within-deme relatedness, and little evidence of recent migration or population admixture. We also discovered unexpectedly high between-year genetic differentiation (F(ST)) for breeding sites, suggesting that breeding groups vary over localized space and time. While environmental factors associated with high-elevation montane habitats apparently play an important role in shaping population differentiation, additional, species-specific biological processes must also be operating to account for observed deviations from temporal, among-year panmixia. Our study emphasizes the population-level insights that can be gained from high-density sampling in space and time, and the highly substructured population biology that may characterize amphibians in extreme montane habitats.

Current conservation mitigation plans often fail to ensure full in-kind habitat replacement for endangered species, which suggests the need for improved methods for calculating mitigation credits. A simple, yet biologically meaningful method for calculating mitigation credits would be to let the number of mitigation credits assigned to a parcel of land scale with the reproductive value of the individuals occupying that parcel. This can be accomplished by dividing the population into 2 or more subdivisions with different reproductive values, calculating the densities of these subdivisions as a function of one or more habitat parameters, and then forming a weighted sum of these densities such that each density distribution is weighted by the reproductive value of its respective subdivision of the population. This weighted sum is the density distribution of reproductive value, and by integrating it over a particular parcel, one can determine the mitigation value of that parcel. We carried out this procedure for a population of California tiger salamanders (Ambystoma californiense), with distance from breeding site as our habitat parameter and the 3 visually identifiable age classes (adults, juveniles, and metamorphs) as our population subdivisions. This led to a density distribution of reproductive value that decreased exponentially with increasing distance from a breeding site. Mitigation strategies derived from this function will be more likely to ensure the persistence of California tiger salamander populations than current approaches, which assign all land within 1.6 km of a breeding site the same mitigation value. Use of the density distribution of reproductive value as a basis for mitigation plans is a procedure that can be applied to all endangered species, and it should improve the quality of mitigation decisions.

Hybridization between differentiated lineages can have many different consequences depending on fitness variation among hybrid offspring. When introduced organisms hybridize with natives, the ensuing evolutionary dynamics may substantially complicate conservation decisions. Understanding the fitness consequences of hybridization is an important first step in predicting its evolutionary outcome and conservation impact. Here, we measured natural selection caused by differential viability of hybrid larvae in wild populations where native California Tiger Salamanders (Ambystoma californiense) and introduced Barred Tiger Salamanders (Ambystoma tigrinum mavortium) have been hybridizing for 50-60 years. We found strong evidence of hybrid vigor; mixed-ancestry genotypes had higher survival rates than genotypes containing mostly native or mostly introduced alleles. Hybrid vigor may be caused by heterozygote advantage (overdominance) or recombinant hybrid vigor (due to epistasis or complementation). These genetic mechanisms are not mutually exclusive, and we find statistical support for both overdominant and recombinant contributions to hybrid vigor in larval tiger salamanders. Because recombinant homozygous genotypes can breed true, a single highly fit genotype with a mosaic of native and introduced alleles may eventually replace the historically pure California Tiger Salamander (listed as Threatened under the U.S. Endangered Species Act). The management implications of this outcome are complex: Genetically pure populations may not persist into the future, but average fitness and population viability of admixed California Tiger Salamanders may be enhanced. The ecological consequences for other native species are unknown.

Genetic introgression from introduced species into native populations is a growing challenge for biological conservation, and one that raises unique practical and ethical issues. Here, we describe the extent of introgression between native California tiger salamanders (Ambystoma californiense) and introduced barred tiger salamanders (A. tigrinum mavortium) relative to habitat, distance from introduction sites, and watershed boundaries. We used ancestry informative markers (AIMs) to characterize the degree of introgression at 85 sites within the range of A. californiense. Eight unlinked markers showed concordant patterns, indicating that different chromosomal segments are introgressing at similar rates. The current distribution of introduced alleles is largely contained in the Salinas Valley, California. Within it, the distribution of nonnative alleles was best explained at a broad geographic scale by the history of introductions, with limited introgression beyond 12 km from multiple independent release sites. The spatial transition from highly admixed to nearly pure native populations was abrupt, suggesting either cryptic barriers to dispersal or locally rapid displacement of natives by an advancing hybrid swarm. At a more ecological level, highly modified perennial breeding ponds had higher introduced allele frequencies than more natural seasonal ponds, suggesting greater invasion success in perennial breeding ponds. Management favoring natural habitat characteristics may substantially decrease the rate of spread of introduced alleles.

Delimiting the boundaries of species involved in radiations is critical to understanding the tempo and mode of lineage formation. Single locus gene trees may or may not reflect the underlying pattern of population divergence and lineage formation, yet they constitute the vast majority of the empirical data in species radiations. In this study we make use of an expressed sequence tag (EST) database to perform nuclear (nDNA) and mitochondrial (mtDNA) genealogical tests of species boundaries in Ambystoma ordinarium, a member of an adaptive radiation of metamorphic and paedomorphic salamanders (the Ambystoma tigrinum complex) that have diversified across terrestrial and aquatic environments. Gene tree comparisons demonstrate extensive nonmonophyly in the mtDNA genealogy of A. ordinarium, while seven of eight independent nuclear loci resolve the species as monophyletic or nearly so, and diagnose it as a well-resolved genealogical species. A differential introgression hypothesis is supported by the observation that western A. ordinarium localities contain mtDNA haplotypes that are identical or minimally diverged from haplotypes sampled from a nearby paedomorphic species, Ambystoma dumerilii, while most nDNA trees place these species in distant phylogenetic positions. These results provide a strong example of how historical introgression can lead to radical differences between gene trees and species histories, even among currently allopatric species with divergent life history adaptations and morphologies. They also demonstrate how EST-based nuclear resources can be used to more fully resolve the phylogenetic history of species radiations.

We used two different experimental approaches to test the hypothesis that thyroid hormone receptor (TR) variation is associated with alternate life cycles modes in ambystomatid salamanders. In the first experiment, the inheritance of TRalpha and TRbeta genotypes was determined for metamorphic and non metamorphic offspring from backcrosses between Ambystoma mexicanum (an obligate metamorphic-failure species) and metamorphic F1 hybrids (A. mexicanum x A. tigrinum tigrinum). The segregation of TR genotype was independent of the expression of life cycle mode phenotype, and neither TR locus was linked to DNA markers that flank a major-effect locus for life cycle mode. In the second experiment, a portion of the ligand-binding domain of TRalpha and TRbeta was cloned and sequenced for DNA samples from 14 different ambystomatid salamander populations, including obligate metamorphic, facultative metamorphic, and obligate metamorphic-failure taxa. Nucleotide sequence variation was found for both TRalpha and TRbeta, with several nonsynonomous substitutions that presumably code for nonconservative amino acid replacements. However, no general relationship was found between TR allelic variation and life cycle mode among populations or species. These data do not implicate TRs as candidate loci involved in the current maintenance or past evolution of alternate life cycle modes in members of the tiger salamander complex.

Natural and artificial hybrids represent an important source of material for developmental and evolutionary studies of urodeles. We review the available literature on hybrid salamanders, emphasizing the unique developmental insights that these organisms provide. Of particular interest is the application of new molecular tools to identify DNA markers for traditional characters in developmental research, and we discuss our own results using Bulk Segregant Analysis to identify RAPD markers for the white phenotype in the axolotl. We pay particular attention to the inferences that can be drawn from the many disparate crosses between ambystomatid salamanders that vary in their metamorphic response. These crossing experiments suggest that 1) metamorphosis is dominant to paedomorphosis, 2) that different ambystomatids use different genetic mechanisms to block metamorphosis and become sexually mature, larval paedomorphs, and 3) metamorphosis may be controlled by a few genetic loci. As increasingly sophisticated molecular approaches are applied to these and other hybrid crossing schemes, it should be possible to understand the mechanistic basis of a wide variety of developmental characters that differentiate urodele species.