Multiscale resistant kernel surfaces derived from inferred gene flow: An application with vernal pool breeding salamanders.

The importance of assessing spatial data at multiple scales when modelling species-environment relationships has been highlighted by several empirical studies. However, no landscape genetics studies have optimized landscape resistance surfaces by evaluating relevant spatial predictors at multiple spatial scales. Here, we model multiscale/layer landscape resistance surfaces to estimate resistance to inferred gene flow for two vernal pool breeding salamander species, spotted (Ambystoma maculatum) and marbled (A. opacum) salamanders. Multiscale resistance surface models outperformed spatial layers modelled at their original spatial scale. A resistance surface with forest land cover at a 500-m Gaussian kernel bandwidth and normalized vegetation index at a 100-m Gaussian kernel bandwidth was the top optimized resistance surface for A. maculatum, while a resistance surface with traffic rate and topographic curvature, both at a 500-m Gaussian kernel bandwidth, was the top optimized resistance surface for A. opacum. Species-specific resistant kernels were fit at all vernal pools in our study area with the optimized multiscale/layer resistance surface controlling kernel spread. Vernal pools were then evaluated and scored based on surrounding upland habitat (local score) and connectivity with other vernal pools on the landscape, with resistant kernels driving vernal pool connectivity scores. As expected, vernal pools that scored highest were in areas within forested habitats and with high vernal pool densities and low species-specific landscape resistance. Our findings highlight the success of using a novel analytical approach in a multiscale framework with applications beyond vernal pool amphibian conservation.

Abundance and phenology patterns of two pond-breeding salamanders determine species interactions in natural populations.

Phenology often determines the outcome of interspecific interactions, where early-arriving species often dominate interactions over those arriving later. The effects of phenology on species interactions are especially pronounced in aquatic systems, but the evidence is largely derived from experimental studies. We examined whether differences in breeding phenology between two pond-breeding salamanders (Ambystoma annulatum and A. maculatum) affected metamorph recruitment and demographic traits within natural populations, with the expectation that the fall-breeding A. annulatum would negatively affect the spring-breeding A. maculatum. We monitored populations of each species at five ponds over 4 years using drift fences. Metamorph abundance and survival of A. annulatum were affected by intra- and interspecific processes, whereas metamorph size and date of emigration were primarily influenced by intraspecific effects. Metamorph abundance, snout-vent length, date of emigration and survival for A. maculatum were all predicted by combinations of intra- and interspecific effects, but often showed negative relationships with A. annulatum metamorph traits and abundance. Size and date of metamorphosis were strongly correlated within each species, but in opposite patterns (negative for A. annulatum and positive for A. maculatum), suggesting that the two species use alternative strategies to enhance terrestrial survival and that these factors may influence their interactions. Our results match predictions from experimental studies that suggest recruitment is influenced by intra- and interspecific processes which are determined by phenological differences between species. Incorporating spatiotemporal variability when modeling population dynamics is necessary to understand the importance of phenology in species interactions, especially as shifts in phenology occur under climate change.

Evolutionary basis of mitonuclear discordance between sister species of mole salamanders (Ambystoma sp.).

Distinct genetic markers should show similar patterns of differentiation between species reflecting their common evolutionary histories, yet there are increasing examples of differences in the biogeographic distribution of species-specific nuclear (nuDNA) and mitochondrial DNA (mtDNA) variants within and between species. Identifying the evolutionary processes that underlie these anomalous patterns of genetic differentiation is an important goal. Here, we analyse the putative mitonuclear discordance observed between sister species of mole salamanders (Ambystoma barbouri and A. texanum) in which A. barbouri-specific mtDNA is found in animals located within the range of A. texanum. We test three hypotheses for this discordance (undetected range expansion, mtDNA introgression, and hybridization) using nuDNA and mtDNA data analysed with methods that varied in the parameters estimated and the timescales measured. Results from a Bayesian clustering technique (structure), bidirectional estimates of gene flow (migrate-n and IMa2) and phylogeny-based methods (*beast, bucky) all support the conclusion that the discordance is due to geographically restricted mtDNA introgression from A. barbouri into A. texanum. Limited data on species-specific tooth morphology match this conclusion. Significant differences in environmental conditions exist between sites where A. texanum with and without A. barbouri-like mtDNA occur, suggesting a possible role for selection in the process of introgression. Overall, our study provides a general example of the value of using complimentary analyses to make inferences of the directionality, timescale, and source of mtDNA introgression in animals.

Evolution of maternal egg size effects in sister salamander species.

Egg size varies genetically and with the maternal environment. It is correlated with and can act as a resource fueling variation in many other key life history traits. This study examined hypotheses about how plastic responses of offspring to yolk variation evolve (and contribute to phenotypic evolution) when maternal investment in egg size evolves. I used a split-clutch, controlled, surgical experiment with a longitudinal (repeated-measures) design to examine the effects of yolk removal on sister salamander species with distinct egg and larval phenotypes. Yolk removal had large effects in the derived larger-egged species, A. barbouri, and greatly reduced effects in A. texanum. Early hatching and smaller larval body size was only found in A. barbouri and survival rates decreased more in A. barbouri. These results provide strong experimental evidence that as female salamanders evolve greater yolk investment in each egg, offspring coevolve an increased magnitude of phenotypic plasticity in response to yolk variation across a suite of life history traits. Yolk therefore acts as an integrator of phenotypes that allows females to modify modules of life history traits together (facilitating adaptation). When organisms invade new environments, complex integrated phenotypes may evolve via correlated responses to increased maternal investment, yet individual traits can be coupled or decoupled to yolk quantity variation in different species.

Genetic and genomic interactions of animals with different ploidy levels.

Polyploid animals have independently evolved from diploids in diverse taxa across the tree of life. We review a few polyploid animal species or biotypes where recently developed molecular and cytogenetic methods have significantly improved our understanding of their genetics, reproduction and evolution. Mitochondrial sequences that target the maternal ancestor of a polyploid show that polyploids may have single (e.g. unisexual salamanders in the genus Ambystoma) or multiple (e.g. parthenogenetic polyploid lizards in the genus Aspidoscelis) origins. Microsatellites are nuclear markers that can be used to analyze genetic recombinations, reproductive modes (e.g. Ambystoma) and recombination events (e.g. polyploid frogs such as Pelophylax esculentus). Hom(e)ologous chromosomes and rare intergenomic exchanges in allopolyploids have been distinguished by applying genome-specific fluorescent probes to chromosome spreads. Polyploids arise, and are maintained, through perturbations of the 'normal' meiotic program that would include pre-meiotic chromosome replication and genomic integrity of homologs. When possible, asexual, unisexual and bisexual polyploid species or biotypes interact with diploid relatives, and genes are passed from diploid to polyploid gene pools, which increase genetic diversity and ultimately evolutionary flexibility in the polyploid. When diploid relatives do not exist, polyploids can interact with another polyploid (e.g. species of African Clawed Frogs in the genus Xenopus). Some polyploid fish (e.g. salmonids) and frogs (Xenopus) represent independent lineages whose ancestors experienced whole genome duplication events. Some tetraploid frogs (P. esculentus) and fish (Squaliusalburnoides) may be in the process of becoming independent species, but diploid and triploid forms of these 'species' continue to genetically interact with the comparatively few tetraploid populations. Genetic and genomic interaction between polyploids and diploids is a complex and dynamic process that likely plays a crucial role for the evolution and persistence of polyploid animals. See also other articles in this themed issue.

Estimating ancestry and heterozygosity of hybrids using molecular markers.

BACKGROUND: Hybridization, genetic mixture of distinct populations, gives rise to myriad recombinant genotypes. Characterizing the genomic composition of hybrids is critical for studies of hybrid zone dynamics, inheritance of traits, and consequences of hybridization for evolution and conservation. Hybrid genomes are often summarized either by an estimate of the proportion of alleles coming from each ancestral population or classification into discrete categories like F1, F2, backcross, or merely "hybrid" vs. "pure". In most cases, it is not realistic to classify individuals into the restricted set of classes produced in the first two generations of admixture. However, the continuous ancestry index misses an important dimension of the genotype. Joint consideration of ancestry together with interclass heterozygosity (proportion of loci with alleles from both ancestral populations) captures all of the information in the discrete classification without the unrealistic assumption that only two generations of admixture have transpired. METHODS: I describe a maximum likelihood method for joint estimation of ancestry and interclass heterozygosity. I present two worked examples illustrating the value of the approach for describing variation among hybrid populations and evaluating the validity of the assumption underlying discrete classification. RESULTS: Naively classifying natural hybrids into the standard six line cross categories can be misleading, and false classification can be a serious problem for datasets with few molecular markers. My analysis underscores previous work showing that many (50 or more) ancestry informative markers are needed to avoid erroneous classification. CONCLUSION: Although classification of hybrids might often be misleading, valuable inferences can be obtained by focusing directly on distributions of ancestry and heterozygosity. Estimating and visualizing the joint distribution of ancestry and interclass heterozygosity is an effective way to compare the genetic structure of hybrid populations and these estimates can be used in classic quantitative genetic methods for assessing additive, dominant, and epistatic genetic effects on hybrid phenotypes and fitness. The methods are implemented in a freely available package "HIest" for the R statistical software ( http://cran.r-project.org/web/packages/HIest/index.html).

Time and time again: unisexual salamanders (genus Ambystoma) are the oldest unisexual vertebrates.

BACKGROUND: The age of unisexual salamanders of the genus Ambystoma is contentious. Recent and ancient evolutionary histories of unisexual Ambystoma were proposed by a few separate studies that constructed phylogenies using mitochondrial DNA markers (cytochrome b gene vs. non-coding region). In contrast to other studies showing that unisexual Ambystoma represent the most ancient unisexual vertebrates, a recent study by Robertson et al. suggests that this lineage has a very recent origin of less than 25,000 years ago. RESULTS: We re-examined the phylogenetic relationship of the unisexuals to A. barbouri from various populations using both mitochondrial markers as well as the complete mitochondrial genomes of A. barbouri and a unisexual individual from Kentucky. Lineage dating was conducted using BEAST and MultiDivTime on a complete mitochondrial genome phylogeny. Our results support a monophyletic lineage for unisexual Ambystoma that shares its most recent common ancestor with an A. barbouri lineage from western Kentucky. In contrast to the Robertson et al.'s study, no A. barbouri individual shared an identical or almost identical cytochrome b haplotype with any unisexual. Molecular dating supports an early Pliocene origin for the unisexual linage (approximately 5 million years ago). We propose that a unisexual-like cytochrome b numt (or pseudogene) exists in the controversial A. barbouri individuals from Kentucky, which was likely the cause of an erroneous phylogeny and time estimate in Robertson et al.'s study. CONCLUSION: We reject a recent origin of unisexual Ambystoma and provide strong evidence that unisexual Ambystoma are the most ancient unisexual vertebrates known to exist. The likely presence of an ancient cytochrome b numt in some Kentucky A. barbouri represents a molecular "fossil" reinforcing the hypothesis that these individuals are some of the closest extant relatives to unisexual Ambystoma.

An examination of intergenomic exchanges in A. laterale-dependent unisexual salamanders in the genus Ambystoma.

The evolutionary longevity of unisexual salamanders in the genus Ambystoma may be attributed to their flexible reproductive system and meiotic intergenomic interactions. More than 20 different unisexual genomic combinations have been found and all the unisexuals live with at least one of the sexual species A. laterale, A. jeffersonianum, A. texanum, and A. tigrinum. Most unisexuals rely on A. laterale orA. jeffersonianum as sperm donors. Intergenomic exchanges were previously reported in A. jeffersonianum-dependent unisexual populations from southern Ontario and are believed to be an important meiotic mechanism that provides genetic diversity. The situations of intergenomic exchanges in many of A. laterale-dependent unisexual populations, however, remain unknown. In this study we collected specimens from populations where unisexuals use A. laterale as sperm donors, including mainly triploid A. 2 laterale--jeffersonianum (or LLJ), and employed genomic in situ hybridization (GISH) to examine the intergenomic exchanges. Five patterns of intergenomic exchanges were detected. Intergenomic exchanges are less frequent and lack association among populations in A. laterale-dependent than in A. jeffersonianum- dependent unisexual populations, but more recombined homeologues were observed in LLJ unisexuals. Our observations show that the patterns and frequencies of intergenomic exchanges are different when unisexuals use different sexual species as sperm donors. We propose a few possible mechanisms that may account for these different observations.

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-2alpha, caspase activation and recruitment domain (CARD) and beta-OH-steroid oxidoreductase. Selection analysis showed evidence of very strong purifying selection on eIF-2alpha, purifying selection within certain viral clades on CARD and positive selection on beta-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.

Hybrid vigor between native and introduced salamanders raises new challenges for conservation.

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.

Intergenomic translocations in unisexual salamanders of the genus Ambystoma (Amphibia, Caudata).

Intergenomic interactions that include homoeologous recombinations and intergenomic translocations are commonly observed in plant allopolyploids. Homoeologous recombinations have recently been documented in unisexual salamanders in the genus Ambystoma and revealed exchanged chromosomal segments between A. laterale and A.jeffersonianum genomes in individual unisexuals. We discovered intergenomic translocations in two widespread unisexual triploids A.laterale--2 jeffersonianum (or LJJ) and its tetraploid derivative A.laterale--3 jeffersonianum (or LJJJ) by genomic in situ hybridization (GISH). Two different types of intergenomic translocations were observed in two unisexual populations and one contained novel chromosomes generated by an intergenomic reciprocal translocation. We also observed chromosome deletions in several individuals and these chromosome fragmentations were all derived from the A. jeffersonianum genome. These observed intergenomic reciprocal translocations are believed to be caused by non-homologous pairing during meiosis followed by breakage-rejoining events. Genomes of unisexual Ambystoma undergo complicated structural changes that include various intergenomic exchanges that offer unisexuals genetic and phenotypic complexity to escape their evolutionary demise. Unisexual Ambystoma have persisted as natural nuclear genomic hybrids for about four million years. These unisexuals provide a vertebrate model system to examine the interaction of distinct genomes and to evaluate the corresponding genetic, developmental and evolutionary implications of intergenomic exchanges. Intergenomic translocations and homoeologous recombinations appear to be frequent chromosome reconstruction events among unisexual Ambystoma.

Phylogeographic concordance in the southeastern United States: the flatwoods salamander, Ambystoma cingulatum, as a test case.

Well-supported, congruent phylogeographic and biogeographic patterns permit the development of a priori phylogeographic and distributional predictions. In the southeastern Coastal Plain of the United States, the common discovery of east-west disjunctions (phylogeographic breaks and species' distributional boundaries) suggests that similar disjunctions should occur in codistributed taxa. Despite the near ubiquity of these disjunctions, the most recent morphological analyses of the flatwoods salamander, Ambystoma cingulatum, indicate that none occur in this low-vagility, Coastal Plain endemic. We conducted molecular and morphological analyses to test whether the flatwoods salamander is an exception to this common biogeographic pattern. Assessing geographic variation in this species is also an important management tool for this threatened, declining amphibian. We demonstrate that flatwoods salamanders, as predicted by comparisons to codistributed taxa, are polytypic with a major disjunction at the Apalachicola River. This drainage is a common site for east-west phylogeographic breaks, probably because repeated marine embayments during the Pliocene and Pleistocene interglacials generated barriers to gene flow. Based on mitochondrial DNA, morphology, and allozymes, we recognize two species of flatwoods salamanders -- Ambystoma cingulatum to the east of the Apalachicola drainage and Ambystoma bishopi to the west. Given this increased diversity, the conservation status of these two taxa may warrant re-evaluation. More generally, these results emphasize that in the absence of taxon-specific data, established comparative patterns can provide strong expectations for designing management units for unstudied species of conservation concern.

Color change and color-dependent behavior in response to predation risk in the salamander sister species Ambystoma barbouri and Ambystoma texanum.

Although many organisms show multiple types of trait responses to predation risk (e.g., shifts in behavior, morphology, color, chemistry or life history), relatively few studies have examined how prey integrate these multiple responses. We studied the joint expression of color and behavioral responses to predation risk in two sister species of salamander larvae that live in habitats with different selection pressures. We examined responses to predation risk in three situations that differed in availability of refuge and substrate color heterogeneity, and thus availability of behavioral options for reducing risk. Relative to Ambystoma texanum, A. barbouri larvae were darker in color and showed a greater range of color change. With no variation in background color or refuge available, both species exhibited color change to better match the available background. The degree of color change showed by both species, however, did not depend on predation risk. Given the option to choose between light and dark substrates, A. texanum exhibited behavioral background matching (i.e., they preferred substrates that matched their own body color), while A. barbouri's substrate preferences did not depend on their initial body color. Instead, A. barbouri responded to risk by showing a strong preference for dark substrates, followed by a change to a darker body color. With refuge available, A. texanum's refuge use was color-dependent; larvae that were well camouflaged spent less time in refuge. In contrast, A. barbouri showed strong refuge use in response to risk, regardless of their body color. Overall, these results reflect how conflicting selection pressures (predation risk, habitat ephemerality, risk of UV damage) and species differences in mean color and ability to change color can govern the interplay of complementary and compensatory behavioral and color responses to predation risk.

Biogeography of the southeastern United States: a comparison of salamander phylogeographic studies.

Most phylogeographic studies of species from the southeastern United States have shown a simple east-west division of mtDNA variation. However, a study of the salamander Ambystoma maculatum resulted in a more complex pattern that includes a close affinity between populations from the Central Highlands of Missouri and Arkansas and the Coastal Plain separated by a genetically distinct central group of populations. We test the generality of this observation by surveying mitochondrial DNA (mtDNA) variation in the closely related species A. talpoideum. An Ambystoma-specific intergenic spacer was amplified and sequenced. The 26 resulting haplotypes varied from 380 to 800 base pairs, and alignments, including the outgroup, required 101 insertions/deletions. Sequence divergence among haplotypes ranged from 0.001 to 0.758. Population subdivision was extensive (theta = 0.64). Phylogenetic analysis of A. talpoideum mtDNA sequence reveals a close relationship between the populations from the Central Highlands and the Coastal Plain. This result is similar to that obtained for A. maculatum, although the A. talpoideum clade is not as well differentiated from its sister clades. We discuss the differences and similarities between the two Ambystoma species and previous studies and call for increased focus on multiple species with similar ecologies as a way to detect subtle biogeographic events.

Small effective population size in the long-toed salamander.

The effective population sizes (Ne) of six populations of the long-toed salamander (Ambystoma macrodactylum) from Montana and Idaho, USA were estimated from allozyme data from samples collected in 1978, 1996 and 1997 using the temporal allele frequency method. Five of the six estimates ranged from 23 to 207 (mean = 123 +/- 79); one estimate was indistinguishable from infinity. In order to infer the actual Ne of salamander populations, we compared the frequency distribution of our observed Ne estimates with distributions obtained from simulated populations of known Ne. Our observed Ne estimate distribution was consistent with distributions from simulated populations with Ne values of 10, 25, and 50, suggesting an actual Ne for each of the six salamander populations of less than 100. This Ne estimate agrees with most other Ne estimates for amphibians. We conclude by discussing the conservation implications of small Ne values in amphibians in the context of increasing isolation of populations due to habitat fragmentation.