Ecosystem effects of intraspecific variation in a colour polymorphic amphibian.

An emerging consensus suggests that evolved intraspecific variation can be ecologically important. However, evidence that evolved trait variation within vertebrates can influence fundamental ecosystem-level processes remains sparse. In this study, we sought to assess the potential for evolved variation in the spotted salamander (Ambystoma maculatum) to affect aquatic ecosystem properties. Spotted salamanders exhibit a conspicuous polymorphism in the colour of jelly encasing their eggs-some females produce clear jelly, while others produce white jelly. Although the functional significance of jelly colour variation remains largely speculative, evidence for differences in fecundity and the morphology of larvae suggests that the colour morphs might differ in the strength or identity of ecological effects. Here, we assessed the potential for frequency variation in spotted salamander colour morphs to influence fundamental physiochemical and ecosystem properties-dissolved organic carbon, conductivity, acidity and primary production-with a mesocosm experiment. By manipulating colour morph frequency across a range of larval densities, we were able to demonstrate that larva density and colour morph variation were ecologically relevant: population density reduced dissolved organic carbon and increased primary production while mesocosms stocked with white morph larvae tended to have higher dissolved organic carbon and conductivity. Thus, while an adaptive significance of jelly coloration remains hypothetical, our results show that colour morphs differentially influence key ecosystem properties-dissolved organic carbon and conductivity.

Polyphenism predicts actuarial senescence and lifespan in tiger salamanders.

Actuarial senescence (called 'senescence' hereafter) often shows broad variation at the intraspecific level. Phenotypic plasticity likely plays a central role in among-individual heterogeneity in senescence rate (i.e. the rate of increase in mortality with age), although our knowledge on this subject is still very fragmentary. Polyphenism-the unique sub-type of phenotypic plasticity where several discrete phenotypes are produced by the same genotype-may provide excellent study systems to investigate if and how plasticity affects the rate of senescence in nature. In this study, we investigated whether facultative paedomorphosis influences the rate of senescence in a salamander, Ambystoma mavortium nebulosum. Facultative paedomorphosis, a unique form of polyphenism found in dozens of urodele species worldwide, leads to the production of two discrete, environmentally induced phenotypes: metamorphic and paedomorphic individuals. We leveraged an extensive set of capture-recapture data (8948 individuals, 24 years of monitoring) that were analysed using multistate capture-recapture models and Bayesian age-dependent survival models. Multistate models revealed that paedomorphosis was the most common developmental pathway used by salamanders in our study system. Bayesian age-dependent survival models then showed that paedomorphs have accelerated senescence in both sexes and shorter adult lifespan (in females only) compared to metamorphs. In paedomorphs, senescence rate and adult lifespan also varied among ponds and individuals. Females with good body condition and high lifetime reproductive success had slower senescence and longer lifespan. Late-breeding females also lived longer but showed a senescence rate similar to that of early-breeding females. Moreover, males with good condition had longer lifespan than males with poor body condition, although they had similar senescence rates. In addition, late-breeding males lived longer but, unexpectedly, had higher senescence than early-breeding males. Overall, our work provides one of the few empirical cases suggesting that environmentally cued polyphenism could affect the senescence of a vertebrate in nature, thus providing insights on the ecological and evolutionary consequences of developmental plasticity on ageing.

Managing invasive hybrids with pond hydroperiod manipulation in an endangered salamander system.

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.

Manejo de híbridos invasores mediante la manipulación del hidroperiodo de los estanques en el sistema de una salamandra en peligro de extinción Resumen La hibridación entre un taxón nativo en peligro y uno invasor introduce nuevos retos para la conservación. Una plaga híbrida de salamandras tigre de California (STC) (Ambystoma californiense), especie en peligro, y salamandras tigre barradas (STB) (Ambystoma mavortium) introducidas ha reemplazado a las poblaciones nativas en una región amplia del centro de California, lo que amenaza la integridad genética de las STC y el sistema de estanques vernales que habitan. Realizamos un experimento de campo a gran escala y con información genética para probar si la reducción del hidroperiodo reproductivo del estanque favorecería al genotipo de las STC nativas. Construimos 14 estanques seminaturales grandes para analizar el efecto de la duración del hidroperiodo sobre la supervivencia y masa larval durante la metamorfosis. Monitoreamos los cambios en la frecuencia de alelos no nativos con una matriz de captura de exones de 5,237 genes y utilizamos una combinación de modelos lineales generalizados y bayesianos a medida para cuantificar los efectos de la duración del estanque sobre la adaptabilidad de las salamandras. Los primeros trabajos en este sistema mostraron la superioridad híbrida bajo varias condiciones y sugirieron que los híbridos están favorecidos en los estanques con modificaciones antropogénicas e hidroperiodos de larga duración artificial. En coherencia con estos primeros resultados, encontramos evidencia abrumadora de la superioridad híbrida. Los hidroperiodos muy cortos redujeron sustancialmente la masa (1.1­1.5 más veces) y la probabilidad de supervivencia (10­13 más veces) de las larvas nativas e híbridas, lo que confirma que el hidroperiodo probablemente ejerce una fuerte presión selectiva en vida silvestre. Identificamos 86 genes, que representan el 1.8% de los 4,723 loci examinados, que respondieron significativamente a la selección basada en el hidroperiodo. Con las condiciones más naturales de nuestro experimento, y en contraste a nuestros primeros trabajos, la supervivencia y el tamaño de las STC nativas durante la metamorfosis siempre fueron menores a las de los híbridos, lo que sugiere que el manejo del hidroperiodo por sí solo no cambiará la selección a favor de los genotipos larvales nativos. Sin embargo, la reducción del hidroperiodo del estanque puede limitar la productividad de los estanques híbridos y complementar otras estrategias para extirpar a los híbridos mientras que mantiene el ecosistema del estanque vernal. Este estudio confirma y amplía los trabajos anteriores que resaltan la importancia del manejo del hidroperiodo para controlar las especies acuáticas invasoras.

Taxonomy and nomenclature of Oophila amblystomatis (Chlorophyceae, Chlamydomonadales).

The unicellular green alga Oophila amblystomatis was named by Lambert in 1905 based upon its association with egg masses of the spotted salamander Ambystoma maculatum. We collected algal cells from Lambert's original egg capsule preparations that were contributed to Phycotheca Boreali-Americana (PBA) in 1905 and subjected them to DNA extraction and PCR with O. amblystomatis-specific 18S rRNA gene primers. DNA amplified from these preparations was cloned and nine clones were sequenced. Along with representative sequences from the Oophila clade and Chlorophyceae, a phylogenetic tree was inferred. Seven sequences clustered within the Oophila clade and two clustered with Chlamydomonas moewusii, which is included in a sister clade to Oophila. By sequencing algal material from the egg capsules of representative type material we can unambiguously characterize O. amblystomatis and define a monophyletic clade centered on this type material. Accordingly, we reject a recent proposal that this species be transferred to Chlorococcum.

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.

The role of environmental variation in mediating fitness trade-offs for an amphibian polyphenism.

Fitness trade-offs are a foundation of ecological and evolutionary theory because trade-offs can explain life history variation, phenotypic plasticity, and the existence of polyphenisms. Using a 32-year mark-recapture dataset on lifetime fitness for 1093 adult Arizona tiger salamanders (Ambystoma mavortium nebulosum) from a high elevation, polyphenic population, we evaluated the extent to which two life history morphs (aquatic paedomorphs vs. terrestrial metamorphs) exhibited fitness trade-offs in breeding and body condition with respect to environmental variation (e.g. climate) and internal state-based variables (e.g. age). Both morphs displayed a similar response to higher probabilities of breeding during years of high spring precipitation (i.e. not indicative of a morph-specific fitness trade-off). There were likely no climate-induced fitness trade-offs on breeding state for the two life history morphs because precipitation and water availability are vital to amphibian reproduction. Body condition displayed a contrasting response for the two morphs that was indicative of a climate-induced fitness trade-off. While metamorphs exhibited a positive relationship with summer snowpack conditions, paedomorphs were unaffected. Fitness trade-offs from summer snowpack are likely due to extended hydroperiods in temporary ponds, where metamorphs gain a fitness advantage during the summer growing season by exploiting resources that are unavailable to paeodomorphs. However, paedomorphs appear to have the overwintering fitness advantage because they consistently had higher body condition than metamorphs at the start of the summer growing season. Our results reveal that climate and habitat type (metamorphs as predominately terrestrial, paedomorphs as fully aquatic) interact to confer different advantages for each morph. These results advance our current understanding of fitness trade-offs in this well-studied polyphenic amphibian by integrating climate-based mechanisms. Our conclusions prompt future studies to explore how climatic variation can maintain polyphenisms and promote life history diversity, as well as the implications of climate change for polyphenisms.

Bacterial Diversity in Egg Capsular Fluid of the Spotted Salamander Ambystoma maculatum Decreases with Embryonic Development.

Egg capsules within egg masses of the spotted salamander Ambystoma maculatum host a symbiosis with the unicellular green alga Oophila amblystomatis. However, this alga is not the only microbe to inhabit those capsules, and the significance of these additional taxa for the symbiosis is unknown. Spatial and temporal patterns of bacterial diversity in egg capsules of A. maculatum have recently begun to be characterized, but patterns of bacterial diversity as a function of embryonic development are unknown. We sampled fluid from individual capsules in egg masses over a large range of host embryonic development in 2019 and 2020. We used 16S rRNA gene amplicon sequencing to examine how diversity and relative abundance of bacteria changed with embryonic development. In general, bacterial diversity decreased as embryos developed; significant differences were observed (depending on the metric) by embryonic development, pond, and year, and there were interaction effects. The function of bacteria in what is thought of as a bipartite symbiosis calls for further research.

Expression cloning of TMEM16A as a calcium-activated chloride channel subunit.

Calcium-activated chloride channels (CaCCs) are major regulators of sensory transduction, epithelial secretion, and smooth muscle contraction. Other crucial roles of CaCCs include action potential generation in Characean algae and prevention of polyspermia in frog egg membrane. None of the known molecular candidates share properties characteristic of most CaCCs in native cells. Using Axolotl oocytes as an expression system, we have identified TMEM16A as the Xenopus oocyte CaCC. The TMEM16 family of "transmembrane proteins with unknown function" is conserved among eukaryotes, with family members linked to tracheomalacia (mouse TMEM16A), gnathodiaphyseal dysplasia (human TMEM16E), aberrant X segregation (a Drosophila TMEM16 family member), and increased sodium tolerance (yeast TMEM16). Moreover, mouse TMEM16A and TMEM16B yield CaCCs in Axolotl oocytes and mammalian HEK293 cells and recapitulate the broad CaCC expression. The identification of this new family of ion channels may help the development of CaCC modulators for treating diseases including hypertension and cystic fibrosis.

Diversity and composition of mixed-ploidy unisexual salamander assemblages reflect the key influence of host species.

Understanding processes that govern and sustain biological diversity is a central goal of community ecology. Unisexual complexes, where reproduction depends on sperm from males of one or more bisexual host species, are rare and the processes driving their diversity and structure remain poorly understood. Unisexual Ambystoma salamanders produce distinct biotypes ('genomotypes') depending on which bisexual species they 'steal' sperm from. This reproductive mode should generate distinct assemblages depending on the locally available bisexual host species. Yet, how availability and relative abundance of multiple bisexual hosts influences composition and diversity of natural unisexual assemblages at local or regional scales remains unknown. We hypothesize that host identity most directly drives local assemblage composition, with host variation associated with increased beta and gamma diversity within unisexuals. We collected genetic samples from Ambystoma salamanders across Pelee Island, Ontario, Canada (2015-2022). Two host species were identified (A. texanum and A. laterale) with nine sites having a single host and one site having both. Unisexual assemblages were grouped into four clusters by similarity, with host identity being a key determinant. Gamma diversity increased as a result of distinct host-specific assemblages forming at different sites on the island (i.e., high beta diversity). Assemblage composition, but not diversity, was correlated with relative host abundance, which may reflect matching niche requirements between host and unisexual forms they produce. Our results demonstrate that diversity and structure of unisexual assemblages are clearly shaped by their host(s) and such systems may serve as models for studying how biotic interactions shape ecological communities.

Terrestrial force production by the limbs of a semi-aquatic salamander provides insight into the evolution of terrestrial locomotor mechanics.

Amphibious fishes and salamanders are valuable functional analogs for vertebrates that spanned the water-land transition. However, investigations of walking mechanics have focused on terrestrial salamanders and, thus, may better reflect the capabilities of stem tetrapods that were already terrestrial. The earliest tetrapods were likely aquatic, so salamanders that are not primarily terrestrial may yield more appropriate data for modeling the incipient stages of terrestrial locomotion. In the present study, locomotor biomechanics were quantified from semi-aquatic Pleurodeles waltl, a salamander that spends most of its adult life in water, and then compared with those of a primarily terrestrial salamander (Ambystoma tigrinum) and a semi-aquatic fish (Periophthalmus barbarus) to evaluate whether terrestrial locomotion was more comparable between species with ecological versus phylogenetic similarities. Ground reaction forces (GRFs) from individual limbs or fins indicated that the pectoral appendages of each taxon had distinct patterns of force production, but GRFs from the hindlimbs were comparable between the salamander species. The rate at which force is produced can affect musculoskeletal function, so we also calculated 'yank' (first time derivative of force) to quantify the dynamics of GRF production. Yank was sometimes slower in P. waltl but there were some similarities between the three species. Finally, the semi-aquatic taxa (P. waltl and P. barbarus) had a more medial inclination of the GRF compared to terrestrial salamanders, potentially elevating bone stresses among more aquatic taxa and limiting their excursions onto land.

FGF8 and SHH substitute for anterior-posterior tissue interactions to induce limb regeneration.

In salamanders, grafting of a left limb blastema onto a right limb stump yields regeneration of three limbs, the normal limb and two 'supernumerary' limbs. This experiment and other research have shown that the juxtaposition of anterior and posterior limb tissue plus innervation are necessary and sufficient to induce complete limb regeneration in salamanders. However, the cellular and molecular basis of the requirement for anterior-posterior tissue interactions were unknown. Here we have clarified the molecular basis of the requirement for both anterior and posterior tissue during limb regeneration and supernumerary limb formation in axolotls (Ambystoma mexicanum). We show that the two tissues provide complementary cross-inductive signals that are required for limb outgrowth. A blastema composed solely of anterior tissue normally regresses rather than forming a limb, but activation of hedgehog (HH) signalling was sufficient to drive regeneration of an anterior blastema to completion owing to its ability to maintain fibroblast growth factor (FGF) expression, the key signalling activity responsible for blastema outgrowth. In blastemas composed solely of posterior tissue, HH signalling was not sufficient to drive regeneration; however, ectopic expression of FGF8 together with endogenous HH signalling was sufficient. In axolotls, FGF8 is expressed only in the anterior mesenchyme and maintenance of its expression depends on sonic hedgehog (SHH) signalling from posterior tissue. Together, our findings identify key anteriorly and posteriorly localized signals that promote limb regeneration and show that these single factors are sufficient to drive non-regenerating blastemas to complete regeneration with full elaboration of skeletal elements.

The reference genome of the Vernal Pool Tadpole Shrimp, Lepidurus packardi.

In this paper, we report on the scaffold-level assembled genome for the federally endangered, California endemic crustacean Lepidurus packardi (the Vernal Pool Tadpole Shrimp). L. packardi is a key food source for other conserved California species including the California Tiger Salamander Ambystoma californiense. It faces significant habitat loss and fragmentation as vernal pools are threatened by urbanization, agricultural conversion, and climate change. This resource represents the first scaffold-level genome of any Lepidurus species. The assembled genome spans 108.6 Mbps, with 6 chromosome-length scaffolds comprising 71% of total genomic length and 444 total contigs. The BUSCO score for this genome is 97.3%, suggesting a high level of completeness. We produced a predicted gene set for this species trained on the Daphnia magna set of genes and predicted 17,650 genes. These tools can aid researchers in understanding the evolution and adaptive potential of alternative reproductive modes within this species.

Multi-organ transcriptomic landscape of Ambystoma velasci metamorphosis.

Metamorphosis is a postembryonic developmental process that involves morphophysiological and behavioral changes, allowing organisms to adapt into a novel environment. In some amphibians, aquatic organisms undergo metamorphosis to adapt in a terrestrial environment. In this process, these organisms experience major changes in their circulatory, respiratory, digestive, excretory and reproductive systems. We performed a transcriptional global analysis of heart, lung and gills during diverse stages of Ambystoma velasci to investigate its metamorphosis. In our analyses, we identified eight gene clusters for each organ, according to the expression patterns of differentially expressed genes. We found 4064 differentially expressed genes in the heart, 4107 in the lung and 8265 in the gills. Among the differentially expressed genes in the heart, we observed genes involved in the differentiation of cardiomyocytes in the interatrial zone, vasculogenesis and in the maturation of coronary vessels. In the lung, we found genes differentially expressed related to angiogenesis, alveolarization and synthesis of the surfactant protein. In the case of the gills, the most prominent biological processes identified are degradation of extracellular matrix, apoptosis and keratin production. Our study sheds light on the transcriptional responses and the pathways modulation involved in the transformation of the facultative metamorphic salamander A. velasci in an organ-specific manner.

Allele-specific expression and gene regulation help explain transgressive thermal tolerance in non-native hybrids of the endangered California tiger salamander (Ambystoma californiense).

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.

Luteolibacter ambystomatis sp. nov., isolated from the skin of an Anderson's salamander (Ambystoma andersoni).

A bacterial strain designated 32AT was isolated from the skin of an Anderson's salamander (Ambystoma andersoni) and subjected to a comprehensive taxonomic study. The strain was Gram-stain-negative, rod-shaped, non-motile, oxidase- and urease-negative, and catalase-positive. 16S rRNA gene sequence comparisons placed the strain in the genus Luteolibacter with highest sequence similarities to Luteolibacter pohnpeiensis A4T-83T (95.2%), Luteolibacter gellanilyticus CB-286403T (95.1%) and Luteolibacter cuticulihirudinis E100T (94.9%). Genomic sequence analysis revealed a size of 5.3 Mbp, a G+C-content of 62.2 mol% and highest ANI values with Luteolibacter luteus (71.2%), Luteolibacter yonseiensis (71.4%) and L. pohnpeiensis (69.5%). In the polyamine pattern, 1,3-diaminopropane and spermidine were predominant. The diagnostic diamino acid of the peptidoglycan was meso-diaminopimelic acid. The quinone system was composed of the major menaquinones MK-9 and MK-10. Major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, the unidentified aminolipid AL2, the unidentified phospholipid PL2 and the unidentified aminophospholipid APL1. The fatty acid profile contained major amounts of iso-C14:0, iso-C16:0, C16 : 0 and C16 : 1 ω9c. In addition, C14 : 0, C15:0, anteiso-C15 : 0, summed feature 2 (C14 : 0 3OH and/or iso-C16 : 0 I), and the hydroxylated fatty acids iso-C14 : 0 3OH, iso-C16 : 0 3OH and C16 : 0 3-OH were detected. Physiologically, strain 32AT is distinguishable from its next relatives. Based on phylogenetic, genomic, physiological and chemotaxonomic data, strain 32AT represents a novel species of the genus Luteolibacter for which we propose the name Luteolibacter ambystomatis sp. nov. The type strain is 32AT (=CCM 9141T=LMG 32214T).

Drivers of distributions and niches of North American cold-adapted amphibians: evaluating both climate and land use.

Species distribution estimates are often used to understand the niche of a species; however, these are often based solely on climatic predictors. When the influences of biotic factors are ignored, erroneous inferences about range and niche may be made. We aimed to integrate climate data with a unique set of available land cover and land use data for the six cold-adapted amphibians of North America (Ambystoma macrodactylum, Anaxyrus hemiophrys, Anaxyrus boreas, Pseudacris maculata, Rana sylvatica, Rana luteiventris) to determine the relative importance of climate and non-climate drivers through the use of ecological niche models for present-day range estimates. We compared climate-only, land use-only, and combination models of climate and land use, derived from two different model selection techniques, to determine which was most likely to drive current distributions of cold-adapted amphibian species. Land use layers included land cover type, human population, vegetation type, ecoregion, and the overall human footprint. The most supported models included both climate and land use, with climate and human footprint variables having the highest permutation importance and percent contribution. Models that incorporated climate and land use data performed best as measured with AIC and AUC, although qualitatively most underestimated the northern range edge, implying potential sampling bias or locations of reduced habitat quality for these species in the northern area of the ranges. There were small differences in overall combination models dependent on the method of model selection. The overall effect sizes of landscape factors within the combination models were small except for one landscape feature: human footprint, which incorporated multiple aspects of anthropogenic change on the landscape, including human population density, travel access, and agricultural impact. This aspect of the landscape was just as important as climate, and counter to what we expected, the association was mostly positive, with a negative response only occurring at very high levels. This highlights the importance of moving beyond climate only species range estimates as land cover, specifically human impact, may be driving the patterns of species' ranges.

Balancing selection and drift in a polymorphic salamander metapopulation.

Understanding how genetic variation is maintained in a metapopulation is a longstanding problem in evolutionary biology. Historical resurveys of polymorphisms have offered efficient insights about evolutionary mechanisms, but are often conducted on single, large populations, neglecting the more comprehensive view afforded by considering all populations in a metapopulation. Here, we resurveyed a metapopulation of spotted salamanders (Ambystoma maculatum) to understand the evolutionary drivers of frequency variation in an egg mass colour polymorphism. We found that this metapopulation was demographically, phenotypically and environmentally stable over the last three decades. However, further analysis revealed evidence for two modes of evolution in this metapopulation-genetic drift and balancing selection. Although we cannot identify the balancing mechanism from these data, our findings present a clear view of contemporary evolution in colour morph frequency and demonstrate the importance of metapopulation-scale studies for capturing a broad range of evolutionary dynamics.

Direct effects influence larval salamander size and density more than indirect effects.

Direct and indirect effects both influence population and community dynamics. The relative strengths of these pathways are often compared using experimental approaches, but their evaluation in situ has been less frequent. We examined how individual and aggregate impacts of direct and indirect effects of species densities, proxies for competition and predation pressure, and habitat variables influenced patterns of larval density and body size of ringed (Ambystoma annulatum) and spotted salamanders (A. maculatum). We surveyed > 150 ponds in Missouri, USA, from 2012 to 2014 to measure the density and body size of each focal species, the density of co-occurring pond food web members, and select habitat features. We used structural equation modeling to quantify the relative importance of direct and indirect pathways on both body size and larval density. Overall, both responses were explained through a combination of direct and indirect effects. However, the magnitudes of direct effects were often greater than indirect effects. Some of the direct and indirect relationships with larval salamander size and density were also consistent with results from experimental studies. Finally, total direct and indirect effects were often weaker due to habitat and density variables negating each other's impacts. Overall, our study shows that direct effects were equivalent to, or more important than, indirect effects. We also demonstrate that the effects stemming from individual relationships can sum to produce net patterns that are negligible in magnitude. Further work on direct and indirect effects with observational data are needed to examine their magnitudes in natural communities.

Demographic effects of phenological variation in natural populations of two pond-breeding salamanders.

Phenology is a key driver of population and community dynamics. Phenological metrics (e.g., first date that an event occurred) often simplify information from the full phenological distribution, which may undermine efforts to determine the importance of life history events. Data regarding full phenological distributions are especially needed as many species are shifting phenology with climatic change which can alter life-history patterns and species dynamics. We tested whether skewness, kurtosis or maximum duration of breeding phenology affected juvenile emigration phenology and survival in natural populations of ringed (Ambystoma annulatum) and spotted salamanders (A. maculatum) spanning a 7-year period at two study locations. We evaluated the relative importance of different phenological metrics in breeding phenology and larval density dependence on emigration phenology and survival. We found that variability in emigration phenology differed by species, with ringed salamanders having a shorter duration and distributions that were more often right-skewed and leptokurtic compared to spotted salamanders. Emigration phenology was not linked to any measure of variability in breeding phenology, indicating phenological variability operates independently across life stages and may be subject to stage-specific influences. Emigration duration and skewness were partially explained by larval density, which demonstrates how phenological distributions may change with species interactions. Further tests that use the full phenological distribution to link variability in timing of life history events to demographic traits such as survival are needed to determine if and how phenological shifts will impact species persistence.

Radiation Force as a Physical Mechanism for Ultrasonic Neurostimulation of the Ex Vivo Retina.

Focused ultrasound has been shown to be effective at stimulating neurons in many animal models, both in vivo and ex vivo Ultrasonic neuromodulation is the only noninvasive method of stimulation that could reach deep in the brain with high spatial-temporal resolution, and thus has potential for use in clinical applications and basic studies of the nervous system. Understanding the physical mechanism by which energy in a high acoustic frequency wave is delivered to stimulate neurons will be important to optimize this technology. We imaged the isolated salamander retina of either sex during ultrasonic stimuli that drive ganglion cell activity and observed micron scale displacements, consistent with radiation force, the nonlinear delivery of momentum by a propagating wave. We recorded ganglion cell spiking activity and changed the acoustic carrier frequency across a broad range (0.5-43 MHz), finding that increased stimulation occurs at higher acoustic frequencies, ruling out cavitation as an alternative possible mechanism. A quantitative radiation force model can explain retinal responses and could potentially explain previous in vivo results in the mouse, suggesting a new hypothesis to be tested in vivo Finally, we found that neural activity was strongly modulated by the distance between the transducer and the electrode array showing the influence of standing waves on the response. We conclude that radiation force is the dominant physical mechanism underlying ultrasonic neurostimulation in the ex vivo retina and propose that the control of standing waves is a new potential method to modulate these effects.SIGNIFICANCE STATEMENT Ultrasonic neurostimulation is a promising noninvasive technology that has potential for both basic research and clinical applications. The mechanisms of ultrasonic neurostimulation are unknown, making it difficult to optimize in any given application. We studied the physical mechanism by which ultrasound is converted into an effective energy form to cause neurostimulation in the retina and find that ultrasound acts via radiation force leading to a mechanical displacement of tissue. We further show that standing waves have a strong modulatory effect on activity. Our quantitative model by which ultrasound generates radiation force and leads to neural activity will be important in optimizing ultrasonic neurostimulation across a wide range of applications.