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.

Trait variation in patchy landscapes: Morphology of spotted salamanders (Ambystoma maculatum) varies more within ponds than between ponds.

The influence of intraspecific trait variation on species interactions makes trait-based approaches critical to understanding eco-evolutionary processes. Because species occupy habitats that are patchily distributed in space, species interactions are influenced not just by the degree of intraspecific trait variation but also the relative proportion of trait variation that occurs within- versus between-patches. Advancement in trait-based ecology hinges on understanding how trait variation is distributed within and between habitat patches across the landscape. We sampled larval spotted salamanders (Ambystoma maculatum) across six spatially discrete ponds to quantify within- and between-pond variation in mass, length, and various metrics associated with their relationship (scaling, body condition, shape). Across all traits, within-pond variation contributed more to total observed morphological variation than between-pond variation. Between-pond variation was not negligible, however, and explained 20-41% of total observed variation in measured traits. Between-pond variation was more pronounced in salamander tail morphology compared to head or body morphology, suggesting that pond-level factors more strongly influence tails than other body parts. We also observed differences in mass-length relationships across ponds, both in terms of scaling slopes and intercepts, though differences in the intercepts were much stronger. Preliminary evidence hinted that newly constructed ponds were a driver of the observed differences in mass-length relationships and morphometrics. General pond-level difference in salamander trait covariation suggest that allometric scaling of morphological traits is context dependent in patchy landscapes. Effects of pond age offer the hypothesis that habitat restoration through pond construction is a driver of variation in trait scaling, which managers may leverage to bolster trait diversity.

Validation of Salamander Dermal Mucus Swabs as a Novel, Nonlethal Approach for Amphibian Metabolomics and Glutathione Analysis Following Pesticide Exposure.

Evaluating biomarkers of stress in amphibians is critical to conservation, yet current techniques are often destructive and/or time-consuming, which limits ease of use. In the present study, we validate the use of dermal swabs in spotted salamanders (Ambystoma maculatum) for biochemical profiling, as well as glutathione (GSH) stress response following pesticide exposure. Thirty-three purchased spotted salamanders were acclimated to laboratory conditions at Washington College (Chestertown, MD, USA) for 4 weeks. Following acclimation, salamanders were randomly sorted into three groups for an 8-h pesticide exposure on soil: control with no pesticide, 2,4-dichlorophenoxyacetic acid (2,4-D), or chlorpyrifos. Before and after exposure, mucus samples were obtained by gently rubbing a polyester-tipped swab 50 times across the ventral and dorsal surfaces. Salamanders were humanely euthanized and dissected to remove the brain for acetylcholinesterase and liver for GSH and hepatic metabolome analyses, and a whole-body tissue homogenate was used for pesticide quantification. Levels of GSH were present in lower quantities on dermal swabs relative to liver tissues for chlorpyrifos, 2,4-D, and control treatments. However, 2,4-D exposures demonstrated a large effect size increase for GSH levels in livers (Cohen's d = 0.925, p = 0.036). Other GSH increases were statistically insignificant, and effect sizes were characterized as small for 2,4-D mucosal swabs (d = 0.36), medium for chlorpyrifos mucosal swabs (d = 0.713), and negligible for chlorpyrifos liver levels (d = 0.012). The metabolomics analyses indicated that the urea cycle, alanine, and glutamate metabolism biological pathways were perturbed by both sets of pesticide exposures. Obtaining mucus samples through dermal swabbing in amphibians is a viable technique for evaluating health in these imperiled taxa. Environ Toxicol Chem 2024;43:1126-1137. © 2024 SETAC.

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.

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.

Gene functions of the Ambystoma altamirani skin microbiome vary across space and time but potential antifungal genes are widespread and prevalent.

Amphibian skin microbiomes can play a critical role in host survival against emerging diseases by protecting their host against pathogens. While a plethora of biotic and abiotic factors have been shown to influence the taxonomic diversity of amphibian skin microbiomes it remains unclear whether functional genomic diversity varies in response to temporal and environmental factors. Here we applied a metagenomic approach to evaluate whether seasonality, distinct elevations/sites, and pathogen presence influenced the functional genomic diversity of the A. altamirani skin microbiome. We obtained a gene catalogue of 92 107 nonredundant annotated genes and a set of 50 unique metagenome assembled genomes (MAGs). Our analysis showed that genes linked to general and potential antifungal traits significantly differed across seasons and sampling locations at different elevations. Moreover, we found that the functional genomic diversity of A. altamirani skin microbiome differed between B. dendrobatidis infected and not infected axolotls only during winter, suggesting an interaction between seasonality and pathogen infection. In addition, we identified the presence of genes and biosynthetic gene clusters (BGCs) linked to potential antifungal functions such as biofilm formation, quorum sensing, secretion systems, secondary metabolite biosynthesis, and chitin degradation. Interestingly genes linked to these potential antifungal traits were mainly identified in Burkholderiales and Chitinophagales MAGs. Overall, our results identified functional traits linked to potential antifungal functions in the A. altamirani skin microbiome regardless of variation in the functional diversity across seasons, elevations/sites, and pathogen presence. Our findings suggest that potential antifungal traits found in Burkholderiales and Chitinophagales taxa could be related to the capacity of A. altamirani to survive in the presence of Bd, although further experimental analyses are required to test this hypothesis.

Age- and dose-dependent susceptibility of axolotls (Ambystoma mexicanum) by bath exposure to Ambystoma tigrinum virus (ATV).

Ranaviruses are large, dsDNA viruses that have significant ecological and economic impact on cold-blooded vertebrates. However, our understanding of the viral proteins and subsequent host immune response(s) that impact susceptibility to infection and disease is not clear. The ranavirus Ambystoma tigrinum virus (ATV), originally isolated from the Sonoran tiger salamander (Ambystoma mavortium stebbinsi), is highly pathogenic at low doses of ATV at all tiger salamander life stages and this model has been used to explore the host-pathogen interactions of ATV infection. However, inconsistencies in the availability of laboratory reared larval tiger salamanders required us to look at the well characterized axolotl (A. mexicanum) as a model for ATV infection. Data obtained from five infection experiments over different developmental timepoints suggest that axolotls are susceptible to ATV in an age- and dose-dependent manner. These data support the use of the ATV-axolotl model to further explore the host-pathogen interactions of ranavirus infections.

Forecasting the flooding dynamics of flatwoods salamander breeding wetlands under future climate change scenarios.

Ephemeral wetlands are globally important systems that are regulated by regular cycles of wetting and drying, which are primarily controlled by responses to relatively short-term weather events (e.g., precipitation and evapotranspiration). Climate change is predicted to have significant effects on many ephemeral wetland systems and the organisms that depend on them through altered filling or drying dates that impact hydroperiod. To examine the potential effects of climate change on pine flatwoods wetlands in the southeastern United States, we created statistical models describing wetland hydrologic regime using an approximately 8-year history of water level monitoring and a variety of climate data inputs. We then assessed how hydrology may change in the future by projecting models forward (2025-2100) under six future climate scenarios (three climate models each with two emission scenarios). We used the model results to assess future breeding conditions for the imperiled Reticulated Flatwoods Salamander (Ambystoma bishopi), which breeds in many of the study wetlands. We found that models generally fit the data well and had good predictability across both training and testing data. Across all models and climate scenarios, there was substantial variation in the predicted suitability for flatwoods salamander reproduction. However, wetlands with longer hydroperiods tended to have fewer model iterations that predicted at least five consecutive years of reproductive failure (an important metric for population persistence). Understanding potential future risk to flatwoods salamander populations can be used to guide conservation and management actions for this imperiled species.

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.

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.

Testing whether adrenal steroids mediate phenotypic and physiologic effects of elevated salinity on larval tiger salamanders.

Salinity (sodium chloride, NaCl) from anthropogenic sources is a persistent contaminant that negatively affects freshwater taxa. Amphibians can be susceptible to salinity, but some species are innately or adaptively tolerant. Physiological mechanisms mediating tolerance to salinity are still unclear, but changes in osmoregulatory hormones such as corticosterone (CORT) and aldosterone (ALDO) are prime candidates. We exposed larval barred tiger salamanders (Ambystoma mavortium) to environmentally relevant NaCl treatments (<32-4000 mg·L-1 ) for 24 days to test effects on growth, survival, and waterborne CORT responses. Of those sampled, we also quantified waterborne ALDO from a subset. Using a glucocorticoid antagonist (RU486), we also experimentally suppressed CORT signaling of some larvae to determine if CORT mediates effects of salinity. There were no strong differences in survival among salinity treatments, but salinity reduced dry mass, snout-vent length, and body condition while increasing water content of larvae. High survival and sublethal effects demonstrated that salamanders were physiologically challenged but could tolerate the experimental concentrations. CORT signaling did not attenuate sublethal effects of salinity. Baseline and stress-induced (after an acute stressor, shaking) CORT were not influenced by salinity. ALDO was correlated with baseline CORT, suggesting it could be difficult to decouple the roles of CORT and ALDO. Future studies comparing ALDO and CORT responses of adaptively tolerant and previously unexposed populations could be beneficial to understand the roles of these hormones in tolerance to salinity. Nevertheless, our study enhances our understanding of the roles of corticosteroid hormones in mediating effects of a prominent anthropogenic stressor.

Assessing Leukocyte Profiles of Salamanders and Other Amphibians: A Herpetologists' Guide.

Assessing numbers of leukocytes in salamanders and other amphibians can be useful metrics for understanding health or stress levels of individuals in a population. In this chapter we describe the procedures for obtaining blood samples from amphibians, preparing blood films for microscopy, counting, and identifying cells. We also provide reference values for amphibian leukocytes for use in interpreting leukocyte data. From our assessment of the published and unpublished literature, "non-stressed" salamanders would have a leukocyte profile where 60-70% of cells are lymphocytes, 17-30% are neutrophils, 1-4% are eosinophils, 4-12% are basophils, and 2-6% are monocytes. In Ambystoma spp., the eosinophil abundance can be notably higher (30% of all white blood cells), for reasons unknown. Finally, the neutrophil-lymphocyte ratio of most non-stressed salamanders tends to be between 0.3 and 0.4 (sometimes less), while the ratios of stressed salamanders tend to be over 1.0.

PREVALENCE OF RANAVIRUS IN SPOTTED SALAMANDER (AMBYSTOMA MACULATUM) LARVAE FROM CREATED VERNAL POOLS IN WEST VIRGINIA, USA.

Ranavirosis is a disease of high concern for amphibians due to widespread documentation of its lethal and sublethal impacts and its high transmission potential across populations and species. We investigated whether spotted salamander (Ambystoma maculatum) ranavirus prevalence and viral load were associated with habitat characteristics, genetic diversity, corticosterone levels, and body size. In 2015 and 2016, we sampled 34 recently created vernal pools in the Monongahela National Forest, West Virginia, USA. We collected tail clippings from 1,128 spotted salamander larvae and waterborne hormone samples from 436 of those larvae, along with eight environmental characteristics of the pools. Over the 2-yr period, we detected ranavirus in 62% of pools, with prevalence ranging from 0% to 63% (mean, 7.68%). Spotted salamander size was positively correlated with ranavirus presence and viral load; however, we did not find associations between ranavirus prevalence or viral load and habitat characteristics, spotted salamander genetic diversity, relatedness, effective number of breeders, or corticosterone levels. The widespread occurrence of ranavirus in the vernal pools illustrates the potential for rapid natural introduction of the pathogen to created wetlands. Managers could consider monitoring local distributions of ranavirus before creation of new vernal pools to guide strategic placement of the wetlands to minimize occurrence and prevalence of this pathogen.

The effects of road salt (NaCl), predation, and competition on the growth and community interactions of spotted salamanders (Ambystoma maculatum) and wood frogs (Lithobates sylvaticus).

Road deicing salts are frequently used in northern regions of the world during the winter and early spring months. As a result, a significant portion of road runoff into surrounding aquatic habitats contains road deicing salts. Previous studies found road salt contaminations in vernal pools that pond-breeding amphibians commonly use, including spotted salamanders (Ambystoma maculatum) and wood frogs (Lithobates sylvaticus). Studies have examined the impact of road salt on both amphibian species, but to our knowledge no previous studies have examined how road salt impacts the interspecific competition between both amphibians. We hypothesized that road salt would negatively impact growth and survivorship of both amphibian species. During the spring and summer of 2017, we conducted an outdoor mesocosm experiment in which we created eight experimental conditions with three main factors: presence/absence of NaCl (1000 mg/L Cl-), presence/absence of interspecific competition between the two amphibian species (A. maculatum and L. sylvaticus), and presence/absence of predatory dragonfly larvae (Family Libellulidae). Our experiment revealed that salt delayed hatching and increased deformity in spotted salamander hatchlings. Additionally, salt reduced salamander survivorship by 62% and frog survivorship by 30%. Wood frog tadpoles and road salt interacted to diminish salamander survivorship a further 80% beyond salt alone, likely through an increase in interspecific competition. Road salt increased the larval period of salamanders and decreased the proportion metamorphosed by the end of the experiment. Dragonfly larvae reduced salamander survivorship by 35%, whereas they increased wood frog tadpole development rates. Dragonfly larvae and salt interacted to alter tadpole denticle size, with salt negating the impact of dragonfly larvae. Thus, we found that salt interfered with aquatic predatory chemical cues. Overall, the results of this study suggest that management strategies should be implemented in order to reduce the impact of road salts on freshwater aquatic ecosystems.

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.

Conservation genomics of urban populations of Streamside Salamander (Ambystoma barbouri).

In Tennessee, populations of the state endangered Streamside Salamander (Ambystoma barbouri) are in decline as their distribution lies mostly within rapidly developing areas in the Nashville Basin. Information regarding the partitioning of genetic variation among populations of A. barbouri and the taxonomic status of these populations relative to northern populations and their congener, the Small-mouthed Salamander (A. texanum), have important implications for management and conservation of this species. Here we combined mitochondrial sequencing and genome-wide single nucleotide polymorphism (SNP) data generated using Genotyping-by-Sequencing (GBS) to investigate patterns of genetic variation within Tennessee populations of A. barbouri, to assess their relationship to populations in Kentucky, and to examine their phylogenetic relationship to the closely related A. texanum. Results from phylogenetic reconstructions reveal a complex history of Tennessee A. barbouri populations with regards to northern populations, unisexual A. barbouri, and A. texanum. Patterns of mitochondrial sequence variation suggest that A. barbouri may have originated within Tennessee and expanded north multiple times into Kentucky, Ohio, Indiana, and West Virginia. Phylogenetic reconstructions based on genome-wide SNP data contradict results based on mitochondrial DNA and correspond to geographic and taxonomic boundaries. Variation in allele frequencies at SNP genotypes, as identified by multivariate analyses and Bayesian assignment tests, identified three evolutionary significant units (ESUs) for A. barbouri within Tennessee. Collectively, these results emphasize the need for prioritizing conservation needs for Tennessee populations of A. barbouri to ensure the long-term persistence of this species.

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.

Morphometric analyses of the vertebrae of Ambystoma (Tschudi, 1838) and the implications for identification of fossil salamanders.

Ambystoma (Tschudi, 1838) represents a speciose clade of salamanders that are found across much of North America. Fossils referred to Ambystoma are reported from early Cenozoic deposits and are common in Quaternary fossil deposits. Most fossils identified as Ambystoma are isolated vertebrae. Both quantitative and qualitative characters were reported as being useful for identifying fossilized vertebrae of Ambystoma below the genus level. However, there is limited information on intraspecific variation in those characters and previous studies noted intracolumnar variation which affects the utility of those characters for fossil identification. A lack of understanding of variation in modern species of Ambystoma casts uncertainty on our ability to identify fossil vertebrae confidently. We aimed to document intraspecific and intracolumnar variation in vertebral morphology among species of Ambystoma and examine the implications for fossil identification. We assembled one of the largest skeletal data sets for Ambystoma and took linear measurements on 15 species. We used 2D geometric morphometric analyses to characterize atlantal shape variation in Ambystoma. We apply those morphometric data in a case study where we identify fossil vertebrae from Hall's Cave, a Quaternary fossil locality in central Texas. We found patterns of intraspecific and intracolumnar variation that have substantial implications for fossil identification. Classification accuracies for species and clades within Ambystoma varied considerably. Overall classification accuracies based on size-adjusted measurements and 2D geometric morphometric landmarks were lower compared with classifications from non-size adjusted linear measurements. We identified fossil vertebrae from our case study as likely belonging to the tiger salamander clade within Ambystoma, but found that some fossils with lower classification probabilities are of uncertain identity. We discuss biogeographic implications for our fossil identifications and comment on challenges and next steps for advancing our understanding of morphological variation in Ambystoma.

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).