Mining expressed sequence tag (EST) microsatellite markers to assess the genetic differentiation of five Hynobius species endemic to Taiwan.

Taiwan harbors five endemic species of salamanders (Hynobius spp.) that inhabit distinct alpine regions, contributing to population fragmentation across isolated "sky islands". With an evolutionary history spanning multiple glacial-interglacial cycles, these species represent an exceptional paradigm for exploring biogeography and speciation. However, a lack of suitable genetic markers applicable across species has limited research efforts. Thus, developing cross-amplifying markers is imperative. Expressed sequence-tag simple-sequence repeats (EST-SSRs) that amplify across divergent lineages are ideal for species identification in instances where phenotypic differentiation is challenging. Here, we report a suite of cross-amplifying EST-SSRs from the transcriptomes of the five Hynobius species that exhibit an interspecies transferability rate of 67.67%. To identify individual markers exhibiting cross-species polymorphism and to assess interspecies genetic diversity, we assayed 140 individuals from the five species across 84 sampling sites. A set of EST-SSRs with a high interspecies polymorphic information content (PIC = 0.63) effectively classified these individuals into five distinct clusters, as supported by discriminant analysis of principal components (DAPC), STRUCTURE assignment tests, and Neighbor-joining trees. Moreover, pair-wise FST values > 0.15 indicate notable between-cluster genetic divergence. Our set of 20 polymorphic EST-SSRs is suitable for assessing population structure within and among Hynobius species, as well as for long-term monitoring of their genetic composition.

Range-wide salamander densities reveal a key component of terrestrial vertebrate biomass in eastern North American forests.

Characterizing the population density of species is a central interest in ecology. Eastern North America is the global hotspot for biodiversity of plethodontid salamanders, an inconspicuous component of terrestrial vertebrate communities, and among the most widespread is the eastern red-backed salamander, Plethodon cinereus. Previous work suggests population densities are high with significant geographic variation, but comparisons among locations are challenged by lack of standardization of methods and failure to accommodate imperfect detection. We present results from a large-scale research network that accounts for detection uncertainty using systematic survey protocols and robust statistical models. We analysed mark-recapture data from 18 study areas across much of the species range. Estimated salamander densities ranged from 1950 to 34 300 salamanders ha-1, with a median of 9965 salamanders ha-1. We compared these results to previous estimates for P. cinereus and other abundant terrestrial vertebrates. We demonstrate that overall the biomass of P. cinereus, a secondary consumer, is of similar or greater magnitude to widespread primary consumers such as white-tailed deer (Odocoileus virginianus) and Peromyscus mice, and two to three orders of magnitude greater than common secondary consumer species. Our results add empirical evidence that P. cinereus, and amphibians in general, are an outsized component of terrestrial vertebrate communities in temperate ecosystems.

Exposure to polystyrene nanoplastics causes immune damage, oxidative stress and intestinal flora disruption in salamander (Andrias davidianus) larvae.

The toxic effects of nanoparticles have been increasingly investigated, but there has been limited research on amphibians, especially those of conservation value. This study examined the effects of different concentrations (0, 0.04, 0.2, 1, 5 mg/L) of polystyrene nanoplastics (PS-NPs, 80 nm) on the short-term exposure (7 d) of Andrias davidianus. Results demonstrated the concentration-dependent enrichment of PS-NPs in the intestine. Histological lesions displayed increased hepatic macrophages with cellular rupture, broken intestinal villi, decreased cuprocytes and crypt depression. Antioxidant- and inflammation-related enzyme activities were analysed, and it was found that hepatic and intestinal MDA content and CAT activity were highest in the N-1 group and SOD activity was highest in the N-0.2 group (p < 0.05). AKP activity continued to decline, and iNOS activity was highest in the N-0.2 group (p < 0.05). il-10, tgf-ß, bcl-w and txnl1 were significantly downregulated in the N-0.2 group, while il-6 and il-8 were markedly upregulated in the N-0.2 group (p < 0.05). Exposing to PS-NPs decreased probiotic bacteria (Cetobacterium, Akkermansia) and increased pathogenic bacteria (Lachnoclostridium). Our results suggest that NPs exposure can have deleterious effects on salamanders, which predicts that NPs contamination may lead to continued amphibian declines. Therefore, we strongly recommend that attention be paid to amphibians, especially endangered species, in the field of NPs.

A skin secretion metabolome analysis of the Greek Dodecanese Lycian salamanders: Preliminary evidence of dietary alkaloid sequestration in urodeles.

Lyciasalamandra species, like most amphibians, secrete a wide array of compounds from their granular and mucous skin glands, including the internally synthesized samandarine alkaloids, making their skin a complex organ performing a variety of functions. Lyciasalamandra helverseni and L. luschani basoglui are insular endemics of the Dodecanese islands of SE Greece, bearing distinct isolated populations, with well-documented phylogenetic profiles. Here, we employ a metabolomics approach, utilizing UPLC-ESI-HRMS/MS data of the skin secretions sampled from a number of specimens found in the islands of Karpathos, Kasos and Kastellorizo, in an effort to reveal aspects of their chemistry and diversity across populations. The results indicated statistically significant variation between all taxa examined, based on various secreted compounds. The underlying factors of variation highlighted by the multivariate analysis were differences in samandarine and other alkaloid content as well as in animal size. Metabolite annotation, based on dereplication tools and most importantly HRMS and HRMS/MS spectra, yielded a number of known samandarine alkaloids, reported for the first time in the currently studied Lyciasalamandra species. We also present documentation for novel members of the samandarine alkaloid family, as well as preliminary evidence for a possible dietary alkaloid sequestration. This work can set the basis for further research of this often-neglected endemic species of the Salamandridae, as well as the structural investigation of the samandarine alkaloid group.

Protein Charge Neutralization Is the Proximate Driver Dynamically Tuning Reflectin Assembly.

Reflectin is a cationic, block copolymeric protein that mediates the dynamic fine-tuning of color and brightness of light reflected from nanostructured Bragg reflectors in iridocyte skin cells of squids. In vivo, the neuronally activated phosphorylation of reflectin triggers its assembly, driving osmotic dehydration of the membrane-bounded Bragg lamellae containing the protein to simultaneously shrink the lamellar thickness and spacing while increasing their refractive index contrast, thus tuning the wavelength and increasing the brightness of reflectance. In vitro, we show that the reduction in repulsive net charge of the purified, recombinant reflectin-either (for the first time) by generalized anionic screening with salt or by pH titration-drives a finely tuned, precisely calibrated increase in the size of the resulting multimeric assemblies. The calculated effects of phosphorylation in vivo are consistent with these effects observed in vitro. The precise proportionality between the assembly size and charge neutralization is enabled by the demonstrated rapid dynamic arrest of multimer growth by a continual, equilibrium tuning of the balance between the protein's Coulombic repulsion and short-range interactive forces. The resulting stability of reflectin assemblies with time ensures a reciprocally precise control of the particle number concentration, encoding a precise calibration between the extent of neuronal signaling, osmotic pressure, and the resulting optical changes. The charge regulation of reflectin assembly precisely fine-tunes a colligative property-based nanostructured biological machine. A physical mechanism is proposed.

PAX6 protein in neuromasts of the lateral line system of salamanders (Eurycea).

PAX6 is well known as a transcription factor that drives eye development in animals as widely divergent as flies and mammals. In addition to its localization in eyes, PAX6 expression has been reported in the central nervous system, the pancreas, testes, Merkel cells, nasal epithelium, developing cells of the inner ear, and embryonic submandibular salivary gland. Here we show that PAX6 also appears to be present in the mechanosensory neuromasts of the lateral line system in paedomorphic salamanders of the genus Eurycea. Using immunohistochemistry and confocal microscopy to examine a limited number of larvae of two species, listed by the United States of America's federal government as threatened (E. nana) or endangered (E. rathbuni), we found that anti-PAX6 antibody labeled structures that were extranuclear, and labeling was most intense in the apical appendages of the hair cells of the neuromast. This extranuclear localization raises the possibility of an as yet undescribed function for PAX6 as a cytoskeleton-associated protein.

Carpal variability and asymmetry in limb reduced Western lesser sirens (Siren nettingi).

Trait functionality can act as a constraint on morphological development. Traits that become vestigialized can exhibit unstable developmental patterns such as fluctuating asymmetry (FA) and variation in populations. We use clearing and staining along with morphometric analyzes to compare FA and allometry of limbs in Western lesser sirens (Siren nettingi) to Ouachita dusky salamanders (Desmognathus brimleyorum). Our results describe new carpal phenotypes and carpal asymmetry in our sample of S. nettingi. However, we found no significant evidence of limb length asymmetry in S. nettingi. The degree of relative limb asymmetry correlates inversely with body size in both of our samples. This work provides strong evidence of increased mesopodal variation within a population of S. nettingi. Our work provides a basis for further study of a broader range of morphological traits across salamanders.

Methylmercury Concentrations More Strongly Associated With Trait Variation Than Food Web Position in Plethodontid Salamanders.

Salamanders serve as bioindicators of mercury (Hg) in both terrestrial and aquatic habitats and are an important link in the food web between low-trophic prey and higher-trophic predators. We investigated the drivers of methylmercury (MeHg) exposure in three common plethodontid salamander species in New York State, USA, including comparisons among regions, habitat types (terrestrial and semiaquatic), and color morphs of Plethodon cinereus (striped and unstriped). Nonlethal tail samples were collected from one terrestrial species (P. cinereus) and two semiaquatic species (Eurycea bislineata and Desmognathus spp.) in the Adirondack Mountains (ADK) and the Finger Lakes National Forest (FLNF) regions. Samples were analyzed for MeHg and stable isotopes, including δ15N and δ13C which are proxies of trophic position and diet, respectively. Despite elevated biota Hg concentrations typically found in the ADK, salamander MeHg concentrations did not differ by region in the terrestrial species and one of the semiaquatic species. In addition, diet and trophic level did not explain MeHg exposure in salamanders. Semiaquatic salamanders exhibited higher MeHg concentrations than terrestrial salamanders in the FLNF only. Within species, only snout-vent length predicted MeHg concentrations in E. bislineata with few other variables significant as predictors of MeHg concentrations in path models. Among P. cinereus individuals in the FLNF, the striped morph had greater MeHg concentrations than the unstriped morph, and food web tracers were not different between morphs. Overall, New York State salamander Hg concentrations were elevated compared to other locations where these species are present. The present study establishes baseline Hg data in salamanders for future assessments of changes in Hg bioavailability to forests of New York State. Environ Toxicol Chem 2024;43:2045-2057. © 2024 SETAC.

Phylosymbiosis shapes skin bacterial communities and pathogen-protective function in Appalachian salamanders.

Phylosymbiosis is an association between host-associated microbiome composition and host phylogeny. This pattern can arise via the evolution of host traits, habitat preferences, diets, and the co-diversification of hosts and microbes. Understanding the drivers of phylosymbiosis is vital for modelling disease-microbiome interactions and manipulating microbiomes in multi-host systems. This study quantifies phylosymbiosis in Appalachian salamander skin in the context of infection by the fungal pathogen Batrachochytrium dendrobatidis (Bd), while accounting for environmental microbiome exposure. We sampled ten salamander species representing >150M years of divergence, assessed their Bd infection status, and analysed their skin and environmental microbiomes. Our results reveal a significant signal of phylosymbiosis, whereas the local environmental pool of microbes, climate, geography, and Bd infection load had a smaller impact. Host-microbe co-speciation was not evident, indicating that the effect stems from the evolution of host traits influencing microbiome assembly. Bd infection is correlated with host phylogeny and the abundance of Bd-inhibitory bacterial strains, suggesting that the long-term evolutionary dynamics between salamander hosts and their skin microbiomes affect the present-day distribution of the pathogen, along with habitat-linked exposure risk. Five Bd-inhibitory bacterial strains showed unusual generalism: occurring in most host species and habitats. These generalist strains may enhance the likelihood of probiotic manipulations colonising and persisting on hosts. Our results underscore the substantial influence of host-microbiome eco-evolutionary dynamics on environmental health and disease outcomes.

Four years monitoring of the endangered European plethodontid salamanders.

The ongoing biodiversity crisis is strongly threatening amphibians, mostly because of their peculiar physiology, their sensitivity to climate change and the spread of diseases. Effective monitoring involving assessments of pressure effects across time and estimation of population trends play a key role in mitigating amphibian decline. To improve implementation of standardized protocols and conservation efforts, we present here a dataset related to one of the amphibian genera whose onservation status is considered the most declining according to the IUCN. We report information on 66 populations of the endangered European cave salamanders, genus Speleomantes, that was collected through a standardized monitoring along a four-year period (2021-2024). Demographics data of the populations and fitness-related data of single individuals are reported. Furthermore, we include 3,836 high quality images of individuals that can allow to perform studies aiming to assess the phenotypic variability within the genus, and to perform long-term capture-mark-recaptured studies.

Genomic Gigantism is not Associated with Reduced Selection Efficiency in Neotropical Salamanders.

Genome size variation in eukaryotes has myriad effects on organismal biology from the genomic to whole-organism level. Large genome size may be associated with lower selection efficiency because lower effective population sizes allow fixation of deleterious mutations via genetic drift, increasing genome size and decreasing selection efficiency. Because of a hypothesized negative relationship between genome size and recombination rate per base pair, increased genome size could also increase the effect of linked selection in the genome, decreasing the efficiency with which natural selection can fix or remove mutations. We used a transcriptomic dataset of 15 and a subset of six Neotropical salamander species ranging in genome size from 12 to 87 pg to study the relationship between genome size and efficiency of selection. We estimated dN/dS of salamanders with small and large genomes and tested for relaxation of selection in the larger genomes. Contrary to our expectations, we did not find a significant relationship between genome size and selection efficiency or strong evidence for higher dN/dS values in species with larger genomes for either species set. We also found little evidence for relaxation of selection in species with larger genomes. A positive correlation between genome size and range size (a proxy of population size) in this group disagrees with predictions of stronger drift in species with larger genomes. Our results highlight the complex interactions between the many forces shaping genomic variation in organisms with genomic gigantism.

Lack of variations in the salamander chytrid fungus, Batrachochytrium salamandrivorans, at its alleged origin: Updating its Japanese distribution with new evidence.

The chytrid fungus Batrachochytrium salamandrivorans [Bsal] is causing declines in the amphibian populations. After a decade of mapping the pathogen in Europe, where it is causing dramatic outbreaks, and North America, where its arrival would affect to the salamander's biodiversity hotspot, little is known about its current status in Asia, from presumably is native. Japan has several species considered as potential carriers, but no regulation is implemented against Bsal spreading. Previous Bsal known presence detected various cases on the Okinawa Island, southwestern Japan. Previous studies on its sister species, B. dendrobatidis presented a high genomic variation in this area and particularly on Cynops ensicauda. Here, we have done the largest monitoring to date in Japan on the Cynops genus, focusing on Okinawa Island and updating its distribution and providing more information to unravel the still unknown origin of Bsal. Interestingly, we have provided revealing facts about different detectability depending on the used molecular techniques and changes in its Japanese distribution. All in all, the Bsal presence in Japan, together with its low variability in the sequenced amplicons, and the lack of apparent mortalities, may indicate that this part of Asia has a high diversity of chytrids.

Shrinking sizes of trout and salamanders are unexplained by climate warming alone.

Decreases in body sizes of animals related to recent climate warming can affect population persistence and stability. However, direct observations of average sizes over time and their interrelationships with underlying density-dependent and density-independent processes remain poorly understood owing to the lack of appropriate long-term datasets. We measured body size of two species common to headwater streams in coastal and Cascades ecoregions of the Pacific Northwest of North America over multiple decades, comparing old-growth and managed forests. We found consistent decreases in median length of Coastal Cutthroat Trout Oncorhynchus clarkii clarkii, but a coexisting species, the Coastal Giant Salamander Dicamptodon tenebrosus, appears to be more resilient to size changes over time. Based on observed trends, adult trout have decreased in length by 6-13% over the last 30 years. Length decreased more in larger compared to smaller animals, suggesting that these effects reflect changes in growth trajectories. Results from a model-selection approach that included hydroclimatic and biological information as covariates in one of our study ecoregions demonstrated that stream temperature alone did not explain observed length reductions. Rather, a combination of density-dependent (animal abundances) and local density-independent factors (temperature, habitat, and streamflow) explained observed patterns of size. Continued decreases in size could lead to trophic cascades, biodiversity loss, or in extreme cases, species extirpation. However, the intricate links between density-independent and density-dependent factors in controlling population-level processes in streams need further attention.

First Report of Endemic Frog Virus 3 (FV3)-like Ranaviruses in the Korean Clawed Salamander (Onychodactylus koreanus) in Asia.

Frog virus 3 (FV3) in the genus Ranavirus of the family Iridoviridae causes mass mortality in both anurans and urodeles worldwide; however, the phylogenetic origin of FV3-like ranaviruses is not well established. In Asia, three FV3-like ranaviruses have been reported in farmed populations of amphibians and reptiles. Here, we report the first case of endemic FV3-like ranavirus infections in the Korean clawed salamander Onychodactylus koreanus, caught in wild mountain streams in the Republic of Korea (ROK), through whole-genome sequencing and phylogenetic analysis. Two isolated FV3-like ranaviruses (Onychodactylus koreanus ranavirus, OKRV1 and 2) showed high similarity with the Rana grylio virus (RGV, 91.5%) and Rana nigromaculata ranavirus (RNRV, 92.2%) but relatively low similarity with the soft-shelled turtle iridovirus (STIV, 84.2%) in open reading frame (ORF) comparisons. OKRV1 and 2 formed a monophyletic clade with previously known Asian FV3-like ranaviruses, a sister group of the New World FV3-like ranavirus clade. Our results suggest that OKRV1 and 2 are FV3-like ranaviruses endemic to the ROK, and RGV and RNRV might also be endemic strains in China, unlike previous speculation. Our data have great implications for the study of the phylogeny and spreading routes of FV3-like ranaviruses and suggest the need for additional detection and analysis of FV3-like ranaviruses in wild populations in Asian countries.

Real-Time Monitoring on the Chinese Giant Salamander Using RPA-LFD.

The Chinese giant salamander (Andrias davidianus), listed as an endangered species under "secondary protection" in China, faces significant threats due to ecological deterioration and the expansion of human activity. Extensive field investigations are crucial to ascertain the current status in the wild and to implement effective habitat protection measures to safeguard this species and support its population development. Traditional survey methods often fall short due to the elusive nature of the A. davidianus, presenting challenges that are time-consuming and generally ineffective. To overcome these obstacles, this study developed a real-time monitoring method that uses environmental DNA (eDNA) coupled with recombinase polymerase amplification and lateral flow strip (RPA-LFD). We designed five sets of species-specific primers and probes based on mitochondrial genome sequence alignments of A. davidianus and its close relatives. Our results indicated that four of these primer/probe sets accurately identified A. davidianus, distinguishing it from other tested caudata species using both extracted DNA samples and water samples from a tank housing an individual. This method enables the specific detection of A. davidianus genomic DNA at concentrations as low as 0.1 ng/mL within 50 min, without requiring extensive laboratory equipment. Applied in a field survey across four sites in Huangshan City, Anhui Province, where A. davidianus is known to be distributed, the method successfully detected the species at three of the four sites. The development of these primer/probe sets offers a practical tool for field surveying and monitoring, facilitating efforts in population recovery and resource conservation for A. davidianus.

Outbreeding reduces survival during metamorphosis in a headwater stream salamander.

Assessing direct fitness effects of individual genetic diversity is challenging due to the intensive and long-term data needed to quantify survival and reproduction in the wild. But resolving these effects is necessary to determine how inbreeding and outbreeding influence eco-evolutionary processes. We used 8 years of capture-recapture data and single nucleotide polymorphism genotypes for 1906 individuals to test for effects of individual heterozygosity on stage-specific survival probabilities in the salamander Gyrinophilus porphyriticus. The life cycle of G. porphyriticus includes an aquatic larval stage followed by metamorphosis into a semi-aquatic adult stage. In our study populations, the larval stage lasts 6-10 years, metamorphosis takes several months, and lifespan can reach 20 years. Previous studies showed that metamorphosis is a sensitive life stage, leading us to predict that fitness effects of individual heterozygosity would occur during metamorphosis. Consistent with this prediction, monthly probability of survival during metamorphosis declined with multi-locus heterozygosity (MLH), from 0.38 at the lowest MLH (0.10) to 0.06 at the highest MLH (0.38), a reduction of 84%. Body condition of larvae also declined significantly with increasing MLH. These relationships were consistent in the three study streams. With evidence of localised inbreeding within streams, these results suggest that outbreeding disrupts adaptations in pre-metamorphic and metamorphic individuals to environmental gradients along streams, adding to evidence that headwater streams are hotspots of microgeographic adaptation. Our results also underscore the importance of incorporating life history in analyses of the fitness effects of individual genetic diversity and suggest that metamorphosis and similar discrete life stage transitions may be critical periods of viability selection.

Alpine salamanders at risk? The current status of an emerging fungal pathogen.

Amphibians globally suffer from emerging infectious diseases like chytridiomycosis caused by the continuously spreading chytrid fungi. One is Batrachochytrium salamandrivorans (Bsal) and its disease ‒ the 'salamander plague' ‒ which is lethal to several caudate taxa. Recently introduced into Western Europe, long distance dispersal of Bsal, likely through human mediation, has been reported. Herein we study if Alpine salamanders (Salamandra atra and S. lanzai) are yet affected by the salamander plague in the wild. Members of the genus Salamandra are highly susceptible to Bsal leading to the lethal disease. Moreover, ecological modelling has shown that the Alps and Dinarides, where Alpine salamanders occur, are generally suitable for Bsal. We analysed skin swabs of 818 individuals of Alpine salamanders and syntopic amphibians at 40 sites between 2017 to 2022. Further, we compiled those with published data from 319 individuals from 13 sites concluding that Bsal infections were not detected. Our results suggest that the salamander plague so far is absent from the geographic ranges of Alpine salamanders. That means that there is still a chance to timely implement surveillance strategies. Among others, we recommend prevention measures, citizen science approaches, and ex situ conservation breeding of endemic salamandrid lineages.

Metagenomic and transcriptomic analysis revealing the impact of oxytetracycline and ciprofloxacin on gut microbiota and gene expression in the Chinese giant salamander (Andrias davidianus).

Excessive antibiotic use has led to the spread of antibiotic resistance genes (ARGs), impacting gut microbiota and host health. However, the effects of antibiotics on amphibian populations remain unclear. We investigated the impact of oxytetracycline (OTC) and ciprofloxacin (CIP) on Chinese giant salamanders (Andrias davidianus), focusing on gut microbiota, ARGs, and gene expression by performing metagenome and transcriptome sequencing. A. davidianus were given OTC (20 or 40 mg/kg) or CIP (50 or 100 mg/kg) orally for 7 days. The results revealed that oral administration of OTC and CIP led to distinct changes in microbial composition and functional potential, with CIP treatment having a greater impact than OTC. Antibiotic treatment also influenced the abundance of ARGs, with an increase in fluoroquinolone and multi-drug resistance genes observed post-treatment. The construction of metagenome-assembled genomes (MAGs) accurately validated that CIP intervention enriched fish-associated potential pathogens Aeromonas hydrophila carrying an increased number of ARGs. Additionally, mobile genetic elements (MGEs), such as phages and plasmids, were implicated in the dissemination of ARGs. Transcriptomic analysis of the gut revealed significant alterations in gene expression, particularly in immune-related pathways, with differential effects observed between OTC and CIP treatments. Integration of metagenomic and transcriptomic data highlighted potential correlations between gut gene expression and microbial composition, suggesting complex interactions between the host gut and its gut microbiota in response to antibiotic exposure. These findings underscore the importance of understanding the impact of antibiotic intervention on the gut microbiome and host health in amphibians, particularly in the context of antibiotic resistance and immune function.

Androgen and glucocorticoid profiles throughout extended uniparental paternal care in the eastern hellbender salamander (Cryptobranchus a. alleganiensis).

The behavioral endocrinology associated with reproduction and uniparental male care has been studied in teleosts, but little is known about hormonal correlates of uniparental male care in other ectotherms. To address this gap, we are the first to document the seasonal steroid endocrinology of uniparental male hellbender salamanders during the transition from pre-breeding to nest initiation, and through the subsequent eight months of paternal care. In doing so, we investigated the correlates of nest fate and clutch size, exploring hellbenders' alignment with several endocrinological patterns observed in uniparental male fish. Understanding the endocrinology of hellbender paternal care is also vital from a conservation perspective because high rates of nest failure were recently identified as a factor causing population declines in this imperiled species. We corroborated previous findings demonstrating testosterone and dihydrotestosterone (DHT) to be the primary androgens in hellbender reproduction, and that cortisol circulates as the most abundant glucocorticoid. However, we were unable to identify a prolactin or a "prolactin-like" peptide in circulation prior to or during parental care. We observed âˆ¼ 80 % declines in both primary androgens during the transition from pre-breeding to nest initiation, and again as paternal care progressed past its first month. In the days immediately following nest initiation, testosterone and DHT trended higher in successful individuals, but did not differ with males' clutch size. We did not observe meaningful seasonality in baseline glucocorticoids associated with breeding or nesting. In contrast, stress-induced glucocorticoids were highest at pre-breeding and through the first two months of care, before declining during the latter-most periods of care as larvae approach emergence from the nest. Neither baseline nor stress-induced glucocorticoids varied significantly with either nest fate or clutch size. Both stress-induced cortisol and corticosterone were positively correlated with total length, a proxy for age in adult hellbenders. This is consistent with age-related patterns in some vertebrates, but the first such pattern observed in a wild amphibian population. Generally, we found that nesting hellbenders adhere to some but not all of the endocrinological patterns observed in uniparental male teleosts prior to and during parental care.

Stage-Specific Demographic Effects of Hydrologic Variation in a Stream Salamander.

AbstractWe lack a strong understanding of how organisms with complex life histories respond to climate variation. Many stream-associated species have multistage life histories that are likely to influence the demographic consequences of floods and droughts. However, tracking stage-specific demographic responses requires high-resolution, long-term data that are rare. We used 8 years of capture-recapture data for the headwater stream salamander Gyrinophilus porphyriticus to quantify the effects of flooding and drying magnitude on stage-specific vital rates and population growth. Drying reduced larval recruitment but increased the probability of metamorphosis (i.e., adult recruitment). Flooding reduced adult recruitment but had no effect on larval recruitment. Larval and adult survival declined with flooding but were unaffected by drying. Annual population growth rates (λ) declined with flooding and drying. Lambda also declined over the study period (2012-2021), although mean λ was 1.0 over this period. Our results indicate that G. porphyriticus populations are resilient to hydrologic variation because of compensatory effects on recruitment of larvae versus adults (i.e., reproduction vs. metamorphosis). Complex life cycles may enable this resilience to climate variation by creating opportunities for compensatory demographic responses across stages. However, more frequent and intense hydrologic variation in the latter half of this study contributed to a decline in λ over time, suggesting that increasing environmental variability poses a threat even when demographic compensation occurs.