Recurrent co-option and recombination of cytokine and three finger proteins in multiple reproductive tissues throughout salamander evolution

The proteomic composition of amphibian gametes is largely a molecular mystery, particularly for Urodeles (salamanders and newts) which have few genomic-scale resources. Lungless salamanders (family Plethodontidae) include approximately two thirds of all extant salamander species and are classic models of vertebrate mating behavior. As part of an extended, multi-stage courtship ritual, male plethodontid salamanders deliver rapidly evolving protein pheromones that modify female behavior and improve male reproductive success. Despite great interest in this set of pre-mating reproductive barriers, limited characterization of plethodontid gametes has prohibited investigation of post-mating pre-zygotic barriers such as sperm-egg recognition. In this study, we performed transcriptomic analyses of testis and ovary using long-read PacBio sequencing and proteomic analyses of sperm using mass spectrometry for two evolutionary divergent plethodontid species, Plethodon shermani and Desmognathus ocoee. In both species, many of the most abundant sperm proteins were paralogs of the courtship pheromones Plethodontid Receptivity Factor (PRF), Plethodontid Modulating Factor (PMF), and Sodefrin Precursor-like Factor (SPF). Sperm-specific paralogs of PMF and SPF are likely the most abundant secreted proteins in P. shermani and D. ocoee, respectively. In contrast, sperm PRF lacks a signal peptide and may be expressed in cytoplasm. PRF pheromone genes evolved independently multiple times through repeated gene duplication of sperm PRF genes and signal peptides recovered by recombination with PMF genes. Phylogenetic analysis of courtship pheromones and their sperm paralogs support that each protein family evolved for these two reproductive contexts at distinct evolutionary time points between 17 and 360 million years ago. As the first molecular characterization of salamander gametes, this study expands our knowledge of amphibian fertilization beyond frogs and provides novel insight into the evolutionary processes by which new, rapidly evolving reproductive proteins may evolve.

Novel Expansion of Matrix Metalloproteases in the Laboratory Axolotl (Ambystoma mexicanum) and Other Salamander Species

Matrix metalloprotease (MMP) genes encode endopeptidases that cleave protein components of the extracellular matrix (ECM) as well as non-ECM proteins. Here we report the results of a comprehensive survey of MMPs in the laboratory axolotl and other representative salamanders. Surprisingly, 28 MMPs were identified in salamanders and 9 MMP paralogs were identified as unique to the axolotl and other salamander taxa, with several of these presenting atypical amino acid insertions not observed in other tetrapod vertebrates. Furthermore, as assessed by sequence information, all of the novel salamander MMPs are of the secreted type, rather than cell membrane anchored. This suggests that secreted type MMPs expanded uniquely within salamanders to presumably execute catalytic activities in the extracellular milieu. To facilitate future studies of salamander-specific MMPs, we annotated transcriptional information from published studies of limb and tail regeneration. Our analysis sets the stage for comparative studies to understand why MMPs expanded uniquely within salamanders.

Comparative development and ocular histology between epigean and subterranean salamanders (Eurycea) from central Texas

The salamander clade Eurycea from the karst regions of central Texas provides an ideal platform for comparing divergent nervous and sensory systems since some species exhibit extreme phenotypes thought to be associated with inhabiting a subterranean environment, including highly reduced eyes, while others retain an ancestral ocular phenotype appropriate for life above ground. We describe ocular morphology, comparing three salamander species representing two phenotypes—the surface-dwelling Barton Springs salamander (E. sosorum) and San Marcos salamander (E. nana) and the obligate subterranean Texas blind salamander (E. rathbuni) - in terms of structure and size of their eyes. Eyes were examined using confocal microscopy and measurements were made using ImageJ. Statistical analysis of data was carried out using R. We also provide a developmental series and track eye development and immunolocalization of Pax6 in E. sosorum and E. rathbuni. Adult histology of the surface-dwelling San Marcos salamander (E. nana) shows similarities to E. sosorum. The eyes of adults of the epigean species E. nana and E. sosorum appear fully developed with all the histological features of a fully functional eye. In contrast, the eyes of E. rathbuni adults have fewer layers, lack lenses and other features associated with vision as has been reported previously. However, in early developmental stages eye morphology did not differ significantly between E. rathbuni and E. sosorum. Parallel development is observed between the two phenotypes in terms of morphology; however, Pax6 labeling seems to decrease in the latter stages of development in E.rathbuni. We test for immunolabeling of the visual pigment proteins opsin and rhodopsin and observe immunolocalization around photoreceptor disks in E. nana and E. sosorum, but not in the subterranean E. rathbuni. Our results from examining developing salamanders suggest a combination of underdevelopment and degeneration contribute to the reduced eyes of adult E. rathbuni.

Landuse and Water Quality Threats to Current and Historical Cryptobranchus Alleganiensis Streams Across Multiple Ecoregions

The hellbender (Cryptobranchus alleganiensis) is a large, long-lived, and fully aquatic salamander species endemic to streams and rivers across 15 states in the Midwest and Eastern United States. Hellbender populations have experienced drastic declines due to habitat degradation, accelerated sedimentation, aquatic contaminants, and infectious diseases. Although declining water quality is often suggested as a major contributing factor to hellbender population declines, few studies have evaluated the presence of aquatic contaminants at sites with current and historical C. alleganiensis populations. We used a novel passive sampling technique to assess the presence and concentration of two herbicides (glyphosate and atrazine) in the water and analyzed heavy metal (cadmium (Cd), mercury (Hg), and lead (Pb)) concentrations in stream sediment samples at 30 sites across a watershed development gradient throughout the C. alleganiensis range in Tennessee (TN) and North Carolina (NC). Our results indicated the presence of atrazine in 23% of our sites. All 30 sites contained Cd and Pb, and 26 sites contained Hg. Levels of glyphosate were too low to detect using our methods. Although percent watershed development was not significantly different among ecoregions, Blue Ridge watersheds were overall less developed than watersheds in the Ridge and Valley and Interior Plateau ecoregions. The model with greatest support suggested that percent watershed development and specific conductivity were negatively correlated with hellbender population status. Because this research focused on assessing the prevalence of commonly encountered aquatic contaminants, our results and study design are broadly applicable to C. alleganiensis populations across the species range.

Variation in Articular Cartilage Thickness Among Extant Salamanders and Implications for Limb Function in Stem Tetrapods

The size and shape of articular cartilage in the limbs of extant vertebrates are highly variable, yet they are critical for understanding joint and limb function in an evolutionary context. For example, inferences about unpreserved articular cartilage in early tetrapods have implications for how limb length, joint range of motion, and muscle leverage changed over the tetrapod water-land transition. Extant salamanders, which are often used as functional models for early limbed vertebrates, have much thicker articular cartilage than most vertebrate groups, but the exact proportion of cartilage and how it varies across salamander species is unknown. I aimed to quantify this variation in a sample of 13 salamanders representing a broad range of sizes, modes of life, and genera. Using contrast-enhanced micro-CT, cartilage dimensions and bone length were measured non-destructively in the humerus, radius, ulna, femur, tibia, and fibula of each specimen. Cartilage correction factors were calculated as the combined thickness of the proximal and distal cartilages divided by the length of the bony shaft. Articular cartilage added about 30% to the length of the long bones on average. Cartilage was significantly thicker in aquatic salamanders (42 ± 14% in the humerus and 35 ± 8 in the femur) than in terrestrial salamanders (21 ± 7% in both humerus and femur). There was no consistent relationship between relative cartilage thickness and body size or phylogenetic relatedness. In addition to contributing to limb length, cartilage caps increased the width and breadth of the epiphyses by amounts that varied widely across taxa. To predict the effect of salamander-like cartilage correction factors on muscle leverage, a simplified model of the hindlimb of the Devonian stem tetrapod Acanthostega was built. In this model, the lever arms of muscles that cross the hip at an oblique angle to the femur was increased by up to six centimeters. Future reconstructions of osteological range of motion and muscle leverage in stem tetrapods and stem amphibians can be made more rigorous by explicitly considering the possible effects of unpreserved cartilage and justifying assumptions based on available data from extant taxa, including aquatic and terrestrial salamanders.

The Asian plethodontid salamander preserves historical genetic imprints of recent northern expansion

The Korean Peninsula, located at the southern tip of Northeast Asia, has never been covered by ice sheets and was a temperate refugium during the Pleistocene. Karsenia koreana, the sole Asian plethodontid salamander species, occurs only on the southern half of the Korean Peninsula and is thought to have found various climatic refugia. Despite its phylogenetic and biogeographic importance, no population-level genetic analysis has been performed on this species. Here we study the population genetic structure of K. koreana using mitochondrial and microsatellite loci to understand the recent historical dispersion process that shaped its current distribution. Overall, the genetic distance between populations correlated well with the spatial distance, and the genetic structure among populations showed signs of a unilateral northward expansion from a southernmost refugium population. Given the distinct genetic structure formed among the populations, the level of historical gene flow among populations appears to have been very low. As the estimated effective population size of K. koreana was also small, these results suggest that the small, restricted populations of K. koreana are extremely vulnerable to environmental changes that may require high levels of genetic diversity to cope with. Thus, special management strategies are needed to preserve these remnant populations.

A Salamander tale: Relative abundance, morphometrics and microhabitat of the critically endangered Mexican salamander Pseudoeurycea robertsi (Taylor, 1939)

Roberts’ False Brook Salamander (Pseudoeurycea robertsi) is a critically endangered plethodontid salamander, endemic to the Nevado de Toluca Volcano (NTV), Mexico. Little is known about the biology and ecology of this species, including its microhabitats. Thus, this study aimed to collect basic information about P. robertsi. We sampled fourteen forested sites in the NTV; to corroborate the correct identification of the species we used genetic data, we assessed the variation in head morphometric measurements and dorsal colouration patterns amongst localities and the microhabitat features associated with P. robertsi presence. Of the four potential salamander species, P. robertsi was the most abundant (89.80%) and widely distributed (approximately within 130 km2) salamander in the NTV. We did not find significant variations in morphometry; however, we found significant differences in dorsal patterns between populations (in the number and size of segments of the dorsal stripe). The average total length for 185 adults was 89.15 mm (38.7–117.9 mm); we found seven patterns of dorsal stripe. We found 98% of P. robertsi individuals under the bark of fallen logs in Abies religiosa and A. religiosa-Pinus sp. forests, with a higher number of detected salamanders in naturally-fallen logs than in cut logs (34% vs. 10%). Thus, keeping well-preserved A. religiosa forests and retaining fallen logs is essential to P. robertsi conservation.

Slender salamanders (genus Batrachoseps) reveal Southern California to be a center for the diversification, persistence, and introduction of salamander lineages

Background The southern California biodiversity hotspot has had a complex geological history, with both plate tectonic forces and sea level changes repeatedly reconfiguring the region, and likely driving both lineage splittings and extinctions. Here we investigate patterns of genetic divergence in two species of slender salamanders (Plethodontidae: Batrachoseps) in this region. The complex geological history in combination with several organismal traits led us to predict that these species harbor multiple ancient mitochondrial lineages endemic to southern California. These species belong to a clade characterized by fine-scale mitochondrial structure, which has been shown to track ancient splits. Both focal species, Batrachoseps major and B. nigriventris, are relatively widely distributed in southern California, and estimated to have persisted there across millions of years. Recently several extralimital populations of Batrachoseps were found in the San Joaquin Valley of California, a former desert area that has been extensively modified for agriculture. The origins of these populations are unknown, but based on morphology, they are hypothesized to result from human-mediated introductions of B. major. Methods We sequenced the mitochondrial gene cytochrome b from a geographically comprehensive sampling of the mitochondrial lineages of B. major and B. nigriventris that are endemic to southern California. We used phylogenetic analyses to characterize phylogeographic structure and identify mitochondrial contact zones. We also included the San Joaquin Valley samples to test whether they resulted from introductions. We used a bootstrap resampling approach to compare the strength of isolation-by-distance in both Batrachoseps species and four other salamander species with which they co-occur in southern California. Results The northern lineage of B. major harbors at least eight deeply differentiated, geographically cohesive mitochondrial subclades. We identify geographic contact between many of these mtDNA lineages and some biogeographic features that are concordant with lineage boundaries. Batrachoseps nigriventris also has multiple deeply differentiated clades within the region. Comparative analyses highlight the smaller spatial scales over which mitochondrial divergence accumulates in Batrachoseps relative to most other salamander species in southern California. The extralimital populations of Batrachoseps from the San Joaquin Valley are assigned to B. major and are shown to result from at least two independent introductions from different source populations. We also suggest that B. major on Catalina Island, where it is considered native, may be the result of an introduction. Some of the same traits that facilitate the build-up of deep phylogeographic structure in Batrachoseps likely also contribute to its propensity for introductions, and we anticipate that additional introduced populations will be discovered.