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.

Repeatability of water loss and gain rates of Plethodon cinereus

Water loss and gain rates of amphibians are important to understanding their ecology, especially for plethodontid salamanders. We report the first estimates of repeatability of relative water loss and gain rates of the two major colour morphs of the Eastern Red-backed Salamander, Plethodon cinereus, in the fall and spring seasons. Repeatability of relative water loss in P. cinereus was >0 in the fall but not in the spring. Repeatability of relative water gain was significant for all salamanders pooled in the fall, and was not repeatable in the spring. There were no apparent differences in repeatability of relative water loss or gain between the two colour morphs. Our results suggest that the repeatability of relative water loss and gain rates varies by season, but not by colour morph.

Ecological Observations on Hybrid Populations of European Plethodontid Salamanders, Genus Speleomantes

Speleomantes are the only plethodontid salamanders present in Europe. Multiple studies have been performed to investigate the trophic niche of the eight Speleomantes species, but none of these studies included hybrid populations. For the first time, we studied the trophic niche of five Speleomantes hybrid populations. Each population was surveyed twice in 2020, and stomach flushing was performed on each captured salamander; stomach flushing is a harmless technique that allows stomach contents to be inspected. We also assessed the potential divergence in size and body condition between natural and introduced hybrids, and their parental species. Previously collected data on Speleomantes were included to increase the robustness of these analyses. In only 33 out of 134 sampled hybrid Speleomantes we recognized 81 items belonging to 11 prey categories. The frequency of empty stomachs was higher in females and individuals from natural hybrid populations, whereas the largest number of prey was consumed by males. We compared the total length and body condition of 685 adult salamanders belonging to three types of hybrids and three parental (sub)species. Three group of salamanders (one hybrid and two parental species) showed significantly larger size, whereas no difference in body condition was observed. This study provided novel ecological information on Speleomantes hybrid populations. We also provided insights into the potential divergence between hybrids and parental species in terms of size and body condition. We discuss our findings, and formulate several hypotheses that should be tested in the future.

Updating salamander datasets with phenotypic and stomach content information for two mainland Speleomantes

European plethodontid salamanders (genus Speleomantes; formerly Hydromantes) are a group of eight strictly protected amphibian species which are sensitive to human-induced environmental changes. Long-term monitoring is highly recommended to evaluate their status and to assess potential threats. Here we used two low-impact methodologies to build up a large dataset on two mainland Speleomantes species (S. strinatii and S. ambrosii), which represents an update to two previously published datasets, but also includes several new populations. Specifically, we provide a set of 851 high quality images and a table gathering stomach contents recognized from 560 salamanders. This dataset offers the opportunity to analyse phenotypic traits and stomach contents of eight populations belonging to two Speleomantes species. Furthermore, the data collection performed over different periods allows to expand the potential analyses through a wide temporal scale, allowing long-term studies.

Effect of temperature on subclinical infection by Batrachochytrium dendrobatidis in three species of plethodontid salamanders

We assessed the effect of temperature on subclinical Batrachochytrium dendrobatidis (Bd) infection in three species of plethodontids. Collected individuals were tested for Bd in the field during the dry and rainy season and randomly assigned to 15° and 18°C treatments for 30 days. We collected 129 salamanders, of these nine individuals tested positive for Bd in the field. At the two temperature trials, 12 individuals that were Bd-negative in the field, tested positive for Bd. Near the end of the temperature trials, 18 of 21 Bd-positive individuals tested negative for Bd. Three individuals that were Bd positive in the field and assigned to the 18°C trial died during the experiment. Our results suggest that at 15°C the most of plethodontids present subclinical infection, whereas at higher temperatures, Bd infection can increase to detectable levels. The processes underlying plethodontids’s recovery from Bd infection warrants further study.