Understanding “Alexandrian” extinctions using genomic DNA from fluid-preserved museum specimens of Desmognathus salamanders

Species that went extinct prior to the genomic era are typically out-of-reach for modern phylogenetic studies. We refer to these as “Alexandrian” extinctions, after the lost library of the ancient world. This is particularly limiting for conservation studies, as genetic data for such taxa may be key to understand extinction threats and risks, the causes of declines, and inform management of related, extant populations. Fortunately, continual advances in biochemistry and DNA sequencing offer increasing ability to recover DNA from historical museum specimens, including fluid-preserved natural history collections. Here, we report on success in recovering nuclear and mitochondrial data from the apparently-extinct subspecies Desmognathus fuscus carri (Neill 1951), a plethodontid salamander from spring runs in central Florida. The two specimens are 50 years old and were likely preserved in unbuffered formalin, but application of a recently derived extraction procedure yielded usable DNA and partially successful Anchored Hybrid Enrichment sequencing. These data suggest that the populations of D. f. carri from peninsular Florida are conspecific with the D. auriculatus A lineage as suggested by previous authors, but likely represented an ecogeographically distinct genetic segment that has now been lost. Genetic data from this Alexandrian extinction thus confirm the geographic extent of population declines and extirpations as well as their ecological context, suggesting a possibly disproportionate loss from sandy-bottom clearwater streams compared to blackwater swamps. Success of these methods bodes well for large-scale application to fluid-preserved natural history specimens from relevant historical populations, but the possibility of significant DNA damage and related sequencing errors in additional hurdle to overcome.

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.

Patterns of Natural Selection on Mitochondrial Protein-Coding Genes in Lungless Salamanders: Relaxed Purifying Selection and Presence of Positively Selected Codon Sites in the Family Plethodontidae

There are two distinct lungless groups in caudate amphibians (salamanders and newts) (the family Plethodontidae and the genus Onychodactylus, from the family Hynobiidae). Lunglessness is considered to have evolved in response to environmental and/or ecological adaptation with respect to oxygen requirements. We performed selection analyses on lungless salamanders to elucidate the selective patterns of mitochondrial protein-coding genes associated with lunglessness. The branch model and RELAX analyses revealed the occurrence of relaxed selection (an increase of the dN/dS ratio = ω value) in most mitochondrial protein-coding genes of plethodontid salamander branches but not in those of Onychodactylus. Additional branch model and RELAX analyses indicated that direct-developing plethodontids showed the relaxed pattern for most mitochondrial genes, although metamorphosing plethodontids had fewer relaxed genes. Furthermore, aBSREL analysis detected positively selected codons in three plethodontid branches but not in Onychodactylus. One of these three branches corresponded to the most recent common ancestor, and the others corresponded with the most recent common ancestors of direct-developing branches within Hemidactyliinae. The positive selection of mitochondrial protein-coding genes in Plethodontidae is probably associated with the evolution of direct development.

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.

Conservation status of the Georgia Blind Salamander (Eurycea wallacei) from the Floridan Aquifer of Florida and Georgia

The Georgia Blind Salamander (Eurycea wallacei) is a poorly understood stygobitic plethodontid salamander found in the Floridan Aquifer of Florida and Georgia, USA. Its distribution is poorly delimited and little information is available on the ecology and life history of the species. We summarize existing information on the Georgia Blind Salamander and report new findings with regard to distribution, conservation status, and aspects of its ecology and life history from recent surveys in Florida and Georgia, including anecdotal observations made by local cave divers. We also present data on the prevalence of Batrachochytrium dendrobatidis and discuss potential measures and challenges involved in conservation. Georgia Blind Salamanders are known from at least 35 localities within five United States Geological Survey (USGS) HUC8 watersheds in Florida and Georgia, all within the groundwater of the Upper Floridan Aquifer. Some populations may be large, as cave divers have observed >100 salamanders in a single dive at three localities and reported densities up to 10 salamanders per square meter. We confirm through direct observation that the Dougherty Plain Cave Crayfish (Cambarus cryptodytes) is a predator of E. wallacei. Although we found no evidence that any populations of the Georgia Blind Salamander have been infected by amphibian chytrid fungus, clear threats remain and include: (1) An “at risk” ranking by USGS of the Floridian Aquifer for contamination; (2) overharvesting of groundwater; (3) the presence of an emergent infectious amphibian disease in surface amphibians in the region (e.g., amphibian chytrid fun- gus); and (4) a lack of data on the basic biology and ecology of the species. We recommend the following conservation actions: (1) Better regulations and improved methods for retaining pesticide and fertilizer contaminants on the surface; (2) regulatory review of agricultural crops grown in the region and the establishment of systems and crops that do not require center pivot irrigation; (3) regular monitoring for disease across the range of the species; and (4) establishment of multiple assurance colonies such that captive specimens genetically represent at least half of the known populations.

Predicting and mapping Plethodontid salamander abundance using LiDAR-derived terrain and vegetation characteristics

Aim of the study: Use LiDAR-derived vegetation and terrain characteristics to develop abundance and occupancy predictions for two terrestrial salamander species, Plethodon glutinosus and P. kentucki, and map abundance to identify vegetation and terrain characteristics affecting their distribution.Area of study: The 1,550-ha Clemons Fork watershed, part of the University of Kentucky’s Robinson Forest in southeastern Kentucky, USA.Materials and methods: We quantified the abundance of salamanders using 45 field transects, which were visited three times, placed across varying soil moisture and canopy cover conditions. We created several LiDAR-derived vegetation and terrain layers and used these layers as covariates in zero-inflated Poisson models to predict salamander abundance. Model output was used to map abundance for each species across the study area.Main results: From the184 salamanders observed, 63 and 99 were identifdied as P. glutinosus and P. kentucki, respectively. LiDAR-derived vegetation height variation and flow accumulation were best predictors of P. glutinosus abundance while canopy cover predicted better the abundance of P. kentucki. Plethodon glutinosus was predicted to be more abundant in sites under dense, closed-canopy cover near streams (2.9 individuals per m2) while P. kentucki was predicted to be found across the study sites except in areas with no vegetation (0.58 individuals per m2).Research highlight: Although models estimates are within the range of values reported by other studies, we envision their application to map abundance across the landscape to help understand vegetation and terrain characteristics influencing salamander distribution and aid future sampling and management efforts.Keywords: Zero-inflated Poisson model; Kentucky; Cumberland plateau; Plethodon glutinosus; Plethodon kentucki.

Acute physiological response by the plethodontid salamander Eurycea cirrigera (Southern Two-lined Salamander) to predation stress from alarm chemicals and predator kairomones

Plethodontid salamanders may reduce predation risk via behavioral responses to predator kairomones and alarm chemicals from injured salamanders. However, it not known whether such predator cues prompt acute physiological responses, which may enhance arousal and the physical ability to escape from a predator. I examined whether predator chemical cues elicit an acute cardiac response in Eurycea cirrigera (Green, 1831) (Southern Two-lined Salamander). I compared heart rates before and after exposure to the odor of the large predatory Pseudotriton ruber (Sonnini de Manoncourt and Latreille, 1801) (Red Salamander) and exposure to alarm chemicals from homogenized skin of conspecifics. For two controls, I compared heart rates before and after exposure to the odor of live conspecifics and the odor of the large non-predatory Plethodon mississippi Highton in Highton, Maha and Maxson, 1989 (Mississippi Slimy Salamander). Compared with resting values, heart rates significantly increased in response to predator kairomones (mean rate increased 10.9% after 2 min and 12.7% after 5 min) and alarm chemicals from conspecifics (mean rate increased 12.0% after 2 min and 14.5% after 5 min). In contrast, heart rates after exposure to each control odor did not significantly differ from resting values. Results demonstrate an acute cardiac response to chemical cues indicative of either a predator or a predation event.

Natural history of the critically endangered salamander Pseudoeurycea robertsi

Mexico is one of the most diverse countries that is losing a large amount of forest due to land use change, these data put Mexico in fourth place for global deforestation rate, therefore, Mexico occupies the first place in number of endangered species in the world with 665 endangered species. It is important to study amphibians because they are among the most threatened vertebrates on Earth and their populations are rapidly declining worldwide due primarily to the loss and degradation of their natural habitats. Pseudoeurycea robertsi is a micro-endemic and critically endangered Plethodontid salamander from the Nevado de Toluca Volcano and to date almost nothing is known about its natural history therefore, we survey fourteen sites of the Nevado de Toluca Volcano a mountain that is part of the Trans-Mexican Volcanic Belt, Mexico. We carry out the most exhaustive sampling scheme of this species throughout the Nevado de Toluca Volcano to know the number of individuals and the microhabitat features associated with the presence of P. robertsi. Likewise, we carry out a morphometric study and coloration measurements of P. robertsi individuals and we determine the potential distribution of P. robertsi and the other 3 species of pletodontids present in the NTV using ecological niche modeling and to determine the most important habitat features associated with the presence of salamander species, as well as to know the niche overlap among salamander species. This information will help raise conservation strategies for this micro-endemic and critically endangered salamander.

Natural history of the critically endangered salamander Pseudoeurycea robertsi

Mexico is one of the most diverse countries that is losing a large amount of forest due to land use change, these data put Mexico in fourth place for global deforestation rate, therefore, Mexico occupies the first place in number of endangered species in the world with 665 endangered species. It is important to study amphibians because they are among the most threatened vertebrates on Earth and their populations are rapidly declining worldwide due primarily to the loss and degradation of their natural habitats. Pseudoeurycea robertsi is a micro-endemic and critically endangered Plethodontid salamander from the Nevado de Toluca Volcano and to date almost nothing is known about its natural history therefore, we survey fourteen sites of the Nevado de Toluca Volcano a mountain that is part of the Trans-Mexican Volcanic Belt, Mexico. We carry out the most exhaustive sampling scheme of this species throughout the Nevado de Toluca Volcano to know the number of individuals and the microhabitat features associated with the presence of P. robertsi. Likewise, we carry out a morphometric study and coloration measurements of P. robertsi individuals and we determine the potential distribution of P. robertsi and the other 3 species of pletodontids present in the NTV using ecological niche modeling and to determine the most important habitat features associated with the presence of salamander species, as well as to know the niche overlap among salamander species. This information will help raise conservation strategies for this micro-endemic and critically endangered salamander.