Plethodon Cinereus Research Articles

The Identification of 2,4-diacetylphloroglucinol as an Antifungal Metabolite Produced by Cutaneous Bacteria of the Salamander Plethodon cinereus

Beneficial bacteria that live on salamander skins have the ability to inhibit pathogenic fungi. Our study aimed to identify the specific chemical agent(s) of this process and asked if any of the antifungal compounds known to operate in analogous plant-bacteria-fungi systems were present. Crude extracts of bacteria isolated from salamander skin were exposed to HPLC, UV-Vis, GC-MS, and HR-MS analyses. These investigations show that 2,4-diacetylphloroglucinol is produced by the bacteria isolate Lysobacter gummosus (AB161361), which was found on the red-backed salamander, Plethodon cinereus. Furthermore, exposure of the amphibian fungal pathogen, Batrachochytrium dendrobatidis (isolate JEL 215), to different concentrations of 2,4-diacetylphloroglucinol resulted in an IC50 value of 8.73 microM, comparable to crude extract concentrations. This study is the first to show that an epibiotic bacterium on an amphibian species produces a chemical that inhibits pathogenic fungi.

ANTIPREDATOR ADAPTATIONS AND PREDATOR AVOIDANCE BY TWO COLOR MORPHS OF THE EASTERN RED-BACKED SALAMANDER, PLETHODON CINEREUS

Populations of eastern red-backed salamanders, Plethodon cinereus, exhibit color polymorphism, and two color morphs are common: the red-striped morph and the lead-phase morph. A recent hypothesis attributes the maintenance of the polymorphism to selection on behavior and physiology. We conducted a series of laboratory experiments and a field study to determine whether striped and lead-phase salamanders exhibit different antipredator responses and whether predators differentially attacked the color forms. In predation trials, red-striped salamanders spent significantly more time in an “all trunk raised” posture and tended to remain immobile, whereas lead-phase salamanders were significantly more mobile. In field collected museum specimens of individual P. cinereus, we found more lead-phase salamanders with autotomized tails compared to red-striped salamanders, possibly indicating different attack rates on the two forms. The results from this study demonstrate that striped and lead-phase salamanders of P. c...

Habitat relationships of eastern red-backed salamanders ( Plethodon cinereus) in Appalachian agroforestry and grazing systems

Woodland salamander responses to either traditional grazing or silvopasture systems are virtually unknown. An information-theoretic modelling approach was used to evaluate responses of red-backed salamanders ( Plethodon cinereus) to silvopasture and meadow conversions in southern West Virginia. Searches of area-constrained plots and artificial coverboards that were distributed across a gradient of agricultural conversion and grazing intensity, including hardwood silvopastures, hay meadows, forest edges, and reference forests yielded 2823 salamanders between May 2004 and November 2005. Salamander presence and abundance were positively associated with increasing cover of herbaceous vegetation and negatively associated with the intensity of agricultural disturbance. Although salamander presence and abundance appeared to be negatively influenced by agricultural disturbance when compared to reference forest conditions, the occurrence of red-backed salamanders within agriculturally modified habitats indicates this species may be more resilient to forest conversion than previously thought. This study suggests that herbaceous vegetation retained within some agricultural treatments, in combination with artificial cover, may at least partially mitigate the loss of forest canopy for red-backed salamanders.

Seeing What We Want to See: Confirmation Bias in Animal Behavior Research

AbstractConfirmation bias is the tendency of observers to see what they expect to see while conducting scientific research. Although confirmation bias has been well‐studied by psychologists in the context of qualitative judgments, it has been much less studied with respect to the kinds of quantitative observations made by behavioral biologists. We carried out two experiments that used multiple observers of the aggression and foraging behaviors of red‐backed salamanders (Plethodon cinereus) to determine whether behavioral observations were influenced by the a priori expectations of observers. In both experiments, one group of observers was given a specific set of expectations with respect to sex differences in salamander behavior, while a second group was given the opposite set of expectations. In one experiment, observers collected data on variable sets of live salamanders, while in the other experiment, observers collected data from identical videotaped trials. Across experiments and observed behaviors, the expectations of observers did appear to bias observations, but only to a small or moderate degree. Confirmation bias never accounted for more than 13% of the observed variation in behavior, and was generally equivalent to <20% of the mean value of each variable. The estimated magnitude of confirmation bias was quite similar for men and women, suggesting no relationship between observer gender and susceptibility to confirmation bias. We believe that these results are largely optimistic with respect to confirmation bias in behavioral ecology, in that they suggest the bias may often be small relative to individual variation in behavior, even for relatively inexperienced observers.

Common Cutaneous Bacteria from the Eastern Red-Backed Salamander Can Inhibit Pathogenic Fungi

The objective of this study was to characterize the cutaneous antifungal bacterial flora of a plethodontid salamander, Plethodon cinereus, and to relate our results to aspects of amphibian ecology. Scanning electron micrographs of salamander skin indicated a wide distribution of bacteria across the skin. Polymerase Chain Reaction/Denaturant Gradient Gel Electrophoresis (PCR/DGGE) analyses of 16S rRNA gene fragments revealed that a transient community of bacteria was rinsed off and that a resident epibiotic community remained on the skin. Resident bacteria were isolated from the skin of P. cinereus on a low nutrient medium and challenged against a pathogenic ascomycete fungus collected from dead salamander eggs. The strong antifungal bacteria, identified by sequencing of the 16S rRNA gene (∼1400 bp), were related to the genera Lysobacter, Pseudomonas, Chryseobacterium, and Bacillus. Patterns of the cutaneous bacterial flora varied among individual salamanders, as revealed by DGGE, although analysis of DGGE bands from salamander skin revealed the presence of at least one species of uncultured β-proteobacteria on virtually every salamander. These bacteria were closely related to an isolated antifungal Janthinobacterium lividum strain. Antifungal skin bacteria may form mutualistic ecological relationships with amphibian species and help protect them from pathogenic fungi.

Comparison of Three Methods for Surveying Amphibians in Forested Seep Habitats in Washington State

We compared the effectiveness of three amphibian survey methods (trapping, light-touch, and destructive sampling) in seeps because the efficiency of these methods in this kind of habitat has not been evaluated previously. Our study sites were located on managed forests in southwest Washington State. Trapping involved setting up an array of funnel traps across the seep, light-touch is a visual-encounter method facilitated by overturning and replacing moveable cover objects, and destructive sampling consisted of searching the seep surface by excavating the top 15 cm of soil and dismantling woody debris. Trapping and light-touch were compared through six, three-week periods, whereas the non-repeatable destructive sampling was compared with trapping and light-touch only during the final sampling period. Light-touch detected more species (P = 0.007), more coastal tailed frogs (Ascaphus truei; P < 0.001), and similar numbers of Columbia torrent salamanders (Rhyacotriton kezeri; P = 0.123) and western red-backed salamanders (Plethodon vehiculum; P = 0.152) compared to trapping. When compared to other survey methods during the final sampling period, destructive sampling detected more species (P = 0.001) and more torrent salamanders (P= 0.005) than trapping, but detected similar numbers of species (P = 0.15) and torrent salamanders (P = 0.21) as light-touch. Light-touch was less expensive in material costs and required fewer visits, but more time (77 vs. 19 person-minutes) per survey session than trapping. Destructive sampling had the same material costs as light-touch, but required more time per survey session (690 person-minutes) than either of the other two methods. Where a repeatable method is required, light-touch seems preferable to trapping because it enumerates a higher percentage of species and individuals, has fewer potential survey biases, and can provide data on within-seep amphibian use.

Effects of roads on patterns of genetic differentiation in red-backed salamanders, Plethodon cinereus

Roads can fragment animal populations by reducing gene flow, which can lead to drift and the loss of genetic diversity. One of the principle signatures of reduced gene flow is increased genetic differentiation in isolated populations, and evidence that roads contribute to such differentiation has been reported for several species. We used microsatellites to examine whether six roads led to increased genetic differentiation in red-backed salamanders (Plethodon cinereus). These six roads included one divided interstate highway, one undivided four-lane highway, and four secondary roads. We found that the genetic distance between plots that were bisected by the interstate highway was significantly greater than the genetic distance between equidistant plots on the same side of the highway. However, for the five smaller roads, plots across the road were no more genetically distinct than were plots on the same side of the road. Bayesian clustering methods also supported both of these findings. The optimal clustering of plots for the interstate highway consisted of two clusters that corresponded to the two sides of highway. For the other five sites, the optimal grouping consisted of a single cluster containing all of the plots. Our findings suggest that gene flow across very large roads is rare and that bisected red-backed salamander populations are likely to diverge from one another. For smaller roads, our results imply that the indirect effects of roads on genetic population structure are probably less of a pressing concern for terrestrial salamanders than are the direct effects of mortality and habitat alteration.

MALE RED-BACKED SALAMANDERS CAN DETERMINE THE REPRODUCTIVE STATUS OF CONSPECIFIC FEMALES THROUGH VOLATILE CHEMICAL SIGNALS

Previous research suggests that female red-backed salamanders (Plethodon cinereus )i n Virginia court biennially whereas males court annually. Therefore, males may face a choice to court either gravid or nongravid females. Because gravid females represent an immediate insemination opportunity (whereas nongravid females do not), male red-backed salamanders may be under selection to be able to distinguish the reproductive status of conspecific females. We conducted an experiment to determine if males could discriminate between gravid and nongravid conspecific females through volatile chemical signals. Focal males were allowed to establish territories in testing arenas for 5 d and then were exposed to three treatments in a randomized order: volatile chemical signals from gravid females, nongravid females, and a control (blank filter paper). Randomization tests revealed that focal males exhibited significantly more aggressive behavior when they were exposed to volatile chemical signals from nongravid females than when they were exposed to those from gravid females and the control. We infer that male red-backed salamanders can determine the reproductive status of conspecific females through volatile chemical signals, which may influence their social associations.

Edge Effects of Gated and Ungated Roads on Terrestrial Salamanders

ABSTRACTRoads through forest habitats reduce the abundance of many animal species. These reductions are often referred to as edge effects and their causes include roadkill, degradation of forest habitat, and changes in biotic interactions. Which of these causes are operating can have important implications for management. Terrestrial salamanders in the southern Appalachians have previously been shown to be subject to edge effects from forest roads that are open to traffic. In this study, I examined edge effects on red‐backed salamanders (Plethodon cinereus) along forest roads that were either open or gated to prevent vehicle entry. I also included roads that varied in the width of the gravel surface, the width of the roadside verge, and the magnitude of habitat gradients at the forest edge. I found that ungated roads were associated with consistent edge effects on salamanders, whereas no detectable edge effects were found for gated roads. Road width was as good a predictor of the magnitude of edge effects as was the presence of a gate, though the width of the roadside verge was largely unrelated to the magnitude of edge effects. Gradients in habitat variables (soil moisture, temp, leaf litter thickness) were not closely related to the magnitude of edge effects. These results demonstrate that narrow, gated roads do not typically produce edge effects on terrestrial salamanders of the same magnitude as wider, ungated roads. In addition, the apparent importance of road type or road width and the relative unimportance of habitat characteristics suggest that traffic‐related factors may be a substantial contributor to edge effects on terrestrial salamanders. These findings provide some support for the closing of redundant forest roads as a low‐cost method for diminishing the negative effects of roads on forest ecosystems.

Influence of Headwater Site Conditions and Riparian Buffers on Terrestrial Salamander Response to Forest Thinning

Abstract Although thinning of young, even-aged forests may accelerate the development of characteristics associated with mature forests, in the short term it may negatively affect some taxa, including terrestrial salamanders. Preexisting site conditions, including down wood, and forest management measures, such as riparian buffers, may moderate these effects, but these relationships are poorly understood. To explore whether down wood and riparian buffer widths might influence short-term responses to thinning, we sampled salamanders using ground searches before and during the first 2 years after experimental thinning at two 45- to 65-year-old headwater forest sites in western Oregon that differed in down wood volume. Prethinning distributions of terrestrial salamanders overlapped one- and two-tree height riparian buffers, and except for red-backed salamanders, overlapped very little with narrower streamside or variable-width buffers. At the site where down wood volume was low, captures of ensatina (Ensatina eschscholtzii Gray) and western red-backed salamanders (Plethodon vehiculum Cooper) both declined by 40% in thinned areas. In contrast, captures of ensatina and Oregon slender salamanders (Batrachoseps wrighti Bishop) were not significantly affected by thinning at the site where down wood volume was high. Our results suggest that site conditions, such as down wood volume, and riparian buffers may influence the effect of thinning on terrestrial salamanders, and demonstrate the tight linkage among management of aquatic, riparian, and upslope resources in headwater forests.

Preliminary Study of the Effects of Headwater Riparian Reserves with Upslope Thinning on Stream Habitats and Amphibians in Western Oregon

Abstract We conducted a preliminary examination of the responses of stream amphibians and instream habitat conditions to alternative riparian buffer zones with forest thinning upslope. Pre and posttreatment surveys were carried out on 68 headwater stream reaches (including 23 unthinned reference reaches) at 11 sites in western Oregon. Streams were in managed conifer stands, 40 to 80 years old, where the thinning treatment reduced stands from 600 trees per hectare (tph) to 200 tph. Treatments consisted of four widths of riparian buffers approximately 6, 15, 70, and 145 m on each side of streams. Over three study years, 3,131 individuals of 13 species were detected. For the more common instream and bank species analyzed, capture rates persisted posttreatment with no negative treatment effect from thinning with any of the buffer widths. More animals were detected after thinning in treatment reaches compared to reference reaches for rough-skinned newts (Taricha granulosa Skilton) occurring on stream banks, and for instream coastal giant salamanders (Dicamptodon tenebrosus Baird and Girard). Treatment effects on instream habitat parameters were not detected. Interannual variation was evident for western red-backed salamanders (Plethodon vehiculum Cooper), and several habitat conditions including pool-riffle ratio, stream spatial intermittency, stream width, and down wood. Overall, riparian buffers with moderate upslope thinning (200 tph) seemed to have retained the aquatic vertebrate community along channels among sites in the first 2 years posttreatment; however, several limitations of the study reduce the inference of the findings, and these preliminary results are best interpreted as hypotheses for further investigation.

IDENTIFICATION OF THE NATURAL BACTERIAL MICROFLORA ON THE SKIN OF EASTERN NEWTS, BULLFROG TADPOLES AND REDBACK SALAMANDERS

Amphibian populations in several regions of the world appear to be declining due to infectious diseases. While many studies have attempted to identify the pathogens associated with specific declines, very few studies have attempted to identify the natural microflora that is present on amphibian skin. Knowledge of the natural flora that healthy individuals carry may, in some cases, provide valuable information for understanding disease outcomes. In this study, we isolated the natural bacterial flora found on the skin of apparently healthy adult eastern newts (Notophthalamus viridescens), larval bullfrogs (Rana catesbieana) and redback salamanders (Plethodon cinereus) living in natural field sites in Virginia. We positively identified five bacterial species from newts, three bacterial species from bullfrogs and four bacterial species and one yeast from redback salamanders. In a parallel study, we examined the physiological profiles of bacterial communities at six sites with newt and bullfrog tadpole populat...

JUVENILE MOVEMENTS AND POTENTIAL INTER-AGE CLASS ASSOCIATIONS OF RED-BACKED SALAMANDERS

For many taxa, little is known about whether territoriality of adults affects the behavior of juveniles. Laboratory research has shown that aggression of adult territorial red-backed salamanders (Plethodon cinereus) decreases with familiarity, but it is not clear whether juveniles are randomly or nonrandomly associated with adult or parental territories. Using data from mark-recapture surveys, we compared the number of captures of first-year juveniles and adults of P. cinereus to ambient meteorological data. We found no evidence for temporal separation of foraging; weather conditions did not affect the proportion of first-year juveniles captured. We then conducted nearest-neighbor analyses to evaluate whether first-year juveniles were spatially associated with or disassociated from adult home ranges. Juveniles were neither closer to nor further away from adults than would be expected by random chance. Nearest-neighbor distances also were not significantly different from the mean radius of adult home ranges. There was no evidence that the distribution of adult home ranges affected the distribution of juveniles, at least after recent rainfall events; there is a possibility that interactions between age classes may occur under drier conditions.

Detection of the Sexual Identity of Conspecifics through Volatile Chemical Signals in a Territorial Salamander

AbstractTerritorial red‐backed salamanders (Plethodon cinereus) have been shown to use nonvolatile chemical signals in both territorial defense and to convey a variety of information to conspecifics. We investigated whether or not red‐backed salamanders could determine the sexual identity of conspecifics through volatile chemical signals, and we explored their use in the context of territorial defense. We exposed male and female red‐backed salamanders to four experimental treatments (i.e. filter papers that had been scent marked by male or female conspecifics for 1 and 5 d) and two control treatments (i.e. unscented filter papers for 1 and 5 d tests). The focal salamanders were prevented from physically accessing the scent marked filter papers and, presumably, some of the substrate scent marks had volatile components that were detected and interpreted by the focal salamanders. Both male and female red‐backed salamanders spent significantly more time in threat displays when they were exposed to volatile chemical signals from same‐sex conspecifics than they did toward similar signals from opposite‐sex conspecifics. A similar statistical pattern was observed for the amount of chemosensory sampling exhibited by focal red‐backed salamanders. From these results, we infer that red‐backed salamanders can determine the sexual identity of conspecifics through volatile chemical signals, some of which may be used in territorial defense. Further, such airborne pheromones may influence the spatial organization of salamander territories on the forest floor.

Ecological and genetic evidence that low-order streams inhibit dispersal by red-backed salamanders (Plethodon cinereus)

While many studies have examined the barrier effects of large rivers on animal dispersal and gene flow, few studies have considered the barrier effects of small streams. We used displacement experiments and analyses of genetic population structure to examine the effects of first-order and second-order streams on the dispersal of terrestrial red-backed salamanders, Plethodon cinereus (Green, 1818). We marked red-backed salamanders from near the edges of one first-order stream and one second-order stream, and experimentally displaced them either across the stream or an equal distance farther into the forest. A comparison of return rates indicated that both streams were partial barriers to salamander movement, reducing return rates by approximately 50%. Analysis of six microsatellite loci from paired plots on the same side and on opposite sides of the second-order stream suggested that the stream did contribute to genetic differentiation of salamander populations. Collectively, our results imply that low-order streams do influence patterns of movement and gene flow in red-backed salamanders. We suggest that given the high density of first-order and second-order streams in most landscapes, these features may have important effects on species that, like red-backed salamanders, have limited dispersal and large geographic ranges.

Future forests and indicator-species population models

In this paper, we provide an overview of a project that we initiated to explore the utility of spatially-explicit metapopulation models linked to dynamic landscape models as a way of incorporating biological indicators into sustainable forest management. We developed models for three indicator species as case studies; brown creeper (Certhis americana), redbacked vole (Clethrionomys gapperi) and red-backed salamander (Plethodon cinereus) in a northern Ontario landscape. Results from the project to date suggest that there are significant advantages to models that are spatially-explicit and dynamic in their treatment of both populations and landscapes. Dynamic landscape metapopulation (DLMP) models allow a manager to track population change through time in response to a changing landscape and a fluctuating environment. These DLMP models may be used to predict the impact of current and alternative forest management strategies on population sizes of a suite of species chosen to indicate the health of forest ecosystems. Key words: biological indicators, population viability analysis, population modeling, dynamic landscape modeling, sustainable forest management, brown creeper, red-backed salamander, red-backed vole

Location‐specific sympatric morphological divergence as a possible response to species interactions in West VirginiaPlethodonsalamander communities

1. The competitive interactions of closely related species have long been considered important determinants of community composition and a major cause of phenotypic diversification. However, while patterns such as character displacement are well documented, less is known about how local adaptation influences diversifying selection from interspecific competition. 2. We examined body size and head shape variation among allopatric and sympatric populations of two salamander species, the widespread Plethodon cinereus and the geographically restricted P. nettingi. We quantified morphology from 724 individuals from 20 geographical localities throughout the range of P. nettingi. 3. Plethodon nettingi was more robust in cranial morphology relative to P. cinereus, and sympatric localities were more robust relative to allopatric localities. Additionally, there was significantly greater sympatric head shape divergence between species relative to allopatric communities, and sympatric localities of P. cinereus exhibited greater morphological variation than sympatric P. nettingi. 4. The sympatric morphological divergence and increase in cranial robustness of one species (P. nettingi) were similar to observations in other Plethodon communities, and were consistent with the hypothesis of interspecific competition. These findings suggest that interspecific competition in Plethodon may play an important role in phenotypic diversification in this group. 5. The increase in among-population variance in sympatric P. cinereus suggests a species-specific response to divergent natural selection that is influenced in part by other factors. We hypothesize that enhanced morphological flexibility and ecological tolerance allow P. cinereus to more rapidly adapt to local environmental conditions, and initial differences among populations have allowed the evolutionary response of P. cinereus to vary across replicate sympatric locations, resulting in distinct evolutionary trajectories of morphological change.

Toxicological responses of red-backed salamanders ( Plethodon cinereus) to subchronic soil exposures of 2,4-dinitrotoluene

Dinitrotoluenes are used as propellants and in explosives by the military and as such have been found at relatively high concentrations in the soil. To determine whether concentrations of 2,4-dinitrotoluene (2,4-DNT) in soil are toxic to amphibians, 100 red-backed salamanders ( Plethodon cinereus) were exposed to either 1500, 800, 200, 75 or 0 mg 2,4-DNT/kg soil for 28 days and evaluated for indicators of toxicity. Concentrations of 2,4-DNT were less than targets and varied with time. Most salamanders exposed to concentrations exceeding 1050 mg/kg died or were moribund within the first week. Salamanders exposed to soil concentrations exceeding 345 mg/kg lost >6% of their body mass though no mortality occurred. Overt effects included a reduction in feed consumption and an increase in bucco-pharyngeal oscillations in salamanders. These results suggest that only high soil concentrations of 2,4-DNT have the potential to cause overtly toxic effects in terrestrial salamanders.

Impact of urban fragmentation on the genetic structure of the eastern red-backed salamander

Urban development is a major cause of habitat loss and fragmentation. Few studies, however, have dealt with fragmentation in an urban landscape. In this paper, we examine the genetic structure of isolated populations of the eastern red-backed salamander (Plethodon cinereus) in a metropolitan area. We sampled four populations located on a mountain in the heart of Montreal (Quebec, Canada), which presents a mosaic of forested patches isolated by roads, graveyards and buildings. We assessed the genetic structure of these populations using microsatellite loci and compared it to the genetic structure of four populations located in a continuous habitat in southern Quebec. Our results indicate that allelic richness and heterozygosity are lower in the urban populations. Exact differentiation tests and pairwise FST also show that the populations found in the fragmented habitat are genetically differentiated, whereas populations located in the continuous habitat are genetically homogeneous. These results raise conservation concerns for these populations as well as for rare or threatened species inhabiting urban landscapes.

Fine-scale population differentiation and gene flow in a terrestrial salamander (Plethodon cinereus) living in continuous habitat

Several recent studies have shown that amphibian populations may exhibit high genetic subdivision in areas with recent fragmentation and urban development. Less is known about the potential for genetic differentiation in continuous habitats. We studied genetic differentiation of red-backed salamanders (Plethodon cinereus) across a 2-km transect through continuous forest in Virginia, USA. Mark-recapture studies suggest very little dispersal for this species, whereas homing experiments and post-Pleistocene range expansion both suggest greater dispersal abilities. We used six microsatellite loci to examine genetic population structure and differentiation between eight subpopulations of red-backed salamanders at distances from 200 m to 2 km. We also used several methods to extrapolate dispersal frequencies and test for sex-biased dispersal. We found small, but detectable differentiation among populations, even at distances as small as 200 m. Differentiation was closely correlated with distance and both Mantel tests and assignment tests were consistent with an isolation-by-distance model for the population. Extrapolations of intergenerational variance in spatial position (sigma(2)<15 m(2)) and pair-wise dispersal frequencies (4 Nm < 25 for plots separated by 300 m) both suggest limited gene flow. Additionally, tests for sex-biased dispersal imply that dispersal frequency is similarly low for both sexes. We suggest that these low levels of gene flow and the infrequent dispersal observed in mark-recapture studies may be reconciled with homing ability and range expansion if dispersing animals rarely succeed in breeding in saturated habitats, if dispersal is flexible depending on the availability of habitat, or if dispersal frequency varies across the geographic range of red-backed salamanders.