In May 2023, herpetologists from six countries converged at the 8th Conference on the Biology of Plethodontid Salamanders in Hammond, Louisiana to share their latest cutting-edge research. The conference was hosted by the Department of Biological Sciences at Southeastern Louisiana University. Dr. Richard Bruce was the honoree. The presentations covered a diverse array of topics from gene expression to behavior to speciation, illustrating the value of plethodontid salamander biology to a range of disciplines. This special issue of Herpetologica includes 16 papers highlighting a range of research on plethodontids and encouraging new approaches to tackle old and new questions.

Studies of reproductive behavior can help reveal patterns of sexual isolation and clarify species boundaries. In northern Georgia, Blue Ridge Two-lined Salamanders (Eurycea cf. wilderae) and Brown-backed Salamanders (Eurycea aquatica) are syntopic but do not hybridize. Within this region, Eurycea cf. wilderae males exhibit a reproductive polymorphism, with “searching” males that appear adapted for terrestrial courtship and “guarding” males that appear adapted for aquatic mate-guarding. To understand better how inter- and intraspecific reproductive variation might contribute to sexual isolation between E. aquatica and E. cf. wilderae, we staged T-maze trials and courtship trials, and we conducted preliminary analyses of sex hormones from noninvasive fecal samples. We found that males of both species preferred conspecific female scents to heterospecific female scents, but that searching male E. cf. wilderae were not better than guarding males at locating a conspecific female scent. We also found evidence for strong propensity asymmetry, with male and female E. cf. wilderae much more likely than E. aquatica to engage in courtship behaviors in the lab. Still, we observed some instances of heterospecific courtship through spermatophore transfer. Finally, we found evidence for higher fecal testosterone concentrations in males than females and higher concentrations in guarding male than in searching male E. cf. wilderae. Together, our data provide further insight into the reproductive ecologies of and the nature of reproductive isolation between Eurycea aquatica and Eurycea cf. wilderae.

Agonistic behavior, a critical component of territoriality, is widespread throughout terrestrial plethodontid salamanders, though geographic variation in this behavior is poorly understood. Using color polymorphic Eastern Red-backed Salamanders (Plethodon cinereus), we conducted same-sex laboratory trials to compare aggressive and submissive behaviors between two genetically distinct groups in northern Ohio. For each group, we tested 120 individuals from 3 populations. We predicted that the genetic group with a single color morph would exhibit a higher degree of agonistic and territorial behavior compared to the genetic group with two morphs as an adaptive consequence of altered social dynamics in monomorphic populations. Contrary to our prediction, laboratory trials demonstrated that residents from the polymorphic group were significantly more aggressive than residents from the monomorphic group. This finding was corroborated by a strong residency effect in the polymorphic group, an effect that was weaker in the monomorphic group. Our results suggest that individuals in the polymorphic group are more aggressive and territorial than individuals in the monomorphic group, signifying a greater propensity to secure a territory and defend it against intruders. Our results add important insight into how geographic variation and differences in morph frequency can be attributed to behavioral differences.

Since being described in the year 2000, little ecological information has accumulated for South Mountains Gray-Cheeked Salamanders (Plethodon meridianus), a narrowly distributed salamander in southern Appalachia. In this study, we used repeat surveys to model occupancy and abundance of P. meridianus along a transect running from the approximate range center to range edge. We found a strong effect of elevation and distance to streams as predictors of occupancy and abundance. The number of days since rain had a strong impact on detection probability, likely due to its effect on salamander surface activity (i.e., time outside of burrow), as opposed to observer error. Treating elevation as a proxy for distance to the range edge, we found that occupancy and abundance declined toward the range edge, supporting the center-periphery hypothesis. Our findings matched that of similar previous studies: at low elevations in dry habitat, salamanders are only found along the cooler microhabitat of streams, whereas at high elevations, salamanders are more widespread across the landscape.

The trophic niche is one of the most important ecological traits for any species, providing information about trophic position in the food web, habitat preferences, and interspecific interactions. In this study, we describe the autumn diet of two sympatric species of salamander from central Italy—Italian Cave Salamanders (Speleomantes italicus) and Fire Salamanders (Salamandra salamandra), assessing whether competition for the same prey occurs. Furthermore, we combine our data with those obtained for the same population during a different season (spring) and assess potential seasonal variation in the diet of these two salamander species. The overlap of the trophic niche between the two species was limited, with S. italicus consuming smaller and fast-moving prey (e.g., Diptera, Collembola), and S. salamandra consuming slow and often elongated prey (e.g., Gastropoda, Haplotaxida, Diplopoda). Seasonal differences in diet composition were observed in both species between spring and autumn; in autumn, both species narrowed their trophic niche breadth. This seasonal divergence is likely due to the variation of prey phenology and availability. The consumption of prey of different size was likely the main factor allowing the coexistence of these two salamander species. In both species we observed a general increase in the proportion of generalist individuals in autumn, most likely because of a reduction in prey availability. In addition to providing information on the trophic niche of these species, our study represented a further step that helps unravel the dynamics promoting the coexistence of potentially competing species.

Foraging in animals ranges from a sedentary, ambush mode to more active patterns. Among the plethodontid genus Desmognathus, early observations have indicated that small species and small members of large species may forage more actively than large individuals. Therefore, we tested the hypothesis that foraging within and across the genus depends on body size. We chose species ranging from the largest (D. amphileucus) to the small members of the D. ocoee complex (D. ocoee and D. perlapsus). We also included two species intermediate in size, D. monticola and D. folkertsi. We analyzed foraging of 570 individual salamanders, collecting them at night and determining whether they were foraging while partially within a refuge, indicating an ambush posture, or outside of a refuge in a more active mode. Using replicate streams, we determined that, for each species or species complex, refuged animals had significantly larger snout–vent lengths (SVLs) than individuals outside of a refuge. Moreover, the proportion of individuals foraging in an ambush posture in a refuge increased with mean SVL of the population sampled with 76% of the variance in proportion of search modes being predicted by mean SVL. The results of a replicated laboratory-based experiment did not match the patterns observed in the field, indicating that the trend of larger individuals to remain in refugia is not a simple reflexive response to refugia. However, the trend of larger individuals to forage while remaining in a sedentary, ambush posture in a refuge in resident streams helps to explain ecological and evolutionary patterns within the tribe Desmognathini.

Headwater streams are critical, fragile ecosystems that supply food and nutrients for an array of organisms that are vital to the overall health of a stream. As the dominant vertebrates in headwater streams of the southern Appalachian Mountains, plethodontid salamanders are critical to healthy headwater communities. However, a neglected feature of these ecosystems is the community of parasites that infect salamanders. The digenetic trematode Metagonimoides oregonensis uses the aquatic snail Elimia proxima as its first intermediate host and the plethodontid salamander Desmognathus amphileucus as its second intermediate host. In a series of southern Appalachian streams, we tested the hypothesis that variance in the prevalence and intensity of infection in salamander hosts is related to respective densities of the snail and salamander species. Infection prevalence was 100% across all streams. There was a strong relationship between the density of the snail host and infection-intensity level in the salamander host. However, the relationship with salamander density was not important, a finding that conflicts with the results of parasitological studies involving lentic and marine systems. The influence of this trematode on the ecology of headwater streams and the resident salamanders has been underappreciated, and a number of aspects of this relationship remain unknown.

Amphibian populations, including those of lungless salamanders (Plethodontidae), are susceptible to population declines, especially considering current and predicted climate change. Like many organisms, plethodontid salamanders exhibit reproductive plasticity to maximize population growth in response to environmental conditions. A better understanding of the external causes of reproductive plasticity can provide more accurate population models through the explicit incorporation of such drivers. To this end, we sampled Southern Pygmy Salamanders (Desmognathus wrighti) along a 1345-m elevational gradient in Great Smoky Mountains National Park, USA, to determine the effects of body size and elevation on reproductive investment. Using Bayesian mixed effects models, we assessed whether body size, elevation, or a combination of these factors best explained the probability an individual was gravid, the number of ova in gravid females, and the number of testis lobes in mature males. We found that the interaction of body size and elevation best explained each reproductive measure. Larger females were generally more likely to be gravid, with annual reproduction in fully mature females at low to mid-elevations and roughly biennial reproduction at high elevations. We also found a positive relationship between body size and ova counts at low and mid-elevations, but despite a reduction in reproductive frequency at higher elevations, we found no effect of body size on ova count. In addition, the addition of testis lobes, which has commonly been used as a proxy of age in plethodontid salamanders, occurred at larger body sizes with increasing elevation. Our findings support previous work showing a relationship between body size and reproductive ecology in desmognathine salamanders while providing insight into the complexity of this relationship in the context of elevational clines. Reproductive ecology significantly influences population dynamics; thus, further refinement of these observed patterns and their underlying causes is necessary to advance plethodontid salamander population ecology.

Multispecies assemblages of Appalachian desmognathans have long intrigued ecologists seeking to understand the structure of ecological communities. Body size and life history in this group correlate with the moisture gradient from stream to forest, with large species being more aquatic and progressively smaller ones being ever more terrestrial. Since the work of N.G. Hairston, Sr. in the early 1980s, the prevailing hypothesis has been that predation and competition among desmognathans themselves have been the driving processes in the development and maintenance of the spatial organization along stream sides, with large, aggressive species forcing smaller ones to occupy less favorable terrestrial niches. Decades of research have confirmed that biotic interactions are important. However, evidence from recent studies has not agreed with the idea that biotic interactions are primary. Herein, I have concluded that the prevailing hypothesis fails to reconcile the currently available data. I have presented an alternative hypothesis that an abiotic factor, specifically the relative abundance of water, has been the primary force driving adaptation of species of Desmognathus longitudinally along moisture gradients by way of stream headwaters. Interspecific interactions, though important aspects of the current ecology of these species, are not the underlying drivers of spatial organization along the moisture gradient. I have further generated specific predictions that will enable this hypothesis to be tested.

Patterns of species distributions and abundance are driven by a combination of species abiotic niches and biotic interactions between members of a community. Joint-species distribution models extend traditional modeling of occurrence or abundance to quantify species corelationships that may result from interspecific interactions. In the southern Appalachian Mountains, stream-associated salamander communities are thought to be structured by competitive and predatory interactions; therefore, spatial patterns in occurrence and abundance and temporal patterns in activity may reflect species associations. To evaluate the role of interspecific interactions in salamander community occurrence and abundance patterns, we conducted repeated point-count and leaf-bag surveys for adult and larval communities, respectively. We estimated adult and larval community occurrence and abundance by using the hierarchical modeling of species communities framework to identify the extent to which residual co-occurrence and correlation in abundance was present within these communities. For both adult and larval communities, we found no evidence that residual co-occurrence or correlation in abundance was present. Instead, results indicated that environmental covariates characterizing both plot- and landscape-level habitat conditions were responsible for observed patterns. In addition, we found no evidence for temporal niche partitioning, suggesting competition does not play a significant role in inter- or intraday activity patterns. Our results contrast with the accepted paradigm that extant biotic interactions drive community composition among stream-associated salamanders. Instead, our findings suggest that the effects of biotic interactions are not evident at the spatial or temporal scales associated with occurrence or abundance.

We collected larvae of the Patch-nosed Salamander, Urspelerpes brucei, and analyzed size-frequency distributions throughout a 12-mo cycle to determine the length of the larval period and size at metamorphosis. The hatchling-size class entered the population across a wide time window, from early winter to late summer, and at a minimal size of 6-mm snout–girdle length (SGL). Examination of both pooled total sample and the samples across six seasonal periods strongly show that the typical larval period is approximately 24 mo, with metamorphosis occurring during late summer to early fall at 18–19 mm SGL. Because oviposition and hatching are extended across several months, it is possible that some animals metamorphose up to 30 mo after hatching. Larval growth in Urspelerpes is 0.5 mm/mo, which is comparable—and even slower—to the slow larval growth seen in all other stream-dwelling plethodontid larvae. Our samples of recently metamorphosed animals and mature adults indicate that males are sexually mature at metamorphosis. This is more difficult to know for females, and we suggest that females may be a mixed group that contains individuals that mature at metamorphosis and individuals that delay maturation for an additional 12 mo. There is no difference in either sex between body size at metamorphosis and at sexual maturation. As in other species in the Spelerpini, the timing of metamorphosis and the timing of sexual maturation appear to be able to evolve independently, with the remarkable result that the early sexual maturation in Urspelerpes results in a tiny adult size despite having a multiyear larval period.

Accurate knowledge of species distributions is necessary for research and conservation. Slimy Salamanders (Plethodon glutinosus complex) are similar in appearance, tend toward small geographic ranges, and have a propensity for hybridization, necessitating genetic analysis and fine-scale sampling for accurate representation of species distributions. Our current understanding of the distributions of some Slimy Salamanders is based on coarse sampling. Several recent studies question the validity of some species, calling for more geographic and genetic sampling to confirm the species in this group and better define their distributions. Toward this, we analyzed the mitochondrial ND2 gene sequences of 434 individual Slimy Salamanders, 412 of which are from 294 localities west of the Mississippi River, USA. We also estimated ancestral ranges to better understand the biogeographic history underlying current species distributions. We found a much larger distribution for P. kiamichi than previously thought, including several contact zones with P. albagula. We confirmed the Ouachita River separates P. kisatchie and P. albagula in southern Arkansas. Texas P. albagula were not recovered as monophyletic, forming two distinct clades, one of which is nested within a large Interior Highlands (Ouachita and Ozark mountains) clade. Our data suggest P. sequoyah from the type locality have the introgressed mitochondria of P. albagula. We discovered three unnamed mitochondrial clades, one of which we hypothesize is nonintrogressed P. sequoyah. Our biogeographic analysis estimates multiple east–west dispersals across the Mississippi River and at least one west–east dispersal. With these findings, we have updated species distributions, identified contact zones and areas of potential contact, and revealed previously unknown evolutionary lineages. This information will be beneficial for further studies on this complex, which have thus far been hampered by unrefined species boundaries. Furthermore, this work has implications for revising the conservation ranks and management of western Slimy Salamanders.

Male Ensatina eschscholtzii do not have a mental gland, a macrogland used by males during courtship that is considered an ancestral character of plethodontids. We tested whether males and females have nonmental courtship glands associated with the integument of the rostrum and mentum (chin). We found that both male and female E. eschscholtzii possess courtship-like integumentary glands (modified granular glands) in the upper labia extending dorsally along the rostrum and lower labia extending ventrally across the mental region. These glands are sexually dimorphic in size (larger in females) and react positively with periodic acid–Schiff. We hypothesize that these glands are involved in courtship through production of pheromones that may or may not be similar to pheromones produced in courtship glands of other plethodontid salamanders. These rostral glands are structurally and chemically unique in comparison with glands of the rostrum that empty along the length of nasolabial grooves (nasolabial glands) of Ensatina, which do not appear to be involved in pheromone production. Both females and males have relatively few traditional mucous glands throughout the skin in comparison with other species of plethodontids, although some clusters are identified at the anterior edges of the labia.

Species are the fundamental unit of biodiversity studies. However, many species complexes are difficult to delimit, especially those characterized by complicated patterns of population structure. Salamanders in the family Plethodontidae often form species by slowly fragmenting across a landscape over space and time. They thus provide many examples of species complexes in which gradual Darwinian evolution has resulted in multiple units of varying degrees of differentiation, including incompletely separated lineages. Herein, we report on a molecular systematic investigation of woodland salamanders in the Plethodon wehrlei group, a group that has recently been split from two species into five species. To quantify patterns of genetic variation, we collected genetic samples from 24 individuals from 20 populations, including all species and representing a carefully selected subset of previous work. From these samples, we obtained genomic data by using anchored hybrid enrichment, resulting in 319 loci averaging 1300 bp in length. Biallelic single-nucleotide polymorphisms were randomly selected from 316 of these loci for some analyses. We examined patterns of genetic structure by using a combination of multivariate statistics and methods based on evolutionary models (such as the Bayesian program STRUCTURE) and found that all of the recognized species formed genetic clusters; however, P. wehrlei and P. punctatus were relatively weakly differentiated and STRUCTURE identified three separate clusters within P. jacksoni. Species trees inferred using the weighted accurate species tree algorithm (wASTRAL), Bayesian phylogenetics and phylogeography (BPP), and TreeMix all recovered the same topology, with P. dixi sister to the other taxa, which included a northern clade (P. wehrlei, P. punctatus, P. pauleyi) and a southern clade (P. jacksoni, with three separate groups). TreeMix only inferred one gene flow event. We evaluated the candidate species by using BPP and the genealogical divergence index (gdi). Although BPP delimited all candidate species with strong support (all posterior probabilities = 1.0), the gdi only strongly supported P. dixi and P. pauleyi, both of which have only been recently described. We discuss the difficult problem of species delimitation in groups that form species via range fragmentation. We also provide a vision for future research, with the aim of better testing and diagnosing the species diversity within the P. wehrlei group.

In some populations of the Two-Lined Salamander (Eurycea bislineata) species complex, males exhibit two alternative reproductive tactics: “searching” and “guarding.” Searching males have secondary sexual characters—including a large, pheromone-producing mental gland—used in terrestrial courtship, whereas guarding males have hypertrophied jaw musculature used in mate-guarding behavior near aquatic nesting sites. Although this polymorphism occurs in at least four evolutionarily distinct lineages, previous histological studies have only focused on soft-tissue differences in one of these species and no studies have evaluated osteological differences between alternative reproductive tactics. Herein, we present new histological and osteological data from three evolutionarily distinct lineages within the E. bislineata species complex. We confirmed that traditional mental glands are restricted to searching males and that caudal courtship glands are present in both male phenotypes, but not in females. We also found variation in other skin glands that warrants further investigation. Cleared and stained specimens and microcomputed tomography data both revealed substantial osteological differences in skull morphology. Compared with searching males, guarding males have increased skull ossification, fewer and larger teeth, and more pronounced otic and squamosal crests. Together, these data expand our knowledge of morphological differences between alternative reproductive tactics, and we discuss the implications of our results for hypotheses regarding phenotypic plasticity throughout the lifetime of an individual male.

The Cumberland Plateau Salamander, Plethodon kentucki, is a cryptic species with respect to the sympatric Northern Slimy Salamander, Plethodon glutinosus. It was first described by Highton and MacGregor (1983) by using allozyme data. In that description, the authors reported extreme levels of genetic differentiation for a single species with a narrow distribution; however, follow-up studies found patterns of genetic variation that were discordant with the allozyme data. In this paper, we describe patterns of genetic variation within P. kentucki by using an anchored hybrid enrichment dataset of 21 individuals sampled from across its known range. We identified four genetic groups with extensive admixture and isolation by distance. We constructed a population tree by using TreeMix and inferred gene flow between two of the four genetic groups. Finally, we used Bayesian phylogenetics and phylogeography (BPP) in conjunction with the genealogical divergence index (gdi) to test species boundaries within P. kentucki. Although BPP suggested that P. kentucki could be four species, gdi indicated that none of the groups were sufficiently independent to constitute separate species. We conclude that P. kentucki is best recognized as a single species with substantial genetic structure within its limited distribution.

Wildfire is an increasingly common disturbance in forested landscapes that can drastically alter local habitats. Under current climate change predictions, wildfires are likely to become more frequent and severe. In regions and ecosystems that have historically infrequent fire return intervals, there is little known about how organisms will respond to the more severe and frequent wildfires predicted under climate change. In the southern Appalachian Mountains, USA, fire has been suppressed and severe burns are historically uncommon. This region boasts immense biodiversity and is considered a biological hot spot for diversity in the salamander family Plethodontidae. These species rely upon cool, moist microclimates that may be impacted more by severe fire than by low-intensity wild or prescribed fire. In 2016, the Chimney Tops Two wildfire burned >6000 ha of Great Smoky Mountains National Park, USA, and left a mosaic of burn severity across the landscape. This presented an opportunity to examine how five plethodontid salamander species respond to and recover from a range of burn severity. Even though the landscape had been recovering for 5 yr at the time of study, populations of Plethodon jordani, Plethodon glutinosus spp., Desmognathus wrighti, Desmognathus imitator, and Eurycea wilderae within the burn boundary had lower abundance than those in unburned habitat. In addition, there was a trend of even lower abundances in more severely burned habitat. Evidence of recovery, as indicated by a relationship between population abundance and distance from the burn boundary, was only present for D. imitator. Finally, body size distributions were different between burned and unburned sites for three of the five species and individuals were larger, on average, in burned sites. This work provides insights into how terrestrial salamander populations may respond to the more severe and frequent wildfires predicted under climate change for the southern Appalachian Mountains region.