Mercury bioaccumulation and Hepatozoon spp. infections in two syntopic watersnakes in South Carolina.

Mercury (Hg) is a ubiquitous environmental contaminant known to bioaccumulate in biota and biomagnify in food webs. Parasites occur in nearly every ecosystem and often interact in complex ways with other stressors that their hosts experience. Hepatozoon spp. are intraerythrocytic parasites common in snakes. The Florida green watersnake (Nerodia floridana) and the banded watersnake (Nerodia fasciata) occur syntopically in certain aquatic habitats in the Southeastern United States. The purpose of this study was to investigate relationships among total mercury (THg) concentrations, body size, species, habitat type and prevalence and parasitemia of Hepatozoon spp. infections in snakes. In the present study, we sampled N. floridana and N. fasciata from former nuclear cooling reservoirs and isolated wetlands of the Savannah River Site in South Carolina. We used snake tail clips to quantify THg and collected blood samples for hemoparasite counts. Our results indicate a significant, positive relationship between THg and snake body size in N. floridana and N. fasciata in both habitats. Average THg was significantly higher for N. fasciata compared to N. floridana in bays (0.22 ± 0.02 and 0.08 ± 0.006 mg/kg, respectively; p < 0.01), but not in reservoirs (0.17 ± 0.02 and 0.17 ± 0.03 mg/kg, respectively; p = 0.29). Sex did not appear to be related to THg concentration or Hepatozoon spp. infections in either species. We found no association between Hg and Hepatozoon spp. prevalence or parasitemia; however, our results suggest that species and habitat type play a role in susceptibility to Hepatozoon spp. infection.

Mercury immunotoxicity in the brown watersnake (Nerodia taxispilota): An in vitro study.

Mercury (Hg) is a heavy metal that enters the environment through natural and anthropogenic means. Once in the environment, Hg can biomagnify in food webs and is known to cause immunotoxic effects to wildlife. Compared with other vertebrates, knowledge of the reptilian immune system is lacking, especially in snakes. Further, even less is known about the impact of environmental contaminants on snake immunity. This gap in knowledge is largely due to an absence of established immune-based assays or specific reagents for these species. In this study, brown watersnakes (Nerodia taxispilota; n = 23) were captured on the Savannah River (Augusta, Georgia, USA), weighed, measured, bled, and released. Peripheral blood leukocytes (24 h old) were enriched and evaluated with an established mammalian in vitro lymphocyte proliferation assay. Enriched leukocytes were then exposed to mercury chloride (HgCl2 ) at 3.75, 37.5, and 75 µM. Total mercury (THg) in whole blood was also quantified. Snake peripheral blood leukocyte enrichment yielded >90% lymphocytes with viabilities averaging >70%. Exposure to HgCl2 resulted in significant dose-dependent suppression of proliferative responses relative to spontaneous proliferation at 37.5 and 75 µM (both p ≤ 0.01) but not 3.75 µM (p = 0.99). Mean ± 1 SE concentration of THg in whole blood was 0.127 ± 0.027 mg/kg (wet weight). Based on the in vitro findings with HgCl2 , snakes in systems with heavy Hg pollution may be at risk of immunosuppression, but N. taxispilota at the site in this study appear to be at low risk.

Metabolic responses to increased temperatures in three semi-aquatic turtle species from the southeastern United States.

Physiological maintenance of ectotherms is largely dependent on temperature. Abrupt changes in environmental conditions can cause shifts in energy budgets, with higher temperatures requiring more energy be devoted to maintenance functions, making less energy available for other activities. As a group, freshwater turtles are of conservation concern; thus, it is important to understand their physiological responses to temperature shifts, particularly given current climate change predictions. We quantified metabolic rates (MR) for three species of semi-aquatic turtles that vary in their degree of terrestriality and propensity to aestivate: chicken turtles (Deirochelys reticularia), eastern mud turtles (Kinosternon subrubrum), and eastern musk turtles (Sternotherus odoratus). Metabolic trials were performed using a flow-through respirometer at three environmentally-relevant temperatures: 25 °C, 30 °C, and 35 °C. As expected, MRs of turtles were significantly and positively associated with increases in mass and temperature. Pairwise comparisons revealed that D. reticularia exhibited significantly higher oxygen consumption rates (VO2) relative to K. subrubrum, which had significantly higher VO2 than S. odoratus. The combination of higher metabolic rates, the tendency to terrestrially aestivate, and declining quantity of high quality thermal habitats may place some semi-aquatic turtles at a disadvantage when considering future climate scenarios. Our findings, when combined with knowledge of other ecological traits, may be a useful tool for predicting relative sensitivities of turtle species to predicted climate change, particularly those species inhabiting seasonal wetlands.

Peripheral blood hematology, plasma biochemistry, and the optimization of an in vitro immune-based assay in the brown watersnake (Nerodia taxispilota).

Reptiles represent a phylogenetic lineage that provides a unique link between ectothermic anamniotes and endothermic amniotes. Compared to mammalian and avian species, our understanding of the reptilian immune system is greatly lacking. This gap in knowledge is largely due to an absence of established immune-based assays or specific reagents for these species. In the present study, brown watersnakes (Nerodia taxispilota) were live-captured in the wild, sexed, weighed, measured, bled via the caudal vein, and released. At 24 hr post-collection, peripheral blood leukocytes were enriched and evaluated with an established mammalian in-vitro lymphocyte proliferation assay. Snake peripheral blood leukocyte enrichment yielded >90% lymphocytes with viabilities averaging 81.5%. Baseline physiologic data for N. taxispilota, including hematology and total solids, leukocyte differentials, cell recovery, and plasma biochemistry, were also collected. Cells cultured with Concanavalin A exhibited significantly increased proliferation at both 72 and 96 hr. These preliminary results show that enriched peripheral blood from wild-caught N. taxispilota provides a sufficient yield of leukocytes that can be cultured and functionally evaluated using a standard mammalian in-vitro immune-based assay.

Snakes as Novel Biomarkers of Mercury Contamination: A Review.

Mercury (Hg) is an environmental contaminant that has been reported in many wildlife species worldwide. The organic form of Hg bioaccumulates in higher trophic levels, and thus, long-lived predators are at risk for higher Hg exposure. Although ecological risk assessments for contaminants such as Hg include pertinent receptor species, snakes are rarely considered, despite their high trophic status and potential to accumulate high levels of Hg. Our current knowledge of these reptiles suggests that snakes may be useful novel biomarkers to monitor contaminated environments. The few available studies show that snakes can bioaccumulate significant amounts of Hg. However, little is known about the role of snakes in Hg transport in the environment or the individual-level effects of Hg exposure in this group of reptiles. This is a major concern, as snakes often serve as important prey for a variety of taxa within ecosystems (including humans). In this review, we compiled and analyzed the results of over 30 studies to discuss the impact of Hg on snakes, specifically sources of exposure, bioaccumulation, health consequences, and specific scientific knowledge gaps regarding these moderate to high trophic predators.

Microbiota of Four Tissue Types in American Alligators (Alligator mississippiensis) Following Extended Dietary Selenomethionine Exposure.

Selenium represents an essential trace nutrient that is necessary for biological functions. Deficiencies can induce disease, but excess can induce toxicity. Selenium deficiency is a major concern in underdeveloped countries, while also posing as a toxic pollutant in waterways surrounding landfills, agricultural areas, and fossil fuel production sites. We examined the microbiome of selenomethionine (SeMet) fed American alligators (Alligator mississippiensis) at the beginning and end of a 7-week exposure experiment. Alligators were randomly divided into three groups: control and 1000 or 2000 ppm SeMet. DNA from before exposure (oral and cloaca swabs) and post-exposure (oral, cloaca, small & large intestines) sampling were extracted and amplified for bacterial 16 s rRNA. While treatment did not seem to have much effect, we observed a predominance of Fusobacteriaceae and Porpyromonodaceae across all tissue types. Cetobacterium and Clostridium are the most abundant genera as potential indicators of the aquatic and carrion feeding lifestyle of alligators.

Close-kin mating, but not inbred parents, reduces hatching rates and offspring quality in a threatened tortoise.

Inbreeding depression, the reduction in fitness due to mating of related individuals, is of particular conservation concern in species with small, isolated populations. Although inbreeding depression is widespread in natural populations, long-lived species may be buffered from its effects during population declines due to long generation times and thus are less likely to have evolved mechanisms of inbreeding avoidance than species with shorter generation times. However, empirical evidence of the consequences of inbreeding in threatened, long-lived species is limited. In this study, we leverage a well-studied population of gopher tortoises, Gopherus polyphemus, to examine the role of inbreeding depression and the potential for behavioural inbreeding avoidance in a natural population of a long-lived species. We tested the hypothesis that increased parental inbreeding leads to reduced hatching rates and offspring quality. Additionally, we tested for evidence of inbreeding avoidance. We found that high parental relatedness results in offspring with lower quality and that high parental relatedness is correlated with reduced hatching success. However, we found that hatching success and offspring quality increase with maternal inbreeding, likely due to highly inbred females mating with more distantly related males. We did not find evidence for inbreeding avoidance in males and outbred females, suggesting sex-specific evolutionary trade-offs may have driven the evolution of mating behaviour. Our results demonstrate inbreeding depression in a long-lived species and that the evolution of inbreeding avoidance is shaped by multiple selective forces.

Examining the Effects of Chronic Selenium Exposure on Traditionally Used Stress Parameters in Juvenile American Alligators (Alligator mississippiensis).

Environmental contaminants, such as the trace element selenium (Se), are a continuing concern to species worldwide due to their potential pathophysiological effects, including their influence on the stress response mediated through glucocorticoids (GCs; stress hormones). Environmental concentrations of Se are increasing due to anthropogenic activities, including the incomplete combustion of coal and subsequent disposal of coal combustion wastes. However, most studies examining how Se affects GCs have been focused on lower trophic organisms. The objectives of this study were to investigate the effects of long-term Se exposure on traditionally used stress parameters and to identify which of these parameters best indicate Se accumulation in liver and kidney of the American alligator (Alligator mississippiensis), a top trophic carnivore found in the southeastern United States and known to inhabit Se-containing areas. Alligators were divided into three dietary treatments and fed prey spiked with 1000 or 2000 ppm of selenomethionine (SeMet) or deionized water (control treatment) for 7 weeks. Following the 7-week treatment protocol, blood and tissue samples were obtained to measure plasma corticosterone (CORT; the main crocodilian GC), tail scute CORT, the ratio of peripheral blood heterophils (H) to lymphocytes (L) as H/L ratio, and body condition. To evaluate which parameter best indicated Se accumulation in the liver and kidney, principal component and discriminant analyses were performed. The only parameter significantly correlated with liver and kidney Se concentrations was scute CORT. Our results suggest that measurement of CORT in tail scutes compared with plasma CORT, H/L ratios, and body condition is the best indicator of Se-exposure and accumulation in crocodilians.

Male Body Size Predicts Reproductive Success But Not Within-Clutch Paternity Patterns in Gopher Tortoises (Gopherus polyphemus).

In many vertebrates, body size is an important driver of variation in male reproductive success. Larger, more fit individuals are more likely to dominate mating opportunities, skewing siring success and resulting in lower effective population sizes and genetic diversity. The mating system of the gopher tortoise (Gopherus polyphemus) has been characterized as both female-defense and scramble-competition polygyny. Mating systems are typically not fixed and can be influenced by factors such as population density, demographic structure, and environmental conditions; however, most populations will have a predominant strategy that results from local conditions. We assessed how male body size influences patterns of paternity and reproductive success in a natural population of gopher tortoises in Florida, United States. Using microsatellites, we assigned parentage of 220 hatchlings from 31 nests collected during 2 reproductive seasons. Larger males were significantly more likely to sire offspring and sired more offspring than smaller males; however, the likelihood of a clutch being multiply sired was unrelated to male body size. We also found evidence of mate fidelity across years. Although paternity patterns in this high-density population are more consistent with defense polygyny, female monopoly by males was incomplete, with both large and small males contributing to multiply sired clutches. Additional behavioral data are needed to clarify the role of female mate selection in paternity outcomes. The context-dependence of mating systems underscores the need to compare parentage patterns across populations and to recognize the potential for more than 1 strategy to be employed within a single population.

Corticosterone in American alligator (Alligator mississippiensis) tail scutes: Evaluating the feasibility of using unconventional samples for investigating environmental stressors.

Baseline plasma corticosterone (CORT) concentrations have been widely used to investigate the effects of stressors in wild and captive crocodilians. However, collecting baseline plasma CORT samples from wild crocodilians may be particularly difficult due to the capture and handling protocols used for large individuals. Thus, it may prove beneficial to use recently modified techniques for extracting CORT deposited in keratinized and non-keratinized tissues to better quantify the effects of long-term stress in crocodilians. In this study, we investigated the feasibility of using American alligator (Alligator mississippiensis) tail scute tissues to quantify CORT by collecting blood and tail scutes from 40 alligators before and after a short-term handling stressor. The objective of the current study was to better understand CORT deposition in crocodilian scutes and whether short-term increases in CORT could be detected. We found that CORT can be reliably extracted from alligator scute tissue and quantified using a commercially available enzyme immunoassay. However, there was a significant increase in scute CORT concentrations following an alligator being exposed to a short-term stressor (p = 0.017), although the magnitude of change was less than observed in plasma samples from the same individuals (p = 0.002). Furthermore, our results indicate that there was a significant effect of body condition on an alligator's post-stressor CORT concentration (p = 0.02). While our study is among the first to experimentally examine the usefulness of tissue CORT in crocodilians, a combination of field and laboratory experiments are needed to better understand deposition rates of CORT in scute tissues and to further validate the usefulness of tissue glucocorticoids for evaluating the effects of stress.

Experimentally Induced Selenosis in Yellow-Bellied Slider Turtles ( Trachemys scripta scripta).

Selenosis, or selenium toxicosis, occurs in wildlife and livestock, usually because of excessive intake of selenium via selenium-containing plants. Although it is known that wild slider turtles can accumulate large amounts of selenium, little is known about how selenium exposure may affect these reptiles. In this study, the authors report histopathologic changes in yellow-bellied sliders ( Trachemys scripta scripta) caused by experimental exposure to selenomethionine. Microscopic changes in kidney and claw tissue were most significant and resembled those reported in birds. Turtles in the selenium treatment groups had acute tubular degeneration and regeneration in the kidney, with hyaline droplets in the high-dose animals, and changes in the claws ranging from epidermal hyperplasia with disorganization and intercellular edema to ulceration, and accumulation of seroheterophilic exudate between the epidermis and cornified layer. Although selenium burdens in this study are comparable with values found in wild slider turtles, more data are needed to determine if similar histopathologic abnormalities arise in wild animals exposed to high levels of selenium.

Dietary Selenomethionine Administration and Its Effects on the American Alligator (Alligator mississippiensis): Oxidative Status and Corticosterone Levels.

Selenium (Se) is an essential nutrient which in excess causes toxicity. The disposal of incompletely combusted coal, which often is rich in Se, into aquatic settling basins is increasing the risk of Se exposure worldwide. However, very few studies have looked at the physiological effects of Se exposure on long-lived, top trophic vertebrates, such as the American alligator (Alligator mississippiensis). During a 7-week period, alligators were fed one of three dietary treatments: mice injected with deionized water or mice injected with water containing 1000 or 2000 ppm selenomethionine (SeMet). One week after the last feeding alligators were bled within 3 min of capture for plasma corticosterone (CORT). A few days later, all alligators were euthanized and whole blood and tail tissue were harvested to measure oxidative damage, an antioxidant-associated transcription factor, and antioxidant enzymes [glutathione peroxidase-1 (GPX1), superoxide dismutase-1 (SOD1), and SOD2] by Western blotting. There was a dose-dependent increase in baseline CORT levels in alligators administered SeMet. Except for blood SOD2 levels, SeMet treatment had no effect (p > 0.05 for all) on oxidative status: oxidative damage, GPX1, SOD1, and muscle SOD2 levels were similar among treatments. Our results illustrate that high levels of Se may act as a stressor to crocodilians. Future studies should investigate further the physiological effects of Se accumulation in long-lived, top-trophic vertebrates.

Effects of selenium exposure on the hematology, innate immunity, and metabolic rate of yellow-bellied sliders (Trachemys scripta scripta).

Selenium (Se) is a naturally occurring essential element that can be toxic to vertebrates at high concentrations. Despite studies that have documented that wild reptile species can accumulate copious amounts of Se, little is known regarding specific toxicologic effects of Se. In this study, 70 juvenile yellow-bellied sliders (Trachemys scripta scripta) were exposed to one of three seleno-L-methionine (SetMet) treatments (control, n = 24; 15 mg/kg, n = 23; and 30 mg/kg, n = 23) via weekly oral gavage for 5 weeks. At the conclusion of the experiment, kidney, liver, muscle, and blood samples were collected for quantitative Se analysis. Turtles in the SeMet treatment groups accumulated significantly higher amounts of Se in all tissue types relative to controls (all p < 0.001). Turtles in the 30 mg/kg SeMet group also accumulated significantly higher amounts of Se compared to the 15 mg/kg group (all p < 0.001). Although toxicity thresholds for reptiles have not been established, Se concentrations in liver tissue from both SeMet treatment groups exceeded reported avian toxicity thresholds for liver tissue. Neither oxygen consumption nor innate bactericidal capacity were impacted by SeMet exposure. However, turtles in the 30 mg/kg SeMet group exhibited anemia, which has been reported in other vertebrates exposed to Se. Furthermore, juvenile T. s. scripta in the 30 mg/kg SeMet group experienced 17% mortality compared to 0% in the 15 mg/kg treatment and control groups. To our knowledge, this study is the first to report dose-dependent Se-associated anemia and mortality in a chelonian species.

Dietary Selenomethionine Administration in the American Alligator (Alligator mississippiensis): Hepatic and Renal Se Accumulation and Its Effects on Growth and Body Condition.

Selenium (Se) is an essential trace nutrient, but in excess, it can induce toxicity. Incomplete combustion of coal produces coal combustion wastes, which are enriched in Se and often disposed of in aquatic basins. While a multitude of studies have investigated Se accumulation in vertebrates, few studies have examined its effects on longer-lived top trophic carnivores, such as the American alligator (Alligator mississippiensis). In this study, alligators were fed one of three Dietary Treatments: mice injected with water (controls) or water supplemented with 1000 or 2000 ppm selenomethionine (SeMet). Dietary Treatment significantly affected Se levels in both the liver (p < 0.0001; raw mean ± SE: 1000 ppm group, 35.20 ± 6.32 ppm; 2000 ppm group, 49.97 ± 4.00 ppm) and kidney (p < 0.0001; raw mean ± SE: 1000 ppm group, 101.60 ± 8.64 ppm; 2000 ppm, 96.38 ± 5.81 ppm), which were significantly higher in alligators fed SeMet than in controls. Post-treatment head length, used to control for size variation, was negatively related to both kidney (p = 0.0142) and liver (p = 0.0010) Se concentrations. Dietary treatment with SeMet significantly reduced body condition (1000 ppm, p < 0.0029; 2000 ppm, p = 0.0075), but it significantly increased growth (1000 ppm, p < 0.0001; 2000 ppm, p = 0.0316). Body condition and growth remained unchanged in control alligators (p > 0.05). Our results demonstrate alligators are capable of accumulating high levels of Se through trophic transfer. The positive effects of accumulation on growth may demonstrate Se essentiality, whereas the negative effects on condition may demonstrate toxicity. Accumulation also was associated with mortality, further demonstrating toxicity. Future studies should further investigate the physiological effects of Se accumulation in long-lived, top-trophic carnivores.

Chronic Ingestion of Coal Fly-Ash Contaminated Prey and Its Effects on Health and Immune Parameters in Juvenile American Alligators (Alligator mississippiensis).

Coal-burning power plants supply approximately 37 % of the electricity in the United States. However, incomplete combustion produces ash wastes enriched with toxic trace elements that have historically been disposed of in aquatic basins. Organisms inhabiting such habitats may accumulate these trace elements; however, studies investigating the effects on biota have been primarily restricted to shorter-lived, lower-trophic organisms. The American alligator (Alligator mississippiensis), a long-lived, top-trophic carnivore, has been observed inhabiting these basins, yet the health or immune effects of chronic exposure and possible accumulation remains unknown. In this study, we investigated how chronic dietary ingestion of prey contaminated with coal combustion wastes (CCWs) for 25 months, and subsequent accumulation of trace elements present in CCWs, affected juvenile alligator immune function and health. Alligators were assigned to one of four dietary-treatment groups including controls and those fed prey contaminated with CCWs for one, two, or three times a week. However, no effect of Dietary Treatment (p > 0.05) was observed on any immune parameter or hematological or plasma analyte we tested. Our results suggest that neither exposure to nor accumulation of low doses of CCWs had a negative effect on certain aspects of the immune and hematological system. However, future studies are required to elucidate this further.

A Stream Multimetric Macroinvertebrate Index (MMI) for the Sand Hills Ecoregion of the Southeastern Plains, USA.

A macroinvertebrate multimetric index is an effective tool for assessing the biological integrity of streams. However, data collected under a single protocol may not be available for an entire region. We sampled macroinvertebrates from the full extent of the Sand Hills ecoregion Level IV of the Southeastern Plains with a standard protocol during the summers of 2010-2012. We evaluated the performance of 94 metrics through a series of screening criteria and built 48 macroinvertebrate multimetric indexs with combinations of the best performing metrics, representing richness, habit, functional feeding guild, sensitivity, and community composition. A series of narrative-response tests for each macroinvertebrate multimetric index was used to find the best performing macroinvertebrate multimetric index which we called the Sand Hills macroinvertebrate multimetric index. The Sand Hills macroinvertebrate multimetric index consisted of the measures Biotic Index, % Shredder taxa, Clinger taxa(2)/total taxa, Plecoptera and Trichoptera richness, and Tanytarsini taxa(2)/Chironomidae taxa. Comparison of the Sand Hills macroinvertebrate multimetric index with existing assessment tools calculated with our data indicated that the Sand Hills macroinvertebrate multimetric index performs at a high level with regard to identifying degraded sites and in its response to stress gradients.

Kinship, inbreeding and fine-scale spatial structure influence gut microbiota in a hindgut-fermenting tortoise.

Herbivorous vertebrates rely on complex communities of mutualistic gut bacteria to facilitate the digestion of celluloses and hemicelluloses. Gut microbes are often convergent based on diet and gut morphology across a phylogenetically diverse group of mammals. However, little is known about microbial communities of herbivorous hindgut-fermenting reptiles. Here, we investigate how factors at the individual level might constrain the composition of gut microbes in an obligate herbivorous reptile. Using multiplexed 16S rRNA gene sequencing, we characterized the faecal microbial community of a population of gopher tortoises (Gopherus polyphemus) and examined how age, genetic diversity, spatial structure and kinship influence differences among individuals. We recovered phylotypes associated with known cellulolytic function, including candidate phylum Termite Group 3, suggesting their importance for gopher tortoise digestion. Although host genetic structure did not explain variation in microbial composition and community structure, we found that fine-scale spatial structure, inbreeding, degree of relatedness and possibly ontogeny shaped patterns of diversity in faecal microbiomes of gopher tortoises. Our findings corroborate widespread convergence of faecal-associated microbes based on gut morphology and diet and demonstrate the role of spatial and demographic structure in driving differentiation of gut microbiota in natural populations.

First case of ranavirus and associated morbidity and mortality in an eastern mud turtle Kinosternon subrubrum in South Carolina.

Ranaviruses are double-stranded DNA viruses that infect amphibians, fish, and reptiles, causing global epidemics in some amphibian populations. It is important to identify new species that may be susceptible to the disease, particularly if they reside in the same habitat as other at-risk species. On the Savannah River Site (SRS) in Aiken, South Carolina, USA, ranaviruses are present in several amphibian populations, but information is lacking on the presence, prevalence, and morbidity of the virus in reptile species. An eastern mud turtle Kinosternon subrubrum captured on the SRS in April 2014 exhibited clinical signs of a ranaviral infection, including oral plaque and conjunctivitis. Quantitative PCR analyses of DNA from liver tissue, ocular, oral, nasal, and cloacal swabs were all positive for ranavirus, and sequencing of the template confirmed infection with a FV3-like ranavirus. Histopathologic examination of postmortem tissue samples revealed ulceration of the oral and tracheal mucosa, intracytoplasmic epithelial inclusions in the oral mucosa and tongue sections, individualized and clusters of melanomacrophages in the liver, and bacterial rods located in the liver, kidney, heart, stomach, and small intestine. This is the first report of morbidity and mortality of a mud turtle with a systemic ranaviral infection.

Use of the NatureServe Climate Change Vulnerability Index as an Assessment Tool for Reptiles and Amphibians: Lessons Learned.

Climate change threatens biodiversity globally, yet it can be challenging to predict which species may be most vulnerable. Given the scope of the problem, it is imperative to rapidly assess vulnerability and identify actions to decrease risk. Although a variety of tools have been developed to assess climate change vulnerability, few have been evaluated with regard to their suitability for certain taxonomic groups. Due to their ectothermic physiology, low vagility, and strong association with temporary wetlands, reptiles and amphibians may be particularly vulnerable relative to other groups. Here, we evaluate use of the NatureServe Climate Change Vulnerability Index (CCVI) to assess a large suite of herpetofauna from the Sand Hills Ecoregion of the southeastern United States. Although data were frequently lacking for certain variables (e.g., phenological response to climate change, genetic variation), sufficient data were available to evaluate all 117 species. Sensitivity analyses indicated that results were highly dependent on size of assessment area and climate scenario selection. In addition, several ecological traits common in, but relatively unique to, herpetofauna are likely to contribute to their vulnerability and need special consideration during the scoring process. Despite some limitations, the NatureServe CCVI was a useful tool for screening large numbers of reptile and amphibian species. We provide general recommendations as to how the CCVI tool's application to herpetofauna can be improved through more specific guidance to the user regarding how to incorporate unique physiological and behavioral traits into scoring existing sensitivity factors and through modification to the assessment tool itself.

Defining the reference condition for wadeable streams in the Sand Hills subdivision of the Southeastern Plains ecoregion, USA.

The Sand Hills subdivision of the Southeastern Plains ecoregion has been impacted by historical land uses over the past two centuries and, with the additive effects of contemporary land use, determining reference condition for streams in this region is a challenge. We identified reference condition based on the combined use of 3 independent selection methods. Method 1 involved use of a multivariate disturbance gradient derived from several stressors, method 2 was based on variation in channel morphology, and method 3 was based on passing 6 of 7 environmental criteria. Sites selected as reference from all 3 methods were considered primary reference, whereas those selected by 2 or 1 methods were considered secondary or tertiary reference, respectively. Sites not selected by any of the methods were considered non-reference. In addition, best professional judgment (BPJ) was used to exclude some sites from any reference class, and comparisons were made to examine the utility of BPJ. Non-metric multidimensional scaling indicated that use of BPJ may help designate non-reference sites when unidentified stressors are present. The macroinvertebrate community measures Ephemeroptera, Plecoptera, Trichoptera richness and North Carolina Biotic Index showed no differences between primary and secondary reference sites when BPJ was ignored. However, there was no significant difference among primary, secondary, and tertiary reference sites when BPJ was used. We underscore the importance of classifying reference conditions, especially in regions that have endured significant anthropogenic activity. We suggest that the use of secondary reference sites may enable construction of models that target a broader set of management interests.