Amphibian Species Research Articles

Microplastics pollution in larvae of toads, frogs and newts in anthropopressure gradient

Amphibians are vital components of global biodiversity and fulfil diverse ecological roles, including insect population control and serving as environmental health indicators. However, there is a concerning decline in amphibian populations globally due to factors such as habitat loss, climate change, and emerging diseases. Concurrently, the issue of microplastic pollution has gained prominence because of its potential to harm aquatic ecosystems. Microplastics, derived from various sources, endanger aquatic organisms, especially amphibians during their sensitive larval stages. In this study, we examined the prevalence of microplastic pollution in the larval stages of 10 amphibian species, covering both tailed (Urodela) and tailless (Anura) amphibians. We observed a significant correlation between amphibian species identity, the extent of artificial land cover, and the degree of microplastic contamination in larvae. Notably, our results indicate interspecific differences in microplastic bioaccumulation. Furthermore, this research is the first to demonstrate a positive correlation between microplastic pollution in amphibian larvae and anthropogenic pressure. Our findings add to the expanding literature underscoring the intricate interplay between human activities and aquatic ecosystem health.

Vernal Pool Amphibian Inventories in the Temperate Forests of Eastern North America: Can Environmental DNA Replace the Traditional Methods?

Amphibian populations have been globally declining since at least 1990. In the temperate forests of eastern North America, vernal pools offer important breeding habitats to many amphibians but are usually not considered for forest management because of their small size and temporary status. The effective monitoring and management of amphibians rely on accurate knowledge of their spatiotemporal distributions, which is often expensive to collect due to the amount of fieldwork required. In this study, we tested whether eDNA metabarcoding could identify the same amphibian communities as the traditional inventory protocols. We collected eDNA samples in twelve vernal pools in the spring of 2019 and identified their communities via metabarcoding. At each pool, three traditional amphibian inventory methods were used: call surveys, trapping, and active search surveys. In total, 13 amphibian species were detected, with most of them being detected using both eDNA and the traditional methods. When comparing the results of eDNA with the traditional methods, we found that species ecology and behavior are key factors of its detectability via a specific method. With its higher taxonomical precision and repeatability, eDNA metabarcoding allows for the inventorying of amphibian species living and reproducing in vernal pools and their vicinity with accuracy. As eDNA metabarcoding is inexpensive compared to the traditional methods, we conclude that eDNA sampling should be considered for integration as a standard monitoring tool, after an initial assessment of amphibian diversity.

Amphibian Diversity: Where everything starts to flood, Cáceres Municipality, North Pantanal, Central-West Brazil

Faunal inventories contribute to our understanding of regional diversity, and are fundamental for policy and decision-making regarding the management and conservation of large natural areas. This study aimed to inventory and compile information on amphibian species occurring in the North Pantanal region, in the municipal limits of Cáceres, Mato Grosso, Brazil. We used three methods to inventory amphibian species: (1) fieldwork, (2) analysis of specimens deposited in scientific collections, and (3) literature reviews. We registered 49 amphibian species in Cáceres. Of them, 48 species belonged to the Anura order and were distributed across eight families and 20 genera, and one species belonged to the Gymnophiona order (Siphonops paulensis). The families Leptodactylidae (20 spp.) and Hylidae (17 spp.) were dominant in terms of richness, accounting for 75.5% of all species found in Cáceres. The remaining families had between four and one species each. The municipality is strongly influenced by non-forested formations (e.g., the Cerrado and Pantanal) and presents a high species richness for a non-forested location in Brazil. Our findings highlight Cáceres as one of the richest areas in amphibian species in the North Pantanal region, expanding our knowledge regarding frog diversity. This study provides a foundation for future conservation strategies and additional assessments of amphibian species in light of potential population declines and other emerging threats.

Predicting amphibian and reptile distributions and species richness across urban parks in the eastern United States

Predicting amphibian and reptile distributions and species richness across urban parks in the eastern United States

Ecological Barriers for an Amphibian Pathogen: A Narrow Ecological Niche for Batrachochytrium salamandrivorans in an Asian Chytrid Hotspot.

The chytrid fungal pathogens Batrachochytrium salamandrivorans (Bsal) and B. dendrobatidis (Bd) are driving amphibian extinctions and population declines worldwide. As their origins are believed to be in East/Southeast Asia, this region is crucial for understanding their ecology. However, Bsal screening is relatively limited in this region, particularly in hotspots where Bd lineage diversity is high. To address this gap, we conducted an extensive Bsal screening involving 1101 individuals from 36 amphibian species, spanning 17 natural locations and four captive facilities in the biodiversity-rich Guangxi Zhuang Autonomous Region (GAR). Our PCR assays yielded unexpected results, revealing the complete absence of Bsal in all tested samples including 51 individuals with Bd presence. To understand the potential distribution of Bsal, we created niche models, utilizing existing occurrence records from both Asia and Europe. These models estimated potential suitable habitats for Bsal largely in the northern and southwestern parts of the GAR. Although Bsal was absent in our samples, the niche models identified 10 study sites as being potentially suitable for this pathogen. Interestingly, out of these 10 sites, Bd was detected at 8. This suggests that Bsal and Bd could possibly co-exist in these habitats, if Bsal were present. Several factors seem to influence the distribution of Bsal in Asia, including variations in temperature, local caudate species diversity, elevation, and human population density. However, it is climate-related factors that hold the greatest significance, accounting for a notable 60% contribution. The models propose that the specific climatic conditions of arid regions, primarily seen in the GAR, play a major role in the distribution of Bsal. Considering the increased pathogenicity of Bsal at stable and cooler temperatures (10-15 °C), species-dependent variations, and the potential for seasonal Bd-Bsal interactions, we emphasize the importance of periodic monitoring for Bsal within its projected range in the GAR. Our study provides deeper insights into Bsal's ecological niche and the knowledge generated will facilitate conservation efforts in amphibian populations devastated by chytrid pathogens across other regions of the world.

Widespread learned predator recognition to an alien predator across populations in an amphibian species

Alien predators are a major cause of decline and extinction of species worldwide, since native organisms are rarely equipped with specific antipredatory strategies to cope with them. However, phenotypic plasticity and learned predator recognition may help prey populations to survive novel predators. Here we examine geographical variation in the learning ability of larval spadefoot toads (Pelobates cultripes) to recognize invasive predatory crayfish (Procambarus clarkii). We compare the learning-mediated behavioural responses of tadpoles from six populations across two regions in Spain (central and southern), with different histories of exposure to the presence of the invasive species. Two of the populations showed innate recognition of chemical cues from the invasive crayfish, whereas three of them learned to recognize such cues as a threat after conditioning with conspecific alarm cues. Learning abilities did not differ among southern populations, but they did among central populations. We assessed patterns of genetic variation within and among these two regions through microsatellite markers and found low genetic divergence among the southern populations but greater differentiation among the central ones. We hypothesize that similar responses to the invasive crayfish in southern populations may have arisen from a combination of extended historical exposure to this introduced predator (~ 50 y) and higher levels of gene flow, as they inhabit a highly interconnected pond network. In contrast, populations from central Spain show lower connectivity, have been exposed to the invasive crayfish for a shorter period of time, and are more divergent in their plastic responses.

Adverse Effect of Metallic Gold and Silver Nanoparticles on Xenopus laevis Embryogenesis.

Exposure to metal nanoparticles is potentially harmful, particularly when occurring during embryogenesis. In this study, we tested the effects of commercial AuNPs and AgNPs, widely used in many fields for their features, on the early development of Xenopus laevis, an anuran amphibian key model species in toxicity testing. Through the Frog Embryo Teratogenesis Assay-Xenopus test (FETAX), we ascertained that both nanoparticles did not influence the survival rate but induced morphological anomalies like modifications of head and branchial arch cartilages, depigmentation of the dorsal area, damage to the intestinal brush border, and heart rate alteration. The expression of genes involved in the early pathways of embryo development was also modified. This study suggests that both types of nanoparticles are toxic though nonlethal, thus indicating that their use requires attention and further study to better clarify their activity in animals and, more importantly, in humans.

Identification of major histocompatibility complex genotypes associated with resistance to an amphibian emerging infectious disease.

Amphibian chytridiomycosis, caused by Batrachochytrium dendrobatidis (Bd), emerged from Asia and spread globally. By comparing functional MHC IIß1 alleles from an Asian Bd-resistant anuran species (Bufo gargarizans) with those of an Australasian Bd-susceptible species (Litoria caerulea), we identified MHC genotypes with a glycine deletion (G90β1) and adjacent motifs in the deepest pathogen-derived peptide-binding groove associated with Bd resistance. Every Bd-resistant individual, but no susceptible individuals, possessed at least one allele encoding the variant. We detected trans-species polymorphism at the end of the MHC IIβ1 sequences in both species. The G90β1 deletion was encoded by different alleles in the two species, suggesting it may have evolved independently in the two species rather than having been derived from a common ancestor. It is possible that MHC adaptations to chytridiomycosis evolved independently among amphibian species. These results are consistent with a scenario by which MHC adaptations that confer resistance to the pathogen have evolved by convergent evolution. Immunogenetic studies such as this are critical to ongoing conservation efforts.

PALLAS’ SPADEFOOT, <i>PELOBATES VESPERTINUS</i> (PALLAS 1771) (AMPHIBIA, PELOBATIDAE), THE SECOND AMPHIBIAN SPECIES TO TOLERATE EXTREME HYPOXIA

Pallas’ spadefoot is a Eurasian species of amphibian that winters for about half a year at a depth to 200 cm, always below the level of freezing, since it does not survive negative temperatures. However, significantly digging into the soil can cause oxygen deficiency. The minimum sufficient for a long-term existence of Pelobates vespertinus and the lethal oxygen content in the air were revealed, as well as metabolic pathways were evaluated under the conditions of normoxia and hypoxia in laboratory experiments. Beside this, the seasonal dynamics of temperatures and oxygen concentrations in sandy loam soils at different depths were determined in a typical wintering biotope of the species (the Oka River valley). Animals have been found to be capable of withstanding a tenfold decrease in oxygen content in soils compared to atmospheric oxygen for more than two months, thereby remaining quite active. When the concentration is decrease 20 times (up to 1%), a state of coma occurs, which is reversible if it lasts not more than a day. This shows a high degree of resistance of the species to stress resulting from reoxygenation and staying in an environment with variable oxygen levels. Thus, P. vespertinus is the second amphibian species following the Siberian frog (Rana amurensis), which can tolerate its complete absence for a long time (several months). Compared to body weight, the spadefoot has small storage organs (fat bodies and liver) and a low content of reserve substances (lipids and glycogen) in tissues, which are usually consumed at a low rate in normoxia at 3°C. During hypoxia, lipid utilisation is suspended and glycogen consumption is increased, indicating a shift from aerobic to predominantly anaerobic metabolism. The cumulative effect of reduced metabolism due to low temperature (3°C) and the activation of glycolysis due to the lack of oxygen provides a high level of resistance to hypoxia, distinguishing Pallas’ spadefoot from other swarming amphibian species studied. The oxygen content in the ground air of typical places at wintering depths (in sandy massifs) is shown to coincide with the atmospheric oxygen. The ubiquitous choice by the species of wintering areas with loose soils is assumed to be associated not with their excellent aeration, but rather with the ease of digging. The results obtained seem to be promising for using the study spadefoot (and possibly other species of the spadefoot genus, as well as burrowing toads) as model for exploring the adaptations to air oxygen deficiency.

No amphibian chytrid fungus Batrachochytrium dendrobatidis detected in four introduced populations of the midwife toad Alytes obstetricans in eastern England

The amphibian chytrid fungus Batrachochytrium dendrobatidis threatens amphibian species globally as the causative agent of chytridiomycosis, with the introduction of non-native species being one of the pathways that the pathogen can spread to naive populations. We have monitored and screened the common midwife toad Alytes obstetricans in four separate populations in eastern England, to investigate the potential threats to local amphibians. Forty-eight toads across all life stages were swabbed between May 2018 and August 2020, and screened for the presence of B. dendrobatidis DNA using qPCR. None of the samples tested were positive, indicating that it is unlikely that any of the A. obstetricans swabbed were infected with B. dendrobatidis. The populations surveyed represent only a small part of the species range in Britain, consequently a more widespread survey is recommended to increase confidence that British A. obstetricans are free of B. dendrobatidis.

Genomic analysis reveals complex population structure within the smooth newt, Lissotriton vulgaris, in Central Europe.

Species with wide-range distributions usually display high genetic variation. This variation can be partly explained by historical lineages that were temporally isolated from each other and are back into secondary reproductive contact, and partly by local adaptations. The smooth newt (Lissotriton vulgaris) is one of the most widely distributed amphibians species across Eurasia and forms a species complex with a partially overlapping distribution and morphology. In the present study, we explored the population genomic structure of smooth newt lineages in the Carpathian Basin (CB) relying on single-nucleotide polymorphisms. Our dataset included new and previously published data to study the secondary contact zone between lineages in the CB and also tested for the barrier effect of rivers to gene flow between these lineages. We confirmed the presence of the South L. v. vulgaris Lineage distributed in Transdanubia and we provided new distribution records of L. v. ampelensis inhabiting the eastern territories of the CB. High genetic diversity of smooth newts was observed, especially in the North Hungarian Mountains and at the interfluves of the main rivers in the South with four distinct lineages of L. v. vulgaris and one lineage of L. v. ampelensis showing a low level of admixture with the spatially closest L. v. vulgaris lineage. Moreover, admixture detected at the interfluve of the main rivers (i.e. Danube and Tisza) suggested a secondary contact zone in the area. Finally, we found that the river Danube has a very weak effect on population divergence, while the river Tisza is a geographical barrier limiting gene flow between smooth newt lineages. As the range boundaries of L. v. vulgaris and L. v. ampelensis in the CB coincide with the river Tisza, our study underpins the influence of rivers in lineage diversification.

Detection of environmental DNA from amphibians in Northern Europe applied in citizen science

AbstractMany species of amphibians in Northern Europe are threatened and the local distributions are rarely described in detail. Application of modern molecular methods provides an important supplementary tool for monitoring the distribution and diversity of amphibians. For this purpose, we designed, tested, validated, and optimized 14 species‐specific assays on genomic DNA extracted from tissue samples to use for quantitative polymerase chain reaction (qPCR) setups targeting mitochondrial DNA from amphibians in freshwater samples. The tests confirmed species specificity for all assays. Considering a systematic definition of the limit of detection for each of the assays, the presented qPCR assays are unlikely to return false positive detection from any co‐occurring species in northern Europe. For field validation, the qPCR assays were applied in a large‐scale nationwide citizen science project in which sampling and qPCR analysis was carried out by high school students. Data from the citizen science project returned the expected results when compared to the known regional distribution of the target species and confirmed the presence of nine out of 14 Danish species of amphibians in the collected freshwater samples. Four out of 2550 qPCR test sets carried out by the high school students required a professional reanalysis in multiple replicates due to initial unexpected results. This emphasizes that efforts from citizen science may generate large amounts of valuable data, as long as the results are carefully scrutinized by experts.

Comparison of amphibian diversity in areas under intensive versus subsistence agriculture in Uganda

Abstract Agricultural intensification has increasingly destroyed natural habitats, resulting in species declines. Insights into the effects of landscape structure on species’ diversity and distribution are needed to effectively conserve biodiversity in agricultural landscapes. The influence of land use practices on amphibian species diversity was investigated in areas of intensive and subsistence agriculture in Namulonge (Uganda, East Africa) using visual encounter surveys while environmental parameters were also recorded. A total of 19 amphibian species were recorded. Species richness varied minimally between intensive and subsistence agriculture plots. However, there was a significant variation in species’ abundance between the two land use areas and the species diversity was reduced in the intensive-agriculture area. The relationship between habitat characteristics and species distribution was statistically significant: in areas with relatively intact natural vegetation cover more species occurred. Such pockets of vegetation provide microhabitat conditions suitable for foraging, breeding and cover. Therefore, the abovementioned observation suggests that there is value at the landscape level to preserve natural/seminatural pockets of vegetation to enhance biodiversity in any agricultural environment.

Comparative genomics of bacteria from amphibian skin associated with inhibition of an amphibian fungal pathogen, Batrachochytrium dendrobatidis.

Chytridiomycosis, caused by Batrachochytrium dendrobatidis (Bd), is a skin disease associated with worldwide amphibian declines. Symbiotic microbes living on amphibian skin interact with Bd and may alter infection outcomes. We completed whole genome sequencing of 40 bacterial isolates cultured from the skin of four amphibian species in the Eastern US. Each isolate was tested in vitro for the ability to inhibit Bd growth. The aim of this study was to identify genomic differences among the isolates and generate hypotheses about the genomic underpinnings of Bd growth inhibition. We identified sixty-five gene families that were present in all 40 isolates. Screening for common biosynthetic gene clusters revealed that this set of isolates contained a wide variety of clusters; the two most abundant clusters with potential antifungal activity were siderophores (N=17 isolates) and Type III polyketide synthases (N=22 isolates). We then examined various subsets of the 22 isolates in the phylum Proteobacteria for genes encoding specific compounds that may inhibit fungal growth, including chitinase and violacein. We identified differences in Agrobacterium and Sphingomonas isolates in the chitinase genes that showed some association with anti-Bd activity, as well as variation in the violacein genes in the Janthinobacterium isolates. Using a comparative genomics approach, we generated several testable hypotheses about differences among bacterial isolates from amphibian skin communities that could contribute to variation in the ability to inhibit Bd growth. Further work is necessary to explore and uncover the various mechanisms utilized by amphibian skin bacterial isolates to inhibit Bd.

Elevational surveys of Sulawesi herpetofauna 1: Gunung Galang, Gunung Dako Nature Reserve.

The Indonesian island of Sulawesi has a unique geology and geography, which have produced an astoundingly diverse and endemic flora and fauna and a fascinating biogeographic history. Much biodiversity research has focused on the regional endemism in the island's Central Core and on its four peninsulas, but the biodiversity of the island's many upland regions is still poorly understood for most taxa, including amphibians and reptiles. Here, we report the first of several planned full-mountain checklists from a series of herpetological surveys of Sulawesi's mountains conducted by our team. In more than 3 weeks of work on Gunung Galang, a 2,254 m peak west of the city of Tolitoli, Sulawesi Tengah Province, on Sulawesi's Northern Peninsula, we recovered nearly fifty species of reptiles and amphibians, more than a dozen of which are either new to science or known but undescribed. The incompleteness of our sampling suggests that many more species remain to be discovered on and around this mountain.

Impact of 2,4-D and glyphosate-based herbicides on morphofunctional and biochemical markers in Scinax squalirostris tadpoles (Anura, Hylidae)

Understanding the effects of pesticides on non-target organisms is essential to assess the impact of these xenobiotics on the environment, allowing for a more informative and safer usage. The present study sought to evaluate the response of Scinax squalirostris tadpoles when exposed to different concentrations of two herbicides, DEZ® (i.e., dichlorophenoxyacetic acid or 2,4-D) and Roundup® Original (i.e., glyphosate). We collected 140 tadpoles between Gosner's 25 and 34 stages in a preservation area of the South American jelly palm Butia odorata. The animals were separated into eight groups and maintained in aquariums: acclimatization control (17 days), exposure control (24 days), and six exposure groups (7 days), including three concentration groups of each pesticide (4 μg/L, 15 μg/L, and 30 μg/L 2,4-D acid equivalent; 65 μg/L, 250 μg/L, and 500 μg/L glyphosate acid equivalent). Markers of body condition (length, body mass, K and Kn index) and oxidative balance (superoxide dismutase, catalase, glutathione S-transferase, TBARS, and carbonyl proteins) were analyzed. After 24 days (17 days of acclimation plus 7 days of exposure), tadpoles exposed to 15 μg/L of 2,4-D and 65 μg/L of glyphosate grew at higher than expected concentrations. They also had less lipoperoxidation than control tadpoles and higher superoxide dismutase, catalase, and glutathione S-transferase activity, specifically at the highest herbicide concentrations (2,4-D: 30 μg/L; glyphosate: 500 μg/L of glyphosate). Only the highest concentration of 2,4-D determined an increase in the levels of carbonyl proteins, indicating oxidative damage induced. DEZ® required more antioxidant defenses and induced a concentration-dependent answer of carbonylated proteins, suggesting oxidative stress and more toxic potential. These results may help government agencies make more conscious decisions regarding the usage of these chemicals and consider a balance between the conservation of amphibian species and agribusiness economic sustenance.

Tissue-specific in vivo transformation of plasmid DNA in Neotropical tadpoles using electroporation.

Electroporation is an increasingly common technique used for exogenous gene expression in live animals, but protocols are largely limited to traditional laboratory organisms. The goal of this protocol is to test in vivo electroporation techniques in a diverse array of tadpole species. We explore electroporation efficiency in tissue-specific cells of five species from across three families of tropical frogs: poison frogs (Dendrobatidae), cryptic forest/poison frogs (Aromobatidae), and glassfrogs (Centrolenidae). These species are well known for their diverse social behaviors and intriguing physiologies that coordinate chemical defenses, aposematism, and/or tissue transparency. Specifically, we examine the effects of electrical pulse and injection parameters on species- and tissue-specific transfection of plasmid DNA in tadpoles. After electroporation of a plasmid encoding green fluorescent protein (GFP), we found strong GFP fluorescence within brain and muscle cells that increased with the amount of DNA injected and electrical pulse number. We discuss species-related challenges, troubleshooting, and outline ideas for improvement. Extending in vivo electroporation to non-model amphibian species could provide new opportunities for exploring topics in genetics, behavior, and organismal biology.

A Critical Review of Amphibian Per- and Polyfluoroalkyl Substance Ecotoxicity Research Studies: Identification of Screening Levels in Water and Other Useful Resources for Site-Specific Ecological Risk Assessments.

With the goal of aiding risk assessors conducting site-specific risk assessments at per- and polyfluoroalkyl substance (PFAS)-contaminated sites, this critical review synthesizes information on the ecotoxicity of PFAS to amphibians in 10 amphibian species and 16 peer-reviewed publications. The studies in this review consisted of spiked-PFAS chronic toxicity experiments with perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA), perfluorohexane sulfonate (PFHxS), and 6:2 fluorotelomer sulfonate (6:2 FTS) that evaluated apical endpoints typical of ecological risk-based decision making (survival, growth, and development). Body mass was the most sensitive endpoint, showing clear and biologically meaningful population level adverse effect sizes (≥20% adverse effects). From these results, we recommend chronic no observed effect concentration (NOEC) screening levels of 590 µg/L for PFOS and 130 µg/L for PFOA. At or above recommended chronic lowest observed effect concentration screening levels of 1100 µg/L PFOS and 1400 µg/L PFOA, there is an increased chance of adverse biologically relevant chronic effects. Biologically relevant adverse effects were not observed for PFHxS and 6:2 FTS, so unbounded NOECs of 1300 µg/L PFHxS and 1800 µg/L 6:2 FTS are recommended. Screening levels are also provided for the concentration of PFAS in an amphibian diet, amphibian tissue, and moss substrate. In addition, we recommend bioconcentration factors that can be useful to predict concentrations of PFAS in amphibians using concentrations in water; these values are useful for food web modeling to understand risks to vertebrate wildlife that prey on amphibians. Overall, the present study provides a guide to the wealth of ecotoxicological research on PFAS conducted by our research group and highlights the need for additional work that would improve the understanding of chemical risks to amphibians. Environ Toxicol Chem 2023;00:1-13. © 2023 SETAC.

Predicting prey diversity with multiple predator effects

Emergent multiple predator effects can result in either risk enhancement or reduction for prey populations. However, whether emergent multiple predator effects influence survival of entire prey assemblages is unknown, which could alter community diversity. We tested whether emergent multiple predator effects would lead to changes in survival of multiple prey species that would subsequently alter prey diversity. We experimentally tested whether larval ringed (Ambystoma annulatum) and marbled salamanders (A. opacum) had additive or multiplicative effects on hatchlings of an assemblage of five amphibian prey species using outdoor mesocosms. We also examined this question using data from a previously published experiment. We extended the multiplicative risk model to estimate prey diversity based on individual prey survival probabilities from each mesocosm. We found that predators generally had additive effects on all prey species, as the expected survival of prey was not different from the observed data, indicating a lack of emergent multiple predator effects. Consistent with this result, observed diversity was not different from expected diversity based on our extension of the multiplicative risk model, further indicating predator species had additive effects. Re-analysis of previously published data showed that when emergent multiple predator effects resulted in risk reduction, diversity changed in a manner consistent that outcome. Overall, we found that additive and multiplicative effects of multiple predators contributed to structuring entire prey communities, including influencing community metrics like diversity. Future work should consider further under what conditions diversity enhancement or reduction would occur.

Effectiveness of rapid sampling assessments for wetland-breeding amphibians

A primary response to the Clean Water Act has been the development of numerous indices of biotic integrity (IBI) using various taxonomic groups to assess the condition of water bodies in the United States. The IBI is a frequently used approach for assessing the ecological integrity of streams; however, it is less commonly applied to wetland systems, despite the scientific and policy needs to assess wetland condition and develop ecological performance goals for wetland monitoring, creation, and restoration. Although a great deal of attention has been given to wetland acreage due to the “no net loss” policy over the past two decades, much less attention has been given to the ecological integrity of these same wetlands. To assess wetland health and function, regulatory agencies and land managers need clear performance standards that are based on the ecology of wetland organisms. To address this need, we sampled 242 wetlands distributed across 45 managed properties in Illinois, U.S.A. over three years (2012–2014) for adult and larval amphibians (12 samples per wetland). Amphibian assemblages make up critical ecological components of many wetland ecosystems and are considered useful bioindicators of environmental change because they are sensitive to various forms of environmental and habitat alteration. We developed an amphibian-based IBI (hereafter aIBI) for the assessment of seasonal wetlands and optimal sampling strategies to best quantify these wetlands (i.e., assessment of time and effort). The aIBI applied a conservation coefficient to all species captured; conservation coefficients were developed through a survey of amphibian experts (n = 16). We captured 25 amphibian species with an average of 7.11 amphibian species (range 1–13) per site. Using the species occurrence data collected from both dipnet and minnow trap surveys over 12 survey periods, we assessed the average percent error in the estimation of aIBIs and species richness. Our results show that a wetland can be accurately evaluated with amphibians in one field season, but that multiple samples during that field season will be necessary. We show that 4–5 surveys using multiple methods distributed throughout the spring and summer are required. This level of effort may be incongruent with existing definitions of ‘rapid assessment’, but it is the necessary level of effort needed to confidently and accurately evaluate wetlands using amphibians as an indicator taxon. Additionally, aIBI provides a more robust framework to assess wetland integrity compared to simple species richness or other similar methodologies.