Interspecific introgression of MHC genes in Triturus newts: Evidence from multiple contact zones.

The major histocompatibility complex (MHC) genes are central to the adaptive immune response in vertebrates. Selection generally maintains high MHC variation because the spectrum of recognized pathogens depends on MHC polymorphism. Novel alleles favoured by selection originate by interallelic recombination or de novo mutations but may also be acquired by introgression from related species. However, the extent and prevalence of MHC introgression remain an open question. In this study, we tested for MHC introgression in six hybrid zones formed by six Triturus newt species. We sequenced and genotyped the polymorphic second exons of the MHC class I and II genes and compared their interspecific similarity at various distances from the centre of the hybrid zone. We found evidence for introgression of both MHC classes in the majority of examined hybrid zones, with support for a more substantial class I introgression. Furthermore, the overall MHC allele sharing outside of hybrid zones was elevated between pairs of Triturus species with abutting ranges, regardless of the phylogenetic distance between them. No effect of past hybrid zone movement on MHC allele sharing was found. Finally, using previously published genome-wide data, we demonstrated that MHC introgression was more extensive than genome-wide introgression, supporting its adaptive potential. Our study thus provides evidence for the prevalence of MHC introgression across multiple Triturus hybrid zones, indicating that MHC introgression between divergent hybridizing species may be widespread and adaptive.

Higher temperature induces oxidative stress in hybrids but not in parental species: A case study of crested newts.

Ectotherms are particularly sensitive to global warming due to their limited capacity to thermoregulate, which can impact their performance and fitness. From a physiological standpoint, higher temperatures often enhance biological processes that can induce the production of reactive oxygen species and result in a state of cellular oxidative stress. Temperature alters interspecific interactions, including species hybridization. Hybridization under different thermal conditions could amplify parental (genetic) incompatibilities, thus affecting a hybrid's development and distribution. Understanding the impact of global warming on the physiology of hybrids and particularly their oxidative status could help in predicting future scenarios in ecosystems and in hybrids. In the present study, we investigated the effect of water temperature on the development, growth and oxidative stress of two crested newt species and their reciprocal hybrids. Larvae of Triturus macedonicus and T. ivanbureschi, and their T. macedonicus-mothered and T. ivanbureschi-mothered hybrids were exposed for 30 days to temperatures of 19°C and 24°C. Under the higher temperature, the hybrids experienced increases in both growth and developmental rates, while parental species exhibited accelerated growth (T. macedonicus) or development (T. ivanbureschi). Warm conditions also had different effects on the oxidative status of hybrid and parental species. Parental species had enhanced antioxidant responses (catalase, glutathione peroxidase, glutathione S-transferase and SH groups), which allowed them to alleviate temperature-induced stress (revealed by the absence of oxidative damage). However, warming induced an antioxidant response in the hybrids, including oxidative damage in the form of lipid peroxidation. These findings point to a greater disruption of redox regulation and metabolic machinery in hybrid newts, which can be interpreted as the cost of hybridization that is likely linked to parental incompatibilities expressed under a higher temperature. Our study aims to improve mechanistic understanding of the resilience and distribution of hybrid species that cope with climate-driven changes.

Evidence for the use of a high-resolution magnetic map by a short-distance migrant, the Alpine newt (Ichthyosaura alpestris).

Newts can use spatial variation in the magnetic field (MF) to derive geographic position, but it is unclear how they detect the 'spatial signal', which, over the distances that newts move in a day, is an order of magnitude lower than temporal variation in the MF. Previous work has shown that newts take map readings using their light-dependent magnetic compass to align a magnetite-based 'map detector' relative to the MF. In this study, time of day, location and light exposure (required by the magnetic compass) were varied to determine when newts obtain map information. Newts were displaced from breeding ponds without access to route-based cues to sites where they were held and/or tested under diffuse natural illumination. We found that: (1) newts held overnight at the testing site exhibited accurate homing orientation, but not if transported to the testing site on the day of testing; (2) newts held overnight under diffuse lighting at a 'false testing site' and then tested at a site located in a different direction from their home pond oriented in the home direction from the holding site, not from the site where they were tested; and (3) newts held overnight in total darkness (except for light exposure for specific periods) only exhibited homing orientation the following day if exposed to diffuse illumination during the preceding evening twilight in the ambient MF. These findings demonstrate that, to determine the home direction, newts require access to light and the ambient MF during evening twilight when temporal variation in the MF is minimal.

Flexibility of intraoral food processing in the salamandrid newt Triturus carnifex: effects of environment and prey type.

Intraoral food processing mechanisms are known for all major vertebrate groups, but the form and function of systems used to crush, grind or puncture food items can differ substantially between and within groups. Most vertebrates display flexible mechanisms of intraoral food processing with respect to different environmental conditions or food types. It has recently been shown that newts use cyclical loop-motions of the tongue to rasp prey against the palatal dentition. However, it remains unknown whether newts can adjust their food processing behavior in response to different food types or environmental conditions. Newts are interesting models for studying the functional adaptation to different conditions because of their unique and flexible lifestyle: they seasonally change between aquatic and terrestrial habitats, adapt their prey-capture mode to the respective environment, and consume diverse food types with different mechanical properties. Using X-ray high-speed recordings, anatomical investigations, behavioral analyses and mechanical property measurements, we tested the effects of the medium in which feeding occurs (water/air) and the food type (maggot, earthworm, cricket) on the processing behavior in Triturus carnifex We discovered that food processing, by contrast to prey capture, differed only slightly between aquatic and terrestrial habitats. However, newts adjusted the number of processing cycles to different prey types: while maggots were processed extensively, earthworm pieces were barely processed at all. We conclude that, in addition to food mechanical properties, sensory feedback such as smell and taste appear to induce flexible processing responses, while the medium in which feeding occurs appears to have less of an effect.

Phylogenomics of the adaptive radiation of Triturus newts supports gradual ecological niche expansion towards an incrementally aquatic lifestyle.

Newts of the genus Triturus (marbled and crested newts) exhibit substantial variation in the number of trunk vertebrae (NTV) and a higher NTV corresponds to a longer annual aquatic period. Because the Triturus phylogeny has thwarted resolution to date, the evolutionary history of NTV, annual aquatic period, and their potential coevolution has remained unclear. To resolve the phylogeny of Triturus, we generated a c. 6000 transcriptome-derived marker data set using a custom target enrichment probe set, and conducted phylogenetic analyses using: (1) data concatenation with RAxML, (2) gene-tree summary with ASTRAL, and (3) species-tree estimation with SNAPP. All analyses produce the same, highly supported topology, despite cladogenesis having occurred over a short timeframe, resulting in short internal branch lengths. Our new phylogenetic hypothesis is consistent with the minimal number of inferred changes in NTV count necessary to explain the diversity in NTV observed today. Although a causal relationship between NTV, body form, and aquatic ecology has yet to be experimentally established, our phylogeny indicates that these features have evolved together, and suggest that they may underlie the adaptive radiation that characterizes Triturus.

Chewing or not? Intraoral food processing in a salamandrid newt.

Food processing refers to any form of mechanical breakdown of food prior to swallowing. Variations of this behaviour are found within all major gnathostome groups. Chewing is by far the most commonly used intraoral processing mechanism and involves rhythmic mandibular jaw and hyobranchial (tongue) movements. Chewing occurs in chondrichthyans (sharks and rays), actinopterygians (ray-finned fishes), dipnoi (lungfishes) as well as amniotes and involves similarities in the patterns of muscle activity and movement of the feeding apparatus. It has been suggested that amniote chewing, which involves the interaction of movements of the mandibular jaw and the muscular tongue, has evolved as part of the tetrapod land invasion. However, little is known about food-processing mechanisms in lissamphibians, which might have retained many ancestral tetrapod features. Here, we identified a processing mechanism in the salamandrid newt, Triturus carnifex, which after prey capture displays cyclic head bobbing in concert with rhythmic jaw and tongue movements. We used high-speed fluoroscopy, anatomical reconstructions and analyses of stomach contents to show that newts, although not using their mandibular jaws, deploy a derived processing mechanism where prey items are rasped rhythmically against the dentition on the mouth roof, driven by a loop motion of the tongue. We then compared patterns and coordination of jaw and tongue movements across gnathostomes to conclude that food processing in this newt species shares traits with processing mechanisms in fish as well as amniotes. This discovery casts salamanders as promising models for reconstructing the evolution of intraoral processing mechanisms at the fish-tetrapod split.

Oxidative cost of interspecific hybridization: a case study of two Triturus species and their hybrids.

Oxidative stress has most recently been suggested as one of the possible mechanisms responsible for reduced fitness of hybrids. To explore possible oxidative cost of hybridization, we examined anti-oxidant defence system parameters (superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase, glutathione reductase, glutathione, SH groups), their interconnectedness (index of integration) and levels of oxidative damage [concentrations of lipid peroxides, TBARS (thiobarbituric acid reactive substances)] in laboratory-reared newt species, Triturus macedonicus and T. ivanbureschi, and their hybrid. Our results showed that parental species differed in anti-oxidant defence system parameters, but not in the levels of integration of the whole system and oxidative damage. Individuals of T. ivanbureschi had higher activities of superoxide dismutase, glutathione S-transferase and concentrations of glutathione. Hybrid individuals of crested newts displayed higher levels of the anti-oxidant defence system (higher superoxide dismutase, catalase, glutathione peroxidase activities and concentrations of SH groups), and a lower overall correlation of anti-oxidant system (lower index of integration) in comparison with both parental species, suggesting that they may possess a less efficient anti-oxidant defence system and a higher investment in maintaining oxidative balance. The higher investment in the anti-oxidant system could divert limited resources away from other functions and affect further hybrid fitness. The presented findings contribute to a better understanding of the anti-oxidant defence system of crested newts and their interspecies differences, and support the hypothesis that oxidative stress is one of the costs of interspecific hybridization.

A signature of dynamic biogeography: enclaves indicate past species replacement.

Understanding how species have replaced each other in the past is important to predicting future species turnover. While past species replacement is difficult to detect after the fact, the process may be inferred from present-day distribution patterns. Species with abutting ranges sometimes show a characteristic distribution pattern, where a section of one species range is enveloped by that of the other. Such an enclave could indicate past species replacement: when a species is partly supplanted by a competitor, but a population endures locally while the invading species moves around and past it, an enclave forms. If the two species hybridize and backcross, the receding species is predicted to leave genetic traces within the expanding one under a scenario of species replacement. By screening dozens of genes in hybridizing crested newts, we uncover genetic remnants of the ancestral species, now inhabiting an enclave, in the range of the surrounding invading species. This independent genetic evidence supports the past distribution dynamics we predicted from the enclave. We suggest that enclaves provide a valuable tool in understanding historical species replacement, which is important because a major conservation concern arising from anthropogenic climate change is increased species replacement in the future.

Advances in methods for estimating stopover duration for migratory species using capture-recapture data.

Many species are migratory, resulting in a life cycle divided into periodic stages occurring in different habitats occupied for a limited amount of time. Estimating the time spent in each habitat is crucial to understanding how individuals modulate their activities and thus to evolutionary ecology and conservation biology. Several methods, including some recent promising advances, can be used to estimate stopover duration as well as arrival and departure probabilities at sites where individuals are monitored using capture-recapture sampling. Our objectives in this study were to (1) describe the available models to estimate stopover duration, (2) illustrate with an original data set what kinds of questions can be addressed using the most recent methods, and (3) to provide in a detailed appendix a practical guide for implementing these methods in E-SURGE software. To illustrate the potential of these models for testing biological hypotheses, we used a capture-recapture data set on marbled newts (Triturus marmoratus). We used time-dependent and time-elapsed-since-arrival effects (using both Markovian and semi-Markov processes for the latter) to model stopover duration and the probability of arriving in and departing from a breeding pond for this species and compared the relative performance of the resulting models. Our findings showed a strong sex effect on stopover duration: females stayed on average 5.63 weeks in a breeding pond whereas males stayed only 3.03 weeks. In both sexes, the retention probability was mainly influenced by the time already spent there. Consequently, individuals of the same sex stayed a similar amount of time in a pond, although they did not arrive simultaneously but successively. The selected data set demonstrated the flexibility of these methods and their potential relevance for applications in evolutionary ecology and conservation.

Investigation of histone H4 hyperacetylation dynamics in the 5S rRNA genes family by chromatin immunoprecipitation assay.

Oogenesis is a critical event in the formation of female gamete, whose role in development is to transfer genomic information to the next generation. During this process, the gene expression pattern changes dramatically concomitant with genome remodelling, while genomic information is stably maintained. The aim of the present study was to investigate the presence of H4 acetylation of the oocyte and somatic 5S rRNA genes in Triturus cristatus, using chromatin immunoprecipitation assay (ChIP). Our findings suggest that some epigenetic mechanisms such as histone acetylation could be involved in the transcriptional regulation of 5S rRNA gene families.

The ontogenetic origins of skull shape disparity in the Triturus cristatus group.

Comparative studies of ontogenies of closely related species provide insights into the mechanisms responsible for morphological diversification. Using geometric morphometrics, we investigated the ontogenetic dynamics of postlarval skull shape and disparity in three closely related crested newt species. The skull shapes of juveniles just after metamorphosis (hereafter metamorphs) and adult individuals were sampled by landmark configurations that describe the shape of the dorsal and ventral side of the newt skull, and analyzed separately. The three species differ in skull size and shape in metamorphs and adults. The ontogenies of dorsal and ventral skull differ in the orientation but not lengths of the ontogenetic trajectories. The disparity of dorsal skull shape increases over ontogeny, but that of ventral skull shape does not. Thus, modifications of ontogenetic trajectories can, but need not, increase the disparity of shape. In species with biphasic life-cycles, when ontogenetic trajectories for one stage can be decoupled from those of another, increases and decreases in disparity are feasible, but our results show that they need not occur.

Kicking Triturus arntzeni when it's down: large-scale nuclear genetic data confirm that newts from the type locality are genetically admixed.

We collected nuclear DNA data (52 markers) with next-generation sequencing for nine Triturus newt specimens, including the holotype and two of the paratypes of T. arntzeni, from the type locality at Vrtovac in eastern Serbia. We compare these data to a reference set composed of the four crested newt species distributed in eastern Serbia namely T. cristatus, T. dobrogicus, T. ivanbureschi and T. macedonicus to determine to which of these species the newts from the type locality of T. arntzeni should be attributed. The majority of alleles in individuals from Vrtovac is derived from T. macedonicus, but a considerable number of T. ivanbureschi alleles is also present; alleles typical for T. cristatus and T. dobrogicus are found at low frequency. Accordingly, we interpret Vrtovac as a T. macedonicus - T. ivanbureschi hybrid population, albeit not composed of F1 hybrids but of genetically admixed individuals derived through multiple generations of backcrossing. The data support the notion that the name T. arntzeni should not be applied to a species newly distinguished in T. karelinii sensu lato (to which the name T. ivanbureschi has been given). We conclude that because of the hybrid nature of the individuals from Vrtovac, the name T. arntzeni should be placed not only in the synonymy of T. macedonicus but also in the synonymy of T. ivanbureschi. In this study we demonstrate that next-generation sequencing can provide high quality data for type material with degraded DNA and therefore can play an important role in taxonomy.

A multimarker phylogeography of crested newts (Triturus cristatus superspecies) reveals cryptic species.

The crested newt Triturus cristatus superspecies is composed of five recognized species. One of these, T. karelinii sensu lato, comprises three geographically structured mitochondrial DNA lineages: 'eastern', 'central' and 'western T. karelinii'. Genetic divergence among these lineages is comparable to that of recognized crested newt species, but morphologically they are indistinguishable. Here, we conduct a multimarker phylogeographical survey to explore the evolutionary independence of these mitochondrial DNA lineages and we include representatives of the other species to guide our interpretation of the results. All markers show distinct patterns when analyzed singly (as a phylogeny or haplotype network) and none of them sort haplotypes fully in line with species or mitochondrial DNA lineage. A multilocus approach (BAPS and *BEAST) on the other hand shows that not only the recognized species, but also the three mitochondrial DNA lineages represent discrete nuclear DNA gene pools. A mismatch is found in the northwest of Asiatic Turkey, where several populations identified as 'central T. karelinii' based on nuclear DNA possesses 'western T. karelinii' mitochondrial DNA. We invoke asymmetric mitochondrial DNA introgression to explain this pattern and support this with a historical biogeographical scenario. The three spatial groups in T. karelinii sensu lato should be regarded as distinct species.

A revised taxonomy of crested newts in the Triturus karelinii group (Amphibia: Caudata: Salamandridae), with the description of a new species.

We present a taxonomic revision of the crested newt Triturus karelinii sensu lato. Based on the presence of discrete nuclear DNA gene pools, deep genetic divergence of mitochondrial and nuclear DNA, and no indication of gene flow, we interpret this taxon as comprising two species: one covering the southern Caspian Sea shore, the Caucasus and the Crimea, i.e. the eastern part of the total range and another covering northern Asiatic Turkey and western Asiatic Turkey plus the southeastern Balkan Peninsula, i.e. the central and western part of the total range. We acknowledge that the central/western species should likely be further subdivided into a central and a western taxon, but we prefer to await a more detailed genetic analysis of the putative contact zone, positioned in northwestern Asiatic Turkey. The name T. karelinii (Strauch, 1870) applies to the eastern species as the type locality is positioned along the coast of the Gulf of Gorgan, Iran. The name T. arntzeni has been applied to the central/western species with Vrtovac, Serbia as the type locality. We show that not T. karelinii sensu lato but T. macedonicus occurs at Vrtovac. Hence, the name T. arntzeni Litvinchuk, Borkin, Dzukic and Kalezic, 1999 (in Litvinchuk et al., 1999) is a junior synonym of T. macedonicus (Karaman, 1922) and should not be used for the central/western species. We propose the name T. ivanbureschi sp. nov. for the central/western species and provide a formal species description.

Histological changes of the skin during postembryonic development of the crested newt Triturus ivanbureschi (Urodela, Salamandridae).

BACKGROUND: Amphibian skin has been studied for many decades, especially the metamorphic changes in the skin of frogs. Less attention has been paid to salamander skin. Here, we describe changes in the skin structure during postembryonic development in a salamandrid species, the Balkan crested newt Triturus ivanbureschi. METHOD: Using traditional histological techniques we examined the skin in the trunk region of three premetamorphic larval stages (hatchling, mid larval and late larval) and two postmetamorphic stages (juvenile, just after metamorphosis, and adult). RESULTS: In larval stages, skin consists only of the epidermis, which gradually develops from the single epithelial cell layer in hatchlings, to a stratified epidermis with gland nests and characteristic Leydig cells at the late larval stage. During metamorphosis, Leydig cells disappear, and the dermal layer develops. In postmetamorphic stages, skin is differentiated on stratified epidermis and the dermis with well-developed glands. Three types of glands were observed in the skin of the postmetamorphic stages: mucous, granular and mixed. Gland composition appears to be stage- and sex-specific, with juveniles and adult female being more similar to each other. In juveniles and adult female, there are a similar proportion of glands in both dorsal and ventral skin, whereas in adult male granular glands dominated the dorsal skin, while mixed glands dominated the ventral skin. CONCLUSION: Our results provide a baseline for future comparative research of skin anatomy in salamanders.

An experimental assessment of detection dog ability to locate great crested newts (Triturus cristatus) at distance and through soil.

Detection dogs are increasingly used to locate cryptic wildlife species, but their use for amphibians is still rather underexplored. In the present paper we focus on the great crested newt (Triturus cristatus), a European species which is experiencing high conservation concerns across its range, and assess the ability of a trained detection dog to locate individuals during their terrestrial phase. More specifically, we used a series of experiments to document whether a range of distances between target newts and the detection dog (odour channelled through pipes 68 mm in diameter) affects the localisation, and to assess the ability and efficiency of target newt detection in simulated subterranean refugia through 200 mm of two common soil types (clay and sandy soil, both with and without air vents to mimic mammal burrows, a common refuge used by T. cristatus). The detection dog accurately located all individual T. cristatus across the entire range of tested distances (0.25 m- 2.0 m). The substrate trials revealed that the detection dog could locate individuals also through soil. Contrary to existing studies with detection dogs in human forensic contexts, however, detection was generally slower for T. cristatus under sandy soil compared to clay soil, particularly when a vent was absent. Our study provides a general baseline for the use of detection dogs in locating T. cristatus and similar amphibian species during their terrestrial phase.

Postglacial species displacement in Triturus newts deduced from asymmetrically introgressed mitochondrial DNA and ecological niche models.

BACKGROUND: If the geographical displacement of one species by another is accompanied by hybridization, mitochondrial DNA can introgress asymmetrically, from the outcompeted species into the invading species, over a large area. We explore this phenomenon using the two parapatric crested newt species, Triturus macedonicus and T. karelinii, distributed on the Balkan Peninsula in south-eastern Europe, as a model. RESULTS: We first delimit a ca. 54,000 km(2) area in which T. macedonicus contains T. karelinii mitochondrial DNA. This introgression zone bisects the range of T. karelinii, cutting off a T. karelinii enclave. The high similarity of introgressed mitochondrial DNA haplotypes with those found in T. karelinii suggests a recent transfer across the species boundary. We then use ecological niche modeling to explore habitat suitability of the location of the present day introgression zone under current, mid-Holocene and Last Glacial Maximum conditions. This area was inhospitable during the Last Glacial Maximum for both species, but would have been habitable at the mid-Holocene. Since the mid-Holocene, habitat suitability generally increased for T. macedonicus, whereas it decreased for T. karelinii. CONCLUSION: The presence of a T. karelinii enclave suggests that T. karelinii was the first to colonize the area where the present day introgression zone is positioned after the Last Glacial Maximum. Subsequently, we propose T. karelinii was outcompeted by T. macedonicus, which captured T. karelinii mitochondrial DNA via introgressive hybridization in the process. Ecological niche modeling suggests that this replacement was likely facilitated by a shift in climate since the mid-Holocene. We suggest that the northwestern part of the current introgression zone was probably never inhabited by T. karelinii itself, and that T. karelinii mitochondrial DNA spread there through T. macedonicus exclusively. Considering the spatial distribution of the introgressed mitochondrial DNA and the signal derived from ecological niche modeling, we do not favor the hypothesis that foreign mitochondrial DNA was pulled into the T. macedonicus range by natural selection.

The balanced lethal system of crested newts: a ghost of sex chromosomes past?

Balanced lethal systems are more than biological curiosities: as theory predicts, they should quickly be eliminated through the joint forces of recombination and selection. That such systems might become fixed in natural populations poses a challenge to evolutionary theory. Here we address the case of a balanced lethal system fixed in crested newts and related species, which makes 50% of offspring die early in development. All adults are heteromorphic for chromosome pair 1. The two homologues (1A and 1B) have different recessive deleterious alleles fixed on a nonrecombining segment, so that heterozygotes are viable, while homozygotes are lethal. Given such a strong segregation load, how could autosomes stop recombining? We propose a role for a sex-chromosome turnover from pair 1 (putative ancestral sex chromosome) to pair 4 (currently active sex chromosome). Accordingly, 1A and 1B represent two variants (Y(A) and Y(B)) of the Y chromosome from an ancestral male-heterogametic system. We formalize a scenario in which turnovers are driven by sex ratio selection stemming from gene-environment interactions on sex determination. Individual-based simulations show that a balanced lethal system can be fixed with significant likelihood, provided the masculinizing allele on chromosome 4 appears after the elimination of the feminizing allele on chromosome 1. Our study illustrates how strikingly maladaptive traits might evolve through natural selection.

Is dispersal guided by the environment? A comparison of interspecific gene flow estimates among differentiated regions of a newt hybrid zone.

How frequently genes pass through a hybrid zone may be influenced by the environment. Accordingly, in long hybrid zones that span more than one environmental setting, different patterns may emerge. The varied conditions allow testing of hypotheses on dispersal as a function of the environment. We reconstruct the amount and direction of gene flow across a heterogeneous hybrid zone of two species of marbled newts (Triturus marmoratus and Triturus pygmaeus), in four widely separated areas of the Iberian Peninsula from one mitochondrial and three nuclear genes. The main variables associated with the position of the contact zone are precipitation, rivers, altitude and relief. In some sections of the contact zone, however, its position is not correlated with any environmental factor and is instead determined by the shortest geographical distance between fixed positions at either side (mountains in the East and river in the West). In areas where the position of the zone is stable, gene flow was bidirectional. External data show that T. pygmaeus has superseded T. marmoratus over a large area and here gene flow was unidirectional. The prediction that a major river would reduce gene flow was not confirmed.

Monitoring endangered freshwater biodiversity using environmental DNA.

Freshwater ecosystems are among the most endangered habitats on Earth, with thousands of animal species known to be threatened or already extinct. Reliable monitoring of threatened organisms is crucial for data-driven conservation actions but remains a challenge owing to nonstandardized methods that depend on practical and taxonomic expertise, which is rapidly declining. Here, we show that a diversity of rare and threatened freshwater animals--representing amphibians, fish, mammals, insects and crustaceans--can be detected and quantified based on DNA obtained directly from small water samples of lakes, ponds and streams. We successfully validate our findings in a controlled mesocosm experiment and show that DNA becomes undetectable within 2 weeks after removal of animals, indicating that DNA traces are near contemporary with presence of the species. We further demonstrate that entire faunas of amphibians and fish can be detected by high-throughput sequencing of DNA extracted from pond water. Our findings underpin the ubiquitous nature of DNA traces in the environment and establish environmental DNA as a tool for monitoring rare and threatened species across a wide range of taxonomic groups.