Claude Miaud

Species detection using environmental DNA from water samples

The assessment of species distribution is a first critical phase of biodiversity studies and is necessary to many disciplines such as biogeography, conservation biology and ecology. However, several species are difficult to detect, especially during particular time periods or developmental stages, potentially biasing study outcomes. Here we present a novel approach, based on the limited persistence of DNA in the environment, to detect the presence of a species in fresh water. We used specific primers that amplify short mitochondrial DNA sequences to track the presence of a frog (Rana catesbeiana) in controlled environments and natural wetlands. A multi-sampling approach allowed for species detection in all environments where it was present, even at low densities. The reliability of the results was demonstrated by the identification of amplified DNA fragments, using traditional sequencing and parallel pyrosequencing techniques. As the environment can retain the molecular imprint of inhabiting species, our approach allows the reliable detection of secretive organisms in wetlands without direct observation. Combined with massive sequencing and the development of DNA barcodes that enable species identification, this approach opens new perspectives for the assessment of current biodiversity from environmental samples.

Persistence of environmental DNA in freshwater ecosystems.

The precise knowledge of species distribution is a key step in conservation biology. However, species detection can be extremely difficult in many environments, specific life stages and in populations at very low density. The aim of this study was to improve the knowledge on DNA persistence in water in order to confirm the presence of the focus species in freshwater ecosystems. Aquatic vertebrates (fish: Siberian sturgeon and amphibian: Bullfrog tadpoles) were used as target species. In control conditions (tanks) and in the field (ponds), the DNA detectability decreases with time after the removal of the species source of DNA. DNA was detectable for less than one month in both conditions. The density of individuals also influences the dynamics of DNA detectability in water samples. The dynamics of detectability reflects the persistence of DNA fragments in freshwater ecosystems. The short time persistence of detectable amounts of DNA opens perspectives in conservation biology, by allowing access to the presence or absence of species e.g. rare, secretive, potentially invasive, or at low density. This knowledge of DNA persistence will greatly influence planning of biodiversity inventories and biosecurity surveys.

Next-generation monitoring of aquatic biodiversity using environmental DNA metabarcoding.

Global biodiversity in freshwater and the oceans is declining at high rates. Reliable tools for assessing and monitoring aquatic biodiversity, especially for rare and secretive species, are important for efficient and timely management. Recent advances in DNA sequencing have provided a new tool for species detection from DNA present in the environment. In this study, we tested whether an environmental DNA (eDNA) metabarcoding approach, using water samples, can be used for addressing significant questions in ecology and conservation. Two key aquatic vertebrate groups were targeted: amphibians and bony fish. The reliability of this method was cautiously validated in silico, in vitro and in situ. When compared with traditional surveys or historical data, eDNA metabarcoding showed a much better detection probability overall. For amphibians, the detection probability with eDNA metabarcoding was 0.97 (CI = 0.90–0.99) vs. 0.58 (CI = 0.50–0.63) for traditional surveys. For fish, in 89% of the studied sites, the number of taxa detected using the eDNA metabarcoding approach was higher or identical to the number detected using traditional methods. We argue that the proposed DNA‐based approach has the potential to become the next‐generation tool for ecological studies and standardized biodiversity monitoring in a wide range of aquatic ecosystems.

Population genetics reveals origin and number of founders in a biological invasion

Propagule pressure is considered the main determinant of success of biological invasions: when a large number of individuals are introduced into an area, the species is more likely to establish and become invasive. Nevertheless, precise data on propagule pressure exist only for a small sample of invasive species, usually voluntarily introduced. We studied the invasion of the American bullfrog, Rana catesbeiana, into Europe, a species that is considered a major cause of decline for native amphibians. For this major invader with scarce historical data, we used population genetics data (a partial sequence of the mitochondrial cytochrome b gene) to infer the invasion history and to estimate the number of founders of non‐native populations. Based on differences between populations, at least six independent introductions from the native range occurred in Europe, followed by secondary translocations. Genetic diversity was strongly reduced in non‐native populations, indicating a very strong bottleneck during colonization. We used simulations to estimate the precise number of founders and found that most non‐native populations derive from less than six females. This capability of invasion from a very small number of propagules challenges usual management strategies; species with such ability should be identified at an early stage of introduction.

Terrestrial movements of the natterjack toad Bufo calamita (Amphibia, Anura) in a semi-arid, agricultural landscape

The movements of 19 natterjack toads ( Bufo calamita ) were monitored by telemetry in a semi-arid agricultural landscape in NE Spain. Toads reproduced in two water pools in fields of barley and an ancient clayquarrying zone filled by rainfall. Thirteen toads were followed during the breeding season (weeks 5-17 of 1997) and 11 toads were followed after reproduction (weeks 18 to 46). During the breeding season four toads moved distances of up to 500 m between breeding sites. No significant differences in movement patterns were found between the sexes, but a tendency was observed for males to show higher terrestrial site fidelity than females. Distances moved after the breeding season were substantial. The distance between the furthest positions recorded was from 164 to 1201 m, the maximum distance observed between daily recordings ranged from 125 to 353 m and the maximum overall distances ranged from 567 to 4411 m. No significant differences were observed between males and females or between small and large toads. The movements of these toads in a semi-arid agricultural landscape in Spain are compared with those observed in England and Germany.

Fidelity to the breeding site in the alpine newt Triturus Alpestris.

In a context of several closed breeding sites (archipelago model), the great majority of alpine newts are sedentary, visiting the same site during two successive years. A homing experiment offered the animals a choice between two breeding sites, one from which they came and the other which was inhabited by another population. This experiment demonstrates the fidelity of a great majority of newts for their site of origin. Fidelity was stronger when sites were distant (150 m) than when nearer (25 m). The speed and the probability of return still varied according to both the distance and the nature of the environment of the release point. The performances were better when the animals were released in the wood than when released in the pasture. The mechanisms of orientation and navigation which should explain such results, as well as the genetic consequences of the fidelity, are examined and discussed.

Integrative approach for landscape-based graph connectivity analysis: a case study with the common frog (Rana temporaria) in human-dominated landscapes

Graph-based analysis is a promising approach for analyzing the functional and structural connectivity of landscapes. In human-shaped landscapes, species have become vulnerable to land degradation and connectivity loss between habitat patches. Movement across the landscape is a key process for species survival that needs to be further investigated for heterogeneous human-dominated landscapes. The common frog (Rana temporaria) was used as a case study to explore and provide a graph connectivity analysis framework that integrates habitat suitability and dispersal responses to landscape permeability. The main habitat patches influencing habitat availability and connectivity were highlighted by using the software Conefor Sensinode 2.2. One of the main advantages of the presented graph-theoretical approach is its ability to provide a large choice of variables to be used based on the study’s assumptions and knowledge about target species. Based on dispersal simulation modelling in potential suitable habitat corridors, three distinct patterns of nodes connections of differing importance were revealed. These patterns are locally influenced by anthropogenic barriers, landscape permeability, and habitat suitability. And they are affected by different suitability and availability gradients to maximize the best possible settlement by the common frog within a terrestrial habitat continuum. The study determined the key role of landscape-based approaches for identifying the “availability-suitability-connectivity” patterns from a local to regional approach to provide an operational tool for landscape planning.

Age, size and growth of the southern crested newt Triturus karelinii (Strauch 1870) in a population from Bozdag (Western Turkey)

We studied the characteristics of bone growth assessed by skeletochronology in a southern crested newt Triturus karelinii (Urodela) population from Western Turkey. The timing and patterns of bone arrested growth were observed using the phalanges of juveniles and adults that were caught in spring at a breeding site. A metamorphosis line was found in the juveniles. In some adults, a classical succession of single lines of arrested growth was observed in about 50% of the cross sections. However, the other adults exhibited a succession of double lines of arrested growth in their phalanges. Due to the arid summer and cold winter climate in the Bozdag region (1200 m a.s.l.), we argue that in this last case, the lines of arrested growth were deposited during both the summer (aestivation) and winter (wintering). Body length, age and growth were similar in males and females. The age of maturity was 3 to 4 years old and longevity was 8 and 11 years in males and females respectively. Body length and age among taxonomically related large bodied newts of the T. cristatus complex were reported from populations experiencing various environmental conditions. Body length and age at maturity were similar to that observed in other newt species. However, longevity seems to be lower than expected in the T. karelinii studied population. We hypothesized that the arid climate of Bozdag could cause a higher mortality risk during the terrestrial phase of the life cycle. Studying more populations exposed to various conditions is clearly needed to assess interpopulational variation of these life-history traits in this newt species.

Variations in age, size at maturity and gestation duration among two neighbouring populations of the alpine salamander (Salamandra lanzai)

We describe the age structures of two neighbouring terrestrial salamander populations. The skeletochronological method was also used on larvae in utero and on new-born individuals. The age of adults was 8‐24 years in population A, while males reached maturity at 3‐5 years old and the youngest females were 6 years old in population B. Males and females from population B were also larger than those in population A. For the first time, lines of arrested growth (LAGs) were also found in the humerus of intra-uterine larvae and new-born individuals, indicating that young can spend up to 3 years in utero (population B) and up to 4 years (population A) before hatching. Growth of adults (fitted by the Bertalanffy model) also exhibited differences in growth coefficient ( k ) and mean asymptotic length (SVLmax) between sexes and populations. Local climatic conditions differed between the two areas of these populations and we hypothesize that the number of rainy days directly influences foraging during the short period of activity (< 3 months), leading to a delay in age at maturity, smaller length and growth rate, and increased gestation duration in the drier environment. The discussion is focused on proximate environmental influences on the variation of length and associated life-history traits in ectotherms, especially in terrestrial salamanders.

To breed or not to breed: past reproductive status and environmental cues drive current breeding decisions in a long-lived amphibian

Iteroparity is an adaptive response to uncertainty in reproductive success. However, spreading reproductive success over multiple reproduction events during a lifetime is constrained by adult mortality and the stochasticity associated with interactions between external factors and physiological states. The acquisition of information about environmental conditions during the growth of progeny and sufficient resources during the non-reproductive period are key factors for breeding success. Consequently, we hypothesized that long-lived animals may skip a breeding opportunity when information about unfavourable environmental conditions is available. In addition, nutritional constraints could prevent an animal from replenishing its reserves sufficiently to invest in the current breeding period. We investigated these questions using capture–recapture data from a 5-year study on a large population of yellow-bellied toads in a forest in north-eastern France. We took advantage of various advances in multi-state capture–recapture models (e.g. unobservable states and mixture models) to test our hypotheses. Our results show that the combined effects of rainfall deficit and the breeding/non-breeding state of individuals during the past breeding season affect breeding probability during the following breeding opportunity. We also found that females breed less frequently than males, suggesting that the overall energy cost of reproduction differs between genders. Finally, the results indicate that toad survival appears to be negatively influenced by rainfall deficits. We discuss the yellow-bellied toad’s reproductive behaviour in term of bet-hedging strategy and life history trait evolution.