Elodie A. Courtois

Widespread presence of the pathogenic fungus Batrachochytrium dendrobatidis in wild amphibian communities in Madagascar

Amphibian chytridiomycosis, an emerging infectious disease caused by the fungus Batrachochytrium dendrobatidis (Bd), has been a significant driver of amphibian declines. While globally widespread, Bd had not yet been reported from within Madagascar. We document surveys conducted across the country between 2005 and 2014, showing Bds first record in 2010. Subsequently, Bd was detected in multiple areas, with prevalence reaching up to 100%. Detection of Bd appears to be associated with mid to high elevation sites and to have a seasonal pattern, with greater detectability during the dry season. Lineage-based PCR was performed on a subset of samples. While some did not amplify with any lineage probe, when a positive signal was observed, samples were most similar to the Global Panzootic Lineage (BdGPL). These results may suggest that Bd arrived recently, but do not exclude the existence of a previously undetected endemic Bd genotype. Representatives of all native anuran families have tested Bd-positive, and exposure trials confirm infection by Bd is possible. Bds presence could pose significant threats to Madagascars unique “megadiverse” amphibians.

Evolution of Sex-Biased Dispersal

Dispersal is central in ecology and evolution because it influences population regulation, adaptation, and speciation. In many species, dispersal is different between genders, leading to sex-biased dispersal. Several theoretical hypotheses have been proposed to explain the evolution of this bias: the resource competition hypothesis proposed by Greenwood, the local mate competition hypothesis, and the inbreeding avoidance hypothesis. Those hypotheses argued that the mating system should be the major factor explaining the direction of such bias. Sociality and the presence of handicap in genders (exaggerated sexual characters or parental care) have recently been proposed to be linked with the direction of this bias. We tested these expected coevolutions using a database of 257 species. Based on phylogenetic approaches, our findings marginally corroborated Greenwood’s hypothesis by showing relationships between the direction of sex-biased dispersal, mating systems, and territoriality. More importantly, our results highlighted that the evolution of this bias was more linked to parental care and sexual dimorphism. These traits were also found to be associated with mating systems, suggesting that sexual asymmetry in morphology and parental care might be the main determinant of the evolution of sex-biased dispersal across species and not mating systems per se, as proposed in Greenwood’s hypothesis.

The trans-riverine genetic structure of 28 Amazonian frog species is dependent on life history

Among the hypotheses formulated to explain the origin of Amazonian biodiversity, two (the riverine-barrier and the river-refuge hypotheses) focus on the role that rivers play as biotic barriers promoting speciation. However, empirical results have both supported and refuted these hypotheses. This is likely due, at least in part, to river-specific hydrologic characteristics and the biology of the focal species. The rivers of the Guiana Shield represent a model system because they have had more stable courses over time than those of the western Amazon Basin, where most tests of riverine barrier effects have taken place. We tested whether life-history traits (body size, habitat and larval development), expected to be important in determining dispersal ability, of 28 frog species are associated with genetic structure and genetic distances of individuals sampled from both banks of the Oyapock River. Thirteen of these species displayed genetic structure consistent with the river acting as a barrier to dispersal. Surprisingly, body size was not correlated with trans-riverine population structure. However, leaf-litter dwellers and species lacking free-living tadpoles were found to exhibit higher river-associated structure than open habitat/arboreal species and those with exotrophic tadpoles. These results demonstrate that rivers play an important role in structuring the genetic diversity of many frog species though the permeability of such riverine barriers is highly dependent on species-specific traits.

Differences in volatile terpene composition between the bark and leaves of tropical tree species

Volatile terpenes are among the most diverse class of defensive compounds in plants, and they are implicated in both direct and indirect defense against herbivores. In terpenes, both the quantity and the diversity of compounds appear to increase the efficiency of defense as a diverse blend of compounds provides a more efficient protection against a broader range of herbivores and limits the chances that an enemy evolves resistance. Theory predicts that plant defensive compounds should be allocated differentially among tissues according to the value of the tissue, its cost of construction and the herbivore pressure on it. We collected volatile terpenes from bark and leaves of 178 individual tree belonging to 55 angiosperm species in French Guiana and compare the kind, amount, and diversity of compounds in these tissues. We hypothesized that in woody plants, the outermost part of the trunk should hold a more diverse blend of volatile terpenes. Additionally, as herbivore communities associated with the leaves is different to the one associated with the bark, we also hypothesized that terpene blends should be distinct in the bark vs. the leaves of a given species. We found that the mixture of volatile terpenes released by bark is different and more diverse than that released by leaves, both in monoterpenes and sesquiterpenes. This supports our hypothesis and further suggests that the emission of terpenes by the bark should be more important for trunk defense than previously thought.

Cryptic diversity in Amazonian frogs: Integrative taxonomy of the genus Anomaloglossus (Amphibia: Anura: Aromobatidae) reveals a unique case of diversification within the Guiana Shield

Lack of resolution on species boundaries and distribution can hamper inferences in many fields of biology, notably biogeography and conservation biology. This is particularly true in megadiverse and under-surveyed regions such as Amazonia, where species richness remains vastly underestimated. Integrative approaches using a combination of phenotypic and molecular evidence have proved extremely successful in reducing knowledge gaps in species boundaries, especially in animal groups displaying high levels of cryptic diversity like amphibians. Here we combine molecular data (mitochondrial 16S rRNA and nuclear TYR, POMC, and RAG1) from 522 specimens of Anomaloglossus, a frog genus endemic to the Guiana Shield, including 16 of the 26 nominal species, with morphometrics, bioacoustics, tadpole development mode, and habitat use to evaluate species delineation in two lowlands species groups. Molecular data reveal the existence of 18 major mtDNA lineages among which only six correspond to described species. Combined with other lines of evidence, we confirm the existence of at least 12 Anomaloglossus species in the Guiana Shield lowlands. Anomaloglossus appears to be the only amphibian genus to have largely diversified within the eastern part of the Guiana Shield. Our results also reveal strikingly different phenotypic evolution among lineages. Within the A. degranvillei group, one subclade displays acoustic and morphological conservatism, while the second subclade displays less molecular divergence but clear phenotypic divergence. In the A. stepheni species group, a complex evolutionary diversification in tadpole development is observed, notably with two closely related lineages each displaying exotrophic and endotrophic tadpoles.

Evolutionary patterns of volatile terpene emissions across 202 tropical tree species

Abstract Plant responses to natural enemies include formation of secondary metabolites acting as direct or indirect defenses. Volatile terpenes represent one of the most diverse groups of secondary metabolites. We aimed to explore evolutionary patterns of volatile terpene emission. We measured the composition of damage‐induced volatile terpenes from 202 Amazonian tree species, spanning the angiosperm phylogeny. Volatile terpenes were extracted with solid‐phase micro extraction and desorbed in a gas chromatography–mass spectrometry for compound identification. The chemical diversity of the terpene blend showed a strong phylogenetic signal as closely related species emitted a similar number of compounds. Closely related species also tended to have compositionally similar blends, although this relationship was weak. Meanwhile, the ability to emit a given compound showed no significant phylogenetic signal for 200 of 286 compounds, indicating a high rate of diversification in terpene synthesis and/or great variability in their expression. Three lineages (Magnoliales, Laurales, and Sapindales) showed exceptionally high rates of terpene diversification. Of the 70 compounds found in >10% of their species, 69 displayed significant correlated evolution with at least one other compound. These results provide insights into the complex evolutionary history of volatile terpenes in angiosperms, while highlighting the need for further research into this important class of compounds.

Widespread occurrence of bd in French Guiana, South America.

The amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) is a purported agent of decline and extinction of many amphibian populations worldwide. Its occurrence remains poorly documented in many tropical regions, including the Guiana Shield, despite the area’s high amphibian diversity. We conducted a comprehensive assessment of Bd in French Guiana in order to (1) determine its geographical distribution, (2) test variation of Bd prevalence among species in French Guiana and compare it to earlier reported values in other South American anuran species (http://www.bd-maps.net; 123 species from 15 genera) to define sentinel species for future work, (3) track changes in prevalence through time and (4) determine if Bd presence had a negative effect on one selected species. We tested the presence of Bd in 14 species at 11 sites for a total of 1053 samples (306 in 2009 and 747 in 2012). At least one Bd-positive individual was found at eight out of 11 sites, suggesting a wide distribution of Bd in French Guiana. The pathogen was not uniformly distributed among the studied amphibian hosts, with Dendrobatidae species displaying the highest prevalence (12.4%) as compared to Bufonidae (2.6 %) and Hylidae (1.5%). In contrast to earlier reported values, we found highest prevalence for three Dendrobatidae species and two of them displayed an increase in Bd prevalence from 2009 to 2012. Those three species might be the sentinel species of choice for French Guiana. For Dendrobates tinctorius, of key conservation value in the Guiana Shield, smaller female individuals were more likely to be infected, suggesting either that frogs can outgrow their chytrid infections or that the disease induces developmental stress limiting growth. Generally, our study supports the idea that Bd is more widespread than previously thought and occurs at remote places in the lowland forest of the Guiana shield.

Taking the lead on climate change: modelling and monitoring the fate of an Amazonian frog

Climate change is expected to have important impacts on biodiversity. However, cases showing explicit links between species decline and climate are scarce, mostly because of a lack of baseline data. Tropical ectotherms with narrow altitudinal ranges are particularly sensitive to climate change; for example the frog Pristimantis espedeus may be at risk, with only nine populations known to date in French Guiana, all on isolated massifs. Ecological niche modelling indicated that these populations could disappear by 2070. To facilitate testing of this prediction we conducted a study to design an efficient, cost-effective monitoring protocol, combining occupancy rate estimations using passive acoustic recorders, and abundance estimations using acoustic repeated counts and capture–mark–recapture. We found the passive recorders to be effective, with a detection probability of 0.8. Two recording sessions were sufficient to estimate occupancy rates reliably. A minimum of 57 surveyed sites were required to detect a decline of 15% in occupancy between two consecutive monitoring events. Acoustic repeated counts and capture–mark–recapture yielded similar density estimates (1.6 and 1.8 calling males per 100 m 2 , respectively). Based on these results we present a protocol based on passive acoustic recording and abundance monitoring to monitor P. espedeus populations.

Consistency of Published Results on the Pathogen Batrachochytrium dendrobatidis in Madagascar: Formal Comment on Kolby et al. Rapid Response to Evaluate the Presence of Amphibian Chytrid Fungus (Batrachochytrium dendrobatidis) and Ranavirus in Wild Amphibian Populations in Madagascar

A recent paper by Kolby et al. [1], surveying for Batrachochytrium dendrobatidis (Bd) and ranavirus in Madagascar, presents results for 508 amphibian specimens and 68 water bodies sampled during a 2-month period of the 2013–14 wet season. Kolby et al. [1] did not detect Bd in any of the samples, presenting evidence that add to our understanding of Bd dynamics in Madagascar. Earlier in 2015, we published “Widespread presence of the pathogenic fungus Batrachochytrium dendrobatidis in wild amphibian communities in Madagascar” in the journal Scientific Reports [2]. We presented rigorous spatial and temporal surveillance data for 4,155 amphibians sampled across a 10-year period, and used two independent molecular diagnostics to demonstrate the occurrence of a molecular signature of Bd infection at multiple locations across the island. We focus here on solely the Bd results, which directly relate to our published study. While the conclusions of Bd’s occurrence and prevalence in Madagascar may appear to conflict between these papers, upon closer investigation the data sets actually complement each other. Our evidence for Bd’s presence and its widespread incidence is based on multi-year monitoring data carried out through the National Monitoring Program [3] and allied survey efforts, occurring in both the wet and dry season. Our data collected during the same time as Kolby et al.’s sampling (2013–14 wet season), is consistent with their recently published results [1,2] (summarized in Table 1). Therefore, Kolby et al.’s conclusion that our data “highly contradict” those reported in their study is inaccurate. In the 2013–14 wet season, we sampled 569 frogs from 8 locations, of which only 3 samples showed a positive signal for Bd. The positive samples were collected from one individual at each of three sites: Antoetra, Ranomafana, and Ankaratra. While both datasets (the sampling reported in Kolby et al. 2015 and the wet season 2013–2014 sampling reported in Bletz et al. 2015) surveyed numerous individuals and locations across the island, there are some differences in sampling locations. More specifically, one of our positive occurrences came from Antoetra, which was not surveyed by Kolby et al [1]. Two of our positives do come from locations surveyed by both groups: Ranomafana and Ankaratra; if we combine the sub-sites within these locations, the prevalence is 0.0043 and 0.0062 respectively, which falls within the prevalence confidence intervals presented in Kolby et al. [1] (Table 1). This same logic is used by Kolby et al. [1] to show the complementarity of their field survey data and Kolby’s previous work showing Bd’s presence in amphibians imported into the US from Madagascar [4]. Both datasets are consistent with the conclusion that Bd had a very low prevalence during the 2013–2014 wet season. Table 1 Summary of published Bd survey data for the 2013–2014 wet season. The low prevalence detected in the wet season may likely be explained by seasonality of Bd. Both papers discuss the possibility of seasonal patterns, where Bd prevalence decreases in the warmer, wetter season and increases in the cooler, dryer season due to climatic or so-far un-described environmental factors. This phenomenon is not unusual and several studies have noted a high degree of seasonal variation in the prevalence of Bd (e.g. [5, 6, 7, 8]). In our published study [2] we present preliminary evidence of a seasonal pattern of Bd, showing that prevalence and/or detection was greater in the dryer, cooler season (May-Oct) than the wetter, warmer season (Nov-April) [2]. This seasonal pattern we documented likely explains the lack of detection by Kolby et al. since they sampled only in the wet (and warmer) season. Kolby et al. [1] supplement their individual sampling with the analysis of filtered water from natural habitats. In this case, the lack of Bd detection might be associated with increased water flow due to increased rainfall during the wet season, which could lower the concentration of Bd zoospores to undetectable levels. Additionally, water-filters of natural water bodies have also been found to be less sensitive than direct sampling of amphibians [9,10]. To better understand seasonal variation as well as other factors such as geographic distribution and host species variation, additional sampling across wet and dry season in a uniform and standardize manner will be important. The major difference between these papers is that we draw from data collected in multiple years and seasons and from a much larger sample of amphibians with further validation using chytrid lineage-based PCR amplification, making our study more comprehensive in nature. Kolby et al. [1] surveyed for a 2-month period, which makes it difficult to make general assumptions about pathogen occurrence from such a small snap shot in time. Our data set thus allows for the conclusion of the “widespread presence of Bd” as we document repeated detections of Bd at geographically distant locations in Madagascar albeit with varying degree of prevalence among sites and seasons. We also have secondary confirmation and validation of Bd’s presence from an independent non-nuclear lineage specific qPCR designed to the Bd mtDNA locus, which is unique to our study. Kolby et al. [1] suggest that Bd in Madagascar cannot yet be described with certainty in part due to the variability of sampling and detection methods. We acknowledge ourselves that our use of various methods may confound some of our findings, such as the seasonal pattern of Bd; however, and importantly, this does not negate or question the evidence for Bd-positive samples collected from Madagascar. While the data presented by both studies indicate a low prevalence of Bd in Madagascar in 2014, we argue that the additional multi-year data we have collected strongly supports the occurrence of one or more Bd lineage(s) in the samples collected from wild Madagascar amphibians. A similar conclusion was also made by Kolby et al. [4] based on their observations of Bd in wild-caught frogs from Madagascar that were imported into the USA. Definitive and final confirmation of Bd in Madagascar awaits histopathology, isolation of a Bd culture, and/or genome sequencing. These additional analyses can clarify whether Madagascar is facing the panzootic, hypervirulent Bd-GPL or a different (possibly endemic) Bd-lineage. More importantly, our results may have serious conservation implications. We presented strong evidence that at least one lineage of Bd exists in Madagascar, with increased prevalence at some locations during the dry season. It remains to be understood if this genotype is virulent with respect to the resident anuran fauna and capable of causing population declines. Using the ‘precautionary principle’ in reacting to suspected introductions of novel emerging infectious diseases infecting wildlife [11] it is essential to initiate conservation actions. Continuing ongoing population monitoring of Madagascars amphibians and pathogen surveillance through the NMP are therefore essential and are a priority of the national amphibian conservation strategy for the country known as ‘A Conservation Strategy for the Amphibians of Madagascar (ACSAM) [12,13]. If it is relatively hypovirulent, it gives conservationists time to engage in mitigation strategies and to plan for the possible (and likely inevitable) arrival of a virulent genotype, which could threaten the diverse, endemic frog communities.

Phenotypic and life-history diversification in Amazonian frogs despite past introgressions

The advent of genomics in phylogenetics and population genetics strengthened the perception that conflicts among gene trees are frequent and often due to introgression. However, hybridization occurs mostly among species that exhibit little phenotypic differentiation. A recent study delineating species in Anomaloglossus, a frog genus endemic to the Guiana Shield, identified an intriguing pattern in the A. baeobatrachus species complex. This complex occurs in French Guiana and Amapá (Brazil) and comprises two sympatric phenotypes contrasting not only in body size, habitat, and advertisement call, but also in larval development mode (endotrophic vs exotrophic tadpoles). However, molecular and phenotypic divergences are, in some cases, incongruent, i.e specimens sharing mtDNA haplotypes are phenotypically distinct, suggesting a complex evolutionary history. Therefore, we genotyped 106 Anomaloglossus individuals using ddRADseq to test whether this phenotype/genotype incongruence was a product of phenotypic plasticity, incomplete lineage sorting, multiple speciation events, or admixture. Based on more than 16,000 SNPs, phylogenetic and population genetic approaches demonstrated that exotrophic populations are paraphyletic. Species tree and admixture analyses revealed a strikingly reticulate pattern, suggesting multiple historical introgression events. The evolutionary history of one exotrophic population in northern French Guiana is particularly compelling given that it received genetic material from exotrophic ancestors but shows very strong genetic affinity with the nearby endotrophic populations. This suggests strong selection on larval development and mating call after secondary contact and hybridization. The case of A. baeobatrachus represents a striking example of introgression among lineages that are phenotypically distinct, even in their larval development mode, and highlights how high-resolution genomic data can unravel unexpectedly complex evolutionary scenarios.