Integrating chytrid fungal parasites into plankton ecology: research gaps and needs.

Chytridiomycota, often referred to as chytrids, can be virulent parasites with the potential to inflict mass mortalities on hosts, causing e.g. changes in phytoplankton size distributions and succession, and the delay or suppression of bloom events. Molecular environmental surveys have revealed an unexpectedly large diversity of chytrids across a wide range of aquatic ecosystems worldwide. As a result, scientific interest towards fungal parasites of phytoplankton has been gaining momentum in the past few years. Yet, we still know little about the ecology of chytrids, their life cycles, phylogeny, host specificity and range. Information on the contribution of chytrids to trophic interactions, as well as co-evolutionary feedbacks of fungal parasitism on host populations is also limited. This paper synthesizes ideas stressing the multifaceted biological relevance of phytoplankton chytridiomycosis, resulting from discussions among an international team of chytrid researchers. It presents our view on the most pressing research needs for promoting the integration of chytrid fungi into aquatic ecology.

Biofilm community composition is changing in remote mountain lakes with a relative increase in potentially toxigenic algae.

Mountain lakes provide clear drinking water to humankind but are strongly impacted by global change. Benthic biofilms are crucial for maintaining water quality in these oligotrophic lakes, yet little is known about the effects of global change on mountain biofilm communities. By combining analyses of metabarcoding data on 16S and 18S rRNA genes with climatic and environmental data, we investigated global change effects on the composition of biofilm prokaryotic and micro-eukaryotic assemblages in a five-year monitoring program of 26 Pyrenean lakes (2016-2020). Using time-decay relationships and within-lake dissimilarity modelling, we show that the composition of both prokaryotic and micro-eukaryotic biofilm communities significantly shifted and their biodiversity declined from 2016 to 2020. In particular, analyses of temporal trends with linear mixed models indicated an increase in the richness and relative abundance of cyanobacteria, including potentially toxigenic cyanobacteria, and a concomitant decrease in diatom richness and relative abundance. While these compositional shifts may be due to several drivers of global change acting simultaneously on mountain lake biota, water pH and hardness were, from our data, the main environmental variables associated with changes for both prokaryotic and micro-eukaryotic assemblages. Water pH and hardness increased in our lakes over the study period, and are known to increase in Pyrenean lakes due to the intensification of rock weathering as a result of climate change. Given predicted climate trends and if water pH and hardness do cause some changes in benthic biofilms, those changes might be further exacerbated in the future. Such biofilm compositional shifts may induce cascading effects in mountain food webs, threatening the resilience of the entire lake ecosystem. The rise in potentially toxigenic cyanobacteria also increases intoxication risks for humans, pets, wild animals, and livestock that use mountain lakes. Therefore, our study has implications for water quality, ecosystem health, public health, as well as local economies (pastoralism, tourism), and highlights the possible impacts of global change on mountain lakes.

The role of abiotic variables in an emerging global amphibian fungal disease in mountains.

The emergence of the chytridiomycete fungal pathogen Batrachochytrium dendrobatidis (Bd), causing the disease chytridiomycosis, has caused collapse of amphibian communities in numerous mountain systems. The health of amphibians and of mountain freshwater habitats they inhabit is also threatened by ongoing changes in environmental and anthropogenic factors such as climate, hydrology, and pollution. Climate change is causing more extreme climatic events, shifts in ice occurrence, and changes in the timing of snowmelt and pollutant deposition cycles. All of these factors impact both pathogen and host, and disease dynamics. Here we review abiotic variables, known to control Bd occurrence and chytridiomycosis severity, and discuss how climate change may modify them. We propose two main categories of abiotic variables that may alter Bd distribution, persistence, and physiology: 1) climate and hydrology (temperature, precipitation, hydrology, ultraviolet radiation (UVR); and, 2) water chemistry (pH, salinity, pollution). For both categories, we identify topics for further research. More studies on the relationship between global change, pollution and pathogens in complex landscapes, such as mountains, are needed to allow for accurate risk assessments for freshwater ecosystems and resulting impacts on wildlife and human health. Our review emphasizes the importance of using data of higher spatiotemporal resolution and uniform abiotic metrics in order to better compare study outcomes. Fine-scale temperature variability, especially of water temperature, variability of moisture conditions and water levels, snow, ice and runoff dynamics should be assessed as abiotic variables shaping the mountain habitat of pathogen and host. A better understanding of hydroclimate and water chemistry variables, as co-factors in disease, will increase our understanding of chytridiomycosis dynamics.

Complex chemical cocktail, containing insecticides diazinon and permethrin, drives acute toxicity to crustaceans in mountain lakes.

Mountain lakes have long been perceived as pristine environments. However, atmospheric deposition of persistent organic pollutants (POPs) have been shown to expose these sensitive ecosystems to chemical pollution. Little is known on how this pollution impacts aquatic ecosystems at high altitudes. We combined passive sampling with liquid and gas chromatography high resolution mass spectrometry (LC- and GC-HRMS) to screen the water of eight lakes in three different regions of the French Pyrenees. In total, we screened for 479 organic chemicals including POPs, polycyclic aromatic hydrocarbons (PAHs), legacy and current-use pesticides, biocides, and musk fragrances. We detected a complex cocktail of 151 individual chemicals and used their toxic unit summation (ΣTU) to assess toxicity for crustaceans and algae. While risks for algae never reached chronic risks, this was always the case for crustaceans. Acute toxic risk thresholds for crustaceans were even exceeded in several of our sites. At sites with acute toxic risk levels (> 0.1 ΣTU) crustaceans were completely absent or showed a low abundance. We conclude that crustaceans were at least partly impacted by the high toxic risks driven by the insecticides diazinon and permethrin. These drugs are widely used to protect livestock from blue tongue disease transmitted by sucking insects, suggesting free roaming livestock as local source. Our results provide important evidence on toxic chemical pollution in relatively remote mountain areas, with important consequences for aquatic mountain ecosystems.

Environment is associated with chytrid infection and skin microbiome richness on an amphibian rich island (Taiwan).

Growing evidence suggests that the origins of the panzootic amphibian pathogens Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal) are in Asia. In Taiwan, an island hotspot of high amphibian diversity, no amphibian mass mortality events linked to Bd or Bsal have been reported. We conducted a multi-year study across this subtropical island, sampling 2517 individuals from 30 species at 34 field sites, between 2010 and 2017, and including 171 museum samples collected between 1981 and 2009. We analyzed the skin microbiome of 153 samples (6 species) from 2017 in order to assess any association between the amphibian skin microbiome and the probability of infection amongst different host species. We did not detect Bsal in our samples, but found widespread infection by Bd across central and northern Taiwan, both taxonomically and spatially. Museum samples show that Bd has been present in Taiwan since at least 1990. Host species, geography (elevation), climatic conditions and microbial richness were all associated with the prevalence of infection. Host life-history traits, skin microbiome composition and phylogeny were associated with lower prevalence of infection for high altitude species. Overall, we observed low prevalence and burden of infection in host populations, suggesting that Bd is enzootic in Taiwan where it causes subclinical infections. While amphibian species in Taiwan are currently threatened by habitat loss, our study indicates that Bd is in an endemic equilibrium with the populations and species we investigated. However, ongoing surveillance of the infection is warranted, as changing environmental conditions may disturb the currently stable equilibrium.

Microbiome function predicts amphibian chytridiomycosis disease dynamics.

BACKGROUND: The fungal pathogen Batrachochytrium dendrobatidis (Bd) threatens amphibian biodiversity and ecosystem stability worldwide. Amphibian skin microbial community structure has been linked to the clinical outcome of Bd infections, yet its overall functional importance is poorly understood. METHODS: Microbiome taxonomic and functional profiles were assessed using high-throughput bacterial 16S rRNA and fungal ITS2 gene sequencing, bacterial shotgun metagenomics and skin mucosal metabolomics. We sampled 56 wild midwife toads (Alytes obstetricans) from montane populations exhibiting Bd epizootic or enzootic disease dynamics. In addition, to assess whether disease-specific microbiome profiles were linked to microbe-mediated protection or Bd-induced perturbation, we performed a laboratory Bd challenge experiment whereby 40 young adult A. obstetricans were exposed to Bd or a control sham infection. We measured temporal changes in the microbiome as well as functional profiles of Bd-exposed and control animals at peak infection. RESULTS: Microbiome community structure and function differed in wild populations based on infection history and in experimental control versus Bd-exposed animals. Bd exposure in the laboratory resulted in dynamic changes in microbiome community structure and functional differences, with infection clearance in all but one infected animal. Sphingobacterium, Stenotrophomonas and an unclassified Commamonadaceae were associated with wild epizootic dynamics and also had reduced abundance in laboratory Bd-exposed animals that cleared infection, indicating a negative association with Bd resistance. This was further supported by microbe-metabolite integration which identified functionally relevant taxa driving disease outcome, of which Sphingobacterium and Bd were most influential in wild epizootic dynamics. The strong correlation between microbial taxonomic community composition and skin metabolome in the laboratory and field is inconsistent with microbial functional redundancy, indicating that differences in microbial taxonomy drive functional variation. Shotgun metagenomic analyses support these findings, with similar disease-associated patterns in beta diversity. Analysis of differentially abundant bacterial genes and pathways indicated that bacterial environmental sensing and Bd resource competition are likely to be important in driving infection outcomes. CONCLUSIONS: Bd infection drives altered microbiome taxonomic and functional profiles across laboratory and field environments. Our application of multi-omics analyses in experimental and field settings robustly predicts Bd disease dynamics and identifies novel candidate biomarkers of infection. Video Abstract.

Scientists' warning of threats to mountains.

Mountains are an essential component of the global life-support system. They are characterized by a rugged, heterogenous landscape with rapidly changing environmental conditions providing myriad ecological niches over relatively small spatial scales. Although montane species are well adapted to life at extremes, they are highly vulnerable to human derived ecosystem threats. Here we build on the manifesto 'World Scientists' Warning to Humanity', issued by the Alliance of World Scientists, to outline the major threats to mountain ecosystems. We highlight climate change as the greatest threat to mountain ecosystems, which are more impacted than their lowland counterparts. We further discuss the cascade of "knock-on" effects of climate change such as increased UV radiation, altered hydrological cycles, and altered pollution profiles; highlighting the biological and socio-economic consequences. Finally, we present how intensified use of mountains leads to overexploitation and abstraction of water, driving changes in carbon stock, reducing biodiversity, and impacting ecosystem functioning. These perturbations can provide opportunities for invasive species, parasites and pathogens to colonize these fragile habitats, driving further changes and losses of micro- and macro-biodiversity, as well further impacting ecosystem services. Ultimately, imbalances in the normal functioning of mountain ecosystems will lead to changes in vital biological, biochemical, and chemical processes, critically reducing ecosystem health with widespread repercussions for animal and human wellbeing. Developing tools in species/habitat conservation and future restoration is therefore essential if we are to effectively mitigate against the declining health of mountains.

Watching sexy displays improves hatching success and offspring growth through maternal allocation.

Male attractiveness can have tremendous effects on the fitness of his offspring via good genes, but also via enhanced maternal allocation of resources. Yet the proximate mechanisms influencing differential maternal allocation in relation to male sexiness are poorly known. Here, we studied the importance of visual stimulation for maternal allocation in the Houbara bustard, a vulnerable bird species bred in captivity to support wild populations. Artificial insemination allowed controlling for potential confounding factors, such as a male's territory quality, social interactions or sperm quality/quantity, probably linked to mate attractiveness. We show that artificially inseminated females stimulated by highly displaying males increased their hatching success, owing to increased fertilization success. The females also increased the allocation of maternal androgens in their eggs, leading to an increase of circulating testosterone and growth rate in chicks. Hence, visual stimulation of the females can promote differential maternal allocation and favour offspring fitness. Our results further suggest that using artificial insemination for species conservation without appropriate stimulation of the breeding females probably has negative impacts on their breeding performance and therefore on population viability.

Environmental Factors and Host Microbiomes Shape Host-Pathogen Dynamics.

Microorganisms are increasingly recognized as ecosystem-relevant components because they affect the population dynamics of hosts. Functioning at the interface of the host and pathogen, skin and gut microbiomes are vital components of immunity. Recent work reveals a strong influence of biotic and abiotic environmental factors (including the environmental microbiome) on disease dynamics, yet the importance of the host-host microbiome-pathogen-environment interaction has been poorly reflected in theory. We use amphibians and the disease chytridiomycosis caused by the fungal pathogen Batrachochytrium dendrobatidis to show how interactions between host, host microbiome, pathogen, and the environment all affect disease outcome. Our review provides new perspectives that improve our understanding of disease dynamics and ecology by incorporating environmental factors and microbiomes into disease theory.

Jailed in the mountains: Genetic diversity and structure of an endemic newt species across the Pyrenees.

The Pyrenees represent a natural laboratory for biogeographic, evolutionary and ecological research of mountain fauna as a result of the high variety of habitats and the profound effect of the glacial and interglacial periods. There is a paucity of studies providing a detailed insight into genetic processes and better knowledge on the patterns of genetic diversity and how they are maintained under high altitude conditions. This is of particular interest when considering the course of past climate conditions and glaciations in a species which is considered site tenacious, with long generation times. Here we analyzed the genetic patterns of diversity and structure of the endemic Pyrenean brook newt (Calotriton asper) along its distribution range, with special emphasis on the distinct habitat types (caves, streams, and lakes), and the altitudinal and geographical ranges, using a total set of 900 individuals from 44 different localities across the Pyrenean mountain range genotyped for 19 microsatellite loci. We found evidence for a negative longitudinal and positive altitudinal gradient of genetic diversity in C. asper populations. The fact that genetic diversity was markedly higher westwards is in accordance with other Pyrenean species. However, the impact of altitudinal gradient on the genetic diversity seems to differ from other species, and mostly from other amphibians. We found that lower altitudes can act as a barrier probably because the lowlands do not provide a suitable habitat for C. asper. Regarding the distinct habitat types, caves had significantly lower values of genetic diversity compared to streams or lakes. The mean FST value was relatively high (0.304) with maximum values as high as 0.771, suggesting a highly structured total population. Indeed, populations were grouped into five subclusters, the eastern populations (cluster 1) remained grouped into two subclusters and the central-western Pyrenees (cluster 2) into three subclusters. The increase of isolation with geographical distance is consistent with the population structure detected. In conclusion, C. asper seems to be adapted to high altitude mountain habitats, and its genetic diversity is higher in the western Pyrenees. In terms of conservation priority, we consider more relevant the populations that represent a reservoir of genetic diversity.

A suite of essential biodiversity variables for detecting critical biodiversity change.

Key global indicators of biodiversity decline, such as the IUCN Red List Index and the Living Planet Index, have relatively long assessment intervals. This means they, due to their inherent structure, function as late-warning indicators that are retrospective, rather than prospective. These indicators are unquestionably important in providing information for biodiversity conservation, but the detection of early-warning signs of critical biodiversity change is also needed so that proactive management responses can be enacted promptly where required. Generally, biodiversity conservation has dealt poorly with the scattered distribution of necessary detailed information, and needs to find a solution to assemble, harmonize and standardize the data. The prospect of monitoring essential biodiversity variables (EBVs) has been suggested in response to this challenge. The concept has generated much attention, but the EBVs themselves are still in development due to the complexity of the task, the limited resources available, and a lack of long-term commitment to maintain EBV data sets. As a first step, the scientific community and the policy sphere should agree on a set of priority candidate EBVs to be developed within the coming years to advance both large-scale ecological research as well as global and regional biodiversity conservation. Critical ecological transitions are of high importance from both a scientific as well as from a conservation policy point of view, as they can lead to long-lasting biodiversity change with a high potential for deleterious effects on whole ecosystems and therefore also on human well-being. We evaluated candidate EBVs using six criteria: relevance, sensitivity to change, generalizability, scalability, feasibility, and data availability and provide a literature-based review for eight EBVs with high sensitivity to change. The proposed suite of EBVs comprises abundance, allelic diversity, body mass index, ecosystem heterogeneity, phenology, range dynamics, size at first reproduction, and survival rates. The eight candidate EBVs provide for the early detection of critical and potentially long-lasting biodiversity change and should be operationalized as a priority. Only with such an approach can science predict the future status of global biodiversity with high certainty and set up the appropriate conservation measures early and efficiently. Importantly, the selected EBVs would address a large range of conservation issues and contribute to a total of 15 of the 20 Aichi targets and are, hence, of high biological relevance.

People, pollution and pathogens - Global change impacts in mountain freshwater ecosystems.

Mountain catchments provide for the livelihood of more than half of humankind, and have become a key destination for tourist and recreation activities globally. Mountain ecosystems are generally considered to be less complex and less species diverse due to the harsh environmental conditions. As such, they are also more sensitive to the various impacts of the Anthropocene. For this reason, mountain regions may serve as sentinels of change and provide ideal ecosystems for studying climate and global change impacts on biodiversity. We here review different facets of anthropogenic impacts on mountain freshwater ecosystems. We put particular focus on micropollutants and their distribution and redistribution due to hydrological extremes, their direct influence on water quality and their indirect influence on ecosystem health via changes of freshwater species and their interactions. We show that those changes may drive pathogen establishment in new environments with harmful consequences for freshwater species, but also for the human population. Based on the reviewed literature, we recommend reconstructing the recent past of anthropogenic impact through sediment analyses, to focus efforts on small, but highly productive waterbodies, and to collect data on the occurrence and variability of microorganisms, biofilms, plankton species and key species, such as amphibians due to their bioindicator value for ecosystem health and water quality. The newly gained knowledge can then be used to develop a comprehensive framework of indicators to robustly inform policy and decision making on current and future risks for ecosystem health and human well-being.

Development and worldwide use of non-lethal, and minimal population-level impact, protocols for the isolation of amphibian chytrid fungi.

Parasitic chytrid fungi have emerged as a significant threat to amphibian species worldwide, necessitating the development of techniques to isolate these pathogens into culture for research purposes. However, early methods of isolating chytrids from their hosts relied on killing amphibians. We modified a pre-existing protocol for isolating chytrids from infected animals to use toe clips and biopsies from toe webbing rather than euthanizing hosts, and distributed the protocol to researchers as part of the BiodivERsA project RACE; here called the RML protocol. In tandem, we developed a lethal procedure for isolating chytrids from tadpole mouthparts. Reviewing a database of use a decade after their inception, we find that these methods have been applied across 5 continents, 23 countries and in 62 amphibian species. Isolation of chytrids by the non-lethal RML protocol occured in 18% of attempts with 207 fungal isolates and three species of chytrid being recovered. Isolation of chytrids from tadpoles occured in 43% of attempts with 334 fungal isolates of one species (Batrachochytrium dendrobatidis) being recovered. Together, these methods have resulted in a significant reduction and refinement of our use of threatened amphibian species and have improved our ability to work with this group of emerging pathogens.

Recent Asian origin of chytrid fungi causing global amphibian declines.

Globalized infectious diseases are causing species declines worldwide, but their source often remains elusive. We used whole-genome sequencing to solve the spatiotemporal origins of the most devastating panzootic to date, caused by the fungus Batrachochytrium dendrobatidis, a proximate driver of global amphibian declines. We traced the source of B. dendrobatidis to the Korean peninsula, where one lineage, BdASIA-1, exhibits the genetic hallmarks of an ancestral population that seeded the panzootic. We date the emergence of this pathogen to the early 20th century, coinciding with the global expansion of commercial trade in amphibians, and we show that intercontinental transmission is ongoing. Our findings point to East Asia as a geographic hotspot for B. dendrobatidis biodiversity and the original source of these lineages that now parasitize amphibians worldwide.

Non-defendable resources affect peafowl lek organization: a male removal experiment.

A lekking mating system is typically thought to be non-resource based with male providing nothing to females but genes. However, males are thought to clump their display sites on areas where they are more likely to encounter females, which may depend on non-defendable resource location. We tested this hypothesis on a feral population of peacocks. In agreement, we found that, within the lek, display site proximity to food resources had an effect on female visitation rate and male mating success. The attractiveness of display sites to male intruders was explained by the distance to the feeding place and by the female visitation rate. We randomly removed 29 territorial males from their display sites. Display sites that were more attractive to male intruders before removal remained highly attractive after removal and display sites closer to the feeding area attracted the attention of intruders significantly more after removal. Similarly, display sites that were more visited by females before removal remained more visited after removal, suggesting again that the likelihood of encountering females is determined by the display site location. Overall, these results are in agreement with non-defendable resources affecting lek spatial organization in the peafowl.

Climate forcing of an emerging pathogenic fungus across a montane multi-host community.

Changes in the timings of seasonality as a result of anthropogenic climate change are predicted to occur over the coming decades. While this is expected to have widespread impacts on the dynamics of infectious disease through environmental forcing, empirical data are lacking. Here, we investigated whether seasonality, specifically the timing of spring ice-thaw, affected susceptibility to infection by the emerging pathogenic fungus Batrachochytrium dendrobatidis (Bd) across a montane community of amphibians that are suffering declines and extirpations as a consequence of this infection. We found a robust temporal association between the timing of the spring thaw and Bd infection in two host species, where we show that an early onset of spring forced high prevalences of infection. A third highly susceptible species (the midwife toad, Alytes obstetricans) maintained a high prevalence of infection independent of time of spring thaw. Our data show that perennially overwintering midwife toad larvae may act as a year-round reservoir of infection with variation in time of spring thaw determining the extent to which infection spills over into sympatric species. We used future temperature projections based on global climate models to demonstrate that the timing of spring thaw in this region will advance markedly by the 2050s, indicating that climate change will further force the severity of infection. Our findings on the effect of annual variability on multi-host infection dynamics show that the community-level impact of fungal infectious disease on biodiversity will need to be re-evaluated in the face of climate change.This article is part of the themed issue 'Tackling emerging fungal threats to animal health, food security and ecosystem resilience'.

Information-content of morphological and behavioural sexual traits in the Palmate newt (Lissotriton helveticus).

The question of why females evaluate more than one sexual trait to choose their mates has received increasing attention in recent years. Here, we investigated the information-content of both morphological and behavioural sexual traits that have been identified as predictors of male reproductive success in the palmate newt, Lissotriton helveticus. We examined the co-variation of multiple traits with one aspect of male quality, the male body condition, using both a correlative study and an experimental diet restriction. We found that the development of the three morphological sexual traits (filament length, hind-foot-web size, and crest size) was positively inter-correlated, and was correlated to body condition. In contrast, courtship activity, an important indicator for male reproductive success, was uncorrelated to male body condition. Our results suggest that females likely obtain redundant information on male condition when evaluating filament length, hind-foot-web size and crest size during mate choice. Contrary to our expectations, display activity was not a reliable indicator of male condition, leaving the information-content of this trait unraveled. Our results further suggest that complex, multiple traits may evolve because redundant message, unreliable signals and, possibly, multiple messages can coexist.

Microscopic aquatic predators strongly affect infection dynamics of a globally emerged pathogen.

Research on emerging infectious wildlife diseases has placed particular emphasis on host-derived barriers to infection and disease. This focus neglects important extrinsic determinants of the host/pathogen dynamic, where all barriers to infection should be considered when ascertaining the determinants of infectivity and pathogenicity of wildlife pathogens. Those pathogens with free-living stages, such as fungi causing catastrophic wildlife declines on a global scale, must confront lengthy exposure to environmental barriers before contact with an uninfected host. Hostile environmental conditions therefore have the ability to decrease the density of infectious particles, reducing the force of infection and ameliorating the impact as well as the probability of establishing an infection. Here we show that, in nature, the risk of infection and infectious burden of amphibians infected by the chytrid fungus Batrachochytrium dendrobatidis (Bd) have a significant, site-specific component, and that these correlate with the microfauna present at a site. Experimental infections show that aquatic microfauna can rapidly lower the abundance and density of infectious stages by consuming Bd zoospores, resulting in a significantly reduced probability of infection in anuran tadpoles. Our findings offer new perspectives for explaining the divergent impacts of Bd infection in amphibian assemblages and contribute to our understanding of ecosystem resilience to colonization by novel pathogens.

What remains from a 454 run: estimation of success rates of microsatellite loci development in selected newt species (Calotriton asper, Lissotriton helveticus, and Triturus cristatus) and comparison with Illumina-based approaches.

The development of microsatellite loci has become more efficient using next-generation sequencing (NGS) approaches, and many studies imply that the amount of applicable loci is large. However, few studies have sought to quantify the number of loci that are retained for use out of the thousands of sequence reads initially obtained. We analyzed the success rate of microsatellite loci development for three amphibian species using a 454 NGS approach on tetra-nucleotide motif-enriched species-specific libraries. The number of sequence reads obtained differed strongly between species and ranged from 19,562 for Triturus cristatus to 55,626 for Lissotriton helveticus, with 52,075 reads obtained for Calotriton asper. PHOBOS was used to identify sequences with tetra-nucleotide repeat motifs with a minimum repeat number of ten and high quality primer binding sites. Of 107 sequences for T. cristatus, 316 for C. asper and 319 for L. helveticus, we tested the amplification success, polymorphism, and degree of heterozygosity for 41 primer combinations each for C. asper and T. cristatus, and 22 for L. helveticus. We found 11 polymorphic loci for T. cristatus, 20 loci for C. asper, and 15 loci for L. helveticus. Extrapolated, the number of potentially amplifiable loci (PALs) resulted in estimated species-specific success rates of 0.15% (T. cristatus), 0.30% (C. asper), and 0.39% (L. helveticus). Compared with representative Illumina NGS approaches, our applied 454-sequencing approach on specifically enriched sublibraries proved to be quite competitive in terms of success rates and number of finally applicable loci.

Mixed reproductive strategies of the Common moorhen on a microscale as revealed by genetic data.

External factors shaping reproductive strategies within a population are still poorly understood. How individuals use space and where they decide to build a nest may influence reproductive strategies, as individuals that are close in space may more frequently interact socially. Here, we investigated a population (n=58 from 15 nests) of the Common moorhen in the Loir river (Western France) using microsatellite data (54 alleles). We found a surprisingly low level of genetic monogamy, a low relatedness among offspring in some nests and a low relatedness between the social parents and the offspring. Nest heterozygosity was highest close to the geographic center of the population. The mating strategies of the Common moorhen were highly variable, despite the social monogamy of the species, and were, to some extent, influenced by the microspatial structure. We discuss how our results contribute to the understanding of parent-offspring and offspring-offspring relationships.