Microplastic pollution calls for urgent investigations in stygobiont habitats: A case study from Classical karst.

Microplastic pollution in karst systems is still poorly studied, despite the presence of protected species and habitats, and important water reserves. Vulnerable key species hosted in these habitats could consume or assimilate microplastics, which can irreversibly damage management efforts, and thus ecosystems functionality. This can be particularly true for subterranean water habitats where microplastic pollution effects on wildlife management programs are not considered. The aim of this study is to provide a case study from the Classical Karst Region, which hosts peculiar habitats and key species protected at European level, such as the olm Proteus anguinus. As this area has been deeply exploited and modified over time, and is adjacent to highways, roads and railways, which could contribute to pollution within the karst system, threatening the ecosystems, it provides a perfect model system. In this study we collected and investigated water and sediment samples from aquatic environments of surface and subterranean habitats hosting several subterranean environment-adapted organisms. Examined particles were counted and characterized by size, color and shape via visual identification under a microscope, with and without UV light. Furthermore, spectroscopic analyses were carried out in order to identify microplastics typology. Microplastics were found in all examined habitats. In water, microplastics concentration ranged from 37 to 86 items/L, in sediments from 776 to 2064 items/kg. Fibre-shape was the main present, followed by fragments and beads, suggesting multiple sources of pollution, especially textile products. Most of the particles were fluorescent under UV light and were mainly transparent, while not-fluorescent ones were especially black, blue or brown. Samples contained especially polyesters and copolymers. These results highlight intense MP pollution in karst areas, with significant impacts on water quality, and potential effects on subterranean environment-dwelling species. We stress the importance of monitoring pollution in these critical environments for biodiversity and habitat conservation: monitoring in karst areas must become a priority for habitat and species protection, and water resources management, improving analyses on a larger number of aquatic surface and subterranean habitats.

Larval development and poor trophic resource availability: Local adaptations and plasticity in a widespread amphibian species.

Theory predicts that, in organisms with complex life cycles, if the earlier-stage limiting factor induces weak later-stage phenotypes, the development of the later-stage trait should evolve to reduce carry-over effects. Local adaptations could thus favour decoupling of later stages. However, decoupling is not always possible. In this study, we used a widespread amphibian, the European fire salamander (Salamandra salamandra), to assess the role of local adaptations to environmental stressful conditions experienced at the larval stage. We exposed 150 larvae from different altitudes to two conditions: rich food and poor food condition. Conditions in early life stages can affect an individual's traits, either as a direct effect or mediated through outcomes in successive life stages. To distinguish between effects of rearing conditions and local adaptation, we searched for a causal model. The causal model detected effects of both food treatment and population origin (altitude) on all life stages. Larvae reared under rich food condition metamorphosed earlier, had higher growth rates and reached smaller size at metamorphosis. Significant differences occurred between larvae of different origin: low-altitude individuals performed poorly under the poor food treatment. Moreover, larvae from higher altitudes were slower with rich food and faster with poor food compared to those from lower altitudes. Our results underline that environmental conditions and local adaptation can interplay in determining the plasticity of larval stages, still adaptations can maximize the growth efficiency of early stages in oligotrophic environments, leading to divergent pathways across populations and environmental conditions.

Global response of conservationists across mass media likely constrained bat persecution due to COVID-19.

Most people lack direct experience with wildlife and form their risk perception primarily on information provided by the media. The way the media frames news may substantially shape public risk perception, promoting or discouraging public tolerance towards wildlife. At the onset of the COVID-19 pandemic, bats were suggested as the most plausible reservoir of the virus, and this became a recurrent topic in media reports, potentially strengthening a negative view of this ecologically important group. We investigated how media framed bats and bat-associated diseases before and during the COVID-19 pandemic by assessing the content of 2651 online reports published across 26 countries, to understand how and how quickly worldwide media may have affected the perception of bats. We show that the overabundance of poorly contextualized reports on bat-associated diseases likely increased the persecution towards bats immediately after the COVID-19 outbreak. However, the subsequent interventions of different conservation communication initiatives allowed pro-conservation messages to resonate across the global media, likely stemming an increase in bat persecution. Our results highlight the modus operandi of the global media regarding topical biodiversity issues, which has broad implications for species conservation. Knowing how the media acts is pivotal for anticipating the propagation of (mis)information and negative feelings towards wildlife. Working together with journalists by engaging in dialogue and exchanging experiences should be central in future conservation management.

Fluorescence Properties of a Novel Isoquinoline Derivative Tested in an Invertebrate Chordate, Ciona intestinalis.

3-Hydroxyisoquinolines (ISOs) and their tautomeric isoquinolin-3-ones are heterocycles with attractive biological properties. Here we reported the revisited synthesis of a highly functionalized ISO that showed blue fluorescence and the characterization of its biological properties in an invertebrate animal model, the ascidian Ciona intestinalis. Larvae exposed to ISO at concentrations higher than 1 µM showed an intense fluorescence localized in the cell nuclei of all tissues. Moreover, exposure to ISO interfered with larval ability to swim; this neuromuscular effect was reversible. Overall, these results suggested that ISOs can have promising applications as novel fluorescent dyes of the cell nuclei.

Long-term drivers of persistence and colonization dynamics in spatially structured amphibian populations.

Many organisms live in networks of local populations connected by dispersing individuals, called spatially structured populations (SSPs), where the long-term persistence of the entire network is determined by the balance between 2 processes acting at the scale of local populations: extinction and colonization. When multiple threats act on an SSP, a comparison of the different factors determining local extinctions and colonizations is essential to plan sound conservation actions. We assessed the drivers of long-term population dynamics of multiple amphibian species at the regional scale. We used dynamic occupancy models within a Bayesian framework to identify the factors determining persistence and colonization of local populations. Because connectivity among patches is fundamental to SSPs dynamics, we considered 2 measures of connectivity acting on each focal patch: incidence of the focal species and incidence of invasive crayfish. We used meta-analysis to summarize the effect of different drivers at the community level. Persistence and colonization of local populations were jointly determined by factors acting at different scales. Persistence probability was positively related to the area and the permanence of wetlands, whereas it was negatively related to occurrence of fish. Colonization probability was highest in semipermanent wetlands and in sites with a high incidence of the focal species in nearby sites, whereas it showed a negative relationship with the incidence of invasive crayfish in the landscape. By analyzing long-term data on amphibian population dynamics, we found a strong effect of some classic features commonly used in SSP studies, such as patch area and focal species incidence. The presence of an invasive non-native species at the landscape scale emerged as one of the strongest drivers of colonization dynamics, suggesting that studies on SSPs should consider different connectivity measures more frequently, such as the incidence of predators, especially when dealing with biological invasions.

Factores a Largo Plazo de la Persistencia y las Dinámicas Colonizadoras en una Población Anfibia Estructurada Espacialmente Resumen Muchos organismos viven en redes formadas por poblaciones locales conectadas por individuos dispersos, llamadas poblaciones estructuradas espacialmente (PEE), en donde la persistencia a largo plazo de la red completa está determinada por dos procesos que actúan a escala local en las poblaciones: extinción y colonización. Cuando múltiples amenazas actúan sobre una PEE, es esencial una comparación entre los diferentes factores que determinan las extinciones y colonizaciones locales para planear acciones de conservación prudentes. Analizamos los factores a largo plazo de las dinámicas poblaciones de varias especies anfibias a escala regional. Usamos modelos de ocupación dinámica dentro de un marco de trabajo bayesiano para identificar los factores que determinan la persistencia y colonización de las poblaciones locales. Ya que la conectividad entre los fragmentos es fundamental para las dinámicas de las PEE, consideramos dos medidas de conectividad que actúan sobre cada fragmento focal: la incidencia de las especies focales y la incidencia de cangrejos de río invasores. Usamos un meta análisis para resumir el efecto de los diferentes factores a nivel de comunidad. La persistencia y la colonización de las poblaciones locales estuvieron determinadas en conjunto por los factores que actúan a diferentes escalas. La probabilidad de persistencia estuvo relacionada positivamente con el área y la permanencia de los humedales; mientras que estuvo relacionada negativamente con la presencia de peces. La probabilidad de colonización fue más alta en los humedales semipermanentes y en sitios con una alta incidencia de especies focales en sitios cercanos; mientras que mostró una relación negativa con la incidencia de los cangrejos de río invasores en el paisaje. Cuando analizamos los datos a largo plazo de las dinámicas de las poblaciones anfibias, encontramos un efecto firme de algunos rasgos clásicos de uso común en los estudios de las PEE, como el área del fragmento y la incidencia de la especie focal. La presencia de una especie invasora no nativa a escala de paisaje surgió como uno de los factores más fuertes para las dinámicas de colonización, lo que sugiere que los estudios sobre las PEE deberían considerar diferentes medidas de conectividad con mayor frecuencia, como lo es la incidencia de depredadores, especialmente cuando se está trabajando con invasiones biológicas.

The good, the bad and the ugly of COVID-19 lockdown effects on wildlife conservation: Insights from the first European locked down country.

The COVID-19 pandemic zoonosis has determined extensive lockdowns worldwide that provide an unprecedented opportunity to understand how large-scale shifts of human activities can impact wildlife. We addressed the impacts of the COVID-19 lockdown on wildlife in Italy, the first European country that performed a countrywide lockdown, and identified potentially beneficial and negative consequences for wildlife conservation and management. We combined a qualitative analysis of social media information with field data from multiple taxa, data from citizen science projects, and questionnaires addressed to managers of protected areas. Both social media information and field data suggest that a reduction of human disturbance allowed wildlife to exploit new habitats and increase daily activity. The field data confirmed some positive effects on wildlife conservation, such as an increase in species richness in temporarily less-disturbed habitats, a higher breeding success of an aerial insectivorous bird, and reduction of road-killing of both amphibians and reptiles. Despite some positive effects, our data also highlighted several negative impacts of the COVID-19 crisis on wildlife. The lower human disturbance linked to lockdown was in fact beneficial for invasive alien species. Results from questionnaires addressed to managers of protected areas highlighted that the COVID-19 lockdown interrupted actions for the control of invasive alien species, and hampered conservation activities targeting threatened taxa. Furthermore, the reduction of enforcement could cause a surge of illegal killing of wildlife. The COVID-19 crisis, besides having deep socio-economic impacts, might profoundly affect wildlife conservation, with potentially long-lasting effects.

Exploring miR-9 Involvement in Ciona intestinalis Neural Development Using Peptide Nucleic Acids.

The microRNAs are small RNAs that regulate gene expression at the post-transcriptional level and can be involved in the onset of neurodegenerative diseases and cancer. They are emerging as possible targets for antisense-based therapy, even though the in vivo stability of miRNA analogues is still questioned. We tested the ability of peptide nucleic acids, a novel class of nucleic acid mimics, to downregulate miR-9 in vivo in an invertebrate model organism, the ascidian Ciona intestinalis, by microinjection of antisense molecules in the eggs. It is known that miR-9 is a well-conserved microRNA in bilaterians and we found that it is expressed in epidermal sensory neurons of the tail in the larva of C. intestinalis. Larvae developed from injected eggs showed a reduced differentiation of tail neurons, confirming the possibility to use peptide nucleic acid PNA to downregulate miRNA in a whole organism. By identifying putative targets of miR-9, we discuss the role of this miRNA in the development of the peripheral nervous system of ascidians.

Continental-scale determinants of population trends in European amphibians and reptiles.

The continuous decline of biodiversity is determined by the complex and joint effects of multiple environmental drivers. Still, a large part of past global change studies reporting and explaining biodiversity trends have focused on a single driver. Therefore, we are often unable to attribute biodiversity changes to different drivers, since a multivariable design is required to disentangle joint effects and interactions. In this work, we used a meta-regression within a Bayesian framework to analyze 843 time series of population abundance from 17 European amphibian and reptile species over the last 45 years. We investigated the relative effects of climate change, alien species, habitat availability, and habitat change in driving trends of population abundance over time, and evaluated how the importance of these factors differs across species. A large number of populations (54%) declined, but differences between species were strong, with some species showing positive trends. Populations declined more often in areas with a high number of alien species, and in areas where climate change has caused loss of suitability. Habitat features showed small variation over the last 25 years, with an average loss of suitable habitat of 0.1%/year per population. Still, a strong interaction between habitat availability and the richness of alien species indicated that the negative impact of alien species was particularly strong for populations living in landscapes with less suitable habitat. Furthermore, when excluding the two commonest species, habitat loss was the main correlate of negative population trends for the remaining species. By analyzing trends for multiple species across a broad spatial scale, we identify alien species, climate change, and habitat changes as the major drivers of European amphibian and reptile decline.

Predation on Amphibians May Enhance Eurasian Otter Recovery in Southern Italy.

Mediterranean freshwaters undergo extreme seasonal variation in water flow, which, exacerbated by water withdrawal for agriculture or hydroelectric purposes, may affect fish communities and thus prey availability for semi-aquatic predators, such as Eurasian otter Lutra lutra. To investigate the role played by food availability on the ongoing recovery of an otter population at the southernmost limit of its Italian range, we assessed otter diet by the analysis of 357 spraints collected from 2014 to 2017 on eight rivers, and both fish and amphibian availability by, respectively, electrofishing and visual encounter surveys. Fish and amphibians formed the bulk of otter diet, the latter resource contributing as much as fish to otter diet in spring. Use by otters of both fish and amphibians depended only fish availability, suggesting that amphibians constituted an alternative resource to be exploited in conditions of fish shortage. Accordingly, electrofishing showed that fish biomass may barely be sufficient to sustain the current otter population. ATR-FT-IR spectroscopy allowed to point out for the first time the occurrence of amphibian eggs in otter spraints, although the co-occurrence of anuran bones did not allow to discriminate between direct and passive predation. Overall results indicate that the expansion or even survival of this small otter population may depend on the effective management of water resources and reinforcement of fish assemblages.

miR-7 Knockdown by Peptide Nucleic Acids in the Ascidian Ciona intestinalis.

Peptide Nucleic Acids (PNAs) are synthetic mimics of natural oligonucleotides, which bind complementary DNA/RNA strands with high sequence specificity. They display numerous advantages, but in vivo applications are still rare. One of the main drawbacks of PNAs application is the poor cellular uptake that could be overcome by using experimental models, in which microinjection techniques allow direct delivery of molecules into eggs. Thus, in this communication, we investigated PNAs efficiency in miR-7 downregulation and compared its effects with those obtained with the commercially available antisense molecule, Antagomir (Dharmacon) in the ascidian Ciona intestinalis. Ascidians are marine invertebrates closely related to vertebrates, in which PNA techniques have not been applied yet. Our results suggested that anti-miR-7 PNAs were able to reach their specific targets in the developing ascidian embryos with high efficiency, as the same effects were obtained with both PNA and Antagomir. To the best of our knowledge, this is the first evidence that unmodified PNAs can be applied in in vivo knockdown strategies when directly injected into eggs.

Teratogenic potential of nanoencapsulated vitamin A evaluated on an alternative model organism, the tunicate Ciona intestinalis.

Nano-encapsulation is a technology used to pack substances in order to enhance their stability and bioavailability, but this packing may interact with living systems, causing unexpected toxicity. Vitamin A (vit A) is a substance that has received attention, because in developed countries, the increasing availability of supplements is leading to its excessive intake. This study aims to compare teratogenic effects caused by exposure to the traditional formulation of vit A versus nano-encapsulated vit A. We used ascidian embryos as an alternative model. Ascidians are marine organisms closely related to vertebrates that share with them a body plan and developmental programme, including the morphogenetic role of retinoic acid (RA). Our data showed that the adverse effects of exposure to the same concentration of the two formulations were different, suggesting that the nano-encapsulation increased the bioavailability of the molecule, which could be better absorbed and metabolised to RA, the effective teratogenic substance.

Thermal equilibrium and temperature differences among body regions in European plethodontid salamanders.

Information on species thermal physiology is extremely important to understand species responses to environmental heterogeneity and changes. Thermography is an emerging technology that allows high resolution and accurate measurement of body temperature, but until now it has not been used to study thermal physiology of amphibians in the wild. Hydromantes terrestrial salamanders are strongly depending on ambient temperature for their activity and gas exchanges, but information on their body temperature is extremely limited. In this study we tested if Hydromantes salamanders are thermoconform, we assessed whether there are temperature differences among body regions, and evaluated the time required to reach the thermal equilibrium. During summers of 2014 and 2015 we analysed 56 salamanders (Hydromantes ambrosii and Hydromantes italicus) using infrared thermocamera. We photographed salamanders at the moment in which we found them and 1, 2, 3, 4, 5 and 15min after having kept them in the hands. Body temperature was equal to air temperature; salamanders attained the equilibrium with air temperature in about 8min, the time required to reach equilibrium was longer in individuals with large body size. We detected small temperature differences between body parts, the head being slightly warmer than the body and the tail (mean difference: 0.05°C). These salamanders quickly reach the equilibrium with the environment, thus microhabitat measurement allows obtaining accurate information on their tolerance limits.

The problem of anthropogenic microfibres in karst systems: Assessment of water and submerged sediments.

A new worrying micropollutant threathens natural environments: the microfibres (MFs). Natural, regenerated and synthetic MFs have been detected in different environments, as well as in organisms. While synthetic MFs are generally detected in microplastic analyses, natural and regenerated MFs are not taken into account, or are wrongly considered plastics. They are generally considered biodegradable even if their degradation processes in ecosystems are poorly known. Their potential faster degradation could release toxic compounds, and their characteristics could led to a long-term accumulation in the environment. Understanding their dangerousness and the possible impact they could have on ecosystems is fundamental for environment conservation. We collected and investigated water and submerged sediment samples in different caves and springs of the Classical Karst Region (NE Italy), rich in protected habitats and species. MFs were analysed via microscopy and spectroscopy. MFs were found in all samples, highlighting pollution in surface and subterranean habitats of the karst system. MF concentration was higher in submerged sediments respect to waters, highlighting an accumulation of MFs over time. Big microfibres were less abundant, and MF amount increased with the decrease in the considered size. More than 80% of fibres were fluorescent under UV light. Fluorescent MFs were especially transparent, while non-fluorescent ones were mainly black and blue. Most MFs were cellulosic, and synthetic MFs represent only 15-22%, highlighting a significant gap between the MF composition detected in natural environments and the global production of synthetic textiles in recent times. Synthetic MFs were more abundant in waters. Our results improve the knowledge on micropollutants in karst environments, laying the foundations for future research. MF pollution monitoring in karst areas must become a priority for species protection, habitat conservation, and waters management, improving analyses on a larger number of aquatic environments, taking into account the ecological connections between surface and subterranean habitats.

Four years monitoring of the endangered European plethodontid salamanders.

The ongoing biodiversity crisis is strongly threatening amphibians, mostly because of their peculiar physiology, their sensitivity to climate change and the spread of diseases. Effective monitoring involving assessments of pressure effects across time and estimation of population trends play a key role in mitigating amphibian decline. To improve implementation of standardized protocols and conservation efforts, we present here a dataset related to one of the amphibian genera whose onservation status is considered the most declining according to the IUCN. We report information on 66 populations of the endangered European cave salamanders, genus Speleomantes, that was collected through a standardized monitoring along a four-year period (2021-2024). Demographics data of the populations and fitness-related data of single individuals are reported. Furthermore, we include 3,836 high quality images of individuals that can allow to perform studies aiming to assess the phenotypic variability within the genus, and to perform long-term capture-mark-recaptured studies.

Alpine salamanders at risk? The current status of an emerging fungal pathogen.

Amphibians globally suffer from emerging infectious diseases like chytridiomycosis caused by the continuously spreading chytrid fungi. One is Batrachochytrium salamandrivorans (Bsal) and its disease ‒ the 'salamander plague' ‒ which is lethal to several caudate taxa. Recently introduced into Western Europe, long distance dispersal of Bsal, likely through human mediation, has been reported. Herein we study if Alpine salamanders (Salamandra atra and S. lanzai) are yet affected by the salamander plague in the wild. Members of the genus Salamandra are highly susceptible to Bsal leading to the lethal disease. Moreover, ecological modelling has shown that the Alps and Dinarides, where Alpine salamanders occur, are generally suitable for Bsal. We analysed skin swabs of 818 individuals of Alpine salamanders and syntopic amphibians at 40 sites between 2017 to 2022. Further, we compiled those with published data from 319 individuals from 13 sites concluding that Bsal infections were not detected. Our results suggest that the salamander plague so far is absent from the geographic ranges of Alpine salamanders. That means that there is still a chance to timely implement surveillance strategies. Among others, we recommend prevention measures, citizen science approaches, and ex situ conservation breeding of endemic salamandrid lineages.

Ethical reasoning and participatory approach towards achieving regulatory processes for animal-visitor interactions (AVIs) in South Africa.

South Africa's wide range of animal facilities offers many different types of Animal-Visitor Interactions, wild animal encounters where animals and visitors come closer than in normal circumstances. The aim of this study was to provide a map of the ethically relevant aspects involved in AVIs in South Africa as a first step towards regulating these activities. A participative approach based on the ethical matrix, a tool which organizes the ethical standings of the stakeholders by three bearing ethical principles (wellbeing, autonomy, fairness), was applied. The matrix was populated through a top-down approach and refined by engaging stakeholders in a workshop and two online self-administrated surveys. The outcome is a map of the value demands concerning Animal Visitor Interactions. This map shows how the ethical acceptability of AVIs is linked to different relevant issues like animal welfare, education, biodiversity conservation, sustainability, human competency, facility mission, impact on scientific research and socio-economic outcomes. In addition, results highlighted the importance of cooperation among stakeholders and suggested that attention for animal welfare can inform decision making and inspire a multidisciplinary approach in implementing a regulatory frame for South African wildlife facilities.

Decline and Extinction of the Italian Agile Frog Rana latastei from Core Areas of Its Range.

Detecting the trends of species and populations is fundamental to identifying taxa with high conservation priority. Unfortunately, long-term monitoring programs are challenging and often lacking. The Italian agile frog Rana latastei is endemic to Northern Italy and adjacent countries, is considered vulnerable by the IUCN, and is protected at the European level. However, quantitative estimates of its decline are extremely scarce. In this study, we document the trends in abundance and distribution of Rana latastei within Monza Park, which currently represents the area closer to the type locality of the species and holds unique genetic features. Wetlands within the park were monitored from 2000 to 2023; counts of egg clutches were taken as a measure of reproductive output and the abundance of breeding females. In 2000, the species occurred over a significant proportion of the park. Total abundance showed strong yearly variation but remained rather constant from 2000 to 2019. However, Rana latastei disappeared from the park around 2021 and was never detected in 2022-2023. The decline is probably related to the joint effect of multiple factors, including the conversion of breeding sites for farming, inappropriate water management, invasive alien species, and severe drought. The local extinction of Rana latastei occurred despite legal protection, highlighting the need for more effective and stringent tools for the conservation of European biodiversity.

Factors determining the dorsal coloration pattern of aposematic salamanders.

Aposematic bright colors have a key role for animal defense and can be expressed through metabolic production or by acquiring pigments from diet. Aposematic coloration can be related to both local adaptations and availability of trophic resources. The European fire salamander (Salamandra salamandra) shows significant color variability and occurs across a broad range of habitats. Here we combined field observations with common rearing experiments to disentangle the role of environmental conditions and local adaptations in determining aposematic coloration of salamander populations. We assessed color variation and measured habitat features and food availability in adults from 25 populations. Furthermore, we reared newborn larvae from 10 populations under different food availability and analyzed color of metamorphs. To assess color pattern, we measured the percentage of yellow covering the body, and the Hue, Saturation and Value of yellow coloration. Adult showed strong variation of color pattern; variation was strongly related to the individual's size, to habitat productivity and to food availability. Under common garden conditions, differences between populations were not anymore evident, and coloration was only affected by resource availability during larval development. Our results suggest that environmental conditions and food availability are more important than local adaptations in determining differences in aposematic color pattern.

Environmental DNA of insects and springtails from caves reveals complex processes of eDNA transfer in soils.

Subterranean environments host a substantial amount of biodiversity, however assessing the distribution of species living underground is still extremely challenging. Environmental DNA (eDNA) metabarcoding is a powerful tool to estimate biodiversity in poorly known environments and has excellent performance for soil organisms. Here, we tested 1) whether eDNA metabarcoding from cave soils/sediments allows to successfully detect springtails (Hexapoda: Collembola) and insects (Hexapoda: Insecta); 2) whether eDNA mostly represents autochthonous (cave-dwelling) organisms or it also incorporates information from species living in surface environments; 3) whether eDNA detection probability changes across taxa with different ecology. Environmental DNA metabarcoding analyses detected a large number of Molecular Operational Taxonomic Units (MOTUs) for both insects and springtails. For springtails, detection probability was high, with a substantial proportion of hypogean species, suggesting that eDNA provides good information on the distribution of these organisms in caves. Conversely, for insects most of MOTUs represented taxa living outside caves, and the majority of them represented taxa/organisms living in freshwater environments (Ephemeroptera, Plecoptera and Trichoptera). The eDNA of freshwater insects was particularly abundant in deep sectors of caves, far from the entrance. Furthermore, average detection probability of insects was significantly lower than the one of springtails. This suggests that cave soils/sediments act as "conveyer belts of biodiversity information", possibly because percolating water lead to the accumulation of eDNA of organisms living in nearby areas. Cave soils hold a complex mix of autochthonous and allochthonous eDNA. eDNA provided unprecedented information on the understudied subterranean cave organisms; analyses of detection probability and occupancy can help teasing apart local eDNA from the eDNA representing spatially-integrated biodiversity for whole landscape.

Assessing Population Trends of Species with Imperfect Detection: Double Count Analyses and Simulations Confirm Reliable Estimates in Brown Frogs.

Most animal species are detected imperfectly and overlooking individuals can result in a biased inference of the abundance patterns and underlying processes. Several techniques can incorporate the imperfect detection process for a more accurate estimation of abundance, but most of them require repeated surveys, i.e., more sampling effort compared to single counts. In this study, we used the dependent double-observer approach to estimate the detection probability of the egg clutches of two brown frog species, Rana dalmatina and R. latastei. We then simulated the data of a declining population at different levels of detection probability in order to assess under which conditions the double counts provided better estimates of population trends compared to naïve egg counts, given the detectability of frog clutches. Both species showed a very high detection probability, with average values of 93% for Rana dalmatina and 97% for R. latastei. Simulations showed that not considering imperfect detection reduces the power of detecting population trends if detection probability is low. However, at high detection probability (>80%), ignoring the imperfect detection does not bias the estimates of population trends. This suggests that, for species laying large and easily identifiable egg clutches, a single count can provide useful estimates if surveys are correctly timed.