Biology subjects

Thalinger, B.

Publications and source records attributed to Thalinger, B..

10 recordsLinked to original sources

bioRxiv2026

From fluke to fragment: a multifaceted method for molecular sex identification and mitochondrial haplotyping from environmental DNA samples

O_LIEnvironmental DNA (eDNA) analyses have become a powerful tool for non-invasive biodiversity monitoring, yet the applicability of population genetic approaches to environmental samples remains largely unexplored. Even when genetic traces originate from a single individual, low target DNA concentrations and amplification or sequencing artefacts can compromise downstream genetic inferences. Here, we present a novel approach for obtaining demographic insights and lineage-level mitogenomic information from aquatic eDNA samples collected near vertebrate individuals. C_LIO_LIPaired eDNA and tissue samples were collected during sperm whale (Physeter macrocephalus) encounters in the Azores. Samples were screened for the presence of vertebrate eDNA and analyzed with a novel molecular sex identification assay. Additionally, long-range PCR was used to amplify up to five mitochondrial DNA fragments ([~]3-4k bp) before subsequent sequencing on an Oxford Nanopore Technologies platform. A stringent three-tier filtering framework capable of identifying true mitogenomic variation across eDNA samples was developed for maximum recovery of genetic diversity at the haplogroup level. By benchmarking eDNA samples via their paired tissues, parameter values were optimized to maximize concordance and minimize spurious variant calls. C_LIO_LISexing was successful for 50% of eDNA samples, with 96% concordance to paired tissues, and marine vertebrate DNA concentration significantly predicted sexing success. Further, Medaka polishing produced high identity mitochondrial consensus sequences (>16 kb) from eDNA samples. Across filtering regimes in the framework, curated SNP panels comprising up to 453 high-confidence mitochondrial SNPs resolved 19 haplogroups, with 93% concordance between eDNA and tissue samples. An intermediate bioinformatics filtering strategy maximized biologically accurate haplogroup recovery while minimizing sequencing artefacts, providing the most reliable lineage-level inferences. C_LIO_LIThis integrative approach demonstrates that targeted nuclear assays combined with long-range mitochondrial sequencing can recover individual-level genetic information from aquatic eDNA. By defining analytical thresholds governing success, the framework advances non-invasive genetic monitoring of populations via eDNA and enables population-level monitoring and conservation of endangered and genetically-vulnerable species. C_LI

genomics↗

bioRxiv2023

BirT: a novel primer pair for avian environmental DNA metabarcoding

Environmental DNA metabarcoding has become a widely used technique to detect animals from environmental samples and is on the brink of being implemented into routine species monitoring. Surprisingly, birds are among the taxonomic groups which until recently, received comparably little attention in eDNA research, a fact that is changing rapidly, with the growing number of air eDNA analyses. Since birds are hardly ever the most abundant species in aquatic or terrestrial habitats, a high specificity of the employed metabarcoding primers is key to limit non-target amplifications and enable reliable detection from environmental samples. Here, we present a novel primer pair (BirT) for metabarcoding of avian environmental DNA. We optimized specificity and fragment length regarding taxonomic resolution and available sequencing technology. Additionally, we evaluated the availability of 12S reference sequences for birds and filled database gaps by generating novel 12S barcodes. Finally, we tested the applicability of this approach using field-collected eDNA samples obtained with three different filter types. These results were compared to visual observations uploaded to eBird (www.eBird.org) during the sampling period. Our results confirm the suitability of the BirT primer pair for bird eDNA metabarcoding with optimized fragment length, no amplification of key non-target groups, and taxonomic resolution provided by the amplified fragment. Albeit there are still substantial gaps in the 12S reference sequence database, the analysis of bird eDNA from water samples resulted in species-level taxonomic resolution for 92% of the detected taxa. All tested filter/filtration combinations delivered similar results for total read numbers per sample (mean: 613,972 {+/-} 340,088 SD) and species detected per sample (mean: 5.5 {+/-} 2.3 SD). Ninety-five percent of the bird detections were highly plausible and 58% confirmed by visual observations. The majority of the detected bird species was closely associated with aquatic habitats confirming the suitability of water samples for the detection of waterfowl and species inhabiting similar ecological niches via eDNA.

molecular biology↗

bioRxiv2023

Unravelling bird nest arthropod community structure using metabarcoding

Bird nests are fascinating microcosms harboring a wide range of arthropods parasitizing the nesting birds or feeding on prey remains, feces, and the nest material. Studies of these communities have been entirely based on emergence traps which collect live organisms out of the nests. The analysis of nest contents and environmental DNA (eDNA) via metabarcoding could expand our knowledge and identify prey, exuviae, and other animal remains in bird nests. Here, we investigated the potential of arthropod remains, nest dust, and feathers to better describe taxonomic diversity accumulated in 20 bird nests collected in Guelph (Canada). We used subsampling strategies and tested two extraction approaches to investigate the distribution of DNA in nests, account for low-quality DNA, and the presence of inhibitory substances. In total, 103 taxa were detected via metabarcoding. Arthropod remains delivered the highest number of taxa (n=67), followed by nest dust (n=29). Extractions with the PowerSoil kit outperformed DNeasy extractions coupled with PowerClean Pro inhibitor removal. The subsamples of the same nest showed 5.5% and 47.1% taxonomic overlap for arthropod remains and PowerSoil extracted nest dust, respectively, indicating a heterogeneous eDNA distribution in nests. Most detected species were either feeding in the nest, i.e., herbivorous / predatory, or bird food. We also detected molecular traces of 25 bird species, whose feathers were likely used as nest material. Consequently, the metabarcoding of bird nest materials provides a more complete picture of nest communities, which can enable future studies on functional diversity and better comparisons between nesting species.

ecology↗

bioRxiv2021

Evaluating 87Sr/86Sr isotope ratios and Sr mass fractions in otoliths of different European freshwater fish species as fishery management tool in an Alpine foreland with limited geological variability

The focus of this study was to assess the potential of otolith microchemistry as a fishery management tool for different European freshwater fish species in an Alpine foreland with a diverse range of different water bodies but low geological variation. 87Sr/86Sr isotope and Sr/Ca ratios in water samples from 26 habitat sites in a pre-alpine catchment region around lake Chiemsee, Germany, an important region for recreational and economic fisheries, were analysed. 87Sr/86Sr isotope ratios and the Sr mass fractions in otoliths of 246 fish out of 16 species were determined using (laser ablation) inductively coupled plasma mass spectrometry ((LA)-ICP-MS). Habitats could be discriminated into three distinct strontium isotope regions (SIGs) and seven clusters with characteristic 87Sr/86Sr isotope and Sr/Ca ratios. The direct comparison of 87Sr/86Sr isotope ratios in water and otolith samples allowed to identify fish that might have been a) migrating b) transferred from other water bodies or c) stocked from fish farms. Sr/Ca ratios in water and the Sr mass fraction in otoliths were highly correlated, although significant differences between species from the same environment could be documented. Sr mass fractions in sagittae of Perca fluviatilis were about 60 % of those in sagittae of Coregonus spp and of lapilli of roach Rutilus rutilus from the same habitats. Different partition factors for water to otolith Sr/Ca mass fractions were determined for different species. Discrimination of fish otoliths by 87Sr/86Sr isotope ratios and Sr mass fractions according to habitat clusters was possible with success rates ranging from 92 % to 100 % for cyprinids, European perch Perca fluviatilis, whitefish Coregonus spp. and European grayling Thymallus thymallus, and was 74 % for salmonids. Otolith microchemistry proved to have great potential to serve as a fishery management tool at smaller spatial scales such as in the studied Alpine foreland when considering the limited variation of 87Sr/86Sr isotope and Sr/Ca ratios, the type and spatial distribution of habitats, and the species and question under investigation. HighlightsO_LIOtolith microchemistry applied in in area with limited geological variability C_LIO_LIFish transferred, stocked or migrated were identified C_LIO_LIRegressions between Sr/Ca ratios in water predict Sr mass fractions in otoliths C_LIO_LISpecies specific Sr discrimination from water into otoliths C_LIO_LIEuropean freshwater fish species assigned to habitat clusters of origin C_LI

ecology↗

bioRxiv2021

Diet composition of reintroduced Red-and-Green Macaws (Ara chloropterus) reflects gradual adaption to life in the wild

Over the last two centuries, the Red-and-green Macaw (Ara chloropterus) has become locally extinct in Argentina. In an attempt to restore its key ecosystem functions as both disperser and regulator of large-seeded plants, a reintroduction project was initiated at the Ibera National Park in northeastern Argentina. The ability of released individuals to find food is crucial, in particular when working with captive-bred animals, as long-term establishment of a self-sustaining population depends on their short-term ability to exploit wild food sources. Monitoring of feeding habits is usually conducted through behavioral observation, but in recent years DNA metabarcoding has emerged as an alternative for obtaining highly resolved data on diet composition. In this study we use a combination of both techniques to characterize the breadth and composition of the reintroduced macaws diet. In addition, we evaluate the efficiency of both direct field observations and metabarcoding of feces as techniques to assess diet composition. Individuals fed on a variety of plant species (n = 49) belonging to a broad phylogenetic spectrum (28 families). Dietary richness estimated by direct observation and metabarcoding was similar, though smaller than the combination of the two datasets as both techniques detected at least 15 species not recorded by the other method. While the total number of detected species was higher for observational data, the rate of species-detection per sampling day was higher for metabarcoding. These results suggest that a combination of both methods is required in order to obtain the most accurate account of the total diversity of the diet of a species. The ability of the reintroduced macaws to successfully exploit local food resources throughout the year indicates a good level of adjustment to the release site, an important step towards the creation of a stable, self-sustaining population of Red-and-green Macaws in Northern Argentina. RESUMENEn el transcurso de los ultimos dos siglos, el Guacamayo Rojo (Ara chloropterus) se ha extinguido en la Argentina. Buscando recuperar su rol ecologico tanto de dispersor como de depredador de semillas de gran tamano, se comenzo un proyecto de reintroduccion de la especie en el Parque Nacional Ibera, en la region noreste del pais. La capacidad para encontrar alimento por parte de los individuos liberados es crucial, particularmente cuando se trabaja con animales provenientes de condiciones de cautiverio, ya que el establecimiento de una poblacion autosuficiente a largo plazo dependera de la habilidad de estos para explotar fuentes de alimento silvestre a corto plazo. El monitoreo de habitos alimenticios se realiza usualmente a traves de observaciones comportamentales. Sin embargo, en los ultimos anos la tecnica del meta-codigo de barras de ADN ha surgido como una alternativa para la obtencion de datos de composicion dietaria con alto nivel de resolucion. En este estudio, utilizamos una combinacion de ambas tecnicas para caracterizar la amplitud y composicion de la dieta de los guacamayos reintroducidos. A su vez, evaluamos la eficiencia de la observacion directa y el codigo de barras genetico de heces como tecnicas para evaluar la composicion de la dieta. Los individuos se alimentaron de una amplia variedad de especies (n = 49), abarcando un amplio espectro filogenetico (28 familias). La riqueza dietaria estimada por observacion directa y por meta-codigo de barras genetico fue similar, aunque menor a la resultante de la combinacion de todos los datos ya que ambas tecnicas detectaron al menos 15 especies no registradas por el otro metodo. Mientras que el numero total de especies detectadas fue mayor para los metodos observacionales, la tasa de deteccion de especies por dia de muestreo fue mayor para el analisis genetico. Estos resultados sugieren que una combinacion de ambos metodos es necesaria para obtener la descripcion mas precisa posible de la diversidad dietaria total de una especie. La capacidad de los guacamayos reintroducidos para explotar recursos alimenticios locales a lo largo del ano estaria indicando un buen nivel de adaptacion al sitio de liberacion, un paso muy importante hacia el establecimiento de una poblacion de Guacamayo Rojo estable y autosuficiente en el norte de Argentina. Palabras claveAra chloropterus, Conservacion, Dieta, Frugivoria, Meta-codigo de barras, Guacamayo Rojo, Reintroduccion, Ecologia trofica LAY SUMMARYO_LIThe Red-and-green Macaw reintroduction project aims to restore this species to Argentina, where it is locally extinct. To assess if reintroduced macaws are successfully adapting to life in the wild, we studied their foraging habits at the Ibera National Park. Their food consumption was observed visually, and their feces were analyzed with molecular methods for traces of the consumed plants. C_LIO_LIMacaws fed from a large diversity of food items, exhibiting a flexible diet which varied with fruit availability in different months. A combination of both methods was required to obtain the most accurate account of the total diversity of the diet of a species. C_LIO_LIThe reintroduced macaws were able to successfully locate and exploit food resources throughout the year, indicating a good level of adjustment to the release site. C_LI

ecology↗

bioRxiv2020

Activity, energy use and species identity affect eDNA shedding in freshwater fish

The quantitative measurement of environmental DNA (eDNA) from field-collected water samples is gaining importance for the monitoring of fish communities and populations. The interpretation of these signal strengths depends, among other factors, on the amount of target eDNA shed into the water. However, shedding rates are presumably associated with species-specific traits such as physiology and behavior. Although such differences between juvenile and adult fish have been previously detected, the general impact of movement and energy use in a resting state on eDNA release into the surrounding water remains hardly addressed. In an aquarium experiment, we compared eDNA shedding between seven fish species occurring in European freshwaters. The investigated salmonids, cyprinids, and sculpin exhibit distinct adaptions to microhabitats, diets, and either solitary or schooling behavior. The fish were housed in aquaria with constant water flow and their activity was measured by snapshots taken every 30 s. Water samples for eDNA analysis were taken every 3 h and energy use was determined in an intermittent flow respirometer. After controlling for the effect of fish mass, our results demonstrate a positive correlation between target eDNA quantities as measured with digital PCR, fish activity, and energy use, as well as species-specific differences. For cyprinids, the model based on data from individual fish was only partly transferable to groups, which exhibited lower activity and higher energy use. Our findings highlight the importance of fish physiology and behavior for the comparative interpretation of taxon-specific eDNA quantities. Species traits should therefore be incorporated into eDNA-based monitoring and conservation efforts.

ecology↗

bioRxiv2020

Endpoint PCR coupled with capillary electrophoresis (celPCR) provides sensitive and quantitative measures of environmental DNA in singleplex and multiplex reactions

The use of sensitive methods is key for the detection of target taxa, from trace amounts of environmental DNA (eDNA) in a sample. In this context, digital PCR (dPCR) enables direct quantification and is commonly perceived as more sensitive than endpoint PCR. However, endpoint PCR coupled with capillary electrophoresis (celPCR) potentially embodies a viable alternative as it quantitatively measures signal strength in Relative Fluorescence Units (RFU). Provided comparable levels of sensitivity are reached, celPCR permits the development of cost-efficient multiplex PCRs, enabling the simultaneous detection of several target taxa. Here, we compared the sensitivity of singleplex and multiplex celPCR to dPCR for species-specific primer pairs amplifying mitochondrial DNA (COI) of fish species occurring in European freshwaters by analysing dilution series of DNA extracts and field-collected water samples. Both singleplex and multiplex celPCR and dPCR displayed comparable sensitivity with reliable positive amplifications starting at two to 10 target DNA copies per {micro}l DNA extract. celPCR was suitable for quantifying target DNA and direct inference of DNA concentrations from RFU was possible after accounting for primer effects. Furthermore, multiplex celPCRs and dPCRs were successfully used for the detection and quantification of fish-eDNA in field-collected water samples, confirming the results of the dilution series experiment and exemplifying the high sensitivity of the two approaches. The possibility of detection and quantification via multiplex celPCR is appealing for the cost-efficient screening of high sample numbers. The present results confirm the sensitivity of this approach thus enabling its application for future eDNA-based monitoring efforts.

molecular biology↗

bioRxiv2020

Metabarcoding, direct stomach observation and stable isotope analysis reveal a highly diverse diet for the invasive green crab in Atlantic Patagonia

The European green crab Carcinus maenas and its sister species C. aestuarii are highly invasive species causing damage to coastal ecosystems and contributing to severe economic losses worldwide. C. maenas was first detected at the Atlantic Patagonian coast in 2001. In this work, we studied the diet of the green crab in a recently invaded location in Golfo Nuevo, using three complementary techniques: direct stomach observation, metabarcoding of gut content and stable isotope analysis. Direct stomach observation and metabarcoding showed that green crabs have a broad omnivorous diet, ingesting most of the phyla present in the study area. Gut content metabarcoding allowed a detailed description of algal diversity and revealed other taxa that went unnoticed in the visual stomach analysis. Stable isotope analysis showed that the major contribution to the crabs diet was from the phytoplankton chain (by bivalve consumption) and not directly from algae. This study approach combining three complementary techniques also allowed us to detect some differences in the diet between sexes, which suggests that male and female crabs are not as ecologically equivalent as previously thought. Besides, we detected sequences corresponding to C. aestuarii suggesting that the green crab Patagonian population is a hybrid of both sister species. These findings are key to understanding the impacts green crabs can have on the local ecosystem.

ecology↗

bioRxiv2020

Lateral and longitudinal fish eDNA distribution in dynamic riverine habitats

Assessing the status and distribution of fish populations in rivers is essential for management and conservation efforts in these dynamic habitats. Currently, methods detecting environmental DNA (eDNA) are being established as an alternative and/or complementary approach to the traditional monitoring of fish species. In lotic systems, a sound understanding of hydrological conditions and their influence on the local target detection probability and DNA quantity is key for the interpretation of eDNA-based results. However, the effect of seasonal and diurnal changes in discharge and the comparability of semi-quantitative results between species remain hardly addressed. We conducted a cage experiment with four fish species (three salmonid and one cyprinid species) in a glacier-fed, fish-free river in Tyrol (Austria) during summer, fall, and winter discharge situations (i.e. 25-fold increase from winter to summer). Each season, water samples were obtained on three consecutive days at 13 locations downstream of the cages including lateral sampling every 1-2 m across the wetted width. Fish eDNA was quantified by species-specific endpoint PCR followed by capillary electrophoresis. Close to the cages, lateral eDNA distribution was heterogenous and mirrored cage placement within the stream. In addition to the diluting effect of increased discharge, longitudinal signal changes within the first 20 m were weakest at high discharge. For downstream locations with laterally homogenous eDNA distribution, the signals decreased significantly with increasing distance and discharge. Generally, the eDNA of the larger-bodied salmonid species was less frequently detected, and signal strengths were lower compared to the cyprinid species. This study exemplifies the importance of hydrological conditions for the interpretation of eDNA-based data across seasons. To control for heterogenous eDNA distribution and enable comparisons over time, sampling schemes in lotic habitats need to incorporate hydrological conditions and species traits.

ecology↗

bioRxiv2020

A validation scale to determine the readiness of environmental DNA assays for routine species monitoring

The use of environmental DNA (eDNA) analysis for species monitoring requires rigorous validation - from field sampling to the analysis of PCR-based results - for meaningful application and interpretation. Assays targeting eDNA released by individual species are typically validated with no predefined criteria to answer specific research questions in one ecosystem. Hence, the general applicability of assays as well as associated uncertainties and limitations, often remain undetermined. The absence of clear guidelines for assay validation prevents targeted eDNA assays from being incorporated into species monitoring and policy; thus, their establishment is essential for realizing the potential of eDNA-based surveys. We describe the measures and tests necessary for successful validation of targeted eDNA assays and the associated pitfalls to form the basis of guidelines. A list of 122 variables was compiled, consolidated into 14 thematic blocks, (e.g. "in silico analysis"), and arranged on a 5-level validation scale from "incomplete" to "operational" with defined minimum validation criteria for each level. These variables were evaluated for 546 published single-species assays. The resulting dataset was used to provide an overview of current validation practices and test the applicability of the validation scale for future assay rating. Of the 122 variables, 20% to 76% were reported; the majority (30%) of investigated assays were classified as Level 1 (incomplete), and 15% did not achieve this first level. These assays were characterised by minimal in silico and in vitro testing, but their share in annually published eDNA assays has declined since 2014. The meta-analysis demonstrates the suitability of the 5-level validation scale for assessing targeted eDNA assays. It is a user-friendly tool to evaluate previously published assays for future research and routine monitoring, while also enabling the appropriate interpretation of results. Finally, it provides guidance on validation and reporting standards for newly developed assays.

molecular biology↗