Bettina Thalinger

Rapid Plant Identification Using Species- and Group-Specific Primers Targeting Chloroplast DNA

Plant identification is challenging when no morphologically assignable parts are available. There is a lack of broadly applicable methods for identifying plants in this situation, for example when roots grow in mixture and for decayed or semi-digested plant material. These difficulties have also impeded the progress made in ecological disciplines such as soil- and trophic ecology. Here, a PCR-based approach is presented which allows identifying a variety of plant taxa commonly occurring in Central European agricultural land. Based on the trnT-F cpDNA region, PCR assays were developed to identify two plant families (Poaceae and Apiaceae), the genera Trifolium and Plantago, and nine plant species: Achillea millefolium, Fagopyrum esculentum, Lolium perenne, Lupinus angustifolius, Phaseolus coccineus, Sinapis alba, Taraxacum officinale, Triticum aestivum, and Zea mays. These assays allowed identification of plants based on size-specific amplicons ranging from 116 bp to 381 bp. Their specificity and sensitivity was consistently high, enabling the detection of small amounts of plant DNA, for example, in decaying plant material and in the intestine or faeces of herbivores. To increase the efficacy of identifying plant species from large number of samples, specific primers were combined in multiplex PCRs, allowing screening for multiple species within a single reaction. The molecular assays outlined here will be applicable manifold, such as for root- and leaf litter identification, botanical trace evidence, and the analysis of herbivory.

Molecular prey identification in Central European piscivores

Diet analysis is an important aspect when investigating the ecology of fish‐eating animals and essential for assessing their functional role in food webs across aquatic and terrestrial ecosystems. The identification of fish remains in dietary samples, however, can be time‐consuming and unsatisfying using conventional morphological analysis of prey remains. Here, we present a two‐step multiplex PCR system, comprised of six assays, allowing for rapid, sensitive and specific detection of fish DNA in dietary samples. This approach encompasses 78 fish and lamprey species native to Central European freshwaters and enables the identification of 31 species, six genera, two families, two orders and two fish family clusters. All targeted taxa were successfully amplified from 25 template molecules, and each assay was specific when tested against a wide range of invertebrates and vertebrates inhabiting aquatic environments. The applicability of the multiplex PCR system was evaluated in a feeding trial, wherein it outperformed morphological prey analysis regarding species‐specific prey identification in faeces of Eurasian otters. Additionally, a wide spectrum of fish species was detected in field‐collected faecal samples and regurgitated pellets of Common Kingfishers and Great Cormorants, demonstrating the broad applicability of the approach. In conclusion, this multiplex PCR system provides an efficient, easy to use and cost‐effective tool for assessing the trophic ecology of piscivores in Central Europe. Furthermore, the multiplex PCRs and the primers described therein will be applicable wherever DNA of the targeted fish species needs to be detected at high sensitivity and specificity.

Maximizing dietary information retrievable from carcasses of Great Cormorants Phalacrocorax carbo using a combined morphological and molecular analytical approach.

Avian carcasses can provide important information on the trophic ecology of birds. Usually, the number of carcasses available for examination is limited and therefore it is important to gain as much dietary information per specimen as possible. In piscivorous birds and raptors, the stomach has been the primary source of dietary information, whereas the gut (intestine) has so far been neglected as it usually contains only a few morphologically identifiable hard parts of prey. Molecular approaches have the potential to retrieve dietary information from the gut, although this has not yet been verified. As well as identifying the prey, it is important to estimate any secondary predation to avoid food web errors in dietary analyses. The assignment of accidentally consumed prey is notoriously difficult regardless of the prey identification approach used. In the present study, morphological and molecular analyses were, for the first time, combined to maximize the dietary information retrievable from the complete digestive tract of Great Cormorants Phalacrocorax carbo sinensis. Moreover, a novel approach based on predator–prey size ratios was applied to these piscivorous birds to minimize the number of samples that might contain secondarily predated prey. The stomach contents of the examined birds were found to provide the most dietary information when morphological and molecular analyses were used in combination. However, compared with the morphological approach, the molecular analysis increased the number of fish species detected by 39%. The molecular approach also permitted the identification of fish DNA in the Cormorant guts. Predator–prey size ratios derived from morphological analysis of fish hard parts can reduce the incidence of potential confounding influence of secondarily predated prey by 80%. Our findings demonstrate that a combination of morphological and molecular approaches maximizes the trophic information retrievable from bird carcasses.

Diet analysis in piscivorous birds: What can the addition of molecular tools offer?

Abstract In trophic studies on piscivorous birds, it is vital to know which kind of dietary sample provides the information of interest and how the prey can be identified reliably and efficiently. Often, noninvasively obtained dietary samples such as regurgitated pellets, feces, and regurgitated fish samples are the preferred source of information. Fish prey has usually been identified via morphological analysis of undigested hard parts, but molecular approaches are being increasingly used for this purpose. What remains unknown, however, is which dietary sample type is best suited for molecular diet analysis and how the molecular results compare to those obtained by morphological analysis. Pellets, feces, and regurgitated fish samples of Great Cormorants (Phalacrocorax carbo sinensis) were examined for prey using both morphological hard part analysis and molecular prey identification. The sample types and methods were compared regarding number of species detected (overall and per sample) as well as the prey species composition and its variability among individual samples. Via molecular analysis, significantly higher numbers of prey species were detected in pellets, feces, and fish samples. Of the three sample types, pellets contained the most comprehensive trophic information and could be obtained with the lowest sampling effort. Contrastingly, dietary information obtained from feces was least informative and most variable. For all sample types, the molecular approach outperformed morphological hard part identification regarding the detectable prey spectrum and prey species composition. We recommend the use of pellets in combination with molecular prey identification to study the diet of piscivorous birds.

The influence of meal size on prey DNA detectability in piscivorous birds

Molecular methods allow noninvasive assessment of vertebrate predator–prey systems at high taxonomic resolution by examining dietary samples such as faeces and pellets. To facilitate the interpretation of field‐derived data, feeding trials, investigating the impacts of biological, methodological and environmental factors on prey DNA detection, have been conducted. The effect of meal size, however, has not yet been explicitly considered for vertebrate consumers. Moreover, different noninvasively obtained sample types remain to be compared in such experiments. Here, we present a feeding trial on abundant piscivorous birds, Great Cormorants (Phalacrocorax carbo), to assess meal size effects on postfeeding prey DNA detection success. Faeces and pellets were sampled twice a day after the feed of large (350–540 g), medium (190–345 g) and small (15–170 g) fish meals contributing either a large (>79%) or small (<38%) share to the daily consumption. Samples were examined for prey DNA and fish hard parts. Molecular analysis of faeces revealed that both large meal size and share had a significantly positive effect on prey DNA detection rate postfeeding. Furthermore, large meals were detectable for a significantly longer time span with a detection limit at ~76 hr and a 50% detection probability at ~32 hr postfeeding. In pellets, molecular methods reliably identified the meal consumed the previous day, which was not possible via morphological analysis or when examining individual faeces. The less reliable prey DNA detection of small meals or meal shares in faeces signifies the importance of large numbers of dietary samples to obtain reliable trophic data.

Quantifizierung von Fischbeständen mittels eDNA in alpinen Fließgewässern

Umwelt-DNA (eDNA) erlaubt das Vorkommen von Fischarten in Gewässern hochsensitiv und schnell zu bestimmen. Inwiefern sich dieser Ansatz jedoch für eine Quanti zierung von Fischbeständen, insbesondere für alpine Fließgewässer, eignet, ist noch weitgehend unbekannt. Der Vergleich zwischen klassischer Bestandserhebung mittels Elektrobe schung und eDNA-Analysen in zwei alpinen Fließgewässern zeigt eine gute Korrelation zwischen Fischbestand sowie eDNA-Signalstärke und lässt das Potenzial von eDNA zur Quanti zierung von Fischbeständen erkennen.

Evaluation of an automated protocol for efficient and reliable DNA extraction of dietary samples

Abstract Molecular techniques have become an important tool to empirically assess feeding interactions. The increased usage of next‐generation sequencing approaches has stressed the need of fast DNA extraction that does not compromise DNA quality. Dietary samples here pose a particular challenge, as these demand high‐quality DNA extraction procedures for obtaining the minute quantities of short‐fragmented food DNA. Automatic high‐throughput procedures significantly decrease time and costs and allow for standardization of extracting total DNA. However, these approaches have not yet been evaluated for dietary samples. We tested the efficiency of an automatic DNA extraction platform and a traditional CTAB protocol, employing a variety of dietary samples including invertebrate whole‐body extracts as well as invertebrate and vertebrate gut content samples and feces. Extraction efficacy was quantified using the proportions of successful PCR amplifications of both total and prey DNA, and cost was estimated in terms of time and material expense. For extraction of total DNA, the automated platform performed better for both invertebrate and vertebrate samples. This was also true for prey detection in vertebrate samples. For the dietary analysis in invertebrates, there is still room for improvement when using the high‐throughput system for optimal DNA yields. Overall, the automated DNA extraction system turned out as a promising alternative to labor‐intensive, low‐throughput manual extraction methods such as CTAB. It is opening up the opportunity for an extensive use of this cost‐efficient and innovative methodology at low contamination risk also in trophic ecology.