Metabarcoding of PalEnDNA as An Efficient Tool to Recover Ancient Bacterial Diversity

Past bacterial diversity of a paleosol was reconstructed using metabarcoding of paleo environmental DNA (PalEnDNA). The paleosol was subsampled from a sediment core which was excavated from a palaeo beach-ridge located 2.6?km away from present sea shore and identified that it was deposited under marine influence ～6000?years ago, using geological proxies. The bacterial community contained 37 bacterial phyla and dominated by Proteobacteria, followed by Bacteroidetes, Firmicutes, Actinobacteria, Verrucomicrobia, and Chloroflexi. The bacterial community was a mix-up of marine and terrestrial population, and thereby diversity was higher than marine populations. The result shows metabarcoding of PalEnDNA can effectively reconstruct past bacterial community structure.     

Detecting invertebrate ecosystem service providers in orchards: traditional methods versus barcoding of environmental DNA in soil

1. The objective of this study was to assess barcoding of environmental DNA as a method for monitoring invertebrate ecosystem service providers in soil samples.
2. We selected 26 invertebrate ecosystem service providers that occur in New Zealand kiwifruit or apple orchards and produced mitochondrial cytochrome c oxidase gene subunit I (cytochrome oxidase I) and/or 28S ribosomal DNA sequences for each. Specific barcode primers were designed for each invertebrate ecosystem service provider and tested, along with generic barcoding cytochrome oxidase I primers, for their ability to detect DNA from invertebrate ecosystem service providers that had been added to sterilized and unsterilized soil samples.
3. Although the specific primers accurately detected the invertebrate ecosystem service providers in more than 96% of the samples, the generic cytochrome oxidase I primers detected only 37% of the invertebrate ecosystem service providers added to the sterilized samples and 2.5% in the unsterilized samples.
4. In a field test, we compared metabarcoding with traditional invertebrate trapping methods to detect the invertebrate ecosystem service providers in 10 kiwifruit and 10 apple orchards. All invertebrate ecosystem service providers were collected in traps in at least one orchard, but very few were identified by metabarcoding of soil environmental DNA.
5. Although the specific primers can be used as a tool for monitoring invertebrate ecosystem service providers in soil samples, methodological improvements are needed before metabarcoding of soil environmental DNA can be used to monitor these taxa.

     

Negative effects of urbanisation on diurnal and nocturnal pollen-transport networks

Pollinating insects are declining due to habitat loss and climate change, and cities with limited habitat and floral resources may be particularly vulnerable. The effects of urban landscapes on pollination networks remain poorly understood, and comparative studies of taxa with divergent niches are lacking. Here, for the first time, we simultaneously compare nocturnal moth and diurnal bee pollen-transport networks using DNA metabarcoding and ask how pollination networks are affected by increasing urbanisation. Bees and moths exhibited substantial divergence in the communities of plants they interact with. Increasing urbanisation had comparable negative effects on pollen-transport networks of both taxa, with significant declines in pollen species richness. We show that moths are an important, but overlooked, component of urban pollen-transport networks for wild flowering plants, horticultural crops, and trees. Our findings highlight the need to include both bee and non-bee taxa when assessing the status of critical plant-insect interactions in urbanised landscapes.     

When to use next generation sequencing or diagnostic PCR in diet analyses

Next‐generation sequencing (NGS) is increasingly used for diet analyses; however, it may not always describe diet samples well. A reason for this is that diet samples contain mixtures of food DNA in different amounts as well as consumer DNA which can reduce the food DNA characterized. Because of this, detections will depend on the relative amount and identity of each type of DNA. For such samples, diagnostic PCR will most likely give more reliable results, as detection probability is only marginally dependent on other copresent DNA. We investigated the reliability of each method to test (a) whether predatory beetle regurgitates, supposed to be low in consumer DNA, allow to retrieve prey sequences using general barcoding primers that co‐amplify the consumer DNA, and (b) to assess the sequencing depth or replication needed for NGS and diagnostic PCR to give stable results. When consumer DNA is co‐amplified, NGS is better suited to discover the range of possible prey, than for comparing co‐occurrences of diet species between samples, as retested samples were repeatedly different in prey detections with this approach. This shows that samples were incompletely described, as prey detected by diagnostic PCR frequently were missed by NGS. As the sequencing depth needed to reliably describe the diet in such samples becomes very high, the cost‐efficiency and reliability of diagnostic PCR make diagnostic PCR better suited for testing large sample‐sets. Especially if the targeted prey taxa are thought to be of ecological importance, as diagnostic PCR gave more nested and consistent results in repeated testing of the same sample.     

Seasonal dynamics of riverine fish communities using eDNA

As fish communities are a major concern in rivers ecosystems, we investigated if their environmental (e)DNA signals vary according to the sampling period or hydromorphological conditions. Three rivers were studied over a year using eDNA metabarcoding approach. The majority of the species (c. 80%) were detected all year round in two rivers having similar hydromorphological conditions, whereas in the river affected by an upstream lake waterflow, more species were detected sporadically (42%). For all the rivers, in more than 98% of the occasional detections, the reads abundance represented <0.4% of the total reads per site and per sampling session. Even if the majority of the fish communities remained similar over the year for each of the three rivers, specific seasonal patterns were observed. We studied if the waterflow or the reproduction period had an effect on the observed dynamics. Waterflow, which influences eDNA downstream transportation, had a global influence in taxonomic richness, while the fishes' reproductive period had only an influence on certain species. Our results may help selecting the best sampling strategy according to research objectives. To study fish communities at local scale, seasons of low waterflow periods are recommended. This particularly helps to restraint effects of external eDNA coming from connections with other aquatic environment (tributaries, lakes, wetlands, sewage effluents, etc.). To obtain a more integrative overview of the fish community living in a river basin, high waterflow or breeding seasons are preferable for enhancing species detection probability, especially for rare species.     

The predator problem and PCR primers in molecular dietary analysis: Swamped or silenced; depth or breadth?

Dietary metabarcoding has vastly improved our ability to analyse the diets of animals, but it is hampered by a plethora of technical limitations including potentially reduced data output due to the disproportionate amplification of the DNA of the focal predator, here termed “the predator problem”. We review the various methods commonly used to overcome this problem, from deeper sequencing to exclusion of predator DNA during PCR, and how they may interfere with increasingly common multipredator-taxon studies. We suggest that multiprimer approaches with an emphasis on achieving both depth and breadth of prey detections may overcome the issue to some extent, although multitaxon studies require further consideration, as highlighted by an empirical example. We also review several alternative methods for reducing the prevalence of predator DNA that are conceptually promising but require additional empirical examination. The predator problem is a key constraint on molecular dietary analyses but, through this synthesis, we hope to guide researchers in overcoming this in an effective and pragmatic way.     

Diet preferences based on sequence read count: The role of species interaction in tissue bias correction

High-throughput sequencing and metabarcoding techniques provide a unique opportunity to study predator–prey relationships. However, in animal dietary preference studies, how to properly correct tissue bias within the sequence read count and the role of interactions between co-occurring species in metabarcoding mixtures remain largely unknown. In this study, we propose two categories of tissue bias correction indices: sequence read count number per unit tissue (SCN) and its ratio form (SCN ratio). By constructing plant mock communities with different numbers of co-occurring species in metabarcoding mixtures and conducting feeding trails on captive sika deer (Cervus nippon), we demonstrate the features of the SCN and SCN ratio, evaluate their correction effects and assess the role of species interactions during tissue bias correction. Tissue differences between species are defined as the differential ability to generate sequence counts. Our study suggests that pure tissue differences among species without a species interaction is not an optimal correction index for many biomes with limited tissue differences among species. Species interactions in mixtures may amplify tissue differences, which is beneficial for tissue bias correction. However, caution must be taken because varied species interactions among communities may increase the risk of worse correction. Correction effects based on the SCN and SCN ratio are comparable, but the SCN is less influenced by control species than the SCN ratio. Based on our study, several suggestions are provided for future animal diet studies or other high-throughput sequencing studies containing tissue bias.     

Deciphering the diet of a wandering spider (Phoneutria boliviensis; Araneae: Ctenidae) by DNA metabarcoding of gut contents

Arachnids are the most abundant land predators. Despite the importance of their functional roles as predators and the necessity to understand their diet for conservation, the trophic ecology of many arachnid species has not been sufficiently studied. In the case of the wandering spider, Phoneutria boliviensis F. O. Pickard‐Cambridge, 1897, only field and laboratory observational studies on their diet exist. By using a DNA metabarcoding approach, we compared the prey found in the gut content of males and females from three distant Colombian populations of P. boliviensis. By DNA metabarcoding of the cytochrome c oxidase subunit I (COI), we detected and identified 234 prey items (individual captured by the spider) belonging to 96 operational taxonomic units (OTUs), as prey for this wandering predator. Our results broaden the known diet of P. boliviensis with at least 75 prey taxa not previously registered in fieldwork or laboratory experimental trials. These results suggest that P. boliviensis feeds predominantly on invertebrates (Diptera, Lepidoptera, Coleoptera, and Orthoptera) and opportunistically on small squamates. Intersex and interpopulation differences were also observed. Assuming that prey preference does not vary between populations, these differences are likely associated with a higher local prey availability. Finally, we suggest that DNA metabarcoding can be used for evaluating subtle differences in the diet of distinct populations of P. boliviensis, particularly when predation records in the field cannot be established or quantified using direct observation.