Last Saturday, we went to the rivers Ruhr and Möhne to take eDNA samples. In the coming months, Florian and Jan will investigate how eDNA can help to assess the species community in these two rivers.
Last year we used a freshwater invertebrate mock community to test the standard COI barcoding Folmer primers in a metabarcoding setting and demonstrated that primer bias makes estimation of biomass impossible. Together with our colleagues from France we now tested the same mock samples with a ribosomal marker, which is supposed to show less primer bias due to more conserved primer binding sites. As we show in the new article which was just published in PeerJ, the 16S marker indeed leads to more consistent amplification results than the Folmer primer set when used in DNA metabarcoding.
However, primer bias is still substantial (two orders of magnitude) and thus estimation of biomass still impossible. Furthermore, we are currently lacking comprehensive 16S reference databases for animals, which are available for the COI barcoding marker. First tests show that the COI marker performance can be greatly improved by using metabarcoding optimised primers (we will publish a PrePrint soon). Thus, while the 16S marker might have potential, we focus on using the COI maker for DNA metabarcoding of insects but just not with Folmer primers.
Figure: The tested 16S marker shows less variation in read numbers than the COI Folmer maker, thus allows for better detection of freshwater taxa. However, with improved COI primers better detection rates are possible as well, as first tests demonstrated.
The first step for DNA metabarcoding of invertebrate kick samples from stream ecosystems is sample homogenisation. The samples often contain hundreds of organisms which have to be dried and then homogenised, to assure that the DNA of all specimens in mixed. Only then every individual in the sample can be detected, extracting just a small subset of the ground tissue. We are using the IKA ULTRA-TURRAX Tube Drive control system for tissue grinding, as discussed in a previous video. In this new video we are detailing the complete process from sample handling, drying over night and grinding using invertebrate kick samples from Finland.
We just published a short article in the Barcode Bulletin.
In the march issue (pages 10-12), Florian and Jan write about how different cryptic Deleatidium mayfly species from New Zealand show strongly different responses to anthropogenic stressors in the stream and the cosequences this has for ecosystem assessments.
We are starting a new project:
Gunnar Jacobs (who is also working for Emschergenossenschaft, the local water board) starts his PhD thesis. He will investigate the occurrence, distribution and dispersal ability of the “Emscher sculpin” (Cottus cf. rhenanus) in the river Emscher and its tributaries.
The Emscher sculpin is a fish which survived the major industrial pollution of rivers in the Emscher catchment during the last 100 years in the upper reaches of a few streams. Specimens from these relic populations are now used to repopulate ecologically restored streams and rivers in the region.
Gunnar will use eDNA (environmental DNA) to study the occurrence and distribution of the Emscher sculpin and how fast it can disperse and repopulate streams after the reintroduction. Using eDNA means that that Gunnar will filter water from the stream and analyze the sculpin DNA in it, which also means that he does not need to catch the fishes. Gunnar’s work will greatly help to understand the Emscher sculpin and the Emscher ecosystem and to subsequently make protection of the ecosystem easier.
Welcome, Gunnar! We are looking forward to the exciting project!
Together with our colleges from France we just have published our first PrePrint, where we test the potential of the 16S marker for DNA metabarcoding. We were able to show that freshwater insects are more consistently amplified with the developed 16S primer than with standard Folmer COI barcoding primer. While the taxonomic resolution of the 16S marker remains unknown for insects, the ribosomal marker might be a viable alternative metabarcoding marker when local reference databases can be easily established. For now we will however continue using the COI marker, due to availability of good reference databases and optimised freshwater COI markers which show similar performance.
Together with colleagues from the University of Tehran, we just published a paper on the diversity of amphipods in the highly endangered Irano-Anatolian and Caucasus biodiversity hotspots. So far, only five morphospecies had been known from the streams and rivers of these regions. By using molecular techniques, we could identify 42 mostly unknown species, showing that also the scarcely studied freshwater ecosystems of the region harbour a great biodiversity and deserve protection.
Florian was recently interviewed by Radio Leonardo. He talked about how new molecular tools can help to revolutionize and improve ecological water quality assessment of streams and rivers.
Link to the interview (podcast 25.02.2015, Florian’s part starts at minute 25):