Emre Keskin

Recently, biological invasions are considered to be the most serious threats to the biodiversity, which has already been adversely affected by anthropogenic activities. Molecular tools become the most applied method in early warning, species level identification and management of invasive species, and many official authorities and environmental organizations, especially countries from North America, Europe, Australia and Far East started to use these molecular tools as standard procedures. However, Turkey seems to be not integrated with these molecular tools used in management of invasive species.

The use of DNA-based methods for detection of invasive species from environmental samples (e.g. plankton tows, benthic sediment cores, ballast water samples, etc.) is latest innovation and gaining widespread acceptance rapidly. The latest enhance in these methods is the environmental DNA (eDNA) approach and this term dates back to 1987 and used to define a method for extracting microbial DNA from sediments, however the applicability progress on other subjects emerged at the beginning of the 2000s. Environmental DNA was used to define DNA that can be isolated from environmental samples (like soil, water, air), without necessity to target organisms. It is characterized by a complex mixture of genomic DNA from many different organisms and found degraded. Total eDNA includes cellular DNA from living cells and organisms, extracellular DNA from natural cell death and destruction of cell structure. Identifying many taxon at once using complex and degraded DNA with markers targeting nucleotide sequences shorter than DNA barcodes is considered to be the latest scientific innovation on this subject and defined as “eDNA metabarcoding”.

In this study, we aimed to identify invasive species Carassius gibelio, Clarias gariepinus, Orechromis niloticus and Pseudorasbora parva from Upper Sakarya Basin using the most advanced method in the world, eDNA metabarcoding, and revealing their population structures. This method will be used for the first time in Turkey and will provide us important data on evaluation of eDNA metabarcoding approach in early warning and management of invasive species in Turkish freshwaters.

Proposed project includes organizing a standard sampling protocol for eDNA studies; designing specific primers for target species; identifying known and possible unknown invasive species at species level using specific primers together with universal fish primers and determining parameters effecting quality and quantity of eDNA by evaluating the molecular data gathered from results of this study. In this context, two different sampling protocols will be used to collect water samples from 10 different stations selected on Upper Sakarya Basin, at 4 different seasons with 3 repetition (excluding negative and positive controls); eDNA’s will be isolated from these samples; mini DNA barcode regions of the target invasive species will be amplified with PCR; also an alternative mitochondrial region (cytochrome b) will be amplified using universal fish primers and DNA sequencing will be conducted. Then nucleotide sequences will be compared to reference sequences for a species level identification and will be evaluated statistically to determine the significance of the results.

On the grounds that, this will be the first eDNA study of Turkey and one of the few on international basis, our project has an original value fundamentally. Also it is designed as the first eDNA metabarcoding study in terms of study area and target species. The project intend to standardize field protocol of sampling and storage of eDNA samples, points to consider in choosing sampling stations, effects of seasonal factors, sampling repetitions, designing positive and negative controls, optimizing transportation of water samples from field to laboratory and laboratory storage conditions and sharing these knowledge in COST Action to standardize an international protocol which is not published yet. In the end, results will be evaluated and following the approval of the approaches’’ validity, management solutions regarding to invasive freshwater species in Upper Sakarya Basin will be proposed accordantly with Turkeys’ management strategy. Besides, results of the study will be crucial as it will give an updated information about latest population structure of target species in the Upper Sakarya Basin, especially Pseudorasbora parva which is lacking published data.

Scientific and Technological Research Council Of Turkey (TUBİTAK)

2014-2017