Development of workflows for metabarcoding of mass-samples A case study on Diptera Dissertation Vorgelegt von: Jan-Frederic Struwe aus Meschede Bonn, August 2018 Zur Erlangung des Doktorgrades (Dr. rer. Nat.) der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn 1 Angefertigt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn. Die Dissertation wurde am Zoologischen Forschungsmuseum Alexander Koenig (ZFMK) in Bonn durchgeführt. Erstgutachter: Prof. Dr. Johann Wolfgang Wägele Zweitgutachter: Prof. Dr. Thomas Bartolomaeus Kommissionsmitglied (fachnah): Prof. Dr. Bernhard Misof Kommissionsmitglied (fachfremd): apl. Prof. Dr. Ullrich Wüllner Tag der Promotion: 25.06.2019 Erscheinungsjahr: 2020 2 Publication: Searching for the Optimal Sampling Solution; PLOS ONE Gossner MM, Struwe J-F, Sturm S, Max S, McCutcheon M, Weisser WW, Zytynska SE (2016) Searching for the Optimal Sampling Solution: Variation in Invertebrate Communities, Sample Condition and DNA Quality. PLoS ONE 11(2): e0148247. https://doi.org/10.1371/journal.pone.0148247 3 „Krautsalat? “ - Felice Kremer - 4 Content Publication: Searching for the Optimal Sampling Solution; PLOS ONE ....................................................... 3 1 Introduction ...................................................................................................................................................................... 1 1.1 Background .............................................................................................................................................................. 1 1.2 Diptera and their relevance in ecosystems ................................................................................................. 2 1.2.1 The diversity and influence of Diptera ................................................................................................. 2 1.3.2 The challenges ................................................................................................................................................ 4 1.2.2 The benefits of monitoring Diptera ....................................................................................................... 7 1.3 DNA barcoding for biodiversity assessments ............................................................................................ 8 1.3.1 The concept ...................................................................................................................................................... 8 1.4 Hybridisation based target enrichment ....................................................................................................... 9 1.4.1 The exclusive selection of a genomic region ...................................................................................... 9 1.4.2 Designing specific oligonucleotide probes ...................................................................................... 10 1.5 The necessity to build up a sequence reference data pool ................................................................ 11 1.6 The advantage of automated sampling ...................................................................................................... 11 1.7 The Biodiversity Exploratories ..................................................................................................................... 12 1.8 Outlining the project goals .............................................................................................................................. 13 2 A Diptera sequence reference list and COI discriminability ...................................................................... 15 2.1 Introduction .......................................................................................................................................................... 15 2.2 Material and Methods ....................................................................................................................................... 15 2.2.1 Material acquisition and processing ................................................................................................... 15 2.2.2 Testing universality and discriminative properties of COI ....................................................... 17 2.3 Results ..................................................................................................................................................................... 20 2.3.1 Material acquisition and library growth ........................................................................................... 20 2.3.2 The universality and discriminative properties of COI .............................................................. 21 2.4 Discussion .............................................................................................................................................................. 25 2.4.1 Building up a sequence reference list for German Diptera ....................................................... 25 2.4.2 Universal application of COI .................................................................................................................. 25 2.4.3 The capability to discriminate species .............................................................................................. 26 2.4.4 Conclusion ..................................................................................................................................................... 30 3. Test of conservation liquids for traps ................................................................................................................ 31 3.1 Introduction .......................................................................................................................................................... 31 3.2 Material and Methods ....................................................................................................................................... 31 3.2.1 Experimental setup ................................................................................................................................... 31 3.2.2 Arthropod identification and classification ..................................................................................... 32 3.2.3 Measure of quality for morphological species determination ................................................. 32 3.2.4 Species identification through DNA barcoding .............................................................................. 32 3.3 Results ..................................................................................................................................................................... 35 3.3.1 Condition of the samples ......................................................................................................................... 36 3.3.2 Order level ..................................................................................................................................................... 37 3.3.3 Species level analyses ............................................................................................................................... 38 3.4 Discussion .............................................................................................................................................................. 41 3.4.1 The usage of different conservation liquids for traps ................................................................. 41 3.4.2 Conclusion and recommendations ...................................................................................................... 44 4 Mixed species DNA-samples ................................................................................................................................... 46 4.1 Introduction .......................................................................................................................................................... 46 4.2 Material and Methods ....................................................................................................................................... 46 4.2.1 Material acquisition and sample preparation ................................................................................ 46 4.2.2 Next Generation Sequencing ................................................................................................................. 49 4.2.3 Data processing ........................................................................................................................................... 50 4.2.4 The influence of sample treatment, species number and species quantity ....................... 51 4.3 Results ..................................................................................................................................................................... 51 4.3.1 Differences in the chimera identification ......................................................................................... 52 4.3.2 Identification of species ........................................................................................................................... 54 4.3.3 Analyzing DNA input ratios .................................................................................................................... 55 4.4 Discussion .............................................................................................................................................................