NEWSLETTER December 2019

Editorial At the inspiring iBOL congress In this issue: organized by Torbjørn Ekrem in by Agnès Bouchez Trondheim (Norway) last June more than 30 participants from · Editorial DNAqua-Net gathered, many of them presenting talks and posters · Announcements in the various sessions, including 5 young scientists funded with ITC · Working groups Conference Grants. It’s stimulating and great to see young scientists · General reports from ITCs taking a more and · STSMs more active roles in all DNAq- ua-Net related activities! · ITC grants The current Grant Period 4 constitutes the “integration phase”. It’s fascinating how DNAqua-Net It is characterized by a particular shift, with methods leaving the www.dnaqua.net has now reached its full maturity, resulting in so many outcomes. labs where they have been devel- About 50 short research ex- oped, and being tested in real life, changes (STSMs) already took involving more and more stake- place leading to fruitful and in- holders: teresting results with many papers New programs inspired by already published. During the DNAqua-Net have been launched, recent training schools in Bucha- testing the application of molecu- rest, Sweden and France more lar methods for biomonitoring at than 72 trainees from 25 countries a much larger scale: INTERREG participated to build capacities in programs (SYNAQUA, Eco-Alp- metabarcoding. The workshops sWater), SCANDNAnet, GeDNA, held during the last year addressed JDS4, Portugal WFD and more. different topics: High Performance International collaborations Computation and data storage beyond Europe are now well EU COST Action CA15219 (Lyon), sedimentary DNA (Rome), settled, especially with Xiaowei molecular tools to assess new bio- Zhang (Nanjing University, Chi- logical quality elements i.e. micro- na) as well as Eric Stein and Su- Contact organisms and meiofauna (Prague), sanna Theroux (SCCWRP, USA). Prof. Dr. Florian Leese Standardisation University of Duisburg-Essen groundwater biodiversity (Ljublja- : No imple- Aquatic Ecosystem Research na). Furthermore, we engaged with mentation without standards! A Universitaetsstr. 5, various national and international new working group dedicated to 45141 Essen, Germany stakeholders through meetings molecular approaches for moni- email: [email protected] in i.e. Porto and Vienna. This set toring of aquatic ecosystems came phone: +49.201.183.4053 of workshops and meetings has in force at CEN in 2019, led by continued recently in Cyprus with Kristian Meissner (CEN TC 230/ www.dnaqua.net our 5th Management Committee WG28). A task force from WG3 meeting.

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has launched a first preliminary • Hopefully, after a successful on implementation and stand- working item about eDNA sam- application, DNAqua-Net can ardisation of DNA approaches pling in water that is now being formally be extended for one in biomonitoring, e.g. on spring examined in this CEN group. more year to finalise specific biodiversity and others. Other preliminary working items impact tasks! Let’s benefit from the “particle are also being prepared and cur- • While many (trans)national accelerator” effect of DNAqua-Net rently ongoing (sediment sam- projects stimulated by DNAq- that relies on every member and let pling, quality control, phytoben- ua-Net are already productive, us together pursue our ideas! thos sampling and preservation…) new European proposals are on that will hopefully promote future the track: ITN ‘AQUAnOMICS’, Agnès Bouchez implementation. new COST Action ideas focusing (Vice-Chair of DNAqua-Net) Implementation: Both regula- tory stakeholders and private com- panies are beginning to envisage Announcements full use of these new DNA-based tools. In particular, the discussion MC6 meeting in Evian on the implementation in regula- Upcoming events 15 September 2020 tory biomonitoring during a recent WG 1: gap filling workshop in meeting with ECOSTAT in Vien- Bratislava (Feb 3 – 5, 2020) TDWG/GBIF, 23-25 September na looks very promising. 2020, Washington Efforts have to be continued to- COST Mobilise Conference Feb- wards implementation, that is why ruary 2020, Poland our last Grant Period, the “contin- WG 5: stakeholder workshop in uation phase”, will have a strong Paris (March 10, 2020), France. focus on this objective. We now Attached to ECOSTAT Meeting have to consider the sustainability of our efforts beyond the current CETAF 2020 + Belgian empow- funding period of DNAqua-Net, ering biodiversity, May 2020 and definitely, there are many ideas Belgium and possibilities: Society for the Preservation of • A new international scientific Natural History Collections, meeting under the brand of June 2020, Scotland “DNAqua-Net” will have its first 1st DNAqua-Net International venue 15 to 18 September 2020 Conference (September 16-18, in Evian (France), and will be 2020), France proposed to the international th scientific community every two 4 Central European Symposium years! for Aquatic Macroinvertebrate Research – CESAMIR2020, • The journal we launched, which will be held on July 5-10, MBMG (https://mbmg.pensoft. 2020 in Slovakia: https://www. net), is continuing his route with aquabol.sk/cesamir2020/ more and more great papers, waiting for yours to follow!

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New Projects and UTAD, and by the Europe- Life-DNAquatic: https://bit.ly/2t- an project EnvMetaGen as well jvbyz Bosnia-Herzegovina: national as EDP Biodiversity Chair to ePIKE: https://bit.ly/2rWS1M5 Ministry of Civil Affairs decided CIBIO-InBIO. to support the project proposal eDNA – promises and challenges: Sweden titled “A pilot project on DNA : The Swedish Environ- https://bit.ly/2Pv84tA (in swedish) metabarcoding and morpho- mental Protection Agency and logical identification of benthic the Swedish Agency for Marine Reports diatoms on the travertine barriers and Water Management have of the River Una”. This is a direct approved this year eight research Workshops and output of the STSM of Jasmina projects to facilitate the devel- Training Schools Kamberovic and was prepared opment of DNA-methods in by Belma Kalmujic-Sroil, Agnès environmental monitoring (2019- WG2: Microorganisms & Bouchez and others. 2021). Four of these projects have been done in collaboration with Meiofauna Meeting Portugal: In Portugal, research- with DNAqua-Net: “Barcoding (Czech Republic) ers are collaborating with the of freshwater taxa for improved Portuguese Environment Agency assessment of biodiversity The workshop on microorganisms (APA) to evaluate the applica- (FRESHBAR)” (Maria Kahl- and meiofauna was organized in tion of molecular methods in ert, SLU), “Life-DNAquatic” Czech Republic on November 19 the ecological monitoring of (Micaela Hellström, AquaBiota and 20, 2018. freshwaters under the Water Water Research), “ePIKE - Test- The workshop consisted of two Framework Directive (WFD). ing the applicability of eDNA parts, one on prokaryotes (led The work involves more than for monitoring pike (Esox lucius by Marketa Mareckova) and on 150 macroinvertebrate samples L) abundance” Anti Vasemägi, meiofauna (led by Ursula Eisend- collected in Central and North- SLU, “eDNA – promises and le-Flöckner). The goal was to sum- ern Portugal (2018 and 2019) challenges (Per Sundberg, Goth- marize possibilities for including by researchers from the Univer- enburg University)”. Find more microorganisms in routine water sity of Coimbra (UC) and the information on the projects’ quality monitoring and to use the University of Trás-os-Montes webpages: https://bit.ly/35xCUaK outcome to calculate the respective and Alto Douro (UTAD), which (in Swedish only). indices. will be identified using mor- The results of the workshop are Germany phological approaches by UC : The German Federal currently being summarized by and UTAD, and using DNA Environmental Agency funded a drafting a paper that is expected metabarcoding by the research 3.5-year project to scrutinize the to be submitted by the end of this center CIBIO-InBIO. The first power of DNA-metabarcoding year to Science of the Total Envi- results will be available in early for Water Framework Directive ronment. 2020. This project greatly bene- compliant Stream Ecosystem The introduction to the meet- fited from DNAqua-net, which Assessment. Grant recipients: ing was provided by Karel Simek, created the momentum to devel- Florian Leese & Daniel Hering who talked about the microbial op these studies and contributed (University of Duisburg-Essen) interactions in a water column with inputs for study design and Further Projects observed by fluorescence in situ methodological developments. hybridization techniques. Each of Financial support for this work FRESHBAR: https://bit.ly/2sxirnI the participants had a short pres- has been provided by APA to UC entation about his/her perspective

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and results on this matter and later barcodes). Its use for freshwater detection and quantifica- a discussion and conclusions were biomonitoring is particularly inter- tion. made. The talk also focused on esting in the context of the Water • Detail the principal bioinformat- the molecular and bioinformatic Framework Directive (WFD), ics steps applied on metabarcod- tools, selection of most appropriate where thousands of aquatic ecosys- ing data in order to obtain molec- approaches for data collection and tems are monitored annually across ular taxonomic inventories (with analyses with regard to the use of Europe. As part of this assessment, practical exercises on “Mothur” these data in routine biomonitor- benthic diatoms are routinely used software). ing. to evaluate the ecological status of streams using quality indices Twenty-five participants origi- based on composition and relative nating from 9 countries (Croatia, abundance of diatom taxa, iden- Cyprus, France, Germany, Hun- tified at the species level. Recent gary, Lebanon, Portugal, Spain, developments of diatom DNA me- United Kingdom) participated in tabarcoding offer the possibility to this training school. While the full use this DNA-based identification steps of the DNA metabarcod- approach for diatom biomonitor- ing approach were decrypted and ing. The objectives of the training discussed theoretically, practical school were to: training was performed to offer the opportunity to participants to • Introduce the different steps of get acquainted with field diatom the DNA metabarcoding for sampling (benthic communities diatom biomonitoring of streams, from lake Geneva), molecular lab from the sampling of benthic di- methods (e.g. DNA extraction) atoms, laboratory methods, anal- and the use of bioinformatics ysis of metabarcoding data to the software (e.g. Mothur). By this final quality index calculation. way, all participants had the op- • Present the limitations and ad- portunity to “demystify” diatom Participants of the M&M Workshop vantages of the molecular ap- DNA metabarcoding and to gain proach, with strong emphasis on all knowledge necessary for the WG2: ‘DNA-based Diatom the methodological and biological completion of their own metabar- biomonitoring’ training biases which may affect diatom coding projects. school Thonon (France), 11-15 February 2019 Over the past decade, genetic tools have been intensively developed for biodiversity assessments of natural communities. As part of them, DNA metabarcoding allows identifying species in environ- mental communities, based on their genetic variability and using short DNA sequences (or DNA International participants (trainers and trainees) of the Thonon Training School

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WG2: New molecular and for applied use with a focus Wikipedia pages created or methods to assess biodiver- on aquatic ecosystem assessment, modified by the course: sity. Potentials and pitfalls of from species collection to the DNA barcoding DNA Metabarcoding use of diversity in environmen- Microbial DNA barcoding (Uppsala, Sweden) tal legislation. The focus was on Algae DNA barcoding DNA (meta)barcoding and aquatic Fish DNA barcoding An Intensive course week between organisms. Gained knowledge DNA barcoding in diet 25-29th of March 2019 was organ- was examined by creating public assessment ized in Uppsala by Maria Kahlert Wikipedia pages and a collabora- Aquatic macroinvertebrate (working group 3). Afterwards, the tive Open Access publication of all DNA barcoding course period was extended with participants. Open Access publication by the distance homework form 18th of Twenty-eight participants from course (with a picture of us!) March to 31st of May 2019. The 14 countries joined successfully, https://bit.ly/2PsziB3 course was mainly held at the SLU of which 21 were Ph.D. students. Course syllabus: campus, with one excursion to Eleven teachers participated, of https://bit.ly/2PHMjXO SciLifeLab. The course was co-or- which 4 came from the DNAq- ganized and co-funded by FoSW ua-Net community (Germany, the WG2 & WG3: Sedimentary with RS Ecology and the COST Netherlands, France, Sweden). DNA: sampling, preservation network DNAqua-Net. and extraction (Rome, Italy) The WG 2-3 Workshop: Sedimen- tary DNA: sampling, preservation and extraction was held in Rome from 10 to 11 June 2019 at Orto Botanico in Trastevere, Rome, It- aly. It was organized by the Water Research institute (IRSA-CNR) in collaboration with the NGO Mi- cro&co (organisers: Jan Pawlowski, Kat Bruce, Stefano Fazi, Marketa Marečková, Kristel Panksep) and it was attended by 22 participants from 13 countries. Stefano Fazi opened the work- shop, describing its objective, or- Participants of the Ph.D. course PNS0169 at the Swedish University of Agricultural Sciences (SLU) in Uppsala, Sweden. ganization and expected outcomes. Jan Pawlowski and Kat Bruce This course offered a compre- The course got positive reviews gave an overview of the WG2 and hensive overview of the paradigm from the participants and on WG3 prospective. Kristel Panksep shift from traditional morpholo- average, the general opinion about reported on the questionnaire that gy-based to novel molecular organ- the course, its organization as well was circulated among the partic- ism identification approaches. as its contents were rated as very ipants to gather the information We covered the use of molec- good. about the currently used methods ular tools in both basic research and strategies.

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The morning ended with brief Kat Bruce, on the two outputs Training written individual presentations of all of the workshop: communication participants on their own contribu- 1) the best-practice guidelines for On 12 and 13 November 2019 tions and perspectives for the sub- sediment DNA sampling and COST Academy organized a ject. In the afternoon, two parallel extraction (led by Kat Bruce); training on written communica- working groups focused on: 2) Review paper (led by Jan Paw- tion conducted by Ana Rotter. As 1) Identifying key issues regarding lowski and Stefano Fazi). After a there is far more information out sampling strategy; lunch in the garden, small work- there than people have time to 2) Identifying key issues regarding ing groups drafted specific points read and digest, written commu- sample preservation and DNA of the review paper by filling out nication is becoming an essential extraction. the outline that circulated before tool for all scientists. First of all, it the meeting and preparing a is our duty to communicate about The day ended with a plenary technical sketch of the best-prac- our research and second, we have section with groups’ reports and tice guidelines. to be the source of information for general discussion, followed up clarifying emerging topics, when by a beer in Trastevere and a nice The workshop ended with a vis- it to the Accademia Nazionale dei they arise. However, the way we dinner! communicate is of extreme impor- The second day started with a Lincei. The Accademia was found- ed in 1603 by Federico Cesi, being tance as our field of expertise is in very interesting plenary lecture by constant competition with other John Pearman - Cawthron Insti- the oldest scientific academy in the world and among its first members engaging topics. Here are a couple tute New Zealand and a plenary of take-home messages: discussion, moderated by Galileo Galilei. 1. Tailor the communication activity to the specific audi- ence, depending on what you are trying to achieve. Once you identify and understand your audience, the rest is much easier. Think about their starting level of knowledge and about the questions they might be asking. Answer them in the communica- tion already. 2. When preparing your message, ask yourself: why should people read it? It is likely that you will reach your target audience once you identify the topic in your message that the target audience Workshop on Sedimentary DNA in Rome, Italy will take personally: disease, children, pristine nature, ...

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3. Active voice is clearer and more 6. Avoid using jargon. By that, you DACH UGM engaging than the passive one. will create a distance between (Innsbruck, December 4th-5th) Use it, when possible. you and your target audience DNAqua-Net member from which will prevent you from 4. Journalists answer to the pub- Austria, Michael Traugott, hosted delivering your main message. lic (their target audience) and a first large User Group Meeting not you. Once you have given 7. Press releases and notifications (UGM) in German for the end-us- them the facts, it is up to them for journalists should not be ers from the countries of Austria, to tailor the story and select the too long – 1-page max, as they Germany and Switzerland. DNAq- direction they will finalize it are busy and receive a lot of ua-Net co-hosted the meeting and around. It might be a happy co- information. Train yourself not funded the participation of several incidence that their highlighted to try to tell EVERYTHING keynote-speakers (Florian Alter- information is exactly what you as you will never be able to. So matt, Kristian Meissner, Bernd wanted to say, but it is not often filter your message based on the Hänfling, Didier Pont, Florian the case. most important message you Leese) to provide the end-users, es- 5. The top paragraph is the most are delivering in that particular pecially from governmental agen- important for the pitch. Some- circumstance. cies, service-providers and industry times even the title as busy with a state-of-the-art of eDNA journalists scroll only titles. applications. The great interest in the tech- niques by end-users was obvious with more than 150 very active participants. While the potential is seen by all, major concerns from end-users are still questions re- garding the standardization of the method in order to better interpret its results. Given the success of the meeting, a second User Group Meeting will be held early 2021 in Berlin (German Federal Environ- More than 150 stakeholders participated in mental Agency), co-organised by the DACH User Group Meeting on DNA-based Excellent organisers: Michael Traugott and his methods. team from University of Innsbruck University of Duisburg-Essen.

Participants at DACH User Group Meeting

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Action DNAqua-Net (CA15219), supported by the COST (Euro- pean Cooperation in Science and Technology) program “. Belma Kalamujic Stroil report- ed on short term scientific missions (STSMs), which still are quite popular in our Action. Until now 8 STSMs have been granted in this GP and the last call is currently under evaluation (5 applications). Marlen Vasquez reported on the ITC Conference Grants (ITC CGs). This networking item is still Audience at MC5 underutilized and DNAqua-Net MC5 meeting in Limassol After the really nice start, members are urged to propose this opportunity to eligible candidates. th Sarah Kückmann continued with On September 30 2019 the DN- After a first coffee break all WG Aqua-Net Management Commit- a financial overview of the last Grant Period 3 (GP3). In the end, leaders reported on their current tee (MC) gathered at Cyprus Uni- activities (please see the following versity of Technology (CUT) in 99.77% of the budget was spent. Florian Leese then switched to reports). the beautiful old town of Limassol. Florian showed some important After the welcome address the current GP4, the ‘Integration Phase’. Three items are currently parts of the mid-term evaluation of with the first overview of current our COST Action. We were grated activities by Florian Leese, the delayed: WG3 report, WG4 report and the glossary. The glossary was ‘very good’, thanks to all of you. Senate of the Faculty of Geochem- Only the delays in WG reports ical Sciences and Environmental started by WG2 in a Workshop lead by Maria Kahlert in Uppsala, and Exploitation were weaker Sciences of CUT, Prof. Theodoros points but both will be fixed with- Zachariadis, gave a heartily wel- Sweden (see a report of this meet- ing above). Volunteers for translat- in this grant period (at least four come to all of us. Stakeholder Workshops, two of Last our Local Organizer, ing this are always welcome. Please send an email to Sarah if you want them upcoming). Marlen Vasquez, introduced her- The MBMG journal also made self and gave an overview of the to participate here. upcoming activities for the 3-day The two delayed reports are in Meeting. Thanks a lot to Marlen, progress. Katerina and the whole team, for Up to now more than 40 this perfectly organized confer- publications with direct input ence. from DNAqua-Net are published. As proof of the still-growing Florian highlights the possibility network of DNAqua-Net, we had to apply for open access funds if several new faces who introduced authors from at least 3 different themselves to the Committee. DNAqua-Net countries are in- Great to have all of you on board volved. The following acknowl- and welcome to the community! edgment is required: “This article Xiaowei Zhang giving an overview of DNA- is based upon work from COST based bioassessment in China.

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good progress but further academ- Should we develop curated, ic authors are needed. Please con- high quality datasets for aquatic tact Florian or Alexander Weigand invertebrates in Europe? if you want to participate. Alexander Weigand presented For GP5 nearly the whole the topic and led the discussion. budget will be spent for the final The following were noted: conference in Evian from Septem- 1. The WG recognizes that curated ber 15th 2020 (MC6) till Septem- Happy organisers after three successful meeting-days datasets will be very beneficial ber 18th 2020 because it will only for reliable identification of be 6 months long. aquatic organisms using DNA At last some ideas for sustaining invited Peter Gilchrist (Jacobs, metabarcoding. the network were discussed and UK) to provide the perspective of the administrative part of MC5 a global company on DNA-based 2. Curating of a full open database was closed. methods, challenges and opportu- could be an ultimate goal, but nities. is seen as unrealistic due to the Once more it became obvious continuous addition of conflict- how important it is that DNAq- ing data. ua-Net is a truly international 3. A bioinformatic tool for au- network that goes beyond Europe tomated detection to identify and beyond academia. conflicting species assignments Thanks to all of you who made in BOLD was presented by the meeting such a success, espe- Filipe O. Costa. This tool will Picnic at the seaside organized by local cially Marlen and her team. be good to detect good entries organizer See you next year in (Grade A and B) versus potential France! errors in BOLD. It can be an excellent starting point to create WG reports high-quality datasets. 4. Datasets will also need contin- WG1 report uous updates to accommodate Working Group 1 reported on changes in and new progress at the annual meeting in genetic diversity. For example, Evening Programme: City tour Limassol and held a one-day group there has to be a designated team meeting on October 1st in the same of editors responsible for the The afternoon session was curation of such datasets, which under the motto “…Europe and place. At this meeting, the follow- ing topics were discussed: are easier to control compared to BEYOND”. We had three excel- full datasets. lent plenary speakers to inform about the application of DNA- Main results from gap-analysis 5. Datasets created in BOLD can based methods in China (Xiao- paper published in STOTEN easily be given a DOI and be wei Zhang, Nanjing University), A major deliverable for the group used as background for publica- Canada (Donald Baird, University has been the gap analysis paper. tions (and thereby cited). They of New Brunswick) and the US Open access version is available can also be used directly when (Susanna Theroux, SCCWRP here: https://doi.org/10.1016/j.scito- using mBR AVE. California). Furthermore, we had tenv.2019.04.247

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6. Curation needs resources (€ and Bratislava workshop for filling ovic Institute (Croatia) in 2019). hrs) both for establishment and the gaps in the barcode library First, Cécile and Ana pre- continuation. Would stakehold- We are planning a workshop to sented together the preservation ers be willing to fund such work? fill the gaps in the barcode refer- experiment and first results. This ence library for European aquatic 7. Taxonomic datasets should have experiment was based on a 2017 invertebrates and would like to priority before regional (e.g. na- survey identifying preservation invite anyone interested to partici- tional) datasets are produced. practices for diatom environmen- pate or provide material to register tal samples. Based on the survey’s 8. Journals that publish studies us- their interest by filling in this sim- results, three different preservation ing DNA metabarcoding should ple survey: http://bit.ly/2EEuFgV. methods were identified as repre- require details on the reference The workshop aims to fill in sentatives for current usage and libraries that are used - proved, barcode gaps in the BOLD data- compared on six different environ- updated and reliable datasets can base for the species from our “wish mental samples (four freshwater be of high value for supporting list”. However, other species, espe- river phytobenthos samples, two results and outputs published. cially those important for national marine phytoplankton samples). monitoring programs, that lack Strategy for filling the gaps in barcodes will also be considered. the barcode library The deadline for registration After a discussion we agreed is 20th December 2019. that number one priority should The workshop will be held in be filling up the information on Bratislava, Slovakia on February missing species and species groups 3-5, 2019, at the Slovak Academy that are widespread and monitored of Sciences. We hope to be able to in multiple countries, with second invite up to 12 participants, but Cécile Chardon (France) presenting the preservation experiment priority should be species or species the final number of reimbursed groups from poorly represented participants will largely depend Each sample was extracted in regions. (e.g. EPT from ITC). on the budget available next year. two replicates, six different times Please contact the Working Group throughout a 1-year period and, leaders for more information and finally, all samples were sequenced access to the wish list. in a single run. DNA extracts were Torbjørn & Fedor compared based on DNA quality and quantity as well as on their DNAqua-Net WG2-3 community diversity and structure Diatom workshop obtained from DNA metabarcod- The workshop was attended by 18 ing. Generally, preservation time participants from 12 countries. did not appear as a major issue, The first day, Octoberst 1 , was while preservation methods have Discussion at WG1 Meeting in Limassol dedicated to discussing a preser- a greater, though limited, impact vation experiment that relies on that has to be explored further. 2 STSMs. Ana Baricevic (Croa- A draft paper was then discussed tia) who came to INRA (France) among participants, led by Ana in 2017, and of Cécile Chardon Baricevic and Cécile Chardon. (France) who came to Ruder Bosk-

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sequencing run that will allow to of both your own curated database compare all samples. Participation and the Genbank. Furthermore to the ring-test will be proposed the study showed the results of fish to DNAqua-Net partners working being eaten in a restaurant along on diatoms metabarcoding, within the river. Interpretation of results is the limits of available places. therefore crucial. Jonas Bylemans – Fish eDNA eDNA Fish workshop - “Field in the EcoAlpsWater project: and Laboratory Methods – Eco- Jonas presented an INTEREG Ana Baricevic (Croatia) presenting during the project on eDNA in large, deep workshop logical Assessment” lakes including fish (both sampled The fish workshop was led by with eDNA and sonar) as well as Second, Susanna Theroux (SC- Jelger Herder and consisted of six CWRP, USA) presented standard- bacteria, phytoplankton and phy- interesting presentations related to tobentos, held in central Europe. ization efforts in state of California eDNA and fish diversity. Approx- and beyond. The presentation was The project included six coun- imately 25 to 30 people attended tries and twelve partners. In this followed by a discussion on stand- the workshop. The following pres- ardization efforts in Europe and project different sampling strat- entations were given: egies (integrated sampling versus in the USA, with possible conver- Bernd Hänfling: overview of the gences. Participants agreed that point sampling) will be tested . FSBI 2019 eDNA fish session and Furthermore a primer comparison diatoms’ metabarcoding should be update on the lake fish tool devel- divided in sub-parts for standard- was presented, which focused on opment: at the FSBI there was an the taxonomic resolution, specific- ization rather than a single stand- eDNA fish session. Important con- ard for the whole process, although ity for the target group and taxo- clusions were that eDNA outper- nomic coverage. an umbrella standard could be formed traditional methods. Most complementary. As standards are In conclusion, it was agreed talks were on eDNA-metabarcod- that the best primers depend on extremely needed to convince ing instead of single species detec- stakeholders that these approach- the region/species targeted and tion. Many presentations showed in the database as well as on the es are feasible, we then looked at examples of using metabarcoding each part of the whole diatoms’ length of the fragment (primers data in a semi-quantitative way. that target longer fragments have metabarcoding process to identify The UK-fish tool is under further the points that could be ready for higher resolution when testing development with 126 lakes being them bioinformatically but prob- drafting a preliminary work item sampled. New indices based on or a technical report for CEN ably have lower detection chances eDNA methods are being created due to fact that long fragments are WG28. for lakes in the UK. nd less frequent in highly degraded The second day, October 2 , Daniela Sint: The need for was dedicated to design a single eDNA. curated sequence databases when Rein Brys: eDNA based mon- ring-test to bring information to metabarcoding fish from eDNA two of the matters discussed on itoring and impact assessment of samples: this presentation showed an invasive Caspian goby in a lotic the previous day: DNA extraction the need for curated sequence da- & PCR amplification. At the end, system: Rein presented several in- tabases with local material. Gen- teresting studies. First off all, the a timetable was agreed upon and bank included many errors which tasks were assigned among partic- tests were carried out with fish in resulted in wrong species assign- a fyke net which investigated the ipants. INRA (France) proposed ments. Best is to use a combination funding in 2020 for the final distance from which one could

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pick up the signal with different church documentation as people ale of the first day was to address sampling strategies. In the me- used to donate fish to the church, point 2, with individual presenta- tabarcoding study in the Berwijn with the documentation serving as tions of invited speakers, showing (small stream/river), the authors a base for comparison. the pipelines we use for analysis, found that at the end of summer Kat Bruce: preliminary results the problems we may have, and to there were highest eDNA concen- from an inter-lab comparison exer- start discussions on specific topics. trations. The differences in eDNA cise for fish eDNA metabarcoding: The first speaker was Adam concentrations were measured on during the FSBI a ring test was Petrusek, who introduced an a small spatial scale. The concen- carried out between labs by put- example of a phylogeographic trations of eDNA measured with ting 100 liters of water from a river study using metabarcoding of a ddPCR were similar as those meas- in a children’s swimming pool and ~450bp fragment of COI from ured with eDNA metabarcoding by allowing the participants to macrozoobenthos in the Carpathi- and correlated with data deriving take the samples. Everyone took ans. The presentation showed the from traditional monitoring. The samples using their own methods use of phylogenetically informed further research on spiking sam- and analysed them with their own analyses to resolve ambiguities in ples was also proposed in order to pipelines. The already obtained assigning OTUs to species using get a more accurate quantification. results showed the similarity for directly BOLD or BLAST. The detecting the more common discussion centered on: i. the use species (detected by each pipe- of phylogenies, ii. the reliability of line) but more stochastic results data when quality control can be for rare species. The idea to use a high, e.g. by checking for indels ring test within DNAqua.net was and stop codons in a protein-cod- postponed for now to wait for the ing marker, and iii. the difference further results of this test. between OTUs and ASV (Ampli- con Sequence Variants). The second speaker was Alexan- WG 4 report Rein Brys explaining on the monitoring of der Eiler, who mentioned poten- invasive gobies using The workshop had two aims: tially conflicting scientific and 1. to finalise the analyses for the stakeholders’ interests in the way Micaela Hellström: Stake- data is analysed, depending on the holders benefits from traditional comparisons between pipelines in unsupervised OTU (Opera- questions and on the rationales. inventory methods and eDNA, ASV were mentioned again, to- Why? When? Where? - Summary tional Taxonomic Unit) cluster- ing, as planned from the previ- gether with the difference between from 30+ large surveys. Michae- ous meetings; supervised and unsupervised clus- la presented a large number of tering, the use of scripts to mine eDNA-metabarcoding fish studies 2. to highlight other potential for data, to merge datasets, and from Sweden. The studies fo- problems or ambiguities in data to identify holes in the taxonomy, cused at effects of the barriers on analyses that could be addressed. and the statistical approaches using upstream and downstream fish training datasets to predict and an- communities, which helped deci- October 1st, 9:00-12:30 alyse larger datasets. The preferred sion-makers to remove the existing After a round of introduction pipeline is cutadapt, DADA2, and barriers. It was also possible to on who we are and what we do, Bayesian classifier. reconstruct historic fish commu- a short summary of the previous nities from around 1600s from the meetings was reported. The ration-

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A suggestion for data storage methods and on different datasets. • the group of Alain and Jean-Marc was the potential use of GBIF for The day finished with a general will perform all the analyses and curated metabarcoding datasets. discussion on additional topics, the comparisons; The third speaker was Simon including the effect of pseudogenes • a paragraph on aims and objec- Creer, who mentioned QIIME, and the use of other tools such as tives and one with the take-home USEARCH, VSEARCH, MO- ecotag and obiclean in OBITools, message will be prepared to clari- THUR, and DADA2 as pipelines, mBR AVE, etc. fy the reason for the comparison; introducing a different method to A brief summary was then obtain OTUs, based on ecologi- reported on the literature review • the plan is to clarify the charac- cally and phylogenetically relevant performed on which proportion teristics of the datasets in a short clades, subsampled from the whole of published papers dealing with time, start the analyses and per- data across ecological gradients, High-Throughput Sequencing data form the comparisons at the end used as the input for GMYC (Gen- actually reports accession numbers of the year, so that a first draft of eralised Mixed Yule Coalescent or make the data publicly available. the manuscript can be prepared Model) analyses to identify taxo- The preliminary results are shock- at the beginning of next year. nomic groups akin to species. ing, as it seems that about every The outcome regarding general fifth published metagenome is not recommendations on analyses, October 2nd, 9:00-13:00 available to science. agreed upon by all participants, are The day started with a continu- The main outcome of the WG4 as follows: ation of the talks from the previ- workshop regarding comparisons • ASV should be the base on any ous day. Francesco Ficetola intro- between pipelines for OTUs are as analyses, on which also OTUs duced statistical ways to address follows: can be calculated; the problem of false positives and • a manuscript will be prepared to • ASV should be stored somewhere, false negatives, by using replicated showcase the results of the com- together with the raw data and technical PCRs, independently se- parison; quenced in Illumina, together with the metadata; • the comparison will involve four different levels of negative controls • the participants will produce a relevant methods, USEARCH, to estimate the detection probabili- table with different pipelines, SWARM, BIN, and YAPOTU; ty for each species. describing them and suggesting The second part of the morning • about ten metabarcoding datasets advantages, problems, and limi- was dedicated to a summary of will be selected, to cover differ- tations. the results of the workshop held in ent taxonomic groups, different Lyon on YAPOTU (Yet Another markers, and both actual and Pipeline for OTU) by Alain Franc, mock communities, together with together with a clear comparison ring tests; between the OTUs obtained with • the dataset will be provided by YAPOTU and SWARM. The talk the participants, together with introduced the reasons for de novo additional selected ones from fish OTU picking, the distance-based and diatoms; methods and their limitations, and the rationale of YAPOTU. • the datasets have to be on The results of the comparison non-dereplicated data, for about suggested the need for a more 100.000 reads, maximum complete comparison between 200.000 reads;

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WG 5 report Nagoya protocol – GDPR Nagoya protocol does not apply (Patricia Mergen) General discussion when the data is only used for st Nagoya protocol: https://www.cbd. taxonomic purposes. For monitor- On the first day (October 1 , int/abs/doc/protocol/nagoya-proto- morning) an overview of the ongo- ing within one country, there is col-en.pdf no problem. It only applies when ing activities was presented: More info at: https://absch.cbd.int/ CEN update (Kristian Meissner) samples are transferred to other countries (even if within the EU). CEN: https://www.cen.eu/about/ 173 countries have a national Pages/default.aspx The main reason for this is that focus point on Nagoya protocol, the country of origin needs to be Not much input needed specifi- what are the prerequisites when cally from working group 5, input recognized, especially when for taking samples? example afterwards the samples from the whole DNAqua-Net Development of a scheme/de- community is crucial. Individual are used for commercialization. cision tree on what kind of docu- GDPR http://bit.ly/38T4wJo countries need to give input to mentation needed when sampling CEN Working Group 28. The Does not directly apply for bio- and transferring samples. monitoring if there is no personal work is ongoing, but many coun- Applying Nagoya protocol on tries are still missing. information is collected. digital sequence information (to Public Sector Information Di- For eDNA, the matrix is im- come from the side of CBD) with portant (sediment or water). The rective > extended to open research danger of applying this also on all data (to be implemented by 2021). standardization does not necessari- data (species data, e.g. GBIF) that ly include the type of net, the type is digitally available since it can be of extraction, but rather defining a problem for open access data. within which contours you have to work (for example how to avoid contamination). Sampling methods for eDNA are now submitted to CEN to define the standards. It will be revised shortly. Good idea is to collect infor- mation for standardization within DNAqua-Net. How in terms of global view (ISO) should we aim to get it on that agenda (Florian). CEN stand- ards can be adopted in an ISO lev- el (Kristian), but it is complicated. Conclusion: follow up action: try to collect items that could be of relevance to get the method stand- ardized, but important to have a Daniel Hering leading discussion of WG5 in the public library of Limassol. person willing to bring it forward.

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Joint DNAqua-Net / ECOSTAT different water quality class Drivers meeting – Vienna, May 6th 2019 • Cost savings (depending on the (Daniel Hering) • databases are not complete, not easy to compare with earlier size of the project and the scale). Conclusion: there is no princi- monitoring, protocols not fin- The cost saving and the impor- ple obstacle to use DNA-based ished, no standardization yet, tance of cost depends on the methods in the EU WFD. When specific labs needed, not adopted stakeholder. changing methods the initiative by EU WFD, lack of consistency • Legal framework comes from the country and needs between labs • Give results faster (according to to do the compliance check. On the second day, the possibilities D. Baird, this is a more impor- for future funding were discussed tant driver than cost, but the re- Step 1 – decide on new method as well as the possibilities of two mark was made by J.Herder that Step 2 – compliance check papers and a policy brief that could in case of pollution events, for still be written. For one paper on fish you can much quicker assess Step 3 – approval by ECOSTAT the essential biodiversity variables, the impact than using DNA- it was decided that Pedro Beja and based monitoring) Pre-stakeholder meeting Pieter Boets would try to take the • You can get extra information A pre-stakeholder meeting was lead in this paper. People who are (but regulators are not always held in the morning to start to get interested in supporting the paper waiting for this) an idea about the obstacles that we can contact one of both. • Accuracy/precision/repeatability are currently facing when imple- (DNA based method is better) menting these new DNA-based methods. For this a few speakers Stakeholder meeting in • Early adoption from industry or SME’s were Limassol • Policy invited. This workshop served as • Disappearance of taxonomists an introduction for the stakehold- A stakeholder meeting was or- ganized (led by Kat Bruce and • Influence from key drivers/key er meeting that was held in the countries afternoon session organized by Kat Jan Pawlowski) during the MC5 Bruce and Jan Pawlowski. A few of meeting in Cyprus to define the • Innovation the obstacles that were mentioned drivers and obstacles related to the • welfare (non-invasive in the morning session were: implementation of DNA-based method) techniques in routine monitoring. • Different approaches between • Human safety countries hamper the standard- The following drivers and ob- ization, Different species are not stacles could be derived from this in the databases making it diffi- workshop, although it was clear cult to annotate them. that depending on the perspective (regulator, industry, service-pro- • Reliability of the method, no vider, researcher), the relative standard method, method is importance of drivers and obstacles working only for some matrices varied. A manuscript that will deal and some species, but not for more specifically with these per- everything spectives will be drafted in WG5 • when applying the DNA-based (Florian Leese): method one might end up in a Collecting and discussing drivers and obstacles of DNA-based method uptake by end-users

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• Increased flexibility (e.g. you can • illusion of understanding/lack of of the call’s results will be on take DNA samples whenever you understanding December 20th 2019. All accepted want) • why changing of we have some- STSMs must take place between st • Once one can take more samples thing that works and this new December 21 2019 and March th within a certain limited time- method request a lot of effort 15 2020. frame • loss of comparison with long term • Reputation/risk of the company: data Reports on the one hand the company • Intrinsic variability of the method wants to invest in it for its status, but on the other hand they also Laura Pompei want to avoid to be involved in Title: Detection of non-native any impact and thus they invest fishes from Italy and Turkish fresh- in it a priori, not to have to pay STSMs waters using species specific cPCR, fines afterwards. qPCR and ddPCR Assays Overview Home/Host country: Italy/Turkey Obstacles One of the most popular aspects • pre-existence of traditional of the DNAqua-Net action so far The aim of the STMS was to methods have been the Short Term Scien- acquire the basis of eDNA tools • if new techniques come, which tific Missions (STSMs). Within in order to apply the new skills to are complementary to what al- the period from October 2018 future researches on early detection ready exists, there is the fear that to October 2019 we announced of non-native fish species (NNF) both techniques are needed six calls in total. The novelty in in the aquatic environments of (extra cost) the Grant Period 4 was that the Central Italy. STSM was based on intensive laboratory practice • technology acceptance models first STSM call was dedicated to the applications contributing to (eDNA sampling, DNA isolation, • lack of companies missing the DNAqua-Net’s involvement in qPCR and ddPCR, sequencing) knowledge/capacity to do this Joint Danube Survey 2019 (JDS4). and in-silico bioinformatics (prim- • lack of regulators accepting Total of 38 applications were sub- er design). The main outcome was the method mitted making our STSM evalu- the knowledge transfer to the local • lack of reference databases (but ation board rather busy. With 23 team in Italy for starting the first depending on the target group) applications accepted, the general application of eDNA techniques • loss of jobs for people using the success rate was around 60%. Two in water bodies of Central Italy. In traditional method thirds of successful applicants were particular, the design of specific female researchers. The involve- primers for the detection of Pa- • too sensitive ment of Inclusiveness Target dogobius bonelli, Salmo trutta and • lack of consensus and lack of Countries (ITCs) was again high, Barbus barbus, will allow devel- method transparency with 68% of cases where ITC was opment of protocols for the early • lack of confidence involved either as an applicant’s detection of these highly invasive • difficult to understand country of origin or as a host species in Italian watercourses. (black box?) country (or, in some cases, both). • false results The next call is opened and the deadline for submission is Decem- ber 13th 2019. The announcement

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Kristel Panksep Cécile Chardon preservation could be an outcome Title: Next generation monitoring Title: Towards standardization of of this work after full data analysis. of phytoplankton diversity, with the preservation and storage of Valentina Slavevska special focus on toxic cyanobacte- WFD phytobenthos and phyto- Stamenkovic ria plankton samples for biomonitor- Home/Host country: Estonia/ ing with eDNA metabarcoding Title: Filling a major gap in the Switzerland approaches DNA-barcode reference library for Home/Host country: France/ the keystone aquatic taxa (Malca- The purpose of the visit was to Croatia costraca, Trichoptera) from Repub- foster the collaboration between re- lic of Macedonia (Balkan Biodiver- searchers from Estonian University This STSM is linked to a one- sity Hotspot) of Life Sciences and the Prof. Jan year experiment started in Sep- Home/Host country: The Repub- Pawlowski Group at the University tember 2017 with diatom experts lic of North Macedonia/Poland of Geneva. The main goal of the from DNAqua-Net participating visit was to learn metabarcoding countries (France, Croatia, Spain, The main aim of the STSM was techniques to monitor biodiver- Germany, and Sweden). This ex- to prepare a molecular barcode sity in aquatic ecosystems, with a periment was realized to assess the library of freshwater malacostracan special focus on toxic cyanobacte- impact of preservation conditions and caddisfly fauna in the Repub- ria. Cyanobacterial blooms are one and storage durations of envi- lic of North Macedonia, based of the major threats to freshwater ronmental samples on the eDNA on sequences from the 5’ region ecosystems globally and as the metabarcoding process for diatom of the mitochondrial cytochrome frequency and extent of the blooms communities. This STSM was the coxidase subunit I gene (COI). are predicted to increase, detection opportunity to analyze the data of Macedonia is known for a high and early warning tools of cyano- this experiment and prepare the level of diversity and endemism bacterial blooms become more draft of a scientific paper based on in freshwater taxa. The obtained important than ever before. To these results, as well as to complete results of the STSM represent the improve the methods of detecting the transfer and the adaptation of first reliable assessment of mala- and quantifying cyanobacteria in the protocol for eDNA metabar- costracan and caddisfly diversity aquatic systems, the specific aim of coding developed in INRA Lab and endemism in the water eco- the STSM was to obtain metabar- (France) to the Center for Ma- systems in Republic of Macedo- coding data for a subset of “Euro- rine Research Lab (Croatia). The nia. Six to ten morphospecies of pean Multi Lake Survey – EMLS” team obtained a favorable data set malacostracans from nearly 40 samples that could be compared answering well the initial question sampling sites and approximately 3 and intercalibrated with the results about the impact of the preser- caddisfly specimens of each species of current biodiversity assessment vation of samples on the eDNA from 30 locations were selected for method. metabarcoding process, compar- DNA isolation. Almost 1000 COI ing three methods of preservation sequences were successfully ob- (ethanol, deep-freezing, DNA tained during the finished STSM, preservative) and six storage dura- which are now being submitted to tions (from 1 day to 1 year). Apart BOLD to create the DNA barcode from the publication, a proposition reference library. of a Preliminary Work Item to CEN TC230-WG28 about sample

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Roman Hodunko were obtained during my STSM matched with neither BOLD nor Title: Providing a reliable reference action, now with available BINs. BLAST species records indicates DANN barcode library for the that the Western Carpathian re- gion is not yet sufficiently covered Balkan mayflies as a first step for Magdalena better taxonomy and conservation by barcoding efforts, and suggests Gajdošová practice that there indeed is a considerable cryptic diversity of macrozoo- Home/Host country: Czech Re- Title: Metabar- public/Poland benthos, as hypothesized in the coding of mac- application. rozoobenthic The purpose of the STSM was communities of Sofía Paz to establish a molecular bar- calcareous spring Sedano code library of mayflies (Insecta: fens at Central European bioge- Ephemeroptera) of the Balkan ographic boundary: testing the Title: Filling gaps Region using the standard region hypothesis of local glacial refugia in Mediterranean of COI. Places of the sample Home/Host country: Czech Re- Mollusca biodi- collection were distributed evenly public/Germany versity – the genus over the main river basins of the Okenia: species central part of the Balkans. Many The main aim was to uncover identity and species gap-analysis in probes were collected in proximity and characterize cryptic molecular DNA barcode libraries to the type localities of 11 Balkan diversity of benthic macroinver- Home/Host country: Italy/Spain endemics. The results obtained tebrate fauna of spring fens of allow evaluation of current diver- Western Carpathians, testing the The aim of this STSM was the sity level of the Balkan Region hypothesis that this biogeographic study of the genus Okenia (Mol- mayflies, as well as integrative region has served as an important lusca: Nudibranchia) in the Med- taxonomic and phylogeographical glacial refugium. COI sequences iterranean Sea. Total of 42 speci- investigations of the genera Baetis, of very high quality were acquired mens from the Mediterranean Sea, Nigrobaetis, Ecdyonurus, Electro- from all 41 samples, which, af- including the Italian, French and gena and Rhithrogena from several ter quality filtering and removal Spanish coast, were collected. Be- adjacent regions. 791 specimens of of sequences of non-metazoan sides, 15 additional specimens be- mayflies were selected for analysis (or vertebrate) taxa, resulted in a longing to different taxa were also according to the main purposes of dataset of 8397710 reads compris- studied due to their similarity with the Project. The collections include ing 794 operational taxonomical the Mediterranean samples. The a considerable part of the Balkans, units (OTUs) of mostly aquatic holotype of Okenia cupella, one situated in the principal river invertebrates, 333 of which were specimen of O. angelensis, eight basins, near the type localities of linked to 274 distinct species and specimens from Florida, and one mayflies (nine regions of this type), most of the rest to higher taxa specimen from Canary Island were belonging to endemic species using BOLD. 10 more OTUs studied. Good quality sequences of (Bosnia and Herzegovina, Monte- were additionally linked to species COI, 16S rRNA and H3 regions negro, Albania, and Macedonia). by BLAST. Most of the OTUs were obtained (4, 4 and 13, respec- Besides that, the reach comparative (613) belonged to Insecta, mainly tively). A complete re-description material from Malta, Turkey, the Diptera (404), Coleoptera (56) of the holotype of Okenia cupella Caucasus, and Iran was utilized. and Trichoptera (41). The fact that was done, and expanded distribu- Altogether, 446 COI sequences 57 % of the OTUs could not be tion range of

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O. zoobotryon was established. Illumina sequencing, and subse- were analyzed. It was found that Three potential undescribed quent bioinformatics analyses were even though attempts to use simi- species were expected to be found expected in the months following. lar parameters in the two pipelines when the study is completed. were made, the final outcome was considerably different due to Judit Fekete Haris different execution of certain steps Zafeiropoulos Title: The potential use of eD- in P.E.M.A. and QIIME2. A com- NA-based macrozoobenthic Title parison of the assigned OTUs to : A compar- their taxonomies, and especially of bioassessment in the Hungarian ison of bioinfor- monitoring system those from the mock communities matic pipelines is needed in order to compare the Home/Host country: Hungary/ and sampling Germany two pipelines. Initial findings indi- techniques to ena- cated that mock community taxa ble benchmarking The intention was to use the were identified in the output using of DNA metabarcoding most methods tested, regardless of eDNA method for detection of red Home/Host country: how the reads were merged. How- list dragonfly species from Hun- Greece/Ireland gary (Cordulegaster heros and C. ever, mock community taxa were bidentata) which are both listed in not found using the Naive-Bayes The aim was to develop stable, classifier implemented in QIIME2 the National Biodiversity Moni- complete and accessible bioin- toring Network. Team aimed to against the Midori database, which formatic pipelines for analysis of warrants further investigation. explore the potential of water sam- HTS metabarcoding data, as well pling and eDNA-based assessment as the benchmarking best-practice Aleksandra Bankowska´ in order to validate the presence of approaches for HTS data analysis the larvae of these protected aquat- through comparison of different Title: DNA barcode reference ic macroinvertebrates in a non-in- pipelines. Also, team wanted to library for the water mites of the vasive fashion. The second aim was investigate the effects of different weakly explored Western Car- to discuss the opportunities of us- field techniques on HTS output. pathians ing of eDNA-based monitoring in During the stay, the results of Home/Host country: Poland/ the National Monitoring Network bioinformatic pipeline P.E.M.A. Slovakia for WFD conform analyses (Water (recently developed in IMB- Framework Directive). During the BC-HCMR) were compared using The main purpose of the STSM, the newly developed group different sets of parameters with proposed STSM was to prepare a (Odonata) specific primers were those obtained from QIIME2, molecular barcode library of water tested and optimized in compar- a commonly used bioinformatic mites fauna of Western Carpathi- ison with general primers (BF2/ pipeline. This STSM developed a ans based on sequences from the BR2). The main result of the stay Singularity image of P.E.M.A. that 5 ‘region of the mitochondrial was knowledge gain which would is now available on the Singularity cytochrome c oxidase subunit I enable the possibility of imple- Hub. All the scripts developed dur- gene (COI). DNA was extracted menting the metabarcoding tech- ing this STSM have been included from about 400 individuals, while niques in the Hungarian National in P.E.M.A.’s page on GitHub. sequences were obtained from Monitoring Network. By the end A total of 22,198,605 reads de- around 200 specimens. However, of the STSM, sequencing librar- rived from 73 paired-end samples, not all sequences were of satis- ies were ready to be sent off for including two mock communities, fying quality to be included in

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subsequent analyses. Total of 33 taxonomic names, with 80% being sequences of good quality were of microbial origin. When using used for the analysis and identifi- stricter blast settings (minimum cation in BOLD. Only 10 samples of 8 sequences for clustering), the were identified successfully to the number of OTUs was reduced to species and two to the genus level, 5124, and 685 OTUs could be whereas 21 samples represent spe- assigned to a taxonomic name. cies absent from BOLD database. Of these, 322 were identified Zoltán Csabai (in the background) These results, even on such a small as macroinvertebrates with an during his STSM number of individuals, clearly identification threshold of 95%. Zoltán Csabai showed the importance of future Metabarcoding allowed assigning a research on water mites for filling total of 659 taxa on the taxonomic Title: Linking morphology and the gaps in the DNA barcodes level of order (with a 90% of iden- barcoding: Building a compre- reference library. tity), 432 with a 94% (family level) hensive DANN barcode library and 349 with 97% (species level). of aquatic macroinvertebrates of Many of the identified species Cyprus Marta Sáinz- are known to be very difficult to Home/Host country: Hungary/ Bariáin identify at the larval stage, espe- Cyprus The aim of the proposed STSM Title: cially those belonging to the orders Freshwa- of Diptera, Trichoptera, Plecop- project was to build a DNA ter ecosystems tera, Coleoptera and Ephemer- barcode reference library for the monitoring with optera. Analyses showed that the Cyprus aquatic macroinvertebrate eDNA: metabar- recovered communities correlated fauna by barcoding of morpho- coding vs. tradi- with abiotic factors. PERMANO- logically identified specimens tional taxonomy VA and NMDS analyses showed derived from new collections and Home/Host country: Spain/ that communities from higher conserved samples, thereby facil- Netherlands altitude sampling sites respectively itating the development of DNA smaller or larger rivers formed dis- based water quality assessment The main objective of this tinct clusters. This further proves methods in Cyprus and in the STSM was to perform the bioin- the ability of the used technique to eastern Mediterranean region. formatic analyses of a DNA me- describe ecological conditions, and Total of 28 new samples were tabarcoding dataset generated by facilitates the use for future ecolog- collected and preserved for DNA Illumina sequencing. The sequenc- ical studies and biomonitoring. studies, in addition of 36 previ- es of 114 samples of eDNA from ously collected samples. By mor- 19 Spanish rivers were analyzed to phological identification of 2762 verify the feasibility of this method individuals, 102 species have been as a substitute for the ecological detected (Mollusca 12, Crustacea quality assessment of water bodies 9, Heteroptera 10, Coleoptera 35, by classical taxonomy of aquatic Odonata 21, Trichoptera 15). The macroinvertebrates. Preliminary coverage of the species, compared results showed that after quality to the known fauna of Cyprus filtering, a total of 55383 OTUs were among 50-100%. The occur- were obtained. These OTUs could rence of six species (2 Coleoptera, be assigned to a total of 4432 2 Heteroptera and 2 Trichoptera)

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were reported within this project the Canary Islands which have a specimens from the Nereididae for the first time from Cyprus. At rich biodiversity not yet barcoded family. Bad weather conditions least three individuals (if avail- or comprehensively investigated. affected the sampling success at El able) per species (altogether 466 Based on previous studies with Hierro, so only one specimen be- individuals) were separated and other benthic invertebrates, par- longing to Perinereis cultrifera was labeled for DNA barcoding. The ticular focus was given to known found. It was planned to perform STSM project initiated a wide European cryptic complexes that the barcoding analysis of collected international collaboration not are also reported in the Macarone- specimens in the upcoming period. only between the applicant and his sia such as Eumida sanguinea, Eu- lab members and the host lab, but lalia clavigera, Platynereis dumerilii Belma also involving further taxonomic and Perinereis cultrifera since there Kalamujic Stroil experts and laboratories for molec- was a high probability of finding Title: ular identification. This expertise unknown lineages within those Unveiling concentration may open the door species complexes, even between the macrozooben- not only to create a DNA reference the different islands. Sampling thos diversity of library but exploring plausible was done in the intertidal zone the Skakavac wa- cryptic taxa, extending our knowl- (tide-dependent) in the rocks and terfall hydrosys- edge about the phylogeography of among the seaweed meadows, tem using DNA barcoding some species groups or disentan- within each island. At Fuerteven- Home/Host country: gling long standing taxonomical tura, some specimens Bosnia and problems. belonging to Platynereis dumerilii Herzegovina/Germany were found, while the abundance Marcos and diversity of amphipods and The aim of the proposed STSM Teixeira isopods were very good. Orzola was hands-on knowledge and technology transfer in the field of Title: (Lanzarote) was a fantastic place to Rich and get large quantities of polychaete DNA barcoding through case- hidden biodi- specimens (more than 20) from the study of macrozoobenthos (MZB) versity not yet Perinereis cultrifera and Platynereis specimens collected from the barcoded in the dumerilii species. hydrosystem of the Skakavac wa- Canary archipel- A few other specimens from the terfall, the second tallest waterfall ago (Spain) as an Nereididae, Maldanidae and Capi- in Europe. The poor state of pre- opportunity to enrich the DANN telidae family were also found. served samples somewhat hindered barcode reference library for Euro- Tenerife island was home to several the success of barcoding. However, pean 48 consensus barcodes that com- Home/Host country: cryptic polychaete species such as: Portugal/ clavigera, Perinereis cultrif- ply with BOLD requirements for Spain era, Platynereis dumerilii, Eurythoe acceptable barcode were retrieved. complanata and a few other species The barcodes represented a wide The objective of this grant was still yet to be identified from the array of MZB groups: Plecop- to contribute to the DNA barcode Nereididae family. Grand Canaria tera, Ephemeroptera, Trichoptera, reference library and investigate sampling produced similar results Diptera, Coleoptera, Myriapoda, the biodiversity of European to those from Tenerife. At La Arhynchobdellida and Amphip- Polychaeta by sampling, sequenc- Palma, high quantities of Eulalia oda. Most of the obtained query ing COI barcodes and describing clavigera were found, as well as sequences did not find match any intertidal polychaete species in Platynereis dumerilii and a few barcode reference in BOLD prov-

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ing that the results of this STSM Urban Aufschnaiter was possible in 85 cases in mor- could contribute to filling the Title: Molecular Analysis and phological analysis. Contrary, 172 existing gaps in the database. Method Comparison of Macrozoo- OTUs were identified to species Although a small sample set benthos Samples within the WFD level with molecular analysis only. was analyzed, it gave very interest- Context On average 34.8 species were ing results in terms of haplotype Home/Host country: Austria/ found by both approaches. The diversity. Four analyzed chirono- Germany number of species found only by mid specimens revealed two differ- morphological identification was ent species and the presence of two The purpose of this STSM was low with an average of 6.6. The haplotypes at the same sampling to analyze in detail stream mac- results suggest that combining location. The same scenario was roinvertebrate bioassessment data morphological and molecular Ecdyonurus seen for mayfly genus . obtained from five Upper Austrian approaches may lead to increased Five analyzed specimens stream sites through morphological certainty on species detection. Ad- grouped into two species, with as well as different DNA-metabar- ditional molecular analysis might one of them displaying haplotype coding approaches. The data set be useful in detecting otherwise diversity at the same sampling was generated within an EU Water hard to identify species. site. Haplotype diversity was also Framework Bioassessment project Johannes seen in specimens of the stonefly that was initiated by the adminis- Rusch genus Protonemura. All haplotypes tration of Upper Austrian (Oberös- detected in this STSM were previ- terreich) Government. The STSM Title: eDNA mon- ously absent from BOLD database. provided strong evidence that The fact that such genetic itoring of native sorted bulk samples yield the best and invasive fresh- diversity was seen in a rather small results when interested in mac- number of samples, originating water crayfish in rozoobenthic taxa, yet, it is also the Czech Repub- from the same locations points to the by far most time-consuming the need of continuing the efforts lic – validation of two species-spe- approach. However, also the very cific qPCR assays against a unique to unveil its MZB biodiversity as ‘quick and dirty’ unsorted bulk well as to take further steps in pro- panel of freshwater crayfish species samples yielded results, which were Home/Host country: tecting the living world of this nat- Norway/ quite consistent with the morpho- Czech Republic ural monument. Previous absence logical data set and might thus be of barcodes generated through this a rapid and pragmatic alternative. The purpose of the STSM was STSM from the BOLD database In general, comparison of the further underlines this. to test two species-specific crayfish morphological and the molecular eDNA assays (for Astacus astacus taxa lists showed clear differences and Pacifastacus leniusculus) against between the two methods. a unique panel of DNA isolates Morphological identification from 29 crayfish species. This revealed 141 taxa. The molecular collection of isolates which is avail- approach yielded 292 OTUs (not able in prof. Adam Petrusek’s lab including duplicates). A total of included almost all known crayfish 59 taxa were only included in the taxa from European waters, as well morphological taxa list and 89 numerous crayfish species common were found by both approaches. in the ornamental pet trade. Identification down to species level

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The testing of assay specificity Kálmán Tapolczai STSMs regarding the 4th Joint Danube Survey (JDS4): was carried out to contribute to an Title: Unleashing DNA metabar- improved validation of the assays. coding for biomonitoring: strat- Emre Keskin, Esra Mine Ünal Validation of two, then unpub- egies to avoid major taxonomical and Aysegül Er lished, modifications of qPCR and bioinformatical biases limiting assays was successful for the species Home/Host country: molecular indices capacities Turkey/Slovakia specific detection of two cray- Home/Host country: Hungary/ fish species through testing them France against a unique panel of DNA The aim of DNAqua-Net team extracts from a large collection of was to collect samples of eDNA The proposed STSM was at previously agreed sites within freshwater crayfish species, availa- designed to facilitate writing of ble at prof. Petrusek’s laboratory. the framework of JDS4, as well the joint publication on the data as to train the national teams on Both assays amplified DNA collected through previous stud- isolates of their respective target the methodology of collecting ies. So far, taxonomy-free indices and processing the samples for species. Against the 28 non-target have proved to be an efficient Asta- eDNA analysis. The sampling was isolates, only one assay ( strategy to keep biological infor- cus astacus) displayed non-target performed at seven stations in mation carried by OTUs whose Ukraine, Romania, Bulgaria and amplification. However, as this taxonomic assignment is otherwise occurred in only one of the two Serbia. sampling. For each sam- not possible because of the incom- pling, three replicates were taken, isolates and a reexamination plete reference library. This STSM using Geneious software showed according to the protocols. It was project proposed to apply a similar planned to conduct further steps an amplification of this species strategy on ISU and ESV data, in (Austropotamobius pallipes) with (isolation, PCR, preparation for order to limit the bias associated HTS) in Ankara, Turkey. eDNA the used assay to be highly unlike- to OTU clustering and to improve ly, this amplification is considered isolates will be biobanked at the the reproducibility and standard- ZFMK (Zoological Research a result of contamination. A joint ization of biomonitoring indices publication was drafted during the Museum Alexander Koenig). based on the microalgae group, Additional eDNA samples were STSM and further plans to use the diatoms (Bacillariophyta phylum). eDNA method as a screening tool collected by cooperating projects The results of this STSM were (Interreg MEASURES, IAD and in the Czech Republic were made published in the Frontiers in Ecol- to detect early onset invasions the NGO VigiLIFE) before and ogy and Evolution: doi: 10.3389/ after the JDS4 eDNA survey. of non-native crayfish in highly fevo.2019.00409. susceptible habitats such as urban Further detailed information on ponds where the synergy between applied methodologies and ob- the two working groups can lead tained results of conducted STSMs to further collaboration. could be found at: http://dnaqua.net/stsms/.

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es (Diptera, Chironomidae): Ska- ITC grants dar Lake versus European barcode Publications library” at the iBoL conference. A total of 10 ITC conference Given the large number of publica- grants were awarded in the current tions that appear from our COST GP. Our Action was represented in Action please visit: various conferences with five ECI https://dnaqua.net/publications participations in the 8th Interna- tional Barcode of Life Conference, two in the Symposium for Euro- pean Freshwater Sciences, two in the 18th International Colloquium on Amphipoda and one in the 7th European Phycological Congress. Mrs Athina Papatheodoulou during her poster presentation entitled “Freshwater macroinver- tebrate barcoding in Cyprus. First steps and new records” in the SEFS conference A remaining budget for up to 10 grants are available until end of Dr Tomasz Mamos from the April 2020. University of Lodz after his oral For further information you presentation “Comparative phy- can visit our website or contact the [email protected] logeography of freshwater inver- tebrates with different dispersal potential in northern part of Car- JDS4 pathians” at the iBoL conference. For an overview on the Joint Danube Survey (JDS) activities we refer to: https://www.icpdr.org/main/ publications/danube-watch-2-2019- re-discover-danube-public-involve- ment-awareness-jds4

Mr Piotr Gadawski from the University of Lodz during the e-post and lighting talk “Integra- tive taxonomy of non-biting midg-

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