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1. A SWOT analysis of the IUCN criteria for naturebased solutions to support European Green Deal goals yielded a broad spectrum of information on the MERLIN freshwater restoration case studies. 2. This information provides the necessary ingredients to draft an optimisation strategy for the MERLIN case studies on themes including multiple goals, society, restoration techniques, economy, policy and regulations. 3. This information offers a basis for peer-to-peer learning between the case studies and those in other regions. 4. The optimisation strategies provide new insights and broaden perspectives on restoration actions in individual case studies. 5. Identifying the potential for multiple Green Deal goal achievements will initiate broader dialogues with stakeholders and provide new incentives for restoration with multiple benefits for local people. 6. Capacity building on financial arrangements will create new opportunities for restoration actions. 7. Clear and agile policy arrangements are needed to enhance stakeholder participation in restoration actions. 8. The visioning, planning and evaluation of restoration actions are long-term activities that benefit from adaptive approaches and sound longterm monitoring programs on multiple aspects of biophysical and socio-ecological systems.

MOBILISE WG3 WIKI

Recent advances in molecular biomonitoring open new horizons for aquatic ecosystem assessment. Rapid and cost-effective methods based on organismal DNA or environmental DNA (eDNA) now offer the opportunity to produce inventories of indicator taxa that can subsequently be used to assess biodiversity and ecological quality. However, the integration of these new DNA-based methods into current monitoring practices is not straightforward, and will require coordinated actions in the coming years at national and international levels. To plan and stimulate such an integration, the European network DNAqua-Net (COST Action CA15219) brought together international experts from academia, as well as key environmental biomonitoring stakeholders from different European countries. Together, this transdisciplinary consortium developed a roadmap for implementing DNA-based methods with a focus on inland waters assessed by the EU Water Framework Directive (2000/60/EC). This was done through a series of online workshops held in April 2020, which included fifty participants, followed by extensive synthesis work. The roadmap is organised around six objectives: 1) to highlight the effectiveness and benefits of DNA-based methods, 2) develop an adaptive approach for the implementation of new methods, 3) provide guidelines and standards for best practice, 4) engage stakeholders and ensure effective knowledge transfer, 5) support the environmental biomonitoring sector to achieve the required changes, 6) steer the process and harmonise efforts at the European level. This paper provides an overview of the forum discussions and the common European views that have emerged from them, while reflecting the diversity of situations in different countries. It highlights important actions required for a successful implementation of DNA-based biomonitoring of aquatic ecosystems by 2030.

Roadmap for implementing environmental DNA (eDNA) and other molecular monitoring methods in Finland – Vision and Action Plan for 2022-2025 Technological development in molecular methodology has been extremely fast in the past two decades, and groundbreaking new approaches have been introduced. It is now possible to detect and quantify DNA or RNA of target species or even map the whole species community in environmental samples of water, sediment, soil, air or assemblages of whole organisms. Moreover, the costs of high-throughput sequencing and other advanced molecular methods have decreased and methodological pipelines from sampling to data analysis developed sufficiently to allow large-scale, routine application of the new methods in environmental monitoring. This presents a huge opportunity to improve the coverage, accuracy and cost-efficiency of monitoring, enabling a much more complete picture of biodiversity and the state of the environment and their trends. As the new European Biodiversity Strategy for 2030 and other international policies to halt biodiversity loss and the degradation of habitats are translated into concrete measures, the quality of the monitoring data will play a crucial role in determining their success or failure. In this roadmap commissioned by the Finnish Ministry of the Environment, we assess the state-of-the-art in molecular monitoring methods in Finland within the international context, identify challenges and development areas that remain to be addressed and propose an action plan for promoting the coordinated implementation of molecular methods in national monitoring programs. Apart from the most recent scientific literature, our analysis is based on survey results, direct enquiries and interviews. Participation of the national community of experts from different sectors was enabled and invited at several stages of the roadmap preparation. Internationally, molecular monitoring methods are being actively developed and are routinely implemented in monitoring across different taxa and ecosystems. In Finland, molecular monitoring methods have been tested and piloted by all major institutions responsible for environmental monitoring, and the methods are already applied routinely in the monitoring of individual game species such as the wolf and European and Canadian beaver. However, other areas such as the monitoring of biodiversity, threatened species, non-mammalian invasive species or emerging plant or animal pests remain less developed, and national efforts and expertise are scattered across different organizations. Funding and know-how are perceived as the most important factors limiting molecular monitoring method implementation. We estimate that extensive, routine implementation of a wide range of molecular monitoring methods is conceivable in Finland before 2030. As the primary development areas for reaching this goal, we identify (i) international coordination and standard development, (ii) networking across sectors, (iii) education, (iv) infrastructure, (v) reference sequence libraries and the mapping of whole genomes, and (vi) modelling and analysis tool development. For concrete actions in 2022–2025, we propose (1) a cross-governmental funding instrument, (2) a permanent working group responsible for national and international coordination, (3) a national network and (4) an online platform to enhance interaction and knowledge transfer, as well as (5) a national data management system with collectively agreed data and metadata formats and standards. ---------- Kansallinen tiekartta ympäristö-DNA:n ja muiden molekyylibiologisten seurantamenetelmien käyttöönotolle – visio ja toimenpidesuunnitelma vuosille 2022-2025 Molekyylibiologisten menetelmien teknologinen kehitys on ollut ennennäkemättömän nopeaa kahden viime vuosikymmenen aikana. Uudet menetelmät mahdollistavat kohdelajien DNA:n tai RNA:n havaitsemisen ja runsausmäärityksen tai koko eliöyhteisön kartoittamisen esimerkiksi vesi-, sedimentti-, maaperä- tai ilmanäytteistä tai kokonaisia yksilöitä sisältävistä kokoomanäytteistä. Massiivisen rinnakkaissekvensoinnin ja muiden menetelmien kustannukset ovat merkittävästi laskeneet ja menetelmäketjut näytteenotosta tulosten tulkintaan kehittyneet asteelle, joka mahdollistaa niiden laajamittaisen, rutiininomaisen käytön ympäristön seurannassa. Uusien menetelmien avulla voimme parantaa seurannan kattavuutta, tarkkuutta ja kustannustehokkuutta ja siten täydentää seurannan kautta muodostuvaa kuvaa luonnon monimuotoisuudesta ja sen muutoksista. Tälle tiedolle on suuri tarve – laadukas seuranta on keskeinen edellytys sille, että EU:n uuden biodiversiteettistrategian ja muiden luontokadon ja elinympäristöjen tilan huonontumisen pysäyttämiseen tähtäävien kansainvälisten sitoumusten toimeenpano onnistuu. Tässä ympäristöministeriön tilaamassa tiekartassa arvioimme molekyylibiologisten seurantamenetelmien nykytilaa Suomessa osana laajempaa kansainvälistä kenttää, tunnistamme huomiota vaativia haasteita ja kehityskohteita ja ehdotamme konkreettisia toimenpiteitä molekyylibiologisten seurantamenetelmien koordinoidun käyttöönoton edistämiseksi lähivuosien aikana. Selvityksemme perustuu uusimman tieteellisen kirjallisuuden lisäksi kyselytutkimukseen sekä suoriin tiedusteluihin ja haastatteluihin. Yhteiskunnan eri sektoreita edustava kansallinen asiantuntijayhteisö osallistui tiekartan valmisteluun työn eri vaiheissa. Molekyylibiologisia seurantamenetelmiä kehitetään parhaillaan aktiivisesti ympäri maailmaa eri eliöryhmille ja ekosysteemeille, ja yksittäisiä menetelmiä on useissa maissa otettu myös rutiininomaiseen käyttöön. Suomessa menetelmiä on kehitetty ja pilotoitu kaikissa keskeisissä ympäristön seurantaa koordinoivissa laitoksissa, ja yksittäisten riistaeläinten kuten suden ja kanadan- ja euroopanmajavan seurannassa ne ovat jo rutiinikäytössä. Biodiversiteetin, uhanalaisten lajien, vieraslajien (nisäkkäitä lukuun ottamatta) ja muiden haitallisten lajien kansallisessa seurannassa molekyylibiologisten menetelmien käyttö on kuitenkin vielä kokeiluasteella, ja kehittämishankkeiden ja asiantuntijuuden kenttä on hajanainen. Riittämätöntä rahoitusta ja osaamista pidetään alan asiantuntijoiden keskuudessa tärkeimpinä menetelmien käyttöönottoa rajoittavina tekijöinä. Arviomme mukaan laaja kirjo molekyylibiologisia seurantamenetelmiä olisi mahdollista ottaa laajamittaiseen rutiininomaiseen käyttöön vuoteen 2030 mennessä. Tärkeimmiksi kehityskohteiksi nousivat (i) kansainvälinen koordinaatio ja menetelmien standardointi, (ii) organisaatioiden ja sektoreiden välinen verkostoituminen, (iii) koulutus, (iv) infrastruktuuri, (v) referenssisekvenssikirjastot ja kokonaisten genomien kartoittaminen sekä (vi) malli- ja analyysityökalujen kehittäminen. Konkreettisiksi toimenpiteiksi vuosille 2022-2025 esitämme (1) poikkihallinnollista rahoitusohjelmaa molekyylibiologisten seurantamenetelmien käyttöönottoa edistäville tutkimus- ja kehityshankkeille, (2) pysyvää työryhmää kansallisen ja kansainvälisen koordinaation edistämiseksi, (3) olemassa olevan kansallisen asiantuntijaverkoston laajentamista, (4) internet-pohjaista alustaa vuorovaikutuksen ja tiedonjaon tehostamiseksi sekä (5) kansallista, yhdessä sovittuja data- ja metadatastandardeja noudattavaa molekyylibiologisten seuranta-aineistojen tiedonhallintajärjestelmää.

Efforts to renew marine ecosystem monitoring to include advanced technology and cost-effective methods have been repeatedly called for. The current environmental legislation in European Union (EU) requires also ecosystem monitoring beyond the scope of conventional methods and sampling strategies. Despite several studies showing the benefits of new methods, the progress to adopt the methods in national monitoring programmes under legal requirements has been slow. In this study, we have reviewed the current use of a set of methods in marine monitoring programmes under the EU marine strategy framework directive (MSFD), which calls for a holistic view of the marine environment and thus requires tens of monitoring parameters by different methods. Here we assess how widely the new methods are being adopted in the EU member states implementing the MSFD. Our results show a relatively high adoption rate for certain methods, while others are widely ignored. We compare the results also with the monitoring strategies of the four regional sea conventions. We argue that the adoption of methods in European and regional programmes may positively influence the national use of new methods.

In this talk I explore how to advance molecular methods from tools in the research domain to routine use in national biomonitoring. I outline the necessity of common guidance, networks, international pilot studies and cooperation with officials to achieve the goal of method uptake into routine use. Lastly, I will explain the role that international method standardization plays in speeding up the uptake process of molecular methods into routine biomonitoring.

Bulk macroinvertebrate DNA metabarcoding is proven a useful tool for routine assessment of freshwater ecosystems. Following a small scale study in 2017, using 18 samples from Finland Elbrecht et al. 2017, sampling and metabarcoding efforts were extended to a total of 297 official monitoring samples from Denmark, Sweden, Norway, Iceland, and Finland as part of the SCANDNAnet project. Macroinvertebrates from these samples were morphologically identified and subsequently DNA metabarcoding was performed. While the project was ultimately successful, documenting generally congruence between identification methods, we encountered a wide range of unexpected challenges when scaling sample throughput Meissner et al. 2020. In this technical talk we highlight major roadblocks and potential solutions to improve turnaround time and reliability of future large scale DNA based monitoring efforts. We identify a need for tighter quality control and quality assurance in laboratory procedures, and exemplify the benefits of laboratory automation when processing hundreds of samples within a single week. We further discuss the need for scalable computational infrastructure, reliable sequencing providers and well-curated, purpose driven, macroinvertebrate reference databases for the Fennoscandian region. The lessons learned from the SCANDNAnet project will be indispensable in building a reliable infrastructure for a future DNA based monitoring of macroinvertebrates across Fennioscandia and beyond.

This book represents a synthesis of knowledge and best practice in the field of DNA-based biomonitoring at the time of writing. It has been written with end-users of molecular tools in mind, as well as those who are new to the field in research settings and are looking to gain an overall grounding in the subject area. For each of the main types of sample (water, soil / sediment, bulk invertebrates and diatoms), and for each stage of the field and laboratory processes, we outline key considerations, decisions that need to be made, factors that might influence those decisions, and trade-offs inherent in the choices made. We hope that this will help users, practitioners, and those commissioning DNA-based monitoring programmes to navigate this large field and critically evaluate the strengths and weaknesses of different analysis workflows based on context, project aims and available resources. DNA-based methods for species detection and identification have revolutionised our ability to assess biodiversity in terrestrial, freshwater and marine ecosystems. Starting from the seminal study that used eDNA to detect invasive american bullfrogs in France (Ficetola et al. 2008), research conducted over the last decade has demonstrated the power of these approaches for surveying a wide range of species and groups. Early applications included the use of eDNA to monitor Asian Carp in the USA (Jerde et al. 2013). Following heavy scrutiny, the method was eventually adopted, and is still employed today by the United States Geological Survey (USGS). A flurry of research followed, with tests designed for many threatened and invasive species including New zealand mudsnails (Goldberg et al. 2013), american crayfish (Geerts et al. 2018), gammarids (Blackman et al. 2017), and great crested newts (Biggs et al. 2015). The great crested newt eDNA test has been employed for regulatory monitoring in the UK since 2014. During the same time period, there was a proliferation of research studies that used high-throughput sequencing approaches to describe whole communities of organisms from mixed species and environmental samples, using an approach termed DNA metabarcoding (Taberlet et al. 2012c). As the field developed fast and the approaches were applied to a wide range of research and monitoring objectives, a high level of methodological variation was introduced at all stages of the workflow (Seymour 2019). Thus, while a significant level of consensus on scientific best-practice now exists in many areas, this may not be readily discerned from the now-extensive body of research literature. As environmental practitioners and policy makers are now increasingly starting to integrate DNAbased methods into routine monitoring applications including protected species licensing1, statutory monitoring2 (Hänfling et al. 2016) and environmental impact assessment3, various national and international efforts have been undertaken to standardise methods and integrate them into monitoring frameworks (Pilliod et al. 2019, Loeza-Quintana et al. 2020, Minamoto et al. 2021, Pawlowski et al. 2020a4). In Europe, the EU COST Action DNAqua-Net (Leese et al. 2018) has been working towards incorporating molecular monitoring tools for Biological Quality Elements (BQEs, e.g., fish, macroinvertebrates and phytoplankton- benthos) into the Water Framework Directive (WFD, 2000/60/EC)5 and the Marine Strategy Framework Directive (MSFD, 2008/56/EC)6. Thus, emphasis now shifts from fundamental research to robust and efficient application of DNAbased methods for operational use at large scales. This requires that scientific robustness is balanced with consideration of the practical realities faced by environmental managers. Moreover, there is increased need for strong quality assurance in a setting where non-expert field samplers and commercial laboratories are involved with the generation of data that non-specialist decision-makers then rely on to inform potentially costly action (or non-action). This places increased emphasis on robustness, replicability, traceability and ease-of-use, which may not always be the central focus of studies carried out in the academic research environment. This document aims to summarise the scientific consensus relating to every step of the field and laboratory workflows involved in the most common types of samples and analyses. We do not go into great detail regarding bioinformatics (computational processing of sequence data) and data analysis since these are extensive topics in their own right. We uniquely set the field and lab steps in the context of the practical and logistical constraints faced by environmental managers in terms of cost, logistics, safety, ease-of-use, and quality assurance, highlighting key decisions to be made and the inherent trade-offs associated with the various options. We hope that this will support non-experts, and those new to the field, to navigate the key considerations associated with planning or evaluating monitoring programmes using DNA-based monitoring methods. Additionally, it will aid decision-makers in writing and evaluating tenders and proposals, ensuring that the methods used for a given project are fit-for-purpose and that results are correctly interpreted. Alongside the many areas of emerging consensus, there remain some areas where further research is still required to balance scientific best-practice with the constraints and priorities of end-users. We hope that by shining a light on the importance of these issues, the research community will be encouraged to address them. More generally, we hope to inspire researchers in this now highly-applied scientific field to consider end-user constraints when designing and implementing research projects. This will help to accelerate uptake by users and maximise the impact of research. DNA-based bioassessment methods continue to evolve, and there are several emerging technologies that show exciting promise to move beyond even what is possible today. Examples include in-field sequencing using the MinION device from Oxford Nanopore Technologies (Pomerantz et al. 2018, Davidov et al. 2020, Hatfield et al. 2020), PCR-free metagenomic approaches (Bista et al. 2018, Giebner et al. 2020) and CRISPR for rapid detection of species, which is particularly relevant for invasive an non-native species monitoring (Williams et al. 2019, 2020). We recognise the potential of these methods, but do not consider them in detail here, since they are not yet far enough developed for routine application. EU COST Action DNAqua-Net (CA 15219 a COST (European Cooperation in Science and Technology). DNAqua-Net, European Union Horizon 2020 Published Refereed Current 14.a Mature Multi-organisational International Genetic differentiation Species distributions Method

Over 75% of all lakes and 40% of all rivers in the EU are found in the Nordic countries. Freshwater biomonitoring according to the EU Water Framework Directive (WFD) has been adopted by all Nordic countries and forms an integral part of management efforts to preserve and restore the ecological quality of freshwaters and their ecosystem services. Current identifications of organisms used in WFD biomonitoring are based on expert morphological identification; an approach that is time consuming and prone to errors. Molecular identification methods could alleviate many problems but thus far have not been rigorously tested for use in routine monitoring. Using a questionnaire, we consulted Nordic experts to assess opinions on the applicability of these methods in routine biomonitoring. Further, we validated the practical use of molecular metabarcoding identification for use in freshwater aquatic macroinvertebrate monitoring in all Nordic countries. A total of 297 waterbodies including both lakes and streams were sampled which constitutes the single largest validation test of the method so far. Experts in each country sampled macroinvertebrates according to their national protocols with only minor modifications and samples were analyzed using a standardized laboratory protocol. The results of this study indicate that in most cases identifications using molecular DNA-based methods were highly congruent with traditional expert-based identifications. However, the study also revealed the importance of using unified guidance documents. Several samples showed signs of DNA degradation, affecting reliability of the method. Nordic experts generally recognized molecular identification methods as playing a major role in future biomonitoring, if issues associated with the current lack of unified methodology are resolved. Based on this study, we suggest that concerted Nordic or European efforts towards implementation and standardization of DNA-based methodology should be undertaken to swiftly ensure the use of this promising tool into WFD compliant monitoring. © Nordic Council of Ministers 2020

A multitude of anthropogenic pressures deteriorate the Baltic Sea, resulting in theneed to protect and restore its marine ecosystem. For an efficient conservation,comprehensive monitoring and assessment of all ecosystem elements is of fundamentalimportance. The Baltic Marine Environment Protection Commission HELCOMcoordinates conservation measures regulated by several European directives. However,this holistic assessment is hindered by gaps within the current monitoring schemes.Here, twenty-two novel methods with the potential to fill some of these gaps andimprove the monitoring of the Baltic marine environment are examined. We asked keystakeholders to point out methods likely to improve current Baltic Sea monitoring. Wethen described these methods in a comparable way and evaluated them based ontheir costs and applicability potential (i.e., possibility to make them operational). Twelvemethods require low to very low costs, while five require moderate and two high costs.Seventeen methods were rated with a high to very high applicability, whereas fourmethods had moderate and one low applicability for Baltic Sea monitoring. Methodswith both low costs and a high applicability include the Manta Trawl, Rocket, SedimentCorer, Argo Float, Artificial Substrates, Citizen Observation, Earth Observation, theHydroFIARpH system, DNA Metabarcoding and Stable Isotope Analysis. © 2020 Mack, Attila, Aylagas, Beermann, Borja, Hering, Kahlert, Leese, Lenz, Lehtiniemi, Liess, Lips, Mattila, Meissner, Pyhälahti, Setälä, Strehse, Uusitalo, Willstrand Wranne and Birk. Funding: This work resulted from the BONUS FUMARI project in collaboration with BONUS SEAM project, funded by BONUS (Art. 185), which is jointly funded by the EU, the Academy of Finland and the Swedish Research Council Formas. Furthermore, funding was received from the BmBF project MicroCatch_Balt, grant number 03F0788A and EA was supported by the Red Sea Research Center at KAUST. Data on ARMS and ASUs were obtained by DEVOTES (DEVelopment Of innovative Tools for understanding marine biodiversity and assessing good Environmental Status) project, funded by the European Union under the 7th Framework Programme, “The Ocean of Tomorrow” Theme (grant agreement no. 308392), www.devotes-project.eu.

River ecosystems receive and process vast quantities of terrestrial organic carbon, the fate of which depends strongly on microbial activity. Variation in and controls of processing rates, however, are poorly characterized at the global scale. In response, we used a peer-sourced research network and a highly standardized carbon processing assay to conduct a global-scale field experiment in greater than 1000 river and riparian sites. We found that Earth’s biomes have distinct carbon processing signatures. Slow processing is evident across latitudes, whereas rapid rates are restricted to lower latitudes. Both the mean rate and variability decline with latitude, suggesting temperature constraints toward the poles and greater roles for other environmental drivers (e.g., nutrient loading) toward the equator. These results and data set the stage for unprecedented “next-generation biomonitoring” by establishing baselines to help quantify environmental impacts to the functioning of ecosystems at a global scale. This research was supported by awards to S.D.T. from the Ecuadorian Ministry of Science [Secretaría de Educación Superior Ciencia, Tecnología e Innovación (SENESCYT)] through the PROMETEO scholar exchange program, the Oakland University Research Development Grant program, and a Huron Mountain Wildlife Foundation research grant. N.A.G. was supported by the U.S. Department of Energy’s Office of Science, Biological and Environmental Research. Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the U.S. Department of Energy under contract DE-AC05-00OR22725. We are grateful for open-access-publishing funds from Kresge Library at Oakland University and Queen’s University Belfast. This research was supported by awards to S.D.T. from the Ecuadorian Ministry of Science [Secretaría de Educación Superior Ciencia, Tecnología e Innovación (SENESCYT)] through the PROMETEO scholar exchange program, the Oakland University Research Development Grant program, and a Huron Mountain Wildlife Foundation research grant. N.A.G. was supported by the U.S. Department of Energy’s Office of Science, Biological and Environmental Research. Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the U.S. Department of Energy under contract DE-AC05-00OR22725. We are grateful for open-access-publishing funds from Kresge Library at Oakland University and Queen’s University Belfast.