Roadmap for implementing environmental DNA (eDNA) and other molecular monitoring methods in Finland Vision and action plan for 2022–2025 Veera Norros, Tiina Laamanen, Kristian Meissner, Terhi Iso-Touru, Aapo Kahilainen, Sirpa Lehtinen, Katileena Lohtander-Buckbee, Henrik Nygård, Taina Pennanen, Marja Ruohonen-Lehto, Päivi Sirkiä, Sanna Suikkanen, Mikko Tolkkinen, Eeva Vainio, Sannakajsa Velmala, Kristiina Vuorio and Petteri Vihervaara Finnish Environment Institute Reports of the Finnish Environment Institute 20 | 2022 Reports of the Finnish Environment Institute 20 / 2022 Roadmap for implementing environmental DNA (eDNA) and other molecular monitoring methods in Finland Vision and action plan for 2022–2025 Veera Norros, Tiina Laamanen, Kristian Meissner, Terhi Iso-Touru, Aapo Kahilainen, Sirpa Lehtinen, Katileena Lohtander-Buckbee, Henrik Nygård, Taina Pennanen, Marja Ruohonen-Lehto, Päivi Sirkiä, Sanna Suikkanen, Mikko Tolkkinen, Eeva Vainio, Sannakajsa Velmala, Kristiina Vuorio and Petteri Vihervaara Reports of the Finnish Environment Institute 20 | 2022 Finnish Environment Institute Biodiversity centre Authors: Veera Norros1), Tiina Laamanen1), Kristian Meissner1), Terhi Iso-Touru2), Aapo Kahilainen1), Sirpa Lehtinen1), Katileena Lohtander-Buckbee1), Henrik Nygård1), Taina Pennanen2), Marja Ruohonen-Lehto1), Päivi Sirkiä1), Sanna Suikkanen1), Mikko Tolkkinen3), Eeva Vainio2), Sannakajsa Velmala2), Kristiina Vuorio1), Petteri Vihervaara1) 1) Finnish Environment Institute (SYKE) 2) Natural Resources Institute Finland (Luke) 3) Finnish Environment Institute (SYKE) / AFRY Subject Editor: Terhi Ryttäri Financier/commissioner: Finnish Ministry of the Environment Latokartanonkaari 11, 00790 Helsinki, Finland, Phone +358 295 251 000, syke.fi Layout: Veera Norros Cover photo: Adobe Stock The publication is available on the internet (pdf): syke.fi/publications | helda.helsinki.fi/syke ISBN 978-952-11-5482-9 (PDF) ISSN 1796-1726 (online) Year of issue: 2022 Reports of the Finnish Environment Institute 20/2022 3 Abstract 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. Keywords: biodiversity, environmental monitoring, metabarcoding, next-generation sequencing, monitoring strategy 4 Reports of the Finnish Environment Institute 20/2022 Tiivistelmä 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. Asiasanat: biodiversiteetti, ympäristön seuranta, metaviivakoodaus, uuden sukupolven sekvensointi, seurannan strategia Reports of the Finnish Environment Institute 20/2022 5 Sammandrag Färdplan för ibruktagande av miljö-DNA (eDNA) och andra molekylära övervakningsmetoder i Finland – Vision och handlingsplan för 2022-2025 Den teknologiska utvecklingen av molekylära metoder har varit extremt snabb de senaste två årtiondena och nya tillvägagångssätt har introducerats. Det är nu möjligt att detektera och kvantifiera DNA eller RNA från målarter eller till och med från hela artsamhällen i vatten-, jord-, luft- eller organismprover. Kostnaderna för högkapacitetssekvensering och andra avancerade molekylära metoder har sjunkit och de metodologiska rutinerna från provtagning till dataanalyser har utvecklats till ett stadie som möjliggör ett storskaligt och rutinmässigt ibruktagande av dessa nya metoder inom miljöövervakningen. Detta erbjuder en stor möjlighet till förbättring av täckning, precision och kostnadseffektivitet av övervakningen och möjliggör en mer komplett bild av den biologiska mångfalden och miljötillståndet samt trenderna inom dessa. När den nya europeiska strategin för biologisk mångfald 2030 och andra internationella avtal för att stoppa förlusterna av biologisk mångfald och habitatdegradering ska översättas till konkreta handlingar, kommer kvaliteten av övervakningsdatan att ha en betydande roll för att avgöra huruvida man lyckats eller misslyckats med målsättningarna. I denna färdplan, gjord på uppdrag av miljöministeriet i Finland, bedömer vi utvecklingsnivån av molekylära övervakningsmetoder i Finland i ett internationellt sammanhang, identifierar utmaningar och utvecklingsområden som bör beaktas samt föreslår en handlingsplan för att främja ett koordinerat ibruktagande av molekylära metoder i nationella övervakningsprogram. I tillägg till den senaste vetenskapliga litteraturen baserar sig våra analyser på undersökningsresultat, direkta förfrågningar och intervjuer. Deltagande av nationella experter från olika sektorer möjliggjordes och experterna inbjöds att inverka på förberedelserna av färdplanen under flera skeden. På internationell nivå utvecklas molekylära metoder aktivt och metoderna används rutinmässigt inom övervakning av arter och ekosystem. I Finland har molekylära metoder testats genom pilotstudier av alla institutioner med ansvar för miljöövervakning och molekylära metoder används redan rutinmässigt i övervakningen av enskilda viltarter så som varg samt europeisk och nordamerikansk bäver. Inom andra områden, så som övervakning av biologisk mångfald, hotade arter, invasiva arter, samt växt- och djurskadegörare är metoderna fortfarande mindre utvecklade och resurserna samt expertisen mer spridda bland olika organisationer. Finansiering och kunnande anses vara de viktigaste faktorerna som begränsar ibruktagandet av molekylära metoder. Vi uppskattar att ett omfattande, rutinmässigt ibruktagande av ett brett spektrum av molekylära övervakningsmetoder kan uppnås före 2030 i Finland. De primära utvecklingsområdena för att uppnå detta mål anses vara (i) internationell koordinering och standardutveckling, (ii) nätverkande över sektorsgränserna, (iii) utbildning, (iv) infrastruktur, (v) referensbibliotek för sekvenser och kartläggning av hela genom samt (vi) modellerings- och analysverktygsutveckling. Som konkreta handlingar 2022- 2025 föreslår vi (1) ett myndighetsövergripande finansieringsinstrument, (2) en permanent arbetsgrupp med ansvar för nationell och internationell koordination, (3) ett nationellt expertnätverk och (4) en online plattform för att befrämja kommunikation och kunskapsöverföring, samt (5) ett nationellt datahanteringssystem med överenskomna data- och metadataformat och -standarder. Nyckelord: biologisk mångfald, biodiversitet, miljöövervakning, metabarcoding, next-generation sequencing, övervakningsstrategi 6 Reports of the Finnish Environment Institute 20/2022 Preface This report is the main output of the eDNA roadmap project (“Kansallinen tiekartta eDNA:n ja muiden molekyylibiologisten seurantamenetelmien tehokkaan, luotettavan ja rutiininomaisen käyttöönoton eri vaiheista”), proposed by researchers at the Finnish Environment Institute (SYKE) and commissioned by the Finnish Ministry of the Environment (YM) in December 2020. The roadmap project is one milestone but also a convergence point of several lines of development with SYKE involvement, including the international DNAqua-Net (2017-) and SCANDNAnet (2018-) networks, inclusion of DNA metabarcoding in the national monitoring of the environmental impact of agriculture and forestry (MaaMet), the establishment of an informal eDNA network and the launch of the Finnish Ecosystem Observatory (FEO) project (2020). The roadmap work has been conducted in close collaboration with FEO (funded by YM) and the eDNA pilot project (“A pilot for implementing environmental DNA (eDNA) based methods into environmental and biomonitoring”, funded by SYKE). The original aims of the eDNA roadmap project were to describe the national state-of-the art in the use of molecular monitoring methods, identify the main possibilities and challenges and development needs, formulate medium- and short-term aims and provide a concrete action plan for the next four years. It quickly became obvious that reaching these goals would be impossible without major input from other organizations, particularly the National Resources Institute Finland (Luke; represented in our team by Eeva Vainio, Terhi Iso-Touru, Taina Pennanen and Sannakajsa Velmala). Utilizing particularly the DNAqua-Net and national eDNA networks, we reached out to the expert community, which contributed essential insight into ongoing research and development projects, the overall state of the field and the possibilities and challenges involved in the implementation of molecular monitoring methods. In addition, the community was invited to comment and discuss the first draft of this report, which resulted in significant improvements in the coverage of different topics and viewpoints. We are deeply indebted to all national and international experts involved in the process – respondents of our Webropol survey, participants of our national workshops on March 25th and November 12th 2021 and all others who gave insightful comments or otherwise contributed to the preparation of this report. We would also like to thank eDNA roadmap project supervisor, Senior specialist Joona Lehtomäki from Finnish Ministry of the Environment for all his valuable guidance during this project. Reports of the Finnish Environment Institute 20/2022 7 Contents Abstract ......................................................................................................................................... 3 Tiivistelmä .................................................................................................................................... 4 Sammandrag ................................................................................................................................. 5 Preface .......................................................................................................................................... 6 Executive summary .................................................................................................................... 9 1 Introduction ........................................................................................................................... 15 1.1 Scope and definitions ........................................................................................................ 15 1.2 Policies and legislation ..................................................................................................... 15 1.3 Biodiversity crisis and the new age of biological monitoring .......................................... 16 1.4 Monitoring development needs ........................................................................................ 17 1.5 State-of-the-art and recent development in molecular methodology................................ 18 2 Status assessment of the implementation of molecular methods – Finland against the backdrop of international development ................................................................................. 21 2.1 Data sources ...................................................................................................................... 21 2.1.1 Systematic literature review ..................................................................................... 21 2.1.2 International Webropol survey ................................................................................. 22 2.1.3 Enquiries and interviews .......................................................................................... 22 2.1.4 National workshops on possibilities, challenges and best practices ........................ 23 2.2 Implementation stage of molecular methodology in monitoring...................................... 23 2.3 Possibilities and challenges for implementation ............................................................... 28 3 Vision ...................................................................................................................................... 35 4 General development plan .................................................................................................... 37 4.1 International coordination and standard development ...................................................... 37 4.2 Networking and promoting market development ............................................................. 38 4.3 Education .......................................................................................................................... 39 4.4 Infrastructure, computing resources and databases .......................................................... 40 4.5 Sequence library development .......................................................................................... 41 4.6 Modelling and analysis tool development ........................................................................ 42 5 Concrete action plan 2022-2025 ........................................................................................... 44 5.1 Funding instruments for applied research and development projects and monitoring pilots .......................................................................................... 44 5.2 Permanent working group to coordinate national efforts and involvement in international networks ...................................................................................................... 45 5.3 Expanding and promoting the eDNA network ................................................................. 45 5.4 Online interaction platform (eDNA hub).......................................................................... 45 5.5 National data management system ................................................................................... 46 6 Monitoring the progress of the implementation ................................................................. 47 References ................................................................................................................................. 54 8 Reports of the Finnish Environment Institute 20/2022 Appendices ................................................................................................................................ 58 APPENDIX 1. Literature search, study selection and data extraction procedure and the resulting data set of the systematic literature review (section 2.1.1). .................................... 58 APPENDIX 2. International survey on the current state and future needs of the use of molecular methods in monitoring (section 2.1.2). ................................................................. 68 Reports of the Finnish Environment Institute 20/2022 9 Executive summary The technological leaps achieved in the past 15 years make it possible to detect, identify and quantify DNA or RNA of target species or even map entire species communities from environmental samples. This novel capability extends to water, air, soil, sediment and diverse samples containing a multitude of trapped organisms such as insects. These molecular monitoring methods (MoMM) are revolutionizing the field of biological and environmental monitoring. They offer highly accurate, repeatable and cost-efficient species identification to anyone regardless of operator taxonomic expertise and show great potential for automatization. MoMM have already been implemented in some Finnish national monitoring programs, particularly for individual game species. Further, several pilot projects have been started e.g. in environmental impact monitoring in freshwater systems, marine phytoplankton, fisheries and threatened species monitoring. At the same time, the field is fragmented, with limited links between different organizations and no national coordination. Knowledge and know-how of the new methods is highly scattered in organizations that conduct monitoring and this currently limits the implementation of MoMM more severely than any technical challenges. This roadmap seeks to launch a national discussion on and provide actionable recommendations for the coordinated implementation of MoMM in Finland. Moreover, the roadmap emphasizes that actions should not proceed only on a national level but stresses the need to take an active role also in ongoing international coordination efforts and initiatives. New biological monitoring tools to track biodiversity trends and ecosystem health Data from biological monitoring is the primary source of information for detecting and quantifying biodiversity loss, as well as finding and evaluating ways to halt it. Molecular techniques have great potential to benefit, improve and extend current biological monitoring in all types of habitats. They can produce objective, easily comparable and reproducible species identification, and can be applied also to many currently hard-to-detect and poorly known groups of organisms such as microbes and fungi. Present-day methods include species-specific PCR assays based on DNA barcoding, quantitative PCR methods (qPCR, dPCR, ddPCR) that also determine the abundance of the target species and DNA metabarcoding based on the sequencing of massive numbers of individual DNA molecules from a single sample. DNA can be analysed from environmental samples (eDNA) or samples containing entire organisms, their parts or feces. RNA-based methods provide information on the activity of organisms in the environment. In many cases, molecular methods can be applied similarly to traditional monitoring methods, with taxonomic identification based on comparison of the DNA sequence to a reference sequence database of confirmed specimens. Alternatively, in taxonomy-free methods, assemblages of DNA (or RNA) sequences are compared e.g. by machine learning approaches to reveal changes in biodiversity without species identification. 10 Reports of the Finnish Environment Institute 20/2022 National forerunners in molecular monitoring: game, fisheries, freshwater and more As a basis for our recommendations, we conducted an analysis of the national state-of-the-art in MoMM implementation within the international context. Our analysis was based on the international scientific literature from the past five years, an internationally distributed Webropol survey, direct enquiries and interviews of persons responsible for national monitoring programs and the results of two national workshops. Internationally, MoMM are being very actively developed across the tree of life and for most ecosystems, from the deep sea to mountains and from tropical forest to polar regions. Generally, eDNA- based methods for biodiversity monitoring are the most actively studied and the readiest for routine implementation in aquatic systems, especially for fish and benthic invertebrates. While most scientific studies report significant advantages offered by MoMM, systematic large-scale demonstrations are still scarce. This result was supported also by the Webropol survey: MoMM were estimated to be in the testing or planning stages, with only some examples of routine implementation. In Finland, examples of implementation in routine monitoring include game species monitoring such as for large carnivores and the European and Canadian Beaver. In addition, advanced pilot projects have included environmental impact monitoring in freshwater systems, fishery assessments and soil quality and microbial biodiversity monitoring. Smaller-scale pilots cover a variety of taxa from liverworts to mussels. Compared to the international level, the national readiness of MoMM is high in managed populations such as game and fisheries, but lags behind in biodiversity, threatened species and invasive species monitoring. Added information, reliability, speed, and cost-effectiveness are perceived as the biggest possibilities offered by MoMM. As the most important challenges limiting MoMM implementation, we identified the lack of funding, expertise and method standards, incompleteness of reference sequence libraries, limitations in producing abundance information and the complicated interpretation of results. Figure 1. Timeline for the transition into the new age of biological monitoring. Reports of the Finnish Environment Institute 20/2022 11 The new age of biological monitoring Given the speed of the international method development and the highly promising results from the first MoMM programs and pilots, we envision that reliable, internationally standardized molecular methods are routinely used in national biological monitoring before 2030 (Fig. 1). Traditional taxonomic monitoring complements the new methods, ensuring the continuity of valuable long-term data sets and facets of biodiversity not yet captured by molecular methods. As the new methods gain ground, the role of citizen observations in providing additional taxonomic data increases. Where appropriate, citizen science is also routinely applied to sample collection for MoMM. Like traditional monitoring data, MoMM-based data should be Findable, Accessible, Interoperable and Reusable according to the FAIR Guiding Principles for scientific data management and stewardship. In the new age of biological monitoring, MoMM have developed alongside Earth observation and other remote sensing methodology as well as ecological modelling and machine learning applications. Validated modelling and analysis tools are used to assist the interpretation of DNA-based observations and in sampling design. Large-scale molecular monitoring requires improvements in standardization, knowledge base and transfer of know-how Based on our analysis of the state-of-the-art and the chief roadblocks to MoMM implementation, we have identified six development areas that merit particular attention in the coming years. 1) International coordination and standard development are paramount to ensure comparability of data in time and space, to avoid unnecessary duplication of efforts and to minimize time until full implementation on a national, European and international scale. 2) Networking across sectors is essential to increase awareness and knowledge of the new methods, to establish the connections that form the basis of a viable market – and to provide platforms for critical discussion of when, where and how the new methods should be applied. 3) Education plays a key role in solving the lack of up-to-date know-how, one of the most pressing challenges identified in our status assessment. Further, education is strongly related to quality control and overall comparability. 4) Infrastructure to support MoMM implementation largely exists but is somewhat scattered. Existing infrastructure should be used more efficiently and should be developed, updated and extended in a coordinated manner. 5) Reference sequence libraries should be extended not only in designated projects but as a running task in monitoring programs and pilots. Also, efforts towards mapping whole genomes of organisms across different environments should be increased. 6) Modelling and analysis tools should be applied and developed alongside the MoMM methods to fully utilize the wealth of the information they provide. Concrete actions: transition funding, coordination, networking, data management We propose five concrete actions for 2022-2025 to promote the implementation of MoMM in Finland (Table 1). A1: Directed funding for the transition, particularly from the Ministry of the Environment (YM) and the Ministry of Agriculture and Forestry (MMM), is required to enable goal-oriented development and implementation of the methods. We suggest that future ministerial research and infrastructure funding models should use co-funding and entail a cross-governmental and cross-departmental requirement, namely that developments towards MoMM implementation need to include at least two 12 Reports of the Finnish Environment Institute 20/2022 agencies under the control of different ministries and that developments are tied to the national environmental monitoring strategy framework and implementation program. The need for new biodiversity indicators to monitor and assess the objectives of policies such as the new National Biodiversity Strategy and Action Plan for 2035 provides one concrete goal for research and development efforts (Table 2). A2: The establishment of a permanent working group within the national environmental monitoring strategy implementation program to increase national MoMM coordination and to strengthen the role of Finland in international MoMM related networks and initiatives. A3: Expand and promote the national cross-governmental eDNA network established in 2020 to strengthen its role as a national community of practice that produces opportunities for networking, exchange of information and experiences, education and critical discussion on MoMM implementation. A4: The launch of an open access, subscription based, online platform to enable continuous real-time discussions and co-creative workflows across organizational boundaries and an easy access to MoMM related information, events and commercial services. A5: Develop a national molecular data management system with data and metadata standards and automated links to international databases. Table 1. Action plan for promoting MoMM implementation in 2022-2025. Action Coordination responsibility Suggested timing Cost estimate A1: Directed R&D funding for transition to MoMM YM, MMM (VM, TEM, STM) 2022-2025 1 M€ yearly A2: Establishment of a permanent working group (eDNA embassy) YM 2022 50 k€ yearly A3: Expansion and promotion of the eDNA network SYKE 2022 30 k€ A4: Launch of an online interaction platform (eDNA hub) SYKE 2022-2023 100 k€ A5: Development of a national data management system SYKE 2022-2024 500 k€ Reports of the Finnish Environment Institute 20/2022 13 Table 2. The possibilities and limitations of MoMM in the monitoring and assessment of the objectives of the National Biodiversity Strategy and Action Plan (NBSAP). The primary aim of NBSAP is no net loss of nature in 2020-2035, in other words, a nature positive Finland by 2035. CONSERVATION SUSTAINABLE USE GENES Threat status Protection and restoration Species Habitats Ecosystem functioning Genetic diversity NBSAP objective by 2035 The level of threat to species and habitats is no longer increasing. The coverage of protected areas has reached 30%, one third of which is strictly protected. Restoration measures cover 15% of degraded habitats. Populations of declined indicator species groups of different habitats have increased [20%]. The quantity and quality of habitats un- der land use pressure have increased [10%]. The capacity of ecosystems to provide central ecosystem services has improved [10%]. Genetic diversity has not decreased (effective population sizes of species have not decreased). Possibilities offered by MoMM • Improved detection of threatened species by eDNA-based targeted monitoring (e.g. from water, soil, sediment, air, stomach contents, honey). Sampling feasible also for citizen scientists without taxonomic expertise. • Delimitation of populations of closely related species • Detection of hybridization • Assessment of the impact of restoration using DNA-based community composition (taxonomy- based/taxonomy-free) or presence of indicator species. • Monitoring of some poorly detectable groups feasible only with MoMM • eDNA-based targeted monitoring of indicator species. Sampling feasible also for citizen scientists without taxonomic expertise. • Cost-efficient monitoring of whole communities (incl. estimates of relative abundances) based on metabarcoding • Assessment of habitat quality using DNA-based community composi- tion e.g. in soil, water or air (taxonomy- based/taxonomy- free). • Assessment of habitat quality by DNA-based detection of indicator species and groups (e.g. microbes, fungi). • Assessment of habitat quality based on gene expression (eRNA) • Inference of functional composition: DNA- based community composition and trait data • Assessment of ecosystem functioning based on gene expression (eRNA) • Monitoring genetic diversity of target species (key/umbrella species, threatened species) • Estimation of effective vs. census population size • Delimitation of populations within species / assessing population structure Challenges / limitations of MoMM • Limited information on species abundance • Indirect observation (DNA transported e.g. by water or air) • Gaps in reference sequence libraries • Representative sampling (high local variability in soil; DNA transported e.g. by water or air) • Sufficient reference data needed to establish DNA-based habitat quality measures. • Comparability of MoMM-based and traditional abundance data • Standardized methods crucial for reliable and consistent monitoring • Representative sampling (high local variability in soil; DNA transported e.g. by water or air) • Sufficient reference data needed to establish MoMM- based habitat quality measures. • Representative sampling (high local variability in soil; DNA transported e.g. by water or air) • Sufficient reference data needed to establish MoMM-based measures for ecosystem functioning. • Estimating genetic diversity is by necessity species specific, and therefore significant investments are needed to increase the number of species with sufficient genomic knowledge 14 Reports of the Finnish Environment Institute 20/2022 Reports of the Finnish Environment Institute 20/2022 15 1 Introduction Human activities have completely transformed major parts of natural habitats and affect all life on Earth. The adverse effects of this development for ecosystems and species, including our own, are becoming increasingly evident. National, European and global efforts to conserve biodiversity are fast proceeding (e.g. Global Biodiversity Framework to 2030, EU Biodiversity Strategy for 2030). At the same time, large gaps remain both in the basic mapping and in our ability to monitor changes in biodiversity at relevant scales and resolution. In this chapter, we define the scope of this report and give a general introduction to the aims and challenges of environmental monitoring and the potential that new molecular methods present to the monitoring field. 1.1 Scope and definitions We define monitoring as systematic, repeated observation particularly aimed at detecting current system status and any temporal changes therein. The scope of this report covers all monitoring of outdoor environments that targets biological organisms either directly (e.g. species monitoring) or indirectly as indicators of the general state of the system (e.g. environmental impact assessment). A particular emphasis is placed on efforts that fit under the umbrella of biodiversity monitoring, such as monitoring community composition or the distribution, population size and population structure of rare and threatened species, EU directive species, invasive alien species or species of economic interest. In addition to natural organisms, we also consider genetically modified organism (GMO) monitoring required by European and national legislation and international agreements. Other types of monitoring considered in this report include water, soil and air quality monitoring. We use the umbrella term ”molecular monitoring methods” (MoMM) to refer to all environmental monitoring methods that base their analysis on DNA or RNA. We focus primarily on methods related to species identification. While MoMM are also central in assessing population genetic metrics (e.g. intraspecific genetic diversity, level of inbreeding, population genetic structure and gene flow), we cover intraspecific genetic diversity only very briefly, recognizing that this topic would merit a separate in-depth assessment. Many MoMM covered here are based on the analysis of environmental DNA (eDNA): DNA found in samples of e.g. water, sediment, soil or air. eDNA includes both DNA released from organisms into their environment and DNA bound to microscopic organisms or their parts (as e.g. in the case of biofilm or plankton samples). However, this report is not restricted to eDNA applications but also considers methods based on different types of samples, such as assemblages of whole organisms (e.g. kick-net samples of stream invertebrates), their parts or fecal samples. Different present- day molecular methods are introduced in section 1.5. 1.2 Policies and legislation Biodiversity monitoring is embedded in many policies and legislations. The Convention on Biological Diversity (CBD) has influenced opinions on biodiversity conservation and sustainable use at both the EU and the national level. Currently the CBD is negotiating towards a Global Biodiversity Framework for 2030. The EU Biodiversity Strategy for 2030 has already been adopted, and Finland is currently preparing its own national National Biodiversity Strategy and Action Plan for 2035. These international and national action plans will be implemented through upcoming national legislation. The use of eDNA and other molecular methods is not specifically included in current legislation, but it is a promising methodology to provide the required data that currently is not feasibly attainable 16 Reports of the Finnish Environment Institute 20/2022 through other means. For instance, eDNA has recently been recommended for use as a standardized biodiversity barometer tool of anthropogenic pressures in coastal ecosystems (DiBattista et al. 2020). Similarly, MoMM can be used for reporting on ecosystem condition and state indicators, requested e.g. by the EU Water Framework Directive (European Union 2000) and the EU Marine Strategy Framework Directive (European Union 2008, 2016, 2017) and can provide accurate data for the EU Habitat and Bird Directive (EU 1992, 2009) as well as for national assessments of threatened species (e.g. Red Lists, see Hyvärinen et al. 2019). Routine MoMM implementation would also benefit the monitoring of threatened species from the field samples and according to international agreements such as Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES), monitoring invasive and other harmful species e.g. in adherence to the Ballast Water Management Convention (see Box 1 at the end of this report), and monitoring of GMOs (see Box 2 at the end of this report). The Finnish environmental protection legislation includes several acts that require knowledge of the state of biodiversity all of which could be supported by eDNA methods (Act on Environmental Impact Assessment Procedure 252/2017, Land Use and Building Act 132/1999 (amendment 222/2003 included)). The current revision of the national nature conservation legislation includes three elements: i) an update of the Nature Conservation Act and the Nature Conservation Decree; ii) the drafting of a new act on compensations for damages caused by protected species; and iii) developing a plan for ecological compensation. The Nature Conservation Act and Nature Conservation Decree cause an increased demand for accurate and near real-time data which could be easily satisfied through the routine implementation of MoMM. 1.3 Biodiversity crisis and the new age of biological monitoring Biodiversity loss has been recognized among the most serious threats for human well-being (IPBES 2019). It is difficult to conserve, manage and restore biodiversity if we do not know its state and trends. Long-term monitoring is pivotal to understand ecological changes in nature. Traditional biological methods cannot provide all the information that is needed to reliably measure all levels of biological diversity or provide required extensive taxonomic, spatial and temporal resolution. There is a dire need for harmonized monitoring of various dimensions of biodiversity, such as intraspecific genetic diversity, species populations and abundance, species traits, and species communities (Navarro et al. 2017). Novel methods such as remote sensing and eDNA could enable massive improvements with regards to the spatial extent and resolution of monitoring and provide insightful new knowledge on poorly known taxonomic groups and other facets of biodiversity (Bush et al. 2017). However, an abrupt adoption of new methods carries the risk of producing largely incompatible data. Novel method uptake ideally is carefully planned and encompasses method validation processes and transition periods with stepwise implementation. New monitoring technologies need not necessarily replace traditional long-term monitoring but sometimes help to fill existing information gaps. MoMM implementation have clear potential to enhance current biodiversity monitoring, as well as to improve development of biodiversity indicators. Several international initiatives have recently been launched to promote the transition into the new age of biological monitoring. Internationally, relevant networks supporting MoMM are GEO BON and iBOL. GEO BON is supporting biodiversity monitoring networks at national, regional and global scales and eDNA methods are one important source of harmonized data. Further, iBOL (BIOSCAN) is working to establish an earth observation system that will reveal species, including their dynamics and interactions, based on barcoding. The EuropaBON project (under the umbrella of GEO BON) and the European Biodiversity Partnership (Biodiversa+) are developing harmonized biodiversity monitoring schemes where MoMM will have an important role. In addition to the aforementioned initiatives, the eBioAtlas, a joint cooperation between the IUCN and a UK based company NatureMetrics seek to leverage eDNA assessments of biodiversity through crowd sourced citizen science initiatives. In https://geobon.org/ https://ibol.org/ https://europabon.org/ https://www.biodiversa.org/1759 https://ebioatlas.org/ Reports of the Finnish Environment Institute 20/2022 17 Finland, SYKE has launched a new development project Finnish Ecosystem Observatory (FEO) which aims at modernizing legislative biodiversity and ecosystem monitoring with novel methods such as remote sensing, artificial intelligence, and eDNA and other molecular biological methods. In general, the use of eDNA and other molecular biological methods has been extensively piloted in a range of freshwater, marine and terrestrial ecosystems and is getting sufficiently mature to allow standardization of techniques for certain specific sample types and target groups such as freshwater invertebrates. There is now a strong need for a comprehensive overview of the current situation – how molecular methods are used, their potential and future for biological monitoring. While the focus of this report is on Finland, we discuss also the international status of the field and Finland’s role in the European and global context. Moreover, we strongly believe that both national and international coordination are crucial to avoid unnecessary repetition of efforts and extensive intercalibration across a fragmented field in subsequent years. Thus, we direct this national roadmap also towards a wider international audience interested in international cooperation on the implementation of eDNA and other molecular methods in the next generation of biodiversity and environmental monitoring. 1.4 Monitoring development needs Molecular identification techniques have great potential to benefit, improve and extend current biological monitoring in all types of habitats. Particularly, currently unmonitored changes in patterns of biodiversity in response to global megatrends (e.g. climate change, urbanization, invasive alien species, increasing chemical stress on soils and groundwaters) stand to benefit from the speedy uptake of these methods. Further, their application to more traditional monitoring and assessments would greatly improve the accuracy of monitoring results and ensuing management actions and potentially increase spatial coverage. Molecular methods can produce objective, easily comparable and reproducible species identification, and can be used in large-scale monitoring. They can detect and monitor currently hard-to- detect and poorly known groups of organisms (e.g. aquatic and soil microbes, fungi, certain groups of insects) that are currently excluded from monitoring based on traditional taxonomic identification methods. Furthermore, molecular genetic methods provide the only tools for reliable mapping of intraspecific genetic diversity, the conservation of which is increasingly acknowledged internationally (Convention on Biological Diversity 2020, Hoban et al. 2020). To produce meaningful results, reliable reference databases and species-specific genetic tools for a large range of organisms are needed. Several methods (e.g. metabarcoding) have evolved from the Technology Readiness Level (TRL; European Association of Research & Technology Organisations 2014) of the prototype stage (TRL 3-4) to the TRL level 6-8 where the technical operation has been demonstrated in relevant settings. Despite their demonstrated success and benefits, applications of molecular identification methods have nationally mainly been limited to proof of concept or validation projects (e.g. WFD related monitoring of the ecological status of water bodies and monitoring of soil fauna biodiversity). Currently their uptake into routine use is restricted by the lack of commonly agreed internationally standardized sampling and analysis protocols, the lack of unified meta- and data standards as well as shortcomings of existing reference libraries. There is an urgent need for national cooperation to implement these methods in the future. At present there is little coordination between different research organizations and other end-users. The development of national guidelines without concurrent international coordination of efforts entails the risk of creating noncompatible solutions in a quickly evolving field. To counteract this threat Finland has taken an active role in the standardization of these methods for biological monitoring. In the past few years, these efforts have spawned European work to standardize sampling of eDNA from water and progressed to a first draft of a CEN standard. This indicates the level of international interest and commitment of countries to the future routine implementation of these methods. However, further work https://feosuomi.fi/en/ 18 Reports of the Finnish Environment Institute 20/2022 is needed to ensure unified application and interpretation of molecular methods in European and national legislative monitoring implementation. On a national scale, an informal network of scientists working on molecular method development and research with the aim of subsequent implementation into legislative environmental monitoring has been formed on a purely voluntary, bottom-up basis (i.e. the so-called eDNA network, coordinated by SYKE). The network has quickly become very popular (currently 60 members) and attracts researchers from many (currently eight) national organizations, as well as occasional international visitors. Thus far it has mainly served as a platform for information exchange but has the potential to become more permanent and goal oriented e.g. if formalized under the umbrella of the national environmental monitoring strategy. 1.5 State-of-the-art and recent development in molecular methodology Conventional monitoring methods that rely on expert microscopy identification have inherent drawbacks, such as their dependency on personal identification skills, and are prone to human fatigue and error. Moreover, cryptic taxa are impossible to separate into species using expert-based taxa identification methods. Ideally, molecular taxon identification methods allow better harmonization and provide more accurate estimates of biodiversity. The new molecular biology-based tools overcome some limitations of traditional monitoring and allow using non-invasive sampling, achieve broad taxonomic coverage, have high sensitivity, and can be automated. These methods provide cost-effective means to produce reliable information for monitoring biodiversity in terrestrial and aquatic environments. Figure 2. Different approaches for environmental monitoring based on community data: traditional morphological species determination (here from a kick-net sample of invertebrates), bulk DNA metabarcoding from a homogenized sample and eDNA metabarcoding (here from a water sample). Reproduced from Hering et al. (2018). New molecular techniques in the field and in the laboratory have enabled sampling and identification of much of terrestrial, marine and freshwater biodiversity. These include environmental DNA (eDNA), bulk-sample DNA metabarcoding approaches (Fig. 2) and targeted RNA-based methods (e.g. Valentini et al. 2016; Elbrecht et al. 2017; Mäki & Tiirola 2018) as well as eRNA, useful e.g. for estimating the age of genomic material (Marshall et al. 2021). The eDNA technique uses DNA that is released from Reports of the Finnish Environment Institute 20/2022 19 organisms into their environment, from which a signal of organisms’ presence in the system can be obtained. For example, in aquatic ecosystems, eDNA is typically extracted from sediment or filtered water samples (e.g. Deiner et al. 2016), and this approach is distinguished from bulk DNA metabarcoding, where organisms are directly identified from e.g. complete biological monitoring samples (e.g. Elbrecht et al. 2017). Despite the demonstrated potential of environmental and bulk- sample DNA metabarcoding approaches in recent years, there are still significant bottlenecks to their routine use that need to be addressed (e.g. Pawlowski et al. 2020). While the sampling for MoMM is generally straightforward and in many cases comparable to traditional monitoring methods (Fig. 2, Fig. 3), each subsequent analysis step (e.g. in metabarcoding DNA/RNA extraction, PCR amplification, sequencing, bioinformatic analysis) involves numerous technical choices requiring a high level of expertise and usually laborious testing for each new sample type and taxonomic group. In taxonomy-based approaches, DNA sequences from the monitoring sample are identified against a reference library of sequences from confirmed specimens, resulting in a species list and an estimate of species’ relative abundances reminiscent of traditional methods. However, MoMM also allow fundamentally different approaches (implementation strategies according to Cordier et al. 2021): taxonomy-free methods based on unidentified groupings of similar sequences (Operational Taxonomic Units or Amplicon Sequence Variants), inferring ecological networks e.g. by identifying DNA in feces or gut contents, and studying the presence and activity of genes linked to specific functions using metagenomics or metatranscriptomics. Furthermore, viruses with RNA genomes (vast proportion of plant, insect and fungal viruses) can be detected using RNA-based methods. The type of the ecosystem, study questions and the target taxonomic group affects the choice of which MoMM should be used. Different habitats and taxa require different types of samples and different protocols. For example, in aquatic monitoring DNA or RNA can be isolated from water, sediment, biofilm or from bulk sample (e.g. macroinvertebrates) (e.g. Pawlowski et al. 2020). PCR- based single-species detection methods (qPCR, dPCR, ddPCR) are cheaper, faster, and easier compared to high-throughput next-generation sequencing (NGS)-based metabarcoding, which requires more specialized equipment and higher-level expertise in generating and analyzing sequence data with different bioinformatic tools. NGS offers information about multiple species at a time, useful for monitoring biodiversity or the state of the environment based on the whole community, while single- species detection is suited for a few focal organisms (e.g., rare or invasive species, targeted pathogens) with well validated single-species primers (Pawlowski et al. 2020). For a more detailed review of the methods, see e.g. Garlapati et al. (2019) and Ruppert et al. (2019). 20 Reports of the Finnish Environment Institute 20/2022 Figure 3. Preanalytic conditions are a key point to access high quality data. Here, eDNA samples have been stored on ice immediately after sampling. © Tiina Laamanen, SYKE Reports of the Finnish Environment Institute 20/2022 21 2 Status assessment of the implementation of molecular methods – Finland against the backdrop of international development The recommendations presented in this report are based on a comprehensive analysis of the current state of the implementation of MoMM in Finland within the international context. This chapter presents the data sources, analysis methods and results of this status assessment. 2.1 Data sources To assess the current status of the implementation of MoMM, we started with an evaluation of the international readiness level of MoMM through a systematic review of the scientific literature published within the past five years. Second, we assessed the present-day international status of MoMM implementation by preparing a Webropol survey to the international community of scientists and officials working in this field. Third, to get a comprehensive overview of the Finnish situation, we made direct enquiries to all relevant institutions and conducted interviews with the key persons responsible for national species monitoring programs. Finally, we organized a national workshop for professionals working in different sectors to interactively identify the possibilities and challenges related to MoMM implementation. Data sources are described in more detail in the following subsections (2.1.1-2.1.4) and results relevant for this report are presented in sections 2.2 and 2.3. The full results of the systematic literature review and the Webropol survey will be published in scientific articles (Laamanen et al., in prep.). 2.1.1 Systematic literature review A search of the Web of Science database was performed on April 15th 2021 using relevant search strings, resulting in 320 separate articles published no more than five years before the search. Following the systematic review protocol implemented in the CADIMA tool (https://www.cadima.info), the articles were screened against predetermined study selection criteria primarily based on the abstract but referring to the full text where necessary. These criteria included the requirement that the study discusses the topic of applying/implementing the adopted methodology in monitoring, and that at least some of the analyzed samples were collected from an outdoor environment. Note that the literature review did not cover population genetic studies; we recommend that a corresponding assessment of the state-of-the-art in population genetic monitoring be conducted soon. For a more detailed description of the systematic review procedure as well as a list of the reviewed papers, see Appendix 1. Data was extracted from the selected articles based on the full text. This report uses the information of a sample of 70 original research papers; the full results of the review will be presented in a scientific article (Laamanen et al. in prep.). Based on the extracted data, we assessed the Technology Readiness Level (TRL) of the method presented or applied in each paper from the point of view of its implementation in routine monitoring. In this context, we interpreted the TRLs using the following progressively applied criteria: • TRL5 (Technology validated in relevant environment): The study selection criteria. • TRL6 (Technology demonstrated in relevant environment): The molecular method is compared to the traditional method and shown to have advantages. 22 Reports of the Finnish Environment Institute 20/2022 • TRL7 (System prototype demonstration in operational environment): The molecular method is ap- plied at a medium or large spatial scale (>10 km) and its implementation in monitoring is at least conditionally recommended. • TRL8 (System complete and qualified): The molecular method is directly compared to the traditional method (i.e. with comparable samples) and its implementation is recommended without major condi- tions. • TRL9 (Actual system proven in operational environment): The method is already implemented in an existing monitoring program. 2.1.2 International Webropol survey The Webropol survey to experts was titled “Survey on the current state and future needs of the use of molecular methods in monitoring” and openly accessible online in February-April 2021. We advertised the survey actively within our national and international networks, most notably in the DNAQUA International Conference March 9th-11th 2021 organized by the EU COST action DNAqua-Net. This virtual conference attracted over 1,400 participants worldwide. The survey was mentioned in several presentations, including keynotes; moreover, it was included on the agenda of the associated national workshops organized in 17 European countries: Belgium and Luxembourg, Bosnia-Herzegovina, Cyprus and Greece, France, Germany, Hungary, Italy, Netherlands, Norway, Portugal, Slovakia, Sweden, Switzerland, Turkey and Finland. Our international collaborators translated the originally English survey into national languages to widen the audience reached. The survey consisted of 24 questions divided into two sections: national level questions specifically on the implementation of MoMM and individual level questions on the personal work and involvement of each respondent in this field. All survey questions are listed in Appendix 2. All responses were anonymous. 2.1.3 Enquiries and interviews Based on the recently updated national Strategy for Environmental Monitoring (Ympäristön seurannan strategia / YSS), we identified the following institutions as the key coordinators of national environmental monitoring programs: Finnish Environment Institute (SYKE), National Resources Institute Finland (Luke), Metsähallitus (MH; Parks & Wildlife Finland), the Finnish Museum of Natural History (LUOMUS), Finnish Institute for Health and Welfare (THL), Finnish Food Authority (RV), National Supervisory Authority for Welfare and Health (Valvira), Finnish Meteorological Institute (FMI) and the Aerobiology Unit at the University of Turku (AU). We contacted all these institutes and enquired about the use of molecular methods in recent, ongoing and soon launching monitoring projects and programs conducted or coordinated by the institute. In addition to actual monitoring, we also enquired about and listed research and development projects conducted by these institutes in which MoMM are piloted. As the focus of this report is on the national-scale implementation and not basic scientific research and the earlier stages of method development, all research projects at Finnish universities applying and developing molecular methods were not systematically mapped. However, we selectively listed university-led research projects that produce extensive data comparable to monitoring or specifically aim at developing methods for routine monitoring. Interviews of 30 persons responsible for systematic species monitoring at SYKE, Luke, MH and LUOMUS were conducted as part of the status assessment of systematic species monitoring performed within the Finnish Ecosystem Observatory project in 2021. The interviews were based on a predetermined list of topics and questions, centered on the current state and development needs of monitoring in each group of organisms. The interviews included one question specifically on the use and on the future potential of using molecular methods in species monitoring or piloting new monitoring methods. Reports of the Finnish Environment Institute 20/2022 23 2.1.4 National workshops on possibilities, challenges and best practices On March 25th 2021, SYKE organized a half-day national workshop on the current state, possibilities and challenges of MoMM (“Kansallinen työpaja molekyylibiologisten seurantamenetelmien nykytilasta, mahdollisuuksista ja haasteista”), which was part of the series of national workshops associated with the DNAQUA International Conference and funded by DNAqua-Net. The workshop was directed at a wide audience of professionals in different sectors whose work is related to or relevant for environmental monitoring, molecular methods or both. All but three (RV, Valvira, FMI) of the monitoring institutions listed in section 3.1.3 were represented among the >70 participants, SYKE (32%) and Luke (12%) with the highest number of participants. Universities (23%) and private companies (13%) were also well represented. Following introductory presentations, the participants were requested to identify and discuss different possibilities, challenges and best practices related to the implementation of MoMM. The participants were randomly divided into six groups, and each group collected their ideas on a virtual whiteboard using the Jamboard application. The whiteboards remained accessible and editable to all participants for one week after the workshop. The ideas in the whiteboards were then listed and categorized in order to define and rank general possibilities, challenges and best practices by the number of individual mentions in the whiteboards. On 12th November 2021, SYKE organized another workshop which provided input for this report. The first part of the workshop was intended for an international audience and gathered over 130 participants. In the second part, ca. 60 national participants discussed the development areas identified in the roadmap work so far. The results of the second workshop are not presented below as figures but the issues raised were incorporated in our recommendations in Chapters 3-5. 2.2 Implementation stage of molecular methodology in monitoring The internationally published scientific research within the last five years is heavily dominated by aquatic environments (Fig. 4A). Aquatic environments also provided the only examples of the highest Technology Readiness Level (TRL9, already implemented), which included invasive fish species (Carim et al. 2020) and benthic invertebrates (Aylagas et al. 2018). Another famous example of routine eDNA-based monitoring is the great crested newt (Triturus cristatus) in Great Britain (Biggs et al. 2015), although this case did not happen to be included in our sample of 70 analyzed papers. Fish and invertebrates are the two most actively studied groups (Fig. 4B). The most common type of monitoring in the reviewed papers was biodiversity monitoring, followed by invasive and threatened species monitoring (Fig. 4C). It should be noted that this distribution also reflects the literature search criteria, as “biodiversity” was one of the search terms. Among the TRL classes 5-9 included in our review, most of the studies represented the lower end of the spectrum (Fig. 4), indicating that while the methods are broadly validated in small-scale field studies, systematic large-scale demonstrations are still scarce. Among the well-represented taxa, the technology is the most developed in fish (mode TRL7) and other vertebrates (mode TRL6), while taxonomically universal methods remain the least ready (TRL5 only). 24 Reports of the Finnish Environment Institute 20/2022 A) B) C) Figure 4. Technology Readiness Level (TRL) of molecular monitoring methods based on a sample of 70 original research papers published in 2016-2021, analyzed in the systematic literature review (section 2.1.1). Reports of the Finnish Environment Institute 20/2022 25 The national state-of-the art summarized in Table 3 is broadly consistent with the international situation based on scientific literature. While there is a relatively high number and diversity of pilot projects, there are relatively few examples of routine implementation. These include the following: 1) The monitoring of large carnivore populations (one of the data sources based on fecal DNA) (Luke). The most recent development of non-invasive DNA-methods used in large carnivore monitoring has been conducted in collaboration with Scandinavian countries. The role of non-invasive DNA-methods has been recognized as increasingly important in monitoring large carnivores as the traditional census methods based on snow tracks are becoming unreliable due to climate change. 2) The monitoring of the distributions of the two beaver species (the domestic European beaver and the invasive Canadian beaver) based on DNA extracted from wood chips collected by citizen scientists (Luke; Iso-Touru et al. 2021). In this case, molecular methods improve markedly the status of monitoring, as for this species pair no other sufficiently reliable non-invasive monitoring method is available. The national monitoring of land use effects on freshwater systems (Maa- ja metsätalouden kuormituksen ja sen vesistövaikutusten seuranta (MaaMet)) led by SYKE has also been extended by pilots of DNA-based monitoring of macroinvertebrates and benthic diatoms (e.g. Elbrecht et al. 2017; Meissner et al. 2020; Kahlert et al. 2021; Turunen et al. 2021a) and currently has a DNA metabarcoding sampling method (preservation of benthic invertebrate samples in lakes and rivers) in routine use, operated by consultants. In contrast to the international TRL assessment, terrestrial systems are comparatively well represented among national monitoring projects and pilots, while examples from the Baltic Sea are few compared to the international status of the marine MoMM field. It is also notable that apart from the Canadian beaver, use of molecular methods in the monitoring of invasive alien species has not been implemented despite the encouraging international examples. However, several method tests and pilots using molecular methods in the monitoring of invasive species have been started, e.g. wild boar, signal crayfish (Mäkinen et al. 2021; Luke, UEF, JYU), wolf-dog hybrids, several fish species (Luke) and mud crab (University of Turku). Bacteria and fungi are better represented in the national pilot projects than in the international scientific literature, suggesting forerunner potential in this field. While almost all national institutes responsible for monitoring have taken some steps towards the implementation of MoMM, Luke stands out in the consistency and scale of the implementation in a variety of monitoring programs. We received a total of 171 responses to the international Webropol survey, of which 35 % were from Italy and 19 % from Finland, followed by Greece (10%), France (10%), Sweden (5%), the Netherlands (5%), Germany (3%), Norway (2%), Hungary (2%), Estonia (1%) and Cyprus (1%). We also obtained one response each from Poland, Denmark, Canada, Cameroon, Greece and Latvia. The responses to the individual level questions indicated that most respondents had personal experience in applying molecular methods but were not directly involved in setting up a national strategy for DNA-based monitoring. Both aquatic and terrestrial systems were well represented in the respondents’ work. It is also notable that while taxonomy-based methods were the most popular out of the classes defined by Cordier et al. (2020), other strategies (taxonomy-free, structural, functional) were also mentioned by over half of the respondents (53%). This is in clear contrast with the reviewed scientific literature, where only 10% of the papers applied other than taxonomy-based methods. It seems that the interest in alternative implementation strategies is currently fast increasing. According to the survey results, the overall state of MoMM implementation is roughly similar in Finland compared to other countries (Fig. 5), and consistent with the TRL analysis (Fig. 4) and the summary of national monitoring projects (Table 3). https://www.syke.fi/hankkeet/maamet https://www.syke.fi/hankkeet/maamet 26 Reports of the Finnish Environment Institute 20/2022 Table 3. Summary of recent and ongoing Finnish monitoring campaigns and pilots utilizing molecular methods. Research projects have been interpreted as “pilots” if they are comparable to monitoring in methodology and scale. Taxa for which methods are in the earlier development stages are not listed. Invasive alien species (IAS) and nationally red-listed species (Hyvärinen et al. 2019) are indicated. Species/group System Methods Stage Conducted by Viruses terrestrial, freshwater, marine eDNA metabarcoding, qPCR, eRNA (water, air, wastewater, ticks, fungi, plants, insects) Pilot THL, SYKE, FMI, Luke, universities Bacteria terrestrial, freshwater, marine eDNA metabarcoding, qPCR (soil, water, air, wastewater, ticks) Pilot Luke, SYKE, FMI, THL, RV, universities Endophytic microbes terrestrial eDNA metabarcoding of bacteria and fungi within plant tissues Pilot Luke Benthic diatoms freshwater eDNA (biofilm) metabarcoding Pilot SYKE / Swedish Univ. of Agricultural Sciences Phytoplankton freshwater, marine eDNA metabarcoding Pilot SYKE Liverworts terrestrial Bulk DNA metabarcoding Pilot Univ. Turku, MH, SYKE Vascular plants terrestrial eDNA metabarcoding/metagenomics (airborne pollen) Pilot FMI Fungi terrestrial, freshwater eDNA metabarcoding/metagenomics (soil, water, air) Pilot Luke, SYKE, FMI, RV, universities Freshwater pearl mussel (EN) freshwater eDNA + specific PCR Pilot Univ. Jyväskylä, MH Benthic macroinvertebrates freshwater, marine Bulk DNA metabarcoding Pilot SYKE Soil invertebrates terrestrial eDNA metabarcoding Pilot Luke Arthropods terrestrial Bulk DNA metabarcoding Pilot Universities Glanville fritillary butterfly (EN) terrestrial 240 SNP panel, whole genome re-sequencing Pilot (long- term research) Univ. Helsinki Noble crayfish (EN), signal crayfish (IAS) freshwater eDNA + dPCR Pilot Luke, Univ. Eastern Finland Atlantic salmon (VU), Baltic salmon (VU) freshwater, marine An array of 220k SNPs Routine Univ. Helsinki, Luke Fish freshwater, marine (coastal) eDNA + qPCR, eDNA metabarcoding Pilot Luke, MMM Common frog, moor frog freshwater eDNA + qPCR Pilot Luke, Luomus, MMM Lesser white fronted goose (CR) freshwater eDNA + Sanger sequencing Pilot Kiljuhanhi LIFE, MH, Univ. Oulu Bats terrestrial DNA from feces + metabarcoding Pilot Luomus Brown bear (NT) terrestrial DNA from feces + 96 Single Nucleotide Polymorphism (SNP) panel Forthcom ing pilot (2022) Luke European beaver (NT), Canadian beaver (IAS) terrestrial eDNA (wood chips) + PCR assays Routine Luke European lynx terrestrial DNA from feces + 96 SNP panel Forthcom ing pilot Luke White-tailed deer (IAS) terrestrial DNA from feces + microsatelli-tes Pilot Luke Wolf (EN) and wolf-dog hybrids terrestrial DNA from feces/urine + 96 SNP panel Routine 2022- Luke Wolverine (EN) terrestrial 14 microsatellites and mtDNA control region (579 bp) Pilot Univ. Oulu, Luke Reports of the Finnish Environment Institute 20/2022 27 The rate of routine implementation is estimated to be generally low and the highest for health-related microbial monitoring (sewage, drinking and swimming waters). A clearly higher proportion of respondents stated that the implementation of MoMM is currently in the testing and planning stages, while only a few respondents stated that implementation is not yet planned. Considering their involvement in the field, surprisingly many respondents were unaware of the state of the implementation in their country, demonstrating the need for active networking and knowledge transfer. According to the responses, the implementation of MoMM in managed populations such as game and fisheries appears more advanced in Finland than in other countries. Indeed, several monitoring examples listed in Table 3 involve managed populations, while this type of monitoring forms only a small minority in the reviewed international scientific literature (Fig. 4C; note that this is also affected by the search criteria). By contrast, the national implementation of molecular methods lags somewhat behind the international level in biodiversity, threatened species and invasive species monitoring, which is in agreement with results in Fig. 4 vs. Table 3. For a more thorough discussion on the monitoring of invasive species, pests and pathogens with eDNA/eRNA, see Box 1 at the end of this report. Both nationally and internationally, implementation of MoMM in the monitoring of genetically modified organisms (GMOs) is the least well known. We treat this topic in more detail in Box 2 at the end of this report. 28 Reports of the Finnish Environment Institute 20/2022 A) International status of implementation (excluding Finland) B) National status of implementation Figure 5. The status of the implementation of molecular methods in different fields of monitoring, inter- nationally (A; excluding Finland) and nationally (B), as assessed by the respondents of an international Webropol survey (section 2.1.2). 2.3 Possibilities and challenges for implementation Although different studies have addressed the benefit of implementing MoMM into national monitoring programs compared to current traditional monitoring methods, there are still challenges to the effective use of MoMM at a national level. When the experts participating in our national workshop were asked to estimate the time frame within which molecular methods can be implemented in routine monitoring on a scale of 0 to 10 years, 57 workshop attendees gave the average estimate of 2.8 years, with 11% 6% 25% 9% 7% 7% 14% 40% 32% 16% 30% 34% 16% 9% 19% 24% 8% 15% 19% 8% 5% 6% 9% 4% 8% 7% 8% 9% 24% 30% 46% 38% 34% 61% 63% 0% 20% 40% 60% 80% 100% 120% Biodiversity Environmental status Microbial monitoring (sewage, drinking,… Threatened species Invasive species Managed populations (game, fisheries) GMO Routine implementation Testing phase Planned Not planned I do not know 15% 11% 26% 4% 22% 4% 26% 15% 11% 33% 30% 15% 4% 33% 41% 4% 15% 33% 7% 7% 4% 4% 33% 33% 56% 59% 48% 59% 81% 0% 20% 40% 60% 80% 100% 120% Biodiversity Environmental status Microbial monitoring (sewage, drinking, swimming water) Threatened species Invasive species Managed populations (game, fisheries) GMO Routine implementation Testing phase Planned Not planned I do not know Reports of the Finnish Environment Institute 20/2022 29 individual estimates spanning across the whole range. Clearly, there is high degree of optimism and enthusiasm about the possibilities presented by the new methods, but also important reservations that should be carefully considered. In this section, we discuss the concrete possibilities and challenges of MoMM based on the data sources described in section 2.1. Possibilities Based on the output of the national workshop, by far the greatest possibility offered by MoMM is new information – e.g. on rare and evasive taxa, structural and functional aspects of natural communities and the genetic structure of populations (Fig. 6A). Other commonly mentioned possibilities included more reliable, faster and cost-effective monitoring. Using molecular methods does not require taxonomic expertise and identification is more transparent, repeatable and comparable between different environments and countries. Therefore, molecular methods can be seen as more reliable than traditional monitoring methods. 30 Reports of the Finnish Environment Institute 20/2022 A) Possibilities B) Challenges Reports of the Finnish Environment Institute 20/2022 31 C) Best practices Figure 6. Possibilities (A), challenges (B) and best practices (C) related to molecular monitoring, as identified by ca. 60 experts participating in a national workshop in March 2021 (section 2.1.4). Challenges Based on the survey, lack of funding, expertise and method standards were recognized as the most important limiting factors of the implementation of MoMM both in Finland and internationally (Fig. 7). Lack of expertise was stressed particularly at the national level, possibly partly due to the more diverse backgrounds represented among the Finnish respondents. Indeed, although taxonomic identification expertise is less crucial when using molecular methods, knowledge and skills related to the new methods will be in great demand. It is important to note that this is the case even when the DNA analytics are purchased from external service providers. In the national workshop as well as other networking events, many professionals in the field of biological monitoring have expressed the difficulty in using, selecting or even finding such services due to the lack of the necessary background knowledge. Another challenge emphasized particularly at the national level is the incompleteness of sequence libraries used as reference databases in the identification of DNA sequences detected in samples (Fig. 6B, Fig. 7). Lack of laboratory infrastructure was acknowledged as a limiting factor more often in the international than a national level. IT infrastructures were not seen as the primary factor limiting the implementation. 32 Reports of the Finnish Environment Institute 20/2022 0% 10% 20% 30% 40% 50% 60% 70% Funding Laboratory infrastructure Analytical service providers Know-how or trained personnel Bioinformatics methods and pipelines Sequence libraries IT infrastructure Cost of analysis (e.g. sequencing) Lack of European method standards Other, please specify: All Finland Figure 7. Factors currently limiting the implementation of molecular methods in monitoring, as perceived by the respondents of an international Webropol survey (section 2.1.2). One limiting factor of the implementation of MoMM is that many monitoring programs need to produce quantitative abundances, biomass data or occurrence size estimates. Quantitative data is needed for the environmental status indicators, requested by e.g. the EU Water Framework Directive (European Union 2000) and Marine Strategy Framework Directive (European Union 2008, 2010, 2016, 2017). Similarly, abundances and population sizes are needed for many biodiversity monitoring purposes (e.g. EU Habitat and Bird Directives (EU 1992, 2009), national evaluations of red listed species (Hyvärinen et al. 2019), and national state indicators of biodiversity (e.g. Luonnontila.fi)). Quantitative monitoring data is also the foundation for sustainable management of game and fish populations. Many current eDNA methods do not yet produce reliable quantitative data directly comparable to traditional data, and for this reason they cannot be adopted as an exclusive alternative for the existing, standardized monitoring methods in many ongoing monitoring programs. Enhancing the reliability of quantitative MoMM is under very active international development, and e.g. qPCR-based approaches and high-throughput sequencing without PCR amplification show great promise. As a national example, there is an ongoing pilot project on quantitative estimation based on eDNA of fish species in Luke. Relative sequence abundances have also been successfully used in biodiversity surveys of bulk stream macroinvertebrate samples using DNA metabarcoding (Turunen et al. 2021b). Another example of quantitative MoMM, methods enabling detailed estimation of the number of individuals and territories are those that reveal the identity of individuals or family groups (e.g. from fecal or hair samples), for example the number of wolf packs routinely estimated by Luke. The downside of such methods is that they require either custom-developed genetic markers that vary at the individual or family group level (SNPs or microsatellite markers) or whole genome re-sequencing. Such analyses are therefore laborious and necessarily incur relatively high expenses, making such detailed routine analyses possible only for a small number of species. However, such approaches may become more readily available across species and systems if costs of sequencing continue to lower and genomic resources for non-model organisms continue increasing. Compensating for the remaining lack of quantitative methods, eDNA methods can already offer additional qualitative information on biodiversity. For example, in cases where a target species is spatially or temporally demanding to observe with traditional monitoring methods, presence observation based on eDNA samples may provide great help in targeting the traditional mapping or monitoring methods in the most potential locations. For example, eDNA methods have the potential to increase Reports of the Finnish Environment Institute 20/2022 33 information on biodiversity of single-celled pelagic primary producers of marine and freshwater environments i.e. phytoplankton, since eDNA methods can reveal taxa which are impossible to identify microscopically (e.g. small-sized or cryptic taxa), as well as sparsely occurring taxa (e.g. harmful or introduced/invasive taxa), which may go unnoticed with the standardized method. Thus, the combination of the genetic and traditional standardized microscopic methods will likely give a better assessment of the total phytoplankton diversity. In the future, when genetic methods are developed further, they may also provide quantitative data e.g. for the purposes of environmental status indicator analyses and biodiversity monitoring. In addition to limitations related to quantitative data, implementation of MoMMs in biodiversity monitoring is limited by the lack of information on the life-history phase of the organism, for example, whether it was reproducing or not at the sampling moment. This limitation concerns especially species that migrate, are nomadic or disperse long distances and can be observed in eDNA samples far away from the locations where they reproduce. For example, reporting for the EU Bird Directive and the EU Habitat Directive requires estimates of breeding bird population sizes and locations of breeding and resting places of species of interest, respectively. While MoMMs would in theory suit species mapping surveys and monitoring well, there are currently practical limitations such as too high expenses of analyzing all samples. In addition, while molecular methods may provide great help in identifying individuals that were not identified with traditional methods in the field, it may also be that already the sampling decisions in the field require special taxonomic expertise. An important category of challenges identified in our national workshop is related to the representativeness of DNA-based sampling and the interpretation of the new types of information (Fig. 6B). Many current monitoring programs focus on locally observed biodiversity. By contrast, an eDNA sample composes the aggregation of locally shed DNA traces and DNA transported from remote sources, and the organisms themselves are never directly observed. Although extracellular DNA typically decays in a matter of days, this time frame allows transport across large distances particularly in the air and in many aquatic systems (e.g. Deiner & Altermatt 2014, Goldberg et al. 2015). Specifically, in the river systems eDNA samples also comprise a number of dendric upstream sources. In such cases, utilizing eDNA methods for local anthropogenic impact assessment of biodiversity comparable to traditional methods is challenging. Moreover, much of the eDNA found in water samples is still bound to cells, which further increases its lifespan and thus the potential transport distance. This shortcoming may apply for many assessments of anthropogenic impact in river systems (e.g. those for WFD) and may support the use of bulk DNA metabarcoding over eDNA. Another possible avenue is complementing eDNA with eRNA analysis, allowing estimation of eDNA age and thus improving the reliability of local eDNA detection (Marshall et al. 2021). Best practices Based on the results of the national workshop, collaboration and networking with other experts both nationally and internationally were identified to be important when implementing MoMM (Fig. 6C). The use of commercial services in the analysis of DNA samples was also supported as a good and helpful practice. Standards and practical field/laboratory guides were also mentioned as important practices. In some, particularly microbial systems, next generation molecular methods are already well established in research, thus experience gained in such systems should be utilized more broadly. Moreover, existing infrastructures and databases should be utilized effectively. 34 Reports of the Finnish Environment Institute 20/2022 Reports of the Finnish Environment Institute 20/2022 35 3 Vision In Chapters 1-2, we have set the stage and presented our analysis of the state- of-the-art in the implementation of MoMM. The remaining Chapters 3-6 are future-oriented. Here, we start by outlining our longer-term vision for the role of molecular methods in environmental monitoring. We stress that while the new methods will eventually reduce the costs of monitoring, the primary aim of their implementation should be driven by the need for improved knowledge on biodiversity and the state of the environment and their ability to accurately and quickly detect environmental change facilitating timely application of required management and mitigation measures. Sufficient investment in the transition phase is crucial to ensure the continuity of high-quality data series. As the new methods gain ground, the role of citizen observations in providing additional taxonomic data will increase. At the same time, a minimum set of publicly maintained traditional monitoring efforts performed by professionals should be identified to guarantee the coverage of essential features not captured by the new methods. The commitment of monitoring organizations to follow the FAIR data management principles should cover also MoMM-based data. Based on our analysis of the state of the field, we envision that by 2030, reliable, internationally standardized molecular methods are routinely used in national biological monitoring (Fig. 1). Traditional taxonomic monitoring may complement the new methods, ensuring the continuity of valuable long-term data sets and facets of biodiversity not captured by molecular methods. At the same time, traditional citizen observations are increasing in number, quality and taxonomic coverage, and significant investments in taxonomic and science education and applications assisting data recording are made in recognition of their value. Where appropriate, citizen science is also routinely applied to sample collection for MoMM. Apart from species occurrence and abundance information, MoMM can provide information also on facets of biodiversity that have previously been underrepresented in monitoring schemes. These include the large-scale mapping of intraspecific genetic diversity across multiple species as well as the functions and biochemical processes active within the environment, as can be observed from the presence and activity of groups of functional genes. Sequence libraries have reached a sufficiently high coverage for the reliable application of taxonomy-based methods for most taxonomic groups. Sufficient taxonomic expertise is directed to support continuing efforts of mapping unknown biodiversity and developing sequence libraries. Alongside taxonomy-based methods, taxonomy-free methods are routinely applied to monitor environmental status (e.g. water, sediment, air and soil quality) and environmental impacts of human activities. Additionally, whole genomes of species characteristic of different habitat types are made available at an increasing rate, building capacity towards routine implementation of monitoring of intraspecific genetic diversity and spatial genetic structure of natural populations (Formenti et al. 2022, Lewin et al. 2018). The genomes and DNA-based monitoring data is stored, handled and made available following a national data management plan and using internationally agreed upon metadata standards, following the FAIR (Findable, Accessible, Interoperable, Reusable) Guiding Principles for scientific data management and stewardship (Wilkinson et al. 2016). Most of the data (except for strictly protected species) are georeferenced, openly available and made available through Application Programming Interfaces (APIs) or automatically fed into global databases. Education programs in biological and environmental science and laboratory analytics have been updated to respond to the growing need for expertise in molecular monitoring methods. A significant part of laboratory and bioinformatic analyses for national biological monitoring are carried out by the https://www.go-fair.org/fair-principles/ https://www.go-fair.org/fair-principles/ 36 Reports of the Finnish Environment Institute 20/2022 private sector and there is a well-developed national as well as international market for state-of-the-art molecular analytics and products. Finland is an international forerunner in providing analytical, bioinformatics and consulting services as well as accredited laboratory services through its accredited reference laboratory framework following internationally agreed standards. MoMM are developed alongside Earth observation and other remote sensing methodology as well as ecological modelling and machine learning applications. Validated modelling and analysis tools are used to assist in the interpretation of DNA-based observations and in sampling design. Spatiotemporal approaches enable automatically updated distribution maps based on ecological model simulations, as well as the creation of user-defined future scenarios illustrating e.g. the effect of land use activities and climate change on national biodiversity or the spread and impact of invasive species. Uncertainty is quantified and highlighted in all model outputs. Reports of the Finnish Environment Institute 20/2022 37 4 General development plan Based on our analysis of the state-of-the-art and the chief obstacles standing in the way of the envisioned MoMM implementation, we have identified six development areas that merit particular attention in the coming years: 1) International coordination is paramount to ensure comparability of data in time and space and to avoid unnecessary duplication of efforts and resulting delays in overall method uptake. 2) Networking across sectors is essential to increase awareness and knowledge of the new methods, to establish the connections that form the basis of a viable market for services and products – and to provide platforms for critical discussion on when, where and how the new methods should be applied. 3) Education plays a key role in solving the lack of up-to-date know-how, one of the most pressing challenges identified in our status assessment. 4) Infrastructure exists but should be used more efficiently and codeveloped, updated and extended in a co-creative and coordinated manner between relevant government agencies, universities and other organizations. 5) The coverage of sequence libraries should be continuously improved not only in designated projects but as a running task in monitoring programs and pilots. Additionally, in order to allow for comprehensive mapping of intraspecific genetic diversity, the number genomes published for different organisms would need to be increased. 6) Modelling and analysis tools should be applied and developed alongside MoMM and other new monitoring methodologies to fully utilize their information. 4.1 International coordination and standard development Until recently, there has been little concerted international coordination with respect to implementation of molecular methods into environmental monitoring. While several organizations (e.g. Biodiversity Information Standards, originally Taxonomic Databases Working Group (TDWG)) have long developed guidance for data management, there is little official agreement in the form of international standards. This gap has been realized in Europe and is explicitly addressed for molecular methods used to assess water quality. To fill this gap, in 2018 a working group under the European Committee for Standardization (CEN) was established in the technical committee TC 230 which oversees standards pertaining to water quality. This working group, “WG28 eDNA and DNA methods” is chaired by SYKE as the national representative of the Finnish national standardizing body SFS. WG28 is processing its first work item “Water sampling for capture of macrobial environmental DNA in aquatic environments”, which will be finalized in early 2023. Other standards, concerning the analysis of eDNA from other matrices as well as minimum requirements for data and metadata, are planned for the coming years. 38 Reports of the Finnish Environment Institute 20/2022 Other standards have been developed under the International Organization for Standardization (ISO), such as the standard: “Soil quality — Direct extraction of soil DNA”, ISO 11063:2020. A newly formed working group under ISO TC 331 “Biodiversity” is likely to suggest several general standards for the use of molecular methods in biodiversity assessments. SYKE will attempt to set up a liaison between ISO TC 331 and CEN TC 230 / WG28 in the spirit of the Vienna Agreement (1998), the ISO Council resolution 35/2001, and the CEN Administrative Board resolution 2/2001 to avoid duplication of effort. SYKE has recently established links to the eBioAtlas initiative which originated in the UK through an interaction between the company NatureMetrics and the IUCN. It is in the national interest to both participate and widen the scope of international participation including possibly GeoBON in the eBioAtlas and jointly strive towards the establishment of internationally accepted guidance on the minimal requirements for molecular method and related data use. Similarly, exchanges and more engaged and goal-oriented cooperation between Nordic administrations progressing the implementation as well as with other European forerunners developing molecular methodology are needed to assure a timely uptake of the methods. In Finland this will require formalized fora for information exchange which should be established under the umbrella of the implementation program of the national environmental monitoring strategy. 4.2 Networking and promoting market development As is characteristic for a new and actively growing area, the Finnish field of molecular monitoring is highly scattered, with several individual actors and groups discovering and applying the new methods independently of each other, while the general knowledge of the methods is relatively scarce even among professionals in biological monitoring. Moreover, many researchers and officials in the monitoring field are unaware of molecular biological services that would be available or lack the background knowledge to make use of them. Lack of knowledge and know-how was the second most often mentioned challenge in the implementation of MoMM both in the national workshop (Fig. 6B) and in the international Webropol survey (Fig. 7), and national and international networking was the most often mentioned best practic