Report of the Working Group on the Application of Genetics in Fisheries and Mariculture (WGAGFM)

ICES WGAGFM REPORT 2013

SCICOM STEERING GROUP ON HUMAN INTERACTIONS ON ECOSYSTEMS

ICES CM 2013/SSGHIE:11

REF. SCICOM

Report of the Working Group on the Application of Genetics in Fisheries and Mariculture (WGAGFM)

7–9 May 2013

International Council for the Exploration of the Sea Conseil International pour l’Exploration de la Mer

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ICES. 2013. Report of the Working Group on the Application of Genetics in Fisheries and Mariculture (WGAGFM), 7-9 May 2013. ICES CM 2013/SSGHIE:11. 52 pp.

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The document is a report of an Expert Group under the auspices of the International Council for the Exploration of the Sea and does not necessarily represent the views of the Council.

ICES WGAGFM REPORT 2013 | i

Contents

1 Opening of the meeting ...... 3

2 Adoption of the agenda ...... 4 2.1 ToR a) Produce a review of the identification and use of adaptive gene markers in shellfish aquaculture and for the genetic characterization of wild populations...... 4 2.2 ToR b) Review and consider technological developments in fisheries forensics and management of exploited marine fishes with emphasis on contributions to sustainability and governance ...... 10 2.2.1 Summary ...... 10 2.2.2 Introduction ...... 11 2.2.3 Key technological developments ...... 13 2.2.4 Technology transfer: Issues and Challenges ...... 15 2.2.5 Supportive initiatives, projects, assemblies and legislation tapping into advanced technologies ...... 17 2.2.6 Recommendations ...... 29 2.2.7 References ...... 29 2.3 Tor c) Review on the use of metagenomics and metatranscriptomics as an approach for marine ecosystem management ...... 32 2.3.1 Rationale:...... 32 2.3.2 Environmental sequencing ...... 33 2.3.3 Qualitative and quantitative biodiversity assessment using environmental sequencing ...... 34 2.3.4 Applications of metagenomics to inform the implementation and utilization of an EAFM framework ...... 34 2.3.5 Limitations of using metagenomics for qualitative and quantitative biodiversity assessment ...... 37 2.3.6 Conclusions...... 38 2.3.7 The WGAGFM recommends ...... 38 2.3.8 References ...... 39 2.4 ToR d) Produce an update on SNP-technology assessment...... 40 2.4.1 WGAGFM action list ...... 41 2.4.2 Reference ...... 41

Annex 1: List of Participants...... 42

Annex 2: WGAGFM Agenda ...... 44

Annex 3: WGAGFM Terms of Reference for the next meeting ...... 46

Annex 4: Recommendations ...... 48

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Executive summary

The Working Group on the Application of Genetics in Fisheries and Mariculture (WGAGFM) convened in Reykjavik 7–9 May 2013. Members met to discuss and consider the Terms of Reference (ToR) decided by the ICES Science Committee. The report contains the main issues discussed and the management recommendations for each of these four ToRs. Dorte Bekkevold (Denmark) chaired the meeting, which opened at 09:00 on 7 May and closed at 13.30 on 9 May. The meeting had 15 participants representing eight member nations (Canada, Denmark, Germany, Iceland, Norway, Russian Federation, Spain and UK) and the European Joint Research Centre in Italy. Members discussed developments in genetic methods to trace fish and fish products, which have advanced much over the past few years. Focus was on examining fisheries forensics and DNA-analysis in the context of supporting a shift to a more sustainable exploitation of natural marine resources under current and future fisheries management and governance schemes. This was accomplished by reviewing existing examples demonstrating value and feasibility for control purposes, management and conservation. Members found that the examples demonstrate the methods’ relevance for a suite of management questions and recommend that ICES SCICOM and ACOM push for more standardized use of the methods. Escaped fish from aquaculture are an increasing concern, especially in salmonids where millions of farmed fish each year escape into the wild, where they interact and interbreed with wild fish. Several studies report that hybridization and introgression by escaped farm fish can incur a fitness cost to wild populations, causing increasing concern for the continuing health and viability of wild populations and awareness about conserving native fish gene pools. Molecular quantification has proved valuable for demonstrating introgression by farm fish. However, in many cases, the introgression process is complex, e.g. with respect to escape rates and genetic make-up of escapees, and impacts can therefore be difficult to assess and predict. Also, no general guide-lines exist as to above which level introgression can be expected to impose severe threats to wild populations. There is therefore a growing need for the identification and development of statistical tools to assess and quantify levels of introgression. In response to a request from scientists from the Norwegian Institute for Marine Research WGAGFM members discussed analytical challenges and opportunities. Members concluded that in order to develop and implement reliable management strategies and advice, locally and internationally, it is of importance to review and consider the different options for the analysis of genetic data to quantify the level of introgression. It was therefore recommended that a ToR addressing these issues and contributing advice is planned for 2014. There is increasing pressure for sustainable aquaculture, also in shellfish. Compared to fish aquaculture management issues, shellfish exhibit both similarities and dissimi- larities. Like in fish, many species of shellfish show evidence of local adaptation, but wild/farmed interactions may in some cases be even more difficult to predict e.g. due to differences in dispersive stages. The availability of genomic resources, together with varied farm/wild interaction scenarios means that there is wide scope for implementation of genomic approaches, to address both production and management issues. The WGAGFM therefore recommend expanding an evaluation of such work over the two next years, by also inviting contributions from WGAQUA.

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Metagenetics, and hereunder environmental sequencing, is a powerful method to determine biodiversity, thus offering unprecedented support to the implementation of the marine strategic framework directive and ecosystem based fisheries management. For some applications methods are fully standardized, while for others, especially those requiring quantification of biomass or abundance, more in depth studies are needed for methods to qualify as a management tool. WGAGFM recommend the move towards applying metagenetics to address management of marine resources.

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1 Opening of the meeting

The Working Group on the Application of Genetics in Fisheries and Mariculture (WGAGFM) met in Reykjavik 7–9 May 2013. The Terms of Reference (ToR) were decided by ICES Science Committee in Bergen, Norway, in 2012. Dorte Bekkevold (Denmark) chaired the meeting, which opened at 09:00 on Tuesday, 7 May and closed at 13.30, Thursday, 9 May 2013. The meeting had 15 participants; 14 members and 1 PhD student, representing the European Joint Research Centre and eight member nations (Canada, Denmark, Ger- many, Iceland, Norway, Russian Federation, Spain and UK; Annex 2). An additional member from Canada contributed to the work by correspondence. The meeting was hosted by and held at the Matís ohf., Icelandic Food and Biotech R&D, Vínlandsleið 12, 113 Reykjavík, Iceland. The WG is very grateful to hosts Dr Sarah Helyar and Dr Anna-Kristin Danielsdottir, as well as to the other staff at Matís for their hospitality and kind and efficient assistance throughout. WGAGFM has an established framework for completing its ToR. Prior to the meeting, small ad hoc working groups, under the leadership of one or more persons, are established to prepare position papers related to specific issues in the Terms of Refer- ence. The leader(s) of each ToR is responsible for presenting the position paper in plenary at the meeting and chairing the discussion. Thereafter, volunteers undertake the task of editing and updating position papers according to points raised in the plenary discussions. The ToR leader(s) is responsible for preparing the final report text from their sessions. Prior to the meeting the agenda is circulated to all members.

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2 Adoption of the agenda

2.1 ToR a) Produce a review of the identification and use of adaptive gene markers in shellfish aquaculture and for the genetic characterization of wild populations. John Gilbey, Iveta Matejusova There is an increasing pressure for sustainable aquaculture of many shellfish species in Europe and worldwide. There is evidence of local adaptation in many species of shellfish and locally adapted populations are often characterized by traits allowing them to survive and thrive under heterogeneous environmental conditions (Sanford and Kelly, 2011). Some such traits are also of interest in relation to mariculture production (e.g. those associated with growth rates, survival and disease resistance). The fast developing field of genomics offers a potential to identify the necessary markers linked to such adaptive traits (Stinchcombe and Hoekstra, 2007). Such markers are valuable, not only to ensure sustainable shellfish aquaculture in heterogeneous and changing environments, but also from a marine ecosystem management/biodiversity conservation point of view, where the aim is to identify and protect populations which may be locally adapted (e.g. Riginos and Cunningham, 2005). The recent and continuing development of new genetic screening technologies has the potential to significantly aid the identification of adaptive markers (Morozova and Marra, 2008, Harismendy et al., 2009). Therefore it is important to closely monitor the development in this field and evaluate potential implications for the improvement of aquaculture relevant traits in cultured stocks but also its relevance for population genetic issues concerning wild and interactions between wild and cultured populations. Adaptive variation has been defined as “heritable phenotypic variation that is sorted by natural selection into different environmental niches, so enhancing fitness in specific environments” (Robinson and Schluter, 2000; Carvalho et al., 2003, Garcia de Leaniz et al., 2007). Such variation is the result of heterogeneous local selective pres- sures that act to maximize individual fitness within the different local environments, a process known as local adaptation (Kawecki and Ebert, 2004). Such adaptation results in heritable genetic trait differences between individuals and populations. Typically, and historically, adaptive traits of interest have been targeted in selection programs in both agriculture and mariculture programs using techniques such as Marker Assisted Selection (MAS). MAS is an indirect selection process where the trait of interest is selected on based on a genetic marker linked to it (Ribaut and Hoising- ton, 1998). Historically the most common method for identifying genetic markers linked to functional loci has been Quantitative Trait Loci (QTL) mapping (Lynch and Walsh, 1998). In this process association studies are performed using a number of loci spread across the chromosomes of the organism. Statistical techniques are then utilized to examine associations between loci and traits of interest in multi-generational crossing experiments, pedigree analysis and/or in natural populations and the markers identified can then be utilized in selection programmes. Recent developments in genetic screening resulted in new and more powerful techniques of identifying QTL linked markers using high throughput (or next-generation) sequencing. Such technologies parallelise the sequencing process producing thousands or millions of sequences at once (Shendure and Ji, 2008). This enables very fine scale coverage of the entire genome and so has the potential to identify markers linked to many adaptive loci or specifically linked to traits of interest using techniques such as RAD (Re- striction site Associated DNA) Sequencing (Miller et al., 2006). The technological ad-

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vances and associated reductions in costs of such sequencing efforts have also meant that in many species full genome sequences are becoming available. Such resources allow direct identification of QTL themselves and have the potential to revolutionise both understanding of the genetic architecture underlying the traits of interest and the associated selection efforts upon them. In both the aquaculture and the wild situations adaptive markers have been utilized to examine a number of questions (Table 2.1.1). Genomic resources however differ greatly between the different species in question. In some cases complete genomes have been sequenced, while in others few markers are available. The adaptive markers and genomic resources already available, and new markers under development, have the potential to be used more widely to examine both interactions between mariculture produced and wild populations and help to further develop a sustainable, cost efficient and environmentally friendly mariculture production.

Table 2.1.1. Adaptive marker use in aquaculture – summary of relevant genetic information.

Genomic resources Gene Linkage Genome EST QTL expression Microarray map/s sequence libraries maps Species analysis

Crassostrea gigas - Microsatellites1 Yes3 Yes4 Yes5,6 Yes7,8 Yes9,10

(Pacific oyster) - AFLPs2 Crassostrea - Microsatellites11 Yes12,13 Yes14 Yes15 Yes16 virginica - AFLPs11 (Eastern oyster) - SSCP11 Haliotis rubra - Microsatellites17 Yes18 Yes19 (Blacklip abalone) Haliotis discus - Microsatellites20,21 Yes22 Yes23 Yes24 (Disk abalone) - AFLPs20 - RAPD20 Mytilus edulis - AFLPs25 Yes27 Yes28 (Blue mussel) - Microsatellites26 Mytilus Yes29,30 Yes31,32 Yes31 galloprovincialis (Mediterranean mussel) Dreissena Yes33,34 Yes34 polymorpha (Freshwater mussel) Ostrea edulis - AFLPs35 Yes36 (European flat oyster) Aequipecten -AFLPs37 Yes38 irradians -Microsatellites38 (Atlantic bay scallop) Pecten maximus Yes39 (King scallop)

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Genomic resources Gene Linkage Genome EST QTL expression Microarray map/s sequence libraries maps Species analysis Chlamys farreri - AFLPs40 Yes41-43 Yes44 (Zhikong scallop) Strongylocentrotus - AFLPs45 Yes47 Yes47 nudus Microsatellites46 (Sea urchin) Strongylocentrotus - AFLPs45 Yes48 intermedius (Sea urchin) 1 Hubert and Hedgecook 2004, 2 Li and Guo 2004, 3 Zhang et al. 2012, 4 de Lorgeril et al. 2011, 5 Sauvage et al. 2010, 6 Guo et al. 2012, 7 Romero-Geraldo et al. 2012, 8 Du et al. 2013, 9 Dheilly et al. 2011, 10 Sussarellu et al. 2010, 11 Yu and Guo 2003, 12 Quilang et al. 2007, 13 Wang et al. 2009, 14 Yu et al. 2006, 15 Chapman et al. 2011, 16 Wang et al. 2010, 17 Baranski et al. 2006, 18 Baranski et al. 2008, 19 van der Merwe et al. 2011, 20 Liu et al. 2006, 21 Sekino and Hara et al. 2007, 22 Zhan et al. 2008, 23 Liu et al. 2007, 24 De Zoysa et al. 2011, 25 Lallias et al. 2007, 26 Presa et al. 2002, 27 Delphine et al. 2007, 28 Cubero-Leon et al. 2012, 29 Craft et al. 2010, 30 Venier et al. 2009, 31 Banni et al. 2011, 32 Pantzartzi et al. 2010, 33 Xu et al. 2010a, 34 Xu et al. 2010b, 35 Lallias et al. 2007, 36 Lallias et al. 2009, 37 Wang et al. 2007, 38 Li et al. 2012, 39 Charrier et al. 2012, 40 Wang et al. 2005, 41 Chen et al. 2013, 42 Zhang et al. 2012, 43 Li et al. 2012, 44 Qin et al. 2007, 45 Zhou et al. 2006, 46 Yan et al. 2010, 47 Wei et al. 2011, 48 Yunfei et al. 2011. The number of species in mariculture, the availability of genomic resources now and under development, together with the myriad of general and more localized farm/wild interaction scenarios means that there are potentially a large number of situations that may benefit from examination using adaptive markers and other associated genomic resources. There would thus seem to be a great potential for transfer of expertise from one species/situation to other novel circumstances and species.

WGAGFM action list: The WGAGFM therefore suggest approaching this task in a number of ways over the next two years; 1 ) Use the internal expertise of the WGAGFM members to • identify the full set of genomic tools and techniques available and/or under development • identify specific issues/situations that are being examined using the tools • identify particular issues/situations of concern that may benefit from research using the tools 2 ) Approach the ICES Working Group on Aquaculture (WGAQUA) to • outline the tools available and/or under development • identify issues of concern in the culture situation and those associated with mariculture/wild interactions which may be addressed using the tools 3 ) Approach researchers outside the ICES environment to • identify novel tools and techniques now under development • identify situations in which the genomic tools available and/or under development may be of use The overall and outcome of these efforts will be to bring together information on work already completed using the tools in question (i.e. aiming to expand Table 2.1.1), outline the work that is actually ongoing at present, outline a tool-kit of resources/procedures/approaches that are available and/or under development, and

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identify situations where such tools could potential be of benefit. This information will be detailed in a report to be presented at the WGAGFM in 2014/15.

WGAGFM recommend: That WGAQUA produce a ToR identifying and reviewing cases/scenarios where interactions between shellfish of mariculture and wild origin is of concern, where genomic tools may allow estimation of potential introgression.

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2.2 ToR b) Review and consider technological developments in fisheries forensics and management of exploited marine fishes with emphasis on contributions to sustainability and governance Gary Carvalho, Sarah Helyar, Reinhold Hanel, Daria Zelenina, Geir Dahle, Ian R. Bradbury and Jann Martinsohn.

2.2.1 Summary “Law without enforcement is just good advice” (Abraham Lincoln) Globally sustainable fisheries management continues to be greatly impeded by illegal fishing and also fraud (false labelling) along the supply chain, including in areas covered by the ICES remit. As highlighted by the WGAGFM in its 2009 report, fisheries forensics, the methodical gathering and analysis of evidence that can be presented in

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a legal proceeding, and in particular fisheries forensic genetics, constitutes a powerful asset to fishery control and enforcement, and its application should ideally become routine worldwide. Since 2009 great progress has been made, e.g. concerning the use of genetic barcoding for species identification of marine fish (products), and, importantly, the identification of genetic population structure of marine fish, which underlies origin assignment. Such advances prompted the WGAGFM to revisit the subject with the intention to create an additional impetus for the enhanced integration of forensics and genetics to support control and enforcement. Rather than discussing in detail the technological aspects of this field, here we em- phasize, with judicious reference to recent examples, the availability of fishery forensic applications to support the compliance with legislation and laws, such as the recently reformed Common Fisheries Policy control regulation. Existing legislation which could and should be supported by fisheries forensics is delineated, as are recent high-level events and meetings, where the utility and value of genetic approaches, put into a forensic framework has been highlighted and acknowledged by the stakeholder community. Recently concluded and ongoing major research initiatives such as FishPopTrace and AquaTrace (FP7, EC) have focused attention on the application of cost-effective high throughput DNA-based analyses in fisheries forensics. Indeed evidence is accumulating that (genetic) fishery forensics are readily available, being efficient and cost-effective and giving a highly deterrent impact. However the transfer from research into end-user applications routinely available across nations and regions to control and enforcement authorities lags strikingly and unreasonably behind. We believe ICES, as a major global organization for enhanced ocean sustainability, is ideally positioned to support and facilitate the exploitation the opportunities emerging from progress in forensics, genetics and genomics. As delineated in the recommendations, immediate steps could include the organization of an international event involving geneticists, fisheries policy-makers, fisheries control agencies and stakeholders in the subject. Moreover ICES should encourage Member States to invest in the development of end-user adapted technology platforms and reference databases, as well as to embrace the routine employment of validated DNA-based tools on an ad hoc basis, from catches sampled at ports, market samples and at various stages from capture to consumer. Such practices would greatly contribute to the quest in attaining compliance with rules and laws in the fisheries sector, in line with the common goal of reaching sustainable and profitable fisheries. Enhanced governance coincides with major fishery frameworks such as the currently reformed Common Fisheries Policy.

2.2.2 Introduction In 2009 the ICES WGAGFM reviewed and outlined the status of traceability methods in the fisheries sector based on genetics to support fisheries control and enforcement and the fight against IUU fishing (Carvalho et al., 2009). That report threw light on recent consequential technical/technological progress and argued that new approaches, particularly those based on genetics and genomics, and when integrated into a forensic framework, provide a major opportunity to enhance fisheries control and enforcement, the fight against Illegal, Unreported and Unregulated (IUU) fishing as well as traceability along the supply chain. Rather than merely revisiting the progress made in the field in the meantime, this report examines fisheries forensics and DNA-analysis in the context of supporting a

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shift to more a sustainable exploitation of natural marine resources under current and future fisheries management and governance schemes. To this end emphasis, will be put on applicability and existing examples demonstrating value and feasibility for control purposes, together with implications for management and conservation. Forensics is a field of science dedicated to the methodical gathering and analysis of evidence to establish facts that can be presented in a legal proceeding. Evidence is sometimes required within the fisheries context (“fisheries forensics”), when regulations have been breached, such as illegal fishing activity, mislabelling of a fish product, or under‐sized fish are captured. While there has been no shortage of policies and maritime laws to assist in the governance of our oceans, it has proven much more difficult to enforce maritime law, and to execute prosecutions. In part, con- straints are related to having an appropriate set of tools to yield sufficiently robust evidence in a court of law. As stated by Abraham Lincoln, “Law without enforcement is just good advice”. The quote is as relevant to regulations governing the sustainable management and conservation of fish resources as to any other type of policies. The global level of IUU fishing and supply chain fraud offer financial gains on a scale that attracts organized criminal groups as well as unscrupulous individuals; the tools required to detect and tackle such activities and enforce fishing regulations need to be equally sophisticated. As already argued by WGAGFM in 2009 and others, evidence produced by forensic science can strongly support investigations and deter illegal fishing activities or fraud along the food supply chain (Ogden, 2008; Mar- tinsohn 2011a). IUU fishing remains an important global challenge with highly ad- verse effects on ecosystems as well as the socio-economic well-being of numerous fisheries communities. In European member states alone it is estimated that between 2008 and 2020 the cost of IUU activities will be €10.8 billion, representing a loss of >27,000 jobs in the fisheries sector. Moreover, since fraud along the supply chain, e.g. the selling of fish products under a false label, keeps undermining consumer information and trust, thereby damaging the fisheries industries, forensics constitutes a great asset to control for compliance with existing rules and to enforce laws in cases of infringement. In the fisheries sector two levels of identification are meaningful for control and enforcement: 1) The species level: Unambiguous identification of the true species, 2) Population level: Identification of population (stock)/regional units and assignment of individuals to their respective unit of origin. As has been shown, and will further illustrate here, both levels can be dealt with in a powerful way through genetic and genomic analysis. Efficient governance of marine fisheries is complex and particularly challenging due to the nature of this common natural and exhaustible resource which is shared among numerous individuals, peoples and nations, and since the marine realm is not as readily accessible for assessment and monitoring as its terrestrial counterpart. As mentioned before, additionally illegal activities in the fisheries sector, particularly IUU fishing further burden efficient governance and the move towards fisheries that is at the same time as profitable as it is sustainable. Due to the worldwide boom in marine aquaculture activity (FAO, 2012) the second level of relevance to forensic applications includes the increasingly important distinc- tion between farmed and wild fish, e.g. to identify aquaculture escapees, which might have a considerable impact on wild populations (Genimpact, 2007; Aquatrace 2013 https://aquatrace.eu).

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Worldwide, many nations strive to ensure sustainable fisheries and aquaculture through the development of strong legal frameworks. Within the EU most relevant are the recently implemented and complementing Regulation (EC) No 1005/2008 to prevent, deter and eliminate illegal, unreported and unregulated fishing, the so- called ‘IUU-Regulation’ and Regulation (EC) No 1224/2009 of 20 November 2009 establishing a Community control system. In the USA the so called ‘Lacey Act’, according to which the receipt, acquisition or purchase of fish that was taken, possessed, transported or sold in violation of US laws or regulations, is prohibited. Quite obviously such laws would greatly benefit from the application of advanced analytical technologies, such as those based on genetic and genomic approaches, particularly when put in a forensic context and readily accessible to fisheries control and enforcement authorities. Since marine fisheries have a strong supranational character, ideally such an approach is pursued on a regional multinational or even global level. In this ToR it will be argued that a considerable research-based groundwork has been accomplished to enable the uptake of genetic and genomic analytical approaches into fisheries control, enforcement and traceability schemes covering the entire fish product supply chain. As will be shown, additional challenges, which however are entire- ly surmountable, arise when the application of forensic standards is required. It is encouraging to observe that the awareness of the opportunities for sustainable fisheries and a prosperous fishing industry, which are created by new technological developments, is on the rise. This is shown by numerous recent international multi- stakeholder high-level assemblies and conferences, where particularly (forensic) genetic and genomic approaches has been discussed in the context of fisheries governance (Table 2.2.1). However sustained effort is still required to promote international consistency and coherence with respect to the integration of such approaches into efficient fisheries control, enforcement and traceability schemes. We believe that the International Council for the Exploration of the Sea should spearhead such an effort, as reflected in the recommendations emerging from this document.

2.2.3 Key technological developments Brief overview and critique of existing approaches for control, enforcement and prosecution Most nations with direct access to marine environments have policies in place to manage the exploitation of oceanic resources which are under their jurisdiction. (Car- valho et al., 2009; Martinsohn, 2011a). In addition to relatively long-standing certification procedures to serve as a framework for traceability of fish and fish products, various vessel monitoring and detection systems are in place. For example, all Euro- pean Union vessels above 15 m in length are fitted with a vessel monitoring system (VMS), which relies on satellite navigation and communication technologies. Howev- er, traditional certification procedures are readily susceptible to fraud at various stages along the supply chain, as well as non-functioning or “hidden” signals from the “blue box” associated with VMS. DNA-based procedures provide the only robust approach that can be used throughout the chain from capture to plate, even on highly processed products, and provided appropriate standard operating procedures are in place, can generate traceability with unprecedentedly high levels of accuracy (Nielsen et al., 2012). Many diverse non-genetic approaches have been used both to identify fish species and for stock identification, such as fatty acid analysis, microchemistry, stable isotope analysis, morphology and otoliths (both microchemistry and geomorphometrics). As

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these were reviewed in detail in a previous report from the WGAGFM (Carvalho et al., 2009), and also more recently (Martinsohn et al., 2011a) these are not covered in detail here. Advanced in genetic approaches are considered further both within cited publications, and summarized below.

Focus box 1: Forensic validation of the barcoding gene, COI A key aim of barcoding is the production of COI reference sequences recovered from multiple voucher specimens using standard protocols, resulting in numerous authenticated diagnostic DNA patterns (haplotypes). Given the development of barcoding and associated protocols, the viable application of COI as a forensic marker, alongside existing genes, has expanded its use from being a research tool to applications in law enforcement and regulation. Prior to its use in forensic casework any novel marker or tool needs to be validated. Validation of a genetic marker is designed to determine its reproducibility and limitations by testing its ability to provide accurate results under a variety of conditions – thus enabling its use in providing unequivocal defense or prosecution evidence if required, as well as promoting ease of comparison across laboratories and data sets. Forensic validation of COI included various technical tests of reproducibility, mixed DNA, DNA template con- centration, chemical treatments, substrate variation, environmental conditions and ther- mocycling parameters (Dawney et al., 2007). Strict quality assurance, associated with the standardized nature of protocols and requirement for voucher specimens and associated sample data, provide among the most robust and widely accessible reference sources for the global description and management of natural resources. FishBol, a global network of reference data bases for global DNA barcoding of fishes, currently has data for approximately 30% of all species (9599 from 32,257 est. fish species). Potential forensic applications of fish DNA barcoding include the monitoring of fisheries quotas and by-catch, inspection of fisheries markets and products, the control of trade in endangered species, and improvements in the traceability of fish products.

At the species level, the assignment of individuals to their origin (Individual Assign- ment – IA) relies on a genetic marker that exhibits low intraspecific but high interspecific polymorphism. Options are available to identify specific species, or groups of species such as rt-PCR or Restriction Fragment Length Polymorphism (RFLPs) assays which can be used to differentiate between small numbers of species (for example identifying different gadoid eggs; Taylor et al., 2002), and Microarray technology which has been demonstrated to be suitable to identify and differentiate 30 fish species (Kochzius et al., 2008). However, very few of these methods have been forensically validated, with the exception of Forensically Identifiable Nucleotides (FINS; Jerome et al., 2003; Gil 2007) which can differentiate between small numbers of species, and DNA barcoding (Herbert et al., 2003), which has not only been validated (Dawney et al., 2007), but can also differentiate between a large number of species. While population genetic research has demonstrated that many marine organisms can be separated into more or less genetically distinct groups at the population level (recent reviews in Hauser and Carvalho 2008; Reiss et al., 2009), the identification of the population of origin of an individual or group of individuals can pose significant challenges. Foremost among these is typically the low level of genetic divergence detected among many marine fish populations, and the ensuing associated high levels of variance in various population genetic parameters generated. The impact of such variance is to reduce the certainty for assigning unknown individuals to source population, especially where relatively few DNA markers or loci are employed. While there are DNA markers that can be used, such as microsatellites, many of these

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are difficult to transfer between study areas and laboratories. Thus, there is a requirement to establish a marker‐based framework that is sufficiently informative and robust to deliver evidence within an integrated fisheries forensic framework; for which certain legal standards must be fulfilled to be admissible as evidence before courts of law. Samples need to be handled according to approved protocols - prevention of cross-contamination at the sampling site for example are essential, so handling of samples is one of the most important factors in any forensic analysis. Quality assurance is obtained through Standard Operating Procedures (SOPs), doc- uments containing instructions that are followed to secure routine procedures, standardized protocols and for which no ad hoc modification is acceptable.

2.2.4 Technology transfer: Issues and Challenges Using genetic approaches to provide evidence of fisheries control and enforcement is not a trivial task, particularly when forensic standards are applied. Challenges arise since the transfer of applications, mostly based on and developed in the academic research realm, requires stringent, coherent and concerted efforts involving various stakeholders and end-users. Prior to the use of any DNA-based tool for forensic analysis, it is essential to compile a representative, precise and easily accessible reference database. The reference database needs to encompass diagnostic signatures either at the species level of genetic diversity that are specific to particular spawning populations of stocks of the target species. Such a baseline requires comprehensive sampling across the species range, as well as having sufficiently large sample sizes that are dictated by both the biology of the species and the nature and number of DNA loci employed (Nielsen et al., 2012). Moreover, to comply with the strict procedures required for forensic analysis, it is also important to apply DNA markers that not only exhibit high levels of precision at the species or population levels, but importantly can be readily compared and easily accessible across the scientific and stakeholder communities. At the species level the most reliable approach is the sequencing of the standard mtDNA marker, the cytochrome oxidase I gene (COI), followed by comparison with the FishBol database. While in some circumstances, for example the analysis of highly processed products containing unknown mixed species, it is appropriate to design and employ species- specific tests of identity, in most conditions, it remains possible to employ the usual multispecies DNA barcoding COI marker. The key point here is that it is necessary to avoid the inclusion of more than one species in any single PCR-based test, since such mixtures can generate ambiguous data with sometimes unacceptably low levels of certainty. While the majority of tests at the species level continue to focus on traditional Sanger-based DNA sequencing (Costa and Carvalho, 2007), it is now feasible to employ advanced second generation sequencing procedures of so-called “mini- barcodes” where inherent controls are included that enable the simultaneous testing of samples containing several species (Hajibabael et al., 2011). In some situations, for example where biologically similar species are being compared, or where certain species are suspected of hybridizing, the use of the COI barcode may not be appropriate. For example, in four species commonly found in Europe either as traded products or wild fish are Russian (A. gueldenstaedtii), Persian (A. persicus), Siberian (A. baerii) and Adriatic (A. naccarii) sturgeon. Here it is necessary to employ markers from other mtDNA regions such as cytochrome b and NADH5 and the control region (Birstein et al., 2000). Moreover approximately 30% of A. gueldenstaedtii individuals from the Caspian basin are similar in mtDNA to A. baerii. (Birstein et al., 1998,

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Jenneckens et al., 2000), in such cases, markers from other genetic regions are needed, such as nuclear DNA. At the population level, single nucleotide polymorphisms, or so-called “SNP markers” are considered to be the marker of choice for forensic applications. While valuable population-level information has been generated using previous marker types such as microsatellites, these suffer typically from difficulties for comparison across laboratories and datasets. SNP markers are essentially binary: this means that the various genetic variants or “alleles”, are either present or absent, providing a much more stringent baseline for comparative analyses. Microsatellites, on the other hand, have genetic variants that are determined by their particular size as determined on a gel, and it is well established that on occasions, variation in size of particular alleles may be more a function of running conditions employed in different laboratories, rather than representing genetic variation. SNP databases have already been created for a few marine and anadromous fish species (Seeb et al., 2011), and many others are in the process of being developed. Based on the development of validated population-informative SNP markers, forensic protocols for population identification including appropriate statistical methods for data analysis can be produced (e.g. Nielsen et al., 2012). The resulting forensic assays can then be integrated into appropriate accredited (ISO17025) procedures, and a transferable forensic framework recognized by legal systems throughout the EU. Within the scope of European fisheries enforcement, accessibility to validated protocols and reference data are crucial. It follows therefore that a central, quality assured and accessible database will be required for the application and regular uptake of such technologies. While it may be prudent and cost-effective to have regional baselines covering specific fisheries and species ranges, the significant global transfer of many mislabelled species or populations, as well as the shifts in the range of distribution of wild species, highlights the need for careful cross-referencing across any such regional DNA repositories. Additionally, to allow stakeholders to make an informed decision on whether and how to integrate (forensic) genetics and genomics into fisheries control and enforcement, inherent costs and benefits should be considered, to objectively assess the value of specific technologies. Generally speaking, a Cost Benefit Analysis (CBA) is a tech- nique designed to determine the feasibility of a project, plan or policy, by carefully weighing its costs and benefits using a common monetary unit (Pearce et al., 2006). Interestingly, CBA is an integral part of policies at EU level in the Environmental Protection (European Commission, 2008), and according to the consolidated version of the treaty establishing the European Community, in preparing its policy on the environment, the Community shall also take account of the potential benefits and costs of action or lack of action (Title XIX – Environment; Art. 174 (European Union, 2006)). The current steep fall in costs for genetic and genomic technology, especially for DNA analysis, do indicate that the methods discussed here are cost-effective. Also in the frame of the FP7 project, FishPopTrace (https://fishpoprace.jrc.ec.europa.eu), a CBA on the use of DNA based technologies for fishery control and enforcement was carried out by contacting more than 80 control authorities and fishery ministries worldwide, to assess the use and costs of DNA-based analysis in the context of fisheries control. The outcome demonstrated convincingly in cases employing (forensic) DNA analysis that indeed the benefits of using DNA based analytical technologies for fisheries control and enforcement overcome the operational costs, further empha- sizing the value of the technologies presented here (Guillen et al., Manuscript in preparation). Moreover, these technologies feature an added value in that they pro-

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vide benefits to fisheries management that go beyond control, enforcement and traceability in the context of sustainability and resilience of fish stocks in the face of environmental change (Waples et al., 2008).

2.2.5 Supportive initiatives, projects, assemblies and legislation tapping into advanced technologies

2.2.5.1 Relevant projects extending our knowledge base and capacity

Species level: The concept of DNA barcoding and its relevance to fisheries A key application of DNA forensics to fisheries is to identify unambiguously the species of individual adults or other life-history stages, fish products and trace samples from scales, otoliths, blood or other tissues. While genetic approaches have been used for decades to identify and discriminate species across the animal and plant world, it was the proposal to standardize procedures and to compile a globally accessible reference database that has impacted fisheries management and conservation most significantly (Costa and Carvalho, 2007): the so-called DNA barcoding approach. This approach (Hebert et al., 2003) was based on the premise that the sequence analysis of a short fragment of a single gene (e.g. cytochrome c oxidase subunit 1; COI), enables unequivocal identification of many animal species. Hence, analogous to the barcodes used in commercial products, the DNA barcode would provide a standardized tool for fast, simple, robust and precise species identification. The benefits of more precise species identification using high throughput DNA-based methods became recognized quickly. DNA barcoding differs in many ways from conventional taxonomic identification tools and approaches, over which it offers several advantages. It permit the identification of species from fragments, and from any life-history stage, as well as the standardization of a universal master key in a format that reduces ambiguity and enables direct comparison of specimens to a global reference database. The benefits and need for such DNA-based approaches in fisheries management was demonstrated readily by work conducted on routine processing of icthyoplankton (egg) s samples from commercial fish in UK waters (Fox et al., 2005). The study revealed that over 60% of eggs were misidentified when phenotypic characters were used. Eggs from haddock and whiting may have been reported as cod eggs in previous surveys, possibly leading to an inflation of stock assessments in the Irish Sea. Moreover, early stage haddock eggs were detected in the Irish Sea, indicating the presence of a spawning stock of this species previously unknown to that region. In a context of environmental change induced, , for instance, by global warming, the ability to identify rigorously fish species at all life-history stages from egg to adult is particularly useful to assess changes in geographic distribution ranges, spawning grounds and nursery areas. Despite several initiatives for fish barcoding, the worldwide coverage The coverage of species regionally differs substantially, with areas like Africa and South America being significantly underrepresented.

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Focus Box 2: Barcoding fish along the West African Coastline - a hot spot of illegal fishing. Due to its outstanding significance as a source of fish for the European market especially the West African area requires increased attention. The European Union has consistently had the largest foreign presence off Africa, with EU fish harvest there increasing by a factor of 20 from 1950 to 2001 (Kaczynski and Fluharty 2002). In addition, pirate fishing vessels from foreign fishing ports are abundant in West African waters catching fish of the highest commercial value illegally. The disastrous effects of this extensive IUU fishing activity are further enhanced by exceedingly high volumes of discards. Indeed, as for many commercial fleets, dumping of 70 to 90% of their haul as by-catch occurs frequently (UNEP, FAO 2002). The EU also remains the biggest market for fish products from West Africa (Brashares et al. 2004). However, traceability is poor to non-existent. In many instances, it is difficult if not impossible for importers, processors, retailers and even more so consumers to determine whether the fish they are buying has been caught legally or not. Such gaps in our coverage was highlighted recently by the disclosure that between 2000 to 2011, the People’s Republic of China appear to have declared only 9% of their landings, a substantial proportion of which comes from Western African territorial waters. The potential for IUU activity, and the associated need for enhanced monitoring and forensic testing to combat such activities, is of ut- most importance. While some European processors and retailers are exploring the ways to avoid buying IUU fish, a legal framework must also be put in place to support such efforts. Considering the status of threat of many of the intensely fished fish stocks along the West African coast, a rapid assessment of the genetic diversity of the most important species of that region is highly recommended. Western African species regularly show up at the European market as illegal surrogates of popular and high-priced European seafood products. This was the case for tropical sole species marketed as European sole Solea vulgaris (Rehbein et al. 2009) and recently culminated with Prickly pufferfish (Ephippion guttifer) fillets being imported into the European Union (Ger- many and Italy) labelled as African monkfish (Lophius spp.). One important measure is to provide tools for a correct identification of fish and fisheries products (mainly fillets) to a species level as a first step to prove geographical origin outside European waters. A joint Moroccan-German research project starting in 2013 within the frame of the Moroc- can-German Programme of Scientific Research (PMARS) addresses this issue and aims at an assessment of genetic barcodes for all commercially valuable fish species of Morocco, many of them not yet listed in any genetic database. Within the scientific innovation programme of the German Federal Ministry of Food, Agriculture and Consumer Protection this approach shall be extended to all commercially valuable fish species of the Central Eastern Atlantic as part of a larger project on „Adaptation and Development of Innovative, Non-Invasive Moni- toring and Evaluation Systems for Fisheries Research“. DNA sequence databases like Gen- Bank are only sporadically tied to vouchered specimens, while, especially for forensics purposes, taxonomically validated DNA barcodes are essential. Therefore, best practice for DNA barcoding is to sequence vouchered specimens.

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Focus Box 3: Genetic Species Identification to fight the trade with illegal caviar products Genetic approaches are employed to fight the illegal trade with caviar products and in response to the dramatic sturgeon population decline, demonstrating the power and versatility of DNA- technology for control, enforcement and in support of sustainable fisheries management. The genus Acipenser contains 17 of the 25 caviar-producing fish commonly termed sturgeon. All Acipenser species are now listed under CITES Appendices I or II, with the intention of control- ling international trade and promoting the implementation of sustainable management policies. Regulations within the EU, the world’s largest caviar importer as well as a major producer, include a strict labelling system for all caviar products detailing the species name and country of origin. Such regulations are an attempt to restrict the caviar trade to products derived from CITES quotas, approved farms or licensed repackaging companies (European Commission, 2006). Despite such regulation, the value of caviar drives a multi-million euro black market economy in which caviar from non-sustainable sources is known to be widely traded under mislabelled packaging. To address the issue of illegal trade it is essential that there exists a robust, legally valid (forensic) method of authenticating the caviar labelling system, enabling customs officials and trade authorities to test products in trade on a routine basis or as part of intelligence led investigations. Ideally, such a testing system should allow traceability of a product to source, either the wild geographic origin, or the farm where caviar was produced. In the first instance, it is vital to be able to accurately determine the species of origin of traded caviar in order to assess the validity of the product label. A broad range of analytical tools have been examined for their utility in caviar identification (Rehbein et al., 2008), with the most promising technology for general application found to be DNA-based methods. In fact, DNA identification has been used for caviar identification at a species level for over a decade and is regularly employed to support enforcement action in countries such as Germany, the UK and the USA. A good example of application of genetic knowledge in forensics is genetic identification of eight sturgeon species in Russia (Mugue et al., 2008). At present this sturgeon species-identification system is regularly applied in Russia for control and enforcement purposes – for CITES regulation, for law-enforcement authority’s needs. All the sturgeon products including caviar that undergo export or import procedures should have a CITES permission and the specific belong- ing of the stuff should correspond to declared. Illegally caught caviar confiscated during crime- fighting and surveillance measures is checked for the species origin as well. However problem of distinguishing Russian (A. gueldenstaedtii), Persian (A. persicus) and Adriatic (A. naccarii) sturgeon have not solved yet that limits Europe’s ability to enforce existing regulations concerning the caviar trade. The resulting need to discover, test and validate new molecular markers capable of species identification in this group has recently been addressed by the SturSnip project (https://stursnip.jrc.ec.europa.eu) (Ogden et al.; 2013).

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2.2.5.2 Population Level: The FP7 project FishPopTrace FishPopTrace was an international EC FP7 project (2008–2011) aimed at the construction of a Pan-European framework, built on advanced technologies, for product traceability and policy related monitoring, control and surveillance (MCS) in the fisheries sector. Pursuing a holistic approach, FishPopTrace contributed to fisheries management and conservation measures in line with the global attempt to move towards sustainable fisheries. Marine species, in common with all living organisms, are fragmented, to varying degrees, into a series of locally interbreeding populations. The extent to which such populations differ biologically (“population diversity”) and their distribution in time and space are among the most important drivers of species’ survival and persistence in the face of environmental change. Fisheries biologists and managers have emphasized such thinking since the early 20th century, though there remains typically a mismatch between biological populations and fisheries management units (Reiss et al., 2009). Thus, it remains difficult to devise strategies to relate population diversity to variable harvesting regimes, and even more difficult to conserve overexploited stocks. Further uncertainty in striving for sustainability is the high level of illegal, unregulated and unreported fishing (IUU), estimated globally to cost the industry €10-20 billion, and prior to 2010, €1.1 billion worth of illegal fish was imported into the EU annually. Specifically FishPopTrace, aimed to address such challenges to sustainability within the context of the Common Fisheries Policy to: 1. Develop a range of cost-effective and reliable tools for identifying, monitoring and tracing marine fish populations in four representative European species (cod, herring, hake and common sole); 2. Promote fisheries governance by ensuring that the most effective tools can be applied to forensic standards, and thereby be legally supportive for prosecution and enforcement; 3. Foster technology transfer of outputs in relation to enforcement and conservation policies of the EU Common Fisheries Policy (CFP) and associated socio- economic consequences. Using the key traceability tool, DNA single nucleotide polymorphisms (SNPs; single genetic variants) it was possible to correctly assign fish to populations from more areas and with higher certainty than previously possible, reaching standards which can be used in a court of law (Nielsen et al., 2012). Based on use of the most highly distinct genes among populations, it was possible to develop “minimum assays with maximum power” with from 10-30 SNPs. These assays were developed to target several pertinent needs for traceability tools in European fisheries management. For example, it was possible using fast, efficient and forensically robust SNP tools to discriminate between cod from Canada, North Sea, Baltic Sea and Northeast Arctic populations, between North Sea and North Atlantic herring, between sole from the Irish Sea and Thames and between hake from the Mediterranean and Atlantic areas. By applying high differentiation single nucleotide polymorphism assays, in four commercial marine fish, on a pan-European scale, 93–100% of individuals could be correctly assigned to origin in policy-driven case studies. Case-targeted single nucleotide polymorphism assays were created and forensically validated, using a centrally maintained and publicly available database. Such outputs demonstrate how the application of gene-associated markers have potential to revolutionize origin assignment and become highly valuable tools for fighting illegal fishing and mislabelling worldwide. Moreover the use of a marker system such as SNPs, which is essentially based on the presence or absence of large numbers of single genetic variants means that data can be compiled from sources in a much more reliable and high throughput way than previous genetic approaches. The SNP approach thereby ena-

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bles the generation of baseline and on-going additions for subsequent genetic monitoring.

2.2.5.3 The Legacy of FishPopTrace: Extending and transferring research outputs The use of DNA technology to identify fish species and thereby detect illegality has been widely demonstrated in the UK and internationally but until recently, within species, there has been no possibility to identify the population of origin of fish. From 2011, EC Regulation No. 1224/2009 (Art 58)1 requires all fish products to be labelled with species and geographic origin information, establishing a verifiable claim that can be tested for authenticity. Article 13 of the same regulation explicitly required EU Member States to undertake pilot studies of novel traceability tools by 2013. May 2011 saw the completion of FishPopTrace, a UK-coordinated €4M EU-FP7 research project to elucidate population structure and design DNA traceability systems for four species of commercial fish: cod, herring, sole and hake. These two developments provided the framework for the transfer and application of DNA traceability tools for self-regulation, monitoring and enforcement of the fishing industry at a scale not previously possible. The UK fisheries body, the Department for Food, Environment and Rural Affairs (DEFRA) was among the first in Europe to comply with the requirement to test novel tools in the context of IUU. The project aimed to exploit and extend the research outputs of FishPopTrace to achieve the aim of delivering robust, practical traceability tools that are accessible by industry and government agencies alike. The work, which is ongoing, focuses on the developmental validation and transfer of newly discovered DNA markers from a research setting to an applied testing environment in which genetic assays may be used for monitoring or for forensic analysis to support law enforcement. A proof of concept study within FishPop- Trace has demonstrated the feasibility of such work; the challenge is to undertake the necessary R&D and establish systems for implementing the science within the UK fishing industry to support policy and legislation. Several activities are embedded within the project including, a refinement of FishPopTrace research outputs to focus specifically on fisheries issues relevant to the UK; collaboration with UK governmental, non-governmental and industry representatives responsible for fish product authenticity to ensure the delivery of practical solutions which meet stakeholder needs; the transfer and refinement of traceability assays to address UK fisheries authenticity issues; performance of validation studies to convert research methods into approved Standard Operating Procedures; demonstration of novel tools for investigating suspected IUU fishing and mislabelling in the marketplace. The project represents among the first within European waters that aims to bridge the gap between R&D of advanced tools with forensic applications and their uptake by the industry as tools for enforcement and regulation of fisheries policies.

2.2.5.4 NERC Sustainable Marine Bioresources – population structuring of cod around the UK. A project funded in 2008 by the Natural Environment Research Council (NERC, UK), and part of the UK NERC- Fisheries Advisory Bodies initiative, Sustainable Marine Bioresources, focused on the integration of genetic (SNP) data on cod with advanced population modelling. Using single nucleotide polymorphism (SNP) data it was possible to delineate the geographic limits of three population units of Atlantic cod (Gadus morhua) in northwest European waters. Two of the populations co-habit

1 http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2009:343:0001:0050:EN:PDF

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the North Sea, and trawl survey data showed differing trends in their abundances. A spatial model of these units was used as a framework to simulate population dynamics under spatial patterns of harvesting. Competition between units during the pelag- ic juvenile stages in the model led to suppression of the more localized northern North Sea (Viking) unit by the more widespread (Dogger) unit, and it’s premature extinction under some spatial patterns of fishing. Fishery catch limits for cod are set at the scale of the whole North Sea without regard to such subpopulation dynamics. The approach offered a tractable method to quantify adjustments to regional fishing mortality rates to strike a balance between maximising sustainable yield and conserving vulnerable populations.

2.2.5.5 Reference data and information sources For several species there has already been substantial effort invested in research to provide tools that can be applied to address relevant management issues. An important outcome of these projects is not just the protocols that can be validated, but the creation of a maintained database of the reference data, accessible to inspection and enforcement institutions which can be utilized to enforce fisheries regulations, which is, in the context of notorious non-compliance with existing rules urgently required. At a species identification level there are publically accessible databases at which can be consulted. Although the NCBI database (http://www.ncbi.nlm.nih.gov/) has a large number of sequences that can be accessed, it should be noted that the sequences that are contained within the database have not been validated, nor in many cases have the species to which they are assigned been verified. This can cause potential misidentification, particularly in cases concerning cryptic species. While the Fish Barcode of Life Initiative (FISHBOL; http://www.fishbol.org/) is a global effort to coordinate the construction of a global database of standardized reference sequences for all fish species (currently 9599), that are derived from voucher specimens with an authoritative taxonomic identification. It is important to note that the COI region used for this identification has undergone the process of forensic validation (Dawney et al., 2007). Additional advantages of accessing a large centralized, validated database for species identification are the increased species coverage, and the increased likelihood that any issues will be identified by the larger community of expert users. At a population level, there have again been recent research initiatives that have resulted in the development of accessible databases for the assignment of individuals to the population of origin, of some species. These include the FP7 funded FishPop- Trace (https://fishpoptrace.jrc.ec.europa.eu/) focusing on cod, herring, hake and sole within the Atlantic, Baltic and Mediterranean. Salsea Merge (http://www.nasco.int/sas/salseamerge.htm, FP7 funded), which focuses on Atlantic salmon, has resulted in a database which allows fish captured at sea in the North Atlantic, to be assigned back to the region, river, or tributary of their natal origin. AquaTrace (https://aquatrace.eu/) which uses molecular genetic tools to improve the ability to trace escapee farmed fish in the wild. At an individual level, databases have been set up in both Norway which contains the profiles of all minke whales landed in the period 1997-2012 based on sequences of a part of the mtDNA control region, analysis of 10 microsatellites and a sex- determining marker (Glover et al., 2012). This register permits verification of traded whale products via a match to the register, i.e. monitor any trade with minke whale caught on Norwegian quotas, securing that meat is from legal catches. It has also been used in a number of scientific studies. This is an impressive example of a fully

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functional and robust traceability scheme supported by independent genetic testing which can in many aspects serve as a paradigm for marine fisheries (Martinsohn, 2011a). Recently in Norway, genetic assignment was being used to identify the farm of origin for escaped Atlantic salmon (Salmon salar). To illustrate the effectiveness of such reference databases and application of DNA-based tools, a legal investigation on farm escapees, resulted in a NOK 450 000 fine imposed by the Norwegian police as a direct result of genetic analyses (Glover et al., 2008). Interestingly, in this case a part of the fine was used as economical compensation for the genetic analyses conducted by the laboratory facilities (Glover et al., 2008). This is an important point with regards to costs and benefits inherent to the use of new technologies for fisheries monitoring, control and surveillance (MCS). The above described genetic assignment method is also established for rainbow trout and cod, and it appears that the approach applied is universally applicable (and is likely to be so worldwide). An additional example comes from Russian Sturgeon: it is becoming increasingly important to be able to verify whether caviar has been produced through aquaculture or was harvested from the wild. Since wild stocks continue to decline at an alarming rate, the catch of sturgeon is allowed for the needs of breeding only and thus the trade is completely restricted to aquaculture product. To be able to enforce regulations it is necessary to be able to trace caviar back to its farm of origin. Nuclear genetic markers are already being used in this context in Russia, as part of a "genetic passport" system to license caviar producing farms. A genetic passport contains the number of individual electronic label, the photo of the fish (optional), the fish species detected as mentioned above (“species level”) and results of genotyping for five microsatellite loci. When a caviar producer applies for the export CITES permission he provides the numbers of electronic labels of the “parents” if they have been analysed before or tissue samples for creating genetic passports for new breeders and the results of caviar genotyping are compared with the genetic data for parents. At present “genetic passports” can be ordered by sturgeon breeding farms on a vol- untary basis but the availability of “genetic passports” is an obligatory condition for official registration of the stock in the Russian CITES authority and obtaining an official permission for export caviar and sturgeon embryos abroad as well as gives an advantage in competitive tenders for releasing young fish into the wild. However in future this procedure may become mandatory for all sturgeon farms and it will help in distinguishing legal aquacultural and illegal wild caviar even in determination of origin of caviar from unknown sources. The recently funded FP7 project, AquaTrace (https://aquatrace.eu; 2012) represents an additional program designed to establish DNA-based reference baselines designed to distinguish and assign individuals from wild and captive stocks of sea bream, sea bass and turbot. There is thus there is thus not only an escalation in the need for such tools, but importantly also, increased investment, albeit fragmented in relation to species and geographic regions, in such an advanced tools. However, as emphasized elsewhere, developing the technology is a significant component of the process, but for its effective application, there is a requirement for a review and consensus in the governance of fish resources.

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2.2.5.6 Recent high-level assemblies on the subject Over the last five years several high-level international meetings involving various stakeholders took place where fisheries (forensic) genetics and genomics were presented and discussed and where ICES WGAGFM members were present as experts. This clearly shows that the awareness of opportunities arising through these analytical approaches is continually growing, albeit at slow pace. In the following Table, a selection of such events, which had considerable impact, is listed.

Table 2.2.1. Recent salient international meetings to consider elements of DNA-based tools for the identification and sustainable management of aquatic genetic resources.

Event Date Premise Topic Participants Experts involved

European 17/06/2008 Brussels, Workshop on Policy-makers ICES Commission BE Fisheries Forensics and Scientists WGAGFM Workshop on in support of the Members Fisheries reformed CFP and other Forensics Control Regulation experts (EC 1224/2009) FAO Workshop 09- Rome, IT Workshop on the FAO Staff ICES on Fisheries 10/12/2009 application of Members WGAGFM Forensics Fisheries Forensics Members in the global fight and other against IUU fishing experts International 06- Maputo, International MCS FAO Staff ICES Training 10/09/2010 MZ Network: Third Members; WGAGFM< Workshop Global Fisheries European Members Enforcement Commission and other Training Workshop Staff Members; experts, – Special Session on NOAA Law including Fisheries Forensics. Enforcement Wildlife Staff Members; Forensic Members of experts. national cpontrola and enforcement authorities SLOWFISH 27- Genoa, IT Presentation of the Commissioner JRC Staff – 30/05/2011 European for Fisheries ICES Commission Joint Maria WGAGFM Research Centre Damanaki. Member Reference Report Policy-makers. “Deterring Illegal Fisheries Activities in the Industry. Fisheries Sector” (EUR 24394 EN – 2011) Regional South 12–14 July Marine To introduce University and ICES East Asian 2011 Biology students, fisheries Regional WGAGFM DNA Instutute, biologists, Fisheries Members Barcoding Penang, conservationists and Bodies and and other Workshop Malaysia environmental Government experts managers to the Conservation scope and nature of Agencies DNA barcoding for species identification

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Event Date Premise Topic Participants Experts involved FAO Workshop 11–13 Vigo, ES Workshop on the FAO Staff ICES on Fish Species October application of Members WGAGFM Identification 2011 various approaches Members for fish species and other identification in experts support of sustainable fisheries European 25 January Brussels, Public Hearing on Members of the European Parliament 2012 BE “…” with European Commission Public Hearing presentation on Parliament, Joint fisheries forensics in European Research support of Commission Centre/ICES traceability in the Staff WGAGFM fisheries sector Member ICES Annual 20 Bergen, Session H: Fisheries ICES Science September NO Advances in the Scientists and WGAGFM Conference 2012 traceability of fish Managers Members Session and fish products: and other from species to experts populations European 18–21 Vigo, ES Workshop with National ICES Fisheries November participation of EU Correspondents WGAGFM< Control Agency 2012 Workshop on the of all EU Members. (EFCA) Implemnation of the Member States. workshop on IUU Regulation. – EFCA Staff. the Special Session on implementation Forensic Genetics of Regulation and Genomic for EC) 1005/2008 Fisheries Control, (“IUU Enforcement and Regulation) Traceability FAO meeting 28–30 UN Workshop to FAO regional ICES of the United January Regional consider the scope and HQ staff, WGAGFM Nations State 2013 Pacific and terms of invited experts Members World and reference to describe (geneticists, and other Consultation Asian and monitor global ecologists and experts on Aquatic FAO genetic aquatic managers) Genetic Office in resources – ranging Resources Bangkok from appropriate techniques to potential policy recommendations

2.2.5.7 Existing salient legislation referring to advanced technologies- the need for enhanced governance As discussed above, worldwide legislators attempt to support sustainable fisheries and the protection of the industry and consumers by developing rules and laws which underlie powerful control and enforcement schemes In the following a few of them are listed and briefly documented. In the case of the EU Common Fisheries Control Regulation there is even a direct reference to genetics in support of fish (product) traceability. The International Plan of Action to prevent, deter and eliminate IUU Fishing (IP- OA-IUU): numerous countries have adopted the International Plan of Action to pre-

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vent, deter and eliminate IUU Fishing that has been developed in 2001 within the framework of the Code of Conduct for Responsible Fisheries by the FAO (FAO, 2002). Regulations EC 1224/2009 and EC 1005/2008: The European Union has recently developed two major and complementing legal instruments: in January 2010, Council regulation (EC) No 1005/2008 (1), - the ‘IUU regulation’, entered into force, and in November 2009, Council regulation (EC) No 1224/2009 - the new Control regulation- establishing a Community control system which is meanwhile also implemented. Both regulations place emphasis on detailed catch documentation and traceability for fishery products ‘from ocean to fork’, that is, covering all stages of the supply chain from catch, to landing, transport, processing, and the markets. Traceability is generally acknowledged as being a highly powerful tool in support of monitoring, control and enforcement in the fisheries sector. However, currently it is mainly based on certificates accompanying goods, and labelling of products, both measures which are vulnerable to falsification. Interestingly the new control regulation explicitly refers in Article 13 to “traceability tools such as genetic analysis” as having a potential to improve compliance with the rules of the Common Fisheries Policy. Lacey Act: This US law is very powerful in that it states that any receipt, acquisition or purchase of fish (product) that was taken, possessed, transported or sold in violation of US laws or regulations, is prohibited and subject to prosecution. The Lacey Act was the legislative basis for prosecution and conviction in the notorious illegal import and sale of over 10 million pounds of falsely labelled catfish (Martinsohn, 2011a). The US Endangered species Act stands out as a prime example of how genetic data at the population and species level can be used as evidence in support of legislation (Kelley, 2010). Of particular focus is the concept of stock structure – its detection, spatial and/or temporal distribution and its role in the viability and persistence of natural fish resources in the face of environmental change is core to both to the des- ignation of so-called Distinct Population Segments (DPS), and the associated extinction risk analysis (ERA). The perceived stock structure typically informs the appropriate spatial/temporal scale for the subsequent ERA, with critical decisions about whether to split or lump together various within-species groupings. The concept of stock structure is aimed at capturing the appropriate biological scale of diversity that most effectively encompasses the range of reproductively and biologically distinct assemblages within a given species (Carvalho and Hauser, 1994). Reproduc- tive groupings, so-called “spawning groups” in marine fish are likely to generate marked biological and genetic differentiation across their range – such that the resulting population diversity, also termed “biocomplexity” endows the species with enhanced flexibility and capacity to adapt to environmental change (Hilborn et al., 2003). In relation to the ESA, following a petition of evidence, genetic data are among various lines of evidence typically employed to assess to what degree the best available scientific evidence (ideally from disparate methods) indicates the existence of stock structure and over what spatial or temporal scale should the boundaries of such distinct units be made. To conserve overall levels and patterns of genetic diversity within species as a resource for future adaptive change, the ESA encourages the des- ignation of DPS (Waples, 1991; Kelly, 2010). The US Fish and Wildlife Service (USFWS) in 1996 developed the policy for determination of DPS for protection under the ESA (USFWS, 1996), focusing on 3 elements to designate a DPS: on the basis of their discreteness from other such units, by their perceived “significance” to the respective species they represent, and on the conservation status of a unit. Three significance criteria that are especially relevant in the current context are: (1) the

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persistence of a unit in an ecological setting that is unusual or unique for the taxon; (2) evidence that loss of the unit would result in a significant gap in the range of the taxon; (3) evidence that the unit differs markedly in its genetic characteristics relative to other populations of the same species. Discreteness in general can be estimated by using several approaches such as the use of genetics, life-history data, demographic trends and morphological diversity, whereas significance equates to the degree to which a specific DPS encompasses a unique ecological setting or whose loss would result in a significant gap in the species’ range (e.g. Grunwald et al., 2008 for sturgeon; Arden et al., 2011: for bullhead). A key challenge that faces those in recom- mending the listing of specific DPS is how to identify, and at what hierarchical level do any population groupings identified by specific data (or combined datasets) reach the appropriate boundary of discreteness and significance to the taxon? Compared with the other biological levels typically used by federal agencies in listing endangered or threatened species (species, subspecies), the notion of DPS are in reality more the construct of legal processes rather than biological (Kelly, 2010). As such, there is no common threshold or level of discreteness among identified DPS that is universally applicable across all species and scenarios, and it becomes crucial to examine critically the evidence that indicates an appropriate scale of boundaries and potential significance to the taxon. Since the outcomes of such considerations can have major socio-economic, as well as conservation impacts, it is vitally important that any such genetic evidence is indeed robust and reproducible, especially since circumstances might change, allowing the direct comparison of datasets over time. A recent example to illustrate the execution of such approaches was the petition of two species of river herring (anadromous) to become protected under the ESA (NRDC, 2012). Despite the historical commercial importance of alewife and blueback herring, various factors relating to habitat disturbance and degradation, overexploita- tion, bourgeoning by-catch mortality, and accelerating effects of climate change, have resulted in marked population declines (>90% in some localities from the 1950 to 1970 average) and contraction in geographic range. In 2006 the National Marine Fisheries Service (NMFS) designated river herring as Species of Concern, followed in August 2011 by a petition from the Natural Resources Defence Council (NRDC) to the NMFS to list both alewives and blueback herring, each as threatened throughout all or a significant portion of their range under the Endangered Species Act (ESA). NMFS used among other lines of evidence, genetic information on stock structure to assess whether there are discrete and significant populations of alewives or blueback herring that might warrant separate protections under the joint US Fish and Wildlife Service and NMFS Distinct Population Segments (DPS) policy (61 FR 4722). Upon applying the DPS policy, the evidence gathered will help NMFS to make an informed decision on whether the stock structure can adequately be protected as a single unit or, whether one or more DPS are necessary to best protect certain stock complexes of alewives or blueback herring that represent a discrete and significant unit to the taxon as a whole. In summary, genetic evidence was presented (Carvalho, 2013) to separate alewife and blueback herring as discrete and demographically independent entities, each with their own suite of biological properties that impart differential responses to environmental threats, thereby requiring separate consideration under the ESA. There was compelling evidence from genetic studies and population/regional-diversity in life history, morphology, migratory patterns and behaviour that supported the existence of discrete stock complexes within each of the two river herring species, suitable for consideration under DPS policy: respective populations or groupings thereof, differ

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markedly in their genetic and other biological properties relative to other such units. It was concluded that the level of stock structuring was such that loss of individual components would represent a significant gap in the range of respective species, and as such, the two species should be protected under the ESA.

Magnuson-Stevens Act The Magnuson-Stevens Fishery Conservation and Management Act (MSA), as amended by the Sustainable Fisheries Act of 1996 (Public Law 104-267), established procedures designed to identify, conserve, and enhance Essential Fish Habitat (EFH) for those species regulated under a Federal fisheries management plan. A key part of the Act refers is in place to "prevent overfishing and protect, restore and promote the long-term health and stability of the fishery”. As such, both the long-term aim of genetic studies to assess and enhance levels of population diversity are prerequisites for sustainability of resources, as well as short-term uses of genetic markers for exploring the dynamics and patterns of population structuring for informing appropriate spatial and temporal levels for stock assessment.

Broader perspectives of fisheries forensics – Marine Genetic Resources In addition to the use of DNA-based tools for forensic application to monitor, enforce and deter illegal activities in the fisheries sector, they have additional value in the long-term conservation and sustainability of fish resources. Indeed, it is noteworthy that the International Union for the Conservation of Nature (IUCN) recognizes genes as one of the three primary levels of biodiversity (along with species and ecosystems). Incorporation of population diversity into management instruments and policies will further underpin an ecosystem‐based approach to fisheries through recovery of de- clining stocks and associated resilience in feeding interactions. Conservation of fish stocks has a positive effect not only on the economics and sustainability of the industry, but also on long‐term sustainability of biodiversity. Exploited fish resources, in common with all living organisms, are most often described and instantly recognizable by their species name ‐ cod, herring, hake, or mackerel etc. Indeed, the species is a fundamental level of biological organization that underpins our ability to understand, exploit and manage our natural biological resources. All species, however, whether on land or in the sea, are composed of groups of individuals that interbreed with each other more often than with other such groups, and these so‐called, populations, share many characteristics, making them also recognizable, but at a regional or geographic level. In the management of fish resources it has been known for many decades that although it is important to recognize what species an individual belongs to, it is also crucial to identify and monitor the distribution and dynamics of populations. Ever since Charles Darwin first established the primary mechanism for evolutionary change by natural selection in the mid‐19th century, there has been recognition that it is the nature and extent of differences between local groups or populations that determines the survival and persistence of species in the face of environmental change, through the process of adaptation. It is correspondingly a primary task of many fish biologists and managers to identify populations, and to design strategies to maximize their conservation: in general, the more populations that exist (“population diversity”), the more opportunity there is for fish resources to adapt to such changes as over‐exploitation and climate change. Recent research has demonstrated convincingly that genetics and genomics have come of age and powerfully help to identify the population structure of marine fish and to monitor population dynamics. While such applications meanwhile also

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helped to integrate genetics into fisheries management, more effort, involving a broad scope of scientists as well as stakeholders is needed to universally and fully integrate genetic and genomic information into sustainable fisheries management and conservation of natural marine resources. ICES, as a major and internationally acknowledged scientific fisheries advisory body should play a major role in this challenging endeavour.

2.2.6 Recommendations WGAGFM recommends: • ICES SCICOM to facilitate the organization of a workshop between population geneticists and fisheries policy-makers, fisheries control agencies and stakeholders to explore opportunities arising from fisheries genetics for management issues, such as traceability, IUU activities, and mislabelling. Outputs should include a framework for International Forensic Standards in Fisheries Management. • ICES ACOM and respective WGs to encourage member states to invest in the development of an appropriately robust, cost‐effective and user‐ friendly technology platform with sufficient flexibility in design and application to be employed in the routine testing of traceability of fish/shellfish and products.

2.2.7 References Ardren, W. R., DeHaan, P. W., Smith, C. T., Taylor, E. B., Leary, R., Kozfkay, C. C., Godfrey, L., Diggs, M., Fredenberg, W., Chan, J., Kilpatrick, C. W., Small, M. P., and Hawkins, D. K. 2011. Genetic Structure, Evolutionary History, and conservation Units of Bull Trout in the Coterminous United States, Transactions of the American Fisheries Society, 140(2): 506– 525.

Birstein, V. J., Doukakis, P., and DeSalle, R. 2000. Polphyly of mtDNA lineages in Russian sturgeon: forensic and evolutionary implications. Conservation Genetics, 1: 81–88.

Birstein V. J., Doukakis, P., Sorkin B.,and DeSalle R. 1998. Population aggregation analysis of three caviar-producing species of sturgeons and implications for the species identification of black caviar. Conservation Biol., 12(4): 766–775.

Brashares, J. S., Arcese, P., Sam, M. K., Coppolillo, P. B., Sinclair, A. R. E., and Balmford, A. 2004. Bushmeat Hunting, Wildlife Declines, and Fish Supply in West Africa. Science, 306(5699): 1180–1183.

Carvalho, G. R., and Hauser, L. 1994. Molecular genetics and the stock concept in fisheries. Special Issue of Reviews in Fish and Fisheries Biology. Ed. by G. R. Carvalho and T. J. Pitcher, 4: 351–373.

Carvalho, G., Helyar, S., Bekkevold, D., Volkert, F., Hanel, R.,. McPhee, D., Ford, M., Carlsson, J., Trautner, J., Ogden, R., and Martinsohn, J. 2009. ToR b) Review the current status of traceability methods in the fisheries sector based on genetics. In ICES. 2009. Report of the Working Group on the Application of Genetics in Fisheries and Mariculture (WGAGFM), 1–3 April 2009, Sopot, Poland. ICES CM 2009/MCC:03. 74 pp. Carvalho, G. R. 2013. Petition to list alewife (Alosa pseudoharengus) and blueback herring (Alosa aestivalis) (“River Herring”) under the Endangered Species Act (ESA). Review of Stock Structure and Extinction Risk Analysis Working Group Reports. Prepared for the Centre for Independent Experts, USA.

Costa, F. O. and Carvalho, G. R. 2007. The Barcode of Life Initiative: synopsis and prospective societal impacts of DNA barcoding of Fish. Genomics, Society and Policy, 3: 29–40.

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Dawnay, N., Ogden, R., McEwing, R., Carvalho, G. R., and Thorpe, R. S. 2007. Validation of the barcoding gene COI for use in forensic genetic species identification. Forensic Science In- ternational, 173: 1–6.

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Gil, L. A. 2007. PCR-based methods for fish and fishery products authentication. Trends in Food Science & Technology, 18(11): 558–566.

Glover, K. A., Skilbrei, O. T., and Skaala, Ø. 2008. Genetic assignment identifies farm of origin for Atlantic salmon Salmo salar escapees in a Norwegian fjord. ICES Journal of Marine Sci- ence, 65: 912–920.

Glover, K. A., Øien, H. O., Walløe, L., Lindblom, L., Seliussen, H., and Skaug, H. 2012. The Norwegian minke whale DNA register: a data base monitoring commercial harvest and trade of whale products. Fish and Fisheries, 13: 313–332.

Grunwald, C., Maceda, L., Waldman, J., Stabile, J., and Wirgin, I. 2008. Conservation of Atlantic sturgeon Acipenser oxyrinchus oxyrinchus: delineation of stock structure and distinct population segments. Conservation Genetics, 9: 1111–1124.

Guillen at al. 2013. Applying SNA Analysis for Fisheries Control and Enforcement: A Cost benefit Analysis. Manuscript in preparation.

Hajibabaei M., Shokralla S., Zhou X., Singer, G. A. C., and Baird, D. J. 2011. Environmental Barcoding: A Next-Generation Sequencing Approach for Biomonitoring Applications Us- ing River Benthos. PLoS ONE 6(4): e17497.

Hauser, L. and Carvalho, G. R. 2008. Paradigm shifts in marine fisheries genetics: ugly hypoth- eses slain by beautiful facts. Fish and Fisheries, 9(4), 333–362.

Hebert, P. D. N., Cywinska, A., Ball, S. L., and deWaard, J. R. 2003a. Biological identifications through DNA barcodes. Proc Roy Soc Lond B, 270: 313–321.

Hebert, P. D. N., Ratnasingham, S., de Waard, J. R. 2003b.Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proc Roy Soc Lond B, 270: 96–99.

Hilborn, R., Quinn, T. P., Schindler, D. E., and Rogers, D. E. 2003. Biocomplexity and fisheries sustainability. Proceedings of the National Academy of Sciences of the United States of America, 100: 6564–6568

Jerome, M., Lemaire, C., Verrez-Bagnis, W., and Etienne, M. 2003. ʺDirect sequencing method for species identification of canned sardine and sardine-type products. Journal of Agricul- tural and Food Chemistry, 51(25): 7326–7332.

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Kaczynski, V. M., and Fluharty, D. L. 2002. European policies in West Africa: who benefits from fisheries agreements? Marine Policy, 26(2): 75–93.

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Kochzius, M., Nolte, M., Weber, H., Silkenbeumer, N., Hjorleifsdottir, S., Hreggvidsson, G. O., Marteinsson, V., Kappel, K., Planes, S., Tinti, F., Magoulas, A., Vazquez, E. G., Turan, C., Hervet, C., Falgueras, D. C., Antoniou, A., Landi, M., and Blohm, D. 2008. DNA microar- rays for identifying fishes. Marine Biotechnology, 10(2): 207–217.

Martinsohn, J. T. 2011a. Deterring Illegal Activities in the Fisheries Sector: genetics, genomics, chemistry and forensics to fight IUU fishing and in support of fish product traceability. JRC Reference Reports. Luxemburg, European Commission: 72. ISBN 978-92-79-15905-3.

Martinsohn, J. T., Geffen, A. J., Maes, G. E., Nielsen, E. E., Ogden, R., Waples, R. S., and Car- valho, G.R. 2011b. Tracing fish and fish products from ocean to fork using advanced molecular technologies Food chain integrity: A holistic approach to food traceability, safety, quality and authenticity. J. Hoorfar, K. Jordan, F. Butler and R. Prugger. Cambridge, Woodhead: 259–281.

Mugue, N. S., Barmintseva, A. E., Rastorguev, S. M., Mugue, V. N., and Barmintsev, V. A. 2008. Polymorphism of the Mitochondrial DNA Control Region in Eight Sturgeon Species and Development of a System for DNA-Based Species Identification. Russian Journal of Genetics, 44(7): 793–798.

Nielsen, E., Cariani, A., Mac Aoidh. E., Maes, G., Milano, I., Ogden, R., Taylor, M., Hemmer- Hansen, J., Babbucci, M., Bargelloni, L., Bekkevold, D., Diopere, E., Grenfell, L., Helyar, S., Limborg, M. T., Martinsohn, J. T., McEwing, R., Panitz, F., Patarnello, T., Tinti, F., Van Houdt, J. K. J., Volckaert, F. A. M., Waples, R. S., FishPopTrace Consortium, Carvalho G. R. 2012. Gene-associated markers provide tools for tackling illegal fishing and false eco- certification. Nature Communications, 3: 851. DOI: 10.1038/ncomms1845.

NRDC. 2012. Petition to list alewife (Alosa pseudoharengus) and blueback herring (Alosa aestivalis) (“River Herring”) under the Endangered Species Act (ESA) and to designate Critical Habitat. US Fish and Wildlife Service.

Ogden, R. 2008. Fisheries forensics: The use of DNA tools for improving compliance, traceability and enforcement in the fishing industry. Fish and Fisheries, 9(4): 462–472.

Ogden, R., Gharbi, K., Mugue, N., Martinsohn, J., Senn, H., Davey, J. W., Pourkazemi, M., McEwing, R., Eland, C., Vidotto, M., Sergeev, A. and Congiu, L. 2013. Sturgeon conservation genomics: SNP discovery and validation using RAD sequencing. Molecular Ecology. doi:10.1111/mec.12234.

Pearce, D., Atkinson, G. and Mourato, S. 2006. Cost-Benefit Analysis and the Environment: Recent Developments. Paris: Organisation for Economic Co-Operation and Development.

Rehbein, H., Molkentin, J., Schubring, R., Lieckfeldt, D., and Ludwig, A. 2008. Development of advanced analytical tools for determination of the origin of caviar. Journal of Applied Ich- thyology, 24: 65–70.

Reiss, H., Hoarau, G., Dickey-Collas, M., and Wolff, W.J. 2009. Genetic population structure of marine fish: Mismatch between biological and fisheries management units. Fish and Fish- eries, 10(4): 361–395.

Seeb, J. E., Carvalho, G. R., Hauser, L., Naish, K., Roberts, S., and Seeb, L. W. 2011. Single- nucleotide polymorphisms (SNP) discovery and applications of SNP genotyping in non- model organisms. Special Issue of Molecular Ecology Resources, 11(1): 1–298. Taylor, M. I., Fox C., Rico, I., and Rico, C. 2002. Species specific TaqMan probes for simultaneous identification of (Gadus morhua L.) haddock (Melanogrammus aeglefinus L.) and whiting (Merlangius merlangus L.). Molecular Ecology Notes, 2: 599–601.

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FAO. 2002. Implementation of the International Plan of Action to Prevent, Deter and Eliminate Illegal, Unreported and Unregulated Fishing. (pp. 122). FAO Technical Guidelines for Re- sponsible Fisheries. No. 9. Rome, FAO. FAO 2002.

USFWS. 1996. Substantive Requirements of the Endangered Species Act. US Fish and Wildlife Service.

Waples, R. S. 1991. Pacific salmon, Oncorhynchus spp. and the definition of “species” under the endangered species act. Marine Fisheries Rev., 53: 11–22.

Waples, R. S., Punt, A. E., and Cope, J. M. 2008. Integrating genetic data into management of marine resources: how can we do it better? Fish and Fisheries, 9: 423–449.

List of websites cited Aquatrace: https://aquatrace.eu FishPopTrace: https://fishpoptrace.jrc.ec.europa.eu/ FishBOL: www.fishbol.org

2.3 Tor c) Review on the use of metagenomics and metatranscriptomics as an approach for marine ecosystem management Naiara Rodríguez-Ezpeleta, Aitor Albaina, Jakob Hemmer-Hansen, Dorte Bekkevold, Jackie Lighten

2.3.1 Rationale: Ecosystem Approach to Fisheries Management (EAFM) is a form of fisheries governance framework that combines principles of conventional fisheries management with those aimed at managing whole ecosystems. Central to EAFM is the recognition and incorporation of key ecosystem characteristics, such as complexity, structure, natural variability, and boundaries, as well as encompassing metrics of ecosystem modification and degradation by fisheries, and other land- and sea-based economic activities (Garcia et al., 2003). The functioning of an ecosystem results from the organization of its species communities (with specific abundances and survival, growth, production and reproductive strategies), and their capacity to adapt to the physical environment, and relations with the other communities (predator–prey relationships). Understand- ing these relationships, which form the trophic network, is essential to understanding possible reactions of the ecosystem to exploitation regimes. Implementing an EAFM framework is tightly related to maintaining good environmental status (often abbreviated GES). In European Union member countries, the Marine Strategy Framework Directive (MSFD, 2008/56/EC) is aimed at achieving or maintaining GES in European waters by 2020. Here, GES is defined by 11 descriptors, of which maintenance of biodiversity is a cornerstone (Cochrane et al., 2010). Under the MSFD concept, each descriptor is defined by a set of criteria, which each in turn are associated with a series of indicator metrics (Cardoso et al., 2010). Among the 11 descriptors, four are particularly relevant to EAFM: (1) Biological diversity, (2) non- indigenous species, (3) exploited fish and shellfish and (4) foodwebs. The assessment of each of the indicators requires rigorous and regularly applied measuring techniques, which is often not easily achieved due to methodological or budget limitations (Borja, Elliot 2013). This has led researchers to investigate new and cost-effective monitoring techniques (Frolov, Kudela, Bellingham 2013), that involve the application of new molecular techniques to assess indicators of ecosystem function and health.

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Here, we review the potential of genomics based techniques to provide rapid, accurate and cost-effective measurements of ecosystem function and health. In particular, we focus on the application of environmental sequencing based approaches to study the MSFD indicators related to implementing an ecosystem approach to fisheries management. For the potential of genomics techniques in marine monitoring in general, we refer to Bourlat et al. (2013).

2.3.2 Environmental sequencing Environmental sequencing consists of sequencing genetic material from an environmental sample (water, soil, gut content, etc.) without previous isolation of a particular organism (Eisen, 2007). This approach to batch sequencing is convenient for the characterization of microbial community diversity when species or individuals cannot easily be separated. Yet, more recently this approach has been applied to batch sequencing of multicellular eukaryotic aquatic species (Adura et al., 2011), demonstrating how the capture of community level diversity metrics may be implemented in fisheries management strategies. Depending on the genetic material sequenced (DNA or RNA) and method used (sequencing a given gene or whole genetic material), we distinguish between metagenetics, metagenomics and metatranscriptomics.

2.3.2.1 Metagenetics – addresses 'who is there?' Metagenetics, also called metabarcoding (Taberlet et al., 2012), is based on sequencing one or a few genes by using a primer sequence that amplifies all or specific taxonomic groups (e.g. bacteria, eukaryotes, nematodes). The purpose of the method is the identification of the species present in the sample, which requires a reference library to which the obtained sequences will be compared to ascertain taxonomic classifications of sequenced organisms. The reference library should contain sequences for the target gene from, ideally, all the species expected in the environmental sample. This method has mostly been applied to bacteria and microbial eukaryotes, for which the 16S and the 18S ribosomal RNA subunit genes are the markers of choice, respectively (Streit, Schmitz 2004; Stoeck et al., 2010). Yet, the application of this method has extended beyond the microbial world to meiofauna (e.g. Tang et al., 2012) and macroorganisms (Bik et al., 2012). For animals, the most commonly used markers are the mitochondrial gene cytochrome oxidase I (COI), 12S ribosomal RNA gene (Hebert, Ratnasingham, deWaard 2003; Machida, Kweskin, Knowlton 2012) and nuclear 28S and 18S ribosomal RNA genes (Machida, Knowlton 2012).

2.3.2.2 Metagenomics – addresses 'what can they do?' Metagenomics is based on sequencing the whole DNA present in an environmental sample. This approach enables the identification of the genes present in the sample and consequently, the metabolic activity of the organisms that compose it (Yu, Zhang 2012). The analysis of metagenomic data entails comparison of the sequences obtained against a reference database of high-level functions of biological systems such as the Kyoto Encyclopedia of Genes and Genomes (KEGG). Instead of focusing on the species present in the sample, this approach aims to understand the metabolic capacity of the whole community.

2.3.2.3 Metatranscriptomics – addresses 'what are they doing?' Metatranscriptomics is based on sequencing RNA from an environmental sample. If RNA from a single marker is sequenced, this approach allows the identification of the living (active) organisms in the sample (Wemheuer, Wemheuer, Daniel 2012). DNA is stable for some time outside cells, meaning that metagenetics and metagenomics

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approach can also detect extracellular DNA and/or DNA from parts of dead organisms. In contrast, RNA is very unstable and is degraded as soon as the organism dies. Whereas the DNA sequence (genome) of an individual organism does not change with time, RNA (transcriptome) is only present for the genes that are active at a particular moment, meaning that the RNA content changes with time depending on developmental stages and environmental conditions. By sequencing the total RNA, the detection of metabolic pathways that are active at the sampling moment is achieved (Yu, Zhang 2012), which is useful in comparing the differential expression of genes between variable environmental conditions (Marchetti et al., 2012), or developmental stages.

2.3.3 Qualitative and quantitative biodiversity assessment using environmental sequencing The application of environmental sequencing, in quantifying taxonomic diversity and community level metabolic activity, holds great potential in MSFD. Specifically, the approach offers increased sensitivity in quantification of numerous MSFD indicator metrics at less financial cost, some of which cannot be attained at all from traditional methods. Metagenetics is, from the aforementioned three technologies, the most widely used as it provides a fast and cost-efficient estimation of biodiversity. Yet, for some of the indicators included in the MSFD, a qualitative assessment as well as a quantitative assessment of biodiversity is required. Correlating number of molecules sequenced for each organism with number of individuals or biomass of that particular organism is not an easy task. This correlation requires the number of sequences obtained for a given species in a metagenomics experiment to be proportional to the number of individuals (or biomass) for that species in the sample. Yet, there are several factors, both biological and technical, that prevent this statement to be universally true, at least with most currently used methods (see Section 5).

2.3.4 Applications of metagenomics to inform the implementation and utilization of an EAFM framework Here we examine the potential of metagenomics and metatranscriptomics in quantifying essential ecosystem indicators used to measure GES under an EAFM framework. Within the MSFD, the descriptors identified as directly related to the EAFM are: 1) biological diversity, 2) non-indigenous species, 3) exploited fish and shellfish and 4) foodwebs. All four descriptors are associated with indicators that require qualitative and/or quantitative assessment of biodiversity (Bourlat et al., 2013). When using traditional species identification methods, such assessments are, however, often limited. For example, accurate species identification is difficult or impossible at early developmental stages (egg or larvae), when working on semi-digested specimens (e.g. prey species from stomach contents) or in presence of cryptic species (organisms that appear identical but are reproductively and genetically distinct; (Knowlton 1993)., Metagenetics is a cost-effective alternative to traditional taxonomy in these situations as it is based on comparing DNA sequences to a reference library and not the requirement morphological integrity for accurate species identification. Moreo- ver, metagenetics allows identification of small organisms (e.g. bacteria), small parts of macroorganisms (e.g. fish scales) or even free DNA in the environment (so called eDNA) by sequencing environmental samples e.g. sediments or water. Here, we provide some examples where metagenomics has been used or could be used to quantify indicators associated with the four essential EAFM descriptors. The concepts and examples described below would apply to similar initiatives such as Oceans Policy in

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Australia, Oceans Act and Oceans Strategy in Canada, Oceans Act in USA, and Water Framework Directive (WFD, 2000/60/EC) in the EU.

2.3.4.1 Biological diversity In the MSFD framework a description of biological diversity can be made through quantifying species distributions, the sizes, condition and distributions of populations, the extent and condition of habitats, and the community structure of the ecosystems. Quantification of distributions through presence/absence measurements is relatively straightforward with DNA based methods, particularly with respect to unicellular organisms or early life stages of multicellular organisms sampled directly with a water sample (Wood et al., 2013). Recently, these methods have also been applied to multicellular organisms through monitoring of eDNA. While most approaches have focused on applying eDNA monitoring to terrestrial and freshwater systems (e.g. Thomsen et al., 2012a; Takahara et al., 2013), it has recently also been demonstrated that eDNA based surveys of marine biodiversity may perform equally or better than traditional biodiversity surveys (Thomsen et al., 2012b). It has been shown that DNA degrades within a few days in seawater (Paul et al., 1987; Thomsen et al., 2012b), suggesting that the detected species should reflect fairly recent and local occurrences. Relatively limited dispersal capacity of free-floating DNA should be considered a major advantage when using eDNA for monitoring purposes. So far, studies applying eDNA for the monitoring of biodiversity of multicellular taxa have focused on detecting the presence/absence of species, and quantification of individuals or biomass has rarely been attempted (Takahara et al., 2012). Moreover, most studies have so far targeted a few species and therefore examples of metagenetic approaches are still relatively sparse (Thomsen et al., 2012b).

2.3.4.2 Non-indigenous species Within in the MSFD framework, the quantification of non-indigenous (particularly invasive) species abundances, distributions, and ecosystem impacts are of key importance, and the use of eDNA may be applicable in such circumstances. For example, eDNA has been used to detect the presence of bluegill sunfish (listed as invasive in the Invasive Alien Species Act of Japanese Law) in ponds (Takahara, Minamoto, Doi 2013). Interestingly, DNA from this species was detected in ponds where specimens where not visually observed. In this case, eDNA sampled from the water column was used to target one gene of a particular target species, as well as attempts to quantify the abundance of this species. Similarly, Mahon et al. (2013) described distributions of six non-native cyprinid species in a North American freshwater system. They further reported a positive relationship between numbers of positive eDNA samples and numbers of individual fish following collection of all individuals within the study site, suggesting that the approach could also be applied in a qualitative assessment of non-indigenous species. This method has also proven efficient in detecting invasive species in ballast and open water environmental samples (Harvey, Hoy, Rodriguez 2009). Environmental DNA sequencing can therefore be used as an early warning system to detect the presence of an invasive species.

2.3.4.3 Exploited fish and shellfish Studying exploited fish and shellfish, in the MSFD framework, implies understanding the pressure of a given fishing activity, the reproductive capacity of the stock and population age and size distribution. The spawning-stock biomass is often assessed

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by the daily egg production method (counting eggs); yet, this method is not suitable for species whose eggs are difficult to identify visually. In these cases, molecular methods allow accurate species identification (e.g. Fox et al., 2005; Fox et al., 2008). Similarly, the biomass of adults of a given species can also be assessed by sequencing genetic material present in the water column. For example, Thomsen et al. (2012) and Takahara et al. (2012) found significant positive correlations between DNA quantity and biomass of two target species, suggesting that eDNA could be used for biomass estimation. However, relationships between DNA quantity estimates and absolute numbers of individuals may be complex and difficult to determine under conditions relevant to management. So far, no studies have used eDNA to estimate the abundance of macroorganisms in marine systems.

2.3.4.4 Foodwebs Studying foodwebs, in the MSFD framework, implies understanding productivity of low key species or trophic groups, proportion of selected species at the top of the foodwebs and abundance/distribution of key trophic groups/species. Visual identification of gut contents is the main strategy applied to foodweb studies in aquatic ecosystems. Yet, although visual identification can be performed for whole items ingested by fishes or other marine organisms, semi-digested preys are nearly impossible to identify. Molecular methods now offer an alternative of rapid and unambiguous identification of species present in gut contents (see reviews by Symondson 2002; Albaina et al., 2012, Fox et al., 2012). However, the application of DNA based methods to assess species diet is not straight-forward and there are some specific key factors to consider when attempting identification or even quantification of prey remains:

Complexity of diet samples As stomach complexity (size and nature of the contents) is variable, a combination of various methods may often be required. For example, when facing the analysis of large bodied species with large stomachs, visual identification of large remains should be performed, reducing the amount of tissue for subsequent DNA extraction. Alternatively, small pieces of tissue from large items should be dissected pre- extraction to ease species identification. Conversely, when dissecting micro- organisms (like most of the zooplankton components), extracting DNA from whole individuals might be considered.

Quantification of prey items (see Section 5 for broader considerations) We refer here to the combined effect of (1) the time since prey ingestion and (2) the specific detectability of prey DNA along the digestive process. While water tempera- ture (directly affecting the metabolic rate and prey species DNA degradation), is the main factor that must be taken into account when determining detectability curves over digestion time, estimating the time of ingestion (and thus the exposition time of DNA to digestive fluids) is also of huge relevance, when attempting to estimate the amount of prey ingested. However, respectively, performing experimental studies to determine detectability of prey along temperatures gradients (and involving known quantity of ingested preys or not; e.g. Albaina et al., 2010, Deagle et al., 2010, Marshall et al., 2010, Hunter et al., 2012) and, applying DNA fragment length analysis have been successfully applied to overcome, or at least reduce, each of these two sources of uncertainty. Apart from this, other factors, such as mixed diets or predator size should be considered when quantifying prey ingestion.

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Predator DNA avoidance: If the DNA within the sample contains a small number of sequences in relatively high concentrations, then less concentrated sequences are often not amplified because the PCR favours the dominant DNA types. This is a particular problem in molecular diet studies, where predator DNA is often present in great excess of food-derived DNA (e.g. Vestheim and Jarmar, 2008). To curtail such problems these authors developed so-called 'blocking primers', whereby the predator's own DNA is blocked from amplification by the addition of a modified predator-specific blocking primer. Blocking primers have now become standard in studies applying sequencing analysis to gut contents (i.e. Deagle et al., 2009; 2010).

Scavenging and secondary predation: The fact that a predator tests positive for a target prey species does not necessarily mean that it killed that prey animal or even that it intended to eat it. Alternative routes by which prey DNA might get into the guts of predators include secondary predation and scavenging and have to be considered case by case (King et al., 2008).

2.3.5 Limitations of using metagenomics for qualitative and quantitative biodiversity assessment Metagenomics approaches hold great promise for practical application in an ecosystem management context. However, the molecular and statistical methods are still in development and currently available methods may be associated with limited accuracy and precision especially for generating quantitative estimates of species abundances. Below, we review main factors that may limit genomics based inference on qualitative and especially quantitative ecosystem indicators. Most metagenomic studies that aim to estimate the relative proportions of species and individuals assume a positive, and measurable, relationship between the amount of DNA sequence and the number of individuals (or biomass) of a particular species (or taxonomic group) at a particular sample site. Yet, biological and technical factors that may limit inference include effects of numbers of DNA strands from each individual and species repre- sented in a sample.

2.3.5.1 Biological factors Sequence amount will be affected by at least two different biological factors; multicel- lularity and the presence of multiple gene copies per cell. Unlike microbes, multicellular organisms have a variable number of cells per individual which largely (but not only) depends on their developmental stage. If using sequence counts to estimate numbers of individuals in a sample, a bigger specimen will contribute more to the count than a large number of small ones (Pompanon et al., 2012). Although preventing correlating amount of DNA with individuals, multicellu- larity is not an issue when considering biomass rather than counts of organisms. Even when considering biomass, because not all cells/organisms have the same number of copies of a gene, using sequence counts to extrapolate number of cells (therefore biomass) can also be problematic. For example, in animals, the mitochondrial gene COI is frequently used as the marker of choice. Yet, each cell (even within an organism) has a variable number of mitochondria and each mitochondrion has a variable number of copies of the genome.

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2.3.5.2 Technical factors Apart from biological factors affecting metagenomics-based sequence data, a range of technical factors will also affect the accuracy of results. Below, we list some of the most critical biological factors. Converse to these, most technical factors can be addressed and corrected under controlled experiments. Hence, they need not pose in- surmountable obstacles to reliable estimates of species compositions. First, DNA extraction efficiency can be uneven across species or taxa analysed. For example, cells from organisms with thick cell walls or organisms being part of a bio- film are difficult to break and therefore release lower amounts of DNA than actually present in a sample. Although probably not strong enough to prevent a qualitative biodiversity assessment, this effect could result in erroneous estimates of relative proportions of DNA in a sample. Second, the DNA of some organisms may amplify better with a specific set of markers and primers than others. For example, a primer may have a better match for one taxonomic group than for others, which could result in erroneous estimates of relative proportions of DNA in a sample or even in failure to identify some taxa. Third, there may be biases in the amplification and sequencing steps that cannot be predicted and controlled for a priori. These biases can be reduced by performing several amplification reactions with fewer amplification cycles each. Finally, existing sequence repositories and DNA barcode libraries such as the BOLD database (www.barcodinglife.com) contain over two million sequences, of which almost 130,000 are formally described animals, over 42,000 are formally described plants and about 2,500 are formally described fungi and protists. However, these libraries may not contain reference sequences for in cryptic species and for poorly described taxonomic groups (Bourlat et al., 2013).

2.3.6 Conclusions Environmental sequencing is a powerful method for taxonomic and metabolic biodiversity of virtually any sample containing biological material. Hence, by assisting the measuring of relevant indicators, this method offers unprecedented support to the implementation of the MSFD and therefore, an EAFM. Yet, these methods are being applied to an increasing number of systems, and although metagenetics is the most widely used approach, a raise in the applications that rely on metagenomics and metatranscriptomics approaches is expected in the upcoming years. While for some applications, the environmental sequencing methods are standardized and can be directly applied, for some others, especially those requiring quantification of biomass or abundance, more analyses are needed for an appropriate calibration of the method. In all cases, a careful examination of the biological question and results expected is required in order to determine if an environmental sequencing approach is required and, if so, to evaluate which method would be most suitable. In light of this, WGAGFM should revisit the advances on the development of metagenetics, metagenomics and metatranscriptomics in connection with the implementation an ecosystem based fisheries management approach.

2.3.7 The WGAGFM recommends • that SCICOM and relevant WGs support a move to metagenetics being examined and considered as a method for generating standardized data to

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fulfil the requirements of indicators defined in the Marine Strategy Framework Directive • that SCICOM push for WGAGFM contributing to the Good Environmental Status working group of the Marine Strategy Framework Directive advising the European Commission on the opportunities offered by metagenetics.

2.3.8 References Adura, A., Planes, S., and Garcia-Vazquez, E. 2011. Beyond biodiversity: Fish metagenomics. PLoS ONE 6(8): e22592.

Albaina, A., Fox, C. J., Taylor, N., Hunter, E., Maillard, M., and Taylor, M.I. 2010. A TaqMan real-time PCR based assay targeting plaice (Pleuronectes platessa L.) DNA to detect predation by the brown shrimp (Crangon crangon L.) and the shore crab (Carcinus maenas L.)— assay development and validation. Journal of Experimental Marine Biology and Ecology 391: 178−189.

Albaina, A, Taylor, M. I., and Fox, C. J. 2012. Molecular detection of plaice remains in the stomachs of potential predators on a flatfish nursery ground. Marine Ecology Progress Series, 444: 223–238.

Bik, H. M., Porazinska, D. L., Creer, S., Caporaso, J. G., Knight, R., and Thomas, W. K. 2012. Sequencing our way towards understanding global eukaryotic biodiversity. Trends in Ecology and Evolution, 27: 233–243.

Borja, A., and Elliot, M. 2013. Marine monitoring during an economic crisis: The cure is worse than the disease. Marine Pollution Bulletin, 68: 1–3.

Bourlat, S. J., Borja, A., Gilbert, J. et al. 2013. Genomics in marine monitoring: new opportunities for assessing marine health status. Marine Pollution Bulletin. In press.

Cardoso, A.C., Cochrane, S. K. J., Doerner, J et al. 2010. Scientific Support to the European Commission on the Marine Strategy Framework Directive - Management Group Report. Publications Office of the European Union.

Cochrane, S. K. J., Connor, D. W., Nilsson, P. et al. 2010. Marine Strategy Framework Directive. Guidance on the interpretation and application of Descriptor 1: Biological diversity. Re- port by Task Group 1 on Biological diversity for the European Commission’s Joint Re- search Centre, Ispra, Italy.

Eisen, J. A. 2007. Environmental Shotgun Sequencing: Its Potential and Challenges for Studying the Hidden World of Microbes. PLoS Biology 5:e82.

Fox, C. J., Taylor, M. I., Dickey-Collas, M. et al. 2008. Mapping the spawning grounds of North Sea cod (Gadus morhua) by direct and indirect means. Proc. R. Soc.Lond. B. 275: 1543– 1548.

Fox, C. J., Taylor, M. I., Pereyra, R., Villasana-Ortiz, M. I., and Rico, C. 2005. TaqMan DNA technology confirms likely overestimation of cod (Gadus morhua L.) egg abundance in the Irish Sea: implications for the assessment of the cod stock and mapping of spawning areas using egg based methods. Mol. Ecol. 14: 879–884.

Frolov, S., Kudela, R. M., and Bellingham, J. G. 2013. Monitoring of harmful algal blooms in the era of diminishing resources: A case study of the U.S. West Coast. Harmful Algae, 21-22: 1–12.

Garcia, S. M., Zerbi, A., Aliaume, C., Do Chi, T., and Lasserre, G. 2003. The ecosystem approach to fisheries. Issues, terminology, principles, institutional foundations, implementation and outlook. FAO Fisheries Technical Paper. Rome.

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Harvey, J. B. J., Hoy, M. S., and Rodriguez, R. J. 2009. Molecular detection of native and invasive marine invertebrate larvae present in ballast and open water environmental samples collected in Puget Sound. Journal of Experimental Marine Biology and Ecology, 369: 93–99.

Hebert, P. D. N., Ratnasingham, S., and deWaard, J. R. 2003. Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proceedings Biological Sci- ences, 270: 96–99.

Knowlton, N. 1993. Sibling Species in the Sea. Annual Review of Ecology and Systematics, 24: 189-216.

Machida, R. J., and Knowlton, N. 2012. PCR primers for metazoan nuclear 18S and 28S ribosomal DNA sequences. PloS one 7:e46180.

Machida, R. J., Kweskin, M., and Knowlton, N. 2012. PCR Primers for Metazoan Mitochondrial 12S Ribosomal DNA Sequences. PloS one 7:e35887.

Marchetti, A. S., Durkin, D. M, C. A., Parker, M. S., Kodner, E. B., Berthiaume, C. T., Morales, R., Allen, A. E., Armbrust, E. V. 2012. Comparative metatranscriptomics identifies molecular bases for the physiological responses of phytoplankton to varying iron availability. Proc Natl Acad Sci USA 109:E317–E325.

Pompanon, F ., Deagle, B. E., Symondson, W. O. C., Brown, D. S., Jarman, S. N., Taberlet, P. 2012. Who is eating what: diet assessment using next generation sequencing. Molecular Ecology, 21: 1931–1950.

Stoeck, T., Bass, D., Nebel, M., Christen, R., Jones, M. D. M., Breiner, H-W., Richards, T. 2010. Multiple marker parallel tag environmental DNA sequencing reveals a highly complex eukaryotic community in marine anoxic water. Molecular Ecology, 19(1): 21–31.

Streit, W. R., and Schmitz, R. A. 2004. Metagenomics--the key to the uncultured microbes. Cur- rent Opinion in Microbiology, 7: 492–498.

Taberlet, P., Prud’Homme, S. M., and Campione, E. et al. 2012. Soil sampling and isolation of extracellular DNA from large amount of starting material suitable for metabarcoding studies. Molecular Ecology, 21: 1816–1820.

Takahara, T., Minamoto, T., and Doi, H. 2013. Using environmental DNA to estimate the distribution of an invasive fish species in ponds. PloS one 8:e56584.

Takahara, T., Minamoto, T., Yamanaka, H., Doi, H., and Kawabata, Z. 2012. Estimation of Fish biomass using environmental DNA. PloS one 7:e35868.

Thomsen, P. F., Kielgast, J., Iversen, L. L., and Moller, P. R. 2012. Detection of a diverse marine fish fauna using environmental DNA from seawater samples. PloS one 7:e41732.

Wemheuer, B., Wemheuer, F., and Daniel, R. 2012. RNA-based assessment of diversity and composition of active archaeal communities in the German Bight. Archaea 695826.

Yu, K., and Zhang, T. 2012. Metagenomic and metatranscriptomic analysis of microbial community structure and gene expression of activated sludge. PloS one 7:e38183.

2.4 ToR d) Produce an update on SNP-technology assessment. Paulo Prodöhl, Philip McGinnity, Geir Dahle The nature of new molecular methods implies that this is a ToR with no finite time frame. Although the use of SNP as a genetic tool has accelerated the last decade and Restriction site Associated DNA (RAD) sequencing has been established as the pre- ferred methodology to produce SNPs in non-model organisms, there appear to be more deliberation around selecting the “right” marker. With increasing number of SNP’s and an increasing amount of data linked to each marker, the possibility to se- lect the “perfect marker” increases. Ascertainment bias is still playing a role in handling date, especially on a global scale, and at the moment scientists are using all the

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new information from all available SNP’s to establish the best panels for the specific questions asked, including management, selection or function (Bourret et al 2013)

2.4.1 WGAGFM action list That the WGAGFM revisit annually a ToR on update and evaluation of molecular methods in the world of population genetics.

2.4.2 Reference Bourret, V., Kent, M. P., Primmer, C. R., Vasamagi A., Karlsson, S., Hindar, K., MaGinnity, P., Verspoor, E., Bernatchez, L., and Lien, S. 2013. SNP-array reveals genome-wide patterns of geographical and potential adaptive divergence across the natural range of Atlantic salmon (Salmo salar). Molecular Ecology, 22: 532–551.

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Annex 1: List of Participants

NAME ADDRESS PHONE/FAX E-MAIL

Naiara Rodríguez- Marine Research Phone : +34 667 174 [email protected] Ezpeleta Division, Azti 514 Technalia, Txatxarramendi ugartea z/g E- 48395 Sukarrieta, Bizkaia Jann T.H. Joint Research Phone: +39 0332 78 [email protected] Martinsohn Centre (JRC) 6567 Fax: +39 0332 Institute for the 78 9658 Protection and Security of the Citizen (IPSC) JRC.G.4 - Maritime Affairs Via Enrico Fermi 2749 (TP 051) I-21027 Ispra (Va), Italy Geir Dahle Institute of Marine Phone: +47 55 23 63 [email protected] Research PO Box 49 Fax: +47 55 23 63 1870 N-5817 79 Bergen, Norway Gary R. Carvalho School of Phone: +44 (0)1248 [email protected] Biological Sciences, 382100 Fax: +44 University of (0)1248 371644 Bangor Environment Centre Wales Bangor, Gwynedd LL57 2UW UK Aitor Albaina Lab. Genetics, Fac. Phone: +34- [email protected] Vivanco Science & 946015503 Technology; Dpt. Fax: +34-946013145 Genetics, Physical Anthropology & Animal Physiology; Univ. Basque Country Bº Sarriena s/n 48940 Leioa; Bilbao; Spain Reinhold Hanel Johann Heinrich Phone +49 40 38905 [email protected] von Thünen- 290 Institute, Institute Fax+49 40 38905 for Fishery Ecology 261 Palmaille 9 D- 22767 Hamburg Germany

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NAME ADDRESS PHONE/FAX E-MAIL Dorte Bekkevold Technical Phone: +45 [email protected] University of 35883130 Fax: + 45 Denmark, Vejlsøvej 35883150 39, 8600 Silkeborg, Denmark John Gilbey Freshwater Phone: +44 (0)1224 [email protected] Laboratory, 876544 Fax: +44 Faskally, Pitlochry, (0)1796 473523 Perthshire, PH16 5LB UK Anna Kristin Food Safety & Phone: + 354422 [email protected] Danielsdottir Environment 5014 Vinlandsleid 12 Fax: +354 422 5001 113 Reykjavik, Iceland Sarah Helyar Food Safety & Phone: + 354422 [email protected] Environment 5014 Vinlandsleid 12 Fax: +354 422 5001 113 Reykjavik, Iceland Daria Zelenina Russian Federal [email protected] Research Institute for Fisheries and Oceanography, 107140, 17, V. Krasnoselskaya str., Moscow, Russian Federation Ian Bradbury Fisheries and Phone (709) 772- [email protected] Oceans 3869, Fax. (709) Canada/Pêches et 772-3578 Océans Canada. 80 East White Hills Road, PO Box 5667 St. John's, NL, A1C 5X1, Canada Jakob Hemmer- Technical Phone: +45 [email protected] Hansen University of 35883130 Fax: + 45 Denmark, Vejlsøvej 35883150 39, 8600 Silkeborg, Denmark Jochen Trautner Johann Heinrich [email protected] von Thünen- Institute, Institute for Fishery Ecology Palmaille 9 D- 22767 Hamburg Germany

Kristinn Ólafsson Food Safety & Phone: + 354422 [email protected] Environment 5014 Vinlandsleid 12 Fax: +354 422 5001 113 Reykjavik, Iceland

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Annex 2: WGAGFM Agenda

Tuesday, 7 May 9.00 Welcome by local hosts Anna Kristín Daníelsdóttir and Sveinn Mar- geirsson (Matís CEO). 9.30 Welcome and updates from WG chair Dorte Bekkevold 9.45 - 12.30 Presentation and discussion of position papers for ToRs a–d a ) Produce a review of the identification and use of adaptive gene markers in shellfish aquaculture and for the genetic characterization of wild populations. (leads: Iveta Matejusova/John Gilbey) b ) Review and consider technological developments in fisheries forensics and management of exploited marine fish with emphasis on contributions to sustainability and governance. (leads: Gary Carvalho/Sarah Helyar/Jann Martinsohn) c ) Produce a review on the use of metagenomics and metatranscriptomics as an approach for marine ecosystem management. (lead: Naiara Rodríguez- Ezpeleta) d ) Produce an update on SNP-technology assessment. (leads: Paulo Prodöhl/Phil McGinnity/Geir Dahle). 12.45 - 13.45 Lunch. 14.00 - 16.00 Presentation and plenum discussion of position papers for ToRs a–d (continued) 16.30 – 17.00 Formation of ToR working groups 17.00 – ca. 18.00 Presentation – Kristinn Ólafsson: population genetic analyses of Icelandic salmon

Wednesday, 8 May 9.00 Morning assembly w. updates on activities and practical information 9.15 – 12.15 Parallel work sessions on ToRs a-d 12.45 – 13.45 Lunch 14.00 - 15.30 Work in groups on ToRs a-d (continued) 15.30-16.30 Discussion of request from the Norwegian Institute of Marine Re- search to WGAGFM on “Addressing statistical and analytical methods for quantifying genetic introgression of farmed escaped salmon in native populations as part of an international management implementation strategy”. 16.30 – 17.00 Status of work in ToRs groups – each ToR lead gives an update

17.00 – ca 18.00 Presentation – Jakob Hemmer-Hansen: Genetically based management of cod in the Western Baltic and plaice in the Skagerrak

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Thursday, 9 May 9.00 Morning assembly 9.15 – 12.15 Presentation of ToR reports/recommendations 12.15 - 13.30 Suggestions for new ToRs for 2014, future meeting venue, A.O.B. 13.30 End of meeting

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Annex 3: WGAGFM Terms of Reference for the next meeting

The Working Group on Application of Genetics in Fisheries and Mariculture (WGAGFM), chaired by Dorte Bekkevold, Denmark, will meet in Faro, Portugal, 7–9 May 2014 to: a ) Produce a review of the identification and use of adaptive gene markers in shellfish aquaculture and for the genetic characterization of wild populations b ) Review and consider methods for integrating genomic methods with marine fisheries management c ) Review and consider molecular methods for quantifying genetic introgression of farmed fish in native populations d ) Produce an update on SNP-technology assessment.

WGAGFM will report by 31 March 2014 (via SSGHIE) for the attention of Science Committee.

Supporting Information

Priority The current activities of this Group will lead ICES into issues related to the ecosystem affects of fisheries and mariculture, especially with regard to the application of the Precautionary Approach. Consequently, these activities are considered to have a very high priority.. Scientific Term of Reference a) justification The application of genomic analysis is rapidly developing as a tool in shellfish mariculture. The aim is to review and consider the full set of genomic tools and techniques that are being used/examined in relation to shellfish mariculture, and to identify particular issues of concern in relation to management of wild shellfish populations, that may benefit from research using such genomic tools. To better integrate WG expertise it is an aim to approach the ICES Working Group on Aquaculture (WGAQUA) to outline the tools available and/or under development and to identify issues of concern in the culture situation and those associated with mariculture/wild interactions which may be addressed. Term of Reference b) Technological advances have facilitated the development of genomic tools for the study of non-model organisms. These developments offer new opportunities for bridging traditional gaps between population/conservation genetics and fisheries management. Yet, significant challenges still remain, for example in relation to assessing connectivity in and outside spawning seasons on local geographical scales and over single generations, and in relation to estimating population sizes; all important information in many management scenarios. Thus, it is timely to review the information that can, and cannot, at present be provided by genetic approaches, especially in relation to pertinent information required by fisheries management. The aim is to provide a general evaluation of opportunities, costs and time frames with modern population genomic approaches, and to include examples on a case-by-case basis. It is an aim to invite members of one or more ICES ACOM working groups to contribute to the ToR.. Term of Reference c) Fish strains used in aquaculture are commonly characterized by genetic changes associated with domestication and breeding selection, compared with their wild counterparts. Several studies over the last decade report that hybridization and introgression by escaped farmed fish may incur a fitness cost to wild

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populations, causing increasing awareness and concern about both conserving native fish gene pools and to the continuing health and viability of the wild populations. Consequently, there is an increasing need for the identification and development of statistical tools to assess and quantify the degree of genetic introgression of escaped or released farm fish. Given the dynamic nature and inherent complexity, quantifying genetic introgression using standard population genetic methods is difficult representing an ongoing challenge. The aim is to review studies estimating levels of introgression, number of possible sources contributing to farmed escapees, and impact on populations; to summarize existing tools, assumptions involved, and any studies evaluating success; to identify desired model parameters and parameter space to be evaluated using simulations; to simulate introgression into wild populations and evaluate tool effectiveness, and finally to provide recommendations on usage of analysis tools and their limitations. Term of Reference d) Issues pertaining to ascertainment bias, cost, SNP choice, ease of analyses, screening platform, technical aspects related to genotyping, data management, and broader technological and statistical approaches should be further considered by members of this working group on an ongoing basis. Resource The research programmes which provide the main input to this group are requirements already underway, and resources are already committed. The additional resource required to undertake additional activities in the framework of this group is negligible. Participants The Group is normally attended by some 20–25 members and guests. Secretariat None. facilities Financial No financial implications. Linkages to There are no obvious direct linkages with the advisory committees. advisory committees

Linkages to other SIMWG, WGEVO, WGBIODIV, WGAQUA committees or groups

Linkages to other Linkage with the EC Joint Research Centre at Ispra, Italy. organizations

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Annex 4: Recommendations

Recommendation Adressed to

1. A ToR identifying and reviewing cases/scenarios where WGAQUA interactions between shellfish of mariculture and wild origin is of concern, where genomic tools may allow estimation of potential introgression is produced 2. Organize a workshop between population geneticists and ICES SCICOM fisheries policy-makers, fisheries control agencies and stakeholders to explore opportunities arising from fisheries genetics for management issues, such as traceability, IUU activities, and mislabelling. Outputs should include a framework for International Forensic Standards in Fisheries Management. 3. Encourage member states to invest in the development of a ICES ACOM technology platform with sufficient flexibility in design and application to be employed in the routine testing of traceability of fish and shellfish and products. 4. Support a move to metagenetics being considered as a method ICES SCICOM, SGIMT, for generating standardized data to fulfil the requirements of WGBIODIV, SIBAS indicators defined in the Marine Strategy Frame-work Directive. 5. Support that WGAGFM contribute to the Good Environmental ICES SCICOM Status working group of the Marine Strategy Framework Directive advising the European Commission on the opportunities offered by metagenetics.