FAIR CT97 3882 Concerted Action on Identification

FAIR CT97 3882

Concerted Action on Identification, Management and Exploitation of Genetic Resources in Brown Trout (Salmo trutta).

Consolidated Final report for the period from 1-1-98 to 31-12-99

Type of contract: Concerted Action Total cost: 350,000 EURO EU contribution: 350,000 Euro Commencement date: 1-1-98 Duration: 2 years Completion date: 31-12-99

EU contact: DG XIV, Fisheries. Fax: (+32-2) 2957862

Coordinator: Michael Møller Hansen, Senior Scientist, M.Sc., Ph.D., Danish Institute for Fisheries Research, Vejlsøvej 39, DK-8600 Silkeborg, Denmark. Tel. +45 89 213100. Fax. +45 89 213150. E-mail [email protected]. Partners: Queen’s University of Belfast, Northern Ireland, UK. Norwegian Institute for Nature Research, Norway University of Girona, Spain. University of Berne, Switzerland. INRA, France. Marine Research Institute and Institute of Freshwater Fisheries, Iceland. Universidad de Santiago de Compostela, Spain. Aristotle University of Thessaloniki, Greece. University of Göteborg, Sweden. University of Porto, Portugal. Institute of Marine Research, Norway. Université Montpellier II, France. Wageningen Agricultural University, the Netherlands. Stockholm University, Sweden. Zoological Society of London, England, UK. University of Munich, Germany. Finnish Game and Fisheries Research Institute, Finland. University of Stirling, Scotland, UK. External experts: Prof. Louis Bernatchez ,University of Laval, Canada. Dr. Tiit Paaver, Estonian Agricultural University, Estonia. Dr. Alexander Osinov, Moscow State University, Russia Ms. Ewa Wlodarczyk, Sea Fisheries Institute, Poland. Dr. Roman Wenne, Sea Fisheries Institute, Poland. Prof. Inci Togan, Middle East Technical University, Turkey. Prof. Fred Allendorf, University of Montana, Missoula, USA Dr. Robin Waples, National Marine Fisheries Service, Seattle, USA Dr. Craig Primmer, University of Helsinki, Finland Dr. Steven Weiss, Veterinary University of Vienna, Austria

1 Contents

Abstract ...... 4 I. Objectives of the CA...... 7 II. Description of work...... 7 Summary of activities, 1998 ...... 7 Summary of activities, 1999 ...... 8 Description of work in relation to tasks and subtasks...... 9 III. Results and deliverables...... 12 1) Survey of brown trout population genetics research activities...... 13 2) Recommendations for future studies ...... 15 3) Recommendations for genetic marker nomenclature and harmonisation of the use of techniques...... 19 4) Recommendations for management/conservation of genetic resources of the species ...... 31 5) Evaluation of the potential of the species for aquaculture with emphasis on the genetic resources available...... 32 6) Brown trout bibliography ...... 32 7) World Wide Web site for the CA...... 32 8) Raw data and data bases on genetic markers, available on the WWW site...... 32 9) Workshops of the CA (1998 and 1999)...... 33 10) Mid-term evaluation...... 34 11) Progress report...... 36 12) Final report...... 36 13) First (1998) and Second (1999) Iberian Trout Meetings ...... 36 14) Other results and deliverables...... 37 IV. Dissemination of research results...... 37 V. Future actions...... 38 VI. The role of the CA in coordinating research efforts and activities ...... 38 ANNEX 1: Program − First Workshop on Identification, Management and Exploitation of Genetic Resources in Brown Trout (Salmo trutta)...... 40 ANNEX 2: Proceedings of the First Workshop on Identification, Management and Exploitation of Genetic Resources in Brown Trout (Salmo trutta)...... 48 Introduction...... 48 a) Potentials and different merits of genetic markers ...... 49 b) Conservation of brown trout, preliminary discussion...... 56 c) Stocking impact assessment...... 57 d) Potential of brown trout for aquaculture...... 61 e) Review of brown trout phylogeography ...... 66 f) Local adaptation in brown trout populations...... 68 g) Brainstorm on the internet features of the CA...... 71 h) Preliminary discussion on management recommendations for conservation of genetic resources in brown trout...... 72 i) Genetic marker nomenclature and harmonisation of the use of markers ...... 72 j) Preliminary suggestions for topics at the next workshop...... 79 ANNEX 3: Summary of Iberian trout genetics meeting ...... 81 ANNEX 4: Program − Second Workshop on Identification, Management and Exploitation of Genetic Resources in Brown Trout (Salmo trutta)...... 92

2 ANNEX 5: Proceedings of the Second Workshop on Identification, Management and Exploitation of Genetic Resources in Brown Trout (Salmo trutta)...... 103 Introduction...... 103 a) Presentation of ongoing research activities...... 104 b) Endangered populations (responsible person: Patrick Berrebi)...... 110 c) Sea trout; genetic population structure and conservation (responsible person: Michael M. Hansen) ...... 114 d) Comparative data analysis (responsible person: Andy Ferguson)...... 119 e) Conservation of brown trout ...... 133 f) Behavioural studies of salmonid fishes (Responsible person: Ian Fleming)...... 137 g) Suggestions for future studies (Responsible person: Michael M. Hansen)...... 139 h) Is there a life after the CA? (Responsible person: Michael M. Hansen) ...... 139 ANNEX 6: Report of the Second Iberian Brown Trout (Salmo trutta) Genetics Meeting...... 140 ANNEX 7: Forms and questionnaires of the CA...... 144 ANNEX 8: Overview of participants and scientific teams ...... 157 ANNEX 9: Brief individual reports by participants, describing their input to the objectives and deliverables of the CA...... 159 ANNEX 10: Travel reports of the CA...... 198

Enclosed as separate document:

ANNEX 11: Conservation Genetic Management of Brown Trout (Salmo trutta) in Europe (Laikre et al.). Report by the Concerted Action on Identification, Management and Exploitation of Genetic Resources in Brown Trout (Salmo trutta).

3 Abstract

I. Objectives

The specific objectives of the CA can be summarised as follows: • to promote collaboration among laboratories that are active in research on population genetics of brown trout • to co-ordinate this research and when convenient harmonise the use of genetic markers • to bring together complementary expertise from all parts of the EU and other countries • to review and evaluate the status of the research with a focus on conservation/management of genetic resources of the species and the potential of the species for aquaculture. • to compile overviews of relevant literature, available genetic markers and data from published and unpublished studies.

II. Description of work

1998

The CA commenced 1 January 1998. During the first two months questionnaires were constructed and a survey of the research activities of the participating laboratories was undertaken. The Troutconcert home page was constructed, and the information on research activities and publications were made accessible on this site. A second questionnaire was later distributed specifically on the use of microsatellite loci. This was done in order to provide a list of well- functioning microsatellite loci that could be used by the participating laboratories, and the information was made available at the web site. The first workshop of the CA took place 29 June - 5 July in Silkeborg, Denmark. Approximately half of the workshop was devoted to presentation of research activities of the participating laboratories, whereas the other half concentrated on specific theme sessions. Another meeting took place 21 - 23 September in Lugo, Spain, involving primarily the Iberian participants of the CA. Finally, participants visited other laboratories of the CA. The main part of this travel activity focused on calibration of markers, transfer of technology and joint data analysis.

1999

In 1999 the work on writing a report on conservation and management of genetic resources in brown trout was undertaken. A group of participants, the “Conservation Group”, attended meetings 24 - 27 February at the University of Berne, Switzerland, and 3- 7 November at the Aristotle University of Thessaloniki in order to discuss and write the report. Dr. Linda Laikre, Stockholm University, Sweden edited the report. The report titled “Conservation Genetic Management of Brown Trout (Salmo trutta) in Europe” was printed in December 1999 and also made available via the internet. Allozyme “raw data” were collected from CA participants and external experts and

4 made available on the WWW site. In addition, calibrations of allozyme, microsatellite and mitochondrial DNA markers, respectively, were conducted. The second workshop of the CA took place 28 June – 4 July in St. Feliu de Guixols, Spain. The meeting was hosted by Prof. Carles Pla and Dr. Jose-Luis Garcia-Marin, University of Girona. The meeting contained some discussion sessions where the development and future of the CA was discussed. However, most of the meeting was devoted to scientific sessions. Similar to 1998 a meeting took place (2-4 December 1999 at the University of Porto) involving primarily the Iberian participants of the CA. Finally, travel activities took place among the laboratories of the CA. The purposes of the visits were diverse, ranging from technology transfer and joint data analyses to editing reports and planning and writing EU project proposals.

III. Achievements

• Two workshops took place, in 1998 and 1999, respectively. Both workshops were attended by > 50 persons, representing 28 laboratories from 20 countries. In addition to presentation of research activities of the participants, the workshops covered a wide range of topics, such as conservation, aquaculture, phylogeography, use of molecular markers for population studies etc. • A Troutconcert homepage (http://www.qub.ac.uk/bb/prodohl/TroutConcert/TroutConcert.htm) was established. It presently includes: A description of the background and aims of the CA A list of all participants with e-mail addresses A survey of the research of the participants, including their publications on brown trout population genetics A brown trout bibliography containing more than 1800 references A "molecular marker page" with information and recommendations regarding the use of molecular markers for brown trout studies. A population genetics and fisheries biology glossary Links to other important web sites (including the CORDIS site; click on the FAIR logo) Genetic "raw" data: Allozyme data collected by the participants of the CA. A collection of photos of different trout morphs • A report was written with the aims of reviewing/describing knowledge on genetic diversity and threat status of brown trout in Europe and provide recommendations for management and conservation of genetic resources in the species (Laikre et al: Conservation Genetic Management of Brown trout (Salmo trutta) in Europe). An on-line version of the report was also made accessible via the internet (http://www.dfu.min.dk/ffi/consreport/index.htm) • A survey of research activities of the participants was conducted. The results were made accessible on the Troutconcert homepage. • Recommendations for future studies were given. • Specific recommendations were given concerning the use of mitochondrial DNA and microsatellite markers. Also, data were collected on primer sequences, available microsatellite loci etc. This information was made accessible on the Troutconcert homepage. • Calibrations of microsatellite allele sizes and mitochondrial DNA markers were conducted. • A brown trout bibliography was compiled and made accessible on the Troutconcert homepage. • An electronic discussion list was established. • Travels took place among laboratories in order to learn new molecular techniques, calibrate markers, analyse data and discuss future projects. In addition, two “satellite” workshops took place that involved primarily the Iberian CA participants.

5 IV. Future actions

Many participants have expressed regrets that the CA would end by 1999, as over time this could mean a loss of a significant part of the momentum in terms of collaboration and exchange of ideas that has been gained during the past two year. The CA therefore suggests to have a third TROUTCONCERT workshop three years after the end of the CA in order to update links between research groups. It has been decided by then to ask the Commission for funding for this workshop. Dr. Paulo Prodöhl, Queen’s University of Belfast has agreed to maintain and update the WWW site and the electronic discussion list.

6 I. Objectives of the CA.

The Concerted action on identification, management and exploitation of genetic resources in brown trout (Salmo trutta) "TROUTCONCERT" (EU FAIR CT 97-3882) was initiated with the main objective to coordinate the research on brown trout population genetics that goes on within the EU and in countries affiliated with the EU. However, as important research on this species takes place also in countries outside the EU, external experts from Canada, Poland, Turkey, Russia and Estonia participated in the workshops as well. Finally, for the 1999 workshop we invited external experts from Finland, Austria and USA to give talks on topics regarding conservation and management of salmonid fishes and evolutionary dynamics and use of molecular markers for population studies.

The objectives of the CA can be summarised as follows:

• to promote collaboration among laboratories that are active in research on population genetics of brown trout (Salmo trutta) • to co-ordinate this research and when convenient harmonise the use of genetic markers • to bring together complementary expertise from all parts of the EU and other countries • to review and evaluate the status of the research with a focus on conservation/management of genetic resources of the species and the potential of the species for aquaculture. • to compile overviews of relevant literature, available genetic markers and data from published and unpublished studies.

We met these objectives • by arranging workshops (one in 1998 and another in 1999) • by smaller meetings and exchange visits among laboratories • by distribution of questionnaires to the participants • by writing a report on conservation of genetic resources in the brown trout • by establishing an electronic discussion list • by establishing a TROUTCONCERT home page on the World Wide Web, which among other things contain general information about the CA, information about the research activities of the participants, lists and recommendations about molecular markers for brown trout, an overview of literature on brown trout ecology and genetics and a compilation of allozyme "raw data" from studies on brown trout.

II. Description of work.

Summary of activities, 1998 The CA commenced 1 January 1998 and the electronic discussion list was established immediately thereafter. During the first two months questionnaires were constructed in order to make a survey of the research activities and publications of the participating laboratories, and the questionnaires were retrieved in March-April. In the meanwhile, the first version of the TROUTCONCERT home page was constructed, and the information on research activities and publications were made accessible on this site.

In addition to the first questionnaire a second questionnaire was later distributed specifically on the use of microsatellite loci. This was done in order to provide a list of well-functioning microsatellite

7 loci that could be used by the participating laboratories. A number of other initiatives were also taken. These included the establishment of a "DNA reference sample collection" that was distributed to all participants working with microsatellites in order to test loci and calibrate markers.

The first workshop of the CA took place 29 June - 5 July in Silkeborg, Denmark. Approximately half of the workshop was devoted to presentations of research activities of the participating laboratories, whereas the other half concentrated on specific theme sessions.

Another meeting took place 21-23 September in Lugo, Spain (Hosted by Dr. Paulino Martinez), involving specifically the Iberian participants of the CA. There are many groups working on brown trout population genetics in this region and coordination of the research and increased collaboration are consequently particularly important tasks. The workshop program is attached as Annex 1 and detailed summaries of both the Silkeborg and Lugo meetings are attached as Annex 2 and 3.

Finally, participants visited other laboratories of the CA. The main part of this travel activity focused on transfer of technology and joint data analysis.

Summary of activities, 1999 In 1999 the main part of the work on writing a report on conservation and management of genetic resources in brown trout was undertaken. A group of participants, the “Conservation Group”, with particular interest and expertise in conservation attended a meeting 24 - 27 February at the University of Berne, Switzerland, hosted by Dr. Carlo Largiader. The outline of the report was discussed and planned and the first sections were written during the meeting. Dr. Linda Laikre, Stockholm University, Sweden agreed to edit the report. A preliminary draft of the report was distributed to TROUTCONCERT participants in June and discussed at the workshop in St. Feliu, Spain, in July. The “Conservation Group” met 3- 7 November at the Aristotle University of Thessaloniki, Greece (Hosted by Prof. Costas Triantaphyllidis and Dr. Apostolos Apostolidis), to finalise the report. Immediately after the meeting the draft report was distributed to a number of fisheries managers in different European countries for review. After having received their input and suggestions for alterations the report was printed by a publishing firm in December 1999. Also, the report was made available on the internet in an on-line version and as pdf file.

The second workshop of the CA took place 28 June – 4 July in St. Feliu de Guixols, Spain. The meeting was hosted by Prof. Carles Pla and Dr. Jose-Luis Garcia-Marin, University of Girona. The meeting contained some discussion sessions where the development and future of the CA was discussed. However, most of the meeting was devoted to scientific sessions. During and after the meeting a harmonisation and calibration of allozyme allele nomenclature was conducted.

Similar to 1998 a meeting took place involving specifically the Iberian participants of the CA, but of course open to other CA participants as well. The meeting was hosted by Drs. Paulo Alexandrino and Agostinho Antunes and took place 2-4 December 1999 at the University of Porto, Portugal.

Finally, travel activities took place among the laboratories of the CA. The purposes of the visits were diverse, ranging from technology transfer and joint data analyses to editing reports and planning and writing EU project proposals.

8 Description of work in relation to tasks and subtasks.

Task 1. Annual workshops

Sub-task 1.1 Organisation of workshops. The first workshop of the CA took place 29 June - 5 July 1998 at the conference facilities of the Freshwater Centre, Silkeborg, Denmark. The workshop was organised by the coordinator and other staff at the Danish Institute for Fisheries Research in collaboration with the members of the project management group (see Annex 1 for program of the workshop).

The second workshop of the CA took place 28 June – 4 July in St. Feliu de Guixols, Spain. The workshop was organised by Prof. Carles Pla and Dr. Jose-Luis Garcia-Marin, University of Girona, and the coordinator, in collaboration with members of the project management group (see Annex 4 for program of the workshop).

Sub-task 1.2 Review and discussion of the state of the art of brown trout population genetics. The review and discussion of the state of the art of the research took place during both the 1998 and 1999 workshops. For the 1998 workshop all participating groups were allocated 20 minutes each to present their research on brown trout population genetics. This was essentially the same information as the participants had provided for the survey of research activities. However, the oral presentation gave the opportunity to explain the different aspects in more detail and it gave the other participants the possibility to ask questions. Second, a theme session was devoted to the merits and potentials of different genetic markers for studying brown trout population genetics (but also with a view to other salmonid species). Third, another theme session summarised phylogeographical studies on brown trout. Fourth, a session gave an overview of brown trout stocking activities in Europe and the intricacies of detecting introgression using genetic markers. Fifth, a session gave an overview of local adaptations in brown trout and the difficulties in identifying adaptations. We refer to Annex 2 for summaries of these sessions.

At the 1999 workshop some external experts and guests were given the opportunity to present their research activities on brown trout population genetics, similar to the presentations given by CA participants at the 1998 workshop. A theme session concentrated specifically on the genetic structure of sea trout. Sea trout is the anadromous life-history form of brown trout, and as sea trout populations in different rivers are potentially linked by gene flow the genetic structure may be different compared to resident or land-locked populations, where gene flow among populations is limited or absent. A whole day was devoted to presentations and discussions on the topic "comparative data analyses". This was a follow-up on the theme session on potentials and merits of different genetic markers that took place at the 1998 workshop. However, the "comparative data analyses" session focused primarily on microsatellite markers; their specific mutational properties and how this affect data analysis, and the many new research opportunities that this class of marker has to offer. Finally, a theme session took place that focused on behavioural ecology studies on brown trout and other salmonids and how to link and integrate population genetics and behavioural ecology.

Summaries of sessions and presentations are given in the “workshop proceedings” (Annex 2 and 5).

9 Sub-task 1.3 Recommendations for management/conservation of genetic resources of the species. At the 1998 workshop two sessions were devoted to conservation and management issues. One session focused on the conservation status of brown trout in Europe and the other session focused on the practical aspects of how to give specific management and conservation recommendations.

At the 1999 workshop one theme session focused on some specific cases of endangered brown trout populations. Second, a whole day was devoted to a theme session on management and conservation of genetic resources in brown trout. An invited guest speaker gave a presentation on the concept "Evolutionary Significant Units" (ESU) and how this has been applied to conservation of salmonid fishes in USA. Next, another invited guest speaker gave a presentation on a system for prioritising Pacific salmon populations for conservation and how the system could be implemented in the case of the brown trout. Finally, a draft version of the report "Conservation genetic management of brown trout (Salmo trutta) in Europe" had been distributed to all workshop participants and the contents of the report and how to progress with the work was discussed.

Sub-task 1.4 Assessment of the potential of the species for aquaculture with emphasis on the genetic resources available. The potential of brown trout for aquaculture production was addressed in a theme session at the 1998 workshop, and a summary is given in the workshop proceedings (Annex 2).

Sub-task 1.5 Recommendations for genetic marker nomenclature and harmonisation of some of the most commonly applied techniques. The issue of recommendations for genetic marker nomenclature and harmonisation of techniques was addressed in a specific theme session at the 1998 workshop. It was decided to concentrate on microsatellites and Polymerase Chain Reaction-Restriction enzyme Fragment Length Polymorphism (PCR-RFLP) analysis of mitochondrial DNA. Based on the session and a questionnaire on the use of microsatellites that was previously distributed to the participants, some specific recommendations were given. This is described in detail in the 1998 workshop proceedings (Annex 2), and information on the markers has also been put on the web site. In 1998 it was also decided to establish a "reference sample" of DNA from five brown trout representing three phylogeographical races. In 1998 and 1999 samples of DNA were distributed to laboratories that use microsatellite markers for brown trout. The samples were analysed by each laboratory using their “standard battery” of microsatellite loci and the results were compiled and compared at the 1999 workshop. Finally, during and after the 1999 workshop a calibration and harmonisation of allozyme allele nomenclature was conducted. This turned out to be necessary in order to make data sets comparable that were provided by different laboratories for the collection of allozyme “raw” data. The results are described in III. Results and deliverables, 3) Recommendations for genetic marker nomenclature and harmonisation of the use of techniques.

Task 2. Survey of research activities and literature

Sub-task 2.1 Distribution of questionnaires to all participants to record research activities. The questionnaire on research activities was written by Dr. Carlo Largiader, University of Berne and distributed to all participants (see Annex 7 for a compilation of form and questionnaires of the CA). The questionnaires were later retrieved by the coordinator. Next, as a part of the subcontract work of the Queen's University of Belfast, Dr. Paulo Prodöhl made a more detailed harmonisation of the format that the information was provided in and made the information accessible on the web site (see http://www.qub.ac.uk/bb/prodohl/TroutConcert/TroutConcert.htm).

10 Sub-task 2.2 Survey of literature on data bases, compilation of brown trout bibliography. As a part of the subcontract work of the Queen's University of Belfast, Dr. Paulo Prodöhl and other staff compiled a brown trout bibliography and made it accessible on the web site. (see http://www.qub.ac.uk/bb/prodohl/TroutConcert/TroutConcert.htm).

Task 3. Establishment of World Wide Web site and data bases Sub-task 3.1 Establishment of centralised WWW site. As a part of the subcontract work of the Queen's University of Belfast, Dr. Paulo Prodöhl established a WWW site/homepage for the CA. It is accessible at the following address: http://www.qub.ac.uk/bb/prodohl/TroutConcert/TroutConcert.htm. The first version was established 15 June 1998 and it has since then been updated.

Sub-task 3.2 Establishment of data bases for genetic markers and raw data In 1998 information was collected on available and well-functioning microsatellite loci as well as suitable primers for PCR-amplification of mitochondrial DNA segments. This information was made available on the web site. It turned out not to be feasible to collect data on mtDNA PCR- RFLP analysis, as different groups have analysed different segments and used different restriction enzymes. In the case of microsatellites there were also problems with different laboratories using different loci. Also, many laboratories are still in the start-up phase in applying microsatellite markers, so there are few finished data set to collect. It was therefore decided to concentrate efforts on collecting allozyme raw data (for this class of marker there are many data set available).

Collection of allozyme raw data took place in 1999. First, the principles for this, including a harmonisation of allele designations, were discussed at the 1999 workshop. Next, a form for submission of data was distributed to all participants. As a part of the subcontract work of the Queen's University of Belfast, Dr. Paulo Prodöhl collected and organised all the submitted data and made them available on the web site (http://www.qub.ac.uk/bb/prodohl/TroutConcert/TroutConcert.htm).

Task 4. Mobility among laboratories Several travel activities took place among laboratories during the two years of the CA. More details are given below in the description of sub-tasks and in the travel reports (Annex 8).

Sub-task 4.1 Technology transfer. Technology transfer has been the purpose of several travel activities, such as visits by Drs. Oystein Skaala and Kevin Glover (Institute of Marine Research, Norway) to Dr. John Taggart’s laboratory at the University of Stirling in order to get aquainted with the use of microsatellites for parentage assignment, and visits by Drs. Linda Laikre (University of Stockholm, Sweden), Sophie Launey (INRA, France) and Alistair Duguid (Queen’s University of Belfast, Northern Ireland) to the Danish Institute for Fisheries Research, Denmark, in order to work with microsatellites and DNA extracted from old scale samples.

Sub-task 4.2 Calibration of genetic markers in different laboratories Some calibration of genetic markers took place during travels among laboratories, such as calibration of mtDNA markers (more specifically, PCR-RFLP analysis of the ND-1 segment)

11 during a visit by Dr. Linda Laikre to the Danish Institute for Fisheries Research. Similarly, a calibration of allozyme markers was conducted during the First Iberian Trout Meeting (see Annex 3). However, calibration of microsatellite loci did not take place during visits. Instead, reference samples were distributed to all interested participants who then analysed the samples in their respective laboratories. Later on, the results were collected and compared (see sub-task 1.5).

Sub-task 4.3 Joint data analyses and planning of future projects. Joint data analyses and planning of future projects and collaboration were the objectives of several travel activities (see Annex 8). It is worth pointing out that a series of meetings involving participants from NINA, Norway, the Zoological Society of London, UK and the Agricultural University of Wageningen, the Netherlands, eventually resulted in a project application for the Fifth Framework of the European Commission.

III. Results and deliverables.

The planned main deliverables of the CA were as follows:

1) Survey of brown trout population genetics research activities 2) Recommendations for future studies 3) Recommendations for genetic marker nomenclature and harmonisation of the use of techniques 4) Recommendations for management/conservation of genetic resources of the species 5) Evaluation of the potential of the species for aquaculture with emphasis on the genetic resources available 6) Brown trout population genetics bibliography 7) World Wide Web site for the CA. 8) Raw data and data bases on genetic markers, available on the WWW site 9) Two workshops (in 1998 and 1999) 10) Mid-term evaluation 11) Progress report 12) Final report

In addition to these results and deliverables two more major meeting also took place, i.e. the

13) First (1998) and Second (1999) Iberian Trout Meetings.

Finally, some other results and initiatives of the CA are described under the heading

14) Other results.

The results and deliverables will be discussed in detail in the following. However, the recommendations for genetic marker nomenclature and harmonisation of the use of techniques, the two workshops of the CA and the two "Iberian Trout Meetings" will only be treated briefly. Instead, we refer to Annex 2, 3, 5 and 6.

12 1) Survey of brown trout population genetics research activities.

A survey of brown trout population genetics research by Troutconcert participants was undertaken during the first three months of the CA. A questionnaire (see Annex 7) was distributed to all participants and later recovered by the coordinator. The purpose of the survey was primarily to give an overview of the specific projects carried out by the different groups and to make this information publicly available on the Troutconcert home page. This would help both Troutconcert participants and other groups outside the CA to obtain information on ongoing projects and research interests of the participants. Knowledge on ongoing projects would both help to avoid significant overlap of projects and would also be a significant help in identifying groups with expertise in specific methods (for instance molecular techniques) and for finding future collaboration partners. Consequently, the questionnaire was designed primarily to be as informative on individual projects as possible.

50%

40%

30%

20%

10%

0% Pop. Stock. Phyl. Cons. Demo. Rep. Hyb. Quant. Map.

Fig. 1. The occurrence of different research topics in projects on brown trout population genetics presently undertaken by Troutconcert participants. Pop. = Genetic differentiation and population structure. Stock. = Stocking impact and introgression. Phyl. = Phylogeography. Cons. = Conservation and management. Demo. = Demographical genetics and temporal variation. Rep. = Reproduction biology and parentage assignment. Hyb. = Natural hybridisation. Quant. = Quantitative genetics. Map. = Gene mapping and development of markers. Frequencies add up to more than 100% as each project may cover more than one research topic.

In addition to research activities the participants were also asked to list publications on brown trout genetics. This request did not only concern refereed scientific papers but also technical reports and M.Sc. and Ph.D. theses. Theses and technical reports are rarely distributed above the national level, even though they may contain information of general relevance.

The resulting project summaries of the survey take up too much space (≈ 100 pages) to be incorporated in this progress report, but they can be viewed on the Troutconcert web page: http://www.qub.ac.uk/bb/prodohl/TroutConcert/participants.htm#ParticipantsTopPage. (Click on the "hot" names of the participants to go to the description of their research activities).

13 60%

50%

40%

30%

20%

10%

0% Alloz. Micros. mt.R scn. Minis. mt.s. Morph. Model. Chromo. rDNA

Fig. 2. Methodologies used in projects on brown trout population genetics presently undertaken by Troutconcert participants. Alloz. = Allozyme electrophoresis. Micros. = Microsatellites. mt.R. = Mitochondrial DNA restriction fragment length polymorphism (RFLP). scn. = Single copy nuclear DNA. Minis. = Minisatellite single locus probes. mt.s. = Mitochondrial DNA sequencing. Morph. = Analysis of morphology. Model. = Theoretical modelling and computer simulations. Chromo. = Chromosome analysis. rDNA = Ribosomal DNA. Frequencies add up to more than 100%, as more than one technique was applied in some studies.

It was not the intention of the survey to obtain information for estimating detailed statistics on sources of funding, number of man-months devoted to brown trout population genetics research etc. However, some general information can be extracted from the survey. First of all, it is notable that nearly all projects are funded by national sources. Only three projects have been funded by the EU, and none of these focus specifically on brown trout.

Second, it was obvious and certainly not surprising that genetic differentiation and population structure, stocking impact and introgression, phylogeography and conservation and management were the topics targeted in most projects (Fig. 1). However, it was surprising that very few studies focused on quantitative variation and gene mapping. This may reflect the lack of interest so far in evaluating the suitability of brown trout for selective breeding and aquaculture production.

Third, even though a number of molecular techniques are now available for the study of brown trout populations, allozyme electrophoresis is still a widely used (and very useful) technique (applied in more than 50% of all studies; Fig. 2). Restriction enzyme analysis of mitochondrial DNA (in particular the so-called PCR-RFLP approach) is applied routinely in several laboratories of the CA. In contrast, even though analysis of microsatellite DNA is used in several studies, the number of laboratories using these markers is relatively small (but probably increasing). Only a few studies involve theoretical modelling and computer simulations, and there is apparently a partly unexploited potential for combining advanced theoretical modelling with molecular approaches to population studies.

14 In total, the survey stresses the general impression that there is considerable research activity into brown trout population genetics and that this research is of high quality. However, the research has so far almost exclusively been funded from national sources and there has been limited collaboration among groups at the international level.

2) Recommendations for future studies

The issue of recommendations for future studies was discussed both by electronic communication and at the 1998 and 1999 workshops. It would be of benefit to both the scientific field in general, the researchers themselves and the funding agencies to focus efforts on themes that have so far only been poorly resolved or themes that are clearly important and subject to considerable, but poorly coordinated research activities. The discussions resulted in the following suggested topics:

A. Genetic interactions between wild and domesticated trout populations B. Suitability of brown trout for aquaculture production C. Local adaptations in brown trout populations D. Extended review of conservation genetic status of brown trout E. Identification of evolutionary significant units for effective conservation F. Temporal genetic variability G. Effective population size

A. Genetic interactions between wild and domesticated trout populations. Several studies have been aimed at assessing the genetic impact of escaped farmed Atlantic salmon on wild salmon populations. There can be no doubt that many Atlantic salmon populations are threatened by the repeated escapes of farmed fish and that it is very important to study the problems. It should be stressed, however, that these kinds of problems are by no means restricted to Atlantic salmon. Many indigenous brown trout populations are endangered due to stocking with domesticated trout (see, for instance, section c of the workshop proceedings (Annex 2) on stocking impact assessment). Also, a variety of European fish species, for instance whitefish (Coregonus spp.), Arctic charr (Salvelinus alpinus), grayling (Thymallus thymallus), Northern pike (Esox lucius), cod (gadus morhua) and turbot (Scophthalmus maximus) are likely to be genetically affected by stocked or escaped domesticated conspecifics. It would be very useful to have a model species, in which the general problems of gene flow from domesticated to wild fish population could be studied. Atlantic salmon is not the optimal model organism due to lower genetic variability in comparison to most other salmonid fishes (which sometimes renders application of genetic markers problematic), and because little is still known of the oceanic phase of its life-cycle. In contrast, brown trout exhibits considerably more genetic variability and is much more variable in terms of patterns of life-history. Also, as brown trout does not have an obligatory marine phase (as Atlantic salmon), it would be possible to conduct experiments in closed systems, such as a lake with different tributaries (see Skaala et al. (1996), Can. J. Fish. Aquat. Sci. 53: 2027-2035, for an example of the use of brown trout as a model species for studying genetic interactions between wild and domesticated fish in a closed system). Finally, as described in section c of the workshop proceedings the origin of the stocked fish relative to the stocked populations differs considerably in different stocking situations. In Southern Europe all domesticated trout belong to the Atlantic phylogeographical race, but the stocked populations belong to the Mediterranean or other phylogeographical races. This gives the opportunity to study patterns of introgression when considerable genetic differences exist between donor and recipient populations due to several

15 100,000 years of reproductive isolation. In Northern Europe both stocking material and recipient populations belong to the same phylogeographical group. Consequently, this gives the opportunity to study patterns of introgression where genetic differences between donor and recipient populations are relatively small and for the most part due to the altered selection regimes that exist in hatcheries relative to the wild.

In conclusion, we recommend future studies using brown trout as a model species for studying genetic interactions between wild and domesticated fish. Such a project should include both situations where stocking material and stocked populations are genetically highly divergent (as in Southern Europe) and genetically more similar (Northern Europe). The experiments should be designed in a way that makes it feasible to compare the impact and genetic introgression in the different stocking situations.

B. Suitability of brown trout for aquaculture production. Knowledge on the potential of brown trout for aquaculture production must still be considered incomplete (see section d of the workshop proceedings [Annex 2]). Very few studies have been undertaken to estimate heritabilities of traits that are important for production in aquaculture and only limited work has been done to attempt selective breeding. As a consequence, most comparisons of the performance of brown trout in aquaculture relative to other salmonid fishes (Atlantic salmon, brown trout) have actually been a comparison between populations that have not been subject to selective breeding (brown trout) and strains of other species that have been subject to selective breeding programmes. Thus, the true potential of brown trout for production in aquaculture may have been underestimated.

We recommend studies that focus on the suitability of brown trout for selective breeding and aquaculture production. This project should incorporate quantitative genetics, marker assisted selection and estimates of molecular genetic variation (including variation in important coding genes, such as Major Histocompatibility Complex genes [MHC]) and biological traits and properties of wild populations potentially suitable for aquaculture productions.

C. Local adaptations in brown trout populations. As described in the section of the workshop proceedings on local adapatations (Annex 2, section f), this is an issue that is extremely important in all aspects of conservation and general understanding of the significance of the genetic differences among populations. However, there is still a lack of a study demonstrating local adaptations with reciprocal transplantations of pairs of brown trout populations. Also, it needs to be studied what happens in the F1 and F2 generations, as lowered fitness in the ”foreign” environment may be difficult to detect through only one generation. This should be done both with populations of the same phylogeographic group and with populations belonging to different groups.

Consequently, the CA recommends further studies on the significance of local adaptations in brown trout populations. Also, it should be noted that a good experiment on local adaptations is complementary and crucial to interpretations of results obtained in the recommended studies A and B.

Both in the case of studies A and C we want to stress the importance of conducting long-term studies. The dynamics of introgression (A) and survival of transplanted populations (C) should be

16 followed through several trout generations. Otherwise, in the case of A, the results of the study will only yield information on the dynamics of introgression in the F1 generation, whereas the really important effects (in particular outbreeding depression) would be anticipated to take place in the F2 and following generations. In the case of C it is also necessary to follow the transplanted populations through several years/trout generations. First, to make sure that the populations are exposed to a wide range of environmental conditions (for instance, both harsh and mild winters). Second, to be able to monitor possible genetic changes that take place in response to the altered selective regimes that the tranpslanted populations experience.

D. Extended review of conservation genetic status of brown trout Within the framework of the CA an overview of the conservation genetic management situation of the brown trout in Europe has been provided (see the report "Conservation genetic management of brown trout (Salmo trutta) in Europe", Annex 10). In this overview it is concluded that the brown trout is subjected to many types of human activities known to cause losses of biological diversity at the gene level. Such activities include harvest, stocking, destruction of migratory routes (i.e., through dam constructions and water regulations), and various forms of pollutions. Documentation of practical examples of these activities resulting in loss of brown trout populations or loss of diversity within populations exist. However, no serious attempts have been made, neither at the national or the international level, to delineate what has actually been lost, and what is at risk of being lost in terms of local brown trout populations and the genetic resources they represent. We suggest that more systematic reviews, covering all regions of Europe, are undertaken that aim at clarifying the threats to the genetic resources of brown trout in Europe.

E. Identification of evolutionary significant units for effective conservation Deciding what to conserve is the basic and crucial issue in any conservation activity. Following the recognition of the biological diversity within species, the species focus in conservation has become outdated. Conserving species is not enough; their evolutionary potential, represented by the genetic variability within and between populations must also be conserved. Several workers have discussed the topic of what should be considered the appropriate units for conservation. A consensus of these studies is that conservation efforts should focus on evolutionary lineages within species (Moritz 1999. Hereditas 130:217-228). Such evolutionary groupings within species are called "Evolutionary Significant Units" or ESUs (Waples 1995. American Fisheries Society Symposium 17:8-27). The concept has been applied primarily to the Pacific salmon, but it is applicable to any species.

In most practical situations decisions on conservation efforts will not be based on biological aspects only. Economic, social, and legal aspects are important factors in the process. The concept of "Operational Conservation Units" (OCUs; Dodson et al. 1998. Canadian Journal of Fisheries and Aquatic Sciences 55:312-323) have been introduced to describe the unit of conservation that results from the interaction between biological and socio-economic factors.

Currently, there is no straightforward answer to the question on how brown trout ESUs or OCUs within Europe should be defined. At the same time identification of ESUs within the natural range of the species is necessary in order to develop a European conservation strategy for the brown trout. Methods that have been applied to Pacific salmon to identify ESUs include collection of genetic and ecological data, and some modifications are needed before they are applicable to brown trout. Further, additional empirical studies are needed to generate the necessary genetic and ecological data.

17 F. Temporal genetic variability A large amount of data has been generated on the frequency and geographical distribution of different alleles at genetic marker loci in natural brown trout populations in Europe. However, information regarding the temporal dynamics of those marker alleles is still relatively sparse despite the fact that such information is essential for conservation and sustainable management.

Typically, studies of the genetic structure of populations include sampling at one particular occasion only, i.e. no temporal replication has been conducted. This situation is by no means unique to brown trout, but reflects a general phenomenon. Several workers have compared gene frequencies from natural populations of several species, including brown trout, sampled at 2-3 occasions. For most species, however, few studies exist that systematically follow genetic changes within a population through extended periods of time. This fairly limited knowledge of the extent of temporal variation of genetic markers influences the interpretation of observed spatial patterns; it is largely unclear if they are stable over time.

In a couple of recent studies the genetic composition of several year classes have been reported for a few natural brown trout populations (Jorde & Ryman 1996. Genetics 143:1369-1381; Laikre et al. 1998. Evolution 52:910-915). In those studies material was collected over more than a ten year period, and the main results were that the existence of genetic change over time was established and that estimates of the effective population size indicated that the effective size of populations in lakes of similar size may vary considerably and may be relatively small.

Other recent studies of long-term temporal variation in Atlantic salmon have been based on analyzing microsatellite markers from old scale samples and comparing the results to contemporary data from the same populations. Nielsen et al. (1999. Evolution 53:261-268) and Tessier and Bernatchez (1999. Molecular Ecology 8:169-179) studied the genetic composition of Atlantic salmon populations over several decades and, in contrast to the results from brown trout populations mentioned above, they found that the genetic structure of populations was remarkably stable over time.

It is obvious that extended studies of temporal genetic variability in brown trout populations (as well as populations of other species) are needed to increase the understanding of the genetic dynamics of natural populations. A better understanding for these processes is necessary for adequate management and conservation measures. For instance, it is extremely difficult to monitor and evaluate the effects of various activities on the biodiversity at the gene level if the magnitude of the "normal" variation in genetic composition over time is unknown.

G. Effective population size This suggestion for future studies is closely linked with suggestion F on temporal genetic variation. The genetically effective population size (Ne) is a key parameter in evolutionary, ecological, and conservation genetics. For instance, the effects on a population from the primary evolutionary forces migration, mutation, selection, and genetic drift are dependent on the effective size. Similarly, the concept of effective population size represents a central topic in the rapidly growing field of conservation biology as it determines the rate of inbreeding and loss of genetic variability.

In spite of the fundamental importance of the concept of effective size, our empirical knowledge of this quantity is typically very poor. The effective size is unknown for almost all natural populations,

18 and the likely effects on this quantity resulting from various ways of manipulating a population are, at best, vague. The two major reasons for our limited information are i) the difficulty in obtaining the demographic information necessary for application of much of the existing theory for estimation of Ne, and ii) a pronounced lack of analytical models on allele frequency dynamics pertinent to populations that deviate from the highly simplistic life histories typically dealt with in population genetics theory.

Recently, the effective population sizes of some natural brown trout populations have been estimated using new developments of the so called temporal method that base the assessment of Ne on temporal shifts of allele frequencies at selectively neutral loci rather than on demographic parameters that are very difficult to estimate (Jorde & Ryman 1996. Genetics 143:1369-1381; Laikre et al. 1998. Evolution 52:910-915). Because of the small number of populations analyzed, however, it is impossible to draw any general conclusions regarding the effective sizes of natural brown trout populations. It is imperative that the knowledge of effective populations sizes are developed further both by theoretical studies that aim at refining the analytical models for estimating effective size in populations with varied demographic characteristics, and by empirical studies that generate direct estimates from natural populations. In that context it should be mentioned that hypervariable DNA markers such as mini- and microsatellites can be used for directly monitoring reproductive success of individuals and thereby provide information that can be used for direct estimates of Ne. As shown in previous studies, the brown trout is an excellent model organism for these kinds of studies.

3) Recommendations for genetic marker nomenclature and harmonisation of the use of techniques.

A. Genetic marker nomenclature and harmonisation of the use of markers For a detailed description of the recommendations for genetic marker nomenclature and harmonisation of the use of techniques we refer to the 1998 workshop proceedings (Annex 2, section i) and the Troutconcert homepage http://www.qub.ac.uk/bb/prodohl/TroutConcert/TroutConcert.htm.

In order to be able to compare results obtained in different laboratories it is of course necessary to use the same terminology. Specifically in terms of genetic markers it is important to have a common nomenclature in order to be able to compare results from different studies and laboratories and to avoid confusion caused by, for instance, two laboratories independently give the same name to two different microsatellite loci. Even more important, it turns out that several different microsatellite loci and different segments of mitochondrial DNA are presently being used as genetic markers by groups working with brown trout. The use of different markers makes it difficult to compare results obtained by different laboratories, and meta-analyses of data are virtually impossible. Consequently, it is necessary to give recommendations for use of a specified battery of markers by all groups. Specifically in the case of microsatellites there are technical problems with some loci; some exhibit so-called null-alleles and others are simply just difficult to PCR amplify. By giving a list of microsatellite loci that have been found to work reliably in different laboratories, other laboratories that are in the start-up phase of applying microsatellites do not need to "re-invent the wheel", i.e. test a large number of loci before they decide which to use.

19 By putting the recommendations on the web site we have ensured that they are not only accessible to Troutconcert participants, but also to other groups working on the genetics of brown trout or other salmonid fish species.

B. Calibration of mitochondrial DNA markers (RFLP analysis of the ND-1 segment) A calibration of mitochondrial DNA markers, more specifically the NADH-dehydrogenase subunit 1 coding region (ND-1) of the mitochondrial genome, was conducted for brown trout in the Baltic/Scandinavian region. The calibration involved Dr. Linda Laikre (University of Stockholm, Sweden), who did the actual calibration work in collaboration with non-TROUTCONCERT participants Professor Torbjörn Järvi and Leif Johansson (the Swedish Institute for Freshwater Research of the National Board of Fisheries), Ms. Ewa Wlodarczyk (Marine Research Institute, Gdynia, Poland) and Dr. Michael M. Hansen (Danish Institute for Fisheries Research, Denmark). Briefly, samples representing all different mtDNA haplotypes observed in the laboratories of these participants were collected. Next, they were PCR amplified (using primers equal to or homologous to those designed by Cronin et al. 1993. Canadian Journal of Fisheries and Aquatic Sciences, 50, 708-715). Finally, the samples were digested with five restriction enzymes that are known to detect variability in this mtDNA segment, and electrophoresed on agarose gels, which were visualised under UV light by ethidium-bromide staining. The results concerning approximate sizes of fragments are given below in Table 1 and composite haplotypes are listed in Table 2.

Table 1. Approximate sizes of lengths of restriction fragments resulting from the digestion of the ND-1 coding region using five restriction enzymes.

Restriction Ava II Hinf I Alu I Hae III Hpa II enzyme MorphABABABCABCDAB Fragment 920 1000 660 660 660 660 660 660 660 720 size (bp) 750 560 510 500 500 500 520 520 410 410 540 540 480 450 460 460 290 290 500 500 390 390 390 390 460 210 210 440 290 280 280 280 250 170 230 230 230 210 210 210 180 180 150 150 200 200 200 200 200 200 130 130 200 170 120 110 110 110 110

20 Table 2. Composite mtDNA haplotypes resulting from the digestion of the ND-1 coding region using five restriction enzymes. Restriction morphs are denoted by capital letters as defined in Table 1.

Comment Ava II Hinf I Alu I Hae III Hpa II AAAAA Only in Denmark A A A C A BAACB AABCA AAABA ABABB Only in Gotland AABDA (Sweden) Only in Denmark A A C C A

All together, the results show that most of the haplotypes observed in Sweden, Poland and Denmark are identical, at least as defined by the five restriction enzymes used for analysing the ND-1 segment. It is also reassuring that haplotypes assumed to be identical, assessed from comparisons with data from the literature, in fact turned out to be identical when side-by-side comparisons were conducted.

C. Calibration of microsatellite markers. At the 1998 workshop of the CA it was planned to conduct a calibration of microsatellite markers. This was done by extracting large amounts of DNA from five trout representing three different phylogeographical races: Individual 1: “Marmoratus” race. Individual 2: Mediterranean race. Individual 3: Mediterranean race Individual 4: “Marmoratus” race. Individual 5: Atlantic race.

The DNA was distributed to CA participants that routinely analyse brown trout using microsatellite analysis. These participants analysed the five test samples using their “standard” battery of microsatellite loci for brown trout. The results were collected by the coordinator and discussed at the 1999 workshop of the CA.

The results of the calibration of eight microsatellite loci are listed in Table 3. An additional eight loci had also been analysed, but unfortunately only by one laboratory which of course makes it impossible to compare results among laboratories. Consequently, the results for these loci were not included in the table. It should also be mentioned that the University of Helsinki added a 4bp “tail” to some of the primers in order to circumvent problems caused by the “extra-A” addition by the Taq polymerase.

It is obvious from Table 3 that data cannot be immediately compared from one laboratory to another. This is due to the fact that estimates of allele sizes (in bp) differ among equipment and laboratories. However, it is also obvious that allele sizes are just shifted by 1-9 base pairs, i.e. the size differences (in bp) between alleles are consistent among laboratories and equipment. This means that it is in fact possible to compare results among laboratories once a calibration has been

21 conducted by running the same test samples and record by how many base pairs allele size estimates have been shifted.

There are several possible explanations for the different allele size estimates obtained by the different laboratories. Of course, the addition of a 4bp “tail” to the primers, as mentioned previously, will affect this. Also, inaccuracies in the different equipment used as well as differences in the use of internal and external size marker systems will have an effect. Finally, it may have an effect whether one or the other primer is labelled and, consequently, which of the two single strands of a DNA segment is visualised.

Table 3. Allele sizes (in bp) of microsatellite loci observed in five test samples of brown trout analysed by five different laboratories. Asterisks denote discrepancies in the results.

Lab University of Sea Fisheries NINA, Norway Zoological Institute, Danish Institute for Helsinki, Finland Institue, Poland University of Munich, Fisheries Research, Germany Denamrk Equipment ABI 377 PAGE, silver ABI 310 ABI 377 ALFexpress staining Locus: Str60 1: 97/97 1: 95/95 2: 97/97 2: 95/95 3: 97/97 3: 95/95 4: 97/97 4: 95/95 5: 101/101 5: 99/99 Locus: Str73 1: 160/162* 1: 162/162* 1: 159/161* 2: 144/144 2: 144/144 2: 143/143 3: 144/144 3: 144/144 3: 143/143 4: 160/160 4: 160/160 4: 159/159 5: 148/148 5: 148/148 5: 147/147 Locus: Str15 1: 214/214 1: 216/216 1: 211/211 1: 216/216 2: 218/220 2: 220/222 2: 215/217 2: 220/222 3: 218/220 3: 220/222 3: 215/217 3: 220/222 4: 214/214 4: 216/216 4: 211/211 4: 216/216 5: 220/220 5: 222/222 5: 217/217 5: 222/222 Locus: SsoSL 417 1: 174/200* 1: 169/195* 1: 171/195* 2: 280/280 2: ?? 2: 277/277 3: 280/280 3: ?? 3: 277/277 4: 174/214 4: 169/209 4: 171/211 5: 180/192 5: 177/189 5: 177/189 Locus: SsoSL 438 1: 103/103 1: 111/111 2: 95/95 2: 103/103 3: 95/95 3: 103/103 4: 111/111* 4: 121/121* 5: 103/103 5: 111/111 Locus: Ssa197 1: 135/187 * 1: 136/188* 1: 125/179* 1: 134/186* (tetra) 2: 135/135 2: 136/136 2: 125/125 2: 134/134 3: 135/135 3: 136/136 3: 125/125 3: 134/134 4: 183/211 4: 184/212 4: 175/203 4: 182/210 5: 139/147 5: 140/148 5: 129/137 5: 138/146 Locus: Ssa 171 1: 198/200* 1: 193/195* 1: 196/196* (tetra/di) 2: 236/236 2: 231/231 2: 236/236 3: 236/242 3: 231/237 3: 236/242 4: 198/198 4: 193/193 4: 196/196 5: 240/244 5: 235/239 5: 238/242 Locus: Ssa85 1: 111/117 1: 113/119 1: 106/112 2: 117/121 2: 119/123 2: 112/116 3: 117/117* 3: 117/119* 3: 112/112* 4: 111/111 4: 113/113 4: 106/106 5: 119/119 5: 121/121 5: 114/114

Apart from the differences in allele size estimates there were also some discrepancies in the results concerning scoring of genotypes. For instance, for locus SsoSL 417, individual 1, all laboratories recorded this individual as a heterozygote. However, one laboratory estimated the size difference between alleles to be 24 bp, whereas two other laboratories estimated the diffence to be 26 bp.

22 Another example is locus Str73, individual 1. Two laboratories scored this individual as a heterozygote whereas it was scored as a homozygote by one laboratory. This may be an example of the difficulties in identifying heterozygotes at di-nucleotide repeat microsatellite loci when alleles are separated by only two bp.

The examples of erroneous scoring were discussed at the 1999 workshop, and it turned out that at least some of the problems were due to the fact, that the test individuals were derived from three different phylogeographical races, whereas the laboratories involved in the calibration primarily worked with trout from the Atlantic race. The Mediterranean and marmoratus races sometimes had alleles far outside the size range observed for Atlantic trout. This was particularly the case for the locus SsoSL 417, where some of the Mediterranean and marmoratus alleles were very long and exhibited pronounced stuttering, which made scoring quite difficult. Consequently, this is an example of a locus that is very useful for one phylogeographical race (Atlantic), but problematic for other races (Mediterranean and marmoratus).

D. Polymorphic loci in brown trout and agreed nomenclature of alleles.

During and after the 1999 workshop a calibration and harmonisation of allozyme allele nomenclature was conducted by Prof. Andy Ferguson, Queen’s University of Belfast. This turned out to be necessary in order to make data sets comparable that were provided by different laboratories for the collection of allozyme “raw” data. Table 4 is based on published papers, discussion at the St Feliu meeting, and incorporates written comments received following circulation of the draft. Comments were received from: Jose Luis Garcia-Marin; René Guyomard; Michael Hansen; Kjetil Hindar; Marja-Liisa Koljonen; Paulino Martinez; Alex Osinov; Tiit Paaver; Stefan Palm; Inci Togan; Costas Triantaphyllidis. Locus nomenclature follows the guidelines of Shaklee et al., 1990. Allele nomenclature follows the priority system (similar to that used for species names) i.e. first valid publication with the exception that the ancestral allele in the case of LDH-C1* has been designated as ‘100’. (Considerable confusion exists for this LDH-C1* locus where some workers use ‘90’ and ‘100’ for the two common alleles in NW Europe whereas others use ‘100’ and ‘105’.) Where PhD theses were readily available, these were taken as ‘valid publication’ contrary to the practice in systematics. A conservative system was used i.e. alleles of similar mobility were assumed to be the same unless they have been shown to be different by side-by-side comparisons or by comparison to an outgroup. Information on occurrence for each country should aid workers in further determining homologies for their own alleles.

23 Table 4. Overview of allozyme allele nomenclature in brown trout, including locus and allele synonynoms and occurence of alleles.

Locus Locus synonyms Allele AuthorityA Allele synonyms OccurrenceB NotesC / specification ACP* 100 18 115 18 E(1) 90 24 E(1)

ADH* ODH 100 F(4); CH(4) CH ≡ S.salar 0 -30 / 60 / 85/40 E(2) sAAT-1,2* muscle 100 1 F(3), I(3); CH(4); CH ≡ S.salar EL(4); TR(4) AAT-3,4 45 4 40 / 50 IRL(3); E(1); CH(2) 75 10 70 EL; SU(2); EL(3) 105 25 103 E(1) 119 1 130 / 120 / 140 / 150 IRL(3); DK(3); S(2); F(3), I(3); SU(3)U; E(1); CH(3); FIN(3) 114 22 125 / 130?ref25 I(3); E(1); CH(3) marmoratus 165 22 180 I(3); CH(2) marmoratus 130 25 125? E(1) sAAT-4* liver 100 4 F(4), I (4); CH(4); EL(4); TR(3) AAT-6 25 17 E; S(1); 56 60 / 65 / 50 E(1); CH(1); EL(3) 74 4 87 / 50 / 65 / 80 IRL(3); S(3), 65 is very common F(3); I(3); SU(3); in F samples and is E(3); CH(3); probably 74 - 65 EL(3); TR(3); also in Atlantic EE(3); FIN(3); salmon (ref 22); CH NO(3) ≡ S.salar 85 26 80 / 70 E(2); CH(3) 105 107?ref25 E(1) 115

CK-A1* CPK-1; CK-1, 100 1 F(4); I(4); EL(4); CK-2 TR(4) 75 7 IRL(2); 115 1 125 / 110 IRL(3); GB(3); IRL ≡ S; CH ≡ DK(1); F(4); I(3); S.salar SU(4); E(4); CH(3); S(3); EE(3); NO(3) 150 26 E(1)

24 Locus Locus synonyms Allele AuthorityA Allele synonyms OccurrenceB NotesC / specification CK-A2* CK-2 100 14 EL(4); F(4); CH ≡ S.salar CH(4); TR(3) 50 14 EL(4); TR(2) 120 CH(1)

CK-C1* CK-3 100 F(4); I(4); CH(4 CH ≡ S.salar 85 23 87 CH(1) 90 13 F; CH(3), I(4) 92 23 CH(3),1 95 22 I (4); CH(3) marmoratus 110 ?IRL(1)

CK-C2* 100 CH(4) CH ≡ S.salar 95 CH(1)

DIA-1* DIA 100 3 90 3 95 IRL(2); DK(2); S(2); NO(1) 120 7 IRL(1);

EST-1* Liver 100 F(4); I(4); CH(4) 102 for S. salar 98 22 I(4); CH(3) marmoratus

EST-2* blood 100 2 105 F(4); I(4) 92 2 100 / 90 S(1); SU(3) 110 10 SU(4) 103

EST-5* muscle/liver 100 F(4); I(4) 90 SU(4) 105 9F(4)Corsica

EST-6* 100 90 SU(3)

FBP-1* 100 6 F(4); I(4); CH(3) 150 6 135 F(4);CH(3) CH ≡ S.salar 80 F(2)

FH-1,2* 100 9 F(4); I(4) 10 75 9 70 / 85 / 92 F(2); CH(1) =At. salmon allele 80 84?ref25 I(2); E(1) marmoratus 115 9 104 F(2); CH(1) 130 9 F(3); PL(3); CH(3) 140 9 135 F(3); CH(3) 124 115 or 130? E(1) bGALA-2* GAM-2 100 13 S, E(3) 95 13 S(3), E(1); NO(3) 90 15 S(2), E(1)

BGLUA* BGA 100 15 S, E(3) 150 15 S(3), E(3); NO(3) Atlantic rivers only in E

25 Locus Locus synonyms Allele AuthorityA Allele synonyms OccurrenceB NotesC / specification GLYDH* G2DH 100 17 S, E 85 17 NO(1) One NO locality

GPI-B1* GPI-1 100 F(4), I(4); CH(4); EL(4); TR(4) PGI-1 QO E(3) GPI-A1* 70 SU(3) 25 21

GPI-B2* GPI-2 100 4 F(4); I(4); E(3); CH(4); EL(4); TR(3) PGI-2 QO 20 65 / n / 70 IRL(3); DK(3); SU(2); E(2); TR(2) GPI-A2* 50 15 E(3) 122 4* 130 4* 135 4 110 / 130 / 140 / 200 IRL(3); S(1), * F(4), I(2); E(3); CH(3); NO(2)

GPI-A1* GPI-3 100 4 F(4); I(4); E(4); (=130 for S.salar) CH(4); EL(4); TR(4) PGI-3 80 11 83 / 85 / 90 GB(1); DK(1); E(2); CH(1) GPI-B1* 110 4 103 / 115 / 106 / 105 / IRL(2); GB(3); 120 / 102 DK(1); S(2); SU(2); CH(3); EE(2); FIN(2); NO(1) 115 F(1) =At. salmon 130

G3PDH-2* G3pdh-1 100 1 -100 F(4); I(4); CH(4); CH ≡ S.salar EL(4) Agp-2; AGP 30 25 IRL(1); NO(1) 50 1 75 / 59 / 80 / -150 /- IRL(3); DK(3); IRL ≡ S; 128 / 65 S(3); F(3); I(3); SU(3); CH(3); EL(3); EE(3); FIN(3); NO(3) 120 19 E(1) sIDDH-1* SDH-1 100 0 F(3); I(3); CH(4) -50 15 0 / -100 / 70 S(2); F(3); I(3); Hatchery fish in E; CH(3); EE(3); Atlantic salmon NO(3) allele 200 15 E(3)(Atlantic); Same mobility as SDH-2*100 10 FIN(2) sIDDH-2* 100 F(4); I(4) 170 (F4); I(1) Corsica

26 Locus Locus synonyms Allele AuthorityA Allele synonyms OccurrenceB NotesC / specification mIDHP-1* IDH-1: IDHP-1 100 E(3); F(4); I(4); F fixed; CH ≡ CH(4) S.salar mt form, muscle 85 18 80 E(1); CH(2) or eye 120 CH(1) sIDHP-1* Idh-1; Idh-2; 100 4 F(4); I(4); EL(4) Idh-3; IDHP-2; 60 17 73 S(1) Eye or muscle 160 4 200 / 180 IRL(2); DK(3); S(2); F(3); I(3); E(3); CH(3); NO(3) 0 17 S(1); NO(1) 10 11 30 IRL(2); CH(1) 175 sIDHP-2* Idh-3; Idh-2; 100 4 80 / 70 F(4); I(4); CH(4); EL(4) Idh-4; IDHP-3 130 4 100 / 145 / 160 / 132 / IRL(3); I(3); maybe identical 120 E(2); CH(4); F(4) with a S.salar-allele Strongest in 65 17 70 / 47 / 66 NO(1); CH(4); liver EL(1) Present in eye 80 140 90 22 I(3)

LDH-A1* Ldh-1 100 1 F(4); I(4); CH(4); CH ≡ S.salar EL(4); TR(3) QO 8 n / 240 IRL(3); S(2); SU(2); DK(3); E(4); EL(2); TR(3); NO(2) 25 21 IRL(1)

LDH-A2* Ldh-2 100 18 F(4); I(4); CH(4); CH ≡ S.salar EL(4) 100QL 18 n E(4); EL(4); Mediterranean CH(3); TR(2)

LDH-B1* Ldh-3 100 12 F(4); I(4) CH ≡ S.salar 63 12 40 F(4) Corsica

LDH-B2* Ldh-4 100 F(4); I(4); CH(4); CH ≡ S.salar TR(4) 120 115 / 100 TR(4)

27 Locus Locus Allele AuthorityA Allele synonyms OccurrenceB NotesC synonyms / specification LDH-C1* Ldh-5 100 10 105 *; S(2); F(4); CH ≡ S.salar; = I(4); CH(4); allele in other EL(4); TR(4); salmonids EE(1) 70 74 / 75 / 90/80 S(1); F(3); CH(1) CH=F 90 10 100 *; IRL(3); DK(4); S(3); F(4); I(4); SU(4); CH(4); EL(1); TR(2); EE(4); FIN(3); NO(3) 104 16 105 / 115 E(2) 110 18 *E(3) 120 110(but not above) I(4); CH(4) marmoratus; CH=I

LGL* 100 18 90 18 E(3) southern rivers in E aMAN* 100 15 S, E 70 17 S(2), E; NO(1) 90 15 70 E(3) Mediterranean rivers sMDH-1* Mdh-1, sMDH- 100 3 F(4); I(4); CH(4); CH ≡ S.salar A1* EL(4); TR(4) QO 3n E(1) 50 27 CH(3) sMDH-2* sMDH-A2*; 100 3 F(4); I(4); EL(4); Mdh-2 TR(4) 80 E(1) 152 3 118 / 120 / 125 / 200 / DK(3); S(3); CH ≡ S.salar 175 / 150 F(3); I(3); SU(3); CH(3); EL(1); EE(2); FIN(3); NO(3) 130 E(2) QO 12 n F(4); CH(1) Corsica sMDH-3,4 * MDH-B1,2; 100 1 F(4); EL(4); CH poly but not TR(4) scored Mdh-3,4 50 9 F(2); SU(1) 60 9F(3)Corsica 70 9 F(2); PL(3); E(1) 75 6 80 *; IRL(3); S(3); No side-by-side F(3); E(4); EE(1); runs, probably FIN(2)?; erroneous synonyms 80 1 85 / 88 *; IRL(1); E(hatchery); EL(1); TR(3); NO(2) 125 2 110 / 120 *; IRL(2); S(2); marmoratus F(2); I(3); SU(1); E(3); TR(1); EE(1); NO(1) 134 4*; IRL(2) 83 E(3)

28 Locus Locus synonyms Allele AuthorityA Allele synonyms OccurrenceB NotesC / specification ME* MEL; 100 15 S, E NAD+ form 80 15 S(3); E(1) Hatchery origin in E 120 15 S(2); E(3); NO(3) 140 18 145 E(3) Mediterranean mMEP-1* NADP; MEP-1 100 F(4); I(4); CH(4); CH ≡ S.salar EL(4); TR(4) ME-1 80 70 SU(4); CH(3) Danube in CH 120 150 9F(4)Corsica 70 80? E(1) mMEP-2* Same locus as 100 F(4); CH(4); (At salmon 125?) pm EL(4); TR(4) in At salmon? 90 ? 80 F(3); E(1); TR(3); (At salmon 100?) NO(1) NADP; MEP-2 70 14 60 / 75 / 50 I(1); SU(1); CH(1); EL(3) 45 22 40? I(2); CH(3) CH only found in a mixed Swiss and Danish hatchery strain 95 I(4) carpio s-MEP-2 NADP; MEP-2 100 F(4), I (4); CH: an allele 90 CH(4); EL(4) found in S.salar MEP-3 in E? 97 F(3), I (4) ME-3 105 24 102 I (4); E(1); CH(3) marmoratus 90 18 80? E(3); CH(3)

MPI-2* Pmi-2; MPI, 100 5 90 *F(4); I(4); PMI CH(3) 105 5 100 / 106 / 107 IRL(3); GB(3); * DK(3); S(3); *F(3); I(3); SU(3); FIN(3); NO(3) 107 *F(3); E(3); *105 & 107 are CH(3) different (R Guyomard) 110 16 107 E(2); CH(2) CH not identical to allele 108 found in S.salar 85 86 SU(3) 95 E(1)

PEPB* PEPLGG 100 17 S, E 75 E(3) 120 17 115 S(1), E(1); NO(3) 80 E(1)

PEPLT* 100 15 S, E 70 15 S(3); E(3); NO(2) Atlantic rivers in E

PEPPAP* 100 S, E 95 E(1) 105 E(1) 120 E(3)

29 Locus Locus synonyms Allele AuthorityA Allele synonyms OccurrenceB NotesC / specification 6PGDH-2* 6PGDH-1* 100 8 F(4); I(4); CH(4); CH ≡ S.salar EL(4) 85 8 86 / 65 / 90 DK(1); F(3); I(1); CH(1)

PGM-1* Pgm-2 100 15 F(4); I(4); CH(4); EL(4); TR(4); EE(4) 80 15 90 / -115 E(3); FIN(1) 135 22 I(3) carpio 150 ? m-SOD-1* 100 F(4), I(4); CH(4) CH ≡ S.salar 75 9 50 F(3); CH(1) sSOD-1* SOD-2?, mSOD 100 2 F(4); I(4); CH(4); EL(4); TR(4); EE(4) 35 50 2 75 / 55 S(1); F(1); I(4); marmoratus CH(3); EL(3); TR(4) 80 12 75 F(2); I(1); CH(1) *; CH not identical to allele 83 found in S.salar 125 17 110 / 120 / 135 S(1); F(3); E(3) 70 80? / 75? E(1) 120 125? E(3)

TF-1* 100 6 F(4); I(4); CH(4); TR(3) 75 22 I(4); CH(3) marmoratus 78 22 I(4) carpio 80 6 78 F(3); I(3) Corsica 95 98 / 90 P; CH(2); TR(3) 102 13 98 F(4); I(4); CH(4); 102 migrates faster TR(3) or slower than 100 depending on the buffer used (found in At salmon) 105

A First publication with this allele designation – rules as for species names. References to publications are given below – see TroutConcert reference list for full references.

B Occurrence – country where allele is found and (maximum frequency) Country codes (as per EC designations mostly) – Denmark, DK; Germany, D; Greece, EL; Spain, E; France, F; Ireland, IRL (used here to refer to geographical island of Ireland,not political entity); Italy, I; Austria, A; Portugal, P; Finland, FIN; Sweden S; Great Britain (England, Scotland and Wales), GB; Switzerland, CH; Estonia, EE; Iceland, IS; Norway, NO; Poland, PL; Russia, RU; Turkey, TR; USSR, SU For frequencies: 1 = rare [<0.05]; 2 = low frequency [0.05 – 0.20]; 3 = common [>0.20]); 4 = fixed.

30 CNotes. ≡ means that alleles from these countries have been shown to be the same on side-by-side gel comparisons. * indicates that these alleles have been shown to be different on side-by-side comparisons or by comparison to an outgroup.

References 1 Allendorf et al., 1976 2 Allendorf et al., 1977 3 May et al., 1979 4 Taggart et al., 1981 5 Krieg & Guyomard, 1983a 6 Krieg & Guyomard, 1983b 7 Ferguson & Fleming, 1983 8 Allendorf et al., 1984 9 Krieg, 1984 10 Osinov, 1984 11 Taggart & Ferguson, 1984 12 Krieg & Guyomard, 1985 13 Barbat-Leterrier et al., 1989 14 Karakousis & Triantaphyllidis, 1988 15 Garcia-Marin et al., 1991 16 Martinez et al., 1993 17 Jorde, 1994 18 Garcia-Marin & Pla, 1996 19 Garcia-Marin et al., 1999 20 Henry & Ferguson, 1987 21 Hamilton, 1987 22 Giuffra et al., 1996 23 Largiader et al., 1996 24 Sanz et al. (submitted) 25 Machordom et al. 1999 26 Bouza et al, Mol Ecol (1999) 27 Largiadèr & Scholl 1995

4) Recommendations for management/conservation of genetic resources of the species

As described previously, a group was established consisting of CA participants with particular expertise and interests in conservation. Two meetings were arranged (at the University of Berne, Switzerland, and the Aristotle University of Thessaloniki, Greece, respectively) where the work of writing a report on this issue was planned and discussed and parts of the report were written. Also, this issue was discussed at both workshops of the CA. Dr. Linda Laikre (University of Stockholm, Sweden) edited the report and coordinated the writing tasks. In December 1999 a draft of the report was distributed to 30 persons from different EU countries representing fisheries managers, anglers’ associations and other persons with interests in conservation and management issues. After having received comments and input the report was printed and distributed to CA participants for further distribution within their countries. Also, the report was made available on the internet (http://www.dfu.min.dk/ffi/consreport/index.htm) and advertised on various mailing lists (Evoldir, Fish-Ecology, Aqua-genetics etc.). For the specific management/conservation recommendations we refer to the report itself, Conservation Genetic Management of Brown Trout (Salmo trutta) in Europe, which is enclosed as Annex 10.

31 5) Evaluation of the potential of the species for aquaculture with emphasis on the genetic resources available.

This issue was presented and discussed at the 1998 workshop during a session organised by Dr. Kjetil Hindar, NINA, Norway. We refer to the proceedings of the 1998 workshop (Annex 2) where a thorough treatment of the issue is given in section d): Potential of brown trout for aquaculture.

6) Brown trout bibliography

A brown trout bibliography has been compiled and made accessible on the web site http://www.qub.ac.uk/bb/prodohl/TroutConcert/TroutConcert.htm. It contains more than 1800 references and we trust it will be of use not only to the participants of the CA, but also to other researchers working on genetical and ecological issues related to brown trout and other salmonid fishes.

7) World Wide Web site for the CA. The troutconcert web page was established 15 June 1998 by Dr. Paulo Prodöhl, Queen’s University of Belfast, and has since then been maintained and updated. It has been the most important device for communicating information and results of the CA. It contains the following features:

• A description of the background and aims of the CA • A list of all participants with e-mail addresses • A survey of the research of the participants, including their publications on brown trout population genetics • A brown trout bibliography containing more than 1800 references • A "molecular marker page" with information and recommendations regarding the use of molecular markers for brown trout studies. • A population genetics and fisheries biology glossary • Links to other important web sites (including the CORDIS site; click on the FAIR logo) • Genetic "raw" data: Allozyme data collected by the participants of the CA. • A collection of photos of different trout morphs

We recommend to access the TROUTCONCERT web page on the address: http://www.qub.ac.uk/bb/prodohl/TroutConcert/TroutConcert.htm.

8) Raw data and data bases on genetic markers, available on the WWW site

This deliverable has links with deliverable 3): Recommendations for genetic marker nomenclature and harmonisation of the use of techniques. More specifically, this concerns the issue of data bases on genetic markers (mitochondrial DNA and microsatellite loci), which has already been described under deliverable 3).

Concerning the collection of genetic raw data there was little knowledge on the use of DNA markers in the participating laboratories prior to the beginning of the CA. The survey of research

32 activities and presentations at the 1998 workshop showed that relatively few groups had yet finished studies on brown trout using microsatellites. Furthermore, different loci were in use in the different laboratories and no calibration of allele sizes for specific loci had taken place among laboratories. Mitochondrial DNA markers had been used by several groups. However, the techniques used (sequencing, restriction enzyme analysis of the mitochondrial genome or restriction enzyme analysis of PCR amplified segments) and the segments of the molecule targeted (the whole mitochondrial genome or several smaller segments) were very diverse. It was therefore concluded that at present it was not feasible to collect “raw data” on these classes of markers. In the case of allozyme data, however, there were several data sets that could be collected. The principles for the collection of data and how to define a consistent allele nomenclature (basically, how to know if allele A in laboratory x was identical to or different from allele A observed by laboratory y) were discussed at the 1999 workshop and later on by e-mail. Next, a form was distributed to all CA participants, which should then be filled in with various general information about the samples studied (geographical location, phylogeographic groups etc.) followed by the actual data set. The preferred option was to submit data in as “raw” a format as possible, i.e. individual genotype data. However, some data sets were not available in this format. Therefore, data sets consisting of genotype frequencies and allele frequencies were also accepted. The data sets were collected by Dr. Paulo Prodöhl and co-workers at the Queen’s University of Belfast, who organised the data and made them available at the Troutconcert web site. The data sets include between 5 and 33 loci and encompass more than 11,000 individuals representing more than 200 populations.

9) Workshops of the CA (1998 and 1999)

Two workshops took place during the CA, i.e. one in 1998 and another in 1999. They were both attended by more than 50 persons, representing first of all CA participants but also invited external experts and other guests who paid themselves for their participation.

The 1998 workshop took place at the Freshwater Centre in Silkeborg, Denmark. All participating groups presented their research activities on brown trout and, additionally, a number of theme sessions took place. These theme sessions involved presentations on potentials and merits on different genetic markers, genetic marker nomenclature, conservation of brown trout, stocking impact assessment, potential of brown trout for aquaculture, brown trout phylogeography and local adaptations in brown trout populations. In addition there were some discussion sessions concerning the progress of the CA, the design and contents of the web page and suggestions for topics for the 1999 workshop. For a detailed description of the workshop we refer to the workshop program (Annex 1) and the workshop proceedings (Annex 2).

The 1999 workshop took place in St. Feliu de Guixols, Spain. Some presentations were given by non-CA participants on their research activities on brown trout, in order to supplement the presentations given at the 1998 workshop. However, most of the meeting was devoted to specific theme sessions on the genetic structure of sea trout, comparative data analysis using different molecular markers, conservation genetics of brown trout and behavioural studies of salmonid fishes. In addition, there were some discussion sessions on suggestions for future studies and the general progress of the CA. For a detailed description of the workshop we refer to the workshop program (Annex 4) and the workshop proceedings (Annex 5).

33 10) Mid-term evaluation.

This mid-term evaluation of the CA was conducted in December 1998 by the project management group (Andy Ferguson, Louis Bernatchez, Kjetil Hindar, Carlo Largiader, Carles Pla and Michael M. Hansen [coordinator]). Discussions took place via e-mail. We evaluated the progress of the CA by comparing the planned milestones and deliverables for 1998 to the results actually obtained. In addition to the scientific issues of the CA we also evaluated the efficiency of administration.

Milestones and deliverables.

The milestones and deliverables of the CA in 1998 were as follows:

Milestones:

January Start of CA. Writing and distribution of questionnaires for survey of research activities. March Recovery of questionnaires, preparation of overview for participants. June First workshop. Presentation and discussion of survey activities. Detailed planning of CA, including format of databases and WWW site and principles for harmonising nomenclature and use of genetic markers. June-October Establishment of databases. Data for input will be collected and updated through the remaining period of the CA. October WWW site established. November Brown trout population genetics bibliography completed. December- First progress report. Mid-term evaluation. Involves coordinator and management group.

Deliverables:

• Survey of brown trout population genetics research activities • Recommendations for future studies • Recommendations for genetic marker nomenclature and harmonisation of the use of techniques • Brown trout population genetics bibliography • World Wide Web site for the CA. • First workshop of the CA • Mid-term evaluation • Progress report

As it will appear from the previous sections all milestones and deliverables have been achieved.

The survey of research activities provides a good overview of the ongoing research of the groups participating in the CA and is displayed on the Troutconcert homepage.

The point of recommendations for future studies will be treated once more in the final report (it was also stated in the technical annex of the contract that "recommendations for future studies" would be included in both the progress and the final report). At the first workshop of the CA the research activities of the participants were presented. As this resulted in a large amount of new information being presented during a few days, it was difficult to get a final overview of these activities during the workshop. This rendered it difficult to identify "gaps" in the present research activities and to

34 conclude in which direction(s) the field is moving. Consequently, it was decided to discuss this issue again during the next workshop and give more detailed recommendations in the final report.

The issue of recommendations for genetic marker nomenclature and harmonisation of the use of techniques was discussed in much detail during the workshop. This resulted in specific recommendations for mtDNA and microsatellite markers, the two classes of markers where standardisation was most urgent. The recommendations have been made accessible on the Troutconcert homepage.

The brown trout bibliography is very extensive and is likely to become a really useful tool for the participants. It has been made accessible at the Troutconcert homepage.

Concerning establishment of databases this has so far mainly involved collecting information on genetic markers (microsatellite loci and mitochondrial DNA segments for PCR-RFLP analysis) rather than data sets. Prior to the start of the CA there was little knowledge on the use of molecular markers in the participating laboratories. The survey of research activities and presentations at the workshop showed that few groups had yet finished studies on brown trout using microsatellites, different loci were in use in the different laboratories and no calibration of allele sizes for specific loci had taken place among laboratories. Mitochondrial DNA markers had been used by several groups. However, the techniques used (sequencing, restriction enzyme analysis of the mitochondrial genome or restriction enzyme analysis of PCR amplified segments) and the segments of the molecule targeted (the whole mitochondrial genome or several smaller segments) were very diverse. Consequently, microsatellite and mtDNA "raw" data are not yet fully suitable for meta- analyses and comparisons. This will require both a harmonisation of the use of markers and calibration of markers − two important tasks of the CA. At this point allozyme data are the most suitable data to make available on the web site. Collection of allozyme raw data will take place during 1999.

The first version of the Troutconcert homepage was established 15 June 1998 and has since then been updated. The design and contents of the homepage was discussed during the workshop, and all participants were very satisfied. The later additions of the results of survey of research activities, recommendations for use of markers etc. are also regarded as very satisfactory by the project management group.

The first workshop of the CA took place 29 June - 5 July in Silkeborg, Denmark. Many of the participants had never met before, so this provided a good opportunity for establishing new contacts. The presentation of research activities by all participating groups was very helpful in providing an overview of what is going on within the field. Also, the specific theme sessions went well. There was a good balance between talks and discussions, and even though approximately 50 persons participated it was still possible to have detailed and very constructive discussions. It is certainly the intention to keep the same format and balance between presentations and discussions at the next workshop.

The activities of travels and exchange visits among laboratories have been less than expected, although travel activities have clearly increased during the second half of 1998 and are expected to increase even more in 1999. The reason is no doubt that people have been less prone to visit a laboratory where they don't know people beforehand, but would rather discuss and plan visits during the workshop. Seen in retrospect it would probably have been better if the first workshop

35 had taken place immediately following the start of the CA. However, having a workshop with such a short notice would have left very little time for planning and might thus have caused considerable practical difficulties.

Most parts of the administration have worked smoothly (although the coordinator would also claim that the workload concerning administrative issues has sometimes been rather massive). However, in some cases there have been problems concerning refunding of travel expenses. The conditions for refunding of travel expenses and the sums of money available for daily allowances have been clearly specified by the coordinator and the project management group in a set rules. These rules have been approved by the relevant EU authorities. The travel expenses have been refunded by paying the relevant sum to the institutes of the participants who were then supposed to pay the money to the individual Troutconcert participants. In some cases the institutes refused to pay the money to the participants, as the Troutconcert travel rules (in particular sums for daily allowance) were not in accordance with the national rules on this matter. All problems have now been solved and will hopefully not occur again. However, the coordinator wants to stress that a bit more flexibility on behalf of the administrative staff of the involved institues, including his own, would have saved him and some of the participants from much unnecessary work.

In conclusion, it is the opinion of the project management group that the CA progresses satisfactorily, and that the planned deliveries and milestones have been achieved. The minor problems that have occurred have all been solved, and the CA is developing in the direction that was originally intended.

11) Progress report.

A progress report covering the activities in 1998 was written and submitted in January 1999 and subsequently approved by the Commission.

12) Final report

This is the present document and will not be described further.

Points 13 and 14 concerns some results of the CA that were not actually included in the deliverables:

13) First (1998) and Second (1999) Iberian Trout Meetings

Two “satellite workshops” termed “Iberian Trout Meetings” took place in 1998 and 1999.

The First Iberian Trout Meeting took place at the University of Santiago de Compostela, Lugo, Spain, 21-23 September 1998. Even though there are several groups working on brown trout population genetics in this region of the EU, there has previously been little collaboration and coordination of the research. This meeting brought together the Troutconcert participants of the region as well as other groups that do not participate in the CA (the latter groups at their own expense). The meeting involved both presentations of the research activities of the participants and specific theme sessions. A program and summary of the meeting is given in Annex 3.

36 The Second Iberian Trout Meeting took place 2-4 December 1999 at the University of Porto, Portugal. The meeting was structured into different theme sessions on phylogeography, genetic population structure and conservation of trout in the Iberian region. A program and summary of the meeting is given in Annex 6.

14) Other results and deliverables

Ms. Ewa Wlodarczyk, Sea Fisheries Institute, compiled a glossary covering terms in population genetics, systematics and fisheries biology. The glossary has been made accessible on the web site of the CA.

A collection of photos of trout belonging to different populations and phylogeographical groups has been established and put on the TROUTCONCERT web site. Apart from serving as a "reference collection" for researchers working on brown trout, it is the intention that the photos could serve as an illustration of the tremendous amount of morphological variation that is present within this species.

An e-mail discussion list ([email protected]) was established immediately after the start of the CA. It has since then been decided by the participants that this discussion list should be made accessible to persons outside the CA, whereas messages only of relevance to the CA are distributed simply by including the e-mail addresses of all participants in the mails. The discussion list has been made accessible on the World Wide Web (http://www.mailbase.ac.uk/lists/trout-concerted-action/).

IV. Dissemination of research results.

Dissemination of research results within the CA has taken place during the workshops and the Iberian Trout Meetings (see Annex 2, 3, 5 and 6). Research results have been presented during talks and theme sessions. In addition, during the 1998 workshop a "paper bazaar" was established. All publications of the participating groups were exhibited in a room dedicated to this purpose, and the participants could order papers, which where then photocopied and distributed by staff at the institute hosting the workshop.

The main tool for disseminating results outside of the CA has been the Troutconcert web page. The survey of research activites, recommendations concerning use of molecular markers, genetic “raw data” etc. have been made available on this site, and the web page has been announced on different electronic mailing lists related to population genetics and evolution (EVOLDIR, MICROSAT), aquaculture (AQUA-GENETICS) and general fish biology (FISH-ECOLOGY).

The report on conservation of genetic resources in brown trout (Laikre et al.: Conservation Genetic Management of Brown Trout (Salmo trutta) in Europe) was subject to a round of reviews prior to publishing. Briefly, a draft of the report was distributed to 30 persons/end-users in different European countries, who are all in some way involved with management and conservation of fishes. App. half of these responded with comments and suggestions for alterations. After the printing of the report a number of copies have been distributed to the CA participants (and in some cases external experts representing EU countries that were not involved in the CA). Next, they distribute

37 the report further to national authorities, fisheries managers, anglers’ associations, NGOs and other persons and institutions with an interest in management and conservation of fishes. The report has also been made available in an on-line version, which can be accessed via the internet (http://www.dfu.min.dk/ffi/consreport/index.htm). From the same address the report can be downloaded as a pdf file. The on-line version of the report has been advertised on various mailing lists, including EVOLDIR, FISH-ECOLOGY and AQUA-GENETICS.

V. Future actions.

Even though the CA has now ended a lot of synergy and collaboration has been established that will undoubtedly continue and develop in the future. The future of the more specific features and initiatives developed during the CA was discussed at the 1999 workshop. Many participants expressed regrets that the CA would end after two years, as over time this could mean a loss of a significant part of the momentum in terms of collaboration and exchange of ideas that had been gained during the past two year. After some discussion it was decided that the best way to maintain and update the links between research groups would be to arrange a third TROUTCONCERT workshop three years after the end of the CA. It was also decided by then to ask the Commission for funding for this workshop.

Concerning the Troutconcert electronic discussion group and the web page it was decided to keep both of them. Dr. Paulo Prodöhl, Queen’s University of Belfast, who had constructed the web page and was the “list owner” of the electronic discussion group volunteered for maintaining and updating both facilities.

VI. The role of the CA in coordinating research efforts and activities

The CA has had a significant impact in coordinating research efforts and activities in brown trout population genetics:

• The CA represents the first occasion where nearly all research groups working on brown trout genetics have been assembled to exchange results and ideas. • Research activities of all participating groups have been presented and discussed during the workshops. In addition, a description of the research activities has been written and made accessible on the Troutconcert web site. This has both enabled groups to find new collaboration partners and to avoid unnecessary overlap in research topics. • The CA participants have discussed and given suggestions for future research activities. • In order to harmonise the use of genetic markers suggestions have been given for well- functioning microsatellite loci and PCR primers for mitochondrial DNA analyses. Calibration of genetic markers has also taken place, which will facilitate comparison of results obtained by different laboratories. • A large number of issues related to population genetics, aquaculture and conservation of brown trout have been presented and discussed at the workshops that involved all CA participants. Further, in some cases external experts were invited to give talks for updating or presenting new angles to issues of relevance to all participants. These issues include conservation, phylogeography and use of molecular markers in population genetics.

38 • Allozyme “raw” data have been collected and made available to all CA participants and other interested persons. • Visits have taken place among laboratories, all of which have aimed at coordinating research efforts and activities, such as technology transfer among laboratories and planning of project proposals. In at least two cases the contacts established among laboratories have led to longer postdoctoral visits not funded by the CA (involving the University of Lugo, Spain, the Estonian Agricultural University, Estonia, and the Danish Institute for Fisheries research). • A report has been written on guidelines for management and conservation of brown trout in Europe. The report aims at describing the conservation status of brown trout in Europe and provides suggestions for a coordinated European management and conservation strategy.

In general, all tasks and deliverables of the CA have aimed at coordinating research efforts and activities.

39 ANNEX 1.

Program − First Workshop on identification, management and exploitation of genetic resources in brown trout (Salmo trutta).

29 June - 5 July 1998, Silkeborg, Denmark

40 TROUTCONCERT, First Workshop, 29 June – 5 July in Silkeborg, Denmark.

Program.

29 June: Arrival. Dinner/sandwiches available in the evening.

30 June:

Chairman 9.00 - 12.20 Michael M. Hansen

9.00 – 9.20 Welcome + some practical points etc. (MMH).

9.20 - 9.40 Presentation of research activities (PRA), Danish Institute for Fisheries Research.

9.40 - 10.00 PRA, Institute of Marine Research, Norway.

10.00 – 10.20 Coffee break

10.20 – 10.40 PRA, Estonian Agricultural University.

10.40 - 11.00 PRA, INRA, France.

11.00 - 11.20 PRA, NINA, Norway.

11.20 - 11.40 PRA, Sea Fisheries Institute, Poland.

11.40 - 12.00 PRA, University of Berne, Switzerland.

12.00 - 12.20 PRA, Finnish Game and Fisheries Research Institute.

12.30 – 14.00 Lunch

14.00 – 15.00 "Guided tour" at the AQUA aquarium and the associated park.

15.00 – 15.30 Coffee break.

41 15.30 – 17.00 Discussion on potentials and different merits of genetic markers. Responsible person: Andy Ferguson.

18.30 Dinner.

20.00 Visit at our department and lab.

1 July:

Chairman 9.00 - 10.20 Costas Triantaphyllidis

9.00 – 9.20 PRA, Wageningen Agricultural University, Netherlands.

9.20 - 9.40 PRA, Zoological Society of London, UK.

9.40 - 10.00 PRA, Moscow State University, Russia.

10.00 - 10.20 PRA, Stockholm University, Sweden.

10.20 – 10.40 Coffee break.

Chairman 10.40 - 12.20 Patrick Berrebi

10.40 – 11.00 PRA, Middle East Technical University, Turkey.

11.00 - 11.20 PRA, University of Girona, Spain.

11.20 - 11.40 PRA, University of Stirling, UK.

11.40 - 12.00 PRA, University of Göteborg, Sweden

12.00 - 12.20 PRA, Queen's University of Belfast, UK..

12.30 – 14.00 Lunch.

14.00 – 15.30 Conservation, preliminary discussion. Responsible person: Carlo Largiader.

15.30 – 16.00 Coffee break.

16.00 – 17.30 Stocking impact assessment. Responsible person: Patrick Berrebi.

19.00 – 22.00 Boat tour on the lakes in the Silkeborg area, dinner on board.

2 July:

Chairman 9.00 - 10.20 Einar Eg Nielsen

9.00 – 9.20 PRA, Aristotle University of Thessaloniki, Greece.

42 9.20 - 9.40 PRA, Freshwater Research Institute & Marine Research Institute, Iceland.

9.40 - 10.00 PRA, University of Laval, Canada.

10.00 - 10.20 PRA, Universidad de Santiago de Compostela.

10.20 – 10.40 Coffee break.

Chairman 10.40 - 11.40 Andy Ferguson

10.40 – 11.00 PRA, University of Porto, Portugal.

11.00 - 11.20 PRA, University of Montpellier, France.

11.20 - 11.40 PRA, Ludwig-Maximilians-University, Germany.

11.40 – 12.40 Aquaculture. Introductory discussion. Responsible person: Kjetil Hindar.

12.40 – 13.40 Lunch.

13.40 - 14.00 Photo session.

14.00 – 15.15 Review of brown trout phylogeography (+ discussion). Responsible person: Louis Bernatchez.

15.15 – 15.45 Coffee break.

16.00 – 17.00 Local adaptations in brown trout populations (+ discussion). Responsible person: Bill Jordan.

NB! No dinner arranged at the Freshwater Centre. You can go to various restaurants in the town centre. It is a good idea to make a reservation (you can ask for help in the reception).

3 July:

9.00 – 10.30 Brainstorm on the internet features of the CA. Responsible person: Paulo Prodöhl (NB! This IS meant as a brainstorm, NOT Paulo giving a talk to the other participants).

10.30 – 10.50 Coffee break.

10.50 - 12.00 Preliminary discussion on suggestions for "a Transeuropean strategy for management and conservation of genetic resources in brown trout". Responsible persons: Einar Eg Nielsen, Michael M. Hansen

43 12.30 - 17.00 Picnic and mini-excursion (basically, we will just go to a nice place, have some lunch, and we will electrofish a couple of trout [so you can see what trout look like around here, not for lunch!]).

19.00 Dinner.

4 July:

9.00 – 9.30 rDNA RFLPs in brown trout. Responsible person: Paulino Martinez OR Genetic marker nomenclature...... starts at 9.00.

9.30 - 12.30 Genetic marker nomenclature, harmonisation of the use of markers and principles for calibration of markers among different laboratories. Group work and plenum discussions. Responsible person: Michael M. Hansen

10.30 – 10.50 Coffee break.

Also, if needed, we can continue discussion on previous topics (i.e. we can take up discussions that have not been finalised).

12.30 – 14.00 Lunch.

14.00 – 15.30 Open session. We can discuss any problems or topics that someone wants to raise.

15.30 – 16.00 Coffee break

16.00 – 17.30 Discussion of research activities of the participating laboratories. Initiatives for improving collaboration and for sharing resources (e.g., ”tissue banks”). Are there topics that are clearly not covered by ongoing research? ⇒ Recommendations for future studies.

19.00 Dinner.

5 July:

Departure. Breakfast is of course available, but if needed you will have to arrange your own lunch.

Comments and practical points (in alphabetic order)

E-mail If you want to communicate with somebody by e-mail, we have established a special TROUTCONCERT address: [email protected].

How to get your expenses refunded After you have returned home you send us your spent tickets. You will then get your travel expenses refunded. Also, you will get paid what ever is left of your personal allowance. That is 180 ECU per day minus accommodation, meals and other expenses (boat tour etc.). In other words, we

44 pay the bill at the Freshwater Centre. However, you will have to pay your individual expenses for phone calls etc. at the reception at the Freshwater Centre before you leave.

If you are lost If you are lost in town and want to go back to the Freshwater Centre you can always ask people about the way to "AQUA". Everybody knows where that is.

Informal atmosphere Hopefully, the rather detailed program does not give you an impression of a very formal occasion. It is nice to have a program, but of course we can always make changes in whatever way we want to. This should be just as much a social as a scientific meeting with a very relaxed and informal atmosphere and lots of creative discussions.

Phone numbers If something is wrong some useful phone numbers are listed below: Reception, Freshwater Centre +45 89 212131 Dept. of Inland Fisheries +45 89 213100 Dept. of Inland Fisheries, fax +45 89 213150 Michael, at work +45 89 213145/3148 Michael, private +45 86 845809 Michael, mobile phone +45 22 171427

Photocopying It is of course possible to get papers photocopied during the workshop. We will arrange a photocopying service on a daily basis. If you know by now that you would like to have a copy of a paper from one of the other participants, it might be a good idea to ask this person in advance. She/he could then bring you a copy, or you could get it copied here during the workshop.

Presentation of research activities Each lab has 20 min to present their work. You can spend the 20 min exactly as you want to. You can talk for 15 min and answer questions for 5 min (but there will be plenty of opportunity for talking to each other during the workshop), or you can talk for all 20 min. You can split the 20 min between two persons from the same lab, you can give a broad overview of the work of your lab or you can concentrate on a specific topic.

Silkeborg Silkeborg is a rather small town (app. 50,000 citizens). The Freshwater Centre is situated app. 15 min walk from the town centre, where there are several bars, cafe's etc. There are also several banks and cash dispensers. 2000 years ago, before soccer was invented, the major entertainment around here was killing each other and throwing the corpses into peat bogs. Two of the best preserved peat bog corpses in the world were found in the Silkeborg area, and one of them can be seen at the Silkeborg Museum (certainly worth a look). One of the other attractions of the area is the "AQUA" exhibition aquarium, situated right next to the Freshwater Centre (you have free access to this). Finally, there are two museums of modern art in the neighbourhood. You will get some further info incl. map of the town upon arrival.

45 Soccer World Championship The workshop coincides with the quarter finals of the soccer World Championship. There are televison facilities at the Freshwater Centre and, if needed, matches can be recorded on videotape.

Weather The weather is usually good at this time of year with temperatures between app. 18 - 25o C. However, the temperature may drop in the evening and you should also be prepared for a couple of rainy days. One of the cleanest lakes in Denmark is situated 10 min walk from the Freshwater Centre, and (depending on water temperature) it is very suitable for bathing.

Participants Paulo Alexandrino (Porto, Portugal) Lise-Lotte W. Andersen (DIFRES, Denmark) Bernard Angers (INRA, France) Apostolos Apostolidis (Thessaloniki, Greece) Didier Aurelle (Montpellier, France) Louis Bernatchez (Laval, Canada) Patrick Berrebi (Montpellier, France) Carmen Bouza (Lugo, Spain) Michael W. Bruford (Zool. Society, England) Anna Danielsdottir (Marine Research Institute, Iceland) Alistair Duguid (Belfast, N. Ireland) Andy Ferguson (Belfast, N. Ireland) Jose Luis Garcia-Marin (Girona, Spain) Kevin Glover (IMR, Norway) Riho Gross (Estonian Agr. U., Estonia) Sigurdur Gudjonsson (Marine Research Institute, Iceland) Rene Guyomard (INRA, France) Michael M. Hansen (DIFRES, Denmark) Kjetil Hindar (NINA, Norway) Bill Jordan (Zool. Society, England) Hanne Jørgensen (DIFRES, Denmark) Marja-Liisa Koljonen (Helsinki, Finland) Linda Laikre (Stockholm, Sweden) Carlo Largiader (Berne, Switzerland) Ari Loytynoja (Oulu, Finland/DIFRES, Denmark Paulino Martínez (Lugo, Spain) Dorte Meldrup (DIFRES, Denmark) Karen-Lise Mensberg (DIFRES, Denmark) Einar Eg Nielsen (DIFRES, Denmark) Alex Osinov (Univ. of Moscow, Russia) Tiit Paaver (Estonian Agr. U., Estonia) Stefan Palm (Stockholm, Sweden) Jonas Pettersson (Göteborg, Sweden) Paulo Prodohl (Belfast, N. Ireland) Kornelia Rassmann (Univ. of Munich, Germany) Laura Sánchez (Lugo, Spain)

46 Nuria Sanz (Girona, Spain) Ulrich Schliewen (Univ. of Munich, Germany) Oystein Skaala (IMR, Norway) Rene Stet (Wageningen, the Netherlands) John Taggart (Univ. of Stirling, Scotland) Inci Togan (Middle East Technical Univ., Turkey) Costas Triantaphyllidis (Thessaloniki, Greece) Roman Wenne (Gdynia, Poland) Ewa Wlodarczyk (Gdynia, Poland) Kjartan Østbye (NINA, Norway) Siri Østergaard (DIFRES, Denmark)

47 ANNEX 2

PROCEEDINGS OF THE FIRST WORKSHOP ON IDENTIFICATION, MANAGEMENT AND EXPLOITATION OF GENETIC RESOURCES IN BROWN TROUT (SALMO TRUTTA).

29 June - 5 July 1998, Silkeborg, Denmark

Introduction

The first workshop of the CA took place 29 June - 5 July at the Freshwater Centre, Silkeborg, Denmark. A total of 47 persons representing all laboratories involved in the CA and including all external experts participated in the meeting. Since this was the first time many of the participants met it was considered important to present the research activities of the participating groups, so each group was allocated twenty minutes to present their research. This made up approximately half of the workshop, whereas the other half was dedicated to specific theme sessions. Prior to the workshop specific persons had taken responsibility for the different theme sessions. The sessions were organised either as one talk by the responsible person followed by a plenary discussion, or a number of persons gave smaller talks (10-20 min.) on aspects of the topic of the session.

The titles of the theme sessions were: a) Potentials and different merits of genetic markers (responsible person: Andy Ferguson) b) Conservation of brown trout, preliminary discussion (responsible person: Carlo Largiader) c) Stocking impact assessment (responsible person: Patrick Berrebi) d) Potential of brown trout for aquaculture (responsible person: Kjetil Hindar) e) Review of brown trout phylogeography (responsible person: Louis Bernatchez) f) Local adaptations in brown trout populations (responsible person: Bill Jordan) g) Brainstorm of the internet features of the CA (responsible person: Paulo Prodöhl) h) Preliminary discussion on management recommendations for conservation of genetic resources in brown trout (responsible person: Michael M. Hansen) i) Genetic marker nomenclature and harmonisation of the use of markers (responsible person: Michael M. Hansen)

In addition, there was a discussion on j) Preliminary suggestions for topics at the next workshop.

The detailed program of the workshop is included in the report as Annex 1.

48 a) Potentials and different merits of genetic markers (Reported by A. Ferguson, Queen's University of Belfast, with contributions from Michael M. Hansen, Danish Institute for Fisheries Research, Paulino Martinez, University of Santiago de Compostela, Paulo Prodöhl, Queen's University of Belfast, Rene Stet, Agricultural University of Wageningen and John Taggart, University of Stirling).

OVERVIEW OF DISCUSSION

A wide range of genetic markers has been used for study of brown trout population genetics. As well as molecular genetic markers these include one of the few single locus morphological markers in fish, the fine-spotted brown trout variant described from Norway (Skaala & Jorstad, 1987)). The aim of this session was to consider the strengths and limitations of the different genetic markers available for brown trout and also the inter-linked topic of sampling strategy. Information from other fish species was included where direct information was not available for brown trout.

Allozymes In spite of disadvantages in sample collection, allozymes are still of considerable value in brown trout population genetic studies. In some cases allozymes can reveal as much about population structuring as DNA based approaches. The minimum sample size for a diallelic polymorphism should be 30. Allozymes are also valuable for phylogeographic studies. CK-2*, which has been ignored in some studies, should be routinely screened in Atlantic populations. Before full use can be made in phylogeographical studies, there is a need for nomenclature standardisation based on side- by-side comparison of alleles to check for homologies. Narrow pH range isoelectric focusing may be more appropriate for this than starch gel electrophoresis for some variants.

Mitochondrial DNA Mitochondrial DNA analysis has uses in many types of brown trout genetic studies from parentage identification through to phylogeographical studies although a combination of mitochondrial and nuclear analysis is preferred for many purposes. MtDNA haplotypes can be used as markers for farm stocks in monitoring female breeding in the wild. It would appear that sequencing, at least in some situations, gives little or no advantage over RFLPs based on a large number of restriction enzymes. There appears to be no information at present in the occurrence of nuclear copies of mtDNA in salmonids, as has been reported in birds and mammals. The possible problems of such pseudogenes need to be borne in mind when studies involving mtDNA are undertaken and the problem merits specific targeting.

Nuclear DNA markers Most nuclear DNA studies have been based on variable number tandem repeats (VNTRs) i.e. minisatellites and microsatellites. These are appropriate for parentage identification, population studies and micro-phylogeographical studies. The appropriate number and type of loci and the sample sizes to be used was subject to much discussion during the session, but of course it is dependent on the specific question being addressed. The more highly variable loci are appropriate for parentage studies provided all alleles can be reliably typed. However, concerning the use of VNTR loci for studies of genetic population structure there were two different lines of opinion among the workshop participants. First, one group of participants argued that loci with no more than 8 alleles should be used in population structure studies and sample sizes should be at least 50 for this purpose, ideally 100. The rationale for this recommendation was based primarily on

49 concerns regarding the power of tests for Hardy-Weinberg equilibrium and homogeneity of allele frequencies between samples. Five loci should be a minimum for routine screening (the minimum number for conducting bootstrapping over loci) although it is not clear at present as to how these loci should be decided on, as not all loci are equally informative. For other studies, e.g. estimating heterozygosity, it may be appropriate to use smaller sample sizes and a larger number of loci.

The other group of participants argued that in practice there was not an upper limit concerning recommendations on the number of alleles per locus, except for extreme cases where the number of alleles was close to 2N. Such highly variable loci must be considered extremely rare, at least in the case of microsatellites. Traditional chi-square based statistics are not suitable for analysing highly variable loci. Tests based on numerical resampling procedures (bootstrapping, permutation, Monte- Carlo) should be used instead. The power of tests for deviations from Hardy-Weinberg equilibrium at highly variable loci has been questioned. It must be assumed that the power decreases with increasing number of alleles. On the other hand, in some situations this may be counterbalanced by an increased ability for detecting non-random mating at highly polymorphic loci. If A mates with B and C mates with D, but A does not mate with C and B does not mate with D, this is obviously easier to detect using a locus with 8 alleles rather than a locus with 2 alleles. However, there is clearly a need for analysing the power of tests as a function of number of alleles. Recommended sample sizes, both in terms of loci and individuals, vary according to the specific analyses applied. For some tests and analyses (for instance estimation of F-statistics) a minimum of 5 loci and sample sizes of 50 is recommended. However, for estimating genetic distances between populations (especially using microsatellite statistics such as the (δµ)2 distance) much higher number of loci (>20) are needed, whereas sample sizes do not need to be very high. Loci should be chosen at random and inclusion of only "informative loci" should be avoided. If populations diverge by drift different loci may show different degrees of differentiation, due simply to stochastic effects. The best estimate of genetic differentiation is obtained by calculating the average genetic differentiation over several loci. If only those loci that show the strongest differentiation are analysed, genetic differentiation is overestimated.

Although some studies have used minisatellites there does not appear to be any reason to use these now as microsatellites are technically much more straightforward. However, attention should be paid to the fact that the precise mechanisms of mutations are still poorly understood and may vary among loci and even among alleles at the same locus. A common set of loci should be used in all brown trout population studies so that a comparative database becomes available in the same way as for allozymes. As with other types of markers, attention to the reliability of typing is required to ensure the quality of such a database. Problems of homoplasy may invalidate the use of microsatellites at the macro-phylogeographical level but this possibly could be overcome by sequencing.

New markers Of the new markers currently available or likely to be available in the near future, ribosomal DNA RFLPs appear to offer particular promise for brown trout studies giving the possibility of fixed patterns in individual populations. Major histocompatability (MH) gene polymorphisms are also likely to provide highly variable markers. However, the extent to which selection is likely to be a problem in the use of MH markers in population studies is at present unknown. Indeed the whole issue of selection versus neutrality of markers in such studies needs detailed consideration.

50 Sampling In all sampling, detailed sampling information should be recorded. Sampling should preferably be based on adults. If based on juveniles then information on movements is required. Sampling of siblings may mean that large samples may not be in Hardy-Weinberg equilibrium and this may invalidate many allelic frequency based analyses. Particular emphasis should be given to comparing samples within rivers to establish the geographical extent of sampling required. More modelling is required to establish optimum sampling and analytical procedures. Also much more consideration needs to be given to the methods of data analysis.

COMPARISON OF MARKERS IN THE STUDY OF LOUGH MELVIN SYMPATRIC BROWN TROUT POPULATIONS (Paulo Prodöhl, Queen's University of Belfast)

Due to the availability of extensive background genetic data, the brown trout populations of Lough Melvin provide an ideal benchmark group for a comparative study of minisatellite, microsatellite markers, mtDNA, and allozyme markers in the description of the population genetic structure of brown trout populations. Lough Melvin is a relatively small lake of some 22km2, which occupies a typical glacial-formed valley, situated in north-west Ireland. This particular lake is unique, within Britain and Ireland at least, in having three sympatric morphotypes of brown trout, locally known as gillaroo, sonaghen and ferox (Ferguson & Taggart, 1991).

Allozyme studies carried out over a period of several years have showed major differences in the occurrence and distribution of alleles at several loci indicating that the three types are reproductively isolated. The reproductive isolation at this micro-geographic level is maintained by use of distinct spawning sites through precise homing behaviour.

The examination of mtDNA RFLP variation of Melvin trout revealed the existence of 10 composite mtDNA haplotypes. All ferox surveyed were found to be fixed for the same composite genotype. The occurrence of four unique mtDNA haplotypes in gillaroo and three in sonaghen, in addition to the significant frequency differences among all trout types, adds further convincing evidence that they are genetically distinct populations. (McVeigh et al., 1995)

Although an analysis of the Melvin brown trout population using Jeffreys’ 33.6 minisatellite probe provided some extra evidence for population isolation, the banding patterns obtained were found to be far too complex for routine population screening (Prodöhl et al., 1992). To overcome the problem, several minisatellite single locus probes were developed for brown trout research. Five single locus minisatellite markers were used to examine genetic variation in the Melvin trout types. Each minisatellite locus was moderately polymorphic with 3 to 9 alleles segregating per locus per population. Similarly to other markers, the three populations can be easily differentiated by distinctive frequencies distribution of the main alleles at most loci.

For a series of reasons involving, for instance, the need for high quality DNA extracts, problems with typing, and for being a technically demanding and labour intensive methodology, the minisatellite single locus approach has now being largely replaced by the faster microsatellite approach. Melvin brown trout samples were screened with two microsatellite markers. Similarly to the minisatellite single locus screening, these loci were found to be moderately polymorphic with 4

51 to 10 alleles segregating per locus per population. Again, the three populations could be easily differentiated by distinctive frequency distribution of the main alleles.

A summary comparison, in terms of Fst statistics, for all the assayed molecular markers indicates highly concordant mean values for all nuclear approaches with mtDNA being the overall best marker. This is not entirely surprising. Its one quarter effective population size, relative to nDNA, does result in greater sensitivity to genetic drift and, as such, prone to a larger genetic differentiation. This is particularly relevant to salmonids, where as a result of the precise homing behaviour to specific tributaries, breeding populations are quite limited in size (usually in the hundreds). Therefore, the difference between effective population sizes for nuclear and mtDNA will be significant. In terms of nuclear markers, the minisatellites and microsatellites provided no new information on population structure over that already available from the initial allozyme studies. rDNA RFLPS AS GENETIC MARKERS FOR MANAGEMENT IN THE BROWN TROUT (Paulino Martinez, University of Santiago de Compostela)

Major ribosomal genes (rDNA) constitute a family of moderately repetitive sequences very useful for population and phylogenetic analysis. Ribosomal genes are organized as long tandem arrays of a basic unit. This is made up by the coding region, which holds the major ribosomal genes separated by two internal transcribed spacers (ITS); and the intergenic spacer (IGS), which separares consecutive coding regions. rDNA units show a great dichotomy in their evolutionary rates. While coding regions are very conservative, ribosomal spacers, specially the IGS, exhibit high evolutionary rates. The tandem disposition of rDNA, determine these genes to evolve in a concerted or cohesive manner, which produce homogenization of sequences within populations and divergence between populations.

The most relevant features of the IGS in brown trout are three tandem repetitive motifs of 0.35, 1.0, and 0.2 kb, which show wide intra and interindividual variation. Two of them represent putative regulatory elements for rDNA transcription. The third, in the vicinity of 18S gene, is a mobile element (SINE-like) tandemly arrayed. Also, we have detected polymorphic restriction sites in the IGS due to the presence-absence of specific restriction targets. Finally, a long insertion of 14kb in the 28S gene, was detected in rDNA units of all individuals from a single population. We carried out a population analysis to check the interest of this variation for resource management, and to analyze evolutionary aspects of rDNA genes. The analysis of the closest region to the 28S gene, revealed four different length variants, with population-specific patterns. Another interesting source of variation in the IGS are presence-absence polymorphic restriction sites. So, a variable BglII restriction site, which is absent in the Northern river drainage, was present in all other populations analyzed. This target could represent a river basin-specific marker. In the same way, the SacI site in the middle of the IGS, has evidenced a gradual latitudinal variation. The mobile element in the neighbourhood of the 28S gene, has provided a nearly diagnostic marker for monitoring stocking in Galicia. When applied to the introgressed Asma population, all individuals could be unambiguously classified by using this marker.

These results, although preliminary, demonstrates the large genetic divergence of Galician populations for rDNA, but also the power of concerted mechanisms like unequal crossing-over and gene conversion to homogenize rDNA variants within populations. This circumstance is probably

52 stressed in a species with large population subdivision like brown trout. Our data also suggest the high interest of rDNA variants to mark different population units, according to the hierarchical level considered. The length variants have been shown to be useful for identifying single populations, while the presence-absence polymorphisms could identify larger geographic areas.

MH GENES (Rene Stet, Agricultural University of Wageningen)

Major histocompatibility (MH) genes in fish are comprised of those coding for class I alpha chain and class II alpha and beta chains. Unlike in other cold- and warm-blooded vertebrates the MH genes are not organised in a complex. The class I genes are not linked to class II alpha and beta genes. The latter two, however, have been demonstrated to be closely linked. The molecules the MH genes encode play a pivotal role in the initiation of a specific immune response. In a number of teleostean fishes the MH genes have been shown to be highly polymorphic. This polymorphism is most likely maintained in a population as a result of balancing selection. Thus, MH gene polymorphism is not a neutral marker. However, it would be interesting to compare population analyses based on "neutral markers" and non-neutral markers such as the MH genes. Such an approach would require an expedient technique to detect MH polymorphism. Cloning and sequencing alleles from both class I and class II genes would be an unfeasible approach. However, two alternatives are available: SSCP or satellite markers. SSCP analysis of MH genes has been used in studies of pacific salmonid populations, and has shown differences in allele frequencies between populations. To date, micro- and minisatellite markers have not been used in studies of MH polymorphism. In Atlantic salmon it has been shown that the class I and the class II alpha contain satellite markers. However, in a recent study on farmed Atlantic salmon (Stet and Grimholt FAIR CT97-3643) MH polymorphism, we have identified, in a group of 50 female broodstock fish, 6 microsatellite alleles in a class I gene and 4 minisatellite alleles in a class II alpha gene. If similar repeats are present in brown trout MH genes comparative studies between neutral and selective markers would be possible.

SAMPLING STRATEGIES (John Taggart, University of Stirling)

Obviously, sampling strategies will differ according to the type of questions being addressed (e.g. phylogenetic, macro / micro population differentiation, familial analyses). Three major elements common to all salmonid sampling considerations will be, 1) the number of individuals screened; 2) the type of sample (age structure/ life history stage), and 3) the geographic extent of sampling - a few aspects of which are outlined below.

53 The accuracy of allele frequency estimates and the power of various statistical tests are largely dependent on the sample size screened. Minimum sample sizes of c. 30-50 for population surveys of diallelic polymorphisms have long been recommended (e.g. Allendorf & Phelps, 1981) - though even these modest numbers have not always been reached in published studies. The sample size requirements for more highly variable VNTR loci remains to be fully explored. Though, intuitively, minimum sample sizes in excess of 100 would be expected, for at least some statistical measures (e.g. genetic distances) smaller samples (50-100) may suffice (Chakraborty, 1992; Ruzzante, 1998). The apparent misconception that the now routinely applied population genetic analyses based on exact tests can be reliably carried out on small (sub-optimal) sample sizes needs to be dispelled. This is not the case.

Both the type of sample and geographic extent of sampling must also be considered in light of the known life history of the species / population in question. In the specific case of brown trout population studies this should involve such aspects as the species potentially high fecundity and the possible co-occurrence of two major life history types (resident & migratory). Ideally, sampling should be based on sexually mature adults - on or near known spawning sites. In many (if not most) cases, however, this is an impractical approach. Where based on juveniles, efforts should be made to minimise the demonstrated potential for sampling of only a few families (Hansen et al., 1997). In practical terms this would involve sampling over a large area and including fish of as many different ages as possible (avoiding over representation of any single age class).

It should be recognised that knowledge of both intra-river genetic substructuring and juvenile brown trout movement / migration is minimal. Further exploration of these aspects, together with computer modeling studies, will be necessary to devise and confirm optimum sampling procedures. Undoubtedly there is a need for more detailed description of sampling methods to be given in published studies to enable a more informed interpretation of presented data.

DISCREPANCIES OF ESTIMATES OF GENETIC DIFFERENTIATION AMONG WHITEFISH (COREGONUS SP.) POPULATIONS, USING DIFFERENT KINDS OF ANALYSES OF MITOCHONDRIAL DNA AND MICROSATELLITE MARKERS (Michael M. Hansen, Danish Institute for Fisheries Research)

Two different types of markers were applied, i.e. microsatellites (six loci) and PCR-RFLP analysis of the mitochondrial ND1 and ND5/6 segments, to a study of genetic population structure and phylogeography of whitefish. Genetic differentiation was analysed, using a hierarchical gene diversity analysis. The first level of the hierarchy consisted of major geographical (and presumably phylogeographically important) regions (the Wadden Sea area, inner Danish Fjords and lakes and the Baltic Sea region). The second level consisted of populations within regions. For the mtDNA data two types of analyses (AMOVA) were done: One treating haplotypes as equidistant, and the other taking the molecular distances among haplotypes into account. Also, for the microsatellite data two types of analyses were done: One using "traditional" F-statistics, and the other using ρ- statistics, which assume a stepwise mutation model.

It turned out that both classes of markers as well as the two different analyses applied to each class of marker yielded qualitatively different results concerning the distribution of genetic differentiation

54 at the two levels of the hierarchy. MtDNA Φ-statistics including molecular distances suggested that the main part of genetic differentiation was distributed among populations within groups, whereas Φ-statistics excluding molecular distances suggested that genetic differentiation was approximately the same among populations within groups as among groups of populations. Microsatellite F- statistics also suggested that differentiation was approximately equal at the two levels of the hierarchy, whereas ρ-statistics suggested that differentiation among groups of populations was the most important component.

We assume that the reason for the discrepancies are due to differences in mutation rates and genetic drift acting at the two classes of markers, and, specifically for the microsatellite data, it is important whether an infinite allele or stepwise mutation model is assumed. Mutation rates at microsatellite loci are normally assumed to be much higher than for mtDNA. Therefore, whereas the evolutionary forces (mutation and drift) involved in genetic divergence are the same for both classes of markers, the rates of evolution are different. In the case of mtDNA, mutation is only of major importance at a relatively long time scale (i.e. predating the last glaciation). However, drift is expected to be strong because of the low effective population size compared to nuclear loci, which is likely to increase differentiation among individual populations. For microsatellite loci drift is expected to be of relatively less importance, whereas mutation is expected to be of importance at a shorter time scale than for mtDNA. This could explain the qualitatively different results, as several new mutations at microsatellite loci would be expected to have occurred within the three main group of populations, reflected in a high proportion of genetic diversity distributed among groups of populations, while weak genetic drift (as compared to mtDNA) would tend to render differentiation among populations within groups of less importance, as estimated by ρ-statistics. In contrast, for mtDNA the lower mutation rate would result in few new mutations within the main groups of populations, while strong drift would tend to increase differentiation among populations within groups.

REFERENCES

Allendorf, F.W. and Phelps, S.R. (1981) Isozymes and the preservation of genetic variation in salmonid fishes. Ecological Bulletins. (Stockholm), 34, 37-52.

Chakraborty, R. (1992) Sample size requirements for addressing the population genetic issues of forensic use of DNA typing. Human Biology, 64, 141-159.

Ferguson, A. and Taggart, J.B. (1991) Genetic differentiation among the sympatric brown trout (Salmo trutta) populations of Lough Melvin, Ireland. Biol. J. Linn. Soc, 43, 221-237.

Hansen, M.M., Nielsen, E.E. and Mensberg, K.-L.D. (1997) The problem of sampling families rather than populations: relatedness among individuals in samples of juvenile brown trout Salmo trutta L. Molecular Ecology, 6, 469-474.

McVeigh, H., Hynes, R. & Ferguson, A. (1995) Mitochondrial DNA differentiation of sympatric populations of brown trout, Salmo trutta L., from Lough Melvin, Ireland. Canadian Journal of fisheries and Aquatic Sciences, 52, 1617 - 1622.

55 Prodöhl, P.A., Taggart, J.B. and Ferguson, A. (1992) Genetic variability within and among sympatric brown trout (Salmo trutta) populations: Multi-locus DNA fingerprint analysis. Hereditas, 117, 45-50.

Ruzzante, D.E. (1998) A comparison of several measures of genetic distance and population structure with microsatellite data: bias and sampling variance. Canadian Journal of fisheries and Aquatic Sciences, 55, 1-14.

Skaala, O. and Jorstad, K.E. (1987) Fine-spotted brown trout (salmo trutta): its phenotypic description and biochemical genetic variation. Canadian Journal of fisheries and Aquatic Sciences, 44, 1775-1779.

b) Conservation of brown trout, preliminary discussion (reported by Carlo Largiader, University of Berne)

This session was intended to start a broad discussion on topics related to conservation and sustainable management of brown trout. There is general agreement that the loss genetic diversity in brown trout is still ongoing. The genetic diversity of a species reflects its potential to successfully adapt to a changing environment and thus the conservation of as much genetic diversity as possible, both among and within brown trout populations, is imperative.

Many freshwater resident and anadromous brown trout populations have been managed/exploited by more than one country. Therefore, some co-ordination among all concerned countries is essential to achieve an effective conservation of the genetic diversity of brown trout across its entire native range. The discussion made evident that it will be difficult (but not impossible) to reach a broad consensus on specific conservation strategies and in particular on their realizability in different countries. This is mainly because the socio-economical importance of brown trout and the conservation and fisheries management legislation varies considerably among (and within) countries.

Depending on which species concept is applied, several evolutionary lineages of brown trout that have been identified using molecular markers can be regarded as separate species. It was proposed that within the framework of this CA, species or subspecies status should be assigned to these particular brown trout forms, because in many of the participating countries, the actual conservation units consist of recognised species and/or subspecies. In this context, it is furthermore noteworthy, that the current state of brown trout taxonomy is rather chaotic. For example, four subspecies (cf Lelek 1987) are generally recognised, whereas Kottelat (1997) recently proposed 18 different brown trout species. Therefore, one should be aware of the danger that if a "revision" of brown trout taxonomy based on molecular data is not supported by taxonomists, it may just add to the "nomenclatural chaos".

As was pointed out by several participants, conservation recommendations transmitted to managers should stress the fact that genetic conservation is equally important at any taxonomic level. It should be avoided to raise unintentionally the impression that conservation of genetic diversity is just conserving a few individuals/populations of each evolutionary lineage. It is utterly important to transmit the information that a sustainable management includes per se the conservation of

56 genetic diversity of an exploited population, and that the genetic diversity within a population is important for successful local adaptation. Thus, conservation of genetic diversity at the population level is the basis for preservation at higher levels.

REFERENCES

Kottelat, M. (1997) European freshwater fishes: An heuristic checklist of the freshwater fishes of Europe with an introduction for non systematics and comments on nomenclature and conservation. Biologia, Bratislava, 52 (Supplement 5), 1-271

Lelek, A. (1987) The Freshwater Fishes of Europe, Vol. 9: Threatened Fishes of Europe. AULA- Verlag, Wiesbaden.

c) Stocking impact assessment (reported by Patrick Berrebi, University of Montpellier II)

INTRODUCTION

There is a long tradition in many European countries for rearing and stocking brown trout. The aim of most stocking programmes is to increase population sizes in rivers where the sizes of the native populations have declined due to poor environmental conditions or overexploitation (recreative or commercial fisheries). However, stocking activity may in fact pose a serious threat to the wild populations in the stocked rivers, both ecologically (e.g. competition for food and spawning places between wild and stocked trout) and genetically (e.g. introgression, "swamping" of indigeneous gene pools). Whereas a general treatment of the topic of interactions between wild and stocked fish has been given in several publications, at present there is not a general overview of brown trout stocking procedures in Europe. The following information has been gathered from Troutconcert participants and, in cases where information has been available from the literature, other countries are included as well.

We have obtained detailed information on stocking practices in ten countries, covering both Northern and Southern Europe: Norway, Finland, Estonia, Denmark, Poland, Russia (Northern Europe), Switzerland, France, Spain and Greece (Central and Southern Europe). This division in northern and southern countries corresponds roughly to the major phylogeographical structure of brown trout: Atlantic trout in the north and Mediterranean and other phylogeographical races in the South.

INTENSITY OF STOCKING

The intensity of brown trout stocking activity depends considerably on local traditions. Thus, stocking activity is intense in Poland, the Czech Republic, Switzerland, Denmark, France, Spain, Italy, Slovenia, whereas it is limited in Norway, Estonia, Latvia, Lithuania, Russia and Greece. In some countries, where stocking has traditionally been regarded the keystone of fisheries management, intense stocking activity takes place in essentially all regions (this applies particularly

57 to the situation in France, Spain and the Czech Republic). In most cases, however, the intensity of stocking activity differs considerably among regions within countries, reflecting both the availability of suitable trout habitats and the "need" (i.e. decline of wild populations) for stocking activity.

In Finland, the southern and central parts of the country are the most heavily stocked but also the regions where the wild forms are the most threatened and angling intensive.

In Estonia and Latvia, stocking activity is concentrated in the coastal areas and other regions with good resident trout streams.

In Poland, the Pomerania (the coastal zone in the north-western part of the country) and the Vistula river is subject to intense stocking.

In Denmark, some parts of the country supposed not to contain any surviving indigeneous populations are heavily stocked with hatchery trout. In other parts, stocking of hatchery trout is restricted. Instead, stocking involves realeases of hatchery-reared offspring of local wild trout (most parts of Jutland). Finally, in some rivers and regions with a substantial element of indigeneous populations, stocking is not permitted at all.

In Switzerland, the intensity of stocking is a mosaic due to more than twenty independent political units. The situation is probably quite similar in the UK, where the heterogeneity is mostly due to differences of policies and controls among regions.

Finally, in Greece, stocking activity is limited to the northern part of the country, and in Russia stocking predominantly takes place in regions where native populations are supported by special hatcheries (Baltic sea, Caspian sea and Black sea).

IMPACT ON WILD POPULATIONS

The impact of stocking activity on wild trout populations is generally not well known. Several studies have focused on this issue, but clear-cut conclusions cannot be drawn at present. In some cases it has been found that no introgression has taken place despite intense stocking of hatchery trout into wild populations, whereas in other cases introgression has clearly taken place. In addition, it should be noted that introgression is not the only genetic problem that may occur as a result of stocking. Even in situations where stocking programmes are based on releases of trout of local origin, problems connected to reduced effective population sizes and adaptations to a hatchery environment instead of natural conditions may occur. These latter problems are at present difficult to demonstrate experimentally.

In Southern Europe all domesticated strains used for stocking are derived from the Atlantic phylogeographical race, whereas the stocked populations belong to different phylogeographical races. This means that diagnostic allozyme markers are available for identifying domestic strains and wild populations and for estimating introgression. This has been utilized in several studies and, consequently, the knowledge of introgression as a result of stocking activity is most advanced in Spain, France, Switzerland, Italy, Greece and Slovenia.

58 In northern countries domesticated hatchery strains and wild populations belong to the same phylogeographical race, and there are in most cases no diagnostic allozyme markers available for distinguishing domesticated and wild populations. In some countries it has nevertheless been attempted to assess stocking impact using allozymes, but the data are difficult to interpret or not yet enough developed (Finland, Estonia and Poland). Another option has been to design long-term stocking experiments, where the genetic composition of the stocked populations before and after potential introgression has been monitored (Sweden, Norway). Finally, DNA markers (mtDNA, mini- and microsatellites) may provide new possibilities and increased resolution compared to allozymes. The possibilities of studying introgression using DNA markers are currently being tested in several countries from all parts of Europe.

STOCKING METHODS AND USE OF STOCKING MATERIAL

The methods of stocking differ among countries and regions. For instance, in some countries the main part of stocking activity involves releases of fry, whereas in other countries stocking of subadults or adults is the predominant method. Also, the stocking material may be derived from domestic strains of more or less unknown (but Atlantic) origin, or from local spawners that are caught annually in the rivers targeted for stocking.

"Stocking" of fertilised eggs from domestic (Atlantic) strains is the most important method in Spain. No other countries do the same.

Releases of fry from domestic strains is the main method in France, but an important method also in Spain.

Stocking of subadult and adult domestic trout is the main method used in Poland, and it is also a common practise in Spain, France and Denmark.

Release of smolts from local broodstocks is the main method in Finland, Latvia, Lithuania and Russia. A similar procedure is increasingly being implemented in France, as stocking of trout of local origin is considered more in line with fundamental principles of conservation.

In Estonia, a slightly different stocking strategy is employed. Wild spawners are used for parent fish and their progeny cultivated in fish farm until release at a sub-adult stage. When the stocked trout return during the spawning run, they are caught and used for parent fish in the next generation. This is equivalent to sea ranching as applied in many places in North America.

Stocking of eggs or fry derived from local wild spawners is a widely used stocking strategy in Greece, Denmark, Norway, Latvia, Lithuania, Estonia, Poland and Russia. It is the dominant method in Switzerland since 1991.

Funding for stocking is generally provided by the governments. However, the organisations or institutions actually producing the fish and/or releasing them differ among countries.

Public organisations are responsible for most of the production of stocking material in Poland, Norway, Greece, but also Finland (in case of particularly endangered populations) and to some extent in France.

59 Production of stocking material takes place on a private basis in Denmark, Spain, France, Finland and Switzerland. In Denmark, anglers clubs are responsible for production of all stocking material derived from wild spawners, whereas all domestic strains are maintained by commercial hatcheries.

CONSERVATION

The impact of several decades of stocking activity on indigeneous trout gene pools still remains largely unresolved. Even when efficient markers are not available, scientists can evaluate the impact on natural populations in terms of morphological changes or a decrease of population density. There can be no doubt that many indigeneous trout populations in Europe are threatened, but not always because of stocking. Overfishing, which can be considered as linked to stocking, is considered a serious threat at least in Estonia and Spain.

Stocking is considered as the problem for conservation of wild trout populations in France. Inappropriate stocking policies are considered at least in part responsible for the decrease of wild populations in Norway, Denmark, Finland, Poland, France, Spain and Greece.

RECOMMENDATIONS

Some important features can be extracted from the data obtained in the "Troutconcert" network and literature.

(i) Stocking activity occurs in all countries of Europe, but it appears to be more intense in Southern Europe (France, Spain, Italy, Slovenia).

(ii) The consequences of stocking domesticated trout into wild populations have been well studied in countries where the main natural trout populations belong to the Mediterranean or "marble" (marmoratus) phylogeographical races. This is due to the presence of allozyme diagnostic markers (mainly LDH-5*, TF*, FBP-1*) that can be used to trace introgression from domesticated trout (of Atlantic origin). In "Atlantic" countries, the similarity between natural and domestic trout causes difficulties in obtaining diagnostic markers similar to the allozyme markers applied in Southern Europe. In southern France, interesting results have been obtained using microsatellites with Factorial Correspondences Analyses and Artificial Neural Networks (Montpellier team). However, no simple diagnostic markers are available so far.

(iii) Natural populations of brown trout are considered threatened in all Europe due to anthropogenic effects. These effects consist of pollution, other sorts of habitat degradation (for isntance building of impassable dams and weirs), global climate change is also invoked, and, certainly, stocking. However, it is important to stress that stocking is only a part of the problems, a part which is difficult to assess.

(iv) The methods of stocking used are rather different from one country to another. For the purpose of conservation, the two alternatives are the use of domestic (generally foreign) strains or local broodstocks (if the sites of releases correspond to the origin of the parent fish).

(v) The impact in term of introgression is generally unknown. Some results from French Mediterranean rivers suggest that the efficiency of stocking is very low (less than one per cent of the released domestic fry reach the reproductive age). That means that the benefits for angling,

60 which ultimately depends on an increase of the population size of adult trout, is null. An increase of just a few percent of the population size is hardly detectable by the anglers. However, the cumulative introgression can reach high percentages after the decades or even the century that stocking activity has taken place ("genetic pollution").

(vi) To alter the stocking and management practices, it is necessary to convince the river managers of the inadequacy of the present approach. The money and the control of stocking is generally attributed to governmental institutions, but the choice of the strain used (domestic or derived from local, wild populations), the method applied (i.e. stocking of eggs, fry, subadults or adults) and of the numbers of fish stocked, is often left for anglers organisations or local authorities to decide. The consequences are that insufficient studies are made of the status of the populations prior to stocking, and monitoring of the efficiency and effects of stocking is often inadequate as well. What ultimately happens is that governmental money are used for gradually eradicating natural populations!

To improve the situation, the main question to be answered for each stocked river is: "IS STOCKING REALLY NECESSARY?". To answer this question, basic ecological knowledge on the status of the populations is necessary, which is often the most difficult problem (e.g., are population sizes actually small, what is the reason for the decline of populations and is it possible to solve these problems instead of stocking fish?). Local managers are not able to pay for both stocking and the basic ecological studies that are highly needed. One suggestion could be to stop stocking with domestic strains for a while and use the saved money to analyse the genetics and the demography of the populations. Only when a necessity to suppport a population by stocking (with fish of local origin!) has been demonstrated, releases of fish is an acceptable option.

The other question is "IF STOCKING IS NECESSARY, WHAT KIND OF FISH SHOULD BE STOCKED?" It must be recommended to establish local strains (using wild parent fish from local populations) or, more simply, to stock fertilised eggs from local wild populations. Even though these options may demand more effort than simply stocking hatchery trout, they are far more acceptable from the point of view of conserving biodiversity than stocking of domesticated, exogenous hatchery trout. d) Potential of brown trout for aquaculture (reported by Kjetil Hindar, NINA)

INTRODUCTION

The brown trout is naturally distributed all over Europe, and lives in still- and running water from lowlands to mountains. The species has been transplanted successfully to all continents except Antarctica. Its reproduction has been mastered by humans since the 1760s. Stock enhancement of brown trout populations has involved millions of released fry for a century, and more lately, hundreds of thousands of larger-sized juveniles (including smolts) and sub-adults. Pond-based aquaculture and sea ranching have been on the agenda in several European countries. Yet, the total aquacultural production of brown trout is still small-scale compared to Atlantic salmon and rainbow

61 trout. In 1997, Finland reported producing 25 metric tons of brown trout; other European countries are probably below this level.

By aquaculture, this report understands the captive rearing of fish from eggs to market size. In this definition, we include fish farming where the fish are contained throughout the life cycle, and commercial sea (ocean) ranching where the fish are free-living in that part of the life cycle following smoltification. We exclude artificial production of trout for other purposes such as stock enhancement or conservation.

OBJECTIVE

This report targets four issues which describe the state of knowledge for brown trout aquaculture: 1. The potential of the species for aquaculture 2. Aquaculture-related genetic issues 3. Environment-related genetic issues 4. Constraints/problems that need scientific solutions

POTENTIAL FOR AQUACULTURE

Brown trout aquaculture is possible under a wide range of environmental conditions. The species exists naturally in areas where the air temperature of the warmest month is below 27 oC and that of the coldest month above -17 oC. Lethal water temperature is 25.5-27 oC except for developing eggs, which may have problems above 13 oC. The species can live in freshwater throughout the life cycle, and in brackish and salt water following smoltification (or attainment of a certain body size). Osmotic problems develop at low temperature in full salt water. Brown trout needs well oxygenated water throughout the life cycle.

Several factors would seem to promote brown trout as an interesting species for aquaculture. Among these are: • the good reputation of brown trout for human consumption • the high level of genetic variation (heterozygosity) as estimated by molecular markers • the high level of genetic differentiation between populations • the existence of brown trout populations in a diverse set of environments • the success of brown trout as a transplanted species • a spawning time which can be manipulated • the existence of rapid-growing populations with late maturity • a potentially wide environmental tolerance • the long history of artificial propagation of the species • the long scientific interest in the biology of brown trout • the good knowledge about aquaculture of related species (Atlantic salmon and rainbow trout)

This suggests that brown trout is currently under-utilized as a resource for aquacultural production.

62 AQUACULTURE-RELATED GENETIC ISSUES

Choice of source population. It is clear from a number of experiments with brown trout that different populations vary in their performance with respect to several traits that are interesting for aquaculture. This suggests that a large number of populations should be compared if the full genetic potential of the species were utilized. So far, only a few populations have been tested under aquaculture conditions.

In Norway, a large number of families were tested for tolerance to acid water, but only to the alevin stage. In Sweden and Norway, several populations were compared following smolt releases (sea ranching), but not directly compared in the freshwater stage before release. In France, one synthetic brown trout strain was compared with two Atlantic salmon strains in fresh and salt water for 3 years. In Sweden, six populations were compared in fresh water for 2 ½ years. Collectively, these experiments show that: • brown trout can compete with Atlantic salmon for at least 2 years in terms of aquaculture performance • the variation between trout populations may - and may not - be understood from knowledge about their origin (habitat, life history etc.) • very few populations/experiments show a commercial potential for sea ranching • preliminary results suggest a good potential for selective breeding to increase growth rate • only a large-scale, quantitative genetic experiment can estimate the true aquaculture potential of the species.

Economically important traits. Growth rate, tolerance to salt water, and rate of early maturation seem to be the most important traits for the selection of aquaculture strains. In particular, early sexual maturation seems to be a problem in many experiments. Moreover, there seems to be a conflict between rearing trout to a big size for salt water tolerance, and avoiding sexual maturation because of high growth rate in fresh water.

Selective breeding. As has been shown for Atlantic salmon and rainbow trout (and hundreds of other species), there is a high potential for selective breeding to increase the aquaculture performance of broiwn trout. However, with as much of the genetic variation distributed between populations, it seems that the first step is to make a good comparison of as many source populations as possible (but see Environment-related genetic issues). Some experiments underline this point.

The workshop also discussed very different objectives for selective breeding. For example, it could be possible to encourage the development of aquaculture lines which show severe maladaptation to the surrounding environment, should they escape.

Marker-assisted selection. A large number of tanks/ponds/net-pens are necessary to make a comparison of different populations and families with respect to aquaculture performance. Part of this problem is caused by the high number of groups (full- and half-sib families) that need to be compared, and part stems from the fact that the fish need to be group marked before being reared together. Recent developments in molecular marker techniques (microsatellite and minisatellite DNA), however, have made it possible to rear a very high number of groups together with no external mark.

63 Experiments can therefore be conducted with much more efficient environmental control (and with less use of rearing facilities).

In the future, it is conceivable that a large number of microsatellites can be used to mark chromosome segments and genetic loci that are important for performance (so-called quantitative trait loci). The development of hundreds of microsatellite loci in salmonid species (Atlantic salmon and brown trout, rainbow trout and Pacific salmon, as e.g. in the SALMAP project) will probably show the potential.

Chromosome manipulation. Simple techniques (heat- or pressure shock) can be used to produce triploid fish which do not mature sexually (females) or are not fertile (males). The same techniques can be used together with sex reversal to produce all-female lines (either diploid or triploid), tetraploid fish, or triploid interspecific hybrids. Judging from the problems with early sexual maturity in brown trout (and see below), it seems that sterility is an interesting aspect of trout aquaculture.

Transgenesis. It has been demonstrated that transgenic fish expressing (extra) growth hormone genes for prolonged periods can show a dramatic increase in growth rate. Transgenesis could also be an alternative way of producing sterile fish or other types of fish maladapted to the environment.

ENVIRONMENT-RELATED GENETIC ISSUES

An evaluation of the genetic impact of aquaculture on the environment, relies on knowledge about (1) the genetic structure of the species, (2) the genetic changes occurring in captivity, and on (3) the indirect and direct genetic effects of releasing fish. The genetic effects of accidentally released fish will be similar to the effects of intentionally released fish, other things being equal. In the present report, the relevant genetic issues are discussed in the sections on Stocking impact assessment and Conservation.

In addition to the genetic aspects of the interactions between aquaculture and the surrounding wild populations, a number of other environmental issues need consideration. These issues, which need to be discussed in a forum representing scientists from a number of disciplines, include: • ecological interactions • diseases and parasites • predation and overharvesting • other environmental impact (organic pollution).

CONSTRAINTS/PROBLEMS THAT NEED SCIENTIFIC SOLUTIONS

Constraint no. 1. Some lessons suggest that other salmonid species are more valuable for aquacultural production than brown trout: • rainbow trout grow better in fresh water than brown trout • Atlantic salmon grow better in salt water than brown trout (or show later maturity at the same growth rate)

64 • early sexual maturity appears to accompany rapid growth • it is difficult to produce a high percentage of brown trout smolts with fully developed saltwater tolerance.

Recommendation no. 1. A large-scale, quantitative genetic experiment needs to be carried out to estimate the true aquaculture potential of brown trout (Current knowledge stems partly from comparisons of non- selected strains of brown trout with selected strains of other species). This experiment should make use of molecular genetic possibilities for marking large numbers of families to be reared together.

Constraint no. 2. There are conflicts between choosing the best population for contained culture, choosing the best population for sea-ranching, and ”choosing” the best population for accidental release (i.e. should they escape) to the environment. This conflict partly stems from the different aims of the release, partly from the genetic issues involved, and partly from the scale of the release.

Recommendation no. 2. An evaluation of brown trout aquaculture needs to consider the effects of accidentally released fish from culture operations. These considerations should follow guidelines that appear from conservation strategies and stocking impact assessments.

Constraint no. 3. The genetic effect of a release can be estimated as the rate of interbreeding between the released and the local population multiplied by the genetic difference between them. One strategy is therefore to minimise the rate of interbreeding, another is to minimise the genetic difference between escaped and native fish, should interbreeding occur.

Recommendation no. 3. A precautionary approach to the development of brown trout aquaculture would be to minimise the potential genetic effects by using local sources for developing cultured strains, and to secure physical and biological containment of the cultured fish.

RELEVANT LITERATURE

Alm, G. (1959). Connection between maturity, size and age in fishes. Report of the Institute of Freshwater Research, Drottningholm 40, 5-145.

Dalziel, T. R. K., Kroglund, F., Lien, L. & Rosseland, B. O. (1995). The REFISH (Restoring Endangered Fish In Stressed Habitats) project, 1988-1994. Water, Air, and Soil Pollution 8, 321- 326.

Edwards, D. & Gjedrem, T. (1979). Genetic variation in survival of brown trout eggs, fry and fingerlings in acidic water. SNSF (Ås, Norway) Fagrapport 16/79, 28 pp.

Elliott, J. M. (1994). Quantitative Ecology and the Brown Trout. Oxford: Oxford University Press.

65 Ferguson, A. (1989). Genetic differences among brown trout, Salmo trutta, stocks and their importance for the conservation and management of the species. Freshwater Biology 21, 35-46.

Gjedrem, T. & Gunnes, K. (1978). Comparison of growth rate in Atlantic salmon, pink salmon, Arctic char, sea trout and rainbow trout under Norwegian farming conditions. Aquaculture 13, 135- 141.

Hindar, K., Ryman, N. & Utter, F. (1991). Genetic effects of cultured fish on natural fish populations. Canadian Journal of Fisheries and Aquatic Sciences 48, 945-957.

Jonsson, B. (1982). Diadromous and resident trout Salmo trutta: is their difference due to genetics? Oikos 38, 297-300.

Jonsson, N., Jonsson, B. & Hansen, L. P. (1994). Sea-ranching of brown trout, Salmo trutta L. Fisheries Management and Ecology 1, 67-76.

Krieg, F., Quillet, E. & Chevassus, B. (1992). Brown trout, Salmo trutta L.: a new species for intensive marine aquaculture. Aquaculture and Fisheries Management 23, 557-566.

Näslund, I. & Henricson, J. (1995). Growth and sexual maturation of six stocks of brown trout Salmo trutta L. in culture. Aquaculture Research 27, 815-822.

Ryman, N. (1983). Patterns of distribution of biochemical genetic variation in salmonids: differences between species. Aquaculture 33, 1-21.

Skaala, Ø., Jørstad, K. E. & Borgstrøm, R. (1996). Genetic impact on two wild brown trout (Salmo trutta L.) populations after release of non-indigenous hatchery spawners. Canadian Journal of Fisheries and Aquatic Sciences 53, 2027-2035.

Ugedal, O., Finstad, B., Damsgård, B. & Mortensen, A. (1998). Seawater tolerance and downstream migration in hatchery-reared and wild brown trout. Aquaculture 168, 395-405. e) Review of brown trout phylogeography (reported by Louis Bernatchez, University of Laval)

SUMMARY OF PRESENTATIONS FOR THE PHYLOGEOGRAPHY SECTION

This summary reports on presentations that were made in the context of the phylogeography panel during the Troutconcert meeting that was held last July in Silkeborg, Denmark. Essentially, the content of the panel was constituted of two presentations that summarised the state of knowledge regarding large-scale phylogeographic patterns in the brown trout population complex.

Dr. Jose-Luis Garcia-Marin made a presentation that summarised the large-scale genetic structure of brown trout, as depicted from allozyme data. More specifically, this synthesis included the reanalysis of previously published data on 232 populations from throughout the species distribution range. Populations suspected to have suffered from intense stocking were omitted. The information

66 of eleven polymorphic loci were considered sufficiently comparable among different studies to be analysed together. The data set was analysed by principal component analysis (PCO). PCO-1 and PCO-2 respectively accounted for 37% and 17% of the overall genetic variance. The locus LDH-C* contributed most importantly to PCO-1 whereas CKA1* contributed most to PCO-2. Globally, this allowed to define four (I to IV) major lineages, although the definition of the so-called lineages III and IV was somehow ambiguous. Nevertheless, these four lineages exhibited a distinct pattern of geographic distribution from which Dr. Garcia-Marin proposed a model of colonisation. According to this model, lineage IV which is associated with the Mediterranean drainages did not contribute to the northward recolonization, whereas the three other lineages would have been involved. Thus, during glacial retreat, recolonisation is proposed to have occurred mainly into adjacent areas from (1) a north-western migration from an eastern Mediterranean-Caspian refuge, (2) a northern expansion from a refuge in Atlantic drainages of Iberia and southern France, and (3), a northern and eastern radiation of a refuge centred near the English channel. Extant populations in deglaciated areas are suggested to represent mixed or pure descendants of these migrating groups.

Dr. Louis Bernatchez presented a summary of the actual knowledge of brown trout phylogeographic structure based on mitochondrial DNA variation. Results presented summarised the content of 8 previously published studies in addition to new data. This translated into an analysis of nearly 1800 trout representing 180 populations. RFLP and sequence data were standardised and included in a global analysis involving (1) the quantification of overall diversity, (2) a phylogenetic analysis of relationships among mtDNA haplotypes, (3) an analysis of molecular variance to partition genetic diversity, among groups, and among populations within groups, and (4) and analysis of mismatch distribution from which some evidence for time of population expansion was obtained.

A total of 74 haplotypes were resolved. These grouped into five major phylogenetic groups, each also composed of additional subgroupings. These groupings differed by net sequence divergence estimate varying between 1,2% and 2,2%. Although arguable, those values appear compatible with times of divergence associated with the Pleistocene glaciation events. These five groups each showed a unique geographic pattern of distribution. The « Atlantic » group was the only one found throughout the Atlantic basin, including in Morocco. The « Danubian » group was essentially confined to waters draining into the Black, Caspian, and Aral seas, with the exception of 1 isolated population in Greece. The Mediterranean brown trout populations either belonged to the so-called « Mediterranean » or « Adriatic » groups. This last group was found all the way east to Turkey whereas the former one was not observed beyond Greece. Both groups extended west to Spain. Finally, the « Marmoratus » group was essentially confined to the Marmoratus trout with traces of introgression with brown trout in 2 populations of Greece.

The analysis of molecular variance revealed that nearly 89% of the total genetic variance was imputable to differences among the five major phylogenetic groups. Within each of the five groups, the amount of genetic diversity imputable to population differentiation accounted for percentage varying between 62% and 93%. Consequently, brown is very highly structured genetically, both at the regional and the local scale. Populations clearly tended to be less differentiated in the North, possibly reflecting both the effect of ancestral population admixture in postglacial times, as well as their more recent radiation. The presence of important mountain ranges and isolated drainages may also contribute to stronger patterning in the south.

The mismatch distribution analysis revealed different patterns among major phylogeographic groups. While all of them suggested ancestral population expansion, the timing of such expansion

67 varied from one group to the other. Thus, for the Danubian group, a major expansion was suggested in a time frame varying between 110,000 and 220,000 years ago, depending on the mutation rate used in the calculations. Expansion of the Adriatic group was compatible with a time frame of 50,000-100,000 years. In contrast, values obtained for the Atlantic group suggested a very recent population expansion, in the time frame of 13,000-26,000 years. These values would be compatible with population expansion associated with the last glacier retreat.

Globally, while both allozyme and mtDNA data are not completely congruent, they both reveal the existence of major trout assemblages that are genetically distinct and associated with specific geographic areas. These may represent the basic Evolutionary Significant Units to be considered within the species. Further analyses of these data set should more strictly assess the overall congruence of these different markers. It would also be informative to overlay information of additional nuclear markers such as microsatellite data, as well as sequence data obtained at the nuclear DNA level. More detailed studies within each major areas, especially for southern populations, must also be performed in order to refine our understanding of brown trout evolutionary history on more restricted temporal and geographic scales. Finally, since the model of postglacial colonisation proposed by Dr. Garcia-Marin did not necessarily reach a consensus during the panel discussion, further investigation and debate on this particular aspect is still required. f) Local adaptation in brown trout populations (Reported by Bill Jordan, Zoological Society of London)

INTRODUCTION

The concept of local adaptation pervades the literature on the population biology of anadromous salmonid species, including that for the brown trout (Salmo trutta). However, often the evidence cited for local adaptation is indirect. For example, it is frequently suggested that the presence of local adaptation is required to explain the evolution of homing behaviour. Moreover, for a species such as the brown trout, which displays tremendously high levels of phenotypic diversity, it is a common assumption that at least some of that diversity must be adaptive.

Local adaptation of populations is both an important evolutionary process and an essential consideration in fisheries management. However, robust evidence for the presence of local adaptation is required before the concept can legitimately underpin management policy. Studies on local adaptation can only really be meaningful in the context of a clear definition of adaptation. Such a definition has been provided by Harvey and Pagel (1991).

Adaptation - a derived character which has evolved in response to a specific selective agent.

It is obvious from this definition that unequivocal demonstration of adaptation requires identification of both the character under selection and the agent of selection (preferably shown through experimental manipulation).

Local adaptation involves the component of genetic diversity which is distributed among populations, and evidence from population genetic surveys suggest that the potential for local adaptation is high in the brown trout. For example, in Scotland the value of among-population

68 genetic variance (FST) is an order of magnitude higher for brown trout populations (0.326; Stephen and McAndrew, 1990) than for the closely related Atlantic salmon (0.023; Jordan et al., 1992).

EVIDENCE FOR LOCAL ADAPTATION

The evidence for local adaptation in salmonids in general has been reviewed recently by Taylor (1991) and Elliot (1994), and will not be discussed in detail here. Both studies identified examples of evidence for local adaptation in a broad range of characters: e.g.

Physiology osmoregulatory ability resistance to disease swimming performance

Morphology numerous examples Development developmental rate timing of migration timing of spawning Behaviour agonistic behaviour migratory behaviour*

*In the discussion during this session many studies which have examined migratory behaviour in relation to local adaptation in brown trout were highlighted (e.g. Jonsson 1982; Svärdson and Fagerström, 1982; Huusko et al., 1990; Koljonen and Huusko, 1993; Jonsson et al., 1994).

Case Studies There are two widely cited cases of local adaptation in brown trout:

1. Lough Melvin is a small lake (22km2) in northwest Ireland which has been the subject of intensive work by Andy Ferguson and colleagues for many years. Three brown trout populations (sonaghen, gillaroo and ferox) are found in this lake which show marked differences in morphology, ecology and genetics (see Ferguson and Taggart, 1991)

2. Malcolm Elliot has collected a long time series dataset on the ecology of two brown trout populations in the Lake District of northwest England (Black Brow Beck and Wilfin Beck). He and co-workers have demonstrated differences in migratory behaviour, reproductive biology, variance in body size, and mechanisms of population regulation between these two populations (see Elliot, 1994 for review).

APPROACHES TO STUDYING LOCAL ADAPTATION

QTL markers The term quantitative trait locus (QTL) commonly refers to a gene which influences morphological characters such as size and shape in organisms, although strictly speaking it may refer to a gene influencing any character (physiological, behavioural, developmental, etc.). Usually the function of

69 the gene is unknown and its presence is demonstrated only through close physical linkage to other loci (known as QTL markers; e.g. allozyme, mini- or microsatellite loci) and their association with variation in a quantitative trait. Genome mapping studies are a traditional way of identifying QTLs, and the EU-funded SALMAP project may provide QTL markers which are conserved across salmonid species and which could be used for the study of local adaptation. In addition, a method for the identification of QTL markers from population survey data has recently been developed (Beaumont and Nichols, 1996) which may permit the use of data already collected in a novel analysis.

Candidate Loci An alternative approach to the study of local adaptation is through identification of candidate loci with known function, and which the biology of the organism suggests may have an important role in local adaptation. Some examples of these loci are listed below. In many cases it is easy to see the relationship between the gene function and the characters for which there is evidence for local adaptation in salmonid species.

Hormone and hormone receptor loci - the central role of the endocrine system in determining developmental rate, timing of migration, and timing of spawning suggests that these loci might be important in local adaptation.

Major Histocompatibility Complex (MHC) genes - the role of the MHC in the immune response suggests a potential genetic basis for local adaptation in disease resistance. These loci may also be important in homing through kin recognition (including agonistic behaviour), and inbreeding avoidance during mate choice.

Olfactory receptor genes - these genes produce receptors which are the basis of odour perception and discrimination. The importance of olfaction in homing suggests that temporal changes in levels of expression and among-population variation in allele frequencies of these genes may be involved in olfactory imprinting and homing specificity (i.e. migratory behaviour).

Enzyme loci - LDH-B* in Fundulus heteroclitus, MEP-2* in Atlantic salmon, and LDH-5*, PGI-2* and PGI-3* in brown trout are all well-worked examples of enzyme loci which appear to be involved in local adaptation of fish populations. Further investigation of other enzyme loci using the appropriate statistical analysis may reveal others in brown trout.

DISCUSSION

The discussion in this session revealed that although most participants felt that work on local adaptation in brown trout was very important, the literature contained few examples of relevant studies, and relatively few groups were actively involved in such research. Therefore the instigation of this type of research should be regarded as a priority, as a demonstration of widespread local adaptation in the species would have significant consequences for recommendations on management policy.

References Beaumont, M.A. and R.A. Nichols. 1996. Evaluating loci for use in genetic analysis of population structure. Proc. R. Soc. Lond., 263, 1619-1626.

70 Elliot, J. M.1994. Quantitative Ecology and the Brown Trout. Oxford Univ. Press, Oxford.

Ferguson, A., and J.B. Taggart. 1991. Genetic differentiation among the sympatric brown trout (Salmo trutta) populations of Lough Melvin, Ireland. Biol. J. Linn. Soc., 43, 221-237.

Harvey, P.H. and M. D. Pagel. 1991. The Comparative Method in Evolutionary Biology. Oxford University Press.

Huusko, A., O. Van Der Meer, and M.-L. Koljonen. 1990. Life history patterns and genetic differences in brown trout (Salmo trutta L.) in the Koutajoki river system. Pol. Arch. Hydrobiol., 37, 63-77.

Jonsson, B. 1982. Diadromous and resident trout Salmo trutta: is their difference due to genetics. Oikos, 38, 297-300

Jonsson, N., B. Jonsson, J. Skurdal, and L.P. Hansen. 1994. Differential response to water current in offspring of inlet- and outlet-spawning brown trout Salmo trutta. J. Fish Biol., 45, 356-359.

Jordan, W.C., A.F. Youngson, D.W. Hay, and A. Ferguson. 1992. Genetic protein variation in natural Atlantic salmon (Salmo salar) populations in Scotland: temporal and spatial variation. Can. J. Fish. Aquat. Sci., 49, 1863-1872.

Koljonen, M.-L., and A. Huusko. 1993. Genetic variation of brown trout stocks in the Koutajoki river system. Oulanka Reports, 12, 129-132.

Stephen, A.B. and B.J. McAndrew. 1990. Distribution of genetic variation in brown trout, Salmo trutta L., in Scotland. Aqua. Fish. Manag., 21, 47-66.

Svärdson, G., and A. Fagerström. 1982. Adaptive differences in the long-distance migration of some trout (Salmo trutta L.) stocks. Rep. Inst. Freshwater Res., Drottningholm, 60, 51-80.

Taylor, E.B. 1991. A review of local adaptation in salmonidae, with particular reference to Pacific and Atlantic salmon. Aquaculture, 98, 185-207. g) Brainstorm on the internet features of the CA (responsible person: Paulo Prodöhl, Queen's University of Belfast)

During this session the first version of the Troutconcert WWW site was thoroughly presented. All participants were very satisfied with the design and lay-out, and a number of suggestions were given for further documents and facilities to put on the web site (other than those already planned). It is not intended to give a detailed summary of the discussions here, as the word "brainstorm" should be taken very literally. However, it should be mentioned that the suggestions included a "fish population genetics glossary", and a first version of this has since then been established by Ms. Ewa Wlodarczyk, Sea Fisheries Institute, Poland. Another suggestion that was subject to much discussion (but has not yet been implemented) was the construction of a map, showing the geographical distribution of the phylogeographical races of brown trout that have been identified.

71 This feature would be useful to illustrate the phylogeographical history of brown trout and to stress the importance of conserving the different races. h) Preliminary discussion on management recommendations for conservation of genetic resources in brown trout (responsible person: Michael M. Hansen, Danish Institute for Fisheries Research)

This was also intended as a "brainstorm session" and no detailed summary will be given. The principles of giving recommendations for management and conservation of salmonid fishes were discussed. It was agreed that there are already many publications available that give specific recommendations for conservation of salmonid fishes, and there was no reason to "re-invent the wheel". Consequently, recommendations for managing and conserving brown trout populations should be based on these recommendations, BUT should also take the specific circumstances in brown trout into consideration (for instance, life-history features and the presence of highly divergent phylogeographical races). It was also agreed to establish a "conservation group" that should write the management recommendations. A meeting of this group has later been planned and is going to take place in Berne, Switzerland in late February. i) Genetic marker nomenclature and harmonisation of the use of markers (reported by Michael M. Hansen, Danish Institute for Fisheries Research)

INTRODUCTION

In the session on genetic marker nomenclature and harmonisation of the use of markers it was decided to focus primarily on microsatellites and restriction enzyme analysis of PCR amplified mitochondrial DNA segments. The outcome of the discussions is described in the following sections, which will also appear individually on the Troutconcert web site.

In the case of microsatellites it was agreed to establish a "positive list" of loci that have been found to work reliably in one or more laboratories. A strict priority list can not be established at present, as this will need a more thorough testing of loci in several labs.

In the case of mtDNA it was decided to recommend screening of three different segments, using a minimum set of restriction enzymes. Surely, other restriction enzymes detect variability than those listed, but it was the intention to include preferably the "highly informative" ones, i.e. those detecting common haplotypes and/or having high phylogeographical information value.

NOMENCLATURE FOR MOLECULAR MARKERS.

Allozyme electrophoresis was previously the universal technique for brown trout population genetics studies. However, a large number of different molecular techniques are now being applied to studies of the genetic structure and phylogeography of brown trout populations. Also, in the case of many salmonid fishes, including to some extent brown trout, molecular markers are being used in the context of aquaculture (marker assisted selection, mapping etc.). Finally, molecular markers

72 have become useful tools in experiments addressing questions at the interphase of behavioural ecology and population genetics, where information on parentage of individual fish is needed.

Apart from allozyme electrophoresis, the techniques and markers known to have been used for brown trout include • RFLP analysis of the whole mitochondrial genome • RFLP analysis of PCR amplified mtDNA segments (PCR-RFLP) • sequencing of various mtDNA segments • minisatellite multilocus probes • minisatellite single locus probes • microsatellites • RAPDs (Random Amplified DNA) • ribosomal DNA • various types of analysis (sequencing, RFLP, heteroduplex analysis etc.) of nuclear genes

For some of these markers there is already a well-established nomenclature. This applies to allozyme electrophoresis (1), minisatellite single locus probes (2) and, to some extent, sequencing of mtDNA segments (3). However, for some other of the most commonly applied types of markers and techniques, in particular microsatellites and mtDNA PCR-RFLP analysis, there is clearly a need for establishing an informative nomenclature.

Microsatellites Many microsatellites are currently being developed for brown trout, and, in addition, microsatellites developed for other salmonid species often work in brown trout as well. As several different laboratories are involved in the development of microsatellites and as there is not a specified nomenclature this has resulted in some confusion.

An unofficial nomenclature has in many cases already been adopted. It consists of a three letter code + a number. The three letter code designates the species for which the microsatellite was developed. The first letter is the first letter of the name of the genus, the next two letters are the first letters of the species name (for instance, Salmo trutta would be Str). The number is normally just for reference and not informative in itself. One problem is that different labs may by accident use the same numbers. This is probably one of the reasons why some groups have started to add a four- letter code (capital letters) to the locus names, designating the lab or institute where the microsatellite was developed. For instance, Ssa85DALH would be a microsatellite developed at Dalhousie. Though, obviously, many of the laboratories that develop microsatellites for salmonids are not involved in the concerted action and are therefore "out of reach", the CA recommends the use of a nomenclature as specified above. That is, a three letter code specifying the species for which the microsatellite was developed (e.g. Str) + a number + a four letter code specifying the lab in which the microsatellite was developed (e.g. DALH).

Other useful information that should be mentioned in publications includes • type of repeat (e.g. TG or GATA) • perfect or imperfect status of the microsatellite • GenBank Accession number

73 Specific alleles are usually designated either by size (in bp) or by capital letters. The first option must be considered the most informative, however, it must be stressed that estimated allele sizes should not be taken literally and cannot immediately be compared to allele sizes from the same loci analysed in other laboratories. In order to facilitate comparison of results from different laboratories, the CA recommends calibration of allele sizes by running the same samples in different laboratories.

MtDNA PCR-RFLP analysis The standard nomenclature for mtDNA RFLP data is to designate each restriction morph for each restriction enzyme used, by a capital letter, A, B, C etc. Haplotypes/composite genotypes are then designated by listing capital letters in a row, for instance AABCAC. However, restriction enzymes are usually not listed in any specific order, and in the case of PCR-RFLP more than one segment is usually analysed which may also make things a little complicated. The CA recommends some small adjustments to the existing nomenclature. First, the restriction enzymes used in the screening should be listed in alphabetic order, and restriction morph capital letters should then be listed in that order. Second, if haplotypes consist of restriction morphs from more than one segment, it is recommended to separate the restriction morphs by a “|”. In order to use the same designations for the same restriction morphs, the CA recommends calibration of morphs by exchange of samples among laboratories and side-by-side gel runs of what appears to be the same restriction morphs from trout from different geographical regions.

References (1) Shaklee, J.B., Allendorf, F.W., Morizot, D.C. & Whitt, G.S. (1990). Gene nomenclature for protein-coding loci in fish. Transactions of the American Fisheries Society, 119, 2-15.

(2) Prodöhl, P.A., Taggart, J.B. & Ferguson, A. (1995). A panel of minisatellite (VNTR) DNA locus specific probes for potential application to problems in salmonid aquaculture. Aquaculture, 37, 87-97.

(3) Bernatchez, L., Guyomard, R. & Bonhomme, F. (1992). DNA sequence variation of the mitochondrial control region among morphologically and geographically remote European brown trout Salmo trutta populations. Molecular Ecology, 1, 161-173.

74 MICROSATELLITE LOCI PRESENTLY IN USE FOR BROWN TROUT.

The following list includes a number of microsatellite loci presently used routinely for brown trout by laboratories in the concerted action. Loci that have been found to be technically problematic, for instance exhibiting pronounced stuttering resulting in scoring difficulties, or clearly exhibiting null alleles, have been omitted from the list. We strongly encourage researchers in brown trout population genetics to use loci from this list in order to facilitate comparison of results from different studies.

Locus Source Annealing App. no. alleles Size range Repeat Comments Primer sequences temp. observed (bp) Str15INRA 1 58oC 10 193-225 CT 5'-TGCAGGCAGACGGATCAGGC-3' 5'-AATCCTCTACGTAAGGGATTTGC-3' Str60INRA 1 60oC 9 87-111 GT 5'-CGGTGTGCTTGTCAGGTTTC-3' 5'-GTCAAGTCAGCAAGCCTCAC-3' Str73INRA 1 58oC 11 138-162 GT 5'-CCTGGAGATCCTCCAGCAGGA-3' 5'-CTATTCTGCTTGTAACTAGACCTA-3' Str79INRA 2 60oC Str79INRA-1: 8 118-128 GT Duplicated 5'-GGAAGGGGGGTGTATCAGC-3' Srt79INRA-2: 2 114-116 5'-GGGATTTGGCCTGTATCCG-3' Str85INRA 2 55oC 19 146-200 CT 5'-GGAAGGAAGGGAGAAAGGT-3' 5'-GGAAAATCAATACTAACAA-3' Str543INRA 2 55oC 24 119-169 CT 5'-ATTCTTCGGCTTTCTCTTGC-3' 5'-ATCTGGTCAGTTTCTTTATG-3' Str591INRA 2 55oC 22 146-198 CT 5'-CTGGTGGCAGGATTTGA-3' 5'-CACTGTCTTTCGTTCTT-3' BS131 3 50oC 10 149-177 GT 5'-CACATCATGTTACTGCTCC-3' 5'-CAGCCTAATTCTGAATGAG-3' T3-13 3 54oC 21 175-235 GT 5'-CCAGTTAGGGTTCATTGTCC-3' 5'-CGTTACACCTCTCAACAGATG-3' Str43INRA 3 56oC Str43-1INRA: 3 180-192 GT Duplicated 5'-GTTGTGGGCTGAGTAATTGG-3' Str43-2INRA: 7 141-161 5'-CTCCACATGCATCTTACTAACC-3' Strutta 58 4 56oC 38 102-190 GT 5'-AACAATGACTTTCTCTGAC-3' 5'-AAGGACTTGAAGGACGAC-3' Strutta 12 4 56oC 28 124-216 GT 5'-AATCTCAAATCGATCAGAAG-3' 5'-AGCTATTTCAGACATCACC-3' Ssa197 5 60oC 18 107-177 GTGA 5'-GGGTTGAGTAGGGAGGCTTG-3' (+GT) 5'-TGGCAGGGATTTGACATAAC-3' Ssa171 5 60oC 22 201-243 GTGA alleles 5'-TTATTATCCAAAGGGGTCAAAA-3' +GT separated by 5'-GAGGTCGCTGGGGTTTACTAT-3' both two and four bp

75 Locus Source Annealing App. no. alleles Size range Repeat Comments Primer sequences temp. observed (bp) OmyFgt1TUF 6 60oC 27 187-263 GT 5'-AGATTTACCCAGCCAGGTAG-3' 5'-CATAGTCTGAACAGGGACAG-3' SsoSL417 7 52oC 15 161 - 197 GT 5'-TTGTTCAGTGTATATGTGTCCCAT-3' 5'-GATCTTCACTGCCACCTTATGACC-3' SsoSL438 8 5 x 54oC to 7 103 - 115 GT 5'-GACAACACACAACCAAGGCAC-3' 48oC with 1 5'-TTATGCTAGGTCTTTATGCATTGT-3' degree interval

1) Estoup, A., Presa, P., Krieg, F., Vaiman, D. & Guyomard, R (1993). (CT)n and (GT)n microsatellites: a new class of genetic markers for Salmo trutta L. (brown trout). Heredity 71, 488-496. 2) Presa, P. & Guyomard, R. (1996). Conservation of microsatellites in three species of salmonids. Journal of Fish Biology 49, 1326-1329. 3) Estoup, A., Rousset, F., Michalakis, Y., Cornuet, J.-M., Adriamanga, M. & Guyomard, R. (1998). Comparative analysis of microsatellite and allozyme markers: a case study investigating microgeographic differentiation in brown trout (Salmo trutta). Molecular Ecology 7, 339-353. 4) Poteaux, C. (1995). Interactions génétiques entre formes sauvages et formes domestiques chez la truite commune (Salmo trutta fario L.). 110p. Ph.D. thesis, Université Montpellier II, Montpellier. 5) O'Reilly, P.T., Hamilton, L.C., McConnell, S.K. & Wright, J.M. (1996). Rapid analysis of genetic variation in Atlantic salmon (Salmo salar) by PCR multiplexing of dinucleotide and tetranucleotide microsatellites. Canadian Journal of Fisheries and Aquatic Sciences 53, 2292-2298. 6) Sakamoto, T., Okamoto, N., Ikeda, Y., Nakamura, Y. & Sato, T. (1994). Dinucleotide-repeat polymorphism in DNA of rainbow trout and its application in fisheries science. Journal of Fish Biology, 44, 1093-1096. 7) Slettan, A., Olsaker, I. & Lie, Ø. (1995). Atlantic salmon, Salmo salar, microsatellites at the SSOSL25, SSOSL85, SSOSL311, SSOSL417 loci. Animal Genetics 26, 281-282. 8) Slettan, A. (1995). GenBank Acc. no. Z49134.

76 PCR PRIMERS AND RESTRICTION ENZYMES FOR RFLP ANALYSIS OF BROWN TROUT MITOCHONDRIAL DNA SEGMENTS.

Restriction enzyme analysis of PCR amplified mtDNA segments is a fast and uncomplicated technique that is being used increasingly in studies of brown trout population genetics and phylogeography. In order to facilitate comparison of results from different studies and by different laboratories the Concerted Action strongly recommends the use of the following primer sets (or homologous primers). Also, it is recommended to analyse the amplified segments using, as a minimum, the listed restriction enzymes. All three mtDNA segments have been found to exhibit variation in previous studies of brown trout. It should be noted, however, that the listed set of restriction enzymes has been defined mainly on the basis of studies of brown trout from the Atlantic phylogeographical group. In the case of studies of trout from other phylogeographical groups it is recommended to conduct a screening using a larger number of restriction enzymes.

In order to minimise the number of mismatches, the ND1 and ND5/6 primers listed have been redesigned from rainbow trout sequences (reference 1), but they are homologous to the universal primers previously described by Cronin et al. (1993; ref. 1) and Hall & Nawrocki (1995; ref 2). Also, one of the CytB/D-loop primers has been modified in accordance with the sequence from rainbow trout, whereas the other primer already exhibited perfect match.

The following PCR programme should yield satisfactory amplification on most thermocyclers:

30 cycles of: 30 s at 95oC 45 s at 52oC (alternatively, try annealing temperatures between 50 - 55 oC). 2 min 30 s at 72oC.

The ND5/6 and D-loop primers amplify adjacent segments of the mitochondrial genome. It is a convenient way of analysis to pool the two PCR products prior to restriction enzyme digest. The digested pooled products may be electrophoresed on 1.2% agarose gels in 0.5 x TBE buffer. If PCR products are electrophoresed separately the same electrophoretic conditions are also useful. However, it has also been found that electrophoresis on 2% agarose gels yields satisfactory resolution, and several rows of samples (for instance four rows in a 25 cm long gel) may be run on the same gel. Staining with ethidium bromide and visualisation under UV-light should allow for scoring of fragments at least as small as 200 bp.

77 MtDNA Reference Expected size of Recommended minimum Primer sequences segment amplified segment set of restriction enzymes in rainbow trout ND1 4 2006 bp Alu I, Ava II, Hae III, Hinf Forward primer: I, Hpa II. 5'-GCCTCGCCTGTTTACCAAAAACAT-3' (position 2988 - 3012 in rainbow trout mtDNA sequence) Reverse primer: 5'-GGTATGGGCCCGAAAGCTTA-3' (position 4974 - 4994 in rainbow trout mtDNA sequence) ND5/6 4 2470 bp Ava II, Hae III, Hinf I, Forward primer: Taq I, Xba I. 5'-AATAGCTCATCCATTGGTCTTAGG-3' (position 12873 - 12897 in rainbow trout mtDNA sequence) Reverse primer: 5'-TAACAACGGTGGTTTTTCAAGTCA-3' (position 15319 - 15343 in rainbow trout mtDNA sequence) Cyt B/ D-loop 5, 6 2354 bp Alu I, Asn I, Hinf I, Hpa Forward primer: II, Mbo I, Nci I, Rsa I, Taq 5'-TGACTTGAAAAACCACCGTTGTTA-3' I. (position 15319 - 15343 in rainbow trout mtDNA sequence) Reverse primer: 5'-GTGTTATGCTTTAGTTAAGC-3' (position 1013 - 1033 in rainbow trout mtDNA sequence)

1) Cronin, M.A., Spearman, W.J., Wilmot, R.L., Patton, J.C. and Bickham, J.W. (1993). Mitochondrial DNA variation in Chinook(Oncorhynchus tshawytscha) and chum salmon (O. keta) detected by restriction enzyme analysis of polymerase chain reaction (PCR) products. Can. J. Fish. Aquat. Sci. 50, 708-715. 2) Hall, H.J. and Nawrocki, L.W., 1995. A rapid method for detecting mitochondrial DNA variation in the brown trout, Salmo trutta. J. Fish Biol. 46, 360-64. 3) Zardoya, R, Garrido-Pertierra, A. & Bautista, J.M. (1995). The complete nucleotide sequence of the mitochondrial DNA genome of the rainbow trout, Oncorhynchus mykiss. Journal of Molecular Evolution 41, 924-951. 4) Nielsen, E.E., Hansen, M.M. & Loeschcke, V. (1998). Improved primer sequences for the mitochondrial ND1, ND3/4 and ND5/6 segments in salmonid fishes: application to RFLP analysis of Atlantic salmon. Journal of Fish Biology 53, 216-220. 5) Bernatchez, L. & Danzmann, R.G. (1993). Congruence in control-region sequence and restriction site variation in mitochondrial DNA of brook charr (Salvelinus fontinalis Mitchill). Molecular Biology and Evolution 10, 1002-1014. 6) Bernatchez, L. & Osinov, A. (1995). Genetic diversity of trout (genus Salmo) from its most eastern native range based on mitochondrial DNA and nuclear gene variation. Molecular Ecology 4, 285-297.

78 CALIBRATION OF MICROSATELLITE MARKERS AMONG LABORATORIES

It was agreed that calibration of markers among laboratories is in general needed before it is possible to compare results obtained using the same markers but in different laboratories. However, particularly in the case of microsatellites it is necessary to know if an allele reported from one laboratory corresponds to an allele reported from another. Consequently, it was decided to establish a reference sample of DNA from a few individuals that could be distributed to and run in all laboratories doing microsatellite work. A reference sample consisting of two marmoratus trout, two Mediterranean trout and two Atlantic trout was later established, and aliquots of the DNA were distributed to interested participants. j) Preliminary suggestions for topics at the next workshop.

The group from Girona, Spain, kindly offered to host the next workshop. Consequently, the workshop will take place in Girona, Spain, 28 June - 4 July. The topics listed below were suggested for the next workshop. Some of the topics were already key issues in relation to the whole CA, whereas others were topics of high importance that were identified during the workshop. However, this is only a preliminary list and some changes are likely to occur in the final program for the second workshop of the CA.

Suggested topics/sessions: Sea trout; genetic population structure, conservation. Responsible person: Ewa Wlodarczyk

Endangered populations. Responsible person: Patrick Berrebi

Comparative data analysis. Responsible person: Andy Ferguson

Structure of Framework V. (We have asked DG XIV if it would be possible to send a representative to talk about this issue)

Harvesting of trout populations, effects of regulations. Responsible person: Michael M. Hansen

Quantitative variation. Responsible person: Uli Schliewen.

Aquaculture (part 2). Responsible person: Kjetil Hindar.

Conservation (part 2). Responsible person: Carlo Largiader.

In addition, it was suggested to invite guest speakers to give presentations and lead specific sessions.

79 80 ANNEX 3.

SUMMARY OF IBERIAN TROUT GENETICS MEETING

UNIVERSITY OF SANTIAGO DE COMPOSTELA, LUGO, SPAIN, 21-23 SEPTEMBER 1998.

ORGANISED BY DR. PAULINO MARTINEZ.

AIMS OF THE MEETING

The aim of this meeting was to exchange information and results among research groups working on trout genetics in Iberia (Portugal + Spain), including both Troutconcert participants as well as other research teams. The meeting was undertaken during 21 and 22 September. At Wednesday 23 morning, the Lugo and Girona groups (represented by Nuria Sanz and Jose Luis Garcia-Marín) discussed interpretation of their results and future research cooperation.

The following groups within the CA participated in the meeting: • Porto (Paulo Alexandrino and Agostinho Antunes) • Girona (Jose Luis García-Marín, Nuria Sanz, and Carles Plá) • Lugo (Paulino Martínez, Laura Sanchez, Jaime Castro, Belén G. Pardo, Ana Viñas, and Pablo Presa)

Additional groups not included in the CA: • Zaragoza (Michel Villalta) • Madrid (Juan Suárez) • Oviedo (Juliana Pérez and José Luis Martínez)

The two-day meeting was divided into four sessions devoted to a) population structure of brown trout in Iberia b) management and conservation of trout populations c) genetic markers to study trout populations d) standardisation and homogenisation of genetic variation in trout populations.

PROGRAM

The program of the meeting was the following:

21 September Monday

10.00 Paulino Martínez Presentation of the meeting in the context of the concerted action. Priority of coordination of different brown trout Iberian research groups for conservation resources in this species.

81 10.30 Chairman: Paulo Alexandrino Genetic structure of brown trout in Iberian Peninsula (micro and macrostructural aspects).

• Pablo Presa: Phylogeographic analysis of brown trout in Europe by using microsatellites markers.

• José Luis García-Marín: Phylogeography of brown trout from the Iberian Peninsula using isozymes

• Agostinho Antunes: Preliminary results of brown trout genetic structure in northern rivers from Portugal by using isozymes.

• Paulino Martínez: Microstructural aspects of brown trout at the limit of sea trout distribution (population subdivision, gene flow, model of genetic structure)

13.30 Lunch

16.30 Chairman: Carles Plá Conservation and resource management of brown trout in the Iberian Península.

• Carles Plá: Analysis of stocking programmes and guidelines for resource conservation of brown trout in Iberia

• Paulino Martínez: Failure of stocking programmes in Northwestern Spain.

22 September Tuesday

9.30 Chairman: Paulino Martínez Genetic markers: advantages and disadvantages.

• José Luis García-Marín. Isozyme markers for population structure and resource management analysis

• Paulo Alexandrino: Isoelectric focusing. A resolutive technique for detecting hidden isozyme genetic variation

• Pablo Presa: Microsatellites: Phylogenetic power and statistical approaches for reconstructing phylogenies.

• Jaime Castro: rDNA RFLPs as genetic markers for resource management in brown trout.

• Laura Sánchez: Chromosome polymorphisms in brown trout: their use for population and phylogenetic analysis

• Michel Villalta/ Juan Suárez: mitochondrial RFLPs for analysis of structure in brown trout

82 13.30 Lunch

16.30 Chairman: José Luis García-Marín Harmonization of nomenclature of genetic markers. Main research lines of the different groups from Iberian Península and future coordination.

19.30 Dinner and farewell

SUMMARY OF SESSION 1 "GENETIC STRUCTURE OF BROWN TROUT IN IBERIAN PENINSULA (MICRO AND MACROSTRUCTURAL ASPECTS)". Chairman: P. Alexandrino

PHYLOGEOGRAPHY OF BROWN TROUT FROM THE IBERIAN PENINSULA USING ISOZYMES C. Pla, J.L García-Marín, N. Sanz, University of Girona. Presenter: J.L. Garcia-Marin

This presentation expands the results described in García-Marín & Pla, 1996, Heredity 77:313-323. Allele frequency differences at 33 polymorphic protein-coding loci were compared among 70 collections from naturally spawning brown trout Spanish populations. Other collections were excluded where we suspected large introgression or displacement by exogenous fish on the basis of the LDH-C*90 allele frequency. The Garona River collections were the only where the presence of this LDH-C*90 allele was considered native. The total gene diversity, including 8 additional invariant loci, was high (HT=0.108), but only the 39% of this variation was shared among collections. Similar levels of genetic differentiation were observed between collections within rivers (GCR=20%), between rivers within drainages (GRD=21%) and between the Garona River, the rest of Spanish Atlantic-flowing rivers and the Mediterranean drainage (GDT=20%). The plots based on the two Principal Coordinates (PCO) axes of the matrix of Nei’s distance, clearly differentiated these three sources: Garona River, other Atlantic-flowing rivers and Mediterranean rivers. The Guadalquivir River was the most differentiated among the Atlantic flowing rivers. The third axis distinguished most of the collections from the Tajo and Duero Rivers. No clear patterns were observed for collections taken from rivers northward the Duero River, in the Cantabrian range. These results suggest a marked hydrographical pattern for the large Atlantic rivers and an effect of migration of anadromous fish in the Cantabrian rivers.

No clear hydrographical patterns were observed among the Mediterranean rivers. The wide distribution among rivers of groups identified by the PCO-plots may be associated with several major trout lineages colonising these rivers in the past, as suggested in Greek populations.

83 MICROSTRUCTURAL ASPECTS OF BROWN TROUT AT THE LIMIT OF SEA TROUT DISTRIBUTION (POPULATION SUBDIVISION, GENE FLOW, MODEL OF GENETIC STRUCTURE) P. Martinez, L. Sanchez, J. Castro, B.G. Pardo, Univ. Santiago de Compostela-Lugo Presenter: P. Martinez

OBJECTIVES

-Influence of the anadromous form in the genetic structure of Salmo trutta.

-Microgeographic analysis

* Gene flow estimation * Isolation vs. homing behaviour * Correlation geographic and genetic distances * General model of genetic structure

-Evaluation of impact and outcome of stocking practices in Galicia.

MATERIALS

- 34 enzymatic loci - 4 main river basins from Galicia: three above and one below the limit of sea trout - 53 samples analyzed * 4 hatchery stocks * 2 non-flowing-media (lagoon, reservoir) * 47 native samples from rivers (isolated, free access)

- Preliminary data from Duero and Tajo basins (southwards)

STOCKING

- High divergence between native and hatchery populations * Higher heterozygosity in hatchery stocks (2-3 fold nat.) * Several loci with diagnostic value to evaluate stocking (LDH-C*; G3PDH-2*; IDHP-1*; sMDH-A2*; GPI-B1*) * Northeuropean and mixed origin of Spanish hatchery stocks

- Failure of stocking in Galicia: 8 out of 50 stocked natural populations (Galicia) showed signs of surviving stocked individuals * Non-flowing water bodies highly introgressed (H-W eq.) * Extreme Wahlund effect in river populations * Different stocking outcome in other Spanish regions (?) (hydrology, acidic waters, food availability, ...)

84 GENETIC STRUCTURE

- Influence of the anadromous form: genetic characteristics below the limit of sea trout * Lower genetic diversity * Higher isolation among basins * Factors involved in the distribution of diversity: hydrology, isolation among basins, stocking

- Microgeographic analysis * Large genetic divergence in a small geographic area * Factors responsible of this genetic divergence: Isolation (1/3) vs. homing behaviour (2/3) * Low gene flow among the populations studied: close to 1 with all estimators applied * Correlation between genetic and geographic distances +Significant when all samples included in the analysis +Non-significant when only intrabasin distances were taken into account * Different models of genetic structure according to the hierarchical level considered (islands, stepping-stone)

PRELIMINARY RESULTS OF BROWN TROUT GENETIC STRUCTURE IN NORTHERN RIVERS FROM PORTUGAL BY USING ISOZYMES. P. Alexandrino, A. Antunes, Univ. of Porto Presenter: A. Antunes

This presentation expands the results described in the submitted paper Antunes, A., Alexandrino, P. & Ferrand,N., Ecology of Freshwater Fish.

Allozyme and other protein loci were used for the first time to study the genetic structure of Portuguese brown trout (Salmo trutta) populations. Four out of the 22 protein coding loci examined were found to be polymorphic: CK-A1*, GPI-A2*, MPI-2* and TF*. Up to 247 individuals belonging to three tributaries of the Lima hydrological basin and a hatchery, all located in Northern Portugal, were analysed. Portuguese natural populations are similar to other Northern Iberian populations previously investigated and hatchery fish reveals an autochthonous origin. Samples from Portuguese brown trout were compared to other published data for 25 European populations, and a general perspective of the phylogeographic position of the Portuguese brown trout was obtained. Seven polymorphic loci (CK-A1*, GPI-A2*, GPI-B2*, LDH-C*, MEP-1*, MPI-2* and TF*) were used to perform a cluster analysis that showed a split in the group of Atlantic populations: North and South of Biscay Golf. The Portuguese samples are included in the latter subgroup with other Northern Iberian populations. The relationship between both subgroups suggests that they have diverged recently, probably associated to the glacial periods of the Quaternary.

MACROGEOGRAPHIC PATTERN OF MICROSATELLITE VARIATION IN BROWN TROUT P. Presa, Univ. Vigo, R. Guyomard, INRA, France Presenter: P.Presa

Genetic variation at ten microsatellite loci was investigated in 25 wild brown trout populations collected throughout most of its species range and representing four different putative species of the

85 S. trutta L. complex, S. trutta, S. marmoratus, S. carpio and S. ischchan, two domesticated stocks and two Atlantic salmon samples. Unbiased expected population heterozygosity ranged from 0.12 to 0.41 (average value = 0.327) and explained 45% of the total gene diversity. In all cases, microsatellite variations allowed to recover the four major geographical groups already recognized with mtDNA and protein loci: Atlantic S. trutta (i.e. Atlantic Ocean, North and Baltic sea basins), Western Mediterranean S. trutta, Eastern S. trutta (Black, Caspian and Aral sea basins) and S. marmoratus. Bootstrap values on individuals were very high despite the low number of individuals analysed per population (ca 10) and phylogenies were rather insensitive to sample size.

SUMMARY OF SESSION 2 "MANAGEMENT AND CONSERVATION OF SPANISH POPULATIONS" Chairman: Carles Pla

Carles Pla introduced the session talking about the concept of management and the usefulness of conservation of the autochtonous resources in a local area. He reviewed the Spanish policy concerning the management of brown trout populations according to the political status of the different regional administrations. Since 1978, Spain has been divided in several regional administrations named Autonomous Communities, with different political status. This division has strongly conditioned the management policy of this resource. First, because until 1978 the management had been carried out as a global policy in all Spain by ICONA (Spanish Institute for Wildlife Conservation); second, the different communities have received this management policy at different times. In this sense, reach a global status in Spain for brown trout is very difficult at the moment.

Following the introduction, Carles Pla and Paulino Martinez showed the data concerning this subject related to their areas of study, Catalonia and Galicia respectively. In Catalonia, located in the Northeast of Spain on the Mediterranean, the genetic diversity is high between rivers of different drainage and some times between streams of same drainage. In addition, no differences were found in different samples from the same stream. All these data has been obtained by allozymes and this methodology seems to be useful in order to management the brown trout populations existing in the Catalonian rivers. By contrast, the Mino River, in Galicia (located in the Northwest of Spain on the Atlantic coast), presents a particular characteristic. Data obtained by analysis of ribosomal DNA have shown a high divergence within the river and their tributaries. This supports the existence of different population units within the river.

CONCLUSION: In most cases, when only one population unit is recognised in the river, analysis of allozymes is the faster and cheaper methodology useful to manage the brown trout populations. The only one disadvantage is the scientific sacrifice of the individual. In other cases, when more than one unit are suspected in a population, molecular technologies involving DNA analysis are probably more appropriate.

86 SESSION 3 "GENETIC MARKERS: ADVANTAGES AND DISADVANTAGES" Chairman: Paulino Martínez

COMBINATION OF MUTATION MODELS AND GENETIC DISTANCES FOR MICROSATELLITES P. Presa, Univ. Vigo, R. Guyomard, INRA, France Presenter: P.Presa

Phylogenetic reconstructions from brown trout microsatellite variation were generated with UPGMA and Neighbor-joining methods using genetic distances developped under infinite allele (IAM) or stepwise mutation (SSM) models. Tree topologies differed with respect to the genetic distance and phylogenetic reconstruction method used and the phylogenic relationships between the four major brown trout lineages remains constroversial. The topologies observed within groups were more congruent with the geographic origin of samples when classical genetic distances were used instead of the SSM based genetic distances, illustrating the better performances of the first category of distances over the second one in recovering the correct genetic relationships between closely related populations. rDNA RFLPs AS GENETIC MARKERS FOR RESOURCE MANAGEMENT OF BROWN TROUT Dr. J. Castro. Dpto. Biología Fundamental. Area de Genética. Lugo. Spain

The most relevant features of the IGS in brown trout are three tandem repetitive motifs of 0.35, 1, and 0,2 kb, which show wide intra and interindividual variation. Two of them represent putative regulatory elements for rDNA transcription. The third, in the vicinity of 18S gene, is a mobile element (SINE-like) tandemly arrayed. Also, we have detected polymorphic restriction sites in the IGS due to the presence-absence of specific restriction targets. Finally, a long insertion of 14kb in the 28S gene, was detected in rDNA units of all individuals from a single population.

We carried out a population analysis to check the interest of this variation for resource management, and to analyze evolutionay aspects of rDNA genes. The analysis of the closest region to the 28S gene revealed four different length variants with population-specific patterns. Another interesting source of variation in the IGS is presence-absence polymorphic restriction sites. A variable BglII restriction site, which was absent in the Northern river drainage, was present in all other populations analyzed. This target could represent a river basin-specific marker. In the same way, the SacI site in the middle of the IGS, has evidenced a gradual latitudinal variation. The mobile element in the

87 neighbourhood of the 28S gene has provided a nearly diagnostic marker for monitoring stocking in Galicia. When applied to the introgressed Asma population, all individuals could be unambiguously classified by using this marker.

These results, though preliminary, demonstrates the large genetic divergence of Galician populations at the rDNA level, but also the power of concerted mechanisms like unequal crossing- over and gene conversion to homogenize rDNA variants within populations. This circumstance is probably stressed in a species with large population subdivision like brown trout. Our data also demonstrate the utility of rDNA variants to mark different population units, according to the hierarchical level considered. The length variants have proven useful for identifying single populations, while the presence-absence polymorphisms could be used to distinguish trout from larger geographic areas.

CHROMOSOME POLYMORPHISMS IN BROWN TROUT AND THEIR USE FOR RESOURCE MANAGEMENT Dr. L. Sánchez. Dpto Biología Fundamental. Area de Genética. Lugo. Spain.

The brown trout exhibits a stable karyotype with 2n=80 chromosomes: 10 biarmed pairs and 29 uniarmed. The main NOR-bearing pair 11 can appear as biarmed or uniarmed according to chromosome and individuals. An exhaustive analysis with classical and molecular banding techniques has allowed us to unambiguously identify 17 chromosome pairs. A size polymorphism in the heterochromatic short arms of pairs 8 and 9 was revealed, but the differences have been mostly gradual, which precludes its use as genetic marker. NOR regions have been demonstrated to be highly polymorphic, both in size and position. The main NOR-bearing pair 11 can appear from acrocentric to metacentric in the individuals analyzed. This variation is mainly due to differences between chromosomes rather than between populations and are therefore useful as individual genetic marker. However, a significant variation was detected between the river drainages analyzed showing smaller NORs in southern populations than in northern ones. An unusual NOR-site polymorphism was also detected, mainly in Miño basin, evidencing a high number of new NOR positions apparently as a consequence of a transposition mechanism.

SUMMARY OF DISCUSSION ON ALLOZYMES

This presentation was based on ideas presented in Grant et al, Defining Populations Boundaries for Fishery Management, In: (M. Saalem, ed) Genetics in Sustainable Fishery Management, (in press). And supported by the results of the electrophoretic studies on the Iberian brown trout.

The electrophoretic analysis of enzymatic proteins has been one of the chief sources of information about genetic structuring among fish populations. New DNA technologies have gained preeminece over previous proteins methods, giving the appearance of obsolescence to this older methodology. However, allozyme electrophoresis have proved as useful as newer DNA methods for detecting population differences and for identifying the genetic effects of exogenous hatchery reared fish released into the Iberian brown trout populations (LDH-C* locus). Among others advantages, protein electrophoresis detects, with low expertise, Mendelian variants at identifiable gene loci. Our group has detected 33 informative (polymorphic) nuclear loci to analyse the population structure of Iberian trout. The electrophoretic analysis of this large array of loci is relatively inexpensive when compared to DNA methods. Several world-wide distributed statistical package may be used for

88 analysing genotypic and allelic frequency data to estimate breeding structure within populations and differentiation and migration between populations. Limitations of allozyme techniques include the requirement for about one-gram of fresh or appropriately frozen tissue samples. In addition, the sampling of tissues from different organs often requires lethal sampling, although tissue biopsies (adipose fin) or blood may provide usable data (Transferrin locus) in some studies. Lethal sampling may not be desirable in threatened, usually upstream isolated, trout populations with low numbers of individuals.

ISOELECTRIC FOCUSING. A RESOLUTIVE TECHNIQUE FOR DETECTING HIDDEN ISOZYME GENETIC VARIATION Paulo Alexandrino. Univ. Porto, Portugal.

Isoelectric focusing (IEF) is an electrophoretic separation technique which takes advantage of protein isoelelectric point differences. Usually IEF is performed in thin acrylamide gels using a different range of ampholytes which permits different ranges of pH gradients. Visualization can be achieved by enzyme blotting, general protein staining or immunoblotting.

In this presentation some examples of IEF aplications were presented. In brown trout IEF has until now been used for the analysis of genetic variability at the following proteins: CK, GPI, PGM, TF and PX (non identified plasmatic protein). High variability was found at PX with more than 6 alleles.

The most evident possible advantages of this technique in the analysis of protein loci are: • it increases the capacity of detection of genetic variability (increase the number of alleles detected); • very small amounts of sample are needed; • IEF separations tend to result in highly reproductible patterns which is essential for homogeneity in allelic identification between laboratories.

MTDNA VARIATION IN SPANISH BROWN TROUTS: EVIDENCE OF ALIEN GENOTYPES IN RESTOCKING PROGRAMMES Michel Villalta, Servicio de Investigación Agroalimentaria, Departamento de Sanidad Animal, Diputación General de Aragón, Zaragosa, Spain.

Brown trouts native to the Ebro river system in the Northeast region of Spain provide an excellent example of seriously threatened populations with immediate risk of extinction. To document the genetic relationship among native and restocked brown trout in this geographical location (Mediterannean drainage), we have analysed mitochondrial DNA (mtDNA) variation after selective amplification and RFLP analysis with 7 restriction enzymes. We primarily focused our study on individuals collected from different tributaries of the Ebro river system. Four distinct mtDNA genotypes were identified: AAAAAAA, ABBBAAA, ABBBBBB and BBBBABB. The AAAAAAA genotype was present at maximum frequency in affluent systems or tributaries under low or no restocking action, and therefore was inferred to be a Mediterannean native brown trout genotype. The ABBBBBB and BBBBABB genotypes were characteristic Atlantic stocks currently employed in the restocking programs. Similarly, the ABBBAAA genotype was found in tributaries under intensive restocking but its origin is unknown. In addition, we performed the genetic survey

89 in a local government hatchery involved in a conservation program intended to protect this Mediterannean native brown trout through restocking. Surprisingly, in the hatchery we could find all of the above mentioned mtDNA genotypes in aproximately equal frequency in the Mediterranean native brown trout line. Consequently, this supposedly native stock employed for conservation and exploitation purposes constituted a hybrid population of different macrogeographic genetic variants (Atlantic, Mediterannean and "unknown" origin). We conclude that the genetic integrity of our Mediterannean brown trout taxon is being compromised through inappropiate hatchery broodstock selection and recommend that genetic data be incorporated into management strategy for native brown trout conservation.

SESSION 4 "HARMONIZATION OF GENETIC NOMENCLATURE AND MAIN RESEARCH LINES IN BROWN TROUT GROUPS FROM IBERIA PENINSULA"

Jose Luis García acted as coordinator of the fourth session. In this session, GPI, MDH and PGM electrophoretic variants detected for the Porto, Lugo and Girona groups were run side-by-side in starch gels to check their potential phenotypic homologies. Identical phenotypes were observed for null variants at GPI-B1 and B2 loci, sMDH-B1,2*75 in Girona was related to MDH-3*80 in Lugo. Variation at PGM detected by isoelectrofocusing by the Porto group dissapeared in the starch gels. We provided samples of muscle and eye to run side-by-side isoelectrofocusing at the University of Porto. During the preceding sessions, it was apparent that homogenising genetic nomenclature for other markers was an impossible task. Different groups amplify different regions of mtDNA to characterise RFLP, and only one group had worked on microsatellites (Vigo University) and other in ribosomal DNA and cytogenetics (Lugo University). A summary of research capabilities and research topics of the participant groups was done and listed below.

SUMMARY OF IBERIAN RESEARCH GROUPS WORKING ON TROUT GENETICS

(Name of contact person, molecular and biochemical techniques used and major research topics)

-PAULO ALEXANDRINO Centro de Estudos em Ciência Animal (CECA). ICETA-UP Campus Agrário de VairãoP-4480 Vila do Conde, Portugal. Allozymes ("classical" + Isoelectrofocusing), microsatellites, mtDNA-Dloop sequencing. Population structure, brown trout, phylogenetics, genomic evolution.

-EVA GARCIA Área de Genética Dpto. de Biología Funcional. Facultad de Medicina Universidad de Oviedo, Julián Claveria s/nE -33006 Oviedo, Spain. Allozymes, micro & minisatellites, mtDNA RFLP, FISH. Monitoring of stocking and hatchery stocks. Microgeographic differentiation, genetic control of family relatedness.

90 -ANNIE MACHORDOM/ANA ALMOVOVAR Sección de Ecología. IMIA Dirección. General de Agricultura y Alimentación. Comunidad de Madrid. Finca "El Encin".Apdo 126E-28800-Madrid. Spain. Allozymes, mtDNA RFLP. Monitoring of stocking and hatchery stocks, brown trout phylogenetics.

-PAULINO MARTINEZ/LAURA SANCHEZ. Area de Genética Dpto. Biología Fundamental Facultad de Veterinaria Campus Universitario s/nE-27002-Lugo. Allozymes, rDNA RFLP polymorphisms. Monitoring of stocking and hatchery stocks, brown trout phylogenetics, management programs.

-CARLES PLA/JOSE-LUIS GARCIA-MARINL.I.G.-UdG. Campus de Montilivi s/n17071. Girona, Spain. Allozymes, mtDNA-Dloop sequencing. Monitoring of stocking and hatchery stocks, management programs, brown trout phylogenetics.

-PABLO PRESA Xenetica Evolutiva MolecularFacultade de Ciencias. BioloxiaUniversidade de VigoE-36200 Vigo. Spain. Microsatellites. Brown trout and Salmonidae, phylogenetics, microgeographic differentiation.

-JOSE-ANTONIO SANCHEZ*Area de Genética Dpto. de Biología Funcional. Facultadde MedicinaUniversidad de OviedoJulián Claveria s/nE -33006 Oviedo. Spain. Allozymes, microsatellites, QTL mapping. Monitoring of stocking and hatchery stocks.

-MICHEL VILLALTAJOSE-MARIA BLASCO Servicio de Investigación Agroalimentaria.Diputación General de AragónE-50080 Zaragoza. Spain. mtDNA RFLP. Monitoring of stocking and hatchery stocks, brown trout phylogenetics

* No representative was able to participate in the meeting. The data may be incomplete.

91 ANNEX 4

Program − Second Workshop on identification, management and exploitation of genetic resources in brown trout (Salmo trutta), 28 June – 4 July in St. Feliu de Guixols, Spain.

Organisers:

Carles Pla, University of Girona Jose Luis Garcia-Marin, University of Girona Michael M. Hansen, Danish Institute for Fisheries Research

In collaboration with the City Council of St. Feliu de Guixols

Funded by the EU FAIR Programme (FAIR CT97 3882)

92 Program:

28 June Arrival, registration

29 June

9.00 - 9.30 Welcome and general information (Jose Luis Garcia-Marin & Carles Pla)

9.30 – 10.00 Status of the Concerted Action (Michael M. Hansen)

10.00 – 10.30 Session  presentation of ongoing research activities

10.00 – 10.30 Conservation perspectives of brown trout in Austria and a preliminary report on mtDNA variation in European grayling (Steven Weiss)

10.30 - 11.00 Coffee

11.00 - 12.30 Session  Endagered populations. Responsible person: Patrick Berrebi

12.30 - 15.00 Lunch (includes a walk back and forth to the restaurant)

15.00 - 17.00 Session  Sea trout; genetic population structure, conservation. Responsible person: Michael M. Hansen

15.00 – 16.00 Genetic structure of sea trout populations: A biased review promoting our own results at the expense of others (Michael M. Hansen)

16.00 – 16.30 Genetic structure of brown trout at the limit of the anadromous form (Paulino Martinez & Carmen Bouza)

93 16.30-17.00 Local and introduced stocks of sea trout: A genetic study utilising DNA profiling for family and stock identification (Kevin Glover)

20.30 (- 5.00?) Dinner followed by serious and strictly scientific discussions. Unfortunately, the site of the workshop will be closed at this time of the day and even though this does not sound attractive we will probably have to go to a bar.

30 June

9.00 – 9.40 Session  presentation of ongoing research activities (continued)

9.00 - 9.20 Presentation of brown trout population genetics research at Swedish University of Agricultural Sciences, Umeaa (Jens Carlsson)

9.20 - 9.40 mtDNA and protein variability of Portuguese trout populations and implications for proposed models of post-glacial recolonization (Paulo Alexandrino)

9.40 - 17.00 (excluding breaks) Session  comparative data analysis Responsible person: Andy Ferguson

9.40 – 10.30 Microsatellite polymorphism and mixed-stock analysis in cod and brown trout (Daniel Ruzzante)

10.30 - 11.00 Coffee

11.00 – 11.40 Analysis of DNA from old scale samples. A new tool for conservation of salmonid fishes (Einar Eg Nielsen)

11.40 – 12.30 Mutational properties and analysis of microsatellite data (title not definitive) (Craig Primmer)

12.30 - 15.00 Lunch

94 15.00 – 17.00 Discussion/brainstorm on data analyses. (Andy Ferguson will provide further information)

20.30 Dinner

1 July

Excursion

2 July

9.00 - 10.30 Evolutionary Significant Units (Robin Waples)

10.30 - 11.00 Coffee

11.00-12.30 Conservation priorities (Fred Allendorf)

12.30 - 15.00 Lunch

15.00-17.00 Conservation of brown trout. Includes discussion on management guidelines developed in the CA and discussion on how to prioritise trout populations for conservation. Responsible person: Carlo Largiader.

20.30 Dinner

3 July

9.00 - 10.30 Behavioural studies of salmonid fishes Responsible person: Ian Fleming

10.30 - 11.00 Coffee

11.00 - 12.30 Brainstorm; suggestions for future studies Responsible person: Michael M. Hansen

95 12.30 - 15.00 Lunch

15.00 - 17.00 Is there a life after the CA? What is going to happen afterwards, especially regarding the WWW site? Discussion on how to avoid overlap and unnecessary competition if/when people apply for R&D projects, etc., etc. Responsible person: Michael M. Hansen

4 July

Departure

Comments and practical points (in alphabetic order)

Credit Cards VISA and Mastercard are commonly accepted (American Express may cause problems).

Currency Spanish peseta (PTA). It is within the euro area, so foreing currencies are first transformed to euros and then to PTA. The rate of exchange PTA-euro is fixed at 166PTA=1euro. Currencies outside the euro area: European Union currencies, Swiss Franc, Norwegian Kroners, U.S. and Canadian Dollars should not have problems of exchange. Concerning other foreign currencies (Polish,Russian, Estonian, Turkish,...) there may be problems and it is probably most safe to bring US Dollars (or one of the other currencies listed above).

Hotel Accommodation is at the HOTEL EDEN ROCK, Punta Port Salvi, s/n, 17220 SANT FELIU DE GUIXOLS (Girona), SPAIN. TEL. +34 972320100, FAX +34 972821705.

How to go to St. Feliu The workshop will take place in St. Feliu de Guixols, a small city at the seaside, app. 40 km from Girona. More specifically, the scientific sessions will take place in an old monastery. The main airport is Barcelona Airport (BCN), app. 125 Km southward. There are bus services between Barcelona (Bus station) and St. Feliu., and trains between the Barcelona airport and Barcelona city. It is recommendable to find a direct flight to BCN airport and avoid Madrid Barajas airport. BCN is well connected with most of the main European cities.

The cost of the trip from BCN airport to St.Feliu, including train and bus, is app. 2.000 PTA (12 Euros). Remember that you will need PTAs in order to pay for this (so change some money into PTAs beforehand).

Additional information is available at the web site: http://www.cbrava.es The region for St Feliu is: BAIX EMPORDA

96 How to get your expenses refunded After you have returned home you send your spent tickets to the coordinator (i.e. Michael). You will then get your travel expenses refunded. Also, you will get paid what ever is left of your personal allowance. That is 180 ECU per day minus accommodation, meals and other expenses (excursion etc.). In other words, we pay the bill at the hotel. However, you will have to pay your individual expenses for phone calls etc. at the reception before you leave. Immediately after the workshop has taken place the administration at the Danish Institute for Fisheries Research will be closed for three weeks due to summer holidays, so there may be some waiting time before you receive your money.

Informal atmosphere Just like the first workshop it is the intention to have an informal atmosphere with lots of creative discussions and brainstorms. The workshop programme is flexible and there is room for changes and improvisations. Also, there is much "spare time". This is a deliberate choice as it is the experience from the previous meeting that people want to take this opportunity to arrange smaller meetings, discuss possible exchange visits or future collaboration, or perhaps they just want to get to know each other.

Language The languages spoken by most are Spanish and Catalan (the native language). Very few people speak foreign languages (even English) in connection with various services (e.g. train, bus). However, the symbolic language (hands, faces, etc) is universal! If you have problems finding your way back to the airport, one thing you can do is to find a public square and shout "Viva Real Madrid". Then a lot of nice people will be happy to show you the way to the nearest airport.

Papers If you want reprints/copies of papers from your colleagues, please mail them and ask them to bring the papers to the workshop. It is also a good idea to bring reprints of your own papers that you want to distribute to tohers. However, please note that we will not have a paper “exposition” like we had during the 1998 workshop and that opportunities for photocopying are limited.

Photocopying It is possible to do some photocopying, but not on a larger scale. Therefore, if you have some papers or documents that you want to distribute, please do the photocopying at home and bring the copies to the workshop.

Presentations and sessions The workshop consists of a number of theme sessions. In addition, some people are going to present their research activities on brown trout population genetics and these talks have been collected in a session called “Presentation of ongoing research activities”. Please, stick to the time table. Of course, this is a workshop and not a conference and it is the intention to have a flexible program. However, if there are topics that need be discussed in more detail than the allocated time permits we can always do this by the end of the day. Also, the two last sessions on the last day (“Suggestions for future studies” and “What to

97 do after the end of the CA” ) can easily be shortened, so there is still plenty of time for unfinished discussions, further presentations etc.

Each session is generally 1.5 - 2h. Persons responsible for the sessions are free to arrange them as they want to. Some may want to talk for 1 h and discuss for another hour. Others may ask for volunteers to give short contributions/talks within their sessions. In that case, please arrange a short program for that session with an approximate time schedule and list of speakers/subjects and distribute this to us all via e-mail. Persons who are in charge of specific sessions are also responsible for being chairmen.

Most guest speakers have been allocated 1.5 h. This translates into a presentation of app. 30 - 60 min. (whatever you think is appropriate) followed by a discussion session (30 - 45 min.). Slide and overhead projectors and video will be available in the conference room.

Timetable for the trip Barcelona airport – Sant Feliu de Guixols

NB! Please note that Jose Luis is currently looking into the possibilities of organising common transport from Barcelona to St Feliu by bus. You will be notified by e-mail.

Train from Barcelona-Airport to Walk transfer Bus from Barcelona to Sant Barcelona-Arc de Triomf by Feliu de Guixols (SARFA (RENFE company)* underground company) Departure Arrivalcorridors from Departure Arrival 6.13 6.40 Arc de Triomf 6.43 7.10 railway Station 7.13 7.40 to Bus Station 7.43 8.10 (an old railway station called 8.30 9.50 8.13 8.40 “Estació del 8.43 9.10 Nord”). 9.13 9.40 9.43 10.10 Just few minutes 10.30 11.50 10.13 10.40 10.43 11.10 11.30 12.50 11.13 11.40 11.43 12.10 12.30 13.50 12.13 12.40 12.43 13.10 13.13 13.40 14.00 15.20 13.43 14.10 14.13 14.40 15.00 16.20

98 14.43 15.10 15.13 15.40 16.00 17.20 15.43 16.10 16.13 16.40 17.00 18.20 16.43 17.10 17.13 17.40 18.00 19.20 17.43 18.10 18.13 18.40 19.00 20.20 18.43 19.10 19.13 19.40 20.00 21.10 19.43 20.10 20.30 (last 21.50 one) If you are not able to take the bus at 20.30 then you and us will have a big problem !!! Please inform us or Michael of any problem on this matter asap ! • Timetables of RENFE have changed the May 29th. (Check the web site http://www.renfe.es) • Although used to interconnect the airport with Barcelona city, it is not an specific shuttle service between these two stations. The destination of trains leaving the airport is Mataró, a village several Km far from Barcelona. • There are several train-stations in Barcelona city using the airport line, the first one is Barcelona-Sants, the second is Barcelona-Pl. Catalunya and the third one is Barcelona-Arc de Triomf (Leave the train here !!!). The trains are (usually) modern engines, an information panel is over each door, here you should be aware for: ”PROXIMA PARADA: BARCELONA ARC DE TRIOMF”.(English: next stop:Barcelona Arc de Triomf)

People going directly to Girona (e.g using trains from Europe, enjoy our wonderful Talgo – trains from Paris, Montpellier, Zurich, Milan/Milano/Mitland, etc). The train station and the Bus station are side by side. There is a bus service connecting Girona with St. Feliu de Guixols: first Departure 6.45, then 8.00, 9.15, 10.15, 11.15,..., 20.15, and last one 21.15 (take only 45 minutes from Girona to St.Feliu de Guixols).

Timetable for Trip Sant Feliu de Guixols - Barcelona airport

Bus from St. Feliu de Guixols Walk transfer Train from Barcelona-Arc de to Barcelona. by Triomf to Barcelona-Airport * S= on Saturday underground D= on Sunday corridors from Departure ArrivalBus Station to Departure Arrival Arc de Triomf 6.38 7.02 railway Station

99 7.05 7.31 7.38 8.02 8.08 8.32 8.38 9.02 7.40 (S) 9.00 9.08 9.32 9.38 10.02 8.40 (S and D) 10.00 10.08 10.32 10.38 11.02 9.40 (S and D) 11.00 11.11 11.35 11.38 12.02 12.08 12.32 12.41 13.05 13.08 13.32 12.10 (S and 13.30 D) 13.41 14.05 14.08 14.32 13.10 (S) 14.30 14.38 15.02 15.08 15.32 14.10 (S and 15.30 D) 15.38 16.02 16.08 16.32 16.38 17.02 17.08 17.32 16.10 (S) 17.30 17.38 18.02 18.11 18.35 18.38 19.02 19.08 19.32 18.10 (S and 19.30 D) 19.38 20.02 20.08 20.32 19.10 (S and 20.30 D) 20.38 21.02 21.11 21.35 20.10 (S and 21.30 D) 21.38 22.02 22.08 22.32

100 • Remember: • Timetables of RENFE were changed on May 29th. • Arc de triomf is a railway station also for other trains no making service to the airport • VERY IMPORTANT: On Sunday the speedways to Barcelona are usually collapsed at afternoon and evening. The time needed to go from St. Feliu to Barcelona may be easily x2 or x3. • People leaving from Girona. Timetable of the bus service connecting St. Feliu de Guixols with Girona in July (René, it is now confirmed!): • 1) On Saturday, first departure 7.35, then 9.15, 10.15, 11.15, 12.15, 14.15, 16.15, 18.15 and last one 20.15 (take only 45 minutes). • 2) On Sunday, first departure 10.15, then 12.15, 14.15, 15.15, 16.15, 18.15 and last one 20.15 (because of the high traffic to Girona during the afternoon, please duplicate the time need for the connections at 18.15 and 20.15).

Weather Weather: Usually sunny (but sometimes there are shovers in the afternoon). The temperature is 30C or above during daytime, and 25C or above during night. However, the hotel is situated at a windy spot and it may be a good idea to bring warm clothes for the evening.

List of participants

Paulo Alexandrino, Centro Estudos Ciencia Animal-ICETA/UP, Portugal Fred Allendorf, University of Montana, USA Alvaro Anton, University of the Basque Country, Spain Agostinho Antunes, Centro Estudos Ciencia Animal-ICETA/UP, Portugal Apostolos Apostolidis, University of Thessaloniki, Greece Fabrizio Baumann University of Berne, Switzerland Dorte Bekkevold, Danish Institute for Fisheries Research, Denmark Patrick Berrebi, University of Montpellier II, France Rachel Bouille, University of Berne, Switzerland Carmen Bouza, University of Santiago de Compostela, Lugo, Spain Jens Carlsson, Swedish University of Agricultural Sciences, Sweden Jaime Castro, University of Santiago de Compostela, Lugo, Spain Martí Cortey, University of Girona, Spain Anna Danielsdottir, Marine Research Institute, Iceland Alistair Duguid, Queen’s University of Belfast, Northern Ireland Andy Ferguson, Queen’s University of Belfast, Northern Ireland Ian Fleming, NINA, Norway Jose Luis Garcia-Marin, University of Girona, Spain Kevin Glover, Institute of Marine Research, Norway Riho Gross, Estonian Agricultural University, Estonia Sigurdur Gudjonsson, Institute of Freshwater Fisheries, Iceland Rene Guyomard, INRA, France Michael M. Hansen, Danish Institute for Fisheries Research, Denmark

101 Kjetil Hindar, NINA, Norway Rosaleen Hynes, Queen’s University of Belfast, Northern Ireland Bill Jordan, Zoological Society of London, UK Marja-Liisa Koljonen, Finnish Game and Fisheries Research, Finland Corine Kruiswijk, Agricultural University of Wageningen, Netherlands Linda Laikre, University of Stockholm, Sweden Carlo Largiader, University of Berne, Switzerland Sophie Launay, INRA, France Paulino Martinez, University of Santiago de Compostela, Lugo, Spain Maya Mezzera University of Berne, Switzerland Michael Miller, University of Munich, Germany Einar Eg Nielsen, Danish Institute for Fisheries Research, Denmark Alex Osinov, Moscow State University, Russia Tiit Paaver, Estonian Agricultural University, Estonia Stefan Palm, University of Stockholm, Sweden Henri Persat, Lyon, France Carles Pla, University of Girona, Spain Craig Primmer, University of Helsinki, Finland Paulo Prodöhl, Queen’s University of Belfast, Northern Ireland Kornelia Rassman, University of Munich, Germany Daniel Ruzzante, Danish Institute for Fisheries Research, Denmark Öystein Skaala, Institute of Marine Research, Norway Rene Stet, Agricultural University of Wageningen, Netherlands John Taggart, University of Stirling, Scotland Inci Togan, Middle East Technical University, Turkey Costas Triantaphyllidis, University of Thessaloniki, Greece Fred Utter, University of Girona, Spain Anti Vasemagi, Estonian Agricultural University, Estonia Robin Waples, National Marine Fisheries Service, USA Steven Weiss, Veterinary University of Vienna, Austria Roman Wenne, Sea Fisheries Institute, Poland

102 ANNEX 5

PROCEEDINGS OF THE SECOND WORKSHOP ON IDENTIFICATION, MANAGEMENT AND EXPLOITATION OF GENETIC RESOURCES IN BROWN TROUT (SALMO TRUTTA).

28 June - 4 July 1999, St. Feliu de Guixols, Spain

Introduction

The second workshop of the CA took place 28 June - 4 July 1999 in St. Feliu de Guixols, Spain, kindly hosted by Drs. Carles Pla and Jose-Luis Garcia-Marin, University of Girona, Spain. A total of 52 persons participated in the meeting. They represented laboratories involved in the CA (a total of 39 persons), external experts funded by Troutconcert (8 persons) and persons who participated at their own expense (5 persons). Some time was devoted to presentations of research activities by persons or groups that were not CA participants. However, most of the time was devoted to specific theme sessions.

The titles of the sessions were: a) Presentation of ongoing research activities b) Endangered populations (responsible person: Patrick Berrebi) c) Sea trout; genetic population structure, conservation (responsible person: Michael M. Hansen) d) Comparative data analysis (responsible person: Andy Ferguson) e) Conservation of brown trout (responsible persons: Carlo Largiader and Linda Laikre) f) Behavioural studies in salmonid fishes (Responsible person: Ian Fleming) g) Suggestions for future studies (Responsible person: Michael M. Hansen) h) Is there a life after the CA? (Responsible person: Michael M. Hansen)

The detailed program of the workshop is included in the report as Annex 4.

103 a) Presentation of ongoing research activities

This section includes summaries of presentations aimed at presenting research activities of different groups and new developments and results of general interest.

CONSERVATION PERSPECTIVES OF BROWN TROUT IN AUSTRIA Steven Weiss, Univ. of Veterinary Medicine, Vienna, Austria

As in many other countries, brown trout Salmo trutta is one of the more important components of the sport fishing industry in Austria, and the stocking of hatchery raised fish is used to either supplement or wholly sustain a harvest intensive managment paradigm. In the past there has been little or no consideration given to the source of brood stock or the potential negative effects that such stocking and management strategies may have on native populations.

Despite some growing interest in Austria to protect native populations, as well as gaining a more refined understanding of the efficacy of stocking strategies, the highly privatised legal framework of fishing rights in Austria makes it difficult if not impossible to achieve some of the more progressive breeding policies or conservation-oriented management schemes that are either underway, or being considered elsewhere. Stocking is obligatory wherever fishing licenses are sold, private individuals may raise fish for stocking following whatever breeding practices they wish, and there are presently no laws governing the source of the fish being stocked. As many brood stocks have been founded, or are continually maintained through the importation of domesticated strains of fish from drainages outside of the Danube, there is a continuous introduction of non-native genomes into Austrian waters. Thus, the present situation in Austria is rather grim with respect to the conservation of the native gene pool in all but a handful of isolated populations whereby the present owner of fishing rights does not allow fishing.

In an attempt to identify some populations which may contain relatively intact native populations, a genetic screening project was undertaken to be based on the sequencing of the mtDNA control region as well as the assaying of several diagnostic allozyme loci (TF, LDH -5, MEP1). This work is still in progress but thus far extensive mixture of mtDNA haplotypes that are assumed to be diagnostic of Atlantic and Danubian drainages were found in all flowing waters investigated, and near fixation of Atlantic haplotypes was found in all hatchery stocks. Several populations of brown trout in alpine lakes displayed fixation of Danubian haplotypes but these lakes originally held no fish and are presumed to have been stocked several hundred years ago. While the empirical evidence supports the notion that the presence of Atlantic basin mtDNA haplotpyes in Austria is due to importation of hatchery brood stocks, natural introgression of Rhenian haplotypes into the Danube during or just after the last glacial advance can not be excluded. This question will be addressed to some exent by another research group from Munich (Uli Schleiwen, Kornelia Rassman, and Michael Miller) by evaluating mtDNA diversity in 50 populations of brown trout in Bavaria where tributaries of the Danube, Rhine, and Elbe are located in close proximity to each other. When this data is compared to that collected in Austria a clearer picture may be obtained based merely on the geographical distribution of Atlantic and Danubian haplotypes. However, the question of whether Atlantic basin haplotypes have been sympatric with Danubian haplotypes only recently (the last century) or since the last glacial advance (10-20,000 yrs) will undoubtedly remain unanswered.

104 More objective data addressing this question may be obtained by investigating recombination frequencies between variably linked markers, if such markers can be developed which display basin-specific mutations. mtDNA AND PROTEIN VARIABILITY OF PORTUGUESE BROWN TROUT POPULATIONS AND IMPLICATIONS FOR PROPOSED MODELS OF POST-GLACIAL RECOLONIZATION. Agostinho Antunes & Paulo Alexandrino, University of Porto, Portugal and Steven Weiss, Univ. of Veterinary Medicine, Vienna, Austria

The genetic relationships of brown trout (Salmo trutta L.) populations across Europe have been extensively evaluated through the analysis of protein polymorphism. Based on allelic variation patterns at the LDH-C*, LDH-A2* and CK-A1* loci, four major geographical lineages in unglaciated areas were defined: Mediterranean, Ponto-Caspian, northwestern Atlantic and southwestern Atlantic (García-Marín et al. 1999).

The southwestern Atlantic refuge was mainly composed by northwestern Iberian populations of Spain and Portugal, which are fixed or nearly so for LDH-C*100 and CK-A1*115 alleles (García- Marín et al., 1999; Antunes et al., 1999). Studies of northwestern Iberian Peninsula lacked a key diagnostic locus (TF*) and the genetic characterisation of some Portuguese populations revealed significant frequencies of the TF* 95 allele, not previously found in other Atlantic populations (Antunes et al., 1999). Preliminary results demonstrated that this allele is absent in Asturian populations (Northern Atlantic Spain) and, within Portuguese rivers, its frequency increased southwards. The frequency of CK-A1*115 and MPI-2*100 alleles increased clinaly in Iberian populations from north to northwest, a pattern which is congruent with data presented in Bouza et al. (1999).

Mitochondrial haplotype diversity of Portuguese populations was investigated by sequencing the 5’ end of the mtDNA control region. Five new haplotypes are described for this species, each 2-3 mutational steps away from the common north Atlantic haplotype. Significant population subdivision of mtDNA haplotypes is also apparent. Based on these results, as well as published data on the distribution of both mtDNA haplotypes and allozyme alleles throughout Europe, the postglacial recolonization of northern Europe is re-evaluated. In contrast to a recently proposed model invoking three major glacial refugia (southwest Atlantic, north continental Europe and Ponto- Caspian Basin; García-Marín et al., 1999), we propose that gene flow from refugia in central and eastern continental Europe alone can more easily explain the geographic distribution of mtDNA haplotypes and putatively diagnostic allozyme alleles throughout previously glaciated regions of northern Europe.

Located in the southern limit of the Atlantic distribution of both resident and anadromous brown trout forms, Portuguese brown trout demonstrated a unique genetic architecture within the Atlantic basin. This valuable genetic resource should be protected from introgression with non-endemic strains of hatchery fish.

References Antunes, A., Alexandrino, P. and Ferrand, N. (1999). Genetic characterization of Portuguese brown trout (Salmo trutta L.) and comparison with other European populations. Ecology of Freshwater Fish, 8, 194-200.

105 Bouza, C., Arias, J., Castro, J., Sánchez, L. and Martínez, P. (1999). Genetic structure of brown trout, Salmo trutta L., at the southern limit of the distribution range of the anadromous form. Molecular Ecology, 8, 1991-2002. García-Marín, J. L., Utter, F. M. and Carles, P. (1999). Postglacial colonization of brown trout in Europe based on distribution of allozyme variants. Heridity, 82, 46-56.

PRELIMINARY REPORT ON mtDNA DIVERSITY IN EUROPEAN GRAYLING Steven Weiss, Univ. of Veterinary Medicine, Vienna, Austria

European grayling, Thymallus thymallus is also an important component of many sport fisheries throughout Europe though compared to brown trout it has a more limited distribution geographically, being absent from the Iberian Penninsula as well as the southeastern extremities of Europe such as Greece and Turkey.

In Austria, many grayling populations are in drastic decline. Despite the fact that the causes of these declines are poorly understood, both local governments and fishing organizations have begun to take action in the form of raising and stocking more fish. For this reason, baseline genetic data would be of extreme importance in attempting to identify autochthonous and allocthonous stocks, in the hope of preventing massive importation and stocking of the latter, as has occurred in this region with brown trout.

In cooperation with Henri Persat and his student Robin Eppe, samples from nearly 50 populations throughout Europe are being screened for variation in the mtDNA control region. One goal of this work is to present an intraspecific phylogeny of mtDNA haplotypes which will allow assignment of lineages to major drainage systems. This should provide a rough picture of the location of glacial refugia, routes of post-glacial colonization, and the extent of post-glacial admixture between formally divergent lineages, whether by natural or man-made processes. Such baseline data may serve to identify non-native hatchery strains as well as populations which have be apparently unnaffected by admixture from divergent lineages.

The preliminary results were summarized as follows:

1) there is substantial variation in the mtDNA control region Europe-wide.

2) typically, in unstocked populations, there is little within population variation.

3) while clear groups of divergent haplotypes exist for the Adriatic, Mediteranean, and Danubian basins, populations from Atlantic Ocean draining rivers, and especially those south of the Scandinavian peninsula, do not resolve into a well-defined clade.

4) there are some highly divergent, apparently relict populations located in the Atlantic basin.

5) there is no evidence of massive admixture of northern lineages into the upper Danube basin, as exists for brown trout.

106 6) a centrally located, repetitive stretch of the control region shows rather complex mutational behaviour which does not occur in the homologous stretch in brown trout, suggesting that some kind of functional constraint may be operative in brown trout.

BROWN TROUT POPULATION GENETICS IN UMEAA Jens Carlsson, University of Umeaa, Sweden

Four projects spanning over different geographical scales have been conducted at the Department of Aquaculture (Swedish University of Agricultural Sciences). The first project, called The River Ammerån, focus on genetic structures on a landscape perspective. Several tributaries as well as one location in the main stream were sampled. Three of the localities were resampled one year later. Genetic variability was studied using five microsatellite loci. The results indicate that genetic structure is highly influenced by geological structures (e.g. division into tributaries, multiple stream junctions, rapids and waterfalls). The resampled localities also showed temporal stability. It is argued that distinct units may be predicted from geological structures and that isolation by distance measurements should include geological structures.

The second project, The Nordre Finnvikelv, focuses on the population genetic structure within a small catchment by means of microsatellite polymorphisms. Again geological structures were found to have a profound effect on the genetic structure. Geological features separated all different population fragments that could be detected. Also a pattern resembling isolation by distance was also found. However it is argued that this pattern might be an effect of geological structures.

The third project, The Färsån Stream, uses microsatellite polymorphism to detect population structures within a continuous stream habitat. Seven localities were sampled during two consecutive years. No differences between cohorts within the first sampling year could be detected. However, differences between sampling years were found within the locality closest to the confluence with a large river. It is argued that the temporal stability should not only be assessed between cohorts within a sample but also between years, and even more important when migratory trout is present. The results show differentiation between distal sample localities. Moreover, differentiation increased with increasing distance, indicating isolation by distance both years. It was concluded that the trout of the stream most likely represents a continuously distributed population. It is argued that local homing combined with low, however important, gene flow creates the pattern of isolation by distance.

The fourth project, The Upper Färsån, aims at identifying genetic structures over a micro- geographical scale (within one stream section). Preliminary results were presented on microsatellite polymorphisms. The preliminary results show no relationship between degree of relatedness and geographic distance of individuals. However, more microsatellite loci will be added to further increase the resolution before any conclusive arguments can be made.

The three first projects clearly shows that geological structures influence the genetic structure by means of reducing gene flow. It may also be common that where geological structures are absent continuously distributed populations may be observed.

107 LOCAL, INTRODUCED AND HATCHERY STOCKS OF SEA TROUT: A GENETIC STUDY UTILISING DNA PROFILING. Kevin Glover, IMR, Norway.

Information on the experiences with DNA fingerprinting for family identification was given. For the 22 mostly full sib families used in this study, only two loci (15G10 and 15H8) were needed to have 99.75% identification of offspring to family. The useful loci are included in table 1, which summarises the loci, which have been screened.

Table 1 Locus MgCl Anneal temp °c Cycles Alleles: 22 families conc. Ssa 171 1.0 59 29 Not scored Ssa 197 1.0 61.5 29 9 15G10 1.0 61 29 23 15G6 1.0 61.5 29 16 15H8 1.0 61 29 18

The Ssa series are well used probes for salmon found by the marine gene probe laboratory in Canada. The 15 series are unpublished (as yet) probes designed for salmon by John Taggart but are useful for trout (table 1) also. Further optimisation of PCR conditions for each 15 series probes can be made but time was limited in this stage of the project and an acceptable level of resolution was attained with these conditions.

The uses of DNA profiling are were also discussed in this talk. It is felt that many experiments where family level genetic variation in a closed environment (i.e. one where all parents and progeny are known) exists then DNA profiling can be of use. Families exhibit large amounts of variation and can cause large changes in the results for certain types of genetic experiments (such as those for stock level differences in disease resistance or aggression for example) if family variation is not controlled for.

Individual and family level genetic variation is the basis upon which stock level genetic variation has the ability to develop and so control and observation of family level genetic variation for various factors has the ability to show us where more subtle genetic differences might lie in local adaptations etc. Experiments being carried out and in the planning process at IMR in Bergen are going to utilise the family identification package developed to be able to carry out such experiments.

During the process of semi-optimising the pcr conditions for these probes it was discovered two clear examples where mobility of fragments from forward and reverse labelled primers was significantly different. This emphasises the view that publications should not only state probes used but whether the forward or reverse primer is labelled. This point was also mentioned by several other contributors at TC.

The last observation made from the work on DNA profiling of trout families was that the hatchery in which these experiments had been conducted in had made some “mistakes” in crossing the fish.

108 This not only emphasises the point that careful supervision or choice of workers to create and maintain family crosses should be made, it also casts a spell of doubt onto MANY experiments previously carried out involving stock transplants and crosses where DNA control has not been implemented.

A discussion was also made on the genetic changes under hatchery environments. It was pointed out by the author that the following genetic changes were well documented under hatchery conditions:

Founder effects Inbreeding Genetic drift Direct selection (spawner size etc.)

These genetic changes caused by the hatchery are both easy to be aware of and are possible to do something about (although it is in some cases not possible to obtain a sufficiently high number of spawners). However, the author is more concerned with the area of selection under the hatchery which is termed indirect selection. This is whereby the deaths and growth of fish (i.e. selection) is determined by the environment in which the fish live (hatchery). The hatchery is both a high food environment with high densities of fish. Fish adopting such a life-style as this will prosper more than fish more adapted to a lifestyle where metabolic efficiency and not metabolic capacity is emphasised.

Even though there there is good evidence for indirect selection (for instance differences in aggression and anti-predator behaviour between domesticated and wild salmonids) there are no experiments in the literature stating how family level genetic variation plays a role in survival in the hatchery environment and how one can compare this to the situation in the wild.

From this an experiment was described where fish from selected families were placed under two different feeding regimes. The first feeding regime was normal and matched the normal hatchery feeding whilst the second was trying to mimic a level of food availbale in the wild (it was 25% of the hatchery feeding level). The null hypothesis is that there is no significant difference in family ranking (ranked as mortality and growth of each family) between feeding treatments. This was developed in response to the ability to identify families and the whole host of literature on alternative life history strategies and the idea that some fish like to live in the fast lane whilst some fish adopt a more energy sparing life history strategy which would not be favoured in the hatchery environment. (also metabolic efficiency as opposed to metabolic capacity).

The final idea of this experiment was that if the there was significant difference in the family ranking between feeding treatments it would give good “NEW” evidence to further suggest that hatcheries are encouraging the survival of the “wrong” (IE unadapted to the lower food environment of the wild) genotypes and are causing what I have termed as possible “reverse selection”. This experiment is nearing completion.

109 b) Endangered populations (responsible person: Patrick Berrebi)

The “endangered populations” session focused on several classical dangers able to locally extirpate trout populations. Each potential danger has been illustrated by an example described during a talk. The three following paragraphs give a summary of the three talks.

HIGH INTROGRESSION DUE TO A LONG STOCKING HISTORY IN SLOVENIA Patrick Berrebi, CNRS-University Montpellier 2, Montpellier, France

The marble trout is an endemic form of the Adriatic slope, distributed from Italy to Albania. In Slovenia, the Soca River is the only drainage where this species (or subspecies according to different authors) lives. This river has been heavily stocked since at least 1906 (Povz, 1996). Recent genetic studies (Berrebi, 1995a) have shown that in fact the marble trout of the main stream (Soca river) have been introgressed by several other forms of trout. The northern Atlantic brown trout has been introduced, probably by translocation from Austria and by stocking using a commercial strain (about 30%) and the Danubian form from the closer drainage (about 30%) by fish farming activity. As a result of this the Soca River population is dominated by 65% of introduced form.

A rehabilitation program has now been initiated (Povz et al., 1996). It started in 1993 in the upper basin of the Soca River where hybridisation seemed to be intense. Such a project became feasible in this region for biological, sociologic and economic reasons. The region benefits from a remarkably preserved environment that is officially protected by law (water quality, restricted agricultural and industrial activities, low erosion because of the extensive dense forests, etc.). Fly-fishing for marble trout is one of the major tourist attractions of the region. As this attracts many foreign anglers, the rehabilitation project received complete support from the local regional and national authorities. The angling associations, and particularly the largest of them, the Tolmin Anglers' Society, also support the project because legally they are the managers of half the river courses in Slovenia inhabited by the marble trout.

From 1993 to 1996, during the first phase of the project, five main objectives were defined: (i) to determine the genetic diversity of the populations of autochthonous trout, introduced and hybrid trout; (ii) to look for genetically pure populations of marble trout especially in the headwaters of streams; (iii) to introduce a ban on stocking brown trout Salmo trutta in the upper Soca basin; (iv) to undertake a genetic survey of trout in the hybridisation zone and (v) to publish an action plan using a "watershed" based approach for rehabilitating marble trout.

This type of action plan takes into account not only the management of the trout itself, but also all those activities that could have an impact on the habitat of the species. The last objective was fulfilled at the end of 1996 with the publication of an action plan in English for tourists and in Slovenian for local inhabitants (Povz et al. 1996). In 1996, the third objective was met by a Slovenian government decree prohibiting the stocking of S. trutta in the Soca basin.

The aim of the second phase of the project, which takes place in 1997-2002, is to conduct genetic monitoring of trout in the hybridisation zone and has several new objectives : (vi) the creation of several sanctuary streams containing populations of genetically pure marble trout; (vii) an assessment of the spawning success of marble trout using the standard method described by Rubin (1995); (viii) the conduct of an experimental study on potential competition between marble trout

110 and brown trout, particularly on the spawning sites, and finally (ix) if the results of the above experiments show that marble trout can gradually be outcompeted by hybrid trout, then pure marble trout will be bred for reintroduction, particularly into the hybridisation zone.

CHEMICAL POLLUTION - FALL AND RISE OF THE FINE-SPOTTED TROUT Øystein Skaala, Institute of Marine Research, Bergen Norway.

The fine-spotted brown trout, inhabiting parts of the Hardangervidda mountain plateau, Norway, was rediscovered in 1985 just as it was about to become extinct. The scientific studies and conservation efforts can be grouped into three different time intervals: 1985 - 1991: Rediscovery of population and identification of problems and threats; 1991 - 1993: Developing conservation plan; 1993 - Restoration of trout population, water quality and food chain.

The population inhabits a couple of small lakes at about 1300 m above sea level, in the upper parts of Numedalslågen watercourse. First known written records mentioning the populations date back to 1912 and 1939. According to archaeological excavations however, the large lakes on the Hardangervidda were inhabited by trout as early as 5-6000 years ago.

Electrophoretic studies showed that the population was almost fixed for the fast allele at LDH-5*, and inheritance studies demonstrated that the pigmentation pattern was inherited in a simple Mendelian pattern, like a polymorphic locus.

By 1985 the reproduction of fine-spotted trout had ceased. Later studies, confirmed by earlier field notes, showed that during the early part of the 1980’s water quality had become poorer due to acid rain, while at the same reproduction of the trout population and its prey items, such as Gammarus lacustris and Lepidurus arcticus, stopped. Other threats to this biological diversity were a very small spawning area, small population size, gillnetting and, potentially, the introduction of minnows Phoxinus phoxinus.

A major problem in the early phase of the study was the failure of management authorities to recognise the unique biological diversity represented by the fine-spotted trout, and thus funding to study and conserve it was very limited.

Between 1991 and 1993, a plan was developed to restore and conserve the fine-spotted trout. The measures ranged from cryopreservation of milt, and also setting up a small hatchery production, to a complete restoration of the natural distribution area. As a part of this, a careful sampling programme for water quality and also for benthos was initiated.

In 1993 a careful and limited restocking was initiated, based on brood stock collected in the lakes a few years earlier. Still, the basic idea was to restore the water quality so that the natural recruitment of the population could be restored. Liming by use of helicopter was initiated in parts of the drainage, while other areas were kept untreated as controls. Today we know that without this restocking, the fine-spotted trout on Hardangervidda would have been extinct. Needless to say, the first autumn when we observed the tiny newly hatched fry in the natural spawning area, was a great one!

111 It was also decided to attempt to reintroduce the two most important prey items to restore the food chain for the trout. Also, a programme to sample prey items from other lakes on Hardangervidda, and transfer them individually to the lakes of the fine-spotted trout was initiated.

At present the reproduction of trout has been restored in the main lake, and the abundance of fine- spotted fry and older trout is monitored. The future looks bright, but it still remains to be seen if it is possible to restore the population of prey items and restart the whole biological system.

The rediscovery and conservation of the fine-spotted trout on Hardangervidda rests basically the personal initiative of a few people, and very close co-operation between various institutions such as The Game Police, the local community in Eidfjord, The Institute of Marine Research, The Directorate for Nature Management, Environmental division County of Hordaland, but especially Mr. Åsmund Tysse Environmental division, County of Buskerud.

VERY SMALL TROUT POPULATIONS Patrick Berrebi, CNRS-University Montpellier 2, Montpellier, France

The example of very small population has been taken from the numerous trout populations of Corsica which were analysed mainly using allozymes (Berrebi, 1995b). Such small populations trapped at the top of the drainage are commonly found throughout Europe. They are of interest because they may be considered as relicts of previous populations (Hamilton et al., 1989).

Corsica is thought to have been inhabited by the "Corsican" form probably at the beginning of the Quaternary. This form for which the range is not known, belong to the Adriatic group sensu Bernatchez et al. (1992). More recently, the Mediterranean form invaded the island and has hybridised. Marks of ancient hybridisation with a variable proportion of Corsican and Mediterranean alleles are widespread in the island, except for a few trapped populations (now 6 are known) of pure or almost pure Corsican trout. Even among these 6 populations, two types have been recognised according to an allozymic marker: the East and the West groups. As a result of isolation for long period of time, these populations have developed different allelic frequencies and even allele fixation, and their external morphology can be very different. This last point does not help the visual recognition of this form.

At this time, most of the pure Corsican populations are protected by a stocking ban. In fact this prohibition was not really necessary because the very small size of the tributaries concerned did not encourage stocking. The main threat is in fact poaching. In this region, it is a tradition to pour bleach in the river to harvest the trout, which are very sensitive to this. If such event happens, it is probable that the few hundreds of trapped fish will disappear.

References

Bernatchez, L., R. Guyomard, et al. (1992). DNA sequence variation of the mitochondrial control region among geographically and morphologically remote European brown trout Salmo trutta populations. Molecular Ecology 1: 161-173. Berrebi, P. (1995). Analyse génétique des truites de Slovénie. Rapport final. University Montpellier II: 13p.

112 Berrebi, P. (1995). Etude génétique des truites de Corse. Rapport final 1995, University Montpellier II: 36p. Povz, M. (1995). Status of freshwater fishes in the Adriatic catchment of Slovenia. Biological Conservation 72: 171-177. Povz, M., D. Jesensek, et al. (1996). The Marble trout, Salmo trutta marmoratus, Cuvier 1817 in the Soca River basin, Slovenia, Tour du Valat Publication. Rubin, J. F. (1995). Estimating the success of natural spawning of salmonids in streams. Journal of Fish Biology 46: 603-622.

113 c) Sea trout; genetic population structure and conservation (responsible person: Michael M. Hansen)

This session focused specifically on the anadromous form of the brown trout, i.e. sea trout. The session consisted of a general talk and discussion about the genetic structure of sea trout followed by talks on the genetic structure of sea trout at the southern limit of its distribution, the genetic structure of a sister species of the brown trout, i.e. the Atlantic salmon and the significance of life- history variation in relation to introgression by stocked fish.

GENETIC STRUCTURE OF SEA TROUT POPULATIONS Michael M. Hansen, Danish Institute for Fisheries Research, Denmark

A review was given of current knowledge, but also current dogmas, on the genetic structure of sea trout. The following topics were covered:

• Sea trout - life history, geographical distribution, phylogeography • Differentiation between coexisting resident and anadromous trout? • Genetic population structure of sea trout • Conservation of sea trout

The background of the presentation was that, from the point of view of the presenter, there are a number of problems in understanding the genetic structure of sea trout populations. First of all, there are so far very few published studies that have specifically aimed at studying the genetic population structure of sea trout. In many studies no discrimination has been made between anadromous and landlocked populations and, consequently, conclusions about genetic relationships and gene flow have been based partly on studies of populations where no gene flow is possible. The presenter identified the following dogmas that on closer inspection might not be valid for sea trout:

• There are genetic differences/differentiation between co-existing anadromous/resident trout • There is no correlation between genetic and geographical distance between populations, i.e. no isolation-by distance patterns • There is generally very strong genetic differentiation among populations These issues were given special attention during the presentation.

Sea trout - life history, geographical distribution, phylogeography Brown trout generally exhibits two major life-history forms, i.e. resident and migratory trout. Sea trout is an anadromous form of migratory trout. This means that the spawning and nursery areas are in freshwater, but the foraging areas of adult trout are in the marine environment. The same kind of life-history variation is found in many other salmonid species, such as Atlantic salmon, rainbow trout, cutthroat trout etc. The sex ratio differs between resident and sea trout. In Northern Europe it is commonly found that app. 75% of all sea trout are females whereas 75-100% of all resident trout are males (Jonsson, 1982; Rasmussen, 1986). The geographical distribution of sea trout encompasses drainages flowing into the Atlantic Ocean and the Baltic Sea. In addition, sea trout is present in the Caspian Sea. Sea trout in the Atlantic region all belong to the so-called Atlantic phylogeographical lineage, whereas the Danubian lineage is represented in Caspian sea trout. It is

114 possible that sea trout has also been present in other lineages, in particular the Mediterranean lineage, during colder climatic periods. For instance, it is difficult to explain the occurrence of brown trout in Corsica if the island has not been colonized via the Mediterranean Sea.

Differentiation between coexisting resident and anadromous trout? Because of the differences in morphology and life-history between resident and anadromous trout it has often been assumed that they are reproductively isolated and perhaps even represent different species or subspecies. However, in studies employing genetic markers it has not been possible to reveal any genetic differentiation between co-existing resident and anadromous trout (Hindar et al., 1991; Cross et al., 1992), strongly suggesting that interbreeding between the two forms takes place. There may, however, be some complex situations and exceptions to this rule where reproductive isolation can occur between co-existing ecotypes. For instance, one ecotype could spawn in shallow waters whereas another ecotype could spawn in the same river but in deep waters (cf. Ferguson and Taggart, 1991).

Genetic population structure of sea trout Based on analyses of large data sets it has generally been concluded that genetic differentiation among brown trout populations is very strong and that there is apparently no relationship between genetic and geographical distance between populations (e.g. Ryman, 1983; Ferguson, 1989). However, many if not most of the data included in these analyses are from resident and/or landlocked populations. If impassable barriers exist among populations it is not surprising that genetic drift over time creates very strong genetic differentiation. Also, if there is no gene flow, even between neighboring populations, the genetic composition of populations will drift in different directions irrespective of the geographical distance between populations.

The relatively few studies that have focused specifically on sea trout have revealed that genetic differentiation is in fact much smaller than that observed among resident/landlocked populations (typical FST values for sea trout range from 5-10%). Also, genetic variability is considerably higher than in resident/landlocked populations. Finally, there does seem to be a relationship between genetic and geographical distances between sea trout populations, and gene flow appears to take place mainly according to a stepping-stone model (Moran et al., 1995; Hansen and Mensberg, 1998; Bouza et al., 1999; Østergaard et al., unpublished results). Estimates of genetic differentiation and the observation of isolation by distance in sea trout is quite typical of what has been observed for anadromous populations of other salmonid species (e.g. Allendorf and Leary, 1988; Koljonen et al., 1999; Nielsen et al., 1999). There may be some situations, however, where isolation-by-distance does not occur. More specifically, some data from anadromous populations in Norway (Skaala, 1992) were analysed for isolation-by-distance, but no such patterns could be observed. This could be explained by the fact that rivers inhabited by sea trout were grouped in deep fjords, but geographical distances between fjords were large. This lack of “stepping-stones” between fjords could result in almost complete reproductive isolation of populations from different fjords. A similar situation could occur in some areas in Scotland.

Conservation of sea trout Sea trout are facing the same threats as other brown trout populations, including pollution, destruction of habitats, establishment of impassable dams, over-fishing, introgression by stocked domesticated trout etc. However, the fact that individual sea trout populations are linked by gene flow makes them part of a larger population (a metapopulation according to some definitions of this term). This means that in order to conserve sea trout in a given area it is insufficient to point out just

115 one or a few populations that should be conserved. If populations go extinct that would otherwise serve as “links” or “stepping-stones” between the remaining populations, this means that the genetic population structure is completely altered. The surviving populations become partly or fully reproductively isolated and loss of variability due to lack of gene flow will take place.

References

Allendorf, F.W. and Leary, R.F. 1988. Conservation and distribution of genetic variation in a polytypic species, the cutthroat trout. Conservation Biology 2:170-184. Bouza, C., Arias, J., Castro, J., Sánchez, L. and Martínez, P. (1999). Genetic structure of brown trout, Salmo trutta L., at the southern limit of the distribution range of the anadromous form. Molecular Ecology 8:1991-2002. Cross, T.F., Mills, C.P.R. and de Courcy Williams, M. 1992. An intensive study of allozyme variation in freshwater resident and anadromous trout, Salmo trutta L., in Western Ireland. Journal of Fish Biology 40:25-32. Ferguson, A. 1989. Genetic differences among brown trout (Salmo trutta) stocks and their importance for the conservation and management of the species. Freshwater Biology 21:35-46. Ferguson, A. and Taggart, J.B. 1991. Genetic differentiation among the sympatric brown trout (Salmo trutta) populations of Lough Melvin, Ireland. Biological Journal of the Linnean Society 43:221-237. Hansen, M.M. and Mensberg, K.-L. D. 1998. Genetic differentiation and relationship between genetic and geographical distance in Danish sea trout (Salmo trutta L.) populations. Heredity 81:493-504. Hindar, K., Jonsson, B., Ryman, N. and Ståhl, G. 1991. Genetic relationships among landlocked, resident, and anadromous brown trout, Salmo trutta L. Heredity 66:83-91. Jonsson, B. 1982. Diadromous and resident trout Salmo trutta: Is their difference due to genetics? OIKOS, 38, 297-300. Koljonen, M.-L., Jansson, H., Paaver, T. Vasin, O. and Koskiniemi, J. 1999. Phylogeographic lineages and differentiation pattern of Atlantic salmon (Salmo salar) in the Baltic Sea with management implications. Canadian Journal of Fisheries and Aquatic Sciences 56: 1766-1780. Morán, P., Pendás, A.M., García-Vásquez, E., Izquierdo, J.I. and Lobon-Cervia, J. 1995. Estimates of gene flow among neighbouring populations of brown trout. Journal of Fish Biology 46:93-602. Nielsen, E.E., Hansen, M.M. and Loeschcke, V. 1999. Genetic variation in time and space: Microsatellite analysis of extinct and extant populations of Atlantic salmon. Evolution 53:261- 268. Rasmussen, G. 1986. The population dynamics of brown trout (Salmo trutta L.) in relation to year- class size. Polskie Archiwum Hydrobiologii, 33, 489-508. Ryman, N. 1983. Patterns of distribution of biochemical genetic variation in salmonids: differences between species. Aquaculture 33:1-21. Skaala, Ø. 1992. Genetic population structure of Norwegian brown trout. Journal of Fish Biology 41:631-646.

116 GENETIC STRUCTURE OF BROWN TROUT, SALMO TRUTTA L., AT THE SOUTHERN LIMIT OF THE DISTRIBUTION RANGE OF THE ANADROMOUS FORM Presented by Paulino Martinez, University of Lugo, Spain

Genetic variation at 33 protein loci was investigated in 41 wild brown trout populations from four river basins in Galicia (NW Spain) aimed to analyze the amount and distribution of genetic diversity in a marginal area, located in the distribution limit of the anadromous form of this species. The genetic diversity detected within populations (H between 0 and 6%) lies within the range quoted for this species in previous reports. The Miño, the Southest river basin analyzed, showed a significant lower genetic diversity and the highest genetic differentiation among the river basins studied. The hierarchic gene diversity analysis showed high population differentiation in a restricted area (GST=27%), mostly due to differences among populations within basins (GSC=22%). The reduction of GST observed when the isolated samples were excluded of the analysis (GST=17%) showed the importance of habitat fragmentation on the heterogeneity detected. Gene flow among populations was comparatively evaluated by three indirect methods, which in general revealed low figures of absolute number of migrants per generation, slightly higher than 1. The gene flow among basins reflected a positive relationship with geographic distance. This trend was confirmed by the significant correlation observed between geographic and genetic distances, including all population pairs, which suggests a component of isolation by distance in brown trout genetic structure. Nevertheless, the non-significant intrabasin correlation agrees with the high genetic differentiation observed between some close samples or similarity between distant populations, which demonstrates the complexity of genetic relationships among populations in this species. The model of genetic structure in brown trout is discussed in the light of the results obtained.

PHYLOGEOGRAPHIC LINEAGES AND DIFFERENTIATION PATTERN OF ATLANTIC SALMON IN THE BALTIC SEA WITH MANAGEMENT IMPLICATIONS Presented by Marja-Liisa Koljonen, Finnish Game and Fisheries Research Institute, Finland

The genetic structure and isolation pattern of the Atlantic salmon (Salmo salar L.) throughout its range in the Baltic Sea were examined as a starting point for a conservation strategy for the species in this area. The allozyme variation in seven polymorphic loci was studied on 5125 salmon from 24 rivers in four countries. A clear dichotomy was observed between stock groups from southeastern (Russia, Estonia, Latvia, southern Sweden) and northwestern (northern Finland, northern Sweden) drainage regions, corresponding to the postglacial colonisation of the Baltic Sea by two phylogeographic lineages, one from the east (the Ice Lake Lineage) and one from the west (the Atlantic Lineage). The geographical and genetic distances between stocks was found to fit the one- dimensional "isolation-by-distance" model (p < 0.001). The estimated gene flow ranged from 0 to10 migrants per generation. The total diversity of hatchery stocks was 72% of that of the wild stocks. Genetically similar stock groups, phylogeographic lineages and drainage regions are recommended for use as genetic management units in addition to stock level.

117 PATTERNS OF SUBSPECIFIC ANTHROPOGENIC INTROGRESSION IN SALMONIDS. Fred Utter, University of Washington, Seattle, USA

Clearly demonstrated anthropogenic introgressions between North American trout species (Oncorhynchus spp.) diverged for more than a million years (e.g., Leary et al. 1995) have generated expectations for intraspecific introgressions to commonly occur between introduced and indigenous salmonid lineages. An examination of published information reveals a high variability in such anticipated gene flow that appears to be related to complexity of life history. The complex adaptations of anadromous populations (e.g., freshwater and marine residence, smoltification, juvenile and adult migration) contrasted with simpler requirements of freshwater conspecifics have related to their greater difficulties in translocating to colonized areas (Allendorf and Waples 1996), and also extend to introgressive capabilities. Accordingly, examples of introgression among freshwater populations predominate (e.g., Krieg and Guyomard 1985; Taggart and Fergusson 1986; Garcia-Marin et al. 1998; Poteaux et al. 1998; Largiader and Scholl 1996).

Despite intensive introductions from different lineages, indigenous anadromous populations commonly resist introgression (e.g., Moran et al. 1994; Clifford et al. 1998; Nielsen 1999; Utter et al. 1989; 19.95; Hendry et al. 1996). Within major lineages, anadromous populations appear to be more susceptible to introgression (e.g., Bugart et al. 1995; Utter et al. 1995; Grant et al. 1999). However, measuring the extent and dynamics of such introgressions remains challenging because subgroups within major lineages lie on or beyond the threshold of detection by molecular markers (e.g. Gharrett and Smoker 1993a; b; Utter et al. 1993). These substructures appear to reflect the more rapid evolution of directional selection promoting, for instance, temporal or microgeographic divergence within a molecularly-defined population unit. Consequently, management based on assumed panmixia over a particular region based on even intensive molecular genetic analysis will inevitably erode and prevent reformation of this substructure to the detriment of the genetic variability and productivity of the region.

References

Allendorf, F. W. and R. S. Waples. 1996. Conservation and genetics of salmonid fishes. Pages 238-280 in J. C. Avise and J. L. Hamrick, eds. Conservation genetics: case histories from nature. Chapman and Hall, New York. Bugert, R., C.W. Hopley, C. A. Busack and G. W. Mendel. 1995. Maintenance of stock integrity in Snake River fall chinook salmon. Pages 267-276 in H. L. Schramm and R. G. Piper, eds. Uses and effects of cultured fishes in aquatic ecosystems. American Fisheries Society Symposium 15, Bethesda, Maryland. Clifford, S. L. P. McGinnity and A. Ferguson. 1998. Genetic changes in Atlantic salmon (Salmo salar) populations of northwest Irish rivers resulting from escapes of adult farm salmon. Canadian Journal of Fisheries and Aquatic Sciences 55:358-363. Garcia-Marin, J. L., N. Sanz, and C. Pla. 1998. Proportions of native and introduced brown trout in adjacent fished and unfished Spanish rivers. Conservation Biology 12:313-319. Gharrett, A. J. and W. W. Smoker. 1993a. Genetic components in life history traits contribute to population structure. Pages 197-202 in eds. J. B. Cloud and G. H. Thorgaard, Genetic conservation of slamonid fishes. Plenum Press, New York. Gharrett, A. J. and W. W. Smoker. 1993b. A perspective on the adaptive importance of genetic infrastructure in salmon populations to ocean ranching in Alaska. Fisheries Research 18:45-58.

118 Grant, W. S., J. L. Garcia-Marin and F. M. Utter. 1999. Pages 2772 in S. Mustafa ed, Genetics in Sustainable Fisheries Management. Fishing News Books, Blackwell Science. Oxford. Hendry, A. P., T. P. Quinn and F. M. Utter. 1996. Genetic evidence for the persistence and divergence of native and introduced sockeye salon (Oncorhynchus nerka) within Lake Washington, Washington. Canadian Journal of Fisheries and Aquatic Sciences 53:823-832. Krieg, F. and R. Guyomard. 1985. Population genetics of French brown trout (Salmo trutta L): large geographical differentiation of wild populations and high similarity of domesticated stocks. Genet. Sel. Evol. 17:225-242. Largiader, C. R. and A. Scholl. 1996. Genetic introgresison between native and introduced brown trout (Salmo trutta L.) populations in the Rhone River basin: Molecular Ecology 5: 417426. Leary, R. F., F. W. Allendorf, and G. K. Sage. 1995. Hybridization and introgression between introduced and native fish. Pages 91-101 in H. L. Schramm and R. G. Piper, eds. Uses and effects of cultured fishes in aquatic ecosystems. American Fisheries Society Symposium 15, Bethesda, Maryland. Moran, P., A. M. Pendas, E. Garcia-Vazquez, and J. T. Izquierdo. 1994. Electrophoretic assessment of the contribution of transplanted Scottish Atlantic salmon (Salmo salar) to the Esva River (Northern Spain). Canadian Journal of Fisheries and Aquatic Sciences. 51:248-252. Nielsen, E. E. M. M. Hansen and V. Loeschcke. 1999. Genetic variation in time and. space: microsatellite analysis of extinct and extant populations of Atlantic salmon. Evolution 53:261-268. Poteaux, C., F. Bonhomme and P. Berrebi. 1998. Differences between nuclear and mitochondrial introgressions of brown trout populations from a restocked main river and its unrestocked tributary. Biological Journal of the Linnean Society 63:379-392. Taggart, J. B. and A. Ferguson. 1986. Electrophoretic evaluation of a supplemental stocking programme for brown trout (Salmo trutta L.). Aquaculture and Fisheries Management 17:155-162. Utter, F. M., G. B. Milner, G. Stahl, and D. J. Teel. 1989. Genetic populations structure of chinook salmon in the Pacific northwest, U.S. National Marine Fisheries Service Fishery Bulletin 87:239-264. Utter, F. M., D. W. Chapman and A. R. Marshall. 1995. Genetic population structure and history of chinook salmon of the upper Columbia River. American Fisheries Society Symposium 17: 149- 165. Utter, F. M., J. E. Seeb and L. W. Seeb. 1993. Complementary uses of ecological and biochemical genetic data in identifying and conserving salmon populations. Fisheries Research 18:59-76. d) Comparative data analysis (responsible person: Andy Ferguson)

This session covered a large number of aspects concerning use and properties of molecular markers, different ways of analysing data and the new research possibilities offered both by new statistical developments and new types of molecular markers. The session consisted of a plenary discussion, organised by Andy Ferguson, and a number of longer (60 min.) and shorter (15 min.) presentations.

119 MICROSATELLITE POLYMORPHISM AND MIXED STOCK ANALYSIS IN COD AND BROWN TROUT. Daniel E. Ruzzante, Danish Institute for Fisheries Research, Denmark

The presentation consisted of two parts. During the first part DR talked about the implications of the high levels of polymorphism characteristic of microsatellite DNA regarding required sample sizes and different metrics employed. In particular, the discussion included a description of the behavior of various measures of genetic structure and distance as a function of sample size, number of loci, number of alleles per locus, range in allele sizes (in basepairs) and whether sample sizes are equal or not. The second part of the lecture focused on mixed stock analysis using maximum likelihood methods. In particular the discussion included a description of assumptions and limitations of the Maximum Likelihood method, the majority related to potential sources of bias and lack of precision in the estimates. Two examples were presented, the first dealing with a recently completed study of mixed stock analysis in Atlantic cod (Gadus morhua), and the second dealing with work in progress on mixed stock analysis in brown trout in Denmark.

ANALYSIS OF DNA FROM OLD SCALE SAMPLES. A NEW TOOL FOR CONSERVATION OF SALMONID FISHES Presented by Einar Eg Nielsen, Danish Institute for Fisheries Research, Denmark

Collections of old scales from salmonid fishes can be found in many fisheries institutions in Europe and North America. Such scales have been shown to be useful as a source of DNA, which can be used for analysis of microsatellites.

We here describe the technical procedures in relation to extraction and PCR amplification of DNA from old scales. Further, we describe case stories of the application of old scale DNA analysis for conservation genetics with regard to • identification of native populations • assessment of loss of genetic variation • estimation of effective population size • evaluation of anthropogenic effects on the genetic population structure/mode of migration • the temporal stability of the genetic population structure.

Finally, we describe the perspectives for future genetic studies using old scale DNA. In particular, we evaluate other types of genetic markers such as mtDNA and loci subjected to selection, in association with old scale DNA, as tools for prioritising populations for conservation.

120 MUTATION MODELS AND MICROSATELLITE ANALYSIS. Craig R Primmer, University of Helsinki, Finland.

Over the past few years, microsatellites have been applied as genetic markers for an increasing range of studies, not least in fisheries genetics (O’Connell and Wright 1997). Although very useful for a wide variety of studies (see below), it has become apparent that for some purposes, particularly in the field of population genetics, microsatellites have not proved as useful as previously anticipated (reviewed by Estoup and Angers 1998). One of the main reasons for this failure is the realisation that mutation may play a significant role in determining the genetic (microsatellite) structure of populations, and that the currently available model of microsatellite evolution is not adequate for many population genetic purposes. There is therefore a need for more studies which study the processes involved in microsatellite mutation in order to design an analytical model which more accurately reflects the mode(s) by which microsatellites evolve. This report outlines the current status for microsatellite evolution research and how this relates to their use in population genetic analyses. Finally, it considers some of the applications for which a knowledge of the mode of mutation is not important and microsatellites are therefore clearly still the genetic marker of choice. Microsatellite evolution research Studies of microsatellite evolution can be divided into three categories: theoretical modelling coupled with analyses of allele frequency distributions and two types of empirical approaches. Theoretical studies attempt to model the process of microsatellite evolution by applying assumptions to a range of parameters considered to be important to the mutational process. Computer simulations can then be run and the resulting data compared to observed allele frequency distributions in populations (Valdes et al. 1993; Di Rienzo et al. 1994; Goldstein et al. 1995; Slatkin 1995). The development of accurate models is essential in order to maximise the information gained from microsatellite data. These models however, are reliant on empirical data to provide information confirming that the assumptions of the models are realistic or alternatively, so that the parameters can be altered to more correctly reflect the true situation.

An obvious empirical approach to gain an understanding of the mutation process is to directly analyse and characterise events of de novo mutations in the germline. While in itself appealing, the approach suffers from the general problem associated with all analyses of spontaneous mutations in the vertebrate genome in that they are so rare. The average mutation rate of microsatellites is undoubtedly several orders of magnitude higher than that for nucleotide substitutions in unique non- coding DNA, but is still sufficiently low to obstruct detailed studies in many cases. The vast majority of mutation data therefore concern pooled information from a large number of loci (e.g. Weber and Wong 1993) and there are currently very few non-disease causing loci from any organism where an appreciable number of germline mutation events have been reported (e.g. Primmer et al. 1996, 1998; Sclötterer et al. 1998). If not able to investigate mutation events directly, an alternative approach to studying microsatellite evolution is to analyse the results of past mutations. By sequencing alleles both within and between species, a detailed examination of sequence changes which have arisen from past mutation events can be made (Estoup et al. 1995; Zardoya et al. 1996; Angers and Bernatchez 1997; Primmer and Ellegren 1998). Initially, two mutational alternatives for generating microsatellite polymorphism were considered: Slipped strand mispairing (SSM), which involves intrahelical replication slippage during germ cell replication (Levinson and Gutman 1987); Recombination either due to unequal crossing over or due to gene conversion as has been demonstrated for minisatellites (Jeffreys et al. 1994).

121 Although difficult to demonstrate directly, there is considerable indirect evidence supporting SSM as the primary mechanism by which microsatellite polymorphism is generated. This evidence includes the fact that mismatch repair mutant bacteria and yeast exhibit highly elevated microsatellite mutation rates, while the microsatellite mutation rate of mutants which decrease recombination abilities is completely normal. In addition, the majority of microsatellite mutations observed by analysing pedigree material in a wide range of species result in changes of plus/minus one repeat unit (ru). Mutations due to recombination would not be expected to show such a length bias. Such evidence lead to the adoption of a model of mutation which had initially been proposed as a mutational model for allozymes: the stepwise mutation model (Ohta and Kimura 1973). The basic assumptions of the SMM are: - all mutations result in a change of one repeat unit; - the mutation rate is constant and independent of repeat length; - a mutation is equally likely to cause an increase or decrease in repeat length; - no constraints on allele size. However, empirical studies of microsatellite mutations and microsatellite allele sequence data suggest that a number of these assumptions do not hold for all microsatellite loci. They include factors such as directional mutation (Amos et al. 1996; Primmer et al. 1996, 1998), mutations of greater than one repeat unit (Di Rienzo et al. 1994; Primmer et al. 1996, 1998; Sclötterer et al. 1998), mutation rate variation both within (Jin et al. 1996; Primmer et al. 1996, 1998; Sclötterer et al. 1998) and between loci (Weber and Wong 1993), allele size homoplasy (e.g. Estoup et al. 1995) and allele size constraints (Garza et al. 1995).

Implications for the use of microsatellite data in population genetic analyses Some attempt has been made to utilise the results from these studies to create more complex, and also more realistic, variants of the SMM e.g. a two phase mutation model (TPM) is utilised in some analysis programs such as Bottleneck (Cornuet and Luikart 1996); and Pollock et al. (1998) proposed a genetic distance measure, DGLS which allows for mutation rate variation between loci and for range constraints. However, many additional confounding mutational characteristics have yet to be considered e.g. mutation rate variation between alleles of a single locus, directional bias or sex bias (see references listed above).

Such mutational complexity may lead to inaccurate estimates of genetic structure and/or evolutionary distance when considering different species, or even distantly related populations within a species. Currently, guidelines for determining the limit of evolutionary distance beyond which microsatellites will most likely be of little use are very unclear. Although not yet tested, Estoup and Angers (1998) proposed that an upper limit, above which mutation is likely to have significantly affected the genetic structure of a population, is 1/µ generations, where µ = mutation rate. Assuming an average microsatellite mutation rate of 5 x 10-4 (Weber and Wong 1993), this translates to 2000 generations, which for brown trout, is equivalent to anywhere from less than 6,000 up to 18,000 years divergence, depending on the population life history characteristics. In reality, it is unlikely that such a simple method can accurately estimate the time during which microsatellites retain an evolutionary signal with any great accuracy as such a complex factor may be affected by any number of species and/or microsatellite specific characteristics.

122 Studies for which microsatellites are still the marker of choice There are however, many types of analyses for which microsatellite appear to be the optimal genetic marker as the mode of mutation does not have any significant affect on the results. Often, this is due to the high level of polymorphism often observed at microsatellite loci and the fact that very few, or even no, generations have passed between the groups being considered. Therefore mutation can have had little or no effect in shaping the genetic structure of the groups. Such analyses include: Genetic tagging- where an individual is tracked geographically and/or temporally by means of its multi-locus genotype. A small number of highly variable loci often provides sufficient resolution for most purposes (reviewed by Palsbøll 1999) Individual classification- Individuals are assigned to the reference population from which their multi-locus genotype is most likely to have arisen (currently available methods reviewed by Cornuet et al. 1999). For example, 10 loci, with an average heterozygosity of 0.6, (common for microsatellites) can achieve 100% assignment success for 10 populations each with a divergence of FST = 0.1 (Cornuet et al. 1999). Measuring relatedness- Accurate measure require a large number of highly variable markers therefore, microsatellites currently offer the best possibilities (e.g. Blouin et al 1996) Parentage testing- As for genetic tagging, a relatively small number of highly polymorphic loci will normally give an average exclusion probability of >99% (e.g. Saino et al. 1997).

Conclusions The greatest setbacks thus far in the application of microsatellite to address questions relating to salmonid ecology, and indeed molecular ecology in general, have been in their application to population genetic studies. Potential resolution to these problems requires developing a more accurate model of the process by which microsatellites evolve and/or choosing markers for analyses whose mutational characteristics more closely resemble the currently available models. It is unlikely however, that methods for inter-specific analyses will be sufficiently accurate in the foreseeable future. Nevertheless, it is clear from the range of studies listed above, where knowledge of microsatellite mutational mechanisms is irrelevant, that microsatellites will be successfully utilised for many years to come for a diverse range of purposes, including many in fisheries genetics.

References Amos W, Sawcer SJ, Feakes RW, and Rubinsztein DC. (1996) Microsatellites show mutational bias and heterozygote instability. Nature Genetics 13: 390-391. Angers B, and Bernatchez L. (1997) Complex evolution of a salmonid microsatellite locus and its consequences in inferring allelic divergence from size information. Molecular Biology and Evolution 14: 230-238. Estoup A, Tailliez C, Cornuet JM, and Solignac M. (1995) Size homoplasy and mutational processes of interrupted microsatellites in two bee species, Apis mellifera and Bombus terrestris (Apidae). Molecular Biology and Evolution 12: 1074-1084. Estoup A and Angers B. (1998) Microsatellites and minisatellites for molecular ecology: theoretical and empirical considerations. In: Advances in Molecular Ecology. GR Carvalho (Ed.) IOS Press, Amsterdam. Blouin MS, Parsons M, Lacaille V, Lotz S (1996) Use of microsatellite loci to classify individuals by relatedness. Molecular Ecology 5: 393-401. Cornuet J-M, Piry S, Luikart G, Estoup A and Solignac M. (1999) New methods employing multilocus genotypes to select or exclude populations as origins of individuals. Genetics (in press).

123 Cornuet J-M and Luikart G. (1996) Description and power analysis of two tests for detecting recent population bottlenecks from allele frequency data. Genetics 144: 2001-2014. Di Rienzo A, Peterson AC, Garza JC, Valdes AM, Slatkin M, and Freimer NB. (1994) Mutational processes of simple-sequence repeat loci in human populations. Proceedings of the National Academy of Sciences USA 91: 3166-3170. Garza JC, Slatkin M, and Freimer NB. (1995) Microsatellite allele frequencies in humans and chimpanzees, with implications for constraints on allele size. Molecular Biology and Evolution 12:594-603. Goldstein DB, Linares AR, Cavalli-Sforza LL, and Feldman MW. (1995) An evaluation of genetic distances for use with microsatellite loci. Genetics 139: 463-471. Jeffreys AJ, Tamaki K, MacLeod A, Monckton DG, Neil DL, and Armour JAL. (1994) Complex gene conversion events in germline mutation at human minisatellites. Nature Genetics 6: 136-145. Jin L, Macaubas C, Hallmayer J, Kimura A, and Mignot E. (1996). Mutation rate varies among alleles at a microsatellite locus: phylogenetic evidence. Proceedings of the National Academy of Sciences USA 93: 15285-15288. Levinson G, and Gutman GA. (1987) Slipped-strand mispairing: a major mechanism for DNA sequence evolution. Molecular Biology and Evolution 4: 203-221. O’Connell M and Wright JM. (1997) Microsatellite DNA in fishes. Reviews in Fish Biology and Fisheries 7: 331-363. Ohta T, and Kimura M. (1973) A model of mutation appropriate to estimate the number of electrophoretically detectable alleles in a finite population. Genetics Research 22: 201-204. Palsbøll PJ. (1999) Genetic tagging: contemporary molecular ecology. Biological Journal of the Linnean Society 68: 3-22. Pollock DD, Bergman A, Feldman MW and Goldstein DB. (1998) Microsatellite behaviour with range constraints: parameter estimation and improved distances for use in phylogenetic reconstruction. Theoretical Population Biology 53: 256-271. Primmer CR, Saino N, Møller AP and Ellegren H. (1996) Directional evolution in germline microsatellite mutations. Nature Genetics 13: 391-393. Primmer CR and Ellegren H. (1998) Patterns of molecular evolution in avian microsatellites. Molecular Biology and Evolution 15: 997-1008. Primmer CR, Saino N, Møller AP and Ellegren H. (1998) Unravelling the processes of microsatellite evolution through analysis of germline mutations in barn swallows Hirundo rustica. Molecular Biology and Evolution 15: 1047-1054. Saino N, Primmer CR, Møller AP and Ellegren H. An experimental study of paternity and tail ornamentation in the barn swallow, Hirundo rustica. Evolution 51: 562-570. Schlötterer C, Ritter R, Harr B and Brem G. (1998) High mutation rate of a long microsatellite allele in Drosophila melanogaster provides evidence for allele-specific mutation rates. Molecular Biology and Evolution 15: 1269-1274. Slatkin M. (1995) A measure of population subdivision based on microsatellite allele frequencies. Genetics 139: 457-462. Valdes AM, Slatkin M, and Freimer NB. (1993) Allele frequencies at microsatellite loci: the stepwise mutation model revisited. Genetics 133: 737-749. Weber JL, and Wong C (1993) Mutation of human short tandem repeats. Human Molecular Genetics 2: 1123-1128. Zardoya RD, Vollmer M, Craddock C, Streelman JT, Karl S and Meyer A. (1996) Evolutionary conservation of microsatellite flanking regions and their use in resolving the phylogeny of cichlid fishes (Pisces: Perciformes). Proceedings of the Royal Society of London. Series B. 263:1589- 1598.

124 SUMMARY OF TOPICS DISCUSSED IN THE PLENARY DISCUSSION SESSION Andy Ferguson, Queen’s University of Belfast, UK.

1. DATA QUALITY See summary below by Paulo Prodöhl.

2. SAMPLE SIZES AND STRATEGIES Sampling was discussed in detail last year by John Taggart and he provided further comments this year (see summary below). He pointed out that adequate and representative sampling is a critical aspect of any population genetic study, yet its importance is often minimised or even ignored. John also stated that there are three major elements common to all salmonid sampling considerations: 1) the number of individuals screened; 2) the type of sample (age structure/ life history stage), and 3) the geographic extent of sampling. In many cases people seem to be using similar sample sizes for highly variable microsatellites as for diallelic allozymes. Are there general guidelines that can be used to determine sample sizes for microsatellite, sequencing and mtDNA studies and how are these influenced by the type of data analysis to be undertaken? Chakraborty (1992) discusses in detail sample size requirements for human population genetics using VNTRs. Some relevant statements from that paper include: “it is not feasible to collect samples large enough to encompass all possible genotypes for any VNTR locus in any population”; “a sample of 300 individuals will ensure that all common alleles (frequency > 0.01) will be represented in a sample with at least 95% confidence”; “Because the rare alleles contribute little to heterozygosity or genetic distance, one might conclude that, if we sacrifice the rare alleles and concentrate only on reliable estimation of common allele frequencies, the sample size needed would not be very large”; “Therefore I conclude that for conservative estimates of allele and genotype frequencies at VNTR loci, 100-150 individuals per population may be adequate for such surveys”. In some cases statistical resampling seems to be used as a means of determining significance when small sample sizes are used. Is it valid to carry out bootstrapping / jacknifing when all alleles in the population are not represented in the sample? Has anyone carried out empirical resampling for microsatellites? Are there situations in which it is more important to examine more loci than more individuals e.g. in determining heterozygosity? Zhivotovsky (1999) states that estimation of genetic distance from microsatellites that the number of loci is much more important than sample size and that 5-10 individuals is sufficient but that even 500 loci cannot provide a narrow confidence interval. What is the relationship of sample size required to number of alleles / frequency distribution?

3. GENOTYPE VS ALLELE FREQUENCY DATA The basic information obtained is the genotype of individuals at specific loci. Most data analyses however are based on allele rather than genotype frequencies. However, the allele frequencies only represent the genotype data if in agreement with Hardy-Weinberg. Thus alleles frequencies of 0.5 exactly represent the genotype situation of (homo: het: homo): 25:50:25 but not 30:40:30. What are the problems in testing for H-W e.g. low power of chi-squared / G tests, advantages of exact and pseudo-exact tests? If for microsatellites all genotypes cannot be observed in a realistically sized sample anyway then H-W has to be assumed anyway? Are the assumptions underlying H-W met by trout populations, especially when dealing with highly variable markers? Some people seem to do H-W test and then proceed to ignore significant differences anyway. What about the use of Bonferroni corrections which means that differences are rarely significant anyway except with large sample sizes. Should Bonferroni be used and how? Is it possible to distinguish potential causes of

125 H-W departure? Use of Wahlund effect to detect population admixture. Problems of ‘null’ alleles for microsatellites. Testing for allelic as opposed to genotypic linkage disequilibrium.

4. NEUTRALITY OF MARKERS The universal neutrality of the ‘80s now seems to have been replaced by ‘weak selection’. Is there any such thing as a neutral marker? If not should we be concerned? What are the implications for data analysis? How can selection be tested for? There would appear to be a selective constraint on the upper size limit of microsatellites. For example, it has been shown that in two closely linked microsatellite loci, there was a negative covariance in repeat number between the two loci (Dermitzakis et al., 1998). Selection appears also to occur on the difference between alleles at a locus. Given the importance of spatial organisation in gene regulation such size constraints are not unexpected. ‘Purifying selection’ for microsatellites is, however, no different than purifying selection that acts against particular alleles in coding regions. More important is selection on linked alleles. As some microsatellites are found in intron regions it is to be expected that they are almost certainly influenced by such ‘hitchhiking’ selection although the extent of this will depend on the microsatellite mutation rate (Wiehe, 1998). The idea of microsatellites as truly neutral markers is almost certainly invalid in spite of the many statements to the contrary in the literature. Even small deviations from neutrality will have large effects on population differentiation, and gene flow estimates from allele frequency data are meaningless for markers under very limited selection. Constrained evolution of microsatellite means that some genetic distance measures are non-linear. (See Mitton, 1998, for further discussion.)

5. TEMPORAL STABILITY / CHANGES Importance of temporal stability. How can we detect statistically significant changes in genetic diversity ie in heterozygosity, number of alleles and allele frequency? Using temporal stability to validate sampling protocols.

6. DETERMINING POPULATION STRUCTURE AND INTERPOPULATION GENETIC VARIANCE Many of the newer methods of analysis require information on the evolutionary relationships among alleles. Except for sequence based data, can we use evolutionary relationships among alleles in measuring gene diversity? For microsatellites at least four different mutation models have been suggested. If not step-wise mutation, then statistics which use evolutionary relationships (e.g. Rst) are invalid. What are the implications of different mutation processes at different loci and perhaps different alleles at a locus? What are the problem of homoplasy in distantly related populations. Several of the newer methods require actual mutation rates – what do we know of microsatellite mutation rates? Relative value of different measures of genetic variance and distance - Gst, Rst, ρst, Φst, θ, etc, various genetic distance measures. More importantly what are the underlying assumptions of these different statistics and how far do various markers / brown trout populations meet these assumptions. Congruence - the most powerful statistical test! Importance of and testing for temporal stability. Analyses based on individual genotype rather than allele data. Assignment tests (see summary by Michael Hansen). Do individual rather than population descriptors circumvent the problems of samples sizes with highly variable markers? (For further discussion see, for example, Luikart & England, 1999; Rousset & Raymond, 1997: Ruzzante, 1998; Waples, 1998).

7. ESTIMATION OF Ne AND INBREEDING How can current and historical Ne be estimated? Can inbreeding be detected on the basis of relative lengths of alleles in heterozygotes? Likelihood based methods. Problems of different survival of

126 salmonid families, i.e. although large Ne, considerable variation in survival means that a few families contributes disproportionately to the next generation. Methods for estimating effective number of breeders Nb – determining relationships.

8. ESTIMATING GENE FLOW Is it valid to determine Nm from Fst and analogs, given that the required assumptions are probably not met by most trout populations (in NW Europe at least)? (Populations should be at equilibrium with respect to mutation, migration and genetic drift; also assumes similarity of population sizes, migration rates and symmetrical migration.) Are there better ways of estimating gene flow where the assumptions are valid e.g. assignment tests (i.e identifying immigrant individuals), maximum likelihood, coalescent, Bayesian and Markov chain Monte Carlo methods. Newer methods based on coalescence theory avoid the need to assume symmetry of migration rates or equal population sizes (Beerli, 1998) and in general approaches based on coalescence theory appear to be superior to allele frequency based methods. Coalesence is based on allele genealogies, i.e. allele diversity is traced back through mutations to ancestral alleles with two genealogies coalescing when they have a common ancestor. Individual based analyses potentially allows the analysis of contemporary levels of gene flow as opposed to historical gene flow estimates derived from Fst and other indirect approaches (Estoup and Angers, 1998). Even where valid, limitations of current evolutionary models for particular marker systems, and nonlinearity of the relationship between Fst and Nm, lead to low precision of gene flow estimates and to problems of differentiating between contemporary population structure and population history (Bossart and Prowell, 1998; Templeton, 1998; Waser & Strobeck, 1998).

9. RELATEDNESS AND PARENTAGE Problems of determining relatedness in closed and open populations. Can relatedness be used to determine population even when substantial gene flow exists?

10. SINGLE LOCUS / QUANTITATIVE VARIATION How far does molecular analysis of single locus variation give information on adaptive / quantitative traits? What methods are available to analyse quantitative variation?

11. ANALYSIS PACKAGES / PROGRAMS Any statistic is based on certain assumptions and if the data do not conform then the analysis is suspect. Researchers would not use parametric statistics for data that are not normally distributed etc yet population geneticists gaily plug data into available programs with often apparently little consideration of the underlying assumptions. Strengths and limitations of the different commonly available analysis programs. Are there limitations that are not readily apparent without checking on the models on which the programs are based? Importance and availability of recent statistical methods and programs. A guide of software packages for data analysis is given in Schnabel et al., (1998).

References Beerli, P. (1998) Estimation of migration rates and population sizes in geographically structured populations. In G.R. Carvalho (Ed.) Advances in Molecular Ecology. IOS Press. Bossart, J.L. and Prowell, D.P. (1998) Genetic estimates of population structure and gene flow: limitations, lessons and new directions. TREE 13: 202-206. Chakraborty, R. (1992) Sample size requirements for addressing the population genetic issues of forensic use of DNA typing. Human Biology 64: 141-159.

127 Estoup, A. and Angers, B. (1998) Microsatellites and minisatellites for molecular ecology: Theoretical and empirical considerations. In G.R. Carvalho (Ed.) Advances in Molecular Ecology. IOS Press. Luikart, G. and England, R.P. (1999) Statistical analysis of microsatellite DNA data. TREE 14: 253 – 256. Mitton, J.B. (1998) Molecular markers and natural selection. In G.R. Carvalho (Ed.) Advances in Molecular Ecology. IOS Press. Rousset, F. and Raymond, M. (1997) Statistical analysis of population genetic data: new tools old concepts. TREE 12: 313-317. Ruzzante, D.E. (1998) A comparison of several measures of genetic distance and population structure with microsatellite data: bias and sampling variance. Canadian Journal of Fisheries and Aquatic Sciences 55: 1-14. Schnabel, A., Beerli, P., Estoup, A. and Hillis, D. A guide to software packages for data analysis in molecular ecology. In G.R. Carvalho (Ed.) Advances in Molecular Ecology. IOS Press. Templeton, A.R. (1998) Nested clade analyses of phylogeographic data: testing hypotheses about gene flow and population history. Molecular Ecology 7: 381-397. Waples, R.S. (1998) Separating the wheat from the chaff: Patterns of genetic differentiation in high gene flow species. The Journal of Heredity 89: 438-450. Waser, P.M. and Strobeck, C. (1998) Genetic signatures of interpopulation dispersal. TREE 13: 43- 44. Zhivotovsky, L.A. (1999) A new genetic dstance with application to constrained variation at microsatellite loci. Molecular Biology and Evolution 16: 467-471.

A COMPARISON OF TWO INDIRECT METHODS FOR ESTIMATING AVERAGE LEVELS OF GENE FLOW USING MICROSATELLITE DATA Presented by Kornelia Rassmann, University of Munich, Germany (based on a paper by Gaggiotti, Lange, Rassmann and Gliddon

We compare the performance of Nm estimates based on FST and RST obtained from microsatellite data using simulations of the stepwise mutation model with range constraints in allele size classes. The results of the simulations suggest that the use of microsatellite loci can lead to serious overestimations of Nm, particularly when population sizes are large (N > 5000) and range constraints are high (K < 20). The simulations also indicate that, when population sizes are small (N ≤ 500) and migration rates moderate (Nm ~ 2), violations to the assumption used to derive the Nm estimators lead to biased results. Under ideal conditions, i.e. large sample sizes (ns ≥ 50) and many loci (nl ≥ 20), RST performs better than FST for most of the parameter space. However, FST-based estimates are always better than RST when sample sizes are moderate or small (ns ≤ 10) and the number of loci scored is low (nl < 20). These are the conditions under which many real investigations are carried out and, therefore, we conclude that in many cases the most conservative approach is to use FST.

128 USE OF ASSIGNMENT TESTS BASED ON MICROSATELLITE MARKERS IN STUDIES OF BROWN TROUT. Michael M. Hansen, Danish Institute for Fisheries Research, Denmark

A lot of new statistical tools for analyzing microsatellite data have recently become available (reviewed by Estoup & Angers, 1998). One of these, the so-called “assignment test” (Paetkau et al., 1995; Waser & Strobeck, 1998), has proven particularly useful, as it allows for assigning individuals to populations, based on their multilocus genotypes. Assignment tests can be used in a number of different situations where it is desirable to assess the population of origin of individuals or, conversely, to exclude some populations as being the population of origin of specific individuals.

The principle behind the original assignment test (Paetkau et al., 1995) is quite simple. The likelihood for each individual multilocus genotype to belong to each of the samples is calculated based on the allelic frequencies. An individual is then assigned to the sample in which it has the greatest likelihood of belonging. If assignment tests are performed for individuals from samples from “known” populations the proportion of correctly assigned individuals provides a measure of the genetic distinctness of populations combined with the discriminatory power of the markers used (see, for instance, Paetkau et al., 1995; Estoup et al., 1998). Assignment tests may also be performed, where individuals of “unknown” origin are assigned to a set of baseline populations. This is probably one of the most useful features of assignment tests. For instance, for assessing whether or not a Danish population of Atlantic salmon was indigenous, Nielsen et al. (1997) analysed microsatellite DNA from old scale samples from the original populations and then used this sample along with samples from other populations as baseline samples. Individuals from the present populations was used as an “unknown” sample, and it turned out that most individuals were assigned to the original population, confirming the indigenous status of the present population.

Since the original assignment test was proposed a number of modifications have been made to decrease bias due to, e.g. differences in sample sizes (see Waser & Strobeck, 1998), and some alternative procedures have also been developed (e.g. Ranna & Mountain, 1997). At least three different software packages are available for performing assignment tests and a fourth program (Immanc) can be used for the test by Rannala & Mountain (1997). The programs are the following:

• G-Stat 5.0 (Siegismund, 1995; the version with assignment test is not available by internet) • “Assignment calculator”, available at http://www.biology.ualberta.ca/jbrzusto/Doh.html • Gene Class http://www.ensam.inra.fr/URLB/geneclass/geneclass.html (Cornuet et al., in press) • Immanc http://allele.bio.sunysb.edu/software.html (Rannala & Mountain, 1997)

The Gene Class program is particularly recommendable. It accepts Genepop files as input, it does several different kinds of assignment tests and it includes a very useful option for testing whether or not an individual’s genotype is likely to occur in a specific population.

An example was given on the application of assignment tests for studying stocking effects in brown trout (Hansen et al., 2000). Samples were taken from wild, non-stocked trout in a Danish River, from a stocked subpopulation from the same river and from the hatchery strain used for stocking. The samples were analysed using seven microsatellite loci. First, assignment tests were performed involving only the non-stocked trout and the hatchery strain. Sample sizes were 49 (hatchery strain) and 50 (wild trout) and 92% of the hatchery trout and 94% of the wild trout were assigned correctly,

129 i.e. to the sample they were actually derived from. Next, trout from the stocked section of the river were treated as “unknown individuals” and the hatchery trout and wild, non-stocked trout were used as baseline samples. A total of 93% of sea trout from the stocked section of the river were assigned to the sample of wild, non-stocked trout. In contrast, 54% of resident trout from the same section were assigned to the wild population, whereas 46% were assigned to the hatchery strain. The results suggest differences in survival, homing capabilities and/or reproductive success between stocked trout that become resident and anadromous, respectively.

References

Cornuet JM, Piry S, Luikart G, Estoup A, Solignac M (1999) Comparison of methods employing multilocus genotypes to select or exclude populations as origins of individuals. Genetics, in press. Estoup A, Angers B (1998) Microsatellites and minisatellites for molecular ecology: Theoretical and empirical considerations. In: Advances in Molecular Ecology (ed. Carvalho GR) pp. 55-86. IOS Press, Amsterdam. Estoup A, Rousset F, Michalakis Y, Cornuet J-M, Adriamanga M, Guyomard R (1998) Comparative analysis of microsatellite and allozyme markers: a case study investigating microgeographic differentiation in brown trout (Salmo trutta). Molecular Ecology 7, 339-354. Hansen, M.M., Ruzzante, D.E., Nielsen, E.E. & Mensberg, K.-L.D. (2000) Microsatellite and mitochondrial DNA polymorphism reveals life-history dependent interbreeding between hatchery trout and wild brown trout (Salmo trutta L.). Molecular Ecology, in press. Nielsen, EE, Hansen, MM, Loeschcke V (1997) Analysis of microsatellite DNA from old scale samples of Atlantic salmon: A comparison of genetic composition over sixty years. Molecular Ecology 6, 487-492. Paetkau D, Calvert W, Stirling I, Strobeck C (1995) Microsatellite analysis of population structure in Canadian polar bears. Molecular Ecology 4, 347-354. Rannala B, Mountain JL (1997) Detecting immigration by using multilocus genotypes. Proceedings of the National Academy of Sciences USA 94, 9197-9201. Siegismund, HR (1995) G-STAT, ver. 3.1. The Arboretum, the Royal Veterinary and Agricultural University, Kirkegaardsvej 3A, DK-2970 Hørsholm, Denmark. Waser PM, Strobeck C (1998) Genetic signatures of interpopulation dispersal. Trends in Ecology and Evolution 13, 43-44.

DATA QUALITY Paulo Prodöhl, Queen’s University of Belfast, UK

Microsatellite loci are now recognised as one of the most powerful and potentially useful genetic markers to address a multitude of biological related questions ranging from population structure to the identification of individuals and family groups. Despite its unquestionable usefulness, however, microsatellite screening is not free of problems. On the theoretical side, for instance, very little is known regarding the molecular mechanisms responsible for generating and maintaining the usually high level of variation observed at microsatellite loci. Such knowledge is of fundamental importance for the interpretation of results. Most population genetic models are based on specific assumptions, which should be met by the markers employed (e.g. mutation rate and evolution mode). Published data suggest that many microsatellite loci do not fit to a specific model. Indeed, there is compelling evidence suggesting that different loci exhibit different rates of mutation and thus fit distinct evolutionary models. Surely, these characteristics of microsatellite should be taken

130 in consideration when applying these markers. The objective of this brief review, however, is to focus on technical aspects of microsatellite screening, which is by far the most common source of problems associated with this approach.

- Microsatellites are usually subdivided into categories depending on the size of their motif repeats. Dinucleotides (e.g. CA, GA) are by far the most abundant type of microsatellites. They occur in large numbers in the genome of higher organisms and thus they are relatively easy to clone. More than 80% of all microsatellites available today are dinucleotides. Dinucleotide profiles, however, are usually complicated because of the "artefactual" strand- slippage products of Taq polymerase during PCR amplification, commonly referred to as "shadow" or "stutter" bands. Although these "complicated" patterns can be recognised by an expert eye, in quite a few instances it can lead to erroneous typing.

- The use of tri-, tetra- and pentanucleotide repeats can largely avoid the problem of stutter bands found with dinucleotides. In addition, there is evidence to suggest that tetranucleotides are much more polymorphic than dinucleotides with mutation rates, on average, for tetranucleotides being some four times higher than for dinucleotides.

- Higher repeat motifs (tri-, tetra- and pentanucleotides) are less abundant are hence their cloning and isolation requires considerable extra cost/work.

- New protocols for constructing genomic libraries greatly "enriched" for microsatellites are now available and these offer a powerful alternative for improving the isolation, not only of dinucleotides, but also potentially of “higher” repeat motifs such as tri- and tetranucleotides.

- Most protocols for microsatellite screening are based on end-labelling one of the primers (isotope or fluorescence) prior to PCR and subsequent separation of amplified fragments on gel. One important aspect often ignored by many is that the "allelic" fragments visualised on a gel autorad or other suitable media are basically single stranded fragments. Different end-labelled primers will result in different strands of a particular microsatellite locus being "visible". The rate/speed of mobility of a DNA fragment on a gel is largely dependent on its structure (i.e. base composition). Since alternative DNA strands from the same locus may migrate differently, it is of fundamental importance to state the primer to be end-labelled in order to avoid erroneous interpretations of the results. This is especially important when comparing data from different research groups.

- Although most variation at microsatellite loci is due to loss/gain number of tandem repeats, there is increasing compelling evidence from sequence data suggesting that variation can be due to other factors. This is particular true for highly variable microsatellite loci where "common" insertion/deletions in the flanking region can "imitate" microsatellite variation. Although this is unlikely to be a problem for parentage based studies, it is nevertheless problematic for population analysis.

Data suggest that for most organisms there are different types of microsatellites with different information content (i.e. highly, moderately and low polymorphic loci). Different loci should be considered for particular tasks. For population based work it is more appropriate to chose loci with moderate levels of polymorphisms in order to avoid large sample size requirements.

131 SAMPLING STRATEGIES John Taggart, University of Stirling, UK

Adequate and representative sampling is a critical aspect of any population genetic study, yet its importance is often minimised or even ignored. Currently there is a wide choice of genetic markers available for brown trout research (with differing levels of detectable variation) that are enabling an increasing variety of biological issues to be addressed. It is imperative that appropriate sampling strategies be considered and employed rigorously. My impression from published work is that the practicalities involved in obtaining samples have largely taken precedence over theoretical considerations relating to the studies being undertaken. Thus, in many instances, inappropriate conclusions have been drawn from data analysed from inadequate samples. Obviously, sampling strategies will differ according to the type of questions being addressed (e.g. phylogenetic, macro / micro population differentiation, familial analyses). Three major elements common to all salmonid sampling considerations will be, 1) the number of individuals screened; 2) the type of sample (age structure/ life history stage), and 3) the geographic extent of sampling - a few aspects of which are outlined below. The accuracy of allele frequency estimates and the power of various statistical tests are largely dependent on the sample size screened. Minimum sample sizes of c. 30-50 for population surveys of diallelic polymorphisms have long been recommended (e.g. Allendorf & Phelps, 1981) - though even these modest numbers have not always been reached in published studies. The sample size requirements for more highly variable VNTR loci remains to be fully explored. Though, intuitively, minimum sample sizes in excess of 100 would be expected, for at least some statistical measures (e.g. genetic distances) smaller samples (50-100) may suffice (Chakraborty, 1992; Ruzzante, 1998). The apparent misconception that the now routinely applied population genetic analyses based on exact tests can be reliably carried out on small (sub-optimal) sample sizes needs to be dispelled. This is not the case. Both the type of sample and geographic extent of sampling must also be considered in light of the known life history of the species / population in question. In the specific case of brown trout population studies this should involve such aspects as the species potentially high fecundity and the possible co-occurrence of two major life history types (resident & migratory). Ideally, sampling should be based on sexually mature adults - on or near known spawning sites. In many (if not most) cases, however, this is an impractical approach. Where based on juveniles, efforts should be made to minimise the demonstrated potential for sampling of only a few families (Hansen et al., 1997). In practical terms this would involve sampling over a large area and including fish of as many different ages as possible (avoiding over representation of any single age class). It should be recognised that knowledge of both intra-river genetic substructuring and juvenile brown trout movement / migration is minimal. Further exploration of these aspects, together with computer modelling studies, will be necessary to devise and confirm optimum sampling procedures. Undoubtedly there is a need for more detailed description of sampling methods to be given in published studies to enable a more informed interpretation of presented data.

References Allendorf, F.W. and Phelps, S.R. 1981 Isozymes and the preservation of genetic variation in salmonid fishes. Ecological. Bulletins. (Stockholm) 34: 37-52

132 Chakraborty, R. 1992 Sample size requirements for addressing the population genetic issues of forensic use of DNA typing. Human Biology 64: 141-159 Hansen, M.M., Nielsen, E.E. and Mensberg, K.-L.D. 1997 The problem of sampling families rather than populations: relatedness among individuals in samples of juvenile brown trout Salmo trutta L. Molecular Ecology 6: 469-474 Ruzzante, D.E. 1998 A comparison of several measures of genetic distance and population structure with microsatellite data: bias and sampling variance. Canadian Journal of fisheries and Aquatic Sciences 55: 1-14

e) Conservation of brown trout

This session consisted of presentations by two invited guest speakers. In addition, Linda Laikre, University of Stockholm, and Carlo Largiader, University of Berne, presented a draft of what eventually became the report “Laikre et al.: Conservation Genetic Management of Brown Trout (Salmo trutta) in Europe”. No summary will be given of the latter presentation; instead, we refer to the report.

EVOLUTIONARILY SIGNIFICANT UNITS Robin S. Waples, U.S. National Marine Fisheries Service, Seattle, USA

This session consisted of one presentation by Robin Waples (approximately 45 minutes) and a discussion session that occupied the remainder of the time. Waples is a geneticist with the U.S. National Marine Fisheries Service (NMFS), the agency charged with administering the U.S. Endangered Species Act (ESA) for Pacific salmon. The ESA considers "distinct" populations of vertebrates to be "species" (and hence eligible for legal protection under the Act) but does not explain how distinctness should be evaluated. Waples described how he developed a scientific framework, based on the concept of evolutionarily significant units (ESUs), for evaluating distinct population segments of salmon under the ESA, and this framework formed the basis for the agency's species policy for salmon, which has been used in all ESA listing determinations for Pacific salmon since 1991. The unifying theme of the NMFS species policy is the desire to identify and conserve important genetic resources in nature, thus allowing the dynamic process of evolution to continue largely unaffected by human factors. Waples reviewed case histories in which the NMFS policy has been applied to demonstrate that it is flexible enough to provide guidance on many difficult issues for Pacific salmon, such as anadromy/nonanadromy, variation in life history patterns, and the role of hatchery fish under the ESA.

Waples described how the NMFS ESU policy advocates a holistic approach to defining conservation units, drawing on ecological, environmental, genetic, morphological, life history, and other types of relevant information. He also stressed that defining conservation units is a two step process, with the first being identifying the hierarchical levels of population structure within a species, and the second being determining the appropriate hierarchical level upon which to focus. The first step is (at least in theory) strictly a technical exercise, synthesizing diverse types of biological information as described above. In contrast, there is no single "scientific" answer to the issue of how to choose the appropriate level to focus on in identifying conservation units. This step needs to be informed by science but should be guided by other factors, such as social values,

133 economics, statutory regulations, etc. For example, Waples described how consideration of the legislative and legal history of the ESA leads to the conclusion that most distinct population segments (or ESUs) of salmon should cover fairly large geographic areas and include multiple spawning populations. However, the ESA cannot be expected to be a substitute for sound management of living natural resources, and day-to-day management of the resource (typically carried out by state agencies) should continue to focus on smaller units such as stocks.

Waples suggested that in considering how to finalize the Concerted Action for brown trout, European biologists should consider three major questions:

1. What do you want to accomplish?

A clear statement of goals is necessary before a meaningful conservation strategy can be developed.

2. What is unique about brown trout?

General ideas about conservation strategies can be synthesized from programs that are proposed or in place for other species. However, each species has special considerations, and identification of the unique features of brown trout will facilitate development of effective conservation measures.

3. How best to formulate a global conservation strategy?

Various types of conservation efforts for brown trout are already underway in numerous European countries. These efforts need to be focused and coordinated toward a common goal. An important first step is to identify the biological units that conservation should focus on.

The following is a brief summary of the question and answer session following the presentation.

Q: How has the ESU concept been applied in practice? Has it made a difference?

A: The NMFS ESU policy has been used in all ESA listing determinations for Pacific salmon since 1991. This has resulted in the identification of over 50 ESUs of the seven Pacific anadromous Oncorhynchus species, and about half of these are now listed as threatened or endangered "species" under the ESA.

Q: What happens to populations that don't get designated as ESUs?

A: Under the NMFS ESU policy, all populations are in one ESU or another. Put another way, the species as a whole is divided up into a collection of ESUs that include all populations--so no populations are left out.

Q: What about strongly differentiated populations--how are they treated?

A: Some strongly isolated and very distinctive populations are their own ESUs, but most populations are part of larger, more complex ESUs. The guiding principle is that ESUs should have largely independent evolutionary trajectories.

134 Q: How is life history diversity--particularly freshwater residence vs. anadromy--considered in ESU evaluations?

A: Run or spawn timing and freshwater residence vs anadromy are two major life history features that have had to be considered in many ESU evaluations for Pacific salmon. Genetic information plays a key role in determining how best to form conservation units involving life history diversity. In most cases, genetic data show that different run types (e.g., spring vs fall chinook salmon; summer vs winter steelhead) within a basin are more genetically similar than either is to the same run type in another geographic area. In these cases, an ESU having only summer steelhead or only spring chinook salmon would be an artificial construct since it would include populations with closest relatives outside the group. This type of situation should be avoided in identifying biologically based conservation units because performing an extinction risk analysis is largely meaningless for an artificial group of populations. As a consequence, many salmon ESUs include multiple run-types of chinook salmon or steelhead. In some cases, genetic (and other life history) data indicate that different run types from the same area are substantially different, and in these cases they have been placed in separate ESUs. This has occurred in the upper Columbia and Snake River basins, where there is conclusive evidence that spring and fall chinook salmon are derived from different evolutionary lineages that probably existed in different refugia during that last glacial period.

Evaluation of anadromy/nonanadromy should follow a similar principle: If the two forms share a common gene pool, or are recently derived from a common ancestor, they should be part of the same ESU. As discussed below, treatment of freshwater populations that have been isolated for a considerable period of time is more problematical.

Q: In some areas (e.g., UK), brown trout exist in a series of scattered populations with complex relationships that don't easily conform to geographic ESUs. What can be done in this case?

A: This is a difficult question. Although the genetic structure of anadromous populations may represent an approximate equilibrium between the forces of drift and migration, this will not always be true for resident populations, which may display non-equilibrium dynamics. Even populations that are in close proximity may be strongly isolated and following independent evolutionary trajectories. In such cases, selection of conservation units may have to be guided, at least in part, by more practical considerations that afford opportunities to conserve groups of populations, even though they may not be closely related. If enough such groups of populations can be conserved, then a significant portion of the evolutionary potential of the species as a whole may also be conserved.

CONSERVATION PRIORITIES Fred W. Allendorf, University of Montana, Missoula, USA

This session consisted of a presentation by Fred Allendorf and a discussion. Allendorf is a conservation biologist and geneticist from the University of Montana in the United States. He has been involved in the conservation of salmon and trout for over 20 years, and recently published a

135 paper that provides a system for prioritizing Pacific salmon stocks for conservation (Allendorf et al. 1997; Conservation Biology 11:140-152).

Allendorf's presentation was structured in four parts. First, he talked about the importance of inheritance studies associated with using molecular genetic markers in brown trout and other salmonid fishes because of their polyploid derived-genome. Second, he discussed the importance of recognizing that neutral genetic markers may not accurately reflect the pattern of genetic divergence among populations at loci that are affected by natural selection. For example, two populations that are similar at neutral loci may have important genetic differences at loci that affect local adaptations and therefore are subject to different patterns and intensities of natural selection.

The third part of Allendorf's presentation dealt with identifying appropriate demographic "units" for conservation and priority assignment. He used recent work done in his laboratory with a species that has recently been listed under the Endangered Species Act of the United States (ESA-USA) as an example. Bull trout (Salvelinus confluentus) occupy a naturally fragmented and complex mosaic of stream habitats that are becoming increasingly fragmented by human-related disturbances in western North America. Occurrence of bull trout populations is associated with habitat size (stream width) and patch isolation (stream distance between patches) as predicted metapopulation dynamics. Local demes of bull trout are small enough and exist in variable environments so that they are likely to have relatively short persistence times on an evolutionary time scale. Local populations of bull trout have little genetic variation within them and significant genetic differences among them. Large changes in genotype frequencies between year-classes and excess of heterozygotes within some sampling locations suggest an extremely small effective number of parents in some local populations. However, the relative amount and pattern of genetic variation at nuclear loci and mtDNA suggest that local extinction and recolonization have not occurred historically over short time scales.

The fourth part of Allendorf's presentation discussed the process of setting priorities for brown trout conservation. The ESA-USA mandates that the responsible agencies must develop a system for setting priorities for listing and recovery of species. He presented the priority systems used by the U.S. Fish and Wildlife Service (USFWS) and the National Marine Fisheries Service (NMFS). These systems are based upon several primary criteria, including the magnitude and immediacy of the threat facing a species or conservation unit. Another is the potential biological "value" of the unit being considered. For example, under the USFWS policy, a species in a genus that has no other species is given higher priority than a species in a genus with other species; in addition, a species is given higher priority than a subspecies, and a subspecies is given higher priority than a "distinct population segment". Recently developed NMFS policy also considers the potential cost of any actions and the expected benefits in order to maximize the efficiency of spending limited agency resources. That is, actions with lower cost and greater expected benefits are given higher priority.

Allendorf then reviewed the conclusions from his recent paper on setting priorities on Pacific salmon stocks. The goal of that paper was to provide a mechanism for identifying populations of salmon that would receive high priority for conservation efforts. They first ranked stocks for risk of extinction either by population viability analysis or by a set of surrogate measures. Then they ranked stocks for the biological consequences of extinction for both the species (genetic and evolutionary consequences) and the ecosystem (ecological consequences). Together those rankings

136 allowed stocks to be prioritized for a range of possible actions, with those stocks at highest risk and bearing the greatest biological consequences of extinction receiving the highest priority.

Allendorf's final comments were that perhaps the most important aspect of setting conservation priorities for brown trout is to clearly define the goals and objectives of brown trout conservation efforts.

He pointed out that we have learned from agricultural species, such as corn, rice, and potatoes, that the genetic diversity present in wild populations is an important source of future genetic variation for agriculture. Moreover, research of many people (most of whom were in the room during the presentation) has shown that there is tremendous genetic diversity in brown trout that can only be conserved for potential future use in aquaculture by conserving the diversity of extant wild populations of brown trout. f) Behavioural studies of salmonid fishes (Responsible person: Ian Fleming)

This session consisted of one presentation by Dr. Ian Fleming, NINA, Norway, followed by a plenary discussion.

BEHAVIOURAL STUDIES IN SALMONID FISHES Ian A. Fleming, NINA, Trondheim, Norway.

The session consisted of a presentation by Dr. Fleming followed by discussion. The presentation focused on the linkage between behavioural ecology and molecular genetics in the study of salmonid breeding systems, lifetime reproductive success and life history. Emphasis was placed on how the two approaches together provide a powerful tool to study the evolution and ecology of salmonid fishes. The application of molecular genetics in the absence of behavioural ecology may provide only half the answer, and vice versa. Molecular genetics can allow for the quantification of breeding success, gene flow and family/group survival, while behavioural ecology can identify critical determinants of these parameters, thus providing insight into the dynamics of natural and sexual selection.

Behavioural ecology aims to explain why behaviour is adaptive, i.e. understand how the actions of an organism affect is fitness, while recognising that mechanisms may constrain the outcome. By understanding the adaptiveness of a behaviour, behavioural ecology also aims to predict the expression of the behaviour across a range of environments, populations and species. It does so by combining ideas from behaviour, ecology, evolution and genetics. Thus behavioural ecology includes the study of behavioural mechanisms (e.g., learning and memory, hormones, genetics, chemical and other signals), survival strategies (e.g., foraging, access to resources, predator-prey relations and parasitism), reproductive strategies (e.g., mating systems, sexual selection, alternative breeding tactics and parental care), life histories (e.g., cost of reproduction, reproductive effort, trade-offs, age at maturity, fecundity and lifespan) and population biology (population size, dynamics, migration, hybridisation, co-evolution and use of space).

The remainder of the talk focused on the synergism between behavioural ecology and molecular genetics in the study of reproductive strategies in salmonid fishes. Several topics were identified

137 where such synergism would be highly valuable, these included studies of breeding success, gene flow between cultured and wild fish, effective population size, alternative reproductive tactics, sperm competition, maternal effects on offspring performance and lifetime reproductive success. An overview of the breeding system of salmonid fishes was provided emphasising that it was shaped by natural selection for offspring production and by sexual selection for access to mating opportunities. These evolutionary forces operate with differing intensities in the two sexes to shape their breeding behaviour and tactics. Female breeding success is largely dependent on egg production, access to breeding territories, and nest quality and survival. By contrast, male breeding success is largely determined by access to ovipositing females. The breeding system of brown trout (Salmo trutta) was then contrasted with that of Atlantic salmon (S. salar), its sister species, highlighting the differences in anadromy and frequency of early male maturity as parr, and the implications for the mating system.

Two examples of studies that meshed behavioural ecology with molecular genetics were presented: (1) a study of sexual and natural selection in coho salmon (Oncorhynchus kisutch) during breeding; and (2) a study of the lifetime reproductive success and interactions between farm and wild Atlantic salmon. An experimental investigation of the intensities of natural and sexual selection during reproduction in coho salmon revealed how various selective forces in the breeding environment had likely affected the evolution of body size, secondary sexual characters and sexual dimorphism. This included the role of density-dependent selection in the expression of alternative male tactics. The work involved detailed behavioural observations combined with molecular genetic examinations of paternity.

The second study investigated the ecological and genetic impact of farm Atlantic salmon invading wild populations. Molecular-genetic techniques were used to quantify parentage, identify offspring and follow their lifetime success, and a behavioural-ecology approach to identify the causes and consequences of success/failure. In controlled experimental conditions, the breeding performance of farm salmon was found to be significantly inferior to that of native salmon. This competitive inferiority was higher for males than for females, and was especially high for fish escaping shortly before spawning. Investigations of the offspring showed that those of farm origin typically outgrew those of wild origin, both under artificial and natural conditions. Farm juveniles also differed in aggressive behaviour and dominance relative to wild juveniles, but appear to suffer higher mortality during the earliest life stages, probably due to their lower response to predation risk. Farm-x-wild offspring are behaviourally intermediate between farm and wild juveniles, and show superior growth. In a whole-river experiment, it was shown that the lifetime reproductive success of farm salmon was 16% that of the native salmon. Selection against farm genotypes occurred during breeding and early survival, but was similar thereafter, despite significant ecological differences. The overall productivity of the population, however, appeared depressed. The findings suggested that gene flow from farm to native fish will lead to wild populations changing genetically and behaviourally in the direction of domesticated salmon. Moreover, wild populations may suffer depressed productivity caused by ecological interactions.

It was hoped that these two examples illustrated how the meshing of behavioural ecology and molecular genetics could be used to effectively address questions of both basic and applied importance.

138 g) Suggestions for future studies (Responsible person: Michael M. Hansen)

This was a discussion session aimed at bringing forward more suggestions for future studies. As a result the following suggestions were given:

• Genetic interactions between wild and domesticated trout populations. • Suitability of brown trout for aquaculture production. • Local adaptations in brown trout populations. • Extended review of conservation genetic status of brown trout • Identification of evolutionary significant units for effective conservation • Temporal genetic variability • Effective population size

These suggestions are described in more detail in III. Results and deliverables in the final report, which includes a section termed Suggestions for future studies. h) Is there a life after the CA? (Responsible person: Michael M. Hansen)

This was a discussion session aimed at discussing the future of the initiatives and features established during the CA after the CA has ended. The main conclusions of the discussion are given in V. Future actions in the final report.

139 ANNEX 6.

Report of the Second Iberian Brown Trout (Salmo trutta) Genetics Meeting.

UNIVERSITY OF PORTO, CECA-ICETA; VAIRÃO,

PORTUGAL, 2-4 December 1999.

Organisers:

Paulo Alexandrino

Agostinho Antunes

AIMS OF THE MEETING

Exchange information and results among research groups interested on the genetics of Iberian brown trout populations, including both Troutconcert participants as well as other research teams.

Meeting sessions were devoted to:

1. genetic diversity and phylogeography

2. management and conservation of Iberian brown trout

3. future projects and research cooperation

140 PARTICIPANTS

ALEXANDRINO, Paulo (CECA-ICETA, University of Porto, Portugal) ALMODOVAR, Ana (IMIA, Madrid, Spain). ANTUNES, Agostinho (CECA-ICETA, University of Porto, Portugal) CASTRO, Jaime (University of Santiago de Compostela, Lugo, Spain) FARIA, Rui (CECA-ICETA, University of Porto, Portugal) GARCIA-MARIN, Jose Luis (University of Girona, Spain) MARTINEZ, Paulino (University of Santiago de Compostela, Lugo, Spain) WEISS, Steve (University of Vienna, Austria)

PROGRAM

02 Thursday, December: Arrival

03 Friday, December

10.00 Welcome and presentation of the meeting in the concerted action context 10.30-11.00 Coffee break 11.00-12.30 Proposal of topics for discussion: i) genetic characterisation of Iberian trout populations (homogeneity between the set of markers used by different laboratories); ii) conservation and management strategies; iii) possibility of compiling a conservation oriented publication about Iberian trout populations for the general public and fishery managers; iv) exchange of students and techniques; v) cooperation in future research projects.

12.30-14.30 Lunch

141 14.30-17.00 Discussion of the proposed topics (i). State of the art concerning genetic characterisation of Iberian populations based on allozymes/proteins and mtDNA control region sequences. Geographic distribution of populations already studied. Comparison between different data sets; evaluation of the need of further analysis. 17.00-17.30 Coffee break 17.30-19.30 Discussion of the proposed topic (ii and iii): elaboration of a general publication with a synthesis of the knowledge about Iberian trout populations: including an updated map of distribution, habitat, principal ecological characteristics of the species, diversity (genetic, morphological, ecological), major threats and conservation measures. 20.00 Dinner

04 Saturday, December:

10.30-12.30 Discussion of the proposed topics (iv and v). Future cooperation. Exchange of samples for specific research developed by one particular team (e.g. Lugo). Collaboration between teams for a common scientific topic (e.g. analysis of mtDNA haplotype diversity in Iberian populations). Exchange of techniques (e.g. application of IEF techniques that have been developed by the Porto team by other researchers – Ana Almodovar from Madrid). Discussion about future regular Meetings, projects and possibilities of regional funding. 12.30-14.30 Lunch 14.30-17.00 Excursion and walk around the city

05 Sunday, December: Departure

142 MAIN CONCLUSIONS AND RESULTS

1- Agreement was made to collaborate in the analysis of mtDNA variation in brown trout populations from river drainage’s along the entire coast of the Iberian Peninsula and north into the Bay of Biscay (i.e. including several populations from Atlantic draining French rivers. The purpose of this plan is to clarify the characterisation of the Southwest Atlantic refuge in terms of both present and historical gene flow between Atlantic draining rivers of the region.

A specific plan in terms of the samples needed, and the length of mtDNA sequence was agreed upon and the exchange of samples between laboratories was arranged. For the time being, the effort would be limited to sequencing the entire control region in approximately 10 fish from all available populations. Several analytical/statistical options were discussed related to the ability of such data to answer questions about historical colonisation processes.

2- There was general agreement to publish a conservation-oriented booklet characterising Iberian brown trout populations. The general structure of this publication was discussed which would included a comprehensive distribution map, the diversity of habitats used by brown trout on the Iberian Peninsula, principal ecological characteristics, genetic and phenotypic diversity, the major impacts threatening populations and the conservation measures that are currently active, or could potentially be initiated to preserve and protect brown trout diversity in this region. The Porto team agreed to construct a preliminary draft of the structure of this publication to be distributed to the members of the meeting, and thereafter specific chapters, or tasks would be delegated to the relevant research groups. The publication would be produced in Spanish and Portuguese aimed at informing the interest public, private and governmental authorities.

3- To initiate mobility among laboratories, the Porto lab offered training to those interested in applying Isoelectric focusing techniques. This offer was taken up by Ana Almodovar (Madrid).

4- Several labs agreed to send select samples to Paulino Martinez for his ongoing Europe-wide screening of ITS variation in brown trout populations.

143 ANNEX 7.

FORMS AND QUESTIONNAIRES OF THE CA.

1. Rules and guidelines − Troutconcert mobility funds.

2. Application form − Troutconcert mobility funds.

3. Mobility report − Troutconcert mobility funds.

4. Survey of research activities form.

5. Microsatellite loci survey form.

6. Workshop registration form (used both in 1998 and 1999).

7. Form for collecting allozyme ”raw” data.

8. Form for collecting allozyme ”raw” data  example.

144 Rules and guidelines

TROUTCONCERT – mobility funds

1. First of all, you need to arrange a visit or a meeting with one (or more) of the other laboratories participating in the CA.

2. Next, you fill in the application form and send it by e-mail AND fax to the coordinator. This should be done at least five weeks in advance of the planned visit.

3. The coordinator then decides if the travel falls within the aims of the CA, and (if so) approves the travel (within one week after having received the application).

4. Within five weeks after the visit you need to write a short report, briefly summarising the visit (use the “visit report form”). This report and travel tickets and hotel/accommodation bills (originals, not photocopies) must to be sent to the coordinator by snail mail. In addition, please send an electronic version of the “visit report form” to the coordinator by e-mail (the visit summary will be available for others to read on the WWW homepage). The coordinator will then allocate a corresponding sum from the travel pool to your laboratory. The administration of the institute of the coordinator must transfer the money within a time period of five weeks after the coordinator has received both a travel report and travel tickets and accommodation bills (provided that the five weeks do not coincide with summer or Christmas holidays; in that case the coordinator will inform you of the time schedule). To summarise, the documentation you need to send (if you want to get your money back!) includes:

- flight tickets/ train tickets/ taxi receipts for the travel itself (but not, for instance, bus tickets that you buy during the visit; the daily allowance should cover this sort of expenses) - accommodation bills (hotel or similar)

It is NOT necessary to send bills for meals (included in the daily allowance).

5. Allowable costs for accommodation and daily allowance is covered by a fixed sum of 180 Ecu per day. It is your own choice how you want to administrate this sum (i.e., accommodation prices, prices of meals etc. are your own business). Please note that this sum should also cover taxi transport, bus tickets etc.

6. Expenses for flight tickets should be reduced as much as possible (i.e., tourist/economy class, and include a stay during saturday night)

7. Concerning distribution of mobility funds, the travel pool will be split in two: 113,000 Ecu and 50,000 Ecu. The 113,000 Ecu are for the first 16 months of the CA. During that period no participant will be allocated travel funds exceeding a total of 5,900 Ecu (113,000/19). Any remaining sum of the 113,000 Ecu + the last 50,000 Ecu will be available for travels for the last 8 months of the CA. If you want to travel within these last months you must send an application to the coordinator by month 15. If the costs of the approved travels exceed the total travel funds remaining, the coordinator and the management group will select among the applications, based on the relevance of the aims of the travels and taking previous travel activity by the participants into consideration.

145 8. If two (or more) persons from one laboratory want to participate in a workshop the expenses for one of the persons will be taken from the mobility funds. However, the sum will only be detracted by 50% from the travel sum available for the particular laboratory. It is possible that we need to adjust allocation of travels funds accordingly at a later stage.

146 APPLICATION FORM

TROUTCONCERT – MOBILITY FUNDS

Name:

Laboratory:

Laboratory to visit:

Duration of the visit: From…….. to ………..

Estimated cost of travel (flight tickets etc.) (Ecu):

Estimated living expenses (number of days x 180 Ecu/day) (Ecu):

Purpose of the visit and relevance to the CA:

147 MOBILITY REPORT

TROUTCONCERT – MOBILITY FUNDS

Name:

Laboratory:

Laboratory visited:

Duration of the visit:

From (time and date):

To (time and date):

Total cost of travel (flight tickets etc.) (Ecu):

Total cost of accommodation (Ecu):

Summary of visit:

Please, enclose tickets and accommodation bills

Date: Signature:

148 Survey of research activities.

Laboratory:

Contact person (name and e-mail):

Persons working on brown trout population genetics:

General research topics of the laboratory (List 1 capital letters only!) :

General description of research on brown trout population genetics:

List of projects (brown trout) for the past 3 years (ongoing or finished studies, not planned studies):

Study #1

Researcher: Collaborators (if any): Source of project funding: Topic (List1): Objective: Design: Methodology (List 2): Trout population (List 3): Origin of samples (Country and List 4): Status:

Study #2 etc......

149 Keyword Lists

List1 A: Phylogeography B: Conservation and Management 1: Stocking effects (E.g. Introgression) 2: Stocking efficiency 3: Estimation of effective population size 4: Forensics 5: Genetic monitoring 6: Breeding programs 7: Stock enhancement 0: Other (specify in parentheses) C: Population structure 1: Gene diversity 2: Gene flow 3: Isolation by distance 4: Estimation of effective population size 5: Natural hybridisation 6: Temporal variation 0: Other (specify in parentheses) D: Reproduction biology (e.g. sperm competition) E: Quantitative genetics and gene mapping

List 2 A: Protein electrophoresis B: mtDNA-Sequenzing (specify DNA-segment in parentheses) C: nDNA-Sequenzing (specify DNA-segment in parentheses) D: mtDNA-RFLP E: scnDNA-RFLP E: single locus minisatellite DNA F: single locus microsatellite DNA G: RAPD H: AFLP O: Other (specify in parentheses)

List 3 A: Hatchery stock B: Resident population C: Anadroumous population D: Potamodromous O: Other (specify in parentheses)

150 List 4 A: Atlantic Basin 1: North Sea Basin 2: Baltic Sea Basin B: Black Sea Basin C: Caspian Sea Basin D: Mediterranean Sea Basin 1: Adriatic Sea Basin O: Other (specify in parentheses)

Publications (in alphabetic order. Please arrange the publications as specified in ”Instructions to authors” in Journal of Fish Biology)

Journal papers:

Theses (e.g., PhD and MSc theses):

Reports:

151 Survey of microsatellite loci used for brown trout population genetics.

Laboratory: Contact person + e-mail:

Microsatellite loci used routinely:

Locus Source Annealing Total no. Size Populatio Repeat Comment temp. alleles range ns s observed (bp) studied

Microsatellite loci tested but not used routinely:

Locus Source Annealing Total no. Size Populatio Repeat Comment temp. alleles range ns s observed (bp) studied

152 TROUTCONCERT, First Workshop

Silkeborg, Denmark, 29 June 1998 – 5 July 1998

Registration of participants (to be filled in and returned to the coordinator at [email protected] before 9 March 1998).

Laboratory:

Names of participants:

Special arrangements (for instance, vegetarian):

153 Allozyme ”raw” data form.

TITLE:

CONTRIBUTOR:

PUBLISHED (if possible, please give reference):

PHYLOGEOGRAPHICAL RACE:

GEOGRAPHICAL LOCATION (in the case of wild populations, please give approximate coordinates):

STATUS OF POPULATIONS (hatchery strain/resident/anadromous/potadromous):

STOCKING STATUS:

LIST OF ENZYMES ASSAYED (please give E.C. numbers and presumptive loci in parentheses):

LIST OF POLYMORPHIC LOCI:

LIST OF INDIVIDUAL SAMPLES:

COMMENTS:

TYPE OF DATA (please delete the formats that are NOT used):

Genepop Genotype frequencies Allele frequencies Other (please specify)

DATA:

154 Allozyme ”raw” data form  example

TITLE: Allozyme data, Odder River system, Denmark

CONTRIBUTOR: Michael M. Hansen, Danish Institute for Fisheries Research, [email protected]

PUBLISHED (if possible, please give reference): Hansen, M.M. & Mensberg, K.-L. D. (1996). Founder effects and genetic population structure of brown trout (Salmo trutta) in a Danish river system. Canadian Journal of Fisheries and Aquatic Sciences, 53, 2229-2237.

PHYLOGEOGRAPHICAL RACE: Atlantic.

GEOGRAPHICAL LOCATION (in the case of wild populations, please give approximate coordinates): Odder River, eastern Jutland, Denmark (app. 10o10', 56 o00')

STATUS OF POPULATIONS (hatchery strain/resident/anadromous/potadromous): Mixture of anadromous and resident trout.

STOCKING STATUS: Not stocked.

LIST OF ENZYMES ASSAYED (please give E.C. numbers and presumptive loci in parentheses):

Aspartate aminotransferase (2.6.1.1) (sAAT-1,2*, sAAT-4*) creatine kinase (2.7.3.2) (CK-A1*, CK-A2* ) diaphorase (1.6.2.2) (DIA-1* ) glucosephosphate isomerase (5.3.1.9) (GPI-A1*, GPI-A2*, GPI-B1* ) glycerol-3-phosphate dehydrogenase (1.1.1.8) (G3PDH-2* ) isocitrate dehydrogenase (sIDHP-1*, sIDHP-2* ) lactate dehydrogenase (1.1.1.27) (LDH-A1*, LDH-A2*, LDH-B1*, LDH-B2*, LDH-C1* ) malate dehydrogenase (1.1.1.37) (sMDH-A1*, sMDH-A2*, sMDH-B1,2* ) malic enzyme (1.1.1.40) (MEP-1*, MEP-2* ) mannose-6-phosphate isomerase (5.3.1.8) (MPI-1* ) phosphogluconate dehydrogenase (1.1.1.44) (PGDH-1* ) superoxide dismutase (1.15.1.1) (SOD-1* ).

LIST OF POLYMORPHIC LOCI: sAAT-1,2* sAAT-4* DIA-1* GPI-B1* G3PDH-2* sIDHP-1* LDH-A1* sMDH-A2* MPI-1*

155 LIST OF INDIVIDUAL SAMPLES: Asbaek ...... etc.

COMMENTS:

1. All trout fixed for allele LDH-C1*90. 2. CK-A1* and sMDH-B1,2* were also found to be polymorphic, but due to poor resolution of homo- and heterozygotes they were omitted from the data. 3. The allele "001" at locus LDH-A1* is in fact a null allele.

TYPE OF DATA (please delete the formats that are NOT used):

Genepop

Attach GENEPOP file below. Use the "three digit format" suggested in the GENEPOP manual. Also, please denote each individual by its sample name followed by a comma, such as "asbaek,". Alternatively, you could add a number to each individual, such as "asbaek1, asbaek2, etc. IF you are not able to supply a GENEPOP file (or a similar file with data for each individual genotype) the second best option is to supply a table with genotype frequencies. If this is not possible either, a table of allele frequencies and sample sizes is acceptable.

GENEPOP file:

Allozyme data, Odder River system, Denmark AAT-1,2* AAT-4* DIA-1* GPI-B1* G3PDH-2* IDHP-1* LDH-A1* MDH-A2* MPI-1* POP asbaek, 100100 100100 100100 100100 100100 100100 100100 100152 105105 asbaek, 100100 100100 100100 100100 100100 100100 100100 100100 100105 asbaek, 100140 100100 100100 100100 100100 100100 100100 100100 100105 asbaek, 100100 100100 100100 100100 100100 100160 100100 100100 100105 etc......

156 ANNEX 8.

OVERVIEW OF PARTICIPANTS AND SCIENTIFIC TEAMS

Participant 1: Danish Institute for Fisheries Research, Denmark Scientific team: Einar Eg Nielsen, Daniel E. Ruzzante, Dorte Bekkevold, Michael M. Hansen (project coordinator)

Participant 2: School of Biology and Biochemistry, The Queen’s University of Belfast, N. Ireland, UK. Scientific team: Andrew Ferguson, Paulo Prodöhl (web page and electronic communication coordinator), Rosaleen Hynes, Alistair Duguid.

Participant 3: Norwegian Institute for Nature Research, Norway Scientific team: Kjetil Hindar, Kjartan Østbye, Ian Fleming.

Participant 4: University of Girona, Spain. Scientific team: Carles Pla Zanuy, Jose Luis Garcia-Marin, Nuria Sanz, Marti Cortey.

Participant 5: University of Berne, Switzerland. Scientific team: Carlo Largiader, Fabrizio Baumann, Rachel Bouille, Maya Mezzara.

Participant 6: INRA, France. Scientific team: Rene Guyomard, Sophie Launey.

Participant 7: Marine Research Institute and Institute of Freshwater Fisheries, Iceland. Scientific team: Anna Danielsdottir, Sigurdur Gudjonsson.

Participant 8: Universidad de Santiago de Compostela, Spain. Scientific team: Paulino Martinez, Laura Sanchez, Carmen Bouza, Jaime Castro

Participant 9: Aristotle University of Thessaloniki, Greece. Scientific team: Costas Triantaphyllidis, Apostolos Apostolidis.

Participant 10: University of Göteborg, Sweden. Scientific team: Torgny Bohlin, Jonas Pettersson.

Participant 11: University of Porto, Portugal. Scientific team: Paulo Alexandrino, Agostinho Antunes.

Participant 12: Institute of Marine Research, Norway. Scientific team: Oeystein Skaala, Kevin Glover.

Participant 13: Université Montpellier II, France. Scientific team: Patrick Berrebi, Didier Aurelle.

Participant 14: Wageningen Agricultural University, the Netherlands. Scientific team: Rene Stet, Cornelia Kruiswijk.

157 Participant 15: Stockholm University, Sweden. Scientific team: Nils Ryman, Linda Laikre, Stefan Palm.

Participant 16: Zoological Society of London, England, UK. Scientific team: Michael Bruford, William Jordan.

Participant 17: University of Munich, Germany. Scientific team: Ulrich Schliewen, Kornelia Rassmann, Michael Miller.

Participant 18: Finnish Game and Fisheries Research Institute, Finland. Scientific team: Marja-Liisa Koljonen.

Participant 19: University of Stirling, Scotland, UK. Scientific team: John Taggart.

External experts.

Louis Bernatchez ,University of Laval, Canada. Tiit Paaver, Estonian Agricultural University, Estonia. Alexander Osinov, Moscow State University, Russia. Ewa Wlodarczyk, Sea Fisheries Institute, Poland. Roman Wenne, Sea Fisheries Institute, Poland. Inci Togan, Middle East Technical University, Turkey. Fred Allendorf, University of Montana, Missoula, USA Robin Waples, National Marine Fisheries Service, Seattle, USA Craig Primmer, University of Helsinki, Finland Steven Weiss, Veterinary University of Vienna, Austria

158 ANNEX 9.

BRIEF INDIVIDUAL REPORTS BY PARTICIPANTS, DESCRIBING THEIR INPUT TO THE OBJECTIVES AND DELIVERABLES OF THE CA

Participant 1: Danish Institute for Fisheries Research, Denmark Participant 2: School of Biology and Biochemistry, The Queen’s University of Belfast, N. Ireland, UK. Participant 3: Norwegian Institute for Nature Research, Norway Participant 4: University of Girona, Spain. Participant 6: INRA, France. Participant 7: Marine Research Institute and Institute of Freshwater Fisheries, Iceland. Participant 8: Universidad de Santiago de Compostela, Spain. Participant 9: Aristotle University of Thessaloniki, Greece. Participant 10: University of Göteborg, Sweden. Participant 11: University of Porto, Portugal. Participant 12: Institute of Marine Research, Norway. Participant 13: Université Montpellier II, France. Participant 14: Wageningen Agricultural University, the Netherlands. Participant 15: Stockholm University, Sweden. Participant 16: Zoological Society of London, England, UK. Participant 17: University of Munich, Germany. Participant 18: Finnish Game and Fisheries Research Institute, Finland. Participant 19: University of Stirling, Scotland, UK.

Please note that Participant 5: University of Berne, Switzerland, will report separately to the Swiss government.

159 INDIVIDUAL REPORT FROM TROUTCONCERT PARTICIPANT 1: Danish Institute for Fisheries Research (DIFRES), Department of Inland Fisheries, Silkeborg, Denmark

Five persons from DIFRES participated in the CA: Einar Eg Nielsen (EEN), Daniel Ruzzante (DR), Dorte Bekkevold (DB), Lise-Lotte W. Andersen (LWA) and Michael M. Hansen (MMH; coordinator of the CA). In addition, four persons from the administrative staff were involved in the administration of the CA: Ms Jette Aagaard, Ms Inge Lyngby Jensen, Ms Ann Hesel and Ms Pia Klode. Finally, several other persons from the staff at DIFRES helped with organisation and logistics of the 1998 workshop in Silkeborg.

The input to the CA from DIFRES was as follows:

Objectives

* to promote collaboration among laboratories that are active in research on population genetics of brown trout (Salmo trutta)

MMH edited and organised the project application, which eventually led to the establishment of the CA. Among other things, this involved indentifying and contacting (nearly) all research groups active in brown trout population genetics research. MMH organised two workshops (one in collaboration with Drs. Carles Pla and Jose-Luis Garcia-Marin, University of Girona, Spain) and MMH and EEN participated in both of them, whereas DB, DR and LWA participated in one each. Several persons visited DIFRES during the CA, primarily with the purpose of technology transfer, and two longer postdoctoral visits were arranged as an outcome of the contacts established during the CA.

* to co-ordinate this research and when convenient harmonise the use of genetic markers

MMH took the initiative of collecting information about the research activities of the participating laboratories. Also, he organised workshop sessions aimed at discussing the harmonisation of the use of genetic markers and afterwards he organised a survey of the use of microsatellite markers in the different laboratories. He put the recommendations into writing for inclusion in the final report and for the Troutconcert web page. He also organised the distribution of reference samples to all CA participants that use microsatellites for studies of brown trout in order to perform a calibration of allele sizes. He later collected, organised and described the results which were subsequently discussed at a workshop and put into writing for inclusion in the final report. Finally, in collaboration with Dr. Linda Laikre (Stockholm University, Sweden) and Ms Ewa Wlodarczyk (Sea Fisheries Institute, Poland) the laboratory conducted a calibration of mitochondrial DNA markers (the ND-1 coding segment of the mitochondrial genome).

* to bring together complementary expertise from all parts of the EU and other countries

As stated previously, five persons from DIFRES participated in the workshops. In addition, MMH organised the participation of external experts and guest speakers from European non-EU countries, Canada and USA.

160 * to review and evaluate the status of the research with a focus on conservation/management of genetic resources of the species and the potential of the species for aquaculture

EEN, DR and MMH participated in the two meetings of the "Conservation Group" at the University of Berne, Switzerland and the Aristotle University of Thessaloniki, Greece, respectively. They coauthored the report "Conservation Genetic Management of Brown trout (Salmo trutta) in Europe" and MMH arranged the printing of the report.

MMH collected information on microsatellites used in different laboratories. Further, DIFRES contributed to the collection of allozyme "raw data" that were subsequently made available on the Troutconcert web page.

Deliverables

Workshops MMH organised two workshops (one in collaboration with Drs. Carles Pla and Jose-Luis Garcia- Marin, University of Girona, Spain) and MMH and EEN participated in both of them, whereas DB, DR and LWA participated in one each. MMH, EEN and DR gave a total of eight presentations during the workshops and were responsible for six theme sessions.

Survey/review of brown trout population genetics research activities The group's research activities in brown trout population genetics were presented orally at the 1998 workshop and a written description of research activities was put on the Troutconcert web page. The writing, distribution and collection of questionnaires for the survey of research activities was done by Dr. Carlo Largiader, University of Berne, Dr. Paulo Prodöhl, Queen’s University of Belfast and MMH.

Recommendations for future studies The group provided suggestions for future studies and was responsible for discussion sessions at the workshops, where this issue was discussed. MMH edited the contributions for subsequent inclusion in the final report.

Recommendations for genetic marker nomenclature and harmonisation of the use of techniques MMH was responsible for a workshop session on these issues. Further, he distributed questionnaires in order to collect information on the use of microsatellite loci in different laboratories. Finally, he put the information into writing for subsequent inclusion in the final report and on the Troutconcert web page.

Recommendations for management/conservation of genetic resources of the species EEN, DR and MMH participated in the “Conservation working group” and co-authored the report “Conservation Genetic Management of Brown Trout (Salmo trutta) in Europe. Finally, MMH arranged the printing of the report and made an on-line version of the report, which was made available at the WWW.

161 Evaluation of the potential of the species for aquaculture with emphasis on the genetic resources available The group participated in the discussion of this topic at the 1998 workshop.

Brown trout population genetics bibliography, World Wide Web site for the CA. Raw data and data bases on genetic markers, available on the WWW site. The group provided input for several parts of WWW site, contributed data sets for the collection of allozyme “raw data” and collected information about the use of microsatellite loci in different laboratories.

Mid-term evaluation, Progress report, Final report MMH co-ordinated the mid-term evaluation and edited the progress and final reports.

Administration of the CA Administrative staff at DIFRES (along with the co-ordinator) took care of the administration of the CA. This included booking facilities for workshops, refunding travel expenses for CA participants and external experts, payments for support services (WWW site and printing of reports) and correspondence with the Commission.

162 INDIVIDUAL REPORT FROM TROUTCONCERT PARTICIPANT 2: School of Biology and Biochemistry, The Queen’s University of Belfast, N. Ireland, UK.

Four persons from the School participated in the work of the concerted action: Alistair Duguid (graduate student); Andy Ferguson (professor); Rosaleen Hynes (experimental officer); Paulo Prodöhl (lecturer).

Objectives

* to promote collaboration among laboratories that are active in research on population genetics of brown trout (Salmo trutta)

Duguid, Ferguson and Prodöhl participated in the first TROUTCONCERT workshop and Duguid, Ferguson, Hynes and Prodöhl participated in the second workshop. The results of ongoing work in the Belfast laboratory was presented and discussions held with other participants on areas of common interests in respect of research on brown trout population genetics. Ferguson organised and chaired a session on the comparative value of different genetic markers at the first workshop and on comparative data analysis at the second workshop. Prodöhl contributed a talk to each of these sessions.

Duguid visited the Danish Institute for Fisheries Research to learn techniques of extracting DNA from trout scales and to discuss ongoing research. Exchange of samples for analysis and new primer details has been undertaken with several other participating laboratories including University of Berne (Carlo Largiader), University of Santiago de Compostela, Lugo (Paulino Martinez), University of Girona (Jose Luis Garci-Marin), University of Montpellier II (Patrick Berrebi), INRA (Sophie Launey) and University of Thessaloniki (Costas Triantaphyllidis, Apostolos Apostolidis). Email discussions on brown trout topics have been carried out with most of the other participating laboratories during the course of the concerted action.

Duguid and Ferguson participated in the first working group on conservation genetic management of brown trout in Europe held at University of Berne and Duguid participated in the second working group held at the University of Thessaloniki. Both contributed to the writing of the conservation report.

* to co-ordinate this research and when convenient harmonise the use of genetic markers Prodöhl contributed to discussions and methodology on the harmonisation of microsatellite nomenclature. Hynes contributed data and was involved in discussion on mtDNA-RFLP interpretation and nomenclature. Ferguson coordinated an overview of the allozyme nomenclature and discussion of homologies.

* to bring together complementary expertise from all parts of the EU and other countries Personnel from the School participated in four workshop meetings, in one laboratory exchange and in many email discussions. Input from other members of the CA has been very beneficial in developing our programme of research on brown trout population genetics. Hopefully we have also contributed similarly to programmes in other laboratories.

163 * to review and evaluate the status of the research with a focus on conservation/management of genetic resources of the species and the potential of the species for aquaculture Duguid and Ferguson participated in the first working group on conservation genetic management of brown trout in Europe held at University of Berne and Duguid participated in the second working group held at the University of Thessaloniki. Both contributed to the writing of the conservation report.

* to establish databases on relevant literature, available genetic markers and data from published and unpublished studies. The databases and reports from the CA will be made publicly accessible on the World Wide Web Prodöhl was responsible for the design, implementation and maintenance of the WWW site. In addition all personnel contributed to the information for the site. Data on some 9200 individuals from more than 120 brown trout populations in Britain and Ireland were contributed to the allozyme database.

Deliverables

Workshops Duguid, Ferguson and Prodöhl participated in the first TROUTCONCERT workshop and Duguid, Ferguson, Hynes and Prodöhl participated in the second workshop.

Survey/review of brown trout population genetics research activities We have presented our own research activities on brown trout population genetics both at oral presentations at the sessions arranged at the workshops, written summaries provided for the WWW site, and as contributions to the reports.

Recommendations for future studies We participated actively in the discussions on recommendations for future studies, and provided suggestions for such studies.

Recommendations for genetic marker nomenclature and harmonisation of the use of techniques We were involved in nomenclature and technique harmonisation regarding the use of microsatellite, mtDNA and allozyme markers.

Recommendations for management/conservation of genetic resources of the species We have participated actively in the working group, and in the preparation of the report, on conservation genetic management of brown trout.

Evaluation of the potential of the species for aquaculture with emphasis on the genetic resources available We participated in discussions of this topic at the workshops.

164 Brown trout population genetics bibliography, World Wide Web site for the CA. Raw data and data bases on genetic markers, available on the WWW site. We provided literature details and collated the bibliography (currently with more 1800 references) for presentation on the WWW site. We provided raw data and collated and presented all data on the WWW site.

Mid-term evaluation, Progress report, Final report Ferguson participated in the mid-term evaluation. We provided information as required for the progress and final reports.

165 INDIVIDUAL REPORT FROM TROUTCONCERT PARTICIPANT 3: Norwegian Institute for Nature Research (NINA), Trondheim, Norway

Objectives

* to promote collaboration among laboratories that are active in research on population genetics of brown trout (Salmo trutta) NINA has taken part in both workshops arranged by the TROUTCONCERT. Senior research scientist Kjetil Hindar and graduate student Kjartan Østbye participated in the 1998 (Silkeborg) workshop, whereas Kjetil Hindar and senior research scientist Ian Fleming participated in the 1999 (St. Feliu de Guixols) workshop. In September 1999, Ian Fleming visited the Institute of Zoology, London, for 3 days exchanging ideas, presenting a talk and discussing future collaborative research.

NINA has regular contact with other TROUTCONCERT participants by collaborative research projects (e.g. current EU funded research with Stockholm University), by exchanging students (Kjartan Østbye is currently in Louis Bernatchez’ laboratory at the University of Laval, studying whitefish) and by exchanging material for genetic analyses (e.g. samples of trout and salmon hybrids sent to the University of Santiago de Compostela for chromosome analysis).

* to co-ordinate this research and when convenient harmonise the use of genetic markers NINA participated in the comparative genetic analysis of microsatellite markers in five brown trout. NINA contributed to the allozyme marker harmonisation organised by the Queen’s University of Belfast. NINA regularly uses genetic techniques (allozymes, DNA extraction, mtDNA-RFLPs, mini- and microsatellites) developed by other TROUTCONCERT participants.

* to bring together complementary expertise from all parts of the EU and other countries In the context of TROUTCONCERT, NINA’s strongholds are a long research tradition on brown trout ecology and a more recent focus on behavioural ecology. Also, as Norway is one of the major producers of cultured fish, NINA contributes hands-on experience and experimental results on the interaction between cultured and wild salmonids.

* to review and evaluate the status of the research with a focus on conservation/management of genetic resources of the species and the potential of the species for aquaculture Kjetil Hindar was responsible for presenting aquaculture as a topic for discussion at the first workshop, and also for editing a report on that issue. At the second workshop, Ian Fleming presented some implications of behavioural ecology for conservation and management. NINA also participated in the writing of the conservation report.

* to establish databases on relevant literature, available genetic markers and data from published and unpublished studies. The databases and reports from the CA will be made publicly accessible on the World Wide Web NINA contributed literature listings for the literature database, and allozyme genotype (raw) data for the allozyme database set up by TROUTCONCERT participants at the Queen’s University of Belfast.

166 Deliverables

Workshop NINA sent two participants to both workshops arranged by the TROUTCONCERT. At the first workshop, oral presentations were given by Kjetil Hindar on NINA’s own research activities, as well as on brown trout aquaculture genetics. At the second workshop, an oral presentation was given by Ian Fleming on behavioural ecology in salmonid fishes. NINA researchers participated actively in the discussions at both workshops.

Survey/review of brown trout population genetics research activities NINA presented its research activities within the field of brown trout population genetics at the first workshop, and its activities in behavioural ecology/genetics at the second workshop. Summaries of these activities have been contributed in writing and to the WWW site. NINA also has reviewed studies on brown trout aquaculture and ecological and genetic studies which are relevant for aquaculture activities.

Recommendations for future studies NINA participated in the discussion about future directions of brown trout population genetics research, and suggested topics for future collaborative work including the meshing of behavioural ecology and population genetics. In collaboration with two other laboratories inside the TROUTCONCERT (Agricultural University, Wageningen, and the Zoological Institute, London) and others outside it, NINA actively pursued one of these topics in an application to EU for funding of research.

Recommendations for genetic marker nomenclature and harmonisation of the use of techniques NINA commented on a list of suggested allozyme nomenclature which was distributed through TROUTCONCERT by the Queen’s University of Belfast. Owing to a long history of collaboration, NINA originally adopted allozyme techniques and nomenclature which were developed by the Division of Population Genetics, Stockholm University, and relates genotypes of brown trout found in Norway to those found in Sweden (and elsewhere) by the Stockholm group. This system can to a very large extent be harmonised with the one suggested by Queen’s University.

NINA analysed DNA samples of five individual brown trout extracted by the Danish Institute for Fisheries Research. Specifically, we determined microsatellite genotypes and allele sizes on a capillary-based, automated DNA sequencer, the ABI 310. The results were contributed to the TROUTCONCERT for comparative analyses and discussion at the second workshop.

NINA currently uses PCR-RFLP-analysis of mitochondrial DNA which has been suggested by the Danish Institute for Fisheries Research.

Recommendations for management/conservation of genetic resources of the species NINA provided input to the discussions on management and conservation of brown trout genetic resources at both workshops, and also contributed to the Conservation report. Specifically, NINA provided information on Norwegian legislation pertaining to management of genetic diversity, and more generally to information on brown trout population size, behaviour and ecology, and the potential effects of harvesting.

167 Evaluation of the potential of the species for aquaculture with emphasis on the genetic resources available. NINA (Kjetil Hindar) was responsible for presenting brown trout aquaculture and initiating the discussion of of this issue at the first workshop. We also edited the chapter on aquaculture which accompanied the progress report from the TROUTCONCERT.

Brown trout population genetics bibliography NINA has provided literature listings for the brown trout population genetics bibliography.

World Wide Web site for the CA NINA has provided information on its population genetics laboratory for the WWW site, including personnel, research projects and relevant literature.

Raw data and data bases on genetic markers, available on the WWW site NINA contributed brown trout allozyme raw data from the Voss River, western Norway, covering populations of anadromous, resident and landlocked brown trout from the same river system. The data were originally published by Hindar, Jonsson, Ryman & Ståhl (1991. Heredity 66: 83-91).

Mid-term evaluation As a member of the management group, NINA took part in the mid-term evaluation of the TROUTCONCERT.

Progress report, Final report editorial meeting, Final report NINA has contributed to the Progress and Final reports by responding to questionnaires, by writing sections on aquaculture and behavioural ecology, by contributing to and/or commenting on other sections of the reports, by participating in the management group, and by specifying NINA’s own research activities.

168 INDIVIDUAL REPORT FROM TROUTCONCERT PARTICIPANT 4: University of Girona, Spain

The group from the University of Girona co-organized the second workshop of the CA that was held in Sant Feliu de Guixols, Spain, from 24 June - 4 July of 1999. Carles Pla and José Luis García-Marín arranged the facilities and logistics concerning the workshop, including accommodation of participants. Members of the group have participated in several meetings and task involved on the CA. The following is a description of our input to main activities carried out within the framework of the CA.

1998 Workshop: José Luis García-Marín and Núria Sanz participated in the meeting as representatives of the group. Dr. García-Marín presented the research activities of the group and furthermore presented data of the group related to the postglacial colonitazion of brown trout in Europe.

First Iberian Trout Genetics Meeting (1998): Carles Pla, José Luis García-Marín and Núria Sanz participated in this meeting. Dr. García-Marín and Dr. Pla conducted two sessions and gave three oral presentations.

1999 Workshop: Carles Pla, José Luis García-Marín and Martí Cortey coorganised and participated in this meeting.

Second Iberian Trout Genetics Meeting (1999): José Luis García-Marín was the representative in this meeting.

Furthermore, Dr. García-Marín participated in the meetings that took place in Berne, Switzerland, and Thessaloniki, Greece, in order to discuss and write the report on conservation of genetic resources in brown trout, and he coauthored the report.

169 INDIVIDUAL REPORT FROM TROUTCONCERT PARTICIPANT 6: INRA, Paris, France

The following describes the group’s input to the tasks and sub-tasks of the CA.

Task 1. Annual reports:

Sub-task 1.2: Review and discussion on the state of the art of brown trout population genetics INRA participated to the two annual meetings, in Silkeborg, Danemark and Sant Feliu de Guixols, Spain. One person, René Guyomard, and two persons, R. Guyomard and Sophie Launey, attended to the first and second meeting respectively.

During the first meeting, R. Guyomard gave an overall presentation of the scientific activities of the Laboratory of Fish Genetics (quantitative genetics, population genetics and developmental genetics) and researches in brown trout population genetics at INRA. These information are essentially the same as those provided in the short forms distributed by C. Largiadèr (University of Bern) for the survey of research activities. R. Guyomard also presented recent results on the potential of adjacent microsatellite markers for assessment of introgression rate between populations in the session Stocking impact assessment (Chairman: Patrick Berrebi; 1 july).

Sub-task 1.3: Recommendations for management/conservation resources of the species R. Guyomard participated in the discussion on recommendations for management/conservation resources of the species during the two annual meetings and was present to the first meeting specifically which was devoted to these issues in Bern (25-27 February 99). R. Guyomard participated to the general discussions and to the working group on macro-geographic differentiation. He reviewed the state of art for the description of genetic resources of the Mediterranean S. trutta and S. marmoratus lineages in collaboration with Jose-Luis Garcia-Marin (University of Gerona). This review is included in the report "Conservation Genetic Management of Brown trout (Salmo trutta) in Europe", which was edited by Linda Laikre.

Task 2. Survey of research activities and literature

Sub-task 2.1: Distribution of questionnaires to all participants to record research activities: INRA has answered to questionnaires related to research activities and characteristics of microsatellite loci used in the laboratory in population genetics studies. Information were sent to the coordinator of the concerted action. This information is available on the Concerted Action website.

Task 3. Establishment of data bases for genetic markers and raw data

Sub-task 3.2: Establishment of data bases for genetic markers and raw data R. Guyomard participated to the workshop on the standardisation of protein loci and alleles nomenclature and provides a table of the alleles for all the loci routinely analysed in the laboratory and the tentative correspondence with allele scored in other laboratories. The information has been sent to the coordinator for allozyme standardisation (Andrew Ferguson, Queen’s University, Belfast).

170 Task 4. Mobility among laboratories

Sub-task 4.1 Technology transfer In August 1988, R. Guyomard has visited Dr Carlo Largiadèr (Bern, Switzerland) in order to initiate purification and sequencing of salmonid microsatellite clones. This visit to Bern has lead to the generation of 88 clones containing microsatellites. These clones are available for the brown trout gene mapping project initiated in the laboratory of Fish Genetics.

In December 99, Sophie Launey visited the Danish Institute for Fisheries Research for one week. The purpose of this visit was to learn techniques of extraction and amplification of DNA from old samples (namely scale collections). The technique is now available in our laboratory.

Sub-task 4.3. Joint data analyses and planning of future project The visit in Bern (August 1998) has been dedicated to the finalization of a first manuscript on microsatellite analysis of polyandry and spawning site competition in brown trout (Salmo trutta L.) A common research program on the study of the modalities of gene introgression system where stocking has taken place until recently and its effects on the biology of the native population was also discussed and some specific actions were planned.

171 INDIVIDUAL REPORT FROM TROUTCONCERT PARTICIPANT 7: Marine Research Institute and Institute of Freshwater Fisheries, Iceland

Two persons from the MRI and IFF participated in the work of the concerted action: Anna Kristin Danielsdottir (MRI) and Sigurdur Gudjonsson (IFF).

The input to the CA from MRI and IFF was as follows:

Objectives

* to promote collaboration among laboratories that are active in research on population genetics of brown trout (Salmo trutta) Danielsdottir participated in the first and second TROUTCONCERT workshop. The results of past and ongoing work in Iceland was prepared by Danielsdottir and Gudjonsson and presented at the first workshop. Gudjonsson was also in communication regarding subjects discussed at the meeting. Discussions with other participants of areas of common interests with respect to research on brown trout population genetics. Danielsdottir participated in a session on the name coordination of genetic markers and data analysis at the second workshop.

* to co-ordinate this research and when convenient harmonise the use of genetic markers Danielsdottir and Gudjonsson distributed Icelandic brown trout samples to University of Girona (Jose Luis Garci-Marin), to analyse the whole D-loop region of the mitochondrial DNA of brown trout. Danielsdottir participated in a session on the name coordination of genetic markers (allozyme nomenclature)

* to bring together complementary expertise from all parts of the EU and other countries Danielsdottir from the MRI participated in the workshops and took part in email discussions. Gudjonsson from the IFF took part in email discussions. Meeting experts from the EU and other countries was extremely useful for our current brown trout research programs.

* to review and evaluate the status of the research with a focus on conservation/management of genetic resources of the species and the potential of the species for aquaculture Gudjonsson planned but didn´t succeed in participating in the second working group helt at the University of Thessaloniki, Greece. We followed the discussions and plan to use the results of the conservation report for management and conservation of this species in Iceland.

* to establish databases on relevant literature, available genetic markers and data from published and unpublished studies. The databases and reports from the CA will be made publicly accessible on the World Wide Web We provided progress and status report on management and population genetic studies of brown trout in Iceland. We didn´t provide raw allozyme data to the site yet, but we hope to do so as soon as a manuscript has been completed.

Deliverables

Workshops Danielsdottir participated in the first and second TROUTCONCERT workshop.

172 Survey/review of brown trout population genetics research activities The research activities in Icelandic brown trout population genetics and management were presented orally at the 1998 workshop and a written description of research activities was provided for the Troutconcert web page.

Recommendations for future studies We participated actively in the discussions on recommendations for future studies, and provided suggestions for such studies.

Recommendations for genetic marker nomenclature and harmonisation of the use of techniques Danielsdottir was involved in nomenclature and technique harmonisation regarding the use of allozyme markers.

Recommendations for management/conservation of genetic resources of the species Gudjonsson participated in discussions on conservation genetic management of brown trout.

Evaluation of the potential of the species for aquaculture with emphasis on the genetic resources available The group participated in the discussion of this topic at the 1998 workshop.

Brown trout population genetics bibliography, World Wide Web site for the CA. Raw data and data bases on genetic markers, available on the WWW site. We provided progress and status report on management and population genetic studies of brown trout in Iceland. We didn´t provide raw allozyme data to the WWW site yet, but hope to do so as soon as a manuscript including the data has been completed.

Mid-term evaluation, Progress report, Final report We provided information as required for the progress and final reports.

173 INDIVIDUAL REPORT FROM TROUTCONCERT PARTICIPANT 8: Universidad de Santiago de Compostela, Spain.

Summary. During the two years of this Concerted Action the group of Lugo participated in the two main workshops which took place in Silkeborg (Denmark) and Girona (Spain); was responsible of the organization of the first meeting and participate in the second meeting on brown trout genetic resources in the Iberian Península; and finally participated in the second general meeting in Thessaloniki, where the final document on conservation resources in brown trout was definitively elaborated. Besides, during this period our lab established several contacts with different labs for transfer of technology, interchange of samples (a set of samples of brown trout from Northwestern Spain is available for participants) and information on different aspects of brown trout population structure and conservation. All data from allozymes from Northwestern Spain obtained in our lab were transferred to P. Prodöhl for the general database of allozymes. Finally several important collaborations were developed in the context of this concerted action with the labs of M.M. Hansen, L. Bernatchez, J.L. García-Marín, and P. Alexandrino.

Participation and organization of workshops and meetings

1) First general workshop in Silkeborg Three members of our group participate in this workshop: Drs. Laura Sánchez and Paulino Martínez, coleaders of the group, and Dr. Carmen Bouza. The presentation of the group was made by L. Sánchez and P. Martinez. Also, a contribution to the session devoted to merits of genetic markers for population analysis chaired by Dr. A. Ferguson was made: "rDNA RFLPs as genetic markers for conservation of genetic resources in brown trout" (P. Martínez). During this meeting was established a contact with Dr. M.M. Hansen for a postdoctoral stay of Dr. Bouza in the Danish Institute of Fisheries Research in Silkebork during 9 months. During this visit C. Bouza made an interesting collaboration in the assesment of stocking by parentage assignment by using microsatellites and mitochondrial DNA. A compromise for the organization of a meeting related with genetic estructure and conservation in Iberian Península between the three groups of this area (Girona, Porto, Lugo) was taken. A possible collaboration, which was finally developed at the end of 1999, for a phylogeographic analysis in brown trout by using ITSs of rRNA genes was discussed with Dr. L. Bernatchez.

2) Second general workshop in Girona Three members of the group participated in this meeting: Drs. L. Sánchez, J. Castro and P. Martínez. A presentation was made in the session devoted to the anadromous form of brown trout chaired by Dr. M.M. Hansen: "Genetic structure of brown trout at the limit of the anadromous form"(P. Martínez). Our group also participate in the meeting for harmonization of allozyme nomenclature chaired by Dr. A. Ferguson.

3) First Iberian Meeting in Lugo This meeting was organized by Dr. P. Martínez and took place in the Faculty of Veterinaria of the Universidad de Santiago de Compostela in Lugo, where the lab of the group is located. The meeting lasted two complete days and most of the groups working in brown trout in Iberian Península participated. Both aspects on population structure and phylogeography, as well as on

174 conservation of resources in brown trout where analyzed in Iberian area. Five members of the group participated in this meeting: PhD Student B. G. Pardo and Drs. L. Sánchez, A. Viñas, J. Castro and P. Martínez. Several presentations were made by members of the group: "Genetic structure of brown trout: a microgeographic view" (P. Martínez); "rDNA RFLPs as markers for resource management in brown trout" (Dr. J. Castro); and "Chromosome polymorphisms in brown trout: their use for population and phylogenetic analysis"(Dr. L. Sánchez).

4) Second Iberian meeting in Porto. Two members of the group participated in this meeting: Drs. J. Castro and P. Martínez. The elaboration of a document specifically related with conservation status of brown trout in Iberian area was taken in this meeting. Also, a collaboration was established for a phylogenetic analysis of brown trout in this area by using allozymes (integration of data from all labs in the Península), mtDNA and ITS1 sequences.

5) Second meeting in Thessaloniki for the elaboration of the document on "Conservation genetic management of brown trout". Dr. P. Martínez participated in this meeting, where the final version of this important document, which recover the view of participants on conservation genetics of brown trout, was elaborated.

Interchange of samples In the context of this CA it was possible to obtain samples from very distant locations of our lab for different purposes. Samples for chromosome analysis were received from Drs. K. Hindar and C. Largiader. Also, for the analysis of ITSs we received samples from Drs. M.M. Hansen, A. Ferguson, C. Triantaphyllidis, J.l. García-Marín, and P. Alexandrino. Our lab also sent DNA samples to different labs (A. Ferguson, J.L. García-Marín, P. Alexandrino)

Research collaborations During this action it was possible to establish contact and carried out several research collaborations with different labs, as oultlined before: - Stay of Dr. D. Bouza in the lab of Dr. M.M. Hansen for the analysis of stocking in Danish brown trout populations by using microsatellites and parentage assignment - Phylogeographic analysis of brown trout along its distribution area, and more specifically, in Iberian Peninsula and other regions of interest for conservation, with Drs. L. Bertnatchez and P. Presa. - Homogenization of allozyme data in Iberian Península for phylogenetic analysis and conservation resources in brown trout.

175 INDIVIDUAL REPORT FROM TROUTCONCERT PARTICIPANT 9: Aristotle University of Thessaloniki, Greece.

For each of the objectives of the TROUTCONCERT project we comment briefly the contribution by our laboratory.

Objectives

* to promote collaboration among laboratories that are active in research on population genetics of brown trout (Salmo trutta) Two scientists of our group (Professor C. Triantaphyllidis and Dr A. Apostolidis) have participated in the two workshops held within TROUTCONCERT. We have presented the research conducted at our laboratory and have learnt about ongoing research in other labs. These meetings have provided an opportunity to discuss with people with similar research interests and to apply for two more EU projects.

Costas Triantaphyllidis and Apostolos Apostolidis have participated very actively in the working group on "Conservation genetic management of brown trout in Europe". Two workshop were organised specifically for this working group:

First meeting in Berne. Professor C. Triantaphyllidis participated in this meeting.

Second meeting in Thessaloniki, Greece. This meeting was organised by Professor C. Triantaphyllidis and Dr A. Apostolidis and took place in the Department of Genetics, Development and Molecular Biology, School of Biology, Aristotle University of Thessaloniki, where the lab of the group is located. The meeting lasted four complete days and during these days the editing of the final version of "Conservation genetic management of brown trout in Europe" took place. In addition, on the last day of this meeting a trip was organised to the River Tripotamos, so that the participants had the opportunity to observe sampling in a local native brown trout population.

Interchange of samples In the context of this CA our lab sent DNA samples to four different labs (A. Ferguson - Northern Ireland-U.K., J.L. Garcia-Marin - Spain, Paulino Martinez - Spain, S. Palm - Sweden).

* to co-ordinate this research and when convenient harmonise the use of genetic markers Our lab has participated actively in the work with harmonising genetic markers using allozymes and PCR-RFLP analysis of mitochondrial DNA. Dr A. Apostolidis participated in several meetings of the group within TROUTCONCERT working with harmonisation of allozyme and of PCR-RFLP mtDNA markers, and has provided information on analytical procedures and data generated at our lab. The work has also included the photographic documentation of the different genotypes.

* to bring together complementary expertise from all parts of the EU and other countries Professor C. Triantaphyllidis and Dr A. Apostolidis have participated in total of four workshop- meetings arranged within TROUTCONCERT. During these meeting we had the opportunity to interact with scientists with similar research interests, from both other parts of the EU and from countries outside of EU, many of whom we had not had the chance to meet previously. We have found these interactions most educational and stimulating.

176 * to review and evaluate the status of the research with a focus on conservation/management of genetic resources of the species and the potential of the species for aquaculture Professor C. Triantaphyllidis and Dr A. Apostolidis have participated very actively in the working group on "Conservation genetic management of brown trout in Europe". The work within this group has included literature reviews and construction of overviews on various information of relevance to the topic of conservation genetic management. The conservation working group provided a report, ‘Conservation genetic management of brown trout (Salmo trutta) in Europe’ where participated both professor C. Triantaphyllidis and Dr A. Apostolidis.

* to establish databases on relevant literature, available genetic markers and data from published and unpublished studies. The databases and reports from the CA will be made publicity accessible on the World Wide Web Our lab has provided information on brown trout literature, genetic markers and data from genetic studies of brown trout in Greece. This information has been incorporated into the databases, reports and webpages generated within TROUTCONCERT.

Deliverables

Workshop As noted above, C. Triantaphyllidis and A. Apostolidis have participated in both the annual workshops organised by TROUTCONCERT and in the two workshops of the conservation working group within TROUTCONCERT.

Survey/review of brown trout population genetics research activities We have presented our own research activities on brown trout population genetics both at oral presentations at the seminars arranged at the workshops and written summaries provided for the WWW site, and for reports generated by TROUTCONCERT.

Recommendations for future studies We have participated in the discussions on recommendations for future studies and have provided suggestions for such studies.

Recommendations for genetic marker nomenclature and harmonisation of the use of techniques We have participated actively in the work with harmonisation of allozyme and mtDNA laboratory techniques.

Recommendations for management/conservation of genetic resources of the species We have participated actively in the working group on "Conservation genetic management of brown trout". We have also given the address of three Greek institutions so as to distributed the "Conservation genetic management of brown trout" document. This document will be useful in future programs of stocking activities in brown trout in Greece.

Evaluation of the potential of the species for aquaculture with emphasis on the genetic resources available Despite that we have not been particularly involved in this issue we have participated in the sections regarding this topic presented at the workshops.

177 Brown trout population genetics bibliography, WWW site for the CA. Raw data and data bases on genetic markers available on the WWW site. We have provided literature listings for the population genetics bibliography and we have also supplied data on genetic characteristics of Greek as well as other European brown trout populations for the collection presented at the WWW site.

Mid-term evaluation, Progress report, Final report We have provided requested information for the reports produced on the progress and work within TROUTCONCERT.

178 INDIVIDUAL REPORT FROM TROUTCONCERT PARTICIPANT 10: University of Göteborg Sweden.

PhD-student. Jonas Pettersson participated as the department representative in the TROUTCONCERT- programme. He took part in the workshop in Silkeborg (Denmark) in July 1998. During the Silkeborg meeting he gave a presentation of the trout research at the department of Zoology/Animal Ecology at Göteborg university.

In Silkeborg. J. Pettersson also took part in several unofficial discussions exchanging ideas on future research on trout-genetics with an emphasis on population structure and geneflow. The TROUTCONCERT-programme has been an important source for possible contacts with other labs. It also supplies important information on conservation issues, methods and studies made on trout genetics.

179 INDIVIDUAL REPORT FROM TROUTCONCERT PARTICIPANT 11: CECA-ICETA, University of Porto, Portugal.

Summary. During the two years of the Concerted Action the group of Porto University participated in the two main workshops which took place in Silkeborg (Denmark) and Girona (Spain); they also participate in the first meeting in Lugo (Spain) and organized the second meeting in Porto (Portugal) on brown trout genetic resources in the Iberian Peninsula; the team also participate in the second general meeting in Thessaloniki (Greece) where the final document on conservation resources in brown trout was definitively elaborated. During this period several collaborations, contacts and sample exchanges were established with other teams, namely the scientific collaboration with labs of R. Guyomard, P. Martinez, J.L Garcia-Marin within the CA, and individual collaboration with S. Weiss who, although not included in CA, also participate in the second general workshop.

Participation and organization of workshops and meetings 1) First general workshop in Silkeborg One member (P. Alexandrino) participated in this meeting presenting the situation of brown trout research at the laboratory and methodologies used with a special enphasis on isoelectric focusing techniques for detection of protein variability. A compromise for the organization of a meeting related with genetic structure and conservation in Iberian Peninsula between the three groups of this area (Girona, Lugo and Porto) was taken. 2) First Iberian Meeting in Lugo. Two members of the group participated in this meeting: P. Alexandrino and A. Antunes. Both aspects on population structure and phylogeography, as well as on conservation of resources in brown trout where analyzed in Iberian area. Two presentations were made by members of the group: "Preliminary results of brown trout genetic structure in northern rivers from Portugal by using isozymes" (A. Antunes); "Isoelectric focusing: a relative technique for detecting hidden isozyme genetic variation" (P. Alexandrino). 3) Second general CA workshop in Girona Two members of the group participate in this meeting: P. Alexandrino and A. Antunes. A presentation was made in the session devoted to the presentation of ongoing research activities: "mtDNA and protein variability of Portuguese brown trout populations and implications for proposed models of post-glacial recolonization"(A. Antunes, P. Alexandrino-University of Porto and S. Weiss-University of Vienna). A. Antunes also participate in the meeting for harmonization of allozyme nomenclature chaired by A. Ferguson. 4) Second meeting in Thessaloniki for the elaboration of the document on "Conservation genetic management of brown trout in Europe". A. Antunes participated in this meeting, which recover the participants country view on conservation genetics of brown trout. 5) Second Iberian meeting in Porto. This meeting was organized by P. Alexandrino and A. Antunes, and took place in the University of Porto, CECA-ICETA, Vairão. The elaboration of a document specifically related with conservation status of brown trout in Iberian area was taken in this meeting. Also, a collaboration was established for a phylogenetic analysis of brown trout in this area by using allozymes (integration of data from all labs in the Peninsula), mtDNA and ITS1 sequences.

180 Established databases from published and unpublished studies Our lab have provided data from genetic studies of brown trout in Portugal

Interchange of samples Our lab sent DNA samples to P. Martinez lab for ITS1 sequences. We received samples from P. Martinez, Garcia-Marin and R. Guyomard laboratories.

Research collaborations

During this period it was possible to establish or continue several research collaborations with laboratories within CA or individual researchers associated to activities of the CA: - Stay of Agostinho Antunes in R. Guyomard laboratory included in his PhD programme, for the analysis of microsatellite variation in Portuguese populations; - Collaboration with S. Weiss regarding the analysis of mtDNA diversity in Portuguese populations; - Calibration and harmonisation of genetic data in Iberian Peninsula for phylogenetic analysis and conservation resources in brown trout.

181 INDIVIDUAL REPORT FROM TROUTCONCERT PARTICIPANT 12: Institute of Marine Research, Norway.

A total of two workshops each have been funded by the European Union for K.Glover and O.Skaala. A total of three presentations have been given. A further two research trips have been made by Kevin Glover to the University of Stirling to work with Dr J. Taggart. Summaries of all talks and trips have been given to the organisers of the Troutconcert action group.

28.6.98-4.7.98: Troutconcert workshop in Silkeborg, Denmark. At this workshop one presentation was given by Oystein Skaala on transformed gene pools and their worthiness for conservation. The talk summarised the authors work with stocked and unstocked populations of trout in the Hardangervidda area in Norway and the resultant conservation difficulties based upon whether gene pools are still natural after stocking.

17.4.99-14.5.99: Screening microsatellite DNA at the University of Stirling, Scotland with Dr. J. Taggart This trip was undertaken by Kevin Glover. Aims: To learn the technique of microsatellite DNA screening under the instruction of DR John Taggart. The aim was to develop loci and pcr conditions to get a family identification package up and running for sea trout for later experiments looking at selection in the hatchery and local adaptation. Summary of Achievements: Ability to carry out routine microsatellite DNA screening on fish (pcr, isotopic work and autoradiography). Technical ability to identify offspring routinely to parentage for 22 full sib families with the two loci Ssa 407 and Ssa 410. Mistakes in hatchery practices with implications for experiments of others were discovered. This trip allowed other planned experiments in Norway to be carried out.

28.6.99-6.7.99: Troutconcert workshop in Girona, Spain. At this workshop two presentations were given, one by Kevin Glover and one by Oystein Skaala. Kevin Glover: A presentation of the work on family selection in hatcheries and its implications for the health of wild stocks of fish supplemented by hatchery fish was given. Some of the results obtained whilst in Scotland earlier that year were also presented and its relevance to brown trout genetics. Oystein Skaala: A presentation was given on the fall and rise of the fine-spotted trout in the Hardangervidda in Norway. A population of fine spotted trout were rediscovered in the mid 1980`s but the population had experienced failed reproduction due to acid rain amongst other factors. The talk outlined what measures were necessary in order to conserve this unique variation of trout and the success of the action.

10.10.99-23.10.99: Screening Microsatellite DNA on ABI machine at the University of Stirling, Scotland with Dr J. Taggart. This trip was undertaken by Kevin Glover. Aims: To apply new developed microsatellite DNA markers discovered by Dr. J. Taggart on the ABI machine to routinely screen large amounts of offspring for selection experiments under hatchery conditions. Summary of achievements: After some experimenting we had the ability to use some of the markers originally developed for isotopic pcr to be used for fluorescent primers on the ABI machine. This allowed screening of 240 individuals from a selection experiment on sea trout in the hatchery. This

182 also allows us to be able to carry out several other planned experiments in the future on the topic of selection and local adaptation in sea trout and other salmonids.

183 INDIVIDUAL REPORT FROM TROUTCONCERT PARTICIPANT 13: Université Montpellier II, France.

The group was composed of Patrick BERREBI, senior scientist, CNRS and Didier AURELLE, thesis student during all the time of the Action.

Patrick BERREBI participated to the first general Troutconcert meeting at Silkeborg (June 98, Denmark) and to the second at St Feliu (June 99, Spain), plus the special "conservation" meeting at Berne (Feb. 99, Switzerland). Didier AURELLE participated to the Silkeborg meeting, due to his thesis work, he cannot participate to the others.

Silkeborg meeting : During this meeting, both PB and DA. gave a composite talk of presentation of research activities at the University of Montpellier, France. Then, P.B., managed the "Stocking impact assessment" workshop. P.B. was also chairman during the presentation of research activities (10h-12h session) of Turkey, Spain, United Kingdom and Sweden.

Berne meeting : During this meeting devoted to genetic conservation questions, P.B. participated to the dissertation of a part of the future report : General conservation genetic guidelines for natural and cultured salmonid populations. This text was composed of seven sub topics : (i) Genetic assessment of populations; (ii) General recommendations when genetic data are not available; (iii) Ecological modification with indirect genetic effects; (iv) Selection in nature; (v) Uncertainty principle; (vi) Geographic scale; (vii) Facilitate and enhance communication between scientists and resource managers. After that, P.B. participated to the improvement of the text which gave a report of about one hundred pages.

St Feliu meeting : P.B. managed the session "Endangered populations". Three examples were presented, one illustrating hybridisation after stocking (the marble trout is Slovenia), the second concerned acidic rain in small drainages (fine spotted trout in Norway) and the last illustrated the weakness of very small sized populations of trout (specially those of Corsica).

The Montpellier group participated in the main "electronic" discussions, gave data on microsatellites and allozymes, gave a collection of photographs of southern France wild populations of trout. Exchanges of samples and texts occurred with Spain (garcia-Marin) and Canada (Bernatchez).

Due to the moving of P.B. to California mid-1999, no participation to the Thessaloniki meeting was possible.

184 INDIVIDUAL REPORT FROM TROUTCONCERT PARTICIPANT 14: Wageningen Agricultural University, the Netherlands.

Objectives

*to promote collaboration among laboratories that are active in research on population genetics of brown trout ( Salmo trutta) The group has participated in the two workshops organised during the tenure of Trout Concert. René J. M. Stet attended the fist meeting in Denmark. A presentation was given outlining the current research within the Molecular Immunology Group of the Cell Biology and Immunology group. The focus of the presentation was on the use of Major Histocompatibility Complex (MHC) genes as non-neutral markers. MHC genes play a pivotal role in the initiation of an immune response upon exposure to a pathogen. It was outlined that salmonids contained micro- and minisatellites in the MHC class I and class II A genes. These markers could be used in population studies. Most population studies use neutral markers and the availability of selective markers, such as the MHC genes may expand our knowledge of local adaptation of brown trout populations. René J. M. Stet and a graduate student (Corine Kruiswijk) attended the second workshop in Spain. During this meeting, we developed together with Bill Jordan from the Institute of Zoology a project to be submitted under the 5th framework. The project would address among other the use of MHC markers to study the impact of aquaculture on local natural populations of brown trout. This project was subsequently further developed during a short visit of René J.M. Stet to London, and finally written during a stay of Bill Jordan in Wageningen.

*to co-ordinate this research and when convenient harmonise the use of genetic markers The presentation initiated a collaboration with the Institute of Zoology, London (Bill Jordan). Within the framework of the Trout Concert, a presentation was given at the Institute, and further discussions of research collaborations on the identification and use of MHC markers in brown trout were started. During the second year of the Trout Concert Bill Jordan from the Institute of Zoology, London worked in the Cell Biology and Immunology Group Wageningen for three months to prepare a cDNA library of brown trout, which was subsequently used to isolate and characterise the MHC genes. In addition, the detection of markers in the brown trout was tested using Atlantic salmon MHC marker primers. It was shown that the class II A primers can be used to detect variation of the class II A genes in the brown trout.

*to bring together complementary expertise from all parts of the EU and other countries The two workshops provided an excellent opportunity to present and discuss the work of the Cell Biology and Immunology Group with research groups that showed an interest in using the MHC genes as selective markers, complementing the growing set of inferred neutral markers. This has resulted in an expanding interest of several research groups to use these markers in the future. In addition, a consortium was formed which aim is to write a project proposal to be submitted under the 5th framework, addressing issues of impact of aquaculture on natural populations of brown trout. The initial consortium consisted of René J. M. Stet (Cell Biology and Immunology Group, Wageningen), Bill Jordan (Institute of Zoology, London), and Kjetil Hindar and Ian Fleming (NINA, Trondheim) combining expert knowledge of the molecular biology of the MHC, population genetics of brown trout, and aquaculture.

185 *to review and evaluate the status of the research with a focus on conservation/management of genetic resources of the species and the potential of the species for aquaculture The Cell biology and Immunology Group has provided up-to-date knowledge of the major histocompatibility complex genes in salmonids as markers for fitness. We have actively participated in discussions on the use of such makers to compliment the neutral markers widely used in population genetics. We have highlighted the potential of such selective markers and its potential use in addressing issues of local adaptation, imperative for proper conservation/management of populations. The MHC research in brown trout is still in its infancy, however, its role has been acknowledged by population genetic researchers as a future tool.

*to establish databases on relevant literature, available genetic markers and data from published and unpublished studies. The databases and reports from the CA will be made publicly accessible on the World Wide Web The Cell Biology and Immunology Group has provided information of the Major Histocomaptibility Complex (MHC) genes and its usefulness in population studies. This information has been incorporated in the workshop reports conceived under the Trout Concert.

Deliverables

Workshop The Cell Biology and Immunology Group (René J.M. Stet and Corine Kruiswijk) participated in both annual workshops organised by the Trout Concert.

Survey/review of brown trout population genetics research activities We have presented data on the use of the MHC genes in population studies. The main justification of the use of these genes in population studies is its role in conferring fitness, and could be used to study local adaptations in brown trout populations. This aspect of the biology of this species has received little attention, due to the lack of suitable non-neutral markers.

Recommendations for future studies The Cell Biology and Immunology Group in collaboration with the Institute of Zoology, and Norwegian Institute for Nature Research (all participants of the Trout Concert), together with two Irish research groups have written a proposal (SALIMPACT) on the impact of aquaculture on natural populations of brown trout, and Atlantic salmon using both neutral and MHC markers, which has been submitted under the October 1999 call of the 5th framework. The objectives of the proposed research address issues of the interaction between fish farming and natural populations of brown trout and Atlantic salmon.

186 INDIVIDUAL REPORT FROM TROUTCONCERT PARTICIPANT 15: Stockholm University, Sweden.

For each of the objectives of the TROUTCONCERT project we comment briefly the contribution by the Division of Population Genetics, Stockholm University.

Objectives

* to promote collaboration among laboratories that are active in research on population genetics of brown trout (Salmo trutta) Two representatives, one scientist (Linda Laikre) and one graduate student (Stefan Palm) from the Division of Population have Genetics have participated in the two workshops held within TROUTCONCERT. We have presented the research conducted at our laboratory and have learnt about ongoing research in other labs. This has been very stimulating, and the project has provided an opportunity to interact with people with similar research interests, most of whom we had not had the chance to meet previously.

We have had the opportunity to initiate scientific collaboration with the Danish Institute for Fisheries Research, and Linda Laikre has made three research visits to that institute within the framework of TROUTCONCERT. The purposes of these visits have included the learning of laboratory techniques for analyzing genetic variability in microsatellite loci in brown trout, and to apply the technique to brown trout samples from different localities in Sweden which have been investigated using other genetic techniques. Several ongoing collaboration projects have resulted from the opportunities for contacts that these visits have provided.

Linda Laikre, Stefan Palm and Nils Ryman (professor and research leader at the Division of Population Genetics) have participated very actively in the working group on conservation genetic management of brown trout in Europe. Two workshop were organized specifically for this working group and Linda Laikre and Stefan Palm participated in both these workshops, and Nils Ryman participated in one of them.

* to co-ordinate this research and when convenient harmonise the use of genetic markers The Division of Population Genetics have participated actively in the work with harmonising genetic markers using protein electrophoresis and PCR-RFLP-analysis of mitochondrial DNA. Stefan Palm has participated in several meetings of the group within TROUTCONCERT working with harmonisation of allozyme markers, and has provided information on analytical procedures and data generated at the Division of Population Genetics. Linda Laikre has participated in the group working on harmonisation of mtDNA-markers detected by PCR-RFLP-analysis. This work has included laboratory analysis of selected samples from different laboratories/countries for comparison of haplotypes. The work has also included the photographic documentation of the different detected haplotypes. Apart from participants of TROUTCONCERT, professor Torbjörn Järvi and Leif Johansson at the Swedish Institute for Freshwater Research of the National Board of Fisheries have been involved in this work.

* to bring together complementary expertise from all parts of the EU and other countries Representatives from the Division have participated in a total of four workshop-meetings, and three research visits arranged within TROUTCONCERT. During these meeting we have enjoyed the

187 interaction with many interesting scientists from both other parts of the EU and from countries outside of EU. We have found these interactions most educational and stimulating.

* to review and evaluate the status of the research with a focus on conservation/management of genetic resources of the species and the potential of the species for aquaculture Linda Laikre, Stefan Palm and Nils Ryman have participated very actively in the working group on conservation genetic management of brown trout in Europe. The work within this group has included literature reviews, and construction of overviews on various information of relevance to the topic of conservation genetic management. The conservation working group provided a report, "Conservation Genetic Management of Brown Trout (Salmo trutta) in Europe" which was edited by Linda Laikre.

* to establish databases on relevant literature, available genetic markers and data from published and unpublished studies. The databases and reports from the CA will be made publicly accessible on the World Wide Web The Division of Population Genetics has provided information on brown trout literature, genetic markers and data from genetic studies of brown trout in Sweden. This information has been incorporated into the databases, reports and webpages generated within TROUTCONCERT.

Deliverables

Workshop As noted above, Stefan Palm and Linda Laikre have participated in both the annual workshops organized by TROUTCONCERT, and in the two workshops of the conservation working group within TROUTCONCERT. Nils Ryman has participated in one of the conservation workshops.

Survey/review of brown trout population genetics research activities We have presented our own research activities on brown trout population genetics both at oral presentations at the seminars arranged at the workshops, and written summaries provided for the WWW site, and for reports generated by TROUTCONCERT.

Recommendations for future studies We have participated in the discussions on recommendations for future studies, and have provided suggestions for such studies.

Recommendations for genetic marker nomenclature and harmonisation of the use of techniques We have participated actively in the work with harmonisation of protein electrophoretic and mtDNA laboratory techniques.

Recommendations for management/conservation of genetic resources of the species We have participated actively in the working group on conservation genetic management of brown trout, and one of us (LL) has edited the report provided by this working group.

188 Evaluation of the potential of the species for aquaculture with emphasis on the genetic resources available We have not been particularly involved in the issue of the potential for brown trout aquaculture, but we have participated in the sections of presentations and discussions regarding this topic presented at the workshops.

Brown trout population genetics bibliography, World Wide Web site for the CA. Raw data and data bases on genetic markers, available on the WWW site. We have provided literature listings for the population genetics bibliography, and information on our laboratory for the WWW site. We have also supplied data base information on genetic characteristics of Swedish brown trout populations for the collection presented at the WWW site.

Mid-term evaluation, Progress report, Final report We have provided requested information for the reports produced on the progress and work within TROUTCONCERT.

189 INDIVIDUAL REPORT FROM TROUTCONCERT PARTICIPANT 16: Zoological Society of London, England, UK.

*to promote collaboration among laboratories that are active in research on population genetics of brown trout ( Salmo trutta) Two members of staff from the Institute of Zoology, Zoological Society of London attended the first Workshop in Silkeborg, 1998 (Michael Bruford and Bill Jordan), while Bill Jordan alone attended the St Feliu de Guixols Workshop in 1999 (Michael Bruford was no longer a member of staff at IoZ by this time). Ongoing research in IoZ on the population genetics of brown trout and other salmonid species was presented at the first workshop.

Due to the high degree of shared research interests most collaboration and co-ordination occurred between IoZ and Wageningen Agricultural University (WAG). Numerous discussions on the use of major histocompatibility complex (MHC) genes in brown trout population studies were held during the course of a number of exchanges between laboratories (see below for details). Other laboratories were included in these discussions at the two workshops, and during a small-scale meeting held in London in September, 1999 to plan a proposal to the EU Vth Framework (see below). During one visit by Bill Jordan to WAG (February, 1999), a number of cDNA libraries from brown trout spleen tissue were constructed - these libraries will facilitate isolation of expressed MHC genes from brown trout and have provided preliminary data upon which the application to the EU Vth Framework was based.

As a direct result of TroutConcert, a collaboration was set up between IoZ and the University of Laval, Quebec, Canada. The collaboration entailed two exchange visits between laboratories for the conduct of research on salmonid MHC genes (see below). An application by a Quebecois student for funding for a PhD project to be carried out at IoZ is in review, and other joint applications between IoZ and Laval are planned.

*to co-ordinate this research and when convenient harmonise the use of genetic markers Plans for co-ordinated research on functional genomics and local adaptation were discussed at both workshops and during exchange visits. Samples of brown trout tissues were provided to WAG for work on brown trout MHC genes and other genes involved in disease resistance.

*to bring together complementary expertise from all parts of the EU and other countries IoZ hosted a number of workers on brown trout population genetics during the course of TroutConcert, many of whom gave presentations on their research. These include: René Stet (WAG) April, 1998 & September 1999. Louis Bernatchez (University of Laval) August, 1998 & May 1999. Christian Landry (University of Laval) August, 1999 Ian Fleming (NINA) September, 1999 In addition, other workers on brown trout population genetics who were not directly involved in TroutConcert were included in a meeting in London in September, 1999 to put together a proposal to the EU Vth Framework. These workers were Tom Cross (National University of Ireland, Cork) and Phillip McGinnity (Marine Institute, Westport, Ireland). Bill Jordan (IoZ) made two visits to WAG as part of TroutConcert. During one visit, in February 1999, cDNA libraries from brown trout tissue were constructed for isolation of MHC and other disease resistance-related genes and initial discussions of future joint research. The second visit (November 1999) allowed finalisation a proposal (SALIMPACT) to the EU Vth Framework.

190 Bill Jordan also visited the University of Laval, Quebec during September/October 1999 to discuss possible joint research on brown trout and other salonid species. This visit was funded by the Royal Society, but was arranged as the result of contacts made during the first TroutConcert Workshop.

*to review and evaluate the status of the research with a focus on conservation/management of genetic resources of the species and the potential of the species for aquaculture Bill Jordan presented a brief review of the status of research and led a discussion on local adaptation in brown trout and other salmonids at the first TroutConcert Workshop.

*to establish databases on relevant literature, available genetic markers and data from published and unpublished studies. The databases and reports from the CA will be made publicly accessible on the World Wide Web IoZ contributed to the relevant literature and available genetic marker databases, and has provided sections for both progress and final reports.

Deliverables

Workshop Two members of IoZ staff attended the first Workshop, one attended the second Workshop.

Survey/review of brown trout population genetics research activities Ongoing research in IoZ on the population genetics of brown trout and other salmonid species was presented at the first workshop. IoZ also presented a brief review of local adaptation in salmonid fish at the first Workshop.

Recommendations for future studies On the basis of the review and discussion of research on local adaptation in brown trout, the need for further research in this field was highlighted. Recommendations for such future research were integrated into TroutConcert Progress and Final Reports. A proposal to the EU Vth Framework for funding of a relevant project (SALIMPACT) was drawn up along with other TroutConcert and non-TroutConcert partners and submitted for evaluation under the Quality of Life, Key Action 5, in November 1999.

Recommendations for genetic marker nomenclature and harmonisation of the use of techniques Through discussions with other laboratories involved in salmonid MHC research it was decided that the current lack of knowledge on the structure of the MHC in these species makes any attempt at a common system of nomenclature premature. Further research in this field is obviously required, and is one of the objectives of the SALIMPACT project.

Recommendations for management/conservation of genetic resources of the species As a result of discussions during TroutConcert, the relevance of local adaptation for management policies on stocking and inadvertent release of aquaculture stocks was emphasised. The importance of consideration of local adaptation was highlighted in the Conservation Genetic Management Report.

191 Evaluation of the potential of the species for aquaculture with emphasis on the genetic resources available The importance of genes involved in disease resistance to sustainable aquaculture of brown trout was stressed during TroutConcert Workshops, as was the importance of screening for variation at various classes of genetic marker (neutral and non-neutral) as part of genetic management of aquaculture stocks.

Mid-term evaluation, Progress report, Final report IoZ contributed sections to both progress (e.g.. Local Adaptation in Brown Trout Populations) and final reports.

192 INDIVIDUAL REPORT FROM TROUTCONCERT PARTICIPANT 17: University of Munich, Germany.

For each of teh objectives of the TROUTCONCERT project we comment briefly the contribution of our research group

Objectives

* to promote collaboration among laboratories that are active in research on population genetics of brown trout (Salmo trutta) Dr. Rassmann, Dr. Schliewen and Dipl.Biol. Miller from the research group of Prof. D. Tautz, Ludwigs-Maximilians-Universität Munich, took part in the two TROUTCONCERT workshops in Silkeborg, Denmark, and San Feliu de Guixols, Spain, 1998 and 1999, respectively.We presented and discussed the plans and results of our ongoing research on brown trout genetics within in Germany. Both meetings were highly profitable for our group, since we were for the first time able to discuss unpublished results and establishing fruitful contacts with other scientists working on brown trout genetics.

Through these contacts we were able to establish small scale collaboration with other scientists either by obtaining DNA-samples which otherwise would have been very difficult to obtain for us, or by collaborating in combined analysis of poplation genetic samples from Germany with neighbouring regions (Denmark, Austria). In addition, we had access to unpublished anaylsis- methods.

* to co-ordinate this research and when convenient harmonise the use of genetic markers In order to promote harmonisation of the use of genetic markers, our lab provided information on the use of microsatelitte loci and primers, which were informative in the brown trout samples in our studies. In addition, through the contact with Dr. Hansen we were able to translate Restriction- Fragment Lenght Polymorphism haplotypes with those yielded by other groups working with a different set of enzymes. The first workshop in Silkeborg was very profitable for us, since we decided through the information provided there,which mitochondrial subunits would be useful to study for our work in order to enable comparison with other forthcoming work.

* to bring together complementary expertise from all parts of the EU and other countries Members of our lab benefited from the presence of internationally renowned experts (from Canada, too) at the two afromentioned workshops. The diversity of expertise in different fields of population genetics and brown trout genetics in particular resulted partially in replanning of our research in order to obtain results interpretalbe in a Europe-wide context.

* to review and evaluate the status of the research with a focus on conservation/management of genetic resources of the species and the potential of the species in aquaculture Members of our research group provided country-specific information on the status, threats and legislation of brown trout in Germany for the report „Conservation Genetic Management of Brown Trout (Salmo trutta) in Europe“, which was edited by Swedish participants of TROUTCONCERT.

193 * to establish databases on relevant literature, available genetic markers and data from published and unpublished studies. The databases and reports from the CA will be made publicly accessible on the World Wide Web Members of our lab have provided information on the use of genetic markers (especially microsatellites) which have turned out to be useful for the analysis of brown trout population genetics in Germany.

Deliverables

Workshop As mentioned above, members of our lab have participated in the two main workshops which were arranged by TROUTCONCERT

Survey/review of brown trout population genetics research activities During the workshops and in discussions with some members of TROUTCONCERT, we have presented our results and the concept of planned studies. Further we contributed summaries of our research activities for the internet-homepage of TROUTCONCERT, as well as for internal reports.

Recommendations for future studies During the workshops and in addition with several members of TROUTCONCERT we have discussed questions concerning research potential for future studies.

Recommendations for genetic marker nomenclature and harmonisation of the use of techniques By providing information about the use and success of certain microsatellite primers and RFLP enzymes we furnished the establishment of harmonically used genetic markers for brown trout.

Recommendations for management/conservation of genetic resources of brown trout Since the final results of our research of brown trout population genetics in Germany are expected only by the end of the year 2000, we were not able to provide detailed suggestions on conservation of brown trout and use of certain strains in aquaculture. However, during the workshops we exchanged ideas on identification of highly stocked brown trout populations in Germany, which is a notoriously difficult problem for any project planning in brown trout conservation.

Evaluation of the potential of the species for aquaculture with emphasis on the genetic resources available. Due to the structure of our research interests we were not able to provide useful information for this topic.

Brown trout population genetics bibliography, World Wide Web site for the CA. Raw data and databases on genetic markers, available on the WWW site For the WWW site we provided data about our research interests and on the use of genetic markers in our lab in order to make them publicly available.

Mid-term evaluation, progress report, final report We have provided all necessary information for all general reports during the period of TROUTCONCERT.

194 INDIVIDUAL REPORT FROM TROUTCONCERT PARTICIPANT 18: Finnish Game and Fisheries Research Institute, Finland.

During the two years of this Concerted Action one person (Dr. Marja-Liisa Koljonen) participated in the activities related to the TROUTCONCERT. She took part in the two main workshops which were organised in Silkeborg (Denmark) in 1998 and Girona (Spain) in 1999. She also contributed to writing of the report ”Conservation genetic management of brown trout (Salmo trutta) in Europe”.

During the Silkeborg meeting she gave a presentation on ”Brown trout studies in Finland” and in Girona she gave a presentaion on ”Potential phylogeographic lineages for brown trout in the Baltic Sea area”.

In Girona she also took part in the separate subgroup led by A. Ferguson, which organised the standardisation of allozyme allele interpretation. Several unofficial contacts were created during the meetings and practical collaboration was planed among laboratories around the Baltic Sea area, especially with Estonian, Danish and Swedish participants of the workshop. Allozyme interpretation among labs from these countries has been standardised as result of the workshop recommendations.

195 INDIVIDUAL REPORT FROM TROUTCONCERT PARTICIPANT 19: University of Stirling, Scotland, UK.

The Troutconcert participant from the Institute of Aquaculture, University of Stirling, was Dr. John Taggart. In the following a summary is given of specific contributions to Objectives and Deliverables of the CA

Objectives

* to promote collaboration among laboratories that are active in research on population genetics of brown trout (Salmo trutta) I attended both annual TROUTCONCERT workshops presenting an overview of my current research interests and contributing to other specific sessions. The workshops provided an excellent opportunity to meet other researchers, learn about ongoing research activities in their laboratories and to discuss common interests and problems. The format of the workshops provided adequate time for both formal (presentational) and informal interactions. Old contacts were renewed and, perhaps more importantly, many new contacts were made.

TROUTCONCERT has provided the catalyst for initiation of a joint research project with Dr Øystein Skaala, Institute of Marine Research, Bergen. Following on from initial discussions at the first annual Workshop in Silkeborg, Dr Skaala and Mr Kevin Glover availed of mobility funds on two occasions to visit me at the Institute of Aquaculture to firm out the planned research program and specifically to learn about using microsatellite protocols for parentage assignment in brown trout. The outcome has been a three year research studentship (with joint supervision between the two Institutes) investigating aspects of local adaptation in Norwegian populations of resident and anadromous brown trout.

* to co-ordinate this research and when convenient harmonise the use of genetic markers I participated in the groups working on the harmonisation of isozyme and microsatellite markers in brown trout studies. This was primarily undertaken at meetings arranged during the two Annual workshops and involved nomenclature and locus selection issues. Furthermore, microsatellite primer sets I developed / optimised for Atlantic salmon were made available to other CA participants (including outside experts) for evaluation in brown trout studies.

* to bring together complementary expertise from all parts of the EU and other countries Through attendance at both CA workshops I interacted beneficially with a large number of brown trout biologists having a range complementary expertise. The joint project with Dr Skaala was made possible by the complementary strengths we were able to apply to the problem (i.e. extensive ecological / behavioural knowledge of Norwegian stocks / expertise in microsatellite analyses of salmonids).

* to review and evaluate the status of the research with a focus on conservation/management of genetic resources of the species and the potential of the species for aquaculture No specific input in this area other than through discussions in Workshop sessions

196 * to establish databases on relevant literature, available genetic markers and data from published and unpublished studies. Completed questionnaires and details of my research activities were forwarded to the relevant working groups. I was a member of the sub-group formed to advise on the design / format of the genetic database.

Deliverables

Workshops Both workshops attended and actively participated in.

Survey/review of brown trout population genetics research activities All relevant requests for information on my past and current research into brown trout was provided. Oral presentation on aspects of current research was given at first annual workshop.

Recommendations for future studies Through both oral and written presentations I have participated in discussions on recommendations for future population genetic studies

Recommendations for genetic marker nomenclature and harmonisation of the use of techniques Through involvement in isozyme and microsatellite working groups I have had input into recommendations put forward for the use of these types of genetic markers

Recommendations for management/conservation of genetic resources of the species No specific input in this area other than through discussions in Workshop sessions

Evaluation of the potential of the species for aquaculture with emphasis on the genetic resources available No specific input in this area other than through discussions in Workshop sessions

Brown trout population genetics bibliography, World Wide Web site for the CA. Raw data and data bases on genetic markers, available on the WWW site. All relevant personal data supplied for bibliography, WWW site and genetic databases

Mid-term evaluation, Progress report, Final report Written summary of oral presentation on sampling considerations provided for progress report. Individual final report submitted.

197 ANNEX 10.

TRAVEL REPORTS OF THE CA.

This annex contains travel reports from meetings and visits undertaken during and funded by the CA. It has not been required to write reports covering participation in workshops (the two main workshops in 1998 and 1999, the two “Iberian Trout Meetings” in 1998 and 1999 and the two “Conservation Workshops”, both in 1999). Summaries of the main workshops are given in the workshop proceedings (Annex 2 and 5) and summaries the ”Iberian Trout Meetings” are given in Annex 3 and 6. The outcome of the ”Conservation Workshops” was the report ”Conservation Genetic Management of Brown Trout (Salmo trutta) in Europe”, which is enclosed as Annex 11.

Overview of Mobility Reports:

1. Agricultural University of Wageningen ⇒ Zoological Society of London 2. INRA ⇒ University of Berne 3. Stockholm University ⇒ Danish Institute for Fisheries Research 4. Institute of Marine Research, Bergen ⇒ University of Stirling 5. Institute of Marine Research, Bergen ⇒ University of Stirling 6. Zoological Society of London ⇒ Agricultural University of Wageningen 7. Institute of Marine Research, Bergen ⇒ University of Stirling 8. Agricultural University of Wageningen ⇒ Zoological Society of London 9. Norwegian Institute for Nature Research ⇒ Zoological Society of London 10. INRA ⇒ Danish Institute for Fisheries Research 11. Stockholm University ⇒ Danish Institute for Fisheries Research 12. Queen’s University of Belfast ⇒ Danish Institute for Fisheries Research 13. Stockholm University ⇒ Danish Institute for Fisheries Research

198 MOBILITY REPORT

TROUTCONCERT – MOBILITY FUNDS

Name: René J.M. Stet

Laboratory: Cell Biology and Immunology Group, Wageningen Agricultural University

Laboratory visited: Institute of Zoology, London

Duration of the visit:

From (time and date): 12.00 April 25 1998

To (time and date): 20.00 April 29 1998

Total cost of travel (flight tickets etc.) (Ecu): 90

Total cost of accommodation (Ecu): None

Total amount daily allowance (180 Ecu/day): 900 ECU

Summary of visit:

The purpose of the visit was to investigate possibilities for future collaboration in relation to the isolation and characterisation of Major Histocompatibility Complex (MHC) genes in brown trout. Dr. Jordan and I agreed on the fact that for the isolation of the genes a cDNA library is an essential tool. The cDNA library will be prepared from kidney material provided by Dr. Jordan. The isolation of the MHC will be performed by using anchored PCR on the cDNA library using a combination of a vector primer and an MHC specific primer. The Cell Biology and Immunology Group has employed such a strategy successfully for the isolation of MHC sequences from a number of other species, such as cod, Siberian sturgeon, rainbow trout, large African barbus, and carp. As the MHC sequences of a large number of salmonid species, such rainbow trout and Atlantic salmon, are available primer design for both classes of MHC genes (class I and class II) are greatly facilitated. Further to this project we discussed the availability of brown trout samples from different British populations. These should include anadromous, non-anadromous and

199 mixed populations. Such samples are available, including non-anadromous. This will allow to use the sequence information generated from the cDNA library to analyse the presence of these genes in the different populations. We also discussed the implications of the current knowledge of the MHC genes in salmonids in relation to that of brown trout. One of the major difficulties encountered in analysing MHC polymorphism is the designation of loci and alleles. During the visit I gave a seminar on the Major Histocompatibility Complex genes in fish.

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Date: 4 May 1998 Signature: Rene Stet

200 MOBILITY REPORT

TROUTCONCERT – MOBILITY FUNDS

Name: René Guyomard

Laboratory: Laboratory of fish genetics, INRA, 78352 Jouy-en-Josas, France

Laboratory to visit: Abteilung Populationsbiologie, Zoologisches Institut, Universitaet Bern, 3012 Bern, Switzerland

Duration of the visit:

From (time and date): 19/08/98 at 21h22 (arrival in Bern)

To (time and date): 23/08/98 at 16h49 (departure from Bern)

Total cost of travel (flight tickets etc.) (Ecu): 157 Ecu

Total cost of accommodation (Ecu): 150 Ecu

Summary of visit:

The program of the visit included two aspects: 1) Purification and sequencing of salmonids microsatellite clones 2) Preparation of two manuscripts resulting from a collaboration between the University of Bern (Abteilung Populationsbiologie) and INRA (Laboratoire de génétique des poissons and Station d’hydrobiologie lacustre)

Schedule: 20/08/98 Bacterial cultures 21/08/98 Bacterial cultures and Purification of Plasmid DNA 22/08/98 Purification of Plasmid DNA and sequencing 23/08/98 (morning) Discussion on manuscripts

Purification and sequencing of microsatellites Ninety-one clones contening microsatellites were previously isolated by Carlo Largiader and a PhD student at the University of Bern. My work consisted in amplifying individual colonies from these clones and in purifying Plasmid DNA for sequencing. All the clones were subjected to amplification. Three of them did not led to any bacterial cell proliferation. Plasmid DNA was extracted for the remaining clones using a Quiagen miniprep kit. DNA purity and concentration

201 were estimated by fluorometry. Seventy-two miniprep showed DNA concentration and 260/280 D.O. ratio adequate for automatic sequencing. Insert sizes were estimated for ten clonesand ranged from 350 to 1200 bp (average= 650-700 bp). Two clones were sequenced and both sequences contained a microsatellite motif. The other clones will be sequenced in the Laboratoire de génétique des poissons.

Preparation of manuscripts The Abteilung Populationsbiologie of the Zoologisches Institut, the laboratoire de génétique des poissons and the station d’hydrobiologie lacustre of INRA have been involved in a commun research programs for three years. The main objective of this program is focussed on the study of the modalities of gene introgression system where stocking has taken place untill recently and its effects on the biology of the native population. This program is conducted on a Mediterranean system because native populations of this area show a substantial degree of divergence with the domesticated stocks (which originate from the Atlantic subspecies). This divergence facilitates the detection of introgression and increases the probably of outbreeding. The end of the visit has been dedicated to the finalization of a first manuscript on microsatellite analysis of polyandry and spawning site competition in brown trout (Salmo trutta l.) (Carlo R. Largiadèr, Arnaud Estoup, Frederic Lecerf, Alexis Champigneulle and René Guyomard, submitted to PRSL)

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Date: 09/09/98 Signature: R. Guyomard

202 MOBILITY REPORT

TROUTCONCERT – MOBILITY FUNDS

Name: Linda Laikre

Laboratory: Division of Population Genetic

Laboratory visited: Danish Institute for Fisheries Research, Department of Inland Fisheries

Duration of the visit:

From (time and date): 05.00 a.m. November 30, 1998

To (time and date): 09.00 p.m. December 11, 1998

Total cost of travel (flight tickets etc.) (Ecu): 599.38

Total cost of accommodation (Ecu): 577.96

Summary of visit:

The purpose of the visit was to learn laboratory techniques for analyzing genetic variability in microsatellite loci in brown trout, and to apply the technique to a number of brown trout samples from different localities in Sweden which are currently investigated using other genetic techniques. A further purpose of the visit was to plan the work with calibration of mitochondrial DNA-markers currently used by several labs within the TROUTCONCERT program. It is of interest to compare the haplotypes occurring in brown trout in different regions, and to standardize the nomenclature.

During the two weeks visit a total of 137 brown trout samples from seven different localities were analyzed (see table below). PCR-products (Polymerase Chain Reaction) were produced for these samples using primers for seven different microsatellite loci scored rutinely at the Danish Institute for Fisheries Research. The PCR-products were run on polyacrylamide gels in an ALFexpress DNA sequencing and fragment analysis machine. Genotypes were obtained for between 71 and 137 individuals for the seven micorsatellite loci (see table below). The visit also included introduction to software for handling the fragment size data generated by the ALFexpress.

Continued work with calibration of mitochondrial DNA-markers was planned, and PCR-products were generated for further analysis in Stockholm, Sweden.

203 The visit was very rewarding from a tutorial perspective and also generated valuable information on the type of data that can be obtained on genetic variability in microsatellite loci in these particular populations which are currenlty under study using other genetic markers. A continued collaboration on analysis of microsatellite variability in Swedish brown trout populations are currently planned.

Table. Results from screening for genetic variability in microsatellite loci in Swedish brown trout

No. of No. of No. of No. of No. of Hobs analyzed analyzed detected observed mono- individuals populations alleles genotypes morphic populations Str85INRA 137 7 7 17 - 0.6277 Str60INRA 137 7 5 6 - 0.3723 Str73INRA 136 7 6 17 - 0.5882 SsoSL417 136 7 12 38 - 0.6838 Str15INRA 115 7 7 15 - 0.5913 SsoSL438 71 7 6 13 - 0.6056 Ssa197 71 7 6 9 3 0.3380

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Date: January 11, 1999 Signature: Linda Laikre

204 MOBILITY REPORT

TROUTCONCERT – MOBILITY FUNDS

Name: Kevin Glover

Laboratory: Institute of Marine Research, Bergen, Norway.

Laboratory visited: DEPT Aquaculture, University of Stirling

Duration of the visit: 28 days.

From (time and date): 3pm 15th April, 1999.

To (time and date): 9.30pm 10th May, 1999.

Total cost of travel (flight tickets etc.) (ECU):

Travel was car to the ferry in Bergen, ferry to Newcastle and then the car to Stirling. Return trip was the same. The costs of travel are split up as below.

Car to ferry, car from ferry to Stirling and Car back again: Total of 3 miles, then 185 miles then double because of return. Total of 188 (times two = 366 miles, 1.609 Km to a mile= 588 km in total in car. 3,2 Norwegian kroner per KM= 588x3.2= 1881.2 Norwegian kroner.

Exchange rate on 14/5/99: 1ECU= 8.1825 Norwegian Kroner.

1881.2/8.1825= 229.9 ECU for use of private car.

Ferry ticket from Bergen to Newcastle (return ticket with car): 3830 Norwegian kroner.

3830 kroner = 468 ECU

Total travel costs are 468 and 229.9 = 697.9 ECU

Total cost of accommodation (ECU): 173.72 English pounds.

205 Approx. 12.7 Kroner to the pound. Therefore 173.72 pounds is 2206 Norwegian kroner. That is / by 8.1825 Kroner to the ECU, therefore accommodation costs were 269.6 ECU.

Summary of visit:

Screening of Microsatellite DNA at the University of Stirling (17th April-14th May)

The following is a short report of the trip made by Kevin Glover from the institute of Marine research in Bergen, Norway to the Department of Aquaculture in the University of Stirling, Scotland between the above dates.

Aim of the visit

The main aim of the visit was to learn the technique of microsatellite DNA screening and implement it for family identification of Norwegian sea trout in a hatchery. This was carried out with respect to current experiments being planned and set up at the institute of Maine research. The larger project consists of various experiments but aims to investigate the role that family and stock level genetic variation has on local adaptation and selection regimes in the hatchery and natural environments. The key part of all these experiments relies upon the ability to unambiguously assign individual offspring which are mixed either in a hatchery or a natural environment to family routinely. More specifically it was intended that at the end of the visit all the 36 individual broodstock and a sample of offspring from each family would be genotyped for several or more microsatellite DNA markers in order to be able to unambiguously assign mixed offspring to family and to be able to transfer the skills back to Bergen for implementation into the mentioned experiments. The technique of family identification by DNA is new to the Marine research Institute in Bergen and so it was essential to visit John Taggart in Scotland to learn the technique, find out which loci would work to distinguish the family groups and to check experimental material set up so far in the hatchery.

Methods

Microsatellite DNA screening was carried out on all broodstock (36 individuals) used in current experiments and for one individual from each family group (22 families) in accordance with Taggart & Cairney (1993) unpublished. The following loci were used to screen the broodstock: Ssa 171, Ssa 197, 15G6, 15G10, 15H8. The first three loci are published and well used tetranucleotide loci for salmon whilst the 15 series are new unpublished tetranucleotide loci developed by John Taggart. It is the first time that these three loci have been implemented in family identification work, they were developed for salmon. Some work on optimising the loci and the PCR conditions was carried out in order to increase the ability of scoring the alleles correctly.

206 Results

PCR conditions were used for the following loci:

Locus MgCl conc. Anneal temp °c Cycles Alleles Ssa 171 1.0 59 29 Not scored Ssa 197 1.0 61.5 29 9 15G10 1.0 61 29 23 15G6 1.0 61.5 29 16 15H8 1.0 61 29 18

Although multiplexing was not experimented with, the allele size differences between alleles at loci 15G6 and 15G10 allowed double loading of the gel, thus saving some time. It is possible that with some optimising and experimenting, some of the above loci could be multiplexed which would save time in the long run and thus enable us to screen larger sample numbers.

All the 15 series loci were represented by clear bands on the gel and were relatively easy and accurate to score. Ssa 197 was also similar but some problems were encountered with shadow bands for Ssa 171.

The broodstock were genotyped for all the above loci, but in the time available only 15G10 and 15H8 were screened for the offspring. Data was analysed by FAP (family analysis package). This is a DOS based programme which was written by John Taggart for routine assignment of individuals from full or half sib families to parentage. The data obtained from screening the was analysed in this package.

The results show that 99.25% of all offspring are able to be assigned to family (22 families of which half are full sib and the other half is half sib) by using only two loci: 15G10 and 15H8. These two loci proved to be the most useful. In addition to these, the locus 15G6 was able to be double loaded onto the 15G10 gel (different alleles sizes between loci) to get an extra locus with which to check the matching of offspring in the case of mis-genotyping an individual at one particular locus.

Although 99.25% of offspring should theoretically be assigned to family, some problems were met when it came to checking parentage of them. After checking the data and records it is apparent that some mix ups were made in the fertilisation stage and as a result there was some parental miss- matching. This is tentatively solved by some swapping of certain males with females but more screening shall now take place on the offspring from each tank in order to clear up fully the miss- matched parents.

Summary of Achievements

Ability to carry out routine microsatellite DNA screening and autoradiograpy at the Institute of Marine Research by Kevin Glover.

207 Technical ability to separate all 22 full and half-sib families by two unpublished loci from the university of Stirling: 15G10 and 15H8. All loci screened were useful for family identification but 99.75% separation of offspring can be achieved on just the two loci.

It was discovered that there were some mistakes made in setting up of the family crosses, but it is possible to re-match parents correctly after screening more loci for the offspring than would routinely be needed to assign offspring to family.

The results allow us to carry out other planned experiments aimed towards natural versus artificial selection and the local adaptation concept.

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Date: 19th May 1999 Signature: Kevin Glover

208 MOBILITY REPORT

TROUTCONCERT – MOBILITY FUNDS

Name: Øystein Skaala

Laboratory: Institute of Marine Research, Bergen, Norway

Laboratory visited: Department of Aquaculture, University of Stirling, Scotland

Duration of the visit: 3 days

From (time and date): 20.00 18th April 1999

To (time and date): 19.30 21th April 1999

Total cost of travel (flight tickets etc.) (Ecu):

Travel by car and ferry from Rosendal to Bergen, airline from Bergen to Glascow, car from Glascow to Stirling. Return trip was the same.

Use of private car Rosendal-Bergen and return: NOK 3.20per KM: 3.20 x 230= 736 Ferry 2 x 58= 116 Parking 180 Sum NOK 1032

Airline NOK 6957

Use of private car Glascow-Stirling and return: 112 KM 3.20= 358

Total cost of travel: NOK 8237,- ECU 7.989 / 8.1825= 1006,7

Total cost of accommodation (Ecu): 3 x 180= 540

Total cost ECU(travel and accomodation): 1546,7

209 Summary of visit

The aim of the visit was to meet prof. Alan Teale and Dr. John B. Taggart at University of Stirling Scotland and introduce our split centre stipendiat Kevin Glover. The PhD work of Kevin Glover involves the use of DNA microsatellites in family and stock identification of sea trout Salmo trutta, of which successful implementation depend on tight contact with well established expertice. This technique for family identification in salmonids has not yet been introduced in the lab at the Institute of Marine Research.Therefore, the aim was also to discuss practical and technical details as well as experimental design and milestones of the stipendiat work of Kevin Glover. Prof. Teale is superviser for Kevin Glover and Dr. Taggart is the key person on the technical side, while I myself is the external superviser.

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Date: 27.05. 1999 Signature: Øystein Skaala

210 MOBILITY REPORT

TROUTCONCERT – MOBILITY FUNDS

Name: Dr William Jordan

Laboratory: Institute of Zoology

Laboratory visited: Division of Cell Biology and Immunology, Wageningen Agricultural University, Wageningen, The Netherlands.

Duration of the visit:

From (time and date): 5.00 am, 1st February 1999

To (time and date): 9.00pm, 1 March 1999

Total cost of travel (flight tickets etc.) (Ecu): 215.11

Total cost of accommodation (Ecu): 894.17

Summary of visit:

The purpose of the visit was two-fold: to plan future research collaboration, and to construct cDNA libraries which would be useful for isolation of MHC genes from brown trout.

(a) Research Collaboration: We (Dr René Stet, Dr Geert Wiegertjes and I) continued discussion of a project to assess diversity at MHC loci (and other immune response genes) in brown trout, with particular emphasis on the impact of aquaculture on natural populations. We discussed the aims and programme of work of a proposal to the EC Vth Framework for such a project, and have identified potential partners in other European laboratories. We have made initial contact with these partners and plans for a proposal are progressing. In preparation for such a proposal it was decided that during my visit I would gather some preliminary data on MHC genes in brown trout.

(b) Construction of cDNA library: We therefore constructed cDNA libraries from anterior kidney tissue from brown trout. By isolating MHC genes from a cDNA

211 library we will increase the likelihood of identifying functional loci (rather than pseudogenes). Anterior kidney contains many antigen presenting cells, and is therefore a suitable tissue for isolation of both MHC class I and class II loci. Two cDNA libraries from individual brown trout were constructed during the visit, and initial screening of these libraries using the polymerase chain reaction suggested that they will prove useful for isolating cDNA clones containing MHC genes. Work on this isolation will continue in both London and Wageningen laboratories.

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Date: 11 March 1999 Signature: William Jordan

212 MOBILITY REPORT

TROUTCONCERT – MOBILITY FUNDS

Name: Kevin Glover

Laboratory: Institute of Marine Research, Bergen, Norway.

Laboratory visited: DEPT Aquaculture, University of Stirling

Duration of the visit: 14 days (at 180 ecu a day) = 2520 ECU.

From (time and date): 12.00 midday 10.10.99.

To (time and date): 3.00 afternoon 23.10.99.

Total cost of travel (flight tickets etc.) (ECU):

Bus: 25nrk = 3ecu Aeroplane ticket: 3907nrk = 477.4ecu Train: 4.10 pounds = 4.1x12.7= 52nrk = 6.4ecu Taxi: 4.92 pounds = 4.92x12.7 = 62.5nrk= 7.6ecu Taxi: 65 pounds = 65x12.7= 825.5nrk = 101ecu Aeroplane: (included as return ticket) Bus: 45 nrk = 5.5 ecu

Excahange rate is 12.7nrk is 1 British pound Exchange rate on 14/5/99: 1ECU= 8.1825 Norwegian kroner.

Total travel costs are ECU Travel: 600.1 ECU Susistence: 2520 ECU

Total expenses being applied for are: 3120.1 ECU (nrk 25 530).

Total cost of accommodation (ECU): 234 English pounds = 363 ECU.

Summary of visit:

213 Screening of Microsatellite DNA at the University of Stirling (17th-23rd October)

Aim of the visit

The main aim of the visit was to apply new molecular markers (microsatellites) to identify 240 sea trout to known families. Identifying the sea trout to family was the last stage of an experiment which I have carried out in a hatchery here in Norway designed to look at selection regimes on salmonid fish and how they can be altered by different physical environments. I refer the reader to the summary of my talk in Girona in Spain this year. It was this experiment that was being finished off. With the help of John Taggart we managed to change from using isotopes to the ABI available in Stirling to speed up the screening of the fish.

Methods

Microsatellite DNA screening was carried out on all broodstock (16 individuals) used in the current experiment and for 5 individuals from each family group (8 families) on the ABI machine. It was then performed on 240 individuals from the selection experiment. Fluorescent labelled primers (Ssa 407 and Ssa 410) were used to identify the fish. Both loci are tetranucleotide loci and are very “clean” on both isotopic and fluorescent protocols. Some work was carried out in perfecting the PCR conditions when we changed over from isotopic to fluorescent protocols.

Results

PCR conditions for isotopes and fluorescent primers was very different. There was up to a 7 degree difference in annealing temperature needed to get a decent clean product between the protocols. This has possibly something to do with the fluorescent dye altering the molecule binding to the DNA for amplification. However upon correct temperature manipulation similar quality in allele scoring was achieved with the ABI as with the isotopic\autoradiography method.

Multiplexing was experimented with. It looked promising for the two loci, however, time restrictions stopped us from being able to try out fully the workability of multiplexing these two loci together. The PCR products were mixed together before being loaded onto the gel. All bands were clear on the ABI machine and easily and readily scored. Miss matches to parents were either the result of myself miss reading the allele size on the screen or the result of an erroneous family which had more than the two parents it should have had (hatchery mistakes).

All individuals from the 8 families were assigned to their correct parentage. This allowed the results of the experiment to be calculated. 100% assignment of individuals to family was achieved with the locus Ssa 407. Locus Ssa 410 was used as a saftey check to narrow down the ability to miss genotype an individual to the wrong family.

214 These two loci can probably be used to separate up to 12-15 families together despite there being a lot of allele sharing on locus Ssa 410. With the possibility of multiplexing it is recommended that these two loci are a good starting point for others whishing to identify trout to family groups of known parentage.

The results of the experiment on family selection under two different feeding regimes is now being written up. Preliminary results show that there was no significant difference (in fact almost identical!) in relative performance (as measured by relative growth rate of families within a treatment) of each family between a high and low feeding for 4 and 20 weeks post hatch.

Summary of Achievements

Ability to identify sea trout individuals to family on the ABI machine for loci who were originally developed for isotopes.

Technical ability to separate all 8 full families by Ssa 407 alone. The locus Ssa 410 was also found readily scorable and gave helpful information to check that individuals were being genotypes to family correctly.

The results of my experiment on alternative family selection at two different feeding regimes (domestic selection mechanism?) was found. The results shall be made known at a later stage.

The results allow us to carry out other planned experiments aimed towards natural versus artificial selection, local adaptation and also fitness of different sea trout populations, especially those involving large amounts of individuals to screen.

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Date: 5th November 1999 Signature: Kevin Glover

215 MOBILITY REPORT

TROUTCONCERT – MOBILITY FUNDS

Name: René J.M. Stet

Laboratory: Cell Biology and Immunology Group, Wageningen University, NL

Laboratory visited: Institute of Zoology

Duration of the visit: 2 days

From (time and date): 04 September 1999 - 9.00 hrs

To (time and date): 05 September 1999 - 18.00

Total cost of travel (flight tickets etc.) (Ecu): 207

Total cost of accommodation (Ecu): 134

Total amount daily allowance (180 Ecu/day): 360

Summary of visit:

The purpose of the visit was to discuss a shared-cost project under the fifth framework. The project will focus on an evaluation of the impact of fish farming activities on local brown trout populations in three geographical regions. The partners involved were Bill Jordan (Institute of Zoology, UK), Ian Fleming (representing the Norwegian Institute for Nature Research, NO), both of which are part of the CA and two Irish researchers (Tom Cross and Phil McGinnity), and myself. We had two meetings in which we discussed the general outline of the proposal and a more detailed discussion on the different workpackages. The general outline is to compare brown trout populations in different rivers in the three geographical region using anonymous microsatellite markers and selective marker (i.e. MHC genes). In

216 addition, more targeted field experiments will be set-up to validate the use of comparison neutral versus selective markers. The management team (Bill Jordan and René Stet) will co-ordinate the writing of the proposal, which will be submitted before November 15, 1999.

Please, enclose tickets and accommodation bills

Date: 031099 Signature: Rene Stet

217