Microsatellite markers and management of brown trout Salmo trutta fario populations in southwestern France

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Microsatellite markers and management of brown trout

Salmo trutta fario populations in southwestern France

D. Aurelle, Patrick Berrebi

To cite this version:

Original

article

Microsatellite

markers

and

_management

of brown

trout

Salmo

trutta

fario

_populations

in southwestern France

Didier

Aurelle*

Patrick Berrebi

Laboratoire

_genome

et

_{populations, UPR9060,}

Université

_{Montpellier II,}

cc063,

place Eugene Bataillon,

34095

_Montpellier

cedex _05, France

Abstract - The brown trout

_populating

the western _part of the

_Pyrenees

mountains

(southern France)

constitute a

patchwork

of differentiated forms for two main reasons:

the

region corresponds

to the maximum extension of the modern Atlantic form at the

expense of the ancestral Atlantic one;

stocking

is

commonly practiced.

This situation

renders _urgent in this

_region

the

_analysis

of the genetic resources of wild

_origin

and

the

_impact

of domestic

_{introgression.}

Because

_allozymes

which are

_generally

used in

the Mediterranean

_region

are inefficient in Atlantic

_drainages

in

_detecting

the

_genetic

impact of

_stocking,

we used new kinds of _markers, the VNTR microsatellites. Three

microsatellite

_{loci, moderately}

or

_{highly polymorphic,}

were tested in _comparison with

three

diagnostic allozymic

loci. An

_adapted

index of domestic

_{introgression}

_(IPI)

is

proposed

and was tested for five natural

populations

and three domestic strains.

Finally, geographical

variation is summarised

by

a

phylogenetic

tree. The value of microsatellite markers in the Atlantic

drainage

and the use of such data for

_protection

and management of brown trout

_populations

are discussed.

_©

_{Inra/Elsevier,}

Paris

brown trout

_/

_{conservation-biodiversity}

_/

microsatellites

_/

introgression

/

stocking

*

Correspondence

and reprints

Résumé - Marqueurs microsatellites et _gestion des _populations de truite

com-mune, Salmo trutta fario, dans le sud-ouest de la France. La truite commune

peu-plant

l’ouest des

_Pyrénées

forme un

_puzzle

de formes différenciées dû à deux causes

principales :

la

_région

constitue le maximum d’extension de la forme

_atlantique

mo-derne au détriment de la forme

_{atlantique ancestrale,}

et d’autre _part, les

_pratiques

de

repeuplement

y sont

généralement

très actives. Cette situation rend urgente

l’analyse

de la biodiversité naturelle et de

_{l’introgression}

_par les formes

_domestiques.

Parce que

les _enzymes,

_{généralement employés}

en zone méditerranéenne, sont

quasiment

ineffi-caces _pour détecter

l’impact génétique

des

repeuplements

sur le versant

atlantique,

étude,

trois locus

microsatellites, plus

ou moins

_polymorphes

sont testés en comparai-son avec trois locus

_{enzymatiques diagnostiques.}

Un index de

_{l’introgression d’origine}

domestique

(IPI)

a été

_{spécialement adapté}

à ce _type de données. Il est

_appliqué

sur

cinq populations

naturelles et trois échantillons de

_{pisciculture.}

L’intérêt des

mar-queurs microsatellites utilisés en zone

_atlantique

et

_{l’exploitation}

des résultats pour

la protection et la

_gestion

de la truite commune sont discutés.

_©

_{Inra/Elsevier,}

Paris

truite commune

_/

conservation-biodiversité

_/

microsatellites

_/

introgression

/

repeuplements

1. INTRODUCTION

Salmonids constitute one of the most

_manipulated

fish in

_temperate

coun-tries.

_Moreover,

salmonid

_fish,

and

_especially

the brown trout

_(Salmo

trutta

fario

L.),

present

some

_{interesting biological}

characteristics for the

_study

of

genetic

differentiation:

_they

live in the _upper

_part

of

_rivers,

and exhibit

’hom-ing’

behaviour.

_Therefore,

the different

_populations

are

_relatively

isolated but

can be connected

_{by migration}

_(sometimes

_through

the

_sea).

The first

_genetic

studies, using

allozymes,

confirmed marked

_genetic

differentiation

_(see

_e.g.

Fer-guson,

1989,

or

Guyomard,

_1989,

for French

_{populations).}

_Moreover,

Hamilton et al.

_(1989),

_using

the distribution of the different alleles

_{of LDH5}

_*

_,

_proposed

the existence of two different brown trout forms: an ’ancestral’ form fixed for

the LDH5* 100 allele and a modern one characterised

by

the LDH5*90 allele.

According

to this

_hypothesis,

the ancestral form would have been

_supplanted

by

the modern one in the northern

_part

of the distribution _area; it survived

only

in some less accessible

_parts

of some head-river

_systems.

This

_analysis

was

completed

in France

_using

the transferrin locus

_(Guyomard,

_1981),

_{which, along}

with LDH5* and

_FBPl

_*

_,

was used to

_hypothesise

that the Atlantic form is di-vided into ancestral and modern forms named ’modern Atlantic’ and ’ancestral

Atlantic’

_{distinguishable}

_{by particular allozymic}

markers

_(the

locus LDHS* is

diagnostic;

see

_Poteaux,

1995 and table

_1).

Most trout from the French Atlantic

basin

_belong

to the modern

_type

_(except

from some rivers in

_Brittany

[Guy-omard,

1989]

and,

as will be discussed

_later,

some

_Pyrenean

_rivers).

LDHS*90 was

_probably

absent from Iberian

_populations

_prior

to introduction of

_hatchery

fish

_{(Garcia-Marin}

and

_Pla,

_1996).

In the southwest of

_France,

in the Atlantic

part

of the

Pyrenees,

the two Atlantic forms seem to coexist

_(Poteaux,

_1995;

Berrebi,

1997a).

These

_hypotheses

still raise the

_questions

of the

_origin

and the coexistence of these different forms. This

_complex

of

_{populations provides}

a

_{good example}

of

_{high biodiversity}

and should be

_{protected. However,}

the situation is

_complicated

_{by stocking}

_practices

that can

_genetically

_endanger

original

populations

(Berrebi,

1997b).

This is

_why

it is useful to evaluate the

impact

of

_stocking,

and

_possibly

to search for wild

_populations

to

_protect

them

as _pure sources of

_genotypes.

Domestic trout

_belong

_mainly

to the modern

Atlantic form as shown

_{by enzymatic}

_{(Guyomard, 1989)}

and mitochondrial

deoxyribonucleic

acid

_(DNA)

studies

_(Bernatchez

et

_al.,

_1992).

_{Distinguishing}

domestic and wild trout is _easy in the Mediterranean

_part

of France or where ancestral Atlantic trout is found alone

_(Taggart

and

_Ferguson,

_1986).

In the

area of wild modern Atlantic

_populations,

_however,

_allozymes

are

_inefHcient,

these

_genetic

markers makes them

_appropriate

for different _purposes.

_Firstly,

management

of brown trout

_populations

is

_essential,

as the definition of

purely

wild

populations

for

_protection

or for constitution of new

_strains;

secondly,

the

microsatellites would be useful for the

_analysis

of natural

_{differentiation,}

and

especially

the

relationships

between modern and ancestral Atlantic trout.

Because the

_Pyrenees

basins of southwestern France seem to be the limit of the modern Atlantic trout’s extension into the

_territory

of the ancestral Atlantic

trout,

it is also

_important

to know if the two taxa continue their

_competition

or if an

_equilibrium

has now been reached. Marked

_genetic

_melting

occurs in

the

_boundary

area.

The

_objective

of this paper is to test microsatellite loci in such a situation

with the

_help

of useful

_allozymic

markers. Value assessment of this new tool

in future research in Atlantic

_drainages

is also an

_important

objective

with a

view to a

_{complete description}

of the

_genetic

structure of French

_populations

of brown trout.

2. MATERIALS AND METHODS

2.1.

_Sample

sources

Five natural

_populations

were

_{investigated,}

with a

_sample

size

_varying

from 17 to 28 individuals

_(the

details are

_given

in table II and their localisation in

figure

1).

For the Andurentako

River, only

five individuals were

_available,

due to the small

_population

size.

_{Despite this,}

_they

were

_analysed

because of their

interesting morphological

and

_enzymatic

characteristics

_{(Berrebi, 1997a),}

but the statistical

_parameters

are

_given

_simply

for indication.

_Populations

with no or almost no

_allozymic

modern alleles were

_supposed

not to have been stocked with

_hatchery

fish

_(see

table

_III);

for the Andurentako and Beherekobentako

rivers, angling

associations affirmed that these streams had not been restocked for several years.

Moreover,

three

_hatchery

samples

were

_analysed

_{to compare}

genetic

variability.

2.2. DNA extraction

Total DNA was obtained from muscle or blood

kept

frozen

_just

after

dissection, using

the Chelex extraction

_protocol

described in Walsh et al.

2.3. DNA

_{amplification}

and microsatellite

_analysis

Three microsatellite loci were

_used;

the

_primers,

_repeat,

_sequences and

annealing

temperatures

are

_given

in table IV. Strutta58 was cloned

by

Poteaux

(1995);

MST73 and -15 were cloned

_{by Estoup}

et al.

_(1993).

_Polymerase

chain

reactions

_(PCR)

were

_performed

in 10

_vL

with 0.2 units of

_Taq

_polymerase,

2 mM

_MgCl

₂

_,

dNTP

₍₁₀₀

_vM

_each),

IX reaction buffer and 5

_pmol

of each

primer.

One of 5’ ends of the two

_primers

was

_covalently

linked to fluorescein. The

typical

PCR programme used was: 2 min at

_{94 °C;}

30

_cycles

_comprising

45 s at 92

_°C;

45 s at the

_annealing

_temperature

and 45 s at 72

_°C;

final

_step:

Amplification products

were resolved

_{by electrophoresis}

on 6

%

polyacry-lamide

_{denaturing gels}

in a Pharmacia automated _sequencer

_(ALF).

The results of

_{previous enzymatic}

_analysis

of the same

_samples

were also

used, especially

for the identification of wild and Atlantic forms

_(see

complete

results in

_Berrebi,

_1997a).

_Only

_diagnostic

loci

_(lactate

_{dehydrogenase}

_LDH5

_*

_,

transferrin TF* and fructose

_{biphosphatase}

_FBP1’)

are taken into account here to calculate a

_percentage

of modern and ancestral fish alleles.

2.4. Data

_analysis

The distributions of allele

_frequencies

for the three microsatellite loci are

given

in

_figure

!. For the locus

_58,

the

_high

number of alleles

₍₃₃₎

compared

to the

_sample

sizes

_(generally

<

₃₀₎

does not allow a

_{statistically significant}

esti-mation of allele

_frequencies.

_{Nevertheless,}

these

_frequencies

are

_given

for relative

comparisons

between

samples. They

have also been used for an

_{introgression}

estimation,

which is not intended to

_give

an absolute value of

_{introgression}

(see later).

Unbiased

_{expected heterozygosities}

were calculated

according

to Nei

₍₁₉₇₈₎

_using

the

_computer

programme GENETIX

(Belkhir

et

_al.,

_1996).

The Weir and Cockerham

₍₁₉₈₄₎

estimators of

_Wright’s

indices Fis and Fst

were calculated with the same _programme. The statistical

_significance

of the

observed values was evaluated

_using

500

_permutations

of the

_original

data set.

Reynolds

genetic

distances

_(Reynolds

et

_al.,

₁₉₈₃₎

were used for the

construc-tion

_{of a phylogenetic}

tree with the PHYLIP 3.0 programme

(Felsenstein,

1993)

using

the ’Fitch’ method

_(Fitch

and

_Margoliash,

_1967).

The

_impact

of

_stocking

was evaluated

_using

an

_{introgression}

index to

estimate the

_percentage

of domestic _{genes in} natural

_populations.

Poteaux and Berrebi

₍₁₉₉₇₎

used the

_percentages

of LDHS

*

90,

TF* 100 and FBPI* 100 alleles in

_{enzymatic studies;}

for

_{microsatellites,}

Poteaux

₍₁₉₉₅₎

_proposed

a ’maximal

introgression

index’

_using

alleles shared with domestic stocks. These alleles

can have several

_origins:

ancestral

polymorphism, homoplasy

or

_{introgression.}

In view of the substantial

_proportion

of shared alleles in domestic and natural

samples

in our

data,

we _propose here another

index,

the

_{’weighted introgression}

index’ or IPI

(indice

_pondéré

d’introgression),

which is not

_designed

to

_give

an

accurate estimation of

_{introgression,}

but can be

_used,

in relative

terms,

for

management

purposes.

This index compares the allele

frequencies

between rivers and hatcheries. It

_only

retains those

_supposed

to be more

_informative,

i.e. those whose mean

frequencies

in considered domestic stocks are more than X times the

frequency

in natural

_populations

_(the

_comparison

is done with mean

_frequencies

of natural

_{populations):}

these alleles are

_supposed

to be domestic. Their _presence

in natural

populations

is assumed to be due to

_{introgression}

rather than

homoplasy,

for

_example.

For a more sensitive

_estimation,

we should use some

populations

identified as

_purely

wild.

_However,

even

_populations

with the

modern allele of LDHS* can contain modern wild

fish;

this is

_why

for this first

attempt,

we also included these

_populations.

It should be noted that if a mean

frequency

is calculated over _{too many} river

_populations,

some alleles with a

high

frequency

in one

_population

(not

_significant

for this

_particular

population)

could be considered as

_significant;

this means that the more

_populations

that

with few

_populations,

_however,

_comparisons

between

populations

agree well

with

_{allozyme results,}

which can be considered as

_{quite good, especially}

when

compared

to the Poteaux index

_{(see later).}

Various X

_parameters

can be tested

(IPI

X

).

The

_frequencies

of these alleles are then added over all loci

(ED).

This first addition does not take into account all the other alleles with a

_higher

frequency

in hatcheries but with a ratio lower than X. The addition of the

hatchery

mean

_frequencies

is made on the same alleles

(ED,,,).

For

_example,

with X =

1.1,

the calculation of the _mean

frequency

of retained domestic alleles

gives

0.64 for hatcheries. To reach the theoretical 100

%

value,

we have to divide these relative values

_by

0.64. The same

_weighting

is then

_applied

to the sum

of domestic allele

frequencies

in natural

populations. X

values were tested for

1.1, 1.5,

2 and 3

_{(table V!.}

As mentioned

_earlier,

it is

_only

a first

_trial,

and a

more

_{precise analysis}

of this

_problem

should be

_performed.

The formula is:

where

IPI

X

is the IPI index for the chosen X

parameter

value;

ED is the sum of

frequencies

of domestic alleles in the natural

population

considered

(a

domestic allele is

_designated

as an allele whose

_frequency

in involved hatcheries

3. RESULTS

Allele

_frequencies

are

_given

in table III with the three

_{allozymic diagnostic}

loci for

_comparison.

3.1.

_{Intrapopulation}

_variability

The total number of alleles

_by

locus is 33 for

_Strutta58,

6 for MST73 and 7 for MST15. As

_already

_mentioned,

the

_sample

sizes for

_highly

variable loci

(here

locus

_Strutta58)

should be

_great

_enough

for

_significant

estimations of allele

_frequencies.

The mean unbiased

_{expected heterozygosity}

obtained for these three loci was

_{generally slightly higher}

for the

_hatchery

stocks

_(from

0.70 to

_0.77)

than for river

_samples

_(from

0.60 to

_{0.68, except}

Marcadau with

0.78).

These values are

_higher

than those of Presa

_(1995),

_probably

because the studied loci are different. The lowest value was obtained for the Andurentako

River

_(0.30),

but the

_sample

size

₍₅₎

does not allow a firm conclusion to be drawn. A

_higher

_variability

in domestic stocks had

_already

been found

_by

Poteaux

_{(1995) (both}

for

_enzymatic

and

_{microsatellite)}

and

_by

Presa

₍₁₉₉₅₎

with more microsatellite loci.

3.2.

_Departures

from

_{Hardy-Weinberg equilibrium}

The Fis multiloci calculated and the

_percentages

of

_permutations

with values

_superior

and

_equal

to the observed value are

_given

in table VL None of the

_{hatchery populations}

studied here

_{depart significantly}

from the

Hardy-Weinberg

proportions.

In _contrast, all natural

_populations

_(except

3.3. Differentiation between

populations

The Fst values estimated

by

the 0 of Weir and Cockerham

_(1984),

and the associated

_{probabilities,}

are

_given

in table VIL All

paired comparisons

between

populations

were

_significant

(at

1

_%),

but Fst values between domestic stocks

(from

0.04 to

_0.057)

were

_globally

lower than between natural

_populations

_(from

0.088 to

_0.324)

or between natural and domestic ones.

Allele

_frequency

distributions for natural and domestic

_populations

_(mean

for each

population

type)

are

_given

in

_{figure 2}

for the three loci. For the less variable loci

_{( 15}

and

_78),

there were few differences in terms of

_{presence/absence}

of alleles. For

_example,

on locus

_15,

allele sizes inferior to 220 were

_only

found in

natural

_populations

and not in hatcheries. At locus

58,

differences were

_greater;

allele sizes

_larger

than 152 were found

_only

in nature.

_Conversely,

alleles from

120 to 124 were characteristic of hatcheries.

_Major

differences between these

distributions were

_{quantitative.}

The IPI used alleles such as 224 at locus

15,

which is much more

_frequent

in hatcheries than in nature.

The IPI values are

_given

in table V with four

_examples

of

_parameter

X values. Because we know that the

samples Beherekobentako,

Dancharia and

Andurentako are _pure wild

_samples

(see

LDHS*90

frequencies),

it is

obvious

that the X = 3

_parameter

is the more realistic. This result must be tested

_by

extrapolation

to other

_samples

_(especially

some modern natural

populations).

According

to the IPI

values,

the most

_{introgressed populations}

were

Mar-cadau and Beherobie.

_Moreover,

the Fst estimations between the Marcadau

population

and the three domestic stocks

_analysed

were lower than for the

other

_populations.

The lowest

_{introgression}

indexes were found for Dancharia

and Beherekobentako and for Andurentako

_(as

an indication

_only).

This _agrees well with the

_allozyme

results

_(0-2

%

of modern allele

LDHS

*

90,

which is the characteristic of the ancestral Atlantic

_form),

and confirms that these

pop-ulations

_mainly

_comprise

wild fish. On the other

hand,

the values obtained with the Poteaux index are

_always

almost one and

probably

do not

_clearly

reflect the differences in the

_{introgression}

rates. This shows that the different

forms

_(wild

and domestic modern

_Atlantic)

_compared

here are closer

_(sharing

a

higher

number of

_alleles)

than the Mediterranean and Atlantic ones

_compared

in Poteaux’s

_study

_(1995),

and need a more

_{discriminating}

index.

These results are confirmed

_by

the

_analysis

of the

_phylogenetic

tree

con-structed with microsatellite allele

_frequencies

_{(figure 3),}

which

_clearly

_separates

hatcheries and natural

_populations:

Marcadau is the nearest natural

popula-tion from domestic

stocks,

and Beherekobentako and Dancharia are on the other side of the tree. The marked

_separation

of Andurentako from the other

populations

cannot be taken into account due to the _very small size of the

sample.

4. DISCUSSION

The first results have shown that the structure

given by

microsatellites is similar to that

_{given by allozyme}

markers

_analysed

in Berrebi

_(1997a).

_Only

three loci were used for this

preliminary

study;

more loci would

_probably

would tend to reduce differences due to natural differentiation between wild

populations.

Nevertheless,

a

really diagnostic

locus between wild and domestic

populations

would be more useful.

For the

_{intrapopulation analysis,}

we found a

_{higher intrapopulation}

variabi-lity

in domestic stocks than in natural

_populations.

This is

_probably

linked to the

_{breeding practices}

in

_aquaculture:

fish are

exchanged

between hatcheries and a

_single

stock can contain alleles with

_extremely

diverse

_origins

_(otherwise,

genetic

drift would tend to reduce

_{heterozygosity}

as shown

_by

Allendorf and

Phelps

[1980]

for cut-throat

_trout).

_Conversely,

_population

sizes and their

vari-ations

_{(e.g. bottlenecks)}

can

_explain

a lower

_{heterozygosity}

in natural

samples.

The

_allozymic

marker is

_generally

inefficient in Atlantic basins for

discriminat-ing

wild and domestic fish. The ancestral Atlantic form area is an

_exception.

Table 111 shows that the

populations

of

Andurentako,

Beherekobentako and Dancharia are

_{nearly homozygous}

for the alleles LDH,5*100

(and

with a

fre-quency of allele TF100

varying

from 0.77 to

1,

according

to Berrebi

!1997a!),

of the domestic strains is demonstrated

_by

the absence of LDHS*90 alleles. It is concluded that these

_populations

are

_composed

of

_only

wild trout. The IPI

parameter

gave a similar result which makes it

_possible

to trust the IPI values for the other

populations

in which the

_enzymatic

marker is inefficient.

In such a

_{composite genetic environment,}

the

_great

_variability

of the locus 58

_probably

_represents

an obstacle for the discrimination of these different

forms,

because some alleles _may have been shared several times

_{independently}

(homoplasy).

On the other

_hand,

some less variable loci would

_perhaps

not show

_enough

differences between such close forms. As

_already

mentioned,

the most useful locus would be

_moderately

variable but

_diagnostic.

Nevertheless,

even if the microsatellite loci used are not

_{diagnostic markers,}

the

_global

results

_{given by}

this new kind of marker and

_allozymes

are similar for the ancestral Atlantic form

_(where

_allozymes

are

efficient).

This is

_why

microsatellites are also

_expected

to be efficient for the detection of the wild modern Atlantic form.

Departures

from

_{Hardy-Weinberg}

_expectations

are constant in nature in the direction of

_{heterozygote deficiency}

and can have several

_origins.

Technical

problems

such as the _presence of null

_alleles,

even if

_they

cannot

_completely

be

_rejected,

would not have such an

_impact

_(because

of the too few

_possible

null

_homozygotes

_found).

_Moreover,

_heterozygote

deficiencies were also found

with

_allozymes,

where detection of

_homozygotes

of null alleles is efficient. If two different

_populations

are

_supposed

to coexist in the same

_stream,

a ’Wahlund effect’ could

_explain

the observed Fis. This would be the case, for

example,

if domestic and wild trout are

_present

in the same

_sample,

without

(or before)

complete mixing,

as

already

found

by Largiadèr

and Scholl

(1996).

In our _case, this

_phenomenon

can

_{probably explain}

the

_significant

Fis found for the

_expected

most

_introgressed

_populations:

Marcadau and Beherobie

_(see

table

_V!.

_Departures

from

_panmixia

demonstrated in pure wild

populations

(Beherekobentako,

Dancharia and Beherobie

_streams)

_{by significant}

and even

higher

values of Fis than for the other

_populations

are more difficult to

_explain.

Garcia-Marin and Pla

₍₁₉₉₆₎

and

_Apostolidis

et al.

₍₁₉₉₆₎

in

_{enzymatic studies,}

and Presa

₍₁₉₉₅₎

with

_{microsatellites,}

did not find _any

_significant

Fis in natural

populations.

Poteaux

₍₁₉₉₇₎

also observed such deficiencies in Mediterranean

populations.

At our

_sites,

a

_biological

mechanism _may be the cause: in natural

populations, particularly

in Atlantic

_basins,

a

_{regular cycle}

of

_migration

has been observed. Because adults

_migrate

_upstream

to

_reproduce

_(Delacoste,

1995),

sympatry

of differentiated cohorts can _occur,

_provoking

a Walhund

effect.

_However,

this deficit could also be

_partly

due to some

_past

_stocking

practices,

which would

_always

be detectable if

_hybrids

between domestic and wild fish were

_selectively

eliminated. This has

_yet

to be demonstrated

_(this

explanation

would be more

probable

for

populations

where LDHS*90 is

always

present,

even if it may have been eliminated

_by

drift).

This

_phenomenon

alone is

_probably

not sufficient to

_generate

such a

_deficiency.

More

_likely,

it results from a combination of several different mechanisms.

Our results confirm the

_high

natural

_genetic

_diversity

of brown trout

popula-tions.

_Biodiversity

_(comprising

_genetic

_diversity)

is an

_important

_challenge

for

nature

_{conservation, especially}

in wild

_species

with

_developed

domestic strains in commercial

_exchanges.

The

_question

of the _gene flow between the two

reservoir of useful

genotypes

for future

_improvement

of domestic

_strains,

and the domestic form as

possible ’genetic pollution’

of wild

populations.

From a

scientific

_point

of

_view,

the interaction between two differentiated _{genomes is}

of obvious interest.

In the case of French brown

_trout,

the wild

diversity

is

frequently

used to

increase domestic

_polymorphism

_(which

explains

in

_part

the

_higher

heterozy-gosity

of

_hatchery

_strains).

For this _purpose, wild males are sometimes crossed with domestic females.

The most

_{important impact}

of the coexistence of wild and domestic forms of the same

_species

is the

genetic impact

of domestic trout

heavily

stocked

in rivers inhabited

_by

natural trout. The literature

_gives

some estimations

of the

stocking

effect on French brown trout wild

_{populations. Guyomard}

and

_Krieg

₍₁₉₈₆₎

_gave

_{introgression}

values from 0 to 50

%

in Corsican trout

populations,

Barbat-Leterrier et al.

₍₁₉₈₉₎

found 0 to 40

%

in the continental Mediterranean

region

and Beaudou et al.

₍₁₉₉₄₎

estimate that in the Orb basin

(south

Mediterranean

_region)

only

0.5

%

of the stocked

_juvenile

trout reach the

_reproductive

_stage,

which leads to 10 to 20

%

of such limited cumulative

introgressions

over decades. Berrebi

(1995)

gave a

_large

set of

_examples

in the Mediterranean basins of the French

Pyrenees

with an estimated

_{introgression}

value of 0 to 78

%

of domestic alleles in wild

_populations.

5. CONCLUSION

Knowledge

of domestic

_impact

is valuable in various _ways: definition of zones

of

_protection,

_global

protection

of the

genic biodiversity

of the

_species,

conser-vation of local

adaptation

as

_{proposed by Leary}

et al.

₍₁₉₉₅₎

_(this

would have to be

_{demonstrated),}

_possible

modification of the

_stocking

_practices

accord-ing

to its deduced efficiencies in each

region,

creation of new strains of local

origin,

conservation of

_{morphologically}

differentiated

_entities,

reconstitution of the

_origins

of the natural taxonomic groups and of the

history

of

populations

(colonisations,

migrations).

This kind of

development

has occurred in France

_through

the

_description

of

_geographic

structures of brown trout established

_{by allozymic}

studies. The need to continue similar

investigations

in the Atlantic basins is obvious and the

_present

_paper tries to estimate the

_qualities

of the microsatellite markers in such a context. These

preliminary

results

_clearly

show the

utility

of

microsatellite loci for this purpose.

They

can

_help

to determine if a

population

is

_{highly introgressed}

or not

_(using

an

_{introgression}

index).

In some

_respects

microsatellite loci are also easier to use than

_allozyme

loci and can be obtained

without

killing

the fish

_(a

_piece

of fin

_suffices).

_They

are a

_good

tool for the

management

of natural

_populations

in order to

_highlight

which ones would have

to be

protected. Here,

for

_example,

Behrekobentako or Andurentako could be

protected,

either

by creating

reserves or

_by

_raising

the minimum

_acceptable

size in

_angling.

Microsatellite loci

_probably

represent

an

original

_component

of

brown trout

_diversity.

Nevertheless,

more

_{investigations}

are _{necessary to}

_improve

this

approach.

We first intend to test other loci to find less variable and more informative

(diagnostic

if

_possible)

ones.

Comparisons

should also be made with

populations

Atlantic trouts. This constitutes the

_greatest

technical

_challenge.

And from

a fundamental

_point

of

_view,

_larger

scale

_sampling

and a more detailed

study

will have to be done on natural

_populations

for a better

_{understanding}

of their evolution and

_genetic

characteristics. A

_large-scale

_description

_{by allozymes}

is

nearly

finished all

_along

the

_Pyrenees

chain. The

_{complementary}

_description

by

microsatellites of the Atlantic

_drainages

is

_planned.

ACKNOWLEDGEMENTS

This research was

_{supported by}

the Bureau des Ressources

_Génétiques

_(grant

n°

_95011),

the Conseil

Supérieur

de la P6che

_(grant

n°

₉₆₀₂₇₎

the Minist6re

de

1’Education

Nationale

_(ACC-SV7

n°

_9507127)

and the Club

_Halieutique

In-terdépartemental.

The field captures were made

_by

the local Federations de P6che

kindly

assisted

_by

students from ENSAT

_(Toulouse)

and volunteers from

_Montpellier

II

_University.

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