Genetic analysis by interspecific crosses of the tolerance of Drosophila sechellia to major aliphatic acids of its host plant

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Genetic analysis by interspecific crosses of the tolerance

of Drosophila sechellia to major aliphatic acids of its

host plant

M Amlou, E Pla, B Moreteau, Jr David

To cite this version:

Original

article

Genetic

_{analysis by interspecific}

crosses

of the tolerance of

_Drosophila

sechellia

to

_{major aliphatic}

acids of its

host

_plant

M

_{Amlou, E Pla,}

B Moreteau

JR David

Laboratoire

_{populations, génétique}

et

_évolution,

Centre national de la recherche

_{scientifique,}

91198

Gif sur-Yvette cedex,

France

(Received

3 March

_{1997; accepted}

11

_July

₁₉₉₇₎

Summary -

Toxicity

of hexanoic and octanoic

_acid,

ie, the two

_{major aliphatic}

acids found in _ripe fruits of Morinda

_citrifolia,

was measured on adult flies of

_Drosophila

sechellia,

D

simulans,

F1

_hybrids

and backcrosses. With both

acids,

tolerance was much

higher

in D sechellia than in D simulans while F1 and backcross _progeny exhibited

intermediate characteristics. Tolerance to these two acids in D sechellia appears to be a

_major

mechanism for

_{understanding}

the

_{ecological specialization}

of that

species

to the toxic morinda.

Significant

differences in tolerance were found between sexes,

especially

in F1

_hybrids.

The role of X-linked tolerance genes was,

however,

not obvious from the backcross

_generation,

and most of the

_{interspecific}

difference seems to be autosomal and

polygenic. Attempts

were made to

_introgress

the tolerance of D sechellia into D simulans

by selecting

with either morinda fruit or _pure octanoic acid. Both

_{techniques proved}

to be unsuccessful.

_Introgressed

_genotypes

_{progressively}

returned to the

_apparently

pure

D simulans

phenotype

and tolerance

regressed

to the low value

typical

of that

_species.

This barrier

_{against introgression}

seems _quite similar to the barrier observed in

_hybrid

zones of various animal

_species.

ecological specialization

/

hexanoic acid

_/

octanoic acid

_/

interspecific hybrids

/

barrier to _{introgression}

Résumé - Analyse génétique par croisement interspécifique de la tolérance de

Drosophila sechellia aux deux principaux acides aliphatiques de sa plante hôte. La toxicité de l’acide

_hexanoïque

et de l’acide octanoi’que, deux acides

_aliphatiques

majori-taires dans le

fruit

mûr de Morinda

citrifolia,

a été mesurée chez les adultes de D

sechellia,

de D

_simulans,

les

_hybrides

Fl et les individus issus de rétrocroisements avec les deu!

pa-rents. Pour les deux

_acides,

D sechellia est

_{beaucoup plus}

tolérante que D simulans. Les

FI et les rétrocroisements montrent des

_{caractéristiques}

intermédiaires. La tolérance de

D sechellia à ces deu! acides semble être le mécanisme

_majeur

_permettant de

compren-dre la

_{spécialisation écologique}

de cette

_espèce

sur le morinda. Une

_{différence significative}

de tolérance a été observée entre les mâles et les

_{femelles, spécialement}

chez les Fl. Le

chez D simulans par sélection ont été _entreprises, soit avec le

_fruit

du morinda soit avec

l’acide octanoi’que. Les deux

techniques

se sont soldées par un échec total. Les

génotypes

introgressés

retournent _{progressivement} à un

_phénotype

_pur D simulans et leurs tolérances

régressent également

vers des

_faibles

valeurs de

_{DL50, caractéristiques}

de D simulans. Cette barrière contre

_{l’introgression paraît}

similaire aux barrières rencontrées dans les

zones

_{d’hybridations}

chez

_{différentes espèces}

animales.

spécialisation écologique

/

acide hexanoïque

/

acide octanoïque

/

hybrides

inter-spécifiques

/

barrière à l’introgression

INTRODUCTION

The diversification of

_ecological

niches

_by

reduction of niche breadth is often consid-ered as a _consequence of

interspecific competition

and as a

_major

cause for

maintain-ing biodiversity

in an

_ecosystem

(Hutchinson,

_{1978; Tilman,}

1982),

_although

there are

_exceptions

to this

_general

rule

_{(Connell, 1980).}

A

_{long evolutionary}

time seems,

however,

to be needed for

_increasing

the

_complexity

of food webs and the number of

_{coexisting species.}

The relative

_stability

of

_tropical

_ecosystems,

as

_compared

to

temperate

ones, may

explain

why

more numerous

_species

are

generally

found in

the

_tropics

than in

_temperate

biota

_(Pianka,

_{1974; Pielou,}

_1975);

the

_{Drosophilidae}

family

follows this rule. For

example,

more than 400

_species

are known from the

Afrotropical

region

(Tsacas

et

_al,

_1981),

while

_only

80

_species

are found in

_Europe

(Bdchli

and

_Rocha-Pit6,

_1981).

In

_insects,

numerous cases of

_ecological

_{specialization}

are

_known,

_especially

among

phytophagous species

(Price,

1984;

Harborne,

1989).

Most

investigated

cases,

however,

are restricted to

_{species comparisons,}

without any

possibility

of

genetic

analysis.

There are

_only

a few

_biological

situations amenable to

_genetic

investigation,

including

Papillio species

(Thompson

et

al,

1990),

the

_{fly Rhagoletis}

(Feder

et

_al,

_1990a,

_b)

and

_Drosophila

sechellia

_(R’Kha

et

_al,

_1991).

D

_sechellia,

endemic to the

_Seychelles

_archipelago,

is

_{strictly specialized}

on a

single

resource, the fruit of Morinda

citrifolia

(Tsacas

and

Bdchli,

1981;

Lachaise et

_{al, 1986;}

Louis and

_David,

_1986),

_although

it can be reared on usual

laboratory

food.

_Using

its natural _resource, frozen

_morinda,

it was shown

_{that, compared}

to its

_sibling

D

_simulans,

D sechellia is tolerant to fruit

_toxicity,

that adults are not

repelled

but attracted

_by

the resource, that females

prefer

to

_oviposit

on

_morinda,

and that morinda

_stimulates,

instead of

_{inhibits, oogenesis}

_(R’Kha

et

_{al, 1991, 1997;}

Legal

et

_al,

_1992).

For the

analysis

of such a

_{specialization,}

a

_knowledge

of the

responsible

specific

chemicals is needed. More than 150 different

_compounds

were identified from the

ripe

morinda fruit

_(Farine

et

_al,

₁₉₉₆₎

_among which two

_aliphatic

_acids,

hexanoic and octanoic

acids,

are

_present

in

_large

amounts

_(Legal

et

_al,

₁₉₉₄₎

and are

_mainly

responsible

for the

_typical

smell of this fruit.

_Preliminary

observations

_suggested

that octanoic acid was

mainly responsible

for the

_toxicity

(Farine

et

_al,

_1996).

Higa

and

_Fuyama

₍₁₉₉₃₎

_investigated

_only

hexanoic acid and found that it acted

specifically

on behavioral traits. Data from different

_{investigators}

are however sometimes difficult to _compare since different

_techniques

are used. In the

_present

work we

_analyzed

the

_toxicity

of the acids with

_exactly

the same

_technique

as that

Genetic

_comparisons

were made

_{by comparing parental species,}

D sechellia and

D

_simulans,

their F1

_hybrids

and backcross _progeny. We also tried to

_introgress

the

high

tolerance of D sechellia into the sensitive D simulans. Both acids were found

to be

_highly

toxic for D simulans and _{appear to} be

_responsible

for the

_toxicity

of the natural resource. Tolerances of the

_hybrids

and backcrosses were

_{intermediate,}

suggesting mainly

additive effects. Results of

_{introgression}

_attempts

were

_negative.

MATERIALS AND METHODS

Species

and

_hybrids

Mass cultures of D simulans and D sechellia were established

by

_mixing

isofemale

lines collected in the

_Seychelles

in 1985.

_{Interspecific hybrids}

were

_{produced by}

crossing

females of D simulans with males of D

_sechellia,

since this cross is much easier than the

_reciprocal

one

(Lachaise

et

_{al, 1986;}

R’Kha et

_al,

_1991).

_Hybrid

males

are sterile but females are

_fully

fertile. F1 females were backcrossed to both

_parental

species,

producing

second

generation

progeny,

designated

as backcross

_(BCsim

and

BCsech).

All

_experiments

were carried out at 25°C.

Acid

_toxicity

This trait was measured on adult flies. Larvae were _grown at low

_population

_density,

on an axenic

_{killed-yeast, high-nutrient}

medium

(David

and

_Clavel,

1965).

The use of such a medium

_prevents

_crowding

effects and

_produces

numerous adults in

good

physiological

condition. After emergence, adults were anesthetized with

C0

2

,

distributed into _groups of 20 males or

_females,

and

_kept

on the same medium for

3

_{days. Groups}

were then transferred into air

tight

plastic

vials

_containing

2 mL of

a

3%

sucrose water solution on absorbent _paper. This

_technique

is similar to that

implemented

for

_measuring

alcohol

_toxicity

_(David

et

_al,

_1986).

With hexanoic and octanoic acid we faced a

practical difficulty

since their

solubility

in water _{is very}

low. Solutions of different concentrations could not be

_conveniently

_prepared.

After various

_attempts,

it turned out that a better

_technique

was to

_deposit

a

_given

amount of acid

_(in

_!L)

with a

_micropipette

on the wet absorbent _{paper at} the bottom of each vial. Different doses were used in a

single experiment.

For each

dose,

at least four

_vials,

_ie,

two with 20 males and two with 20

_females,

were used.

Dead adults were recorded after 2

_days.

As usual in

_{toxicity studies,}

a

large

and uncontrolled

variability

was observed.

For

_example,

in the same

_experiment

it was not rare to

_find,

for a

_{given dose,}

a vial

where all the 20 flies were dead after 2

days,

while in a similar vial

80%

of the flies were still alive. Such variations are too

_great

to be due to random

_sampling,

and

may be

explained by

the

difficulty

in

achieving

an even distribution of the toxin

in each vial.

Significant

variations were also observed when

repetitions

of the same

toxicity

test were made

_apparently

under the same

_experimental

conditions. Such

variations seem to be a

general

observation in

_toxicity

studies

_using

Drosophila

adults

_(Chakir

et

al,

1993).

For statistical

analyses

and

_comparisons,

two

_possible

may be calculated and

helps

to characterize different

genotypes.

An increase in dead

fly

number is observed with

_increasing

doses of

_acid,

as illustrated in

figure

1. Such

data can be

_analyzed

_using

Anova and demonstrate

_significant

effects of

_genotypes,

doses and interaction.

Another

_procedure,

more usual in

_{toxicity studies,}

was to estimate the

_toxicity

_by

calculating

the lethal

dose,

killing

50%

of the flies

_(LD50)

in a

_given

time. For each

experiment,

from four up to six different doses were used. The LD50 was

_estimated,

with a linear

_model,

after

_log-probit

data

_{transformation,}

and the

_log

LD50 was back

transformed into microliters. For each

_genotype,

at least five different

_experiments

These two

_procedures

were used in all _cases, and

they

led to

basically

identical conclusions. For the sake of

_{simplicity, only}

data from the second

_procedure

will be

presented

in the results section.

Introgression experiments

We tried to

_introgress

the

_high

tolerance of D sechellia into D simulans

_by

submitting

BCsim flies and further

_generations

to

_selection,

either with natural

morinda or with octanoic acid. Such

_experiments

were difficult because of the _very

low

_percentage

of fertile males in the BCsim

_generation

_(Lachaise

et

_al,

_1986).

With natural

_morinda,

a

_huge

mixed

_population

was established in a

population

room at

25°C,

and observations were made after 4 months and about 8

_generations.

With

octanoic

_acid,

selection was

applied

on adult flies and

_surviving

adults were used

to

_produce

the next

_generation.

More details will be

_given

in the results section.

RESULTS

Comparison

of acid

_toxicity

in

_parent

_species,

FI and backcrosses To

_analyze

and _compare acid

_toxicity

on the five

_genotypes,

data of females and

males were

_pooled

and a

_single

LD50 calculated in each

_experiment.

For some

genotypes,

however,

slight

but

_significant

differences were observed between sexes

and this effect will be considered in the next section.

Average

LD50 are illustrated in

_figure

2. D sechellia was

_highly

tolerant to both acids with an LD50 of 13.06 f 0.10 and 11.75 ! 0.37

_R

_L

for C6 and

_{C8, respectively.}

D simulans was

found,

on the other

hand,

to be very sensitive

(LD50

of 4.20 t 0.09

and 1.42 ± 0.08

_vL

for C6 and

_C8).

This

_major

difference is similar to that found with natural morinda

_(R’Kha

et

_al,

_1991).

Fl

_hybrid

flies exhibited a tolerance intermediate between the

_parents

with an LD50 of 9.06 ! 0.28 and 6.45 ! 0.26

!L

for C6 and

C8, respectively.

These values were not

_{statistically}

different from the

_mid-parent

_{(8.63 !}

0.07 for C6 and 6.58 ! 0.19

_!L

for

_C8).

As

_expected,

backcrosses towards

_{parental species}

increased or decreased the

LD50. With D sechellia values of 11.63 ! 0.20

_R

_L

for C6 and 8.11 ! 0.34

_vL

for C8 were obtained.

_By

contrast tolerance in the simulans backcross was much lower:

5.16 ! 0.13

_fiL

for C6 and 3.37 ! 0.22

_vL

for C8.

For each

acid,

all differences between

genotypes

are

_significant.

_Also,

a

_general

tendency

shows the C8 to be more toxic than the C6 under our

_experimental

conditions,

but the difference varies

_according

to the

_genotype.

In D simulans the difference between acids is

_{highly significant}

_(t

=

23.09,

P _<

0.001)

with a ratio

(C6/C8)

of LD50s

_equal

to 2.97. In D

_sechellia,

the difference is still

_significant

(t

=

2.93,

P =

0.015)

but the ratio is much less

(1.11).

Hybrid

_genotypes

(Fl

and

_backcrosses)

exhibit intermediate ratios which are however more similar to

Differences between sexes

For each

_experiment,

LD50s were calculated

_separately

for males and

_females,

and

average data are

_presented

in table I. In all _cases, females

_proved

to be more tolerant than

_males,

and this

_major

sex effect was evidenced

by

Anova

(table II).

For the

two

_acids,

a

_significant

sex

_by

_genotype

interaction was also observed.

_Looking

at

table

_I,

we see that for

_{C8, only}

a

_single

_difference,

between Fl female and

male,

is

_significant.

For the

_C6,

on the other

hand, significant

differences are observed in

Introgression experiments

A first

_experiment

was carried out with natural morinda. Because of the behavioral attraction of D sechellia adults to morinda and of the

_repulsion

of D

_simulans,

we found it

_possible

to have the two

_{species coexisting}

on two different _resources, banana and

morinda,

in the same

_population

room at 25°C. Resources were

put

in open

jars,

seeded with live

yeast

and set on two different

_tables,

_{approximately}

2 m

apart.

D simulans colonized

exclusively

the banana while D sechellia remained over

the morinda. This coexistence lasted for more than 6 months and

_during

that

_time,

only

two

_hybrid

males were found _{among several hundreds examined.}

_Finally

it

was decided to _suppress banana while the D simulans adults remained in the room.

The

_only

available resource was morinda seeded with live

_yeast.

This

_proved

to be

insufficiently

toxic to kill the D simulans

_population

so that their larvae

developed

on this rotten resource.

Also,

adults of the two

_species

started to mate on the

resource and to

_{produce hybrid}

_progeny. After 6 weeks

_(about

three

_generations)

most of the males could be classified as

introgressed hybrids by

examination of

their

_genitalia.

_{Progressively,}

the

_proportion

of

_hybrid

male

_phenotypes

decreased and a return to one

_{parental species}

was observed. From an

_{evolutionary point}

of

_view,

it could be

_expected

that,

because we were

_using

morinda as the

_unique

resource,

genotypes

of D sechellia would be

_favored,

sensitive D simulans _genes would be eliminated and a return to _pure D sechellia would be observed. In

_practice

the

_{hybrid population}

returned

_{progressively}

to a _{pure D} simulans

_phenotype.

D simulans is known to have a

_{major competitive}

_advantage

over D sechellia

because of its

_higher

egg

production

(R’Kha

et

_al,

_1997).

This

_advantage

_apparently

overcame the

_{probable opposite}

selective _pressure

_{imposed by}

morinda. It was also

supposed that,

because

hybrid

flies were observed for several

months,

the morinda

selection

_{might produce}

resistant D simulans. This D simulans

_population

was

_kept

in

_laboratory

bottles as a mass culture. After a few

_generations,

its tolerance to

octanoic acid was measured and the LD50 was 1.61

(confidence

interval:

1.51-1.71),

practically

identical to that observed _{in pure} D simulans.

The fact that D simulans is

_extremely

sensitive to octanoic acid while D sechellia

is about

_eight

times more tolerant offers favorable conditions for artificial selection.

Starting

from BCsim

flies,

adults were

_exposed

to octanoic

_acid,

the survivors were

used to

_produce

the next

_generation,

which was

_again

selected. Results are

_given

Previous studies

_{(table I)}

indicated

_that,

for BCsim

_females,

the LD50 was

3.5

_!L.

In the first

generation,

selection was

_applied

to females

_only.

All males were

kept

since the

_proportion

of fertile individuals in that

_generation

is _very low. A dose of 4

_!LL

was used and 73

_surviving

females out of 229 were selected to

_produce

the

next

_generation.

The _average survival time of the

reproductive

females was 25.8 h. These females were mated to unselected males of the same

_generation.

In the next

generation,

females

_only

were

again selected,

but with a

_higher

concentration of

5

_V

_L.

_Only

18%

of the females were

kept

with a survival time of 21.1 h. At the

third

_generation,

both sexes could be selected with the same dose. Less than

20%

of adults were

kept

and the survival time was close to 20 h. The

procedure

was

repeated

in G4 and G5

_(see

table

_III)

without _any indication of a better survival.

In G6 the

_selecting

dose was decreased to 4

_[

_tL.

Survival time in females was

_longer

than in Gl

_(36.1

_against

25.8

_h),

but the selection _pressure was

_stronger

(23%

surviving

versus

32%).

No

_significant

_tendency

of an increased tolerance was found.

In the G7

_generation,

the LD50 was

_precisely

measured

_using

several doses. The

observed values were 2.08 and 1.77

_!tL

for females and males

_{respectively.}

These

values are much lower than those observed in the first backcross

_generation

and close

to the values found in pure D simulans. In

spite

of the

strong

directional selection

applied

for several successive

_generations,

the _average tolerance to octanoic acid did not increase but

_{significantly}

_{decreased, regressing}

to the low values

_typical

of D simulans.

DISCUSSION AND CONCLUSION

Contrary

to

_previous

observations

_(Farine

et

_al,

_1996),

we found that hexanoic acid

appear to be involved in the

high

toxicity

of the morinda for all

Drosophila species

tested so

_far,

_except

D sechellia. The

_discrepancy

between our data and those of

Farine et al

₍₁₉₉₆₎

is

_probably

due to technical differences: we counted dead flies

after 2

_days

of treatment instead of less than 1

_h,

and we used

_{large plastic}

vials

instead of small Petri dishes.

On _average, hexanoic acid

_{appeared slightly}

less toxic than octanoic

_acid,

although

the

_{physiological}

basis of that difference is not known. It

_might

be due to

differences in water

_solubility

or in vapor pressure, or in the

_sensitivity

of the

biological

target.

Interestingly

the difference varied

_according

to

_genotype

and

species.

C8 is three times more toxic than C6 for D

simulans,

while the difference

is almost nil in D sechellia.

_Hybrid

_genotypes

in this

_respect

are more similar to

the D sechellia

_parent.

For each

_acid,

tolerance varies

_mainly

in an additive _way

(fig 2),

Fl individuals are close to the

_mid-parent

value and backcrosses are intermediate between Fl

and

_parent.

This conclusion contrasts with

_previous

results obtained with natural morinda

_(R’Kha

et

_al,

_1991),

which showed an almost

complete

dominance of the

high

tolerance found in D sechellia. This

_discrepancy

does not reflect different

genetic

mechanisms

but,

more

_probably

_again,

a technical difference. With morinda

it was difficult to

_manipulate

the

_quantity

of toxin

_and,

in

_fact,

a

_large

amount was

used. This

_quantity

was insufficient to kill D sechellia or F1

_hybrids.

A similar observation may be drawn from

figure

1. A dose of 6

R

L

is sufficient to kill almost

100%

of D simulans while in D sechellia and

_{F1, mortality}

remains below

20%.

Results illustrated in

_figure

2

_suggest

an additive inheritance but do not allow

precise

inference on the number of loci involved in morinda

_tolerance,

and for that

goal,

specific

markers should be used. We tried to check the

possible

occurrence of

tolerance _genes on the X-chromosome but the results are difficult to

_interpret.

Because of the direction of the

parental

cross

(female

D simulans with male

D

_sechellia)

_any tolerance carried

_by

the X-chromosome should be

_expressed

in Fl females but not in males. A

_significant

difference was indeed observed between sexes

in the

_F1,

females

_being

more tolerant than males.

However,

the difference almost

disappeared

in the backcross

_{generations. Moreover,}

a

_{general tendency}

seems to

exist for females to be more tolerant than

males,

and the small sex

by

_genotype

interaction is difficult to

_{explain by}

_specific

_genes on the X-chromosome. We _may

conclude

_that,

even if small effects of the X-chromosome are

_possible,

the

_major

difference between the two

_species

is autosomal.

Among

backcross progeny, a small

_percentage

of males are fertile

(Lachaise

et

_al,

1986)

and it is thus

_possible

to breed successive

_generations

without further

back-crosses. With this

_procedure,

male

_fertility

is selected for and

progressively

restored

to a value

_approaching

100%.

This

_phenomenon,

which was

_analyzed

in D simulans x D mauritiana

_hybrids

_(David

et

_al,

₁₉₇₆₎

also occurs between D simulans and D sechellia

_(unpublished

_{observations).}

_{Interestingly}

the

_{morphological}

traits also

change

and

_{progressively}

return to the

_phenotype

characteristic of the _pure

_species.

More

_precisely,

if the backcross of the Fl females is made with D simulans

_males,

the

_population

will

_{progressively}

return to a _{pure D} si!!lans

_phenotype,

while a

backcross with D sechellia will lead to a return to D sechellia. Such a

_progressive

evolution is easy to observe

by

studying

male

_genitalia,

which are _very different

Lemeunier et

_al,

_1986).

In the backcross

_generation,

a broad

_variability

is observed

(Coyne

and

_{Kreitman, 1986;}

Lemeunier et

_al,

₁₉₈₆₎

with numerous intermediate

phenotypes.

These

intermediate, ie,

introgressed,

genotypes

progressively

disappear

over

_generations,

and this was observed in our

_population

room in which a

_hybrid

swarm was

progressively

replaced

by

a _pure D simulans.

Interestingly,

the tolerance

to

_aliphatic

acids returned to the low level

_typical

of D

simulans,

in

_spite

of the

probable

selection

imposed

by

natural morinda.

In the case of the three

_{Drosophila species}

belonging

to the D simulans

complex

(including

D mauritiana and D

_sechellia),

the

_{introgression}

of

_single

visible recessive

markers is

_{generally possible}

without

_special

trouble

_{(personal observations).}

We

hoped that,

if the tolerance to

_aliphatic

acids in D sechellia was due to a

_single

major

gene,

responsible

for the

higher

tolerance in Fl

flies,

this allele could be

introgressed

into D simulans

_by

our selection

procedure.

This was

obviously

not the

case,

suggesting that, again,

the tolerance in D sechellia has a

_polygenic

basis. Two

kinds of

_hypotheses

may be considered for

explaining

the failure to

introgress

this

polygenic

trait. A first

_explanation

is a stochastic loss related to a small

population

size and also to the small number of chromosomes in

_Drosophila

_(Hospital

et

_al,

1992).

A second

_{interpretation}

is that several alleles of minor

effects, dispersed

over the _genome, were

_actively

counter

_selected,

for

_example

if

_they

were linked to

sterility

genes. In

Drosophila,

male

sterility

genes, revealed in

interspecific

crosses are known to be

widespread

over the _genome

(Coyne

et

al, 1991;

Cabot et

al,

1994;

Davis and

_Wu,

_1996).

_Data,

similar to our

_observation,

_ie,

_difficulty

or

_{impossibility}

to

_introgress

as

heterospecific

_genome, have been

recently

described in Helianthus

(Rieseberg

et

_{al, 1995a, b,}

₁₉₉₆₎

and in

_Anopheles

_(Della

Torre et

_al,

_1997).

The barrier to

_{introgression,}

which _may be observed in

_{laboratory experiments,}

reminds

us of

_hybrid

zones observed _among natural

_populations

of various

_species

(Barton

and

_Hewitt,

_1985;

_Hewitt,

_1989).

The number of loci that are modified

during

the

_speciation

_{process remains} a

subject

of debate

_{(Coyne, 1992).}

In the case of D sechellia

_adapting

to

_morinda,

it seemed a

_priori

more

_{probable that, during}

a

_relatively

short

evolutionary

time,

a

_single

_gene underwent a

_major

mutation

_responsible

for the tolerance. Our data

strongly

contradict this idea and favor the occurrence of several _genes.

_However,

a

difficulty

remains. If

_polygenic

_systems

were selected in D

_{sechellia, why}

was that

impossible

in D

_simulans;

and also

_why,

up to _{now, is} D sechellia the

_only

_species

tolerating

hexanoic and octanoic acid ? ACKNOWLEDGMENTS

We thank S R’Kha for her

participation

in the

population

room _experiment, JC Moreteau for

providing

the statistical program used for

calculating

LD50, and F Hospital for

drawing

our attention to the Helianthus

_{introgression experiments.}

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