Body size and developmental temperature in Drosophila simulans: comparison of reaction norms with sympatric Drosophila melanogaster

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Body size and developmental temperature in Drosophila

simulans: comparison of reaction norms with sympatric

Drosophila melanogaster

Jp Morin, B Moreteau, G Pétavy, Ag Imasheva, Jr David

To cite this version:

Original

article

Body

size

and

_{developmental}

temperature

in

_Drosophila

simulans:

comparison

of reaction

norms

with

sympatric

Drosophila

melanogaster

JP Morin

B Moreteau

G

_Pétavy

AG

Imasheva

2

JR

David

1

Laboratoire de

_{populations, genetique}

et

_evolution,

CNRS,

91198

Gif sur-Yvette cedex, Prance;

2

L!avilov Institute

_of

General

_Genetics,

3 Gubkin

_Street,

117809

_Moscow,

Russia

(Received

10 November

1995; accepted

7

_July

₁₉₉₆₎

Summary - Reaction norms of two size-related traits

_(wing

and thorax

_length)

were

analyzed

in relation to

_growth

_temperature in a French natural

population

of

Drosophila

simulans, using

the isofemale lines method. The

_wing/thorax

ratio was also studied.

Data were

compared

to those of the

_{sibling species Drosophila melanogaster}

from the

same

_locality.

Flies were reared at seven constant temperatures,

representing

the whole thermal range of the two

species. Phenotypic

and

_genetic

variabilities were

analyzed.

For

investigating

the

shape

of the response curves

_(ie,

reaction

_norms)

two methods were used:

analysis

of

slope

variations and

_{polynomial adjustments.}

As

expected

from the relatedness of the two

species,

many similarities were observed.

Notably,

the reaction norms of

_wing

and thorax

_lengths

exhibited a maximum at low _temperature, while the

_wing/thorax

ratio

was a

_{regularly decreasing sigmoid}

curve. Numerous and sometimes _great differences were also observed. At the

_{phenotypic level,}

D simulans was

_generally

more

_variable,

while at

the

_{genetic level,}

it was less variable than D

_{melanogaster.}

Isofemale line heritabilities

varied

_according

to

_growth

_temperature, but with different _patterns in the two

_species.

In both

_species,

sexual

_dimorphism

increased with _temperature, but the average values

and the response curves were different. The reaction norms of

_wing

and thorax

_lengths

were

mainly

characterized

by

different TMSs

(temperatures

of maximum

size)

with lower

values in D simulans. This

_species

was also characterized

by

a much lower

wing/thorax

ratio with a

higher

TIP

(temperature

of inflexion

point).

The

possible adaptive significance

of these variations remains unclear.

Indeed,

TMS variations _suggest that D simulans could be more tolerant to cold than its

_sibling.

On the other

hand,

the lower

_wing/thorax

ratio of D simulans suggests a

warm-adapted species.

Résumé - Taille corporelle et _température de développement chez Drosophila simu-lans : comparaison des normes de réaction avec l’espèce sympatrique Drosophila

melanogaster. Les normes de réaction de la taille du corps

(aile

et

thorax)

et du rapport

ailé/thorax

ont été

analysées

en

_fonction

de la

_température

de

développement

par la

méthode des

_{lignées isofemelles.}

Deux

_populations

naturelles

_{sympatriques françaises}

des

espèces

sceurs

Drosophila

simulans et

_{Drosophila melanogaster}

ont été

comparées.

Les

drosophiles

ont été élevées à sept

températures

constantes

_comprises

entre 12 et 31

°C,

ce

qui recouvre l’ensemble de la _gamme des

températures possibles

pour ces deux

espèces.

La variabilité

phénotypique

entre les individus d’une même

_lignée

a été

_analysée

en

utilisant les

_coefficients

de variation, et la variabilité

_génétique

en utilisant les

_coefficients

de corrélation intraclasse. La

_forme

des courbes de

réponse

(ie,

normes de

réaction)

a été

_analysée

_par deux méthodes : la variation des _pentes et les

ajustements

polyno-miaux. En accord avec la

parenté

des dézix

espèces,

de nombreuses similitudes ont été observées. En

particulier

les normes de réaction de l’aile et du thorax

présentent

un

maximum à basse

température,

tandis _que le _rapport

_aile/thorax

est une courbe

_sigmoïde

décroissante. De nombreuses

différences

ont aussi été

_{observées, parfois}

très importantes. Au niveau

_{phénotypique,}

D simulans est

_{généralement plus}

variable que D

_{melanogaster,}

tandis

qu’au

niveau

_génétique

elle s’est avérée en

_général

moins variable. L’héritabilité

varie avec la

température,

mais avec des modalités

_différentes

dans

chaque espèce.

Dans les deux

espèces,

le

_dimorphisme

sexuel

_(évalué

par le rapport

femelle/mâle)

augmente

avec la

_{température,}

mais les valeurs et les courbes de

réponse

sont

_{différentes.}

Les

normes de réaction de l’aile et du thorax sont

_{principalement différenciées}

par les TTMs

(températures

de taille

_maximale),

avec des valeurs

plus

basses chez D simulans. Cette

espèce

est

_également

caractérisée par un rapport

aile/thorax

inférieur

avec une TPI

(température

de

_point

_{d’inflexion)}

plus

élevée. Ces

_différences

sont

_difficiles

à

interpréter.

En

_effet,

les variations de TTMs

_suggèrent

_que D simulans pourrait être

plus

résistante

au

_froid

que D

_{melanogaster ;}

en revanche le rapport

ailé/thorax

plus faible

de D simulans

suggère

une

_adaptation

à la chaleur.

plasticité phénotypique

/

lignée isofemelle

/

taille de l’aile

_/

taille du thorax

_/

rapport

aile/thorax

INTRODUCTION

Body

size,

which exhibits

_huge

variations among

living

organisms,

has

_long

exerted

a kind of fascination _upon

biologists.

Size variations influence numerous

biological

traits,

such as basal

_metabolism,

duration of

development

or _age at

_maturity

_(Reiss,

1989; Stearns, 1992; Charnov,

1993).

Reciprocally,

size is a

_target

for natural selection and varies as a consequence of environmental pressures. For

example,

the old

_Bergman’s

rule

_describes,

in numerous homeotherm

_species,

an increase of size related to a colder environment.

Finally

size exhibits

large

variations between

individuals of the same

population,

not

_only

due to

_genetic

differences but also due to

_{phenotypic plasticity,}

related to different environmental conditions

_during

development.

In

_Drosophila,

allometric

_{relationships}

are not well

documented,

although

im-portant

size variations exist between

_species

_{(Ashburner, 1989).}

Several

species

including Drosophila melanogaster

and

_Drosophila

simulans exhibit

_genetic

latitu-dinal clines with a

_larger

size under colder climate

_(David

et

_al,

1983;

Capy

et

al,

1993),

these clines

_{presumably being}

linked to

_temperature.

_Laboratory

_experiments

genetic

size variations over

_{time, ie,}

smaller flies at

_high

_temperatures

and

_bigger

ones at low

_temperatures

_(Powell,

_1974;

Cavicchi et

_al,

_1985;

_Partridge

et

_al,

_1994).

These observations remind one of

_{Bergman’s rule,}

_{although Drosophila}

is an

ecto-therm so that we do not know

_why

it should be better to be

_larger

in a colder climate

_(David

et

_{al, 1994;}

_Partridge

et

_al,

_1994).

In natural

_populations,

adult size exhibits a

_{huge variability, presumably}

related

to variations in

_feeding

and thermal conditions

_(Atkinson,

1979;

David et

_al,

1980, 1983;

Coyne

and

_{Beecham, 1987;}

Imasheva et

_al,

_1994;

_Partridge

et

_al,

1994;

Moreteau et

_al,

_1995).

This

_{phenotypic plasticity}

cannot be considered as

completely

neutral. For

_example,

a

_positive

_phenotypic

correlation exists between size and fitness in nature

_(Boul6treau,

_{1978; Partridge}

et

_al,

_1987).

_Moreover,

_Coyne

and Beecham

₍₁₉₈₇₎

demonstrated that size variations were to some extent heritable in

_spite

of a

_large

environmental

_component

due to

_plasticity.

_However,

a

_positive

phenotypic

correlation between

_body

size and adult fitness

components,

together

with the existence of additive

genetic

variance for

_body

_size,

does not

_necessarily

lead to the conclusion that

body

size is the

target

of selection

_(Rausher,

_1992).

Up

to now,

quantitative genetic

variations _among natural

_{populations, including}

latitudinal

_clines,

have

_generally

been

_investigated

at a

single

_temperature

(with

the

_exception

of

_Coyne

and

_Beecham,

_1987),

most often 25 °C

_(David

et

_{al, 1983;}

David and

_Capy,

_1988;

_Capy

et

_al,

_1993).

On the other

_hand,

natural

_selection,

which is

_presumed

to be

_responsible

for the

_clines,

acts at various

_temperatures

in different localities

_and,

in all _{cases, upon}

_highly

variable

_phenotypes.

_Moreover,

temperature

is the most

_important

abiotic factor

_explaining

_geographic

distribution and abundance of

_species

in

_Drosophila

_(David

et

_{al, 1983;}

_Parsons,

_1983;

Hoffmann and

Parsons,

1991).

Thus,

for a better

_{understanding}

of these

_problems,

several

temperatures

must be

_investigated

and

_compared.

In other

words,

we have to

investigate

the

_relationship

between

_{developmental}

_temperature

and

_phenotypes,

ie,

the reaction norms of various traits.

Generally,

authors who were interested in the

_genetics

and evolution of reac-tion norms

_only

considered two environments and

_consequently

linear norms

(Via

and

Lande, 1985, 1987;

Scheiner and

Lyman, 1989,

1991;

De

_{Jong, 1990; Scheiner,}

1993a; Via,

1993).

Gavrilets and Scheiner

₍₁₉₉₃₎

_underlined,

_however,

the

neces-sity

of

_studying

nonlinear norms and

_proposed

to model them

_using

_polynomial

adjustments. Indeed,

when a broad range of environments

(eg,

temperature)

is

in-vestigated,

norms of

_quantitative

traits are as a rule nonlinear

_(David

et

_{al, 1983,}

1990, 1994; Delpuech

et

_al,

_1995).

A recent

_controversy

has

_developed

_concerning

the

_genetics

of

_plasticity.

Various authors have considered that the mean value of a trait and the

shape

of the reaction norm should be

_{distinguished.}

In other

_words,

_genes

_regulating

the

_position

of the curve

(trait

mean value

_genes)

and _genes

_{regulating plasticity}

_(shape

genes)

_might

coexist

_(Bradshaw,

_1965;

Scheiner and

_Lyman,

_{1989, 1991;}

Scheiner et

_{al, 1991;}

Weber and

_Scheiner,

_1992;

_Scheiner,

_1993ab;

Gavrilets and

_Scheiner,

_1993).

But this

_conception

was criticized

_by

Via

(1993, 1994)

who considered it an _unnecessary

complication,

and recent papers have tried to reconcile these two

_approaches

_(Van

Tienderen and

_Koelewijn,

1994;

Via et

_al,

_1995).

significance

of the norm? Is it a _consequence of internal constraints or is it

_adaptive,

ie,

shaped by

natural selection?

It is

_{generally recognized}

_that,

before

_developing

a

_theory

on the evolution of

reaction norms, many more

_empirical

data are

needed,

_relating

the norms with

ecological

adaptations

and life

_history

parameters.

In this

respect,

it will be easier

to _compare different

_species

_(Harvey

and

_Pagel,

₁₉₉₁₎

since a

_larger

evolutionary

time should have

_permitted

a broader

_divergence

of the _norms,

_especially

if

_they

were

shaped by

natural selection. In this _paper, we

investigated

the reaction norms of size traits of a natural

_population

of D simulans from

_France,

and

_compared

the results with those obtained for the

_sibling

D

_melanogaster

from the same

locality

(David

et

_al,

_1994).

We found similarities between the two

_species

_but,

more

interestingly,

numerous

_significant

differences. These differences demonstrate

_that,

within a

_relatively

short

evolutionary

time

(about

2 million

years)

reaction norms have

_diverged.

The

_{possible adaptive}

_significance

of these variations is discussed.

MATERIALS AND METHODS

A D simulans

_population

was collected in a

_vineyard

in Pont de la

_Maye

near Bordeaux

_{(southern France).}

_Variability

of size

_according

to

_temperature

was

analyzed,

and

compared

to a

_population

of D

melanogaster

collected in the same

locality

and

_previously

studied

_(David

et

_al,

_1994).

The isofemale lines method was used. Wild

living

females were collected with banana

_traps

and used to establish 20 isofemale

_lines,

and ten of them were then

randomly

chosen. For

each,

ten

_pairs

of the first

_laboratory

generation

were used as

_parents.

_{They oviposited}

at room

_temperature

(20 ! 2 °C)

for about half a

_day.

A rich

_feeding

_medium,

based on killed

_yeast,

was used for the

_development

_(David

and

Clavel,

1965).

Such a food

_prevents

crowding

effects which could affect

fly

size.

_{Density ranged}

between 100 _{and 200 eggs per} vial. Vials with _eggs were then transferred to one of seven

experimental

constant

_temperatures

_(12,

_{14, 17,}

_21,

_25,

28,

31 °C).

Measured flies thus

_correspond

to the second

_{laboratory generation.}

Such a

_procedure

is a

_necessity

for

_{obtaining enough offspring}

(see

Moreteau et

_al,

1995 for

_discussion).

It also eliminates

possible

maternal effects and

provides

Hardy-Weinberg proportions

within lines. ’

From each line at each

_temperature,

ten females and ten males were

randomly

taken. Their

_wing

and thorax

_lengths

were measured with a micrometer in a binocular

microscope.

Total

_wing

_length

was measured from the articulation on

the side of the thorax to the distal

_tip.

Thorax was measured on a left side

_view,

from the base of the neck to the

_tip

of the scutellum.

_Analyses

were made

_directly

on measurements

_expressed

in mm x

_100,

since a

_preliminary

_analysis

with

log-transformed data failed to show any

scaling

effect.

Statistical

_analyses

and

_{orthogonal polynomial adjustments}

were made with

RESULTS

Variation of

_wing

and thorax

_length:

mean of the ten lines Reaction norms

The _response curves

_(fig

₁₎

show that females are

_larger

than males in both

species

and that D

_melanogaster

is

_larger

than D simulans. In both

_species,

a maximum seems to exist at a low

_temperature.

A

_steep

decrease from this maximum is observed when

_temperature

_increases,

and a shorter one when

_temperature

decreases. In both

_{species, significant}

differences exist between the reaction norms

of

_wing

and thorax.

_Finally

D simulans seems to exhibit its maxima for both traits

at lower

_temperatures

than D

_{melanogaster.}

This

problem

will be

analyzed

further.

Sources of variation

Variations were

_investigated

_{simultaneously}

on the two traits in D sim!alans with MANOVA

_{(table I).}

Sex and

temperature

are the main sources of variation. A

_highly

significant

line effect demonstrates their

_genetic

_{heterogeneity.}

The

temperature-line

interaction,

also

_{highly significant,}

shows that the reaction norms of the

differ-ent lines are not

_parallel

but exhibit different

_{shapes. Finally}

the

sex-temperature

Correlation between sexes and sexual

_dimorphism

Male-female correlations were

analyzed considering

the mean values of each line

(table II).

There was no

_temperature

effect on the coefficients of correlation

(ANOVA,

not

_shown).

_Average

correlation is

_{significantly}

lower for

_wing

in D sim!lans

_{(0.66 !}

0.07 versus 0.91 t 0.05 in D

melanogaster),

but similar for thorax in both

_species

_{(0.71 !}

0.06 and 0.76 !

_0.16).

Sexual

_dimorphism

was calculated at each

_temperature

and for each line as the

female/male

ratio,

and submitted to ANOVA

_{(not shown).}

For

_wing

and

_thorax,

only

the

_temperature

effect was

_significant

while the line effect was also

_highly

significant

in D

_{melanogaster.}

A nested ANOVA

_including

the two

_species

_(not

shown)

demonstrated

_{highly significant}

_species

differences. The two traits

_(wing

and

_thorax)

_provide

the same information. In the two

_species,

the two sexes are more similar when reared at low

_temperature

_{(temperature effect).}

The

_female/male

ratio of D simulans is characterized

_by

lower values than in D

_melanogaster

_(species

effect,

see David et

_al,

₁₉₉₄₎

and

_by

a decrease between 28 and 31 °C

Covariation between

_wing

and

_thorax;

the

_wing/thorax

ratio

Wing—thorax

correlation

The

_wing-thorax

correlation was

_investigated

at the individual

₍₌

within

_lines)

and at the line

₍₌

between line

_means)

levels

_(table

_III).

At the individual

level,

the values did _{not vary}

_{significantly}

with

_temperature;

the _average

_phenotypic

correlations were 0.71 for females and 0.77 for males and were similar to those obtained in D

_melanogaster

_(David

et

_al,

_1994).

For the

_lines,

_average values were

superior

in males

_(0.79

versus

0.66)

but not

_{significantly}

so

(t

_test,

not

_shown).

In D

_{!rcelanogaster,}

values were

_quite

similar: 0.73 in males and 0.78 in females.

Wing/thorax

ratio

Average

curves

(fig 2)

have a

_general

_{decreasing sigmoid shape}

in the two

_species,

but values are much lower in D simulans.

Statistical

_analyses

_(ANOVA,

not

_shown)

demonstrated

_highly

_significant

effects of

temperature

(which

explains

87%

of total

_variation)

and lines. Two-factor interactions were

_significant

as was the

_{triple-factor}

one. Similar conclusions were obtained in D

_melanogaster

_(David

et

_al,

_1994).

On the other

_hand,

the sex effect was not

_significant,

and sexual

_dimorphism

was _very reduced for the ratio in both

species

(see

fig

2).

Phenotypic

and

_{genetic variability}

Within-line

_variability

two

_species

concerned the levels of

_variability.

Values were

_higher

in D simulans at

high

temperatures

for the

_wing

_{(25-31 °C)}

and the

_wing/thorax

ratio

_{(21-31 °C),}

and for the thorax over the whole

_temperature

_range. Mean values for the seven

temperatures

are,

respectively

for

_{wing, thorax,}

and

_wing/thorax

ratio

_{2.16 ! 0.18,}

2.40 !

0.21,

1.58 ! 0.15 in D

_simulans,

and 1.97 !

_0.17,

1.96 t

0.21,

1.40 t 0.15 in D

_{melanogaster.}

Between-line

_variability

The between-line variance was

_{analyzed by calculating}

the coefficient of intraclass correlation

t,

for each sex at each

temperature,

which is an indicator of isofemale line

_heritability

_(Hoffmann

and

_Parsons,

_1988).

Values of t for

_wing

and thorax

are

_given

in table IV. For

_wing

length,

a marked

_species

effect is

observed,

with

These results are illustrated in

_figure

3 as a correlation between male and female

t values. In D

_simulans,

t values for the two traits can be divided into two _groups:

high

values

₍₌

_higher

_{heritability)}

are observed at medium

temperatures

(21,

25,

28 °C)

and low values at extreme

_temperatures

_(12,

_14,

_{31 °C).}

Means of these two groups are 0.34 ! 0.03 and 0.12 ! 0.02

_respectively

and

_{statistically}

different

(Student’s

test,

not

_shown).

In D

_{melanogaster,}

no

_temperature

effect was observed for the

_wing,

but a difference between

_high

and low

_temperatures

was observed for the

_thorax,

with a

_higher

_genetic

_variability

at

_high

_{temperatures.}

Analysis

of the

_shape

of reaction norms:

_slope

variations and derivative curves

Wing

and thorax

For each isofemale

_line,

_length

variation for a

_given

_temperature

interval allows the calculation of a

slope

(ie,

length

variation _per

degree),

by

a linear

intrapolation.

Repeating

this process for successive intervals

produces

an

_empirical

derivative of

the reaction norm.

An ANOVA

_(not

_shown)

was conducted on the

slopes

in D simulans. Results were similar for

_wing

and thorax with a _very

_significant

_temperature

_effect,

demon-strating

nonlinear norms.

_Contrarily

to D

_{melanogaster,}

there was no

_significant

sex effect. No line effect was

_detected,

as in the

_sibling

_species.

In the two

_species

a

clear

_{line-temperature}

interaction shows that derivative curves have different

_shapes

among lines.

Finally,

a

_{highly significant}

_{sex-temperature}

interaction is

present,

which was not found in D

_{melanogaster.}

Average

curves and

_single

line curves are

_given

in

_figure

_4,

for

_wing

in females

only.

In the two

_species,

_average curves

(fig 4a)

show a

_progressive

decrease from

positive

to

_negative

values. These values are

_{significantly}

lower at low

_temperature

in D simulans and not

_{significantly}

_greater

than zero. This means that the

point

where this derivative curve crosses the null

_line,

which

_corresponds

to the

temperature

of maximum size

_(TMS),

is far less obvious in D simulans than in D

_{melanogaster, especially}

for the thorax

_(see

also

_fig

_1).

This observation is confirmed

_by

the examination of the curves of different lines

(fig 4b).

Indeed in D

_{simulans, wing}

_length

never reached the zero value in two

_lines,

and for thorax

length

(not

shown)

the

slope

often crossed the null line several times. Hence in D

simulans,

a TMS can be calculated

_{by using}

the _{average curves,} but not for each isofemale line.

_Average

curves

_point

TMS values at 13.5 °C for

_wing

and at 16 °C for thorax in D

_simulans,

and at 16 and 19 °C

_respectively

in D

_{melanogaster.}

In other words TMS values appear to be lower in D simulans than in D

melanogaster.

For

_comparing

the two

_traits,

_slopes

were standardized and

_expressed

as a

percentage

of the mean

(curves

not

_shown).

With such a transformation

(David

et

_al,

_1994),

the

amplitudes

of variation for the two traits become similar. In D

_melanogaster

the variation _range was

_greater:

the overall

_phenotypic

_plasticity

seems to be less

pronounced

in D simulans.

Wing/thorax

ratio

Slopes

of the

_wing/thorax

ratio were calculated in the same _way and an ANOVA

(not shown)

demonstrated a

_major

effect of

_temperature,

a low sex

_effect,

no line

effect but a

_{significant line-temperature}

interaction.

Average

slope

variations are illustrated in

figure

4c for females. In the two

_species,

average derivative curves are

_U-shaped

_indicating

that the maximum

_phenotypic

plasticity

occurs at intermediate

temperatures,

and also that the

_wing/thorax

ratio varies

_according

to a

decreasing sigmoid

curve

(see

fig

2).

A

_regular

feature in D

at extreme

_{temperatures,}

zero values

_correspond

to the fact that the curve of the

wing/thorax

ratio was horizontal

_(see

_fig

_2).

_Moreover,

the overall

_amplitude

of variation is

_larger

in D simulans.

Analysis

of the

_shape

of the reaction norms:

_polynomial

adjustments

Degree

of

_polynomial

_adjustments

After a theoretical

_study

of linear _norms, Gavrilets and Scheiner

(1993)

suggested

that nonlinear norms should be

_adjusted

to second

_degree

_polynomials,

_according

to the formula

_P(t)

=

go +

g

l

t

+

_g

₂

_t

₂

_(if

we are

_dealing

with

_temperature,

_P(t)

is the

_phenotype

value at

_temperature

_t).

The authors

_proposed

for _go, the

_intercept,

a

genetic

significance fixing

a basic value to the studied

_trait,

while _gl, the

_slope,

could be a

_genetic

_parameter

of

_adaptation

to the

_environment,

and g2 a

_genetic

parameter

of curvature. A second

_degree

_polynomial

_implies

that the derivative curve

(ie,

slope

variation)

is linear. Such was not the case for the three traits

(see

fig

4),

so that at least a third

_{degree adjustment}

should be used.

_Incomplete

polynomials

could also be

_used,

for instance with

no t

2

term. The

_validity

of the various

_adjustments

was assessed

_{by adjusted}

R

2

values,

a _poor

_{adjustment being}

characterized

by

a low

_adjusted

R

2

.

A third

_degree

equation

proved

to be convenient for the

_wing/thorax

ratio. For

_wing

and thorax

_{lengths, considering}

the similar

shapes

in the two

_species,

we

_imposed

a constraint on the

_adjustment,

_ie,

the existence of a

plausible

TMS calculated

by

solving

the

equation

P’(t)

= 0. For

third and fourth

_degrees,

two or three solutions were obtained

_{respectively,}

which needed to be checked to know which one

_corresponded

to the overall maximum.

Finally,

for overall

_homogeneity,

all the

_wing

and thorax curves were

_adjusted

to

fourth

_degree

_polynomials,

even those which were

_compatible

with third

_degree

polynomials.

Also,

similar

_adjustments

were made with the data of D

melanogaster

to _compare the two

_species.

Such

_adjustments

were not made in a

_previous

_paper

(David

et

_al,

_1994).

Wing

and thorax

Even with fourth

_degree

polynomials,

there were still some

inadequate

TMS

values,

for

_instance,

6.3 °C for a male

_wing.

This often occurred from an abnormal value at

a

single

_temperature

(=

_rearing

accident?)

which modified the

_adjustment

_equation

and thus the TMS. Such cases

_represented

six out of the 40

_adjustments

made on

D

_simulans,

but

_only

two of them

_(for

male

_thorax)

deviated from a reasonable

value.

Choosing

a fourth _power

_polynomial

leads to much more

_{heterogeneous g}

_i

parameters

than an

_adjustment

in

t

2

.

For

_instance,

for females

_wing

in D

_simulans,

the ten _go values

_ranged

from 62 to 69 with

the t

2

adjustment,

and from —79 to +93 with

the t

4

_adjustment.

A similar conclusion was obtained for all other

_parameters.

Fortunately,

calculation of critical

_points,

such as TMS

values,

provided

much less variable

_values,

thus

_{confirming previous}

observations on ovariole number

_(Delpuech

et

_al,

_1995).

females

_(ANOVA,

not

_shown).

In D simulans an

_overlap

of TMSs of the two traits was

_observed,

_contrarily

to D

_{melanogaster.}

Mean values are

_given

in table V and

compared

to those of D

_{melanogaster.}

In all cases, TMS values are

_{significantly}

higher

for the latter

_species.

Another

_striking

_species

difference is the

large dispersal

among lines of D simulans

contrasting

with a better

homogeneity

in D

_melanogaster

(see

CVs in table

_V).

_Finally,

in all cases, values of males and females of the same line were

_positively

_correlated,

_suggesting

that

_they

provide,

at least in

_part,

the same

_genetic

information.

As in David et al

_(1994),

values of both sexes were

_averaged

for each trait. A scatter

_plot

of

_wing

and thorax TMS values

_(fig

₅₎

_clearly

contrasted the two

species.

Interestingly,

a

_positive

correlation is found in D

_melanogaster

while a

non-significant

but

_negative

correlation is found for the

_eight

lines of D simulans

(excluding

two lines with aberrant TMS for male

_thorax).

The between-line

heterogeneity

seems to be

_mainly

due to thoracic variations.

Taking

all values into

_{consideration,}

_average curves were also

_adjusted

to the fourth

_degree

and gave TMS values of 13.5 °C

_(females)

and 12.4 °C

_(males)

for the

_wing,

and of 16.1 °C

_(females)

and 13.2 °C

_(males)

for the thorax. These values

are lower than in D

_melanogaster

_{(respectively}

_{15.6 and 14.8 °C for}

_wing,

_{19.2 and} 17.6 °C for

_thorax).

_They

are close to the mean values of the ten lines

_given

in table V and thus characterize the

_species.

_{Interestingly,}

the gi

parameters

of the average curves were similar to the mean values of

_{the g}

_i

of the ten lines.

Wing/thorax

ratio

The _gi

_parameters

of the third

_{degree polynomial}

were _very

_variable;

CVs

_ranged

between 16.5 and

40%

for the four female coefficients

_(mean

CV =

28%)

and

between 22 and

65%

_(mean

CV =

46%)

for males. Curves _were then characterized

by

their

_temperature

of inflexion

_point

_(TIP),

_ie,

the

_temperature

where the second derivative becomes null.

between 19.9 and 22.8 °C

_(mean:

21.1 ! 0.3

_°C)

in females and between 19.9 and 21.3 °C

_(mean:

20.6 ! 0.2

_°C)

in males. There was neither line nor sex effect

(ANOVA,

not

_shown).

In D

_{melanogaster,}

the same

_{adjustments produced}

far more

variable g

i

coeffi-cients: mean CV of

69%

in females and

92%

in

_{males, ie,}

more than twice as

_large

as in D simulans. This also resulted in a much

_greater

_dispersal

of the TIP values

of the different lines

_(see

_fig

_6).

Also the TIPs were on the _average

_{significantly}

lower

_(ANOVA,

not

_shown)

in D

_melanogaster

than in D simulans: 19.0 f 0.9 °C in females and 16.9 t 1.2 °C in males.

A final observation was that for a

_given

_temperature,

the ratio of the

DISCUSSION AND CONCLUSION

Our results need to be discussed from two different

_points

of view: a

_{methodological}

approach

for the

_description

of reaction _norms, and the

_comparative

_evolutionary

biology

of the two

_sibling

_species.

A

_major,

still unsolved

_problem,

will be to decide which

_species

is better

_adapted

to a warmer environment.

How should

_empirical

reaction norms be

investigated?

In

_Drosophila,

_genetic

_plasticity

of

_quantitative

traits such as

_wing

and thorax

length

was first

_investigated

over two environments

_(Scheiner

and

_Lyman,

_1989,

1991;

Scheiner et

_{al, 1991;}

Weber and

_Scheiner,

_1992;

_Scheiner,

_1993a)

and a linear model was used. When a broad _range of environmental conditions is

used,

as such

1994;

Gavrilets and

Scheiner,

1993)

and this raises a

major

problem:

what is the best _way to describe and

_analyse

the

_shape

of the curve? Factors of variation can be identified with ANOVA or

_MANOVA,

as well as numerous interactions which

_demonstrate,

for

_example,

that the norms

significantly

differ _among isofemale lines from the same

_population.

More

_precise

_analyses

are however needed for

describing

the norms, and two kinds of methods may be used:

slope

variations and mathematical

_adjustments.

Analysis

of

_slope

variations was used

_by

David et al

₍₁₉₉₀₎

for

_{demonstrating}

different

_pigmentation

norms in successive abdominal

_segments.

This method can be of

_general

use for

_comparing

different traits or

_species,

and

_significant

differences

may be

easily

demonstrated. Also the overall

_shape

of the reaction norm _may be inferred from the

_shape

of its derivative. In D

_simulans,

and

_contrarily

to

D

_melanogaster

_(David

et

_al,

_1994),

this method was not

_satisfactory

_(problems

in TMS values

_{determination)}

and the

_shapes

of the curves had to be studied

_by

mathematical

adjustments.

An

_adjustment

to a mathematical model should be a better method but numer-ous

_equations

could be chosen. In the

_present

case there was no a

_priori

reason for

guiding

the choice and thus we used a

general

method, ie,

a

polynomial

adjust-ment,

as

_{suggested by}

Gavrilets and Scheiner

(1993)

and Via et al

_(1995).

Because of the

_great

_variability

among the

polynomial

coefficients of various

lines,

it

ap-peared

difficult to

_give

them a

_genetic

_sense,

_contrarily

to what has been

_suggested

(Gavrilets

and

Scheiner,

1993).

These

parameters

are,

however, conveniently

used for

_calculating

critical

_points

of the curves,

especially

the

temperature

of maximum size

_(TMS)

for

_wing

and thorax

_lengths

or the

_temperature

of inflexion

_point

(TIP)

for the

_wing/thorax

ratio. Reaction norms _appear to be better characterized

_by

these

_points,

which are less variable and seem to have a

biological significance,

and

presumably

also a

_genetic

basis. In this

_respect,

we found that TMS values of males

and females of the same line were

_positively

correlated in both

_{species and,}

_among

lines,

thorax and

_wing

TMS values were also correlated in D

_melanogaster

(David

et

_al,

_1994).

_{Interestingly}

in D

_{melanogaster, calculating}

the TMS values either

_by

considering slope

variations or with

polynomial

adjustments

provided

similar re-sults. In D

_simulans,

fourth power

polynomials

had to be used instead of

_quadratic

ones for a better characterization of TMS values. But even in that case, the

adjust-ment could not be

_performed

for some isofemale lines. This _may reflect either true

genetic peculiarities

of these lines or some

_{experimental imprecisions.}

This

prob-lem needs further

_{investigation,}

for

_{example, by analyzing}

the same line over two

successive

_generations.

Similarities between the two

species

Similarities between

_closely

related

_species

are

expected

because of

phylogenetic

constraints and also from a

_{possible similarity}

of their

_ecological

niches

_(Harvey

and

_Pagel,

_1991).

In the

present

study,

numerous similarities were

_observed,

which are

_briefly

summarized below.

In the two

_species

females are

_larger

than

males,

and this could be a

_general

result

is a

_{phenotypically plastic}

trait with minimum values at low

_temperatures

in both

species.

Reaction norms of the three characters

(wing

and thorax

_length

and

wing/thorax

ratio)

are nonlinear and

present

the same sources of variation.

Wing

and thorax both exhibit a maximum at low

temperature.

The response of the

wing/thorax

ratio to

_temperature

is a

_{sigmoid decreasing}

_curve, similar for both sexes.

In all _cases, coefficients of intraclass correlation

_(t)

were

_{significantly}

_greater

than zero,

demonstrating

(Hoffmann

and

Parsons,

1988)

a

_{high heritability}

of the traits. Moreover a

_{regular line-temperature}

interaction indicates

significant

_genetic

variations in the

_shapes

of reaction norms

among

isofemale lines.

The within-line CVs varied with

_temperature

in all _cases, with maxima at

extreme

_{temperatures.}

This is

likely

due to an increase of the

developmental

noise under stressful conditions. In both

_species,

the

_wing/thorax

ratio is less variable

(lower

CVs)

than the traits themselves. This is due to the fact that

_wing

and thorax variations are correlated at the individual level.

Differences between the two

_sibling

_species

Numerous and

_important

differences were found between the two

_species.

These differences demonstrate that canalization

_{during development}

is not _very

_strong

so that the

investigated

traits could

diverge,

either as a _consequence of drift or of

ecological adaptation.

As

_already

known from numerous observations

(see

Capy

et

_al,

₁₉₉₃₎

D simulans is a smaller

_species.

We _{may argue} that

_speciation

was

_accompanied

_by

size _gene

variations,

determining

the

_position

of the reaction norms on the Y axis.

Sexual

_{dimorphism presented}

different reaction norms in the two

_species.

It is unfortunate that we do not have a convenient

evolutionary theory

for sexual

dimorphism

in

_organisms

like

_Drosophila

_{(Charnov, 1993).}

Heritability

of size traits was different in the two

_species,

_contrarily

to what

was found

_{by Capy}

et al

₍₁₉₉₄₎

in a broad _survey of numerous

_populations

reared at a

_single

_temperature

₍₂₅

_°C).

In our

_study

of two

_sympatric

_populations,

D

_{melanogaster appeared}

on the _average more variable than D simulans. In

both

_species

variations of isofemale line heritabilities were observed

_according

to

developmental

temperature,

but with different

_patterns

for different traits. These differences are difficult to

_interpret,

and _many more

_comparative

studies should be undertaken.

At the within-line

level,

phenotypic variability

exhibits a

_major

environmental

component

(Falconer,

1989)

and thus reflects in some _way the

_reactivity

of individu-als to minor variations in culture vials

_(eg,

food desiccation or larval

_{competition).}

This

reactivity

may be estimated

by

considering

the CVs. D simulans

appeared

more variable than D

_melanogaster

for thorax

_length

over the whole

_temperature

range and for the other two traits at

_high

_temperatures

_only.

These results are somewhat

_surprising,

because

phenotypic variability

was

previously

found to be similar in the two

_species

_(Capy

et

_al,

_1994).

A

_problem

remains: are these results

general

to the

_species

or

_specific

to the studied

_populations?

the left in D simulans. As the thermal _ranges are about the same in the two

_species

(Cohet

et

_al,

_1980,

and this

_work),

it was more difficult to calculate TMS values in D simuldns. A careful

_analysis

showed

_that,

besides the

translation,

the

shapes

of the norms were somewhat different in the two

_species.

Within

_species,

a

_significant

line-temperature

interaction demonstrates

_genetic

variations in the curve

_shapes.

Finally,

the

_{heterogeneity}

of TMS values between lines is

_larger

in D simulans than in D

_{melanogaster,}

in

_spite

of a lower

genetic

variability

within each

_temperature

in the former

_species.

These observations argue in favor of a

_{genetic regulation}

of

the reaction norm

_shape.

A last but

_major

difference between the two

_species

concerns the

_wing/thorax

ratio which is much smaller in D simulans and

_presents

_higher

TIPs.

All these differences

_support

a

_general

trend: the more the two

_species

are

compared,

the more

_they

_appear different

_(see

_Capy

et

_{al, 1993,}

1994 for discussion and

_references).

Reaction norms and the thermal

_adaptation

of the two

_species

Since we

investigated

the effects of

developmental

_temperature,

we must ask the

question:

is one

_species

better

_adapted

to a colder or warmer climate?

Answering

this

_question

is

_difficult,

since we have

_conflicting

observations.

Although

the thermal

_laboratory

ranges are similar

(12-31 °C)

in the two

_species

(Cohet

et

_al,

_1980),

_ecological

_surveys

_{(Louis, 1983)}

have shown that D simulans is

generally

more abundant than D

_melanogaster

in warm

_temperate

and

_subtropical

regions,

while it is rare or even absent in cold

_regions

where D

melanogaster

is still

present.

These observations lead to the classical

_{interpretation}

that D simulans is less tolerant to cold than D

_melanogaster

_{(Parsons, 1983).}

So our results are

surprising. Indeed,

even if the

biological meaning

of a TMS is not

_{clearly established,}

we

_expect

that a maximum should be related to some

_optimum

(Parker

and

Maynard-Smith,

1990;

Gabriel and

_Lynch,

1992;

Stearns,

1992).

Could we _suppose,

then,

that D simulans is more

_adapted

to cold than D

melanogaster, contrarily

to what was believed _up to _now, and that reaction norms indicate the direction of

_adaptation?

In

fact,

this

_hypothesis

is not

_unlikely.

_Indeed,

from an

_ecological

point

of

_view,

D

_melanogaster

enters human

_buildings

where it is

_protected

_during

winter,

whereas this is not the case for D simulans

(Rouault

and

David,

1982).

So the latter will suffer lower

_temperatures

than D

_melanogaster

_during

_winter,

and hence will be selected for cold tolerance. Two other

_{arguments support}

this

hypothesis. Firstly,

in D

_{melanogaster,}

males reared at 12 or 13 °C are

sterile,

whereas this is not the case in D simulans

_(David,

_unpublished

_{observations).}

Secondly,

in

_{competition experiments}

at

_{25 °C,}

D

_melanogaster

_generally

eliminates D

_simulans,

while the reverse occurs at

temperatures

below 20 °C

_(Tantawy

and

Soliman,

1967; Montchamp-Moreau,

1983).

Other observations

suggest

however a reverse

_{interpretation.}

The

wing/thorax

ratio,

which is

_{inversely proportional}

to

_wing

_loading

and

_wing

beat

_frequency

(P6tavy

et

_al,

_1992,

₁₉₉₆₎

decreases with

_temperature,

_presumably

in relation with a better muscular

_efficiency

at

_higher

_temperature

_(Reed

et

_al,

_1942).

In other

D

_{melanogaster.}

Moreover the

_TIP,

which

_corresponds

to a maximum of

_plasticity,

is

_higher

in D simulans. Even if the

_{possible relationship}

between the TIP and the

_optimum

_flight

temperature

remains to be

_{investigated,}

this could

_support

the

hypothesis

of a better

_adaptation

of D simulans to a warmer environment. Molecular studies at the

_{within-population}

level have shown that D simulans was

generally

more

_polymorphic

than D

melanogaster

(Aquadro

et

_{al, 1988; Begun}

and

Aquadro,

1991;

Aquadro,

1992).

To

_explain

this

_observation,

the former authors

suggested

that the

_population

effective number is

_higher

in D

_simulans,

due to a

higher migration

rate and a better

dispersal capacity.

In this

_respect

the lower

wing/thorax

ratio in D simulans could be more a

_{dispersal adaptation}

than a

thermal

_{adaptation. However,}

in

_spite

of numerous studies

(Brodsky, 1994)

we do

not know what is the best

_strategy

for

_{dispersal, ie, high speed}

correlated with

_high

wing

loading

and

_relatively

short

_flight

duration,

or vice versa.

In

_conclusion,

the two

sibling species

which are

increasingly investigated

as a model for

_evolutionary

studies,

_{appear very} different when more

_thoroughly

analyzed,

and

_{interpretations}

are difficult.

_Concerning

the evolution of reaction norms and their

possible relationship

with thermal

adaptation,

further

_comparative

studies are

needed,

either on

_{geographic populations}

of the two

_sibling

_species

and on other

_Drosophila

species

clearly

adapted

to warm or cold climates.

ACKNOWLEDGMENT

This work benefited from a _grant from the French Minist6re de l’Environnement

_(EGPN

committee)

to JR David.

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