Association among quantitative, chromosomal and enzymatic traits in a natural population of Drosophila melanogaster

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Association among quantitative, chromosomal and

enzymatic traits in a natural population of Drosophila

melanogaster

M Hernández, Jm Larruga, Am González, Vm Cabrera

To cite this version:

Original

article

Association

_among

_{quantitative,}

chromosomal and

_enzymatic

traits

in

a

natural

population

of

_{Drosophila melanogaster}

M

_Hernández,

JM

_Larruga,

AM González, VM

Cabrera

University of La Laguna, Department of Genetics,

Canary Islands, Spain

(Received

27 March 1992;

accepted

9

February

1993)

Summary - A

_sample

of 1359 males and 1 259 females from a natural

_population

of

Drosophila melanogaster

of the

_Canary

Islands was

_{simultaneously}

examined for

_wing

length,

inversion

_{polymorphisms,}

and gene variation at 10

_allozyme

loci. Correlations and nonrandom associations between those

genetic

traits were estimated. In contrast to previous

studies, large

amounts of

_{linkage disequilibrium}

have been found.

_Frequencies

of significant gametic

associations between linked and unlinked elements were 100% and

25%, respectively,

for chromosome inversions, 81%

6nd

4% for chromosome inversions, and

allozymes,

and 36% and 0% for

pairs

of

_{allozymes. Temporal stability}

in chromosome and

_{allozyme frequencies}

and the _average number of alleles _per locus rule out a recent

bottleneck effect. Mean and coefficient of variation of

_{wing length}

are correlated with the

_degree

of

_{heterokaryotypy}

_{(both negatively)}

and with the

_degree

of

_{heterozygosis}

(positively

for the mean,

negatively

for the coefficient of

variation),

mainly implying

chromosome 3 elements. Individuals with

_wing

length

above

_{(or below)}

a standard deviation from the

population

mean showed characteristics for the other

genetic

traits which resembled those of northern

_{(or southern)}

_populations

of the species.

Drosophila melanogaster

/

wing size

_/

chromosomal inversion

_/

enzyme

/

linkage disequilibrium

Résumé - Associations entre un caractère quantitatif et des caractères

chromo-somiques et enzymatiques dans une _population naturelle de Drosophila melanogaster. Un échantillon de 1 359 mâles et

_{1 259 femelles}

d’une

_population

naturelle de

Drosophila

melanogaster

des îles Canaries a été étudiée _pour la

longueur

de

l’aile,

le

polymorphisme

des inversions et les variations

géniques

à 10 locus

d’allozymes.

Les corrélations et les

associations non aléatoires entre les caractères

_génétiques

ont été estimées.

Contraire-ment à des études

_{précédentes, d’importants déséquilibres}

de liaison ont été trouvés. Les

fréquences

des associations

_{gamétiques significatives}

entre éléments

portés

par le même

chromosome ou _par des chromosomes

dif,j&dquo;érents

sont de 100% et 25% respectivement pour les inversions

chromosomiques,

81% et

_4%

pour les inversions et les

_allozymes,

et

36% et 0% pour les

couples d’allozymes.

La stabilité dans le temps des

_fréquences

chro-mosomiques et

_allozymiques

permet d’écarter un

effet

récent de réduction

d’e,!&dquo;ectif.

La

moyenne et le

_coefficient

de variation de la

_longueur

d’aile sont en corrélation avec le

degré d’hétérocaryotypie

(corrélations

négatives

pour les

2)

et avec le

degré d’hétérozygotie

(corrélation

positive pour la moyenne,

négative

pour le

coefficient

de

variation),

impliquant

principalement

des éléments du chromosome !i. Les individus avec une

longueur

d’aile

supérieure

(ou inférieure)

d’un écart type à la moyenne de la

population

montrent, pour

les autres caractères

génétiques,

des

ca

y

n.ctéristiques qui

les

rapprochent

des

populations

naturelles

nordiques

(ou

méridionales)

de

l’espèce.

Drosophila

melanogaster/

taille de l’aile

_/

inversion _{chromosomique}

_/

enzyme

/

déséquilibre gamétique

INTRODUCTION

Selection effects in

_higher

_organisms

are obvious at

_{morphological, physiological}

and chromosomal levels but harder to detect at the molecular level

_(Lewontin,

_1974;

Nei, 1975; Kimura,

1983).

Theories to connect

_phenotypes

with their

_genotypic

bases differ in the relative

_strength

_given

to

_independence

or

_epistasis

_among the

different sets of genes that determine

phenotypic

traits

(Crow, 1987).

Experimental

approaches

have

_mainly

consisted of the

_study

of correlated response at different

levels of

_variation,

driven

_by

artificial selection on a

_{presumably adaptive}

_trait,

but the

_validity

of the results of artificial selection to

_explain

natural selection is

controversial

_{(Nei, 1971).}

An area of

population genetics

where

changes

of variation at different levels have

been detected is in studies on the

_geographical

structure of natural

_populations.

In the

_species

_Drosophila

_{melanogaster,}

the existence of latitudinal clines has been demonstrated for

_{morphological}

and

_{physiological}

characters

_(Tantawy

and

_Mallah,

1961;

David and

_Bocquet,

_1975;

David et

_{al, 1977; Stalker, 1980;}

Cohan and

Graf,

1985;

Watada et

_al,

_1986;

_Coyne

and

_Beecham,

_1987),

additive

_genetic

variance

of

_viability

_(Kusakabe

and

Mukai,

1984),

chromosome inversion

_polymorphism

(Mettler

et

al, 1977;

Inoue and

Watanabe,

1979;

Stalker, 1980;

Knibb et

al,

1981),

and

_{allozyme frequencies}

_(Schaffer

and

_Johnson,

_1974;

Voelker et

_{al, 1978;}

_Singh

et

al, 1982;

Anderson and

_{Oakeshott, 1984;}

Inoue et

_al,

_1984;

_Singh

and

_Rhomberg,

1987).

Nevertheless,

a clear connection among

morphological,

chromosomal and

enzymatic

clines has not

_yet

been well established

_(Voelker

et

al, 1978;

Stalker,

1980;

Knibb,

1983;

Kusakabe and

_Mukai,

_1984).

This is

_explainable

if it is assumed that natural selection is

_acting

_{simultaneously}

_upon several

_{morphological}

and

physiological

traits that are

largely genetically independent

(David

et

al,

1977),

and

character

_{highly dependent}

on a

specific

_{genetic combination,}

association _among different

_genetic

levels should be detectable if sufficient

_sample

sizes have been

employed

(Brown,

1975; Zapata

and

_Alvarez,

_1987).

To test the

_validity

of this

supposition,

we have characterized the

_variability

in a natural

_population

of D

melanogaster, sampled

in the most favorable season, for

wing

length

as a measure of

_body

_size,

chromosomal inversion

_{polymorphism,}

and 10

_enzymatic

loci. In order

to detect relevant associations _among traits that could have been overlooked in the

past,

the

_sample

was an order of

_{magnitude larger}

than in

_preceding

estimates. From a selective

_point

of

_view,

both clines and seasonal

_changes

can be considered as the effect of short limited directional selection of several

_highly

correlated

envi-ronmental features on the

_phenotypic

variance of the

_populations.

In

_Drosophila,

their more visible effect is a

change

in mean and variance of

_body

size.

_{Nevertheless,}

possible

associations of this trait with others can be

_explained

as well

_by

selection as

_by

historical factors. In an

_attempt

to

_distinguish

between these

_hypotheses,

the

total

_sample

was subdivided into

_subsamples

with mean sizes similar to those of

temperate

and

_tropical

natural

_{populations, reanalyzed}

for the other studied traits and their

_possible

interactions,

and then

compared

with _any

_outstanding

features of

natural southern and northern

_populations

of the

_species

for these same characters.

MATERIALS AND METHODS

A

_sample

of 1 359 males and 1 259 females of

_{Drosophila melanogasterwas}

_collected,

using

crushed grape skin

traps,

in an orchard in the

locality

of

Guimar,

Tenerife

(Canary Islands)

during

the

_vintage

_period,

in

_September

1984,

and the

_following

analyses

were conducted.

Morphological analysis

The

_right

_wing,

whenever

_possible,

or the left

_wing

of each

fly

was dissected

and mounted on a slide. The

_wing

_length

was measured as the linear distance between the intersection of the 3rd

_longitudinal

vein with the

_{wing tip}

and the

anterior crossvein. This measurement is known to be

_genetically

and

phenotypically

correlated with other measurements of

_body

size in D

_melanogaster

_(Reeve

and

Robertson,

1953;

David et

_al,

_1977).

Cytological analysis

For

_karyotype

determination,

wild males were crossed

_individually

with

_virgin

females of the

_Oregon

_strain,

which is

_{homokaryotypic}

for the standard

_(st)

arrangement

of all chromosomal arms in this

_species.

Wild females were first frozen

at -11 ! 1°C for 20 min in order to

_delay

the sperm of wild

males,

and then

transferred to fresh medium _every

_day

to eliminate the fertilized _eggs

_(Mayer

and

Baker,

1983).

After

_{this ,}

females were crossed with males of the

_Oregon

strain. In both cases 7 third-instar larvae from

F

l

were dissected and the

_{salivary gland}

chromosomes observed.

We have followed the

_cytological

nomenclature described in

_Lindsley

and Grell

Enzymatic

analysis

After crosses

_{yielded offspring,}

wild flies were

_{electrophoresed}

in horizontal

starch-gels

and the

_following

enzyme loci

analyzed: 6-phosphogluconate dehydrogenase

( 6-Pgdh,

map

position

1 -

0.6),

glucose-6-phosphate dehydrogenase

( G-6-pdh,

map

position

1 -

_63.0).

_{a-Glycerophosphate}

_{dehydrogenase}

_(a-Gpdh,

_map

_position

2 -17.8),

alcohol

_{dehydrogenase}

_(Adh,

_map

_position

_{2 - 50.1 ),}

hexokinase-C

_(Hk-C,

map

position

2-73.5),

phosphoglucomutase

(Pgrrc,

map

position

3-43.4),

esterase-C

_(Est-C,

_map

_position

3 -

_47.7)

and octanol

_{dehydrogenase}

_(Odh,

_map

_position

3-49.2).

In

addition,

another 2 enzyme

loci,

esterase-6

_(Est-6,

_map

_position

_3-36.8)

and

_{glucose dehydrogenase}

_(Gld,

_map

_position

_3-48.5),

were

assayed only

in males. Gel

_preparation,

_{electrophoretic,}

and _enzyme

_staining

methods were as described

by

ConzAlez et at

_(1982),

_except

for Gld which was stained as in Cavener

(1980).

Statistical

_analysis

Linkage disequilibria

were estimated from

_zygotic

_{frequencies following}

Cockerham and Weir

₍₁₉₇₇₎

and Weir and Cockerham

₍₁₉₇₉₎

_methods,

and the normalized

average

correlation, R,

of

Langley

et at

_(1978).

_Only

the 2 more

frequent

alleles or

rearrangements

were

_used,

_pooling

the rarest with the more common ones

_(Weir

and

Cockerham,

1978).

The

_unique

_exception

was the 3R _{arm, in} which the st and In

(3R)P

arrangements

were

_compared.

For

_pairs

of loci

_involving

a sex-linked

locus,

only

female

_genotypic

_frequencies

were used. We considered as

_coupling

_gametes

those with the 2 most or the 2 least

_frequent

alleles

_and/or

_{arrangements,}

as in

Langley

et at

_(1974).

In order to

_study

the

_possible

_{associations among}

_qualitative

and

_quantitative

traits,

several statistical

_analyses

were carried out. Differences in mean

_wing

length

among the different

genotypic

classes

_involving

the 2 more

_frequent

alleles for each

enzymatic

locus and the 2 common

_cosmopolitan

_{rearrangements}

for each chromo-some arm were tested

_by

an

_analysis

of variance

_(ANOVA)

_plus

_{regression, using}

the breakdown and means

subprograms

from SPSS

(Nie

et

at,

1975).

The

contribu-tion of each factor to the

_genetic

variance of the

_quantitative

trait was estimated

according

to Boerwinkle and

_Sing

₍₁₉₈₆₎

and the

_partition

of this contribution into additive and dominance

_components

_following

the method described

_by

Ruiz et at

_(1991).

The overall

_degree

of

_{heterokaryotypy}

and

_{heterozygosis}

_per individual was,

respectively,

established as the number of chromosome arms or

_enzymatic

loci

studied in the

_heterozygous

state. Then the

_{relationships}

between individual

het-erokaryotypy

or

_{heterozygosis}

and

_wing

length

_among the total male and female

samples

were determined

_by

Pearson’s

_{product-moment}

correlation

(r).

Individuals

with the same

degree

of

heterokaryotypy

and

heterozygosis

were

pooled

in

classes,

and correlation between these classes and their means in

_wing

_length

was calculated

by

Kendall’s coefficient of rank correlation

_(Sokal

and

_Rohlf,

_1981).

In

_addition,

2

_analyses

were conducted to assess associations of variance in

wing

length

with

_{heterozygosity}

at the chromosomal and _gene level. For each locus and chromosome arm, the differences between the coefficients of variation

for the

_{morphological}

character in

_homozygous

and

_heterozygous

_groups were

calculated,

and the Wilcoxon’s

_signed-ranks

test

_(Sokal

and

Rohlf,

1981)

followed to

trait. Correlations between

_{heterokaryotypic}

and

_{heterozygotic}

classes and their

respective

coefficients of variation in

_wing

_length

were also calculated

_by

Kendall’s coefficient of rank correlation

_(Sokal

and

Rohlf,

1981).

The total

_sample

was subdivided into 3 classes: at least 1 standard deviation above the mean

(Class I),

within 1 standard deviation from the mean

_(Class

_II)

and at least 1 standard deviation below the mean

(Class III),

in order that the

upper and lower classes would have a mean

wing

size

respectively

similar to the

northern and southern natural

_populations.

The same kind of association

analysis

as in the total

sample

was carried out on them.

RESULTS

As no

_significant

differences were found in inversion or in

_{allozymic frequencies}

between sexes, data of male and female have been

pooled

whenever

possible.

Quantitative

variation

Wing length

means were 1.539 t 0.003 mm for males and 1.710 f 0.004 mm for females. The same values for Classes

I,

II and III were:

_{1.679 ±0.003,}

1.545 ±0.002

and 1.390 f 0.003 in males and 1.869 f

_0.003,

1.720 t 0.002 and 1.531 ! 0.004 in females

_{respectively.}

Karyotype

variation

In the

_present

_study

_{(table I)}

a

sample

of > 2 500 X chromosomes and 3 700

autosomes from the natural

_population

of Guimar was examined. A total of 38

inversions,

all of them

_paracentric,

was found

compared

with

only

16 detected in a

_previous

_{survey in} the same

locality

where

only

226 chromosomes were

analyzed

(Afonso

et

_al,

_1985).

_Following

the

_nomenclature

of Mettler et al

_(1977),

we have

_{distinguished}

the

_following

inversions: 4 common

_{cosmopolitan;}

3 rare

cosmopolitan;

4 endemic recurrent

_previously

detected in this same

population

(Afonso

et

_al,

_{1985) ;}

and 27 new endemic rare inversions. Three of these new

inversions were found on chromosome X which is

_{usually monomorphic}

in wild

populations

of D

_melanogaster

_(Ashburner

and

_Lemeunier,

₁₉₇₆₎

_although

Stalker

(1976)

also found X

_polymorphism

in American

_samples.

Overlapping

inversions are _very scarce in this

species

(Stalker,

1976;

Zacharopou-los and

_Pelecanos,

_1980).

In this

_{study only}

1 such

_complex

_{rearrangement}

was

found,

that

_being

the common

_cosmopolitan

In

(3L)P

and the endemic In

(3L)6/,C;69F

occurring

in the same chromosome. Another

_overlapping

inversion

had

_previously

been found in this same

locality

but

affecting

the 2L arm

(Afonso

et

al,

1985).

In an inversion

_{distribution,}

8

_(21%)

were found on

_2L,

6

_(16%)

on

_2R,

7

_(18%)

on

3L,

14

(37%)

on 3R arms and 3

(8%)

on chromosome X. The inversion

frequency

per individual in the total

sample

was

_1.15;

it decreased to 1.01 in Class

I and increased to 1.23 in Class III. Individuals were sorted in _groups

according

a

significant

excess of individuals without or with 3 or more inversions and a

corre-sponding

deficit of those with

_only

one was observed

(

X

2

=

_23.06,

5

df,

_p <

_0.001).

This was

_just

what Knibb et al

₍₁₉₈₁₎

_reported

for

_{populations latitudinally}

far

from the

equator,

with fewer than one inversion _per individual.

When

_comparing

the

_cosmopolitan

inversion

_frequencies

with those found in the same season of the

_previous

_year

(Afonso

et

al,

1985),

only

those on the !R arm

showed

_{heterogeneity}

_(x

₂

=

_12.85,

3 df,

p <

_0.01).

_Frequency

of the

_{st arrangement}

increased in 1984

_(0.847)

_compared

to 1983

_(0.758)

at the _expense of a decrease in

the common

In(3R)P

and the rare

In(3R)C

cosmopolitan

inversions.

The more relevant effects of

_partition

for

_wing

_length

on chromosomal

poly-morphism

were an increase in mean

heterokaryotypy

(0.282 ! 0.026)

and in in-version

_frequency

per individual

(1.23)

for small flies

(Class III)

and a decrease

(0.216 ! 0.022)

and

_(1.01)

_respectively

for the

_larger

ones

(Class I).

In a more

detailed chromosome

_by

chromosome

_{analysis, significant}

differences were detected

between Classes I and III for inversion

_frequencies

of 2L and 3R arms,

In(2L)t

(x

2

=

5.36,

1

df,

p <

_0.05)

and

_In(3R)P

₍

_X

₂

=

5.16,

1

df,

p <

_0.05)

_having

lower

frequencies

in Class I than in Class III.

_Furthermore,

Class I shows the

_only

ob-served

Hardy - Weinberg

(HW)

deviation

_(x

₂

=

_5.23,

1 df,

p <

_0.05)

due to a deficit

of homokaryotypes

t/t.

Thus,

long wing

flies have

_{higher frequencies}

of standard

_(st)

rearrangements

and lower inversion

_{heterozygosities}

than those with short

_wings,

which is in

_agreement

with the

_{morphological}

_(Tantawy

and

_{Mallah, 1961;}

David et

al,

1977;

Watada et

_al,

_1986;

_Coyne

and

Beechan,

1987)

and chromosomal

_(Mettler

Isozyme

variation

Table II

_gives

allelic

_frequencies,

and observed and

_{expected frequencies}

_of

heterozy-gotes

for the 10

_enzymatic

loci studied. Loci with

_{significant departures}

from HW

equilibrum

were

_{cx-Cpdh, Hk-C,}

Est-6 and Est-C. In all of them

_significance

was

due to an excess of

_homozygotes,

the overall mean

_{heterozygosity}

observed

_(0.231

!

_0.009)

_{being slightly}

less than the

_expected

value

_{(0.243 !}

_0.006).

The average number of alleles per locus for the total

sample

was

_4.3,

_nearly

twice as

_large

as

the value found

_(2.3)

in

_previous

_screenings

of the same

_locality

_(Cabrera

et

_al,

1982;

Afonso et

_al,

_1985).

This difference is attributable to differences in

_sample

size,

which is 20 times

_greater

in this

_study.

When

_only

rare alleles with

_frequencies

high enough

to be detectable with former

_sample

sizes were

considered,

the average

number of alleles per locus

(2.5)

was similar

along

_years, and did not differ from

that calculated for the total

_species

_(2.8)

_using

the same set of loci taken from the data of

_Choudhary

and

_Singh

_(1987),

who studied 15 worldwide

_populations.

When we _compare the common

allozyme frequencies

found in this

study

with those of a

_{previous sample}

of the same

locality

(Afonso

et

_al,

_1985),

_only

one

comparison

involving

the sex-linked locus

_6-Pgdh

was

significantly heterogeneous

(

X

2

=

_13.72,

1

df,

p <

_0.001).

Contrary

to its effect on chromosomal

_variation,

the

_population

subdivision

according

to

_wing

_length

did not affect the

_enzymatic

mean

heterozygosity

which was similar in all 3 Classes

_(0.227

f

_0.023,

0.234 ! 0.012 and 0.222 ! 0.022 for Class

_I,

II and III

_{respectively).}

_{Nevertheless,}

in a locus

_by

locus

_comparison

there are

significant

differences between Classes for Adh

(

X

2

=

_4.04,

1

df,

_p <

_0.05)

and Est-C

₍

_X

₂

=

_4.08,

1

df,

p <

_0.05),

with

Adh

loo

and

Est-C

lol

_(alleles

100 and 101 for Adh and

_{Est-C loci,}

_{respectively)}

_increasing

in Class I when

_compared

to Class III. It is worth

_mentioning

that these same loci showed clinal variation

in D

_melanogaster

with both alleles

_{having higher}

frequencies

in

_temperate

than

in

_tropical

_populations

_(Singh

and

_Rhomberg,

_1987).

A new

_departure

from HW

equilibrium

was observed in Class I for the Adh locus

_(x2

=

_6.8,

₁

_df,

_p <

_0.01)

due

to a deficit in observed

₉₅₁₉₅

_homozygotes,

which

_parallels

the

_decrease,

_already

mentioned,

of

_t/t

_{homokaryotypes}

in the same Class.

Associations between

_{wing length}

and

_karyotype

When ANOVAs were carried out no

heterogeneity

was found for 2L and !R arms in any sex.

However,

significant

associations were observed for both chromosome 3 arms in

males,

stlst

homokaryotypes having

on _average

_wings

significantly larger

than individuals

_carrying

P inversions

_(tables

_III,

_IV).

_Although

F values were not

significant,

a similar trend was observed for females

(table

III).

Stalker

(1980)

found

significantly

lower

_wing-loading

indices

_(larger

_wings

relative to thorax

_volume)

in

wild flies

_homozygous

for st

_{rearrangements}

in 2R

_and/or

3R arms when

_compared

to flies

_carrying

inversions in these arms. He reached the conclusion that wild flies

with

_high

_frequencies

of st chromosomes are

karyotypically

_northern,

and

selectively

favored

_during

the cold season.

A

_slight

but

_significant

_{negative product-moment}

correlation was observed

(r

=

-0.068,

p <

_0.05)

and females

_(r

=

-0.063,

p <

_0.05).

The same result was obtained

correlating

the different

karyotypic

classes with their

respective

wing

length

means

_using

the Kendall’s

nonparametric

rank test

(table V).

Thus,

st/st

homokaryotypes have,

on _average,

longer wings

than

heterokaryotypes.

When

the same statistical test was

_applied

to correlate

_karyotypic

classes with their

coefficients of

_variation,

a

significant

negative

correlation was

_again

found

(table V),

with the variance

_being

smaller in groups with

_{higher degress}

of

_{heterokaryotypy.}

Associations between

_wing

_length

and

_genotypes

The

_possible

associations between

_wing

_length

and

_genotypic

classes were tested

_by

longest

wings

(tables

VI,

VII).

These results are

_congruent

with the aforementioned fact that individuals with

_long

_wings

_(Class

_I)

had the

_{highest frequency}

of the

Est-C

There was a

_positive

correlation between individual

_{heterozygosity}

and

_wing

length

which reached

_significance

in males

_(r

=

+0.066, p

_<

0.05)

but _not in females

(r

=

+0.025, p

=

0.47).

The _same

relationship

was found between

_{genotypic classes,}

sorted

_by

their

_degree

of

_{heterozygosis,}

when

_they

were correlated with

_wing

_length

mean

_using

the Kendall’s rank test

_{(table VIII).}

_Significant

_negative

correlations between

_degree

of

_{heterozygosity}

and

_wing

_length

coefficients of variation for males

and females are also

_presented

in the same table.

_Therefore,

individuals and classes

with increased levels of

_enzymatic

_{heterozygosity correspond}

to

_longer

_wings

and smaller coefficients of variation.

The

_negative

correlations for

_{heterokaryotypy}

and

_{heterozygosis}

with

_phenotypic

variance were also tested

_using

the

_{non-parametric}

Wilcoxon’s

_signed-ranks

test

(Sokal

and

_Rohlf,

_1981).

In our _case, for each

_analyzed

element

_(chromosome

or

gene)

the total

_sample

was divided in to _{2 groups,}

_homozygotes

and

_{heterozygotes}

for that

element,

and the average coefficient of

variation,

in order to use male and female

_data,

was calculated for each _group. The null

_hypothesis

tested was

that

_{heterozygotes}

have on _average the same level of

_wing

length

variation as

homozygotes.

In 19 out of 25

_possible

tests,

heterozygotes

had a lower coefficient

of variation

_(p

<

_0.005).

_Furthermore,

the same

_analysis

for each

autosome

showed that elements in chromosome 3 are

_{primarily responsible}

for this difference. Ten

out of 12

_possible

tests for chromosome 3 had lower coefficients of variation for

heterozygotes (p

<

_0.005),

whereas

_only

6 out of 10 in chromosome 2 showed the

same

_tendency.

This result is in accordance with the fact that correlations with

wing

size were

_mainly

detected with chromosome 3 elements

_(tables

IV,

VII).

Association between inversion

both cases

significant

disequilibria,

due to an excess of

coupling

_gametes,

were found

(table IX).

Moreover,

in a

previous

_survey of this

locality

(Afonso

et

al,

1985),

sim-ilar

tenden

G

ies

were

detected,

reaching

statistical

significance

for the 3L-3R

pair

in

spite

of the small

_sample

_analyzed.

It

is worth

mentioning

that whenever

_significant

disequilibria

or

_tendency

to

_{disequilibria}

were detected in worldwide natural

pop-.ulations of this

_species,

there was

invariably

an excess of

coupling

_gametes

(Knibb

et

at,

1981).

Only

one of 4

_possible

nonrandom associations between unlinked

_cosmopolitan

inversions was

statistically significant

(table IX),

and

again

coupling

gametes

were

in excess. Thus the overall

_tendency

of the nonrandom associations found was to

favour individuals without inversions or with more than one inversion.

Associations between inversions and

_allozymes

Of the 16

possible

comparisons

between linked

allozymes

and gene

arrangements,

13

_(81%)

were in

linkage disequilibrium,

but

only

1

(4%)

out of 24

possible

combi-nations between unlinked inversions and

_allozymes

were in nonrandom association

(table IX).

Associations between

_allozymes

Of 14

_possible

combinations between

_allozyme

loci located in the same

chromosome,

5

_(36%)

were in

linkage disequilibrium

(table

IX),

3 of them

having

an excess and

2 a deficit of

_coupling

_gametes.

None of the 31 combinations between

allozyme

loci

DISCUSSION AND CONCLUSION

In contrast to

_{previous studies,}

_large

amounts of

_{linkage disequilibrium}

have been found in a natural

_population

of D

_{melanogaster.}

_Frequencies

of nonrandom

as-sociations are

_stronger

between linked than unlinked

_pairs

and also between

larger

than smaller elements.

_{Thus, frequencies}

of

_significant

_gametic

associations between linked and unlinked elements

_respectively

were

100%

and

25%

for chromosome

ar-rangements, 81%

and

4%

for chromosome

_arrangements

and

_allozymes,

and

36%

and

0%

for

_pairs

of

_allozymes.

These values are much

_higher

than those

_previously

reported

in natural

_populations

and

_fairly

similar to those found for

_experimental

populations,

with the

_{important exception}

that no

significant

associations between unlinked

_allozymes

were detected in this

study.

The

relatively large

amount of

linkage

disequilibrium

in

_{experimental populations}

has been

_mainly

attributed to

the small size of these

_populations

_(Langley

et

_{al, 1978;}

_{Laurie-Ahlberg}

and

_Weir,

1979)

although

other causes such as selection

_by

varied environments have also been invoked

_(Birley

and

_Haley,

_1987).

The first

_explanation

does not seem to be the case for our insular

_population.

Gene

_arrangement

and

_allozyme

_frequencies

are,

temporally,

rather

stable,

and the average number of alleles seems to indicate

that the

_population

has not

_recently

suffered

_important

_bottlenecks,

its

_population

size

_being

_comparable

to those of continental

_populations

_(Cabrera

et

al,

1982;

Afonso et

_al,

_1985).

It is well known that

_although

D

_melanogaster

_undergoes

sea-sonal bottlenecks and

_expansions

in

_population

_size,

the

_allozyme

and chromosomal

variation does not seem to be

_strongly

affected

_(Langley

et

al,

1977).

Nevertheless,

the

_population

structure could account for moderate levels of

_{disequilibrium}

gen-erated

_{by population}

subdivision due to

_microspatial

_{heterogeneity}

_(Hoffmann

et

al,

1984).

The

_{significant departures}

from HW

equilibrium

of several loci and the

overall

_deficiency

of mean observed

_{heterozygosity}

could be

_{explained by}

this fact. Another

_{possible explanation}

is that these levels are similar in other natural

pop-ulations but have not been detected before due to

_inadequate

_sample

size

_(Brown,

1975).

The

_{temporal stability}

of the

_linkage

_{disequilibria}

found in our

_population,

and their overall consistence with those

_reported

in other worldwide _surveys

(ta-ble

_X),

strongly

support

the action of

_epistatic

selection.

Discrepancies

in the

_sign

of some nonrandom associations are

explainable

because

they

affected loci and

in-versions

_{genotypically}

correlated with differences in

_wing

size. The

_{positive sign}

for

all

_pairs

of nonrandom associations between _gene

_arrangements

_points

to

_frequency

dependent

selection with a

_minority

advantage

(Yamazaki, 1977).

On the

_contrary,

the number of

_negative

_{linkage disequilibria}

detected in nonrandom associations between inversions and

_allozymes,

11

_(79%)

_{significantly}

_greater

₍

_X

₂

=

4.57,

1

df,

p <

_0.05)

than the

_positive

ones, 3

(21%),

suggesting

some

_type

of

balancing

selec-tion

_(Langley

and

_Crow,

₁₉₇₄₎

rather than a historical effect

_(Nei

and

_Li,

1980).

Finally significant allozyme - allozymes

associations are less common and _may be a

simple

consequence of their association with the same

_inversion,

since when these

linkages

were tested

_according

to Zouros et al

₍₁₉₇₄₎

within chromosome

arrange-ments for the 2

_significant

cases, the associations

disappeared Adh-a-Gpdh

within

2L(st)

(x

2

=

1.20,

1

df,

NS)

and

Est-6-Pgm

within

3L(st)

_arrangements

(x

2

=

_2.59,

The

_significant

correlations found between

_karyotypes

and

_genotypes

with the

quantitative

trait

_suggest

a measurable

_genetic

component

within a natural

pop-ulation in the

_phenotypic

variation of a character with low

heritability

(Prout,

1958;

Coyne

and

_Beecham,

_1987).

_Although

both autosomes seem to influence

wing

length,

the effect of chromosome 3 was

stronger.

Although

there are some

_negative

results

_(Handford,

1980;

Pierce and

_Mitton,

1982)

several

_reports

on the

_relationship

between

_genetic

and

_karyotypic

heterozy-gosity

and

_{morphological}

variation seem to indicate

_that,

as in

_experimental

pop-ulations,

a

_negative

correlation between

_genetic

heterozygosity

and

morphological

Grant,

1984;

Zouros and

Foltz,

1987).

Our results in D

_medanogaster

_agree with this

_supposition

as both in individual or

_{genotypic classes,}

_{heterozygotes}

and

het-erokaryotypes

showed

_{significantly}

less

_{morphological}

variance than

_homozygotes

and

_{homokaryotypes respectively.}

Robertson and Reeve

_(1952a)

also

_reported

the

existence of this

_negative

correlation in the same

_species.

Their

_explanation

for this

phenomenon

was that more

heterozygous

individuals will _carry a

_greater

diversity

of alleles that endows them with a

_greater

biochemical

_versatility

in

_development.

Nevertheless,

other authors

_{(Chakraborty, 1987)}

claimed that these

_negative

cor-relations between

_{heterozygosity}

and

_phenotypic

variance can be

_{explained by}

ad-ditive allelic effects without

_implying

heterosis,

overdominance or associative

over-dominance. In

_fact,

when

_following

the methods of Boerwinkle and

_Sing

₍₁₉₈₆₎

and

Ruiz et al

₍₁₉₉₁₎

to estimate the

_underlying

mechanism that

_might

cause these

associations in our data

_{(table XI)}

_practically

the

_totality

of the variance _among classes

_(u’)

can be

_{explained by}

additive effects

₍

_Q

_a).

An

_apparent

contradiction seems to exist in the

_sign

of the correlations found between

_{heterokaryotypy}

and

_{heterozygosis}

with _averages of

_wing

_length

at in-dividual or

_genotypic

class levels.

Wing

length

_averages are

positively

correlated

with

_{homokaryotypy}

but

_negatively

with

_homozygosis,

but a

significant

_positive

correlation also exists between

_{heterozygosis}

and

_{heterokaryotypy}

both in males

(r

=

+0.968,

p <

_0.01)

and females

_(r

=

+0.988,

p <

_0.01).

The

_explanation

is that

within each

_{heterokaryotypic}

class,

the

_largest

individuals are the most

heterozy-gous.

As there are

_gametic

_{associations between gene}

_arrangements

and

_enzymatic

loci,

both correlated with differences in

_wing

_length,

we tested whether a

_genuine

correlation exists between the Gld and Est-C loci and

_wing

_length

within the

homokaryotypic

individuals for the 3R standard

_arrangement.

It can be seen in table XII that a

_significant

association among

genotypes

and

_wing

_length

exists

The interaction found _among

_wing

_length,

chromosome and

genic

variation fits in very well with the natural

population

patterns.

Northern

_{marginal populations}

have

_longer

_wings,

reduced

_{heterokaryotypy}

and similar

_{heterozygosis}

_compared

to

central and southern

_populations.

_However,

there is a

_discrepancy

with

_experiments

of artificial directional selection. Selection for

_long

_wings

favours

_heterozygous

combinations for chromosomal

_{arrangements,}

whereas selection for short

_wings

generally

fixed

_specific

chromosomal

_arrangements

in

_homozygous

combination

(Prevosti, 1967).

It seems that

_experimental

_{selection goes} further than the seasonal and latitudinal natural selection. Since

_long

size is well correlated with

_high

levels of

heterozygosity

(Robertson

and

_Reeve,

_1952b)

the best way to

delay homozygosity

due to

_inbreeding

is the maintenance of different _gene

_arrangements

that

_physically

preserve

heterozygosity.

On the other

hand,

selection for short

wings

in this

species

favours

_{homokaryotypy}

for non-standard _gene

_arrangements

which are more common in southern

_populations

_(Aguad4

and

Serra,

1980;

Serra and

Oller,

1984).

The

_strong

associations found here among different levels of variation seem to

possess a

_{potentially high adaptive}

_value,

as was manifested when we subdivided the

sample

by

differences in

_wing

size,

resembling

the clinal variation existent for this

quantitative

character in natural

_populations

of the

_species.

Subdivision

_changed

the level of

_{heterokaryotypy}

and

_promoted

differences in the

_frequencies

of some

allozymes

linked to gene

arrangements

as in nature.

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