Multilocus structure of the smooth newt (Triturus vulgaris, Caudata) natural populations

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Multilocus structure of the smooth newt (Triturus

vulgaris, Caudata) natural populations

J Crnobrnja, Ml Kalezić, Non Renseigné

To cite this version:

Original

article

Multilocus

structure

of the smooth

newt

(Triturus

vulgaris,

Caudata)

natural

_populations

J

_Crnobrnja

ML

Kalezi&jadnr;

Tuci&jadnr;

2

1

Institute for Biological

Research "Siniša

Stankovi6",

29 Noverrcbra

_142,

11000

Belgrade;

2

Institute

_{of Zoology, Faculty of Biology,}

Studentski _{trg 16,} 11000

_{Belgrade, Yugoslavia}

(Received

7

_August

_1991;

_accepted

22 June

₁₉₉₂₎

Summary - The

_{linkage disequilibrium}

between

pairs

of 7

polymorphic

loci in 27 natural

populations

of the smooth newt

_{(Triturus vulgaris)}

was examined. Pairwise

linkage disequilibrium

parameters were estimated from

_zygotic

_{frequencies using}

Burrow’s method. The average rate of

significance

of the

_{linkage disequilibrium}

parameter in 27

populations

was about 8%. The variation of

linkage disequilibrium

among

populations

was studied

_{by analysis}

of variance of the correlation coefficients between different loci in zygotes. This

_analysis

did not reveal

_systematic

associations between alleles at different loci

over 27

_populations.

In

_only

one _case, Me

_{(malic enzyme)}

x

_Pgm

_{(phosphoglucomutase),}

were correlation coefficients of the same

_sign

and

_magnitude

in a number of

populations.

linkage disequilibrium

/

multilocus association

_/

_allozyme

_/

Triturus vulgaris Résumé - Structure multilocus de populations naturelles de triton _vulgaire

_(Triturus

vulgaris,

Caudata).

Dans 27

_populations

naturelles de triton

_vulgaire

_{(Triturus vulgaris)}

on a examiné les

_{déséquilibres}

de liaison entre 7 locus

_{polymorphes pris}

2 à 2. Les

déséquilibres

de liaison entre locus ont été estimés à partir des

_fréquences

_zygotiques en utilisant la méthode de Burrows. Le pourcentage moyen de

déséquilibres

de liaison

_{significatifs}

dans les 27

_populations

est

proche

de 8 %. La variation des

_{déséquilibres}

de liaison entre les

populations

a été étudiée par

analyse

de variance des

coefficients

de corrélation des

gènes

non

_alléliques

dans les zygotes. Cette

_analyse

ne montre pas d’associations

systématiques

entre les

_gènes

non

_alléliques

dans les 27

populations.

Dans un seul cas, Me

(enzyme

malique)

x

_Pgm

_{(phosphoglucomutase),}

les

_coefficients

de corrélation ont le même signe

et la même

ampleur

dans

_{plusieurs populations.}

déséquilibre de liaison

_/

association multilocus

_/

_allozyme

_/

Triturus _vulgaris

*

INTRODUCTION

It has been

_suggested

that association between alleles at different loci

_(linkage

disequilibrium,

LD)

might

be a useful indicator of the action of natural selection

(Lewontin, 1974).

However,

several workers have

_pointed

out that such associations could arise

_{by genetic drift,}

non-random

_matings,

founder effects and

_hitchhiking

(see

Hedrick,

1982 for

_review).

_Thus,

the mere _presence

_{of allozyme}

_LD,

for

_example,

does not _appear to be critical in the evaluation of the

_{adaptive significance}

of

allozyme

variation.

Although

it is an

_extremely

difficult task to attribute _any

_pattern

of LD to

a

_particular

_cause, one

_approach

has been to consider evidence from more than

one

_population.

Lewontin

(1974),

for

example,

suggested

that if

significant

LD is

observed which is consistent in the

_magnitude

and

_sign

_{in many}

_populations,

then this

_pattern

can be attributed to selection.

There have been several

_experimental

studies

_designed

to detect _{LD among}

allozymes

in animal and

_plant

_populations

_(reviewed

_by

_Barker,

_1979;

_Brown,

1979).

Disequilibrium

has been found in several animal

_species,

such as salamanders

(Webster,

1973; Good,

1989),

blue mussel

_(Mitton

and

_Koehn,

_1973),

and the fish

(Mitton

and

Koehn,

1975).

Most

studies, however,

have been with several

_species

of

_Drosophila,

and

_particularly

D

_melanogaster

_(eg

_Langley

et

_{al, 1978;}

Laurie-Ahlberg

and

Weir,

1979 and references

_{therein). Although}

_Drosophila

studies have

frequently

shown LD between

_allozymes

and

_inversions,

there is little evidence for stable LD among

allozymes

in

Drosophila

as in other animal

_populations.

We have

_previously

_(Kalezi6

and

Tuci6, 1984;

Gonzales-Candelas et

_al,

in

_press)

described allele

frequencies

as well as different environmental and

_geographical

variables which influenced the

_genetic

structure of Triturus

_{vulgaris populations.}

Here we

_report

a _survey of LD _among 7

_{polymorphic allozymes}

in the same 27

natural

_populations

of the common newt Triturus

_vulgaris.

The main

_objective

of this

_study

was to seek for

_consistency

in the

_magnitude

and direction of LD _among

T

_{vulgaris populations.}

MATERIALS AND METHODS

The

_study

of

_linkage

_{disequilibrium}

was carried out on 16

_populations

of the

nominotypical

subspecies,

5

_populations

of T v

meridionalis,

3

populations

of

T v

_dalmaticus,

and 3

_populations

of T v _graecus. For

_{population localities,}

number of individuals

_surveyed,

and loci abbreviations see Kalezi6

(1983). Among

the 22 loci

_studied,

the

_following

loci were

_moderately

to

_{highly polymorphic}

in most of the

_{analyzed populations:}

acid

_phosphatase

_(Acph-2),

esterase

_(Est-4),

a-glycerophosphatase

(ct-Gpdh-),

malate

_{dehydrogenase}

_(Mdh-2),

_{malic enzyme}

(Me),

octanol

_{dehydrogenase}

_(Odh),

and

_{phosphoglucomutase}

_(Pgm).

Several loci

were

_polymorphic

for more than 2

_{electromorphs.}

In these _cases, the least common

alleles were

pooled

so that there were

_just

2 allelic classes for the estimation of

LD. The

_samples,

with about 40

_individuals,

were

_assayed

at each

_pair

of loci. The estimates of LD were made from

_zygotic

frequencies.

The method of

estimation,

the

_departures

from

_{Hardy-Weinberg equilibrium}

for the

_sample

_frequencies

at each locus and does not

_require

the

_assumption

of random

_mating.

An unbiased estimate of Burrows’ coefficient of LD is:

where PI and p2 are the allele

_frequencies

of the &dquo;1&dquo; alleles at 2

_{loci, respectively,}

f (11/11)

is the

_frequency

of double

_homozygotes

for the &dquo;1&dquo;

alleles,

and

_{f (11/01)}

is the

_frequency

of

_zygotes

_heterozygous

at the first locus and

_homozygous

at the second locus for the &dquo;1&dquo; allele.

The statistical

_significance

of Burrows’ coefficient can be tested as follows

(Langley

et

_al,

_1978):

where

X

2

has an

_{approximate chi-square}

distribution with one

_degree

of freedom

(Cockerham

and

_Weir,

_1977),

N is the number of individuals in the

_sample

and R

a correlation coefficient defined

by

Langley

et al

_(1978):

This coefficient

_corresponds

to the total correlation between genes,

including

within

_gametes

as well as between

_uniting

_gametes

correlations. R

_generally

lies between —1 and

_+1,

but between -0.5 and +0.5

only

when there is no correlation

between

_uniting

_gametes.

The variation of LD _among

_populations

was evaluated

_{by analysis}

of variance of the correlation coefficients of different genes in

zygotes.

To

_analyze

variation

_{of R}

_k

_(k

indexes the kth

_population)

attributable to the differences between

_populations

of different

_subspecies

_(dA)

or differences between

_populations

of the same

_subspecies

(dAB),

a weighted analysis

of variance

_(where

_weights,

_4N

_ik

_,

are the

reciprocals

of

sampling

variances)

was

_performed

(for

the

_analysis

of variance scheme and more

details see

_Langley

et

_{al, 1978,}

_pp

_217-220).

A test of

_dA

= 0 is the usual F ratio test with

_(m

₂

_{- 1)}

and

_(m

_l

_-

₎

₂

_m

_degrees

of freedom

_(m

₂

is the number of

_subspecies).

But,

if

_dA

_B

= 0 then the

weighted

sum of _squares at AB level is

_{approximately}

equal

to a

_chi-square

distribution with

_(m

_l

_-

_m

₂

₎

degrees

of freedom.

RESULTS

Burrows’

_{disequilibrium}

_parameters

were calculated for 318 combinations of

_pairs

of

_polymorphic

loci in 27 smooth newt

_populations.

Since

_pairwise

values of D for

analyzed

loci are too numerous to

_report

_here,

the results are summarized. Table I

gives

only

these locus

_pairs

in each

_population

with

_significant

D values. There

were 24

_significant

D values _among

_polymorphic

loci in these

_populations.

If the

disequilibrium

parameters

were

independent,

then 16

(=

0.05 x

₃₁₈₎

_significant

values would be

_expected

due to chance alone at the 0.05

_probability

level. More

_importantly,

the

_significant

values of D were

nonrandomly

distributed over

(in

12

_populations)

to

33.3%

_(in

2

_{populations).}

The average

percent

of

significant

D values over all 27

populations

is

7.99%.

In 2

_populations,

one of T

v vulgaris

and one of T v

meridionalis,

3 combinations

of locus

_pairs

exhibited

_significant

D values

_{(table I).}

Four

_populations

of T v

for 2 combinations of loci. The Mdh-2 x Me combination showed the most

_frequcnt

significant

value of D

_(in

4

_{populations),}

and in 3

_populations

_significant

D’s for

Acph-2

x

_Odh,

_a-Gpdh

x Odh and l!ldh-2 x Odh were detected. The Odh locus

shows

_{significant disequilibrium}

_parameters

with

_Acph-2,

_a-Gpdh

and Mdh-2 in 3

populations,

and with

_Pgm

and Me loci in and 1

_population

_{(table I).}

Table II shows the results of the

_analysis

of variance of the correlation coefficients based on Burrows’

disequilibrium parameters

over 27

_populations

divided into 4

smooth newt

_subspecies.

In this table

_d

_A

and

_d

_AB

are

_reported

rather than their

squared

values

_(variance

_component

attributable to

_subspecies

differentiation in

_R

_k

_,

and variance

_component

attributable to

_population

variation in

_R

_h

_.,

_{respectively),}

so that the scale is the same as that for R. Often

_d

_A

and

_d

_AB

have actual numerical estimates which are

_negative,

in which case 0.0 is

reported.

P <

_0.05)

and

_{nonsignificant}

variance

_components

at both levels. This indicates a

consistency

in

_R!

over the whole set of 15

_{compared populations}

of T

_{v vulgaris}

and T v meridionalis. The average correlation coefficients were also

_significant

for

Acph-2

x Mdh-2

_(0.068,

P <

_0.01),

_Acph-2

x Odh

_(0.077,

P <

_0.001)

and Est-4

x Me

_(0.044,

P <

_0.05).

However,

in all these cases the

highly significant

dA

B

indicate great variation in

_R!

values _among

_analyzed

_{populations. Although}

R was

not

_{significantly}

different from zero, the

highly significant

dA

for Est-4 x

Mdh-2,

Mdh-2 x Odh and Odh x

_Pgm

indicate differences in

_R

_k

values between T v

vulgaris,

T v meridionalis and T _{v graecus}

(second

loci

pair),

and T v

_vulgaris

and T v meridionalis

(first

and third

_comparisons

of loci

_pairs).

There are 3 combinations

of loci

_pairs

_(Est-4

x

_{a-Gpdh, a-Gpdh}

x Me and Mdh-2 x

_Me)

that show

significant

d

A

and

_significant

d

AB

.

The other

_comparisons

were either

_{insignificant}

_individually

for all parameters or showed

_only

_significant

_d

_AB

values.

DISCUSSION

Two studies of LD in natural and

_{laboratory populations}

of

_{Drosophila melanogaster}

involve the use of

_genotypic

data to calculate Burrows’

_{disequilibrium}

_parameter

and are thus

comparable

to the data

_reported

here.

_Langley

et al

₍₁₉₇₈₎

studied 8 _enzyme loci in some 100

_samples

from natural

_populations

and in 2

_laboratory

populations.

The

_frequency

of cases of

_significant

LD between

_pairs

of loci in natural

populations

was

5.1%

for linked _genes and

6.7%

for loci on different chromosomes. In

the 2

_laboratory

_populations,

the

_frequency

of

_significant

_{disequilibrium}

was much

greater: 37.5%

for linked loci

_pairs

and

10.3%

for unlinked loci.

_{Laurie-Ahlberg}

and Weir

₍₁₉₇₉₎

studied 17 _enzyme loci in 9

_{laboratory populations}

of

_Drosophila

melanogaster

and found

_significant

associations at

_frequencies

_fairly

similar to those of

_Langley

and collaborators in

_{laboratory populations:}

34.5%

and

8.9%

for linked and unlinked

_pairs

of

_{loci, respectively.}

The

_frequency

of

_significant

cases of LD in the smooth newt

_populations

is

_nearly

as

_large

as in the

_{laboratory populations}

of

_Drosophila

for unlinked

_pairs

of loci. The average rate of

_significant

D values in 27

_populations

of Triturus

_vulgaris

amounted to about

8%

_{(table I).}

Since we have no

_genetic

data on the

_positions

of these loci on

chromosomes,

it is reasonable to assume that we are

dealing

with unlinked

_pairs

of

loci. It is also

_important

to note that

_analyses

of LD were based on

_relatively

small

samples

(about

40 individuals _per

_population).

_But,

as has been shown

_by

Brown

(1975)

and Nlarinkovic et al

_(1987),

the

sample

size _necessary to detect

_significant

LD must often be

_{quite large. Thus,}

it is to be

_expected

that more instances of LD

would _{appear in} studies of T

_{vulgaris populations}

with

_{larger sample}

size.

Analysis

of variance of the correlation coefficients between alleles at different loci over 27

populations

of the 4 smooth newt

_subspecies

did not reveal

_systematic

associations across the _range of studied

_species.

In

_only

one case

(Me

x

_Pgm)

were the

_R!

values of the same

_sign

and

_magnitude

in a number of

_populations

(which

gives

rise to

_significant

R and

_{nonsignificant}

_d

_A

and

d

AB

;

table

_II).

This observation could be accounted for

_{by epistatic}

selection. The

lack

of

_consistency

in the

_magnitude

and direction of LD _among

_populations

in all other cases can be consistent with both neutralist and selectionist

hypotheses.

In several _cases, both

Mdh-2,

Mdh-2 x Odh and Odh x

_Pgm

which show

_significant

_d

_A

and

_{nonsignificant}

d

AB

values)

and

_genetic

drift via founder effects or

_population

subdivision

_might

be causes of the observed associations. Evidence

_provided

_by

Gonzales-Candelas et al

_{(in press),}

for the same

_populations

of the smooth newt, that

_frequent

extinction and recolonization were

responsible

for

_genetic

structure of

these

populations,

also indicate that founder effects

_might

be a

_primary

cause of the observed

_frequencies

of

_linkage

_{disequilibrium.}

ACKNOWLEDGMENTS

We thank the _anonymous reviewers for their

helpful suggestions

and comments on the

manuscript.

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