Evolutionary systematic of three species of troglobitic beetles: electrophoretic and morphological evidence

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Evolutionary systematic of three species of troglobitic

beetles: electrophoretic and morphological evidence

B Crouau-Roy

To cite this version:

Original

article

Evolutionary systematic

of three

_species

of

_troglobitic

beetles:

_{electrophoretic}

and

_{morphological}

evidence

B

_Crouau-Roy

1 Laboratoire

souterrain du

_CNRS,

_Moulas, 09200

_{Sa$nt-Girons,}

France

(Received

29

_September

_{1988; accepted} 17

_January

₁₉₉₀₎

Summary - Genic and

_{morphological}

variations were compared for 3 allopatric and endemic

_troglobitic

beetles of _{the genus}

_{Speonomus, by}

use of an

_allozyme

data set

(18

putative

loci)

and 1 based on morphometric characters

(16 morphological variables).

Allozymic

and _{morphometric relationships} were also _compared with some _aspects of the

mating

behaviour, and considered in relation to the

_ecology

and

_biogeography

of these

species. The extent of _agreement between the assessments of _evolutionary

_divergence

at

the _genic and _{morphological} levels is discussed. _{Enzyme analysis} revealed _pronounced

differences in

_degree

of

_genetic

differentiation between the 3 _species: S colluvii is the

most

_divergent

_species with

_large

_genetic distances

_{(D ! 1).}

_Morphometric differentiation

between the 3 _species, assessed on 16 _characters, is _important between the 3 _species,

principally between S _xophosinus and the 2 others. The level of _{congruence is}

_strongly

data

-

set dependent and these results reveal

_independent

trends for the 2 sets of characters.

While S _xophosinus has

_diverged

little from S

_hydrophilus

on the molecular _level, the

morphometric

differentiation between them is

_high.

This pattern may result from different sensitivities of each character set to different _components of the environment.

troglobitic beetle _/ systematic / electrophoretic and _morphometric variations Rksumé - _{Systématique} évolutive de trois _espèces de _{Coléoptères troglobies: analyses}

électrophorétiques et _{morphologiques.} Nous avons comparé les variations génétiques

et

_{morphologiques}

de trois _espèces de

_{Coléoptères troglobies}

_{allopatriques} et

_endémiques

du _genre

_Speonomus

sur la base des données

_allozymiques

(18 locus)

et des caractères

morphométriques

(i6 variables).

Les distances basées sur les données _génétiques et les

divergences

morphologiques et _{biométriques} sont _{également comparées} à certains aspects

du comportement lors de _{l’accouplement} et discutées en

fonction

du contexte

_écologique

et des _{caractéristiques}

_{biogéogmphiques}

de ces _espèces.

_L’analyse

des _{locus, correspondant}

à 13

_protéines

_{enzymatiques,} met en évidence des

_{différenciations}

_importantes entre ces

espèces: S colluvii est

_l’espèce

la

_plus

_divergente avec une distance

génétique

voisine de 1. L’étude _{morphométrique} estimée sur 16 caractères sépare les

_populations

des trois _espèces

selon la _largeur du pronotum et la _longueur des _élytres

_(S

_zophosinus les _{plus petits,}

S

_hydrophilus

les

_plus

_grands),

la

_largeur

des articles antennaires et la

_forme

du 8’ article antennaire. Il

_n’y

a _pas concordance entre les données moléculaires

_{(gènes structuraux)}

et

_{morphométriques:}

S _zophosinus sur le _plan moléculaire a _peu

_divergé

de S

_hydrophilus

*

Present address: Centre de Recherches sur le _{Polymorphisme Gdndtique,} des Populations

alors que la

différenciation

morphométr%que est _importante entre ces _espèces. L’action

différentielle

des pressions de sélection aux _{différents niveaux, protéique} et _{morphologique,}

peut rendre compte de ces d%scordances.

coléoptère troglobie / systématique / variations _{électrophorétiques} et morphométri-ques

INTRODUCTION

Closely

related

_species

_provide

_{opportunities}

to

_study

how the

_evolutionary

_process

operates in natural

_{populations. Nevertheless,}

it is often difficult to

_assign

taxo-nomic ranks or to reconstruct

_{phylogenetic relationships.}

A

_{high degree}

of

indepen-dence is often observed between molecular and

_{morphological}

evolution

_(Gorman

and

_Kim,

_1976;

Sene and

_Carson,

_1977;

Schnell et

al,

1978;

Turner et

_{al, 1979;}

Lessios,

1981;

Berlocher and

_Bush,

_1982;

_Allegrucci

et

_al,

_1987);

this _may be due

to

_varying

selection pressures on different traits

_(Turner

et

_al,

₁₉₇₉₎

or

merely

re-lated to the number of loci

_segregating

for the different characters

_{(Lewontin, 1984).}

Electrophoretic

separation

of enzymes has become a

_powerful

tool for

_{investigating}

systematic

and

_evolutionary

_problems

_{(Avise, 1974).}

_{Morphological}

studies alone

are

_usually

_inadequate

to determine

_evolutionary

_{relationships}

in

_closely

related

species.

Integrative

studies

_{utilizing allozyme variability}

and data from behavioural

breeding

systems in addition to

morphometric

variability

are most

_powerful

in such

complex

groups; then we can estimate the

_importance

of _gene

_{flow, genetic}

drift and selection in

_shaping

_population

structures.

The 3

_species

_{of Bathysciinae}

_(Coleoptera)

of this

_study

_belong

to a

_monophyletic

group of the genus

Speonomus

which contains many

cave-dwelling

species

from the

more

_primitive

to the more

_specialized

(Jeannel, 1924).

Studies on these

_species

have been the

_subject

of

_repeated

_{analyses by}

various

_approaches,

_including

the

study

of

_{morphology (Jeannel,}

_1924;

Juberthie et

_{al, 1980a;}

Juberthie et

_al,

_1981;

Delay

et

_al,

_1983),

_karyology

_(Durand

and

_{Juberthie-Jupeau,}

₁₉₈₀₎

and

_allozymic

variation

_(Crouau-Roy,

_1989a,

_b).

The

_relationship

of these beetles to one another

and to other

_species

_groups of

_Speonomus

has not been

_adequately

resolved. The 3

allopatric

species

S

_hydrophidus,

S

_zophosinus

and S

_{codduvii, narrowly}

endemic to

central

_Pyrenees

_{(fig 1),}

have

_undergone

some

_{ecological divergence}

and _present a

high

degree

of

_{specialization}

to

_underground

life

_{( eg}

life

_cycle

of 1 larval

_stage

with

suppression

of larval

_feeding,

_{Deleurance-Glagon,}

_1963).

These blind and

_wingless

troglobitic

species

occur in caves and

_deep

soil

_(Juberthie

et

_al,

_1980a,

_b)

in the massif Arize

_(S

_hydrophidus

at about 430 - 1440

_m),

in the

_valleys

of Salat and Arac rivers

_(S

_zophosinus

at about 480 - 620

_m)

and on the north face of the

massif des Trois

Seigneurs

(S

colduvii at about 700 m - 1350

m)

at the foothill of the French central

_Pyrenees.

The southern extent of the massif Arize

_(between

the area of S

_hydrophilus

and S

_xophosinus)

is

_composed

of colmated

_metamorphic

rocks which seem to be

_geological

barriers to

_underground

colonization

_by

cave

fauna. Their distribution

_strongly

_suggests

that

_speciation

_{occurred after gene flow}

was

_{interrupted by physical}

barriers: there were

_probably

_{geomorphological}

and

pedological

barriers after climatic shifts

_during

the ice _ages and

_{interglacials}

of the

The

_phylogeny

of these 3

_closely

related

_troglobitic

_(ie,

_obligate

cave

dwelling)

beetles was

_{investigated using}

_isozyme

_{electrophoresis}

and biometric

_analysis.

Patterns of

_allozyme

and biometric variation were

_compared

with some _aspects

of the

_mating

behaviour and

_aspects

of the

_ecology

of these

_organisms

to address the

_question

of

_{intrageneric relationships}

of

_species

within this _group of beetles.

The _present

_{study provides}

answers to the

_following

_questions:

₁₎

how much

genetic

variation is contained in these

_populations?

₂₎

how is the

_genetic

variation

partitioned

within and _among the isolated

populations?

3)

what

_phylogenetic

relationships

can be deduced from the

_isozyme

data and how

_congruent

are

_they

with biometrical based

_{relationships}

at the

_species

level?

₄₎

how do the different

approaches

contribute to our

knowledge

of evolution in this

group?

MATERIALS AND METHODS

Twenty-three populations

of S

_hydrophilvs,

6 of S

_zophosanus

and 5 of S colluvii

were studied for

_{electrophoretic, morphometric}

and behavioural studies.

Electrophoretic analyses

Electrophoresis

was

_performed

in slabs of 12%

_hydrolized

starch for the

_following

leucine

_{aminopeptidase}

_(Lap-1,

EC

_3.4.11.1),

malic enzyme

(Me,

EC

1.1.1.40)

hex-okinases

_(Hk-1,

_{Hk-3, Hk-4,}

EC

_2.7.1.1)

_{phosphohexose}

isomerase

_(Phi-1,

_Phi-2,

EC

_5.3.1.9)

and a

_{glycero-phosphate dehydrogenase}

(a-Gpdh,

EC

_1.1.1.8);

in slabs of

_acrylamide

for fumarase

_(Fum,

EC

_4.2.1.12),

_{hydroxybutyrate}

_{dehydrogenase}

(Hbdh-1,

Hbdh-2,

EC

_1.1.1.30),

acid

_phosphatase

_(Pac-1,

EC

_3.1.3.2)

malate

de-hydrogenase

(Mdh,

EC

_1.1.1.37),

and lactate

_{dehydrogenase}

_(Ldh,

EC

_1.1.1.28),

aldehyde

oxidase

_(Ao,

EC

_1.2.3.1).

Additional loci that could not be scored

un-equivocally

were deleted from further

_analysis.

Loci and alleles were

numbered,

respectively,

in order of

_decreasing

anodal

_{migration. Electrophoresis}

buffers and stains are described in

_Crouau-Roy

_(1986).

Individuals from different

species

were run

together

on the same

_gel

to determine whether

_{corresponding}

_{electromorphs}

had similar mobilities.

_Allozyme

_frequencies

for each

_sample

were derived from the

_{electrophoretic}

results. These data were

employed

to _compute

_genetic

distance estimates

_(Nei,

_1972,

₁₉₇₈₎

which were used for a

_{phenetic averaging}

(UPGMA;

Sneath and

_Sokal,

_1973).

The

apportionment

of

_{genetic diversity}

was determined

using genetic diversity analysis

(Wright,

1965;

Nei,

1977, 1986).

Total _gene

_diversity

(H

T

= 1 - E

xi:

_weighted

average allele

frequencies

over all

_populations)

is sub-divided _{into gene}

_diversity

within

_populations

_(H

_s

_:

_weighted

average over all

pop-ulations of the values

_{1- E xi}

for each

_population)

and _gene

_diversity

_among pop-ulations. Differentiation _among

_populations

is calculated as

F

ST

=

H

T

-

H

S

)/H

T

.

Morphometric analysis

Sample

sizes for all

_{morphological analyses}

were 30 adults per

population.

For S

hydro

P

hidus

only

18

_populations

out of the 23 were examined. The

_following

16

morphological

variables were measured from each

specimen:

length

(L)

and width

(W)

of 7 antennal _segments

_(5th,

_6th,

_{7th, 8th,}

_9th,

10th and

_{length only}

of the

llth),

length

of the tibia of the 3rd

_pair

of

_legs

_(LT),

_length

of the

_elytra

_(LE)

and width of the _pronotum

_(WP).

Measurements were recorded in micrometres.

Because of

_{statistically}

_significant

correlations between values for the _sexes, the

measurements were

_only

made in males. Euclidean distances were calculated on the

basis of the 16

_{morphometric variables;}

a

_dendrogram

was drawn _up

_using

these

distances,

according

to the UPGMA method of cluster

_analysis

_(Sneath

and

_Sokal,

1973).

Data were also

_{analyzed using}

a

_principal

_components

_analysis

_(PCA)

on

the standardized variables.

RESULTS

Electrophoretic

differentiation

Genetic structure of

_populations

_{at enzyme} loci

The allele

_frequencies

for each

_putative

locus

₍₁₈

_loci)

are

_given

in table I. Each locus

_containing

more than 1 variant was considered

_polymorphic.

Fourteen of these were

_polymorphic

(Est-6,

_Lap-1,

_{Phi-1, Phi-2,}

_Pac-1,

_{Hk-3, Hk-4, Hbdh-1,}

Est-1,

Hk-1).

Six loci

_(Mdh,

_{Hbdh-1, Fum, Ao,}

Ldh-2 and

_Phi-2)

are

_diagnostic

for at least 1

_species,

and 2 additional loci

_{(Est-6, Pac-1)}

are

diagnostic

with a

1%

probability

of error. Estimates of the

_proportion

of

_polymorphic

loci _per

_population

(P)

and the _average

_frequency

of

_heterozygous

loci _per individual

_(H)

indicate some

differences in

_{genetic variability}

between the 3

_{species. Speonomus}

colluvii

_displays

the

_highest

overall

_percentage

of

_polymorphic

loci

_(41.2%).

For the other

_2,

_percent

polymorphic

loci are

23.0%

in S

_hydrophalus

and

25.5%

in S

zophosinus. Expected

compared

to

_{expected Hardy-Weinberg}

_proportions

_{by x}

₂

or G-test

(Sokal

and

Rohlf,

1969);

x

2

or G values show

_significant

differences between observed

_genotypic

frequencies

and those

_expected

under

_{Hardy-Weinberg equilibrium.}

The

_genotypic

fixation index

_{(Wright, 1965),}

which measures the relative difference between

expected

(He!P)

and observed

_(H

_ob9

₎

_{heterozygosity,}

shows a

significant deficiency

of

_{heterozygotes}

_(F

_IS

_>

_{0) (table II) (Crouau-Roy, 1988).}

Genetic differentiation between

_populations

The

_genetic

differentiation within the

_Speonomus

species complex

was determined

using genetic diversity analysis

(F-statistics:

Wright,

1965;

Nei,

1977,

1986)

and

a x

2

contingency

analysis

of

_{heterogeneity}

_(Workman

and

_Niswander,

_1970).

Nire loci are variable in some or all

_populations

of each

_species

(table II).

Significant

heterogeneity

in gene

frequencies

X

2

was

observed _among S

_hydrophilus

_populations

at all variable loci.

_Significant

_{heterogeneity}

in allele

_frequencies

was observed

among

populations

of S

zophosinus

(only

at the Est-6

_locus).

For S

_colluvis,

2

variable loci

_(Lap-1

and

_Hk-4)

show

_{significant heterogeneity.}

There is

_significant

genetic

variation between

_populations

in the

_complex

of 3

_species

_{(table III).}

Genetic

diversity

for 5 loci is due

_totally

to the

_{between-species}

_component

_(F

_ST

₎

=

_1;

populations

fixed for different alleles:

_{Hbdh-1, Fum, Ao,}

LDh-2 and

_Phi-2).

For 3 additional loci

_(Mdh,

_Pc-1,

_Me)

almost all the

_diversity

is due to

_{between-species}

variation rather than

_{within-species}

variation

_(F

_ST

=

_0.978,

0.979 and

0.902,

among

species

indicates that a

large portion

of the

_genetic

variability

in

Speonomus

is not _present

_among

the individuals of a

_single

_species.

Extensive

_{isozyme divergence}

between

_species

is further indicated

_by

their low Nei

_{genetic identity}

values.

Nei’s standard

_genetic

distances corrected for

_sample

size between the

popula-tions of each

_species

are listed in table IV. Variances of Nei’s distances

(Nei

and

Roychoudhury,

1974)

were all on the order of

1.7%

-

2.5%

of the distance values.

Beetles from the 3

species

are well differentiated: coefficients of

_genetic

distance

range between 1.021 and 0.400. The

genetic

differentiation observed between the

species

S

_hydrophilus

and S

_zophosinus

is

_significant

_(D

=

0.431)

compared

_to that

observed within each of them

_{(intraspecific}

values of Nei’s index: 0.036 and 0.050

respectively). S

zophosinus

differed

_completely

from S

_hydrophilus

at 5

_isozyme

loci

(Mdh,

Ao,

Ldh-2,

Phi-2,

Est-6)

and differed

_{substantially}

at 2 others

_(Phi-1,

_pac-1).

The distribution of the loci with respect to

_{genetic identity}

exhibits the

_U-shaped

pattern characteristic of

_comparisons

between

_good

_species.

Of 18

loci,

most are

either identical in their allelic

_composition

_(55%)

or

_completely

different

(32%

of

its loci are

_diagnostic

for the

_other).

For S

_colluvii,

the

_genetic

differentiation from the other 2

_species

is _greater and affects an

_important

_part of the genome

(50%

of its loci are

diagnostic:

Ao, Fum,

_{Pac-1, Me, Mdh,}

_Hbdh-1,

_Ldh-2,

a-Gpdh,

Phi-2).

Morphometric

differentiation

The _average values for the 16

_{morphological}

characters are

_given

in table V and

relationships

between

_populations

of the 3

_species

have been tested

by

a Student

test. The S

_hydro

_P

_hilus

and S

_zo

_P

_hosinus

_{populations display}

_significant

differences for all measured

_{morphological}

_characters;

between S

hydrophilus

and S

_colluvii,

4

comparisons

are

significant

(L8,

_{L7, L6,}

L5)

and

_only

2 are

significant

(Lll, W9)

between S

_zo

_P

_hossnus

and S colluvii. In

_particular,

the

_shape

of the 8th antenna!

Table IV _reports, with Nei’s

_distances,

matrices of intra - and

_{interspecific}

Eu-clidean distances on

_morphometric

measurements. These data indicate that vari-ations in

_morphology

are more

_important

between

_species

than

_they

are within

(A,

Nei’s

_distances)

and

_morphometric

data

_(B,

Euclidean

_distances).

On the 2

_bases,

_populations

are clustered into 3 groups

_{corresponding}

to the 3

_species

except for 1

_population

of S

_hydrophilus

which clusters with the _group of S col-luvii

_{populations. Nevertheless,}

the

_morphometric

differences between the 3

al-lopatric species

of

_Speonomus

are discordant with the _pattern

_displayed

by

elec-trophoretic

dissimilarities. While on the molecular

_level,

S

_zophosircus

has

_diverged

less from S

_hydrophilus

than has S

_colluvii,

the

_{morphological}

distance between these 2

_species

is

_greater

_(Euclidean

distance: 263.09 f

_5.68).

The marked

_{dissimilarity}

of S

_zophosarcus

is

_distinctly

illustrated in a

plot

of the 3

_species

(conspecific

pop-ulations

_pooled)

on the 1st 2 axes

_generated

_by

the

_{principal components’ analysis}

(fig

4).

The raw

_data,

and the

_{principal-component}

_scores, are dominated

_by

size:

the 1st

_principal

_component,

_explaining

82% of the

variance,

is associated with gen-eral

_size,

with

_near-equal

contributions from each character. The 3

_species

differ

according

to the

_general

size of the individuals

_(S

_zophosinus

individuals are the

smallest and S

_hydrophilus

individuals the

_largest)

and also

_according

to the width of the 8 and 10 antennal

_segments

_(explained

_by

the 2nd

_principal

_component).

DISCUSSION

In

_systematic

_studies,

the choice of _{parameters may}

_{substantially}

influence the

patterns observed. Since selection

_intensity

is difficult to assess, consideration of

more than a

single

class of traits can allow a more reasonable evaluation of the

accuracy of

systematic

schemes. Several

_{investigations}

have demonstrated that

speciation

can occur with little or no

divergence

at _{structural gene loci}

_coding

1975; Wilson, 1976; Nevo,

1978).

These

_species

of

_Speonomus,

show

_high

_degrees

of

genetic

divergence;

this

_genetic

differentiation does not

_correspond

to the differences in allele

_frequencies

at the

_polymorphic

loci but to the fixation of

_specific

alleles. The _{greater part} of the

_isozyme

loci examined contain a

_{large portion}

of their

genetic diversity

among

species

rather than within

species

(table

III)

despite

the

high

level of within -

_population

variation in some local

_populations

of the 3

_species

(table IV).

Electrophoresis usually

underestimates the amount of

_variability

in a

species

or

_{population. Nevertheless, electromorphs}

can

provide

valuable taxonomic

information and we found 32 and 50% of the loci studied to be

_diagnostic

for any of the 3

_sibling

_species

of

_{Speonomus. Moreover,}

an obvious

_disparity

in size and behaviour

_{during mating}

between the 3

_species

is visible.

_Ethological

criteria have a

great

importance

in

_speciation

events. These

_Speonomus

show within a

_single

_species

a _very low

_{experimental frequency}

of

_matings

_{(Crouau-Roy, 1986)}

in contrast to

the 2 larval _stages of S delarouzei

_{(Juberthie-Jupeau}

and

_Cazals,

_1984).

_Thus,

the occurrence of

_reproductive

isolation between the 3

_species

cannot be shown.

Nevertheless,

during

mating,

constant and sometimes

_important

differences appear between the

_{species; they}

concern the

_length

of

_mating,

the different

_phases

of the

mating

and the

amplitude

and

_frequency

of abdominal movements of males

_during

mating

(Crouau-Roy, 1986).

These different _types of

_mating

behaviour _may enable

one to discriminate the 3

_species.

The consistent differences in

_{electrophoretic differentiation, morphometric}

and behavioural

_differences,

in

_conjunction

with

_{biogeographical}

distributions and

eco-logical

separation,

offer

_convincing

evidence of the _separate

_species

status of S

_hydrophilvs,

S

_zophosinus

and S colluvai. The results of this

_{study applied}

to

the taxonomic

_question

are in

_complete

_agreement

with the

_previously

determined

specific

status of these beetles based on male

_genitalia

_{(Jeannel, 1924),}

and on other

morphological

characters

_(Juberthie

et

_al,

_{1980a, 1981;}

_Delay

et

_al,

_1983).

What is _{the congruence} between biochemical and

_{morphological}

data? There is no _{congruence between} the 2 sets of characters at the

_species

level. Relative

degrees

of molecular

_divergence

between the 3

_species

do not concur with

_degrees

of

_{morphological}

differentiation between the same

_species.

The

_highest

_genetic

identity

values are found _among the

_species

S

hydrophalus

and S

zophosinus.

This

is in contrast to

_{morphological}

and biometrical

_analysis

in which these 2

_species

exhibit the

_greatest

difference

_(table

_IV;

_figs

2 and

_3).

S colluvii and S

_hydrophilus,

morphologically

more

_similar,

show _greater biochemical

_separation

_{(fig 2).}

What factors would account for the observed

_discrepancy

in

_degrees

of differentiation? The

_genetic

differences between these

_species,

at structural _genes measured

_by

electrophoresis

of the _gene

_products,

are the results of mutations accumulated since

the time of

_divergence.

This does not

_imply

that

_they

are critical in

_evolutionary

terms.

_{Electrophoresis}

can measure reduced gene flow or absence of _gene

flow,

but cannot say

anything

about

_changes

at the

_{regulatory level,}

which may be

very

important

in some

_speciation

events

_(Wilson

et

_al,

_1974).

_Application

of

electrophoretic

methods to taxonomic studies

_requires

caution in

interpretation

of

experimental

data. It is not easy to

assign

limits to the

_{electrophoretic variability}

disclosed

_{experimentally}

in relation to

_biological

criteria used in differentiation between

_species.

The processes of evolution of different aspects,

represented by

may have been

substantially uncoupled

and

_proceeded

_{independently}

or at different

rates. For

_example,

S colluvii _{appears to} have evolved

_{biochemically}

much more than

it has

_changed

_{morphotypically,}

in

_particular

from S

hydrophilvs.

The

_incongruence

between extent of

_divergence

in the 2 sets of characters could be the result of different sensitivities of each to different _components of the environment. The 3

_species

studied here differ in their

_genetic

structure

_(Crouau-Roy,

_1989a,

_b)

and in their

_ecological

characteristics

_(Juberthie

et

_{al 1981; Crouau-Roy,}

_1986).

The external

_{morphometrical}

characteristics of

_{Speonomus, traditionally}

used in

systematic

studies,

could be under extensive selective pressures

imposed

by

the local environment. Without

_{heritability data,}

1

_suggestion

is that the

_{morphological}

variation has been more

_{immediately shaped by ecological}

differences between the

species.

Further,

an unknown

_proportion

of the variation exhibited

_by

many of these traits could be environmental rather than

_genetic

in

_origin.

The

_{morphometrical}

data may reflect historical processes but are much more under the influence of

differential selective _pressures

_{(micro -}

and macro-environmental

_influences)

than the biochemical data.

ACKNOWLEDGMENTS

I would

_particularly

like to thank TC Kane for

_helpful

discussions and his valuable criticism of the

_manuscript.

I am

_grateful

to D D’Hulst for the

_computer

_program of the

_{genetic analysis,}

to C Juberthie and B

_Delay

for valuable discussions and assistance in the field

_collecting,

to C Ferre for technical assistance and to F Boineau for

_typing

the

_manuscript.

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