Allozyme assessment of genetic diversity within the relic Sicilian fir Abies nebrodensis (Lojac.) Mattei

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Allozyme assessment of genetic diversity within the relic

Sicilian fir Abies nebrodensis (Lojac.) Mattei

Fulvio Ducci, Roberta Proietti, Jean-Michel Favre

To cite this version:

Original

article

Allozyme

assessment

of

_genetic

_diversity

within the relic

Sicilian

fir

Abies nebrodensis

_(Lojac.)

Mattei

Fulvio Ducci

Roberta Proietti

a

Jean-Michel

Favre

b

a

Istituto

_{Sperimentale per la}

_{Selvicoltura,} viale S.

_Margherita,

80-52100 Arezzo,

Italy

b

Laboratoire de

_biologie

forestière associé Inra, faculté des sciences, BP 239, 54506

_{Vandœuvre-lès-Nancy}

_cedex, France

(Received 9

_April

1997;

accepted

I

_{1 February}

1999)

Abstract -

_Allozyme

markers (11 loci, 32 alleles) have been used to estimate the

_{genetic diversity}

within the

_{unique surviving}

popu-lation of the relic

_species

Abies nebrodensis. Results were

_analysed

in

_comparison

with a reference _system

_composed

of 16 Italian

populations

of A. alba and one

_{representative}

_{provenance of A.}

_cephalonica,

A. _{equi-trojani,} A. bornmuelleriana and A.

nordmanni-ana. These

_{investigations}

allowed us _i) to show that alleles Idh-2a and

_Pgi-1

a have contributed to the differentiation of the A. nebro-densis

_population

from those of the reference _system, ii) to show that the

_{genetic diversity}

within A. nebrodensis is similar to that of

dynamic

silver fir

_{populations growing}

in

_analogous

isolation and

_{progressive drifting}

situations, while,

simultaneously,

a _very

_high

excess of

_homozygotes

is _{detected, iii)} to

_identify

in situ three different zones which

_corresponded

to the

_diversity

core of the

_species,

one site in

_{recolonizing phase}

and one site in an extinction

_phase.

The

_origin

of this

_particular

situation is discussed and silvicultural interventions to relaunch the

_dynamics

of the

_species

are

_suggested.

_{(© Inra/Elsevier, Paris.)}

Abies nebrodensis / mediterranean firs /

_{genetic diversity}

/ allozymes

Résumé -

Évaluation

par

analyse

du

_{polymorphisme alloenzymatique,}

de la diversité

_génétique

au sein de

_{l’espèce relique}

Abies nebrodensis

_(Lojac.)

_{Mattei. Des marqueurs}

_{alloenzymatiques}

_{(11 loci,} 32 allèles) ont _{été utilisés pour évaluer la} diversité

génétique

au sein de la seule

_population

existante de

_{l’espèce relique}

A. nebrodensis. Les résultats,

rapportés

à un

_système

de référence

_composé

de 16

_populations

italiennes d’A. alba et _{d’une provenance}

_{représentative}

d’A. _cephalonica, A. _{equi-trojani,} A. bornmuelleriana et A. _{nordmanianna,} ont

_permis,

(i) de montrer _{que la}

_fréquence

des allèles Idh-2a et Idh-2b _permet de différencier A. nebrodensis des

_populations

du

_système

de référence, (ii) de montrer _{que la diversité}

_génétique

à l’intérieur d’A. nebrodensis est

comparable

à celle des

_populations

du

_système

de référence

_présentant

des situations d’isolement et de dérive

_{génétique comparables,}

alors

_qu’en

même _temps on observe un fort excès

_{d’homozygotes}

_(iii) de mettre en évidence in situ trois zones différentes

représen-tant

_{respectivement,}

_{le noyau de diversité de}

_l’espèce,

un site de

_reconquête

et un site en

_phase

d’extinction.

_L’origine

de cette

situa-tion

_{particulière}

est discutée et des mesures de

_{gestion susceptibles}

de favoriser la

_reprise

de la

_dynamique

de

_l’espèce

sont

pro-posées. (©

Inra/Elsevier, Paris.)

Abies nebrodensis /

sapins

méditerranéens / diversité

_génétique

/

_allozymes

1. Introduction

Abies nebrodensis is an endemic

_species

of

_Sicily

_[20,

22, 26]

represented by

a

_single

relic

_population

of

_only

29 adult trees and about 20 small

_seedlings

_[30]

_growing

*

Correspondence

and

_reprints

favre@scbiol.u-nancy.fr

on the Madonie _range, south of the

_city

of Cefalù

(figure

1).

This

_species

is the southernmost fir in

_Italy

and,

_together

with the

_Peloponnesus

Greek fir

_(A.

Cephalonica),

represents the southernmost

_expression

of

The occurrence in the Madonie

_region

of _many

endemic flora and fauna taxa testifies to the

_{participation}

of A. nebrodensis in a former _{very ancient}

_ecosystem,

which is

_{nowadays widely}

_destroyed

_owing

to intense human _pressure

_{[3, 37, 39]. However,}

the decline of the

species

seems to have occurred in

_relatively

recent times.

Indeed,

it has been established that beams made from fir

were still used in the XVIIth and XVIIIth centuries in

roofing

the churches of several

_{villages (Polizzi}

Generosa,

Petralia

_{Sottana, Isnello)}

located within a

30-40-km circle around _{the Madonie range}

_[26].

This

attests to the existence at the time of

_quite

extensive fir

forest resources

_including

A. alba and A. nebrodensis

populations

as confirmed

_by

Biondi and Raimondo

[4].

At _present, the Sicilian fir is considered as an

endan-gered original

gene

pool [36]

and several international

organizations

such as the Council of

_Europe

_[8],

IUCN

[18],

FAO

_[28]

mentioned A. nebrodensis in their red lists.

_Locally,

action was taken to

_protect

this

_germplasm

following

two directions: an in situ

_protection

of trees

was ensured

_by

the

establishment,

within the Natural Park of

_Madonie,

of a strict Reserve Area

_covering

the

A. nebrodensis

_population

_[10]

and an ex situ

conserva-tion programme is

being

carried out

_by

the Forest

Research Institute of Arezzo

_[30].

After the first inventories made

_by

Morandini in 1964

and 1968

_{[26, 27]}

and a field _survey carried out in

1992,

an

_updated

list of A. nebrodensis trees

_growing

in the Madonie range was drawn up

_and,

for each tree,

topo-graphical, morphological

and

_{phytoecological}

data were

recorded

[30, 37].

Two clonal

_grafted

collections

includ-ing

copies

of 27 of the 29

_compiled

trees were estab-lished in 1992-1993 at the Forest Research Institute of

Arezzo. Two trees were too small to endure scion removal without

_damage.

In _{this paper} we

_investigated

the

_{genetic diversity}

within this material which

_represents

an almost

exhaus-tive collection of the

_species,

_using

_allozyme

markers

which have

_proved

to be accurate in several

_genetic

and

phylogenetic

studies on Abies

_species

_{[1, 5,}

11,

12,

17,

21, 32, 33, 41, 42, 44].

This information is essential to

assess the

_{genetic potential}

of the

_species

in order to

re-establish a

_biological

_dynamics

and decide on

appropri-ate _conservatory actions.

A. nebrodensis was

_compared

to a _group of

_dynamic

populations

of silver fir

_{(A. alba)}

_ranging

from northern

to southern

_Italy

and one

representative

_{provenance of}

four fir

_{species originating}

in the eastern Mediterranean

region

(A. nordmanniana,

A.

bornmuelleriana,

A.

equi-trojani,

A.

_{cephalonica).}

2. Material and methods

2.1. Plant material

The 29 Sicilian adult fir trees are distributed over an area of about 150 ha

_(figure

_1).

This zone can be divided into four main sub-zones

according

to the site

_morphology

and

_{phytoecological}

parameters [23, 24, 37].

1)

The central sub-zone of the lower _part of Vallone

Prato is

_{phytoecologically}

variable.

_Depending

on

orien-tation and

_altitude,

the Sicilian fir trees occur in three

sit-uations:

- in the middle

part

(trees 18-20, 29)

beech

_(Fagion)

with Luzula sicula is

dominant;

- _on the western

side,

Quercus

petraea and

_Q.

pubes-cens are _present with

_{Brachypodium sylvaticum}

and

Juniperus

hemispherica

(trees 2, 14-17, 26-28);

- in the south-eastern

part, fir trees

(nos 7,

8,

12, 13)

are scattered over a wide

_{moving slope}

area.

2)

The

_peripheral

sub-zone of Vallone della Madonna

degli Angeli

which

_mainly

includes

northern-north-east-em

_{slopes, belongs}

to the

_Quercion

ilicis

_{(trees: 21, 22,}

30,31).

4)

The

_peripheral

sub-zone of Monte Pene and Monte Scalone

_ridges

characterized

_by

_very

_windy

_positions

with exposure to the

_north-east,

is covered

_by

mixed

patches

of

_{Geranio-versicoloris-Fagion}

and Cisto-eric-etalia (trees:

1, 4, 6, 9-11);

Only

18 out of the 29 adult fir trees

_{produce pollen}

and/or cones. For this reason we observed two distinc-tive

_populations

in the

_analyses:

- Nebr

1,

representing

the total

_population

of the 27

grafted

trees;

- Nebr

2,

representing

that _part of the

_population

which is

potentially capable

of

_contributing

to stand

regeneration.

This second

_population

is

_composed

of tree

nos

_{1, 2, 6-13, 17-23, 27.}

Nebr 1 and Nebr 2 have been

_compared

to a reference

system

composed

of 16 A. alba

_populations

from

_Italy

(several

have been selected as seed stands

_by

Morandini

and

_Magini

_[29])

and one

_{representative}

_provenance

_[11,

12, 25, 41] of

each of the

_following

Mediterranean fir

species:

A. nordmanniana, A.

bornmuelleriana,

A.

equi-trojani

and A.

_cephalonica

_{(table I).}

All these

popula-tions have been described as

_dynamic,

with

_good

natural

regeneration.

2.2.

_Allozyme

_analysis

Allozyme

analysis

was

_performed

on

_samples

of

about 30-40 buds per tree, collected

during

winter. The

_sample

extraction was carried out after

centrifu-gation

of the

_homogenated

tissues for 10 min at 10 000

g. The

electrophoretic

and

staining procedures

were per-formed

_according

to Conkle et al.

_[7]

and Santi

_[40].

Eight

enzyme

systems

coded for

_by

12 loci were

analysed: glutamic-dehydrogenase

(Gdh,

EC

_1.4.1.2),

glutamic-oxaloacetate-transaminase

(Got,

EC

2.6.1.1),

isocitric-dehydrogenase

(Idh,

EC

_1.1.1.42),

leucine-amino-peptidase

(Lap,

EC

3.4.11.1),

malate-dehydroge-nase

_(Mdh,

EC

_1.1.1.37),

6,posphogluconic-dehydroge-nase

_(6,Pgdh,

EC

_1.1.1.44),

phosphogluconic-isomerase

(Pgi,

EC

_5.3.1.9)

and

_{shikimate-dehydrogenase}

_(Skdh,

EC

_1.1.1.25).

Due to insufficient

_availability

of

_samples,

this last enzyme

system

has

_only

been

_analysed

in the Nebr 1 and Nebr 2

_populations.

The inheritance models of

_isozyme

variants were

described for Abies

_{species by}

Schroeder

[42],

Bergmann

et al.

[1],

Fady

and Conkle

[11],

Pascual et al.

2.3. Statistical

analysis

In order to assess

_genetic

variation within the

popula-tions,

the

_following

_parameters

were used: allelic

fre-quencies,

mean number of alleles per

_locus,

_percentage

of

_polymorphic

loci,

deviation from

_{Hardy-Weinberg}

equilibrium,

observed

_(Ho)

and

_expected

_(He)

heterozy-gosity

and the fixation index

_(Fis),

which were calculat-ed

_{using Biosys-1}

_{[9, 43].}

The Levene’s

_[43]

correction for small size

_samples

was used to _carry out the Chi square test for deviation from the

_{Hardy-Weinberg equilibrium.}

For the

_analysis

of the

_genetic

variation within the Sicilian fir

_population,

the

_genotype

_pattern of each tree

was transformed into

_binary

_language

_(each

allele at

each locus was scored 1 _{for presence} and 0 for

absence).

Data were then

_processed

_using

the NTSYS statistic

soft-ware

_[38]

to _carry out

_{correspondence analysis}

and UPGMA

_clustering.

2.4.

_{Topographical}

distribution of the

_genotypes

In order to visualize in situ the

_genetic

differentiation within the A. nebrodensis

_population,

the clusters estab-lished after the NTSYS

_analysis

were

_plotted

on the

map, tree

_by

tree.

3.

Results

3.1. Genetic variation within the

_populations

of the Abies reference

_system

The 11 loci

_analysed

were

_polymorphic

in at least one

of the 20 reference

_{populations. Thirty-two}

alleles were

observed

_{(table II).}

In A.

alba,

the mean number of alleles

per locus estimated

using pooled

data without

_considering

the

_population

sub-divisions,

was 2.8

_{(table III).}

_Among

the

_populations

it

_ranged

from 2.5 to 1.5 and the percent-age of

polymorphic

loci varied from 36.4 %

(La Verna)

to

90.9 %

_{(San Francesco,}

Santa Maria and Listi

basso).

Lowest values of these

_parameters

were recorded in the northern

_Alpine

_provenances

_(Paularo

and Chiusa

_Pesio).

Values for eastern fir

_{species populations}

were

global-ly

similar,

though varying

within a narrower _range.

The observed

_{heterozygosity}

_(Ho)

_ranged

from 0.108

to 0.248 in the A. alba reference

_populations

and from

0.157 to 0.264 _{among the} eastern Abies

_species.

Positive

values of estimated Fis in all

_populations

_{(table III)}

indi-cated a

_{general heterozygote deficiency}

within the refer-ence _system. The lower deficiencies were observed in

the southern

_populations

_{(Monte Pecoraro, Archiforo,}

Fossa

_Nardello,

List

_alto).

Loci that deviated less

_frequently

from

Hardy-Weinberg equilibrium

were

Idh-2,

_6,Pgd-2,

Gdh-1,

_Pgi-1

and

_Pgi-2

_{(table IV). Idh-2,}

_6,Pgd-2

and Gdh-1 were

characterized

_by

an excess of

_{heterozygosity}

_among the

examined A. alba

_populations.

3.2. A. nebrodensis

_compared

to the reference

_system

Results of table III

_clearly

show

_specific

traits of

genet-ic structure in the Nebr

_{1 population. Compared}

to the

ref-erence _system, the mean number of alleles _per

locus,

% of

polymorphic

loci and Ho were inferior.

_Higher

value of

Fis indicated an increased

_{heterozygote deficiency.}

These

observations were

_particularly

evident when Nebr 1 was

referred to the A. alba

_{pooled population.}

However, when the

_comparison

was made

_individually

with each of the 16

A. alba

_populations

included in the reference _system,

some variations could be observed. The Nebr 1 mean

number of alleles _per locus and % of

_polymorphic

loci

were _very similar to that measured in the A. alba

exten-sive

_populations

of northern and central

_Italy

_(Chiusa

Pesio,

La

_Verna),

while wider

_divergences

were found

with the southern

_populations

_{(Fossa Nardello,}

San

Francesco,

Macchia di Pietra and List

_alto).

He was close to that of several A. alba

_populations

(Chiusa Pesio,

Abeti

_Soprani)

and in some cases

_superior

to northern

_(Paularo)

or small and

_relatively

isolated

populations

(La Verna,

Gariglione).

Neverthless,

Ho in

Nebr 1 was lower than in all the silver fir

_analysed

popu-lations.

Allele

_frequencies

also showed Nebr

_1-specific

traits

(table II).

Idh-2a for instance exhibited a

_higher

frequen-cy in Nebr 1 than in the reference system

while,

con-versely,

Idh-2b was rare. A similar situation was

observed for allele

_Pgi-1a

versus alleles

_Pgi-1b

and c.

The number of rare or absent alleles in Nebr 1

₍₁₅₎

was

_higher

than in the A. alba

_{pooled population}

₍₉₎

although

the wider

_sample

size in this

_species.

However,

the number of absent alleles observed in the silver fir

was about twice as

_high.

Significant

deviations from the

Hardy-Weinberg

equilibrium

were found in five of the

11 examined alleles.

The main characteristics of the

_genetic

structure

observed in Nebr 1 were also found in the Nebr 2

restricted

_population.

The observed differences

con-cerned

_principally

the

_percentage

of

_polymorphic

loci

and the estimated Fis which were inferior in Nebr 2

(table III). However,

among the 11

analysed

loci,

seven

exhibited

_slight

excess of

_{heterozygotes}

_(table

_IV).

Neverthless,

the mean value of estimated Fis remained

positive

(table

III).

Results were similar for Skdh-2

_(Fis:

3.3. Genetic differentiation within the A. nebrodensis

population

and in situ structuration of

_diversity

The 27 A. nebrodensis trees showed different

geno-type in at least one locus.

Seventy-three

_per cent of the

total variance were

_{explained by}

the first five factors of

the

_{correspondence analysis.}

Seven alleles were

signifi-cantly

correlated to these factors:

_Idh-2b,

_6,Pgd-1a

and

1b,

Pgi-1b,

Got-2a and 2b and Got-3a.

The UPGMA

_dendrogram

built

_using

these alleles is

given

in

_figure

2.

_Taking

into account the small size of the

_population,

we

_accepted

a differentiation into three

main clusters. Cluster A included 12 trees

_{(nos 2, 6, 12,}

13, 16, 18-20, 22-25),

cluster B

_grouped

13 trees

(nos 1,

4, 7-11, 15,

17, 21, 26-28)

and cluster

C,

two trees

_(nos

14 and

29).

The Nebr 2

_population

was

_represented

in clusters A

and

_B,

each with 50 % of total trees.

Trees of the different clusters were then

_plotted

on the

map

(figure

1).

It

_appeared

that all of the three clusters

were

_represented

in the central sub-zone

_(bottom

_part

of

Vallone

_Prato).

In contrast, in the

_peripheral

sub-zones

trees

_belonged

to

_only

one of the main

clusters,

namely

cluster A in the

_peripheral

sub-zone of Monte

_Cavallo,

and cluster B in the

_peripheral

sub-zone of Monte Pene-Monte Scalone. Tree 6 on the Monte Scalone crest was

the

_unique

_exception.

The

_peripheral

sub-zone of Vallone della Madonna

degli Angeli

was characterized

_by

the _{presence of} trees

nos 21 and

22,

_belonging

to clusters A and

B,

respective-ly.

The in situ localization of the rare alleles identified in

table

_II,

confirmed this

_unequal

distribution _{of the}

geno-types

within the range of the

species

(table

V). Indeed,

4. Discussion

1)

Many

of the

_{previous allozyme}

studies carried out

on Abies

_{species reported}

a

_deficiency

of

_{heterozygotes}

regardless

of the examined _enzyme

loci,

the number of

analysed populations

or the

_sample

size. This was shown in several

_populations

of A. alba as well as in other Mediterranean fir

_species

such as A.

_cephalonica,

A.

equi-trojani,

A. bornmuelleriana and A. borisii

_regis

_[1,

10, 11, 21, 32, 41, 42, 44].

In A. alba for

_instance,

esti-mated Fis values

_ranging

from 0.140 to 0.280 have been

reported

[44:

northern Italian

_{populations] indicating}

clear excess of

_homozygotes.

_However,

in some

popula-tions,

lower Fis values

_showing

no

_significant

differ-ences from the

_{Hardy-Weinberg equilibrium}

have been observed. This is

_notably

the case of the Calabrian popu-lation of Serra San Bruno in which the

_reported

Fis

val-ues _{range from -0.080}

_[44]

to 0.050

_[32].

For a

_given

population,

differences _among authors could be

_high.

In

Abeti

_Soprani

_(central

_Apennines)

for

instance,

Fis

val-ues

_ranged

from 0.050

_[44]

to 0.188

_(calculated

from

Parducci et al.

_[32]),

but the

_analysed

_loci,

the revealed alleles and the number of

_sampled

trees were different.

The results obtained in this

_study

confirmed the gen-eral trend to

_{heterozygote deficiency}

observed in Abies

species, especially

when

_referring

to small and isolated stands. The occurrence of self

_pollination

in Abies

species

[11, 41],

confirmed in A. alba

[13, 32],

together

with the Walhund effect

_resulting

from the ancient

frac-tioning

of the natural _range of Abies around the Mediterranean

basin,

have

_probably

contributed to the

maintenance,

such

_high

_homozygosity

levels. The artifi-cial

_origin

of the A. alba Tuscan

_populations

can also

have determined

_{higher deficiency}

of

_{heterozygotes}

than

in the natural

_populations

of this

_species.

In addition to low

_{heterozygosity,}

some authors

_([1]

and to some extent,

[44])

confirmed

_by

Parducci et al.

[32]

also observed an

_increasing

variation in

_genetic

parameters from the northern

_Alpine

to the southern Italian A. alba

_populations.

Our results also confirmed such a clinal northern-southern

_gradient

within the

Italian range of this

species,

more

_evidently

when the artificial

_populations

of

Camaldoli,

Campigna

and Vallombrosa were excluded.

In

_conclusion,

the overall

_consistency

of our results with the

_previously

collected

data,

also evident for

genetic

parameters

such as the number of alleles _per

locus, percentage

of

_polymorphic

loci and

He,

shows that the 16 A. alba and four eastern fir

_species

popula-tions

_analysed

can be considered as

_{representative}

of the

general

situation in the Mediterranean Abies

_species,

and thus validates the choice of these

_populations

as a

refer-ence _system in

_assessing

the

_genetic

_diversity

within A.

nebrodensis.

2)

In a

_general

_way, the

_{genetic diversity}

within both

the Nebr 1 and Nebr 2

_populations

was lower than in the reference _system.

_However,

a detailed examination of

the main

_genetic

_parameters

showed similarities with

several A. alba

_populations

_(Paularo,

Chiusa

Pesio,

La Verna and

_Gariglione)

which share with A. nebrodensis

common traits such as

_ecological

_conditions,

altitudinal and/or

_{geographical position, long}

time isolation and

progressive

reduction in tree

_density.

The northern

_{geographic position,}

where

_genetic

diversity

is known to be low

_[1]

_{and exposure} to extreme

climatic conditions

_owing

to altitude and relative isola-tion for

instance,

can

_explain

the results obtained in

Paularo and Chiusa

Pesio,

respectively.

The

_isolating

effects of altitude and local

_topography

can also account

for the

_similarity

of

_genetic

_parameters

in the small-sized

population

of La Verna and A. nebrodensis. The case of

the wider

_population

of

_Gariglione

which

_surprisingly

showed lower

_{genetic diversity}

(He)

than A. nebrodensis and all the other

_analysed

Calabrian

_populations,

could be considered as a result of

_long

time

_geographic

isola-tion as

_already

indicated

_by

Parducci et al.

_[32].

A. nebrodensis collects

_together

the

_specific

traits of these four

_{geographically}

distant

_dynamic

A. _alba

topo-graphic

isolation,

extreme climatic and

_{edaphic growth}

conditions,

anthropic

pressure, and a

_dramatically

reduced number of trees. The main difference concerned the

_genetic

structure, characterized

_by

a

_{really high}

excess of

_homozygotes.

Fis values recorded in both the

Nebr 1 and Nebr 2

_populations

were about twice as

_high

as in the

_pooled

A. alba reference

_population

and even

more in the

Paularo,

Chiusa

_Pesio,

La Verna and

Gariglione populations.

These _very

_high

values of Fis could be attributed to

several causes

_probably

_joint

in their

results,

i.e. too

great a distance between the trees, increased rate of self

pollination

due to

_{high scattering}

of the trees,

genetic

drift,

stochastic selection effect of

_position

of the

_living

trees and of their earlier _parent trees

_{[14, 15].}

In contrast with the estimated Fis the other

_genetic

parameters

(especially

the number of alleles per

locus,

percentage

of

_polymorphic

loci and

_He)

exhibited close

values in the Nebr 1 and Nebr 2

_{populations compared}

to

the four

_comparable

A. alba

_populations

of the reference

system.

This _suggests that

_despite

the relic conditions and very small size of

population

(27 trees),

A. nebro-densis still retains a

_{representative sampling}

of the

genet-ic

_potential

of the former tree

_generations,

when the stand was more extended. The fact that the allele

_pattern

found in Nebr 1 and in Nebr 2 was

_relatively

similar to

those of

_populations

of other

_species

confirmed this

con-clusion.

Indeed,

among the 32 alleles detected within 11 enzyme

systems

only

two

_(Idh-2a

versus Idh-2b and

Pgi-la versus

_Pgi-1b)

turned to

_higher

or lower

_frequency

in

A.

nebrodensis,

whereas

_they

were

_conversely

rare or

frequent

in the reference

_system.

The case of locus Idh-2 was

_{especially interesting}

to

consider.

_Frequency

of allele Idh-2a was two to ten times

and

_Gregorius

_[2]

_studying

45

_{European populations}

of A. alba

_including

six from

_Calabria,

showed inverse variation from north to south of Idh-2a and Idh-2b

fre-quencies,

the former

_being

lowest in the south.

_They

interpreted

this result as a _{consequence of} a lower

ther-mostability

of this allele.

_Thus,

the maintenance within

A. nebrodensis of a

_{high frequency}

of Idh-2a

(present

in

all trees but

_one)

could be due to the cold climatic condi-tions

_(altitude,

northern

_slopes)

of the Madonie

site,

which reduce the selection

_against

this

_negatively

ther-mo-influenced allele. The same

_explanation

can be

_given

to account for the results of

_Longauer

_{[21] who}

also found

_high

Idh-2a

_frequencies

_(0.70)

in Calabrian popu-lations of A. alba close to A. nebrodensis.

Finally,

compared

to the reference _system, the relic

population

of A. nebrodensis

_appeared

to be character-ized

_by

both

_relatively

normal

_{genetic diversity}

_(i.e.

number of alleles _per

locus,

number of

_polymorphic

loci,

expected heterozygosity)

and increased

_deficiency

of

heterozygotes. According

to

_Gregorius

and

_Bergmann

[ 14]

the

_higher

_frequency

of some

_alleles,

as well as

homozygosity,

could be

_{explained by}

the

_adaptation

to

peculiar

local environmental

_conditions,

_indicating

thus that the

_{genetic diversity}

within A. nebrodensis is

com-patible

with a

_possible

_restoring

of

_dynamics

in the

pop-ulation.

3)

The in situ identification of trees

_according

to the UPGMA

_clustering

of the A. nebrodensis

_population

showed a

_{heterogeneous}

distribution of

_genetic

variation

in connection with the

_high

microenvironmental

diversi-ty of the

_species

area. Such an

_{explicative relationship}

between

_genetic

variation and microenvironmental

diversity

has also been

_suggested

_by

Müller-Starck

_[31]

for altitude

_Alpine

_{spruce stands.}

Within the Sicilian _{fir range,} three different situations

can be described. On both the southern

_peripheral

crest

zones of Monte Cavallo and Monte Pene-Monte

Scalone,

which are

_subjected

to extreme environmental

conditions,

especially freezing

cold

winds,

trees

belonged

to

_only

one of the three identified clusters. The

future

_long-term

survival of these trees is

doubtful,

and

although

some

_seedlings

are _present around tree 1 on the Monte Pene

_summit,

an efficient

_recolonizing

_{process is}

very

unlikely.

These sites can be therefore considered as

in an extinction

_phase.

The situation in the central zone of Vallone Prato is

quite

different. The three _genotype

_clusters,

as well as

the five rare alleles

observed,

were

_represented

_among

the trees _present on this site. Vallone Prato can be thus

considered as the

_diversity

core of the

_population,

con-taining

the main _part of the gene

pool

and hence

consti-tutes the

_priority

zone for in situ conservation.

It is

_important,

however,

to note that this central zone

also conceals

_high

microenvironmental and

phytoecolog-ical

_diversity

which can _represent, on the one hand

suit-able conditions to maintain

_{genetic diversity,}

but on the

other adverse conditions for natural

_regeneration

and

population

increase.

_Along

the _upper limits of the

Vallone,

as on the

_{surrounding ridges}

of Monte

_Scalone,

trees are

_exposed

to drastic

_edaphic

conditions

_(deep

intensively

eroded

_{rocky slopes)}

which are unsuitable for

regeneration.

In lower locations the soil conditions are

better,

but trees are

_subjected

to

_increasing

beech

cop-pice competition.

The evolution of the beech

_coppice

into

_high

forest formation was

_predicted

as

_early

as 1960

by

Hoffmann

_[16]

and

_recently

confirmed

_by

Raimondo

et al.

[37].

The environmental

conditions,

especially

the

light

conditions,

created

_by

the

_expansion

of the

_coppice,

today aged

40-45 years

[16]

are unsuitable for cone

pro-duction,

germination

and

_development

of the fir

seedlings.

In some cases the fir trees are in

_danger

of

being surpassed by

the

_coppice.

_Examples

of such a

regression

and sometimes

_complete

substitution of Abies

by

beech have been observed in several Calabrian forests

[6].

This evolution within the

_diversity

core of the

popula-tion poses a threat to the future of the fir trees.

Silvicultural interventions should be

_rapidly

realized in order to stimulate the fir

_reproductive

maturation and

regeneration.

Selective

_thinning

of the beech

_coppice,

for

al.

_[6]

and Lovino and

_Menguzzato

_[19]

to _{preserve the}

A. alba

_populations

of the Calabrian forest.

The northern zone of Vallone della Madonna

_degli

Angeli

harbours the

_only

two adult trees at _present able

to _regenerate

_{living seedlings}

with wide survival poten-tial

_(trees

21 and

_22).

_They

_represent the

_unique

_part of the

_population

which can be considered in

_expansion.

They belong

to clusters A and

_B,

_{respectively.}

Tree 22

possess the Got-2a rare allele. These trees

_possibly

derive from Vallone Prato and find in this new site

favourable

_ecological

conditions.

Indeed,

in the

_opening

of this small

_valley,

_{phytoecological}

conditions are

dif-ferent from those of Vallone Prato. As in the

_typical

Mediterranean oak/fir succession

[34, 35],

A.

nebroden-sis is associated there with

_Quercus

ilex and a mixture of

mountain and sub-mountain harwoods and shrubs such

as Fraxinus ornus, Arbutus

unedo,

Ilex

aquifolium,

Acer

campestre, etc.

_{[ 16,}

_26]

and finds

_appropriate

conditions

to re-start an

_{expansion phase.}

Therefore,

in contrast with Vallone

Prato,

any

sylvi-cultural intervention should be avoided in this zone.

Only

the establishment of small diffusion cores

com-posed

of

_propagation

material issued from the three

clus-ters could

_possibly

be considered.

_Special

interest could also be

_given

to the diffusion of

_seedlings

born from rare

allele carrier mother trees.

In this _way, the

_{genetic diversity}

_patterns within this

potential expansion

zone of the

_species

could be

improved.

Acknowledgements:

This work has been

_partially

funded

_by

the Italian-French framework of scientific

co-operation

’Galileus’ and

_by

the EC research _programme

’Mediterranean Firs and Cedars’. The authors wish to

thank Professor Riccardo

Morandini,

former Director of

the Forest Research Institute of

_Arezzo,

for his com-ments and

_suggestions.

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