Genetic profiles from coat genes of natural Balearic cat populations: an eastern Mediterranean and North-African origin

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Genetic profiles from coat genes of natural Balearic cat

populations: an eastern Mediterranean and

North-African origin

M Ruiz-Garcia

To cite this version:

Original

article

Genetic

_profiles

from

coat

_genes

of natural Balearic

cat

_populations:

an

eastern

Mediterranean

and North-African

_origin

M

Ruiz-Garcia

1

1

Instituto

de

_Genetica,

Universidad de Los

_Andes,

Calle 18, Carrena, 1E,

Bogota DC, Colombia;

2

CICEEM Avd

_Virgen

Montserrat 207 ! lQ. Barcelona

_{08026, Spain}

(Received

6 November 1991;

accepted

6

August

1993)

Summary - A detailed

_study

of 7 cat

populations

(Felis

silvestris

_catus)

in the 3

_principal

Balearic islands has been carried out. These

_populations

are Mahon

(474

cats),

Villacarlos

(226 cats),

Mercadal and

Alayor

(104 cats)

and Ciudadela

_{(510 cats)}

in

_Minorca,

Palma

Majorca

(475 cats)

in

_Majorca

and Ibiza

city

(210 cats)

and San Antonio

₍₆₃

_cats)

in Ibiza. The gene

frequencies

derived from the

_{phenotypic frequencies}

of a number of loci

_coding

for coat colour and _pattern, hair

_length

and one skeleton

_anomaly

were studied with the

following implied

mutant allele: 0

_(Orange;

sex-linked

_allele);

a

(Non-agouti); t

b

(Blotched

tabby);

d

_{(Dilution); l (Long hair);}

S

_{(White spotting);}

W

_{(Dominant white);}

c’

_(Siamese);

and M

_(Manx).

The range of

frequency

values for each of the loci studied is the

following:

0: 0.16-0.30; a: _0.72-0.87;

t

b

:

_0.0-0.35; d: _0.14-0.44; 1: _0.0-0.27; S: _0.14-0.30; W:

0.0-0.017; cs: _0.12-0.31; M: 0.0-0.026. In some

populations

in Minorca a

significant

excess of

homozygotes

was detected for the 0 locus which

_might

be due to the influence of some

evolutionary

agent.

Though

the

_{genetic heterogeneity}

of the Balearic cat

_populations

is

substantially

lower than that observed for other island mammals and the theoretical _gene flow between these Balearic cat

_populations

is

_noticeably

_stronger than that observed for other

_populations

of mammals in these islands as well as in other

_islands,

there is a

statistically significant genetic heterogeneity

between most of the loci studied and between the

genetic profiles

of the 7 cat

_populations.

Some alleles

_(d,

_S,

W and

t

b

)

even show a clinal

disposition.

An

_analysis

of the contribution of each locus to the gene

diversity

observed between the Iberian and Balearic cat

_populations

shows that the

_largest

_part of this

diversity

is due to the

t

b

allele.

_{Generally speaking,}

all the _genetic

_{profiles analyzed}

show

stronger

genetic

influences of eastern Mediterranean and North-African cat

_populations

than of western

European

cat

populations. However,

of the 7 cat

_{populations studied,}

that of Palma shows a

_slightly

_stronger influence of western

_European

cat

_populations

while the central and eastern

_populations

of Minorca

_(Mahon,

Villacarlos and

_particularly

Mercadal and

_Alayor)

seem to have followed a

characteristically

different

evolutionary path

the Mediterranean sea which have not _yet been

_thoroughly

studied. The

_{possible origin}

of other species of mammals and the historical and commercial movements of the human

beings

in these islands

_might

be

_parallel

to the model

_proposed

for the cat

populations

of the Balearic islands.

cat

_/

_{population genetics}

_/

coat colour genes

/

genetic heterogeneity

/

gene flow Résumé - Profils génétiques de _populations naturelles de chats des Baléares sur la

base de gènes de _{pelage :} une _provenance de Méditerranée orientale et _d’Afrique du Nord. Une étude détaillée de 7

_populations

de chats

_(Felis

silvestris

_catus)

a été

réalisée dans les

_{3 principales}

îles Baléares. Ces

_populations

sont Mahon

_{(l!74 chats),}

Villacarlos

_{(226 chats),}

Mercadal et

_Alayor

_{(104 chats)}

et Ciudadela

_{(510 chats)}

à

Minorque,

Palma de

Majorque

(475

chats)

à

_Majorque

et Ibiza-ville

_{(210 chats)}

et San Antonio

_{(63 chats)}

à Ibiza. Les

_{fréquences géniques}

dérivées des

_{fréquences phénotypiques}

de

quelques

loci codant pour la couleur et le dessin de la

robe,

la

_longueur

du

_poil

et

une anomalie

squelettique

ont été étudiées pour les allèles mutés suivants: 0

(orange:

allèle lié au

_sexe),

a

_{(Non Agouti), t}

b

(Moucheté tacheté),

d

_(Dilution),

1

_{(Long poil),}

S

(Tacheté

blanc),

W

_{(Blanc dominant),}

cg

_(Siamois)

et M

_(Manx).

L’étendue de variation

des

_{fréquences géniques}

est la suivante: O: 0,16-0,30; a: _0,72-0,87;

t

b

:

_0,0-0,35; d:

0,1l,-0,4/,;

l:

0,0-0,27;

S:

0, 14 -0,30; W: 0,0-0,017;

c’:O, 12-0,31 ;

Nl: 0,0-0,026. Chez

certaines

_populations

de

_Minorque,

un excès

significatif d’homozygotes

a été détecté au

locus 0 dû à

_{l’influence d’un facteur sélectif.}

Bien que

l’hétérogénéité génétique

des chats

des Baléares soit notablement

_inférieure

à celle observée chez d’autres

_Mammifères

îliens

et que le

flux génique théorique

entre ces

_populations

_félines

des Baléares soit notablement

plus fort

que ce _qui est observé pour d’autres

populations

de

_Mammifères

de ces îles et

d’autres

_îles,

il existe une

_{hétérogénéité génétique}

_{statistiquement. significative}

entre la

plupart

des locus et entre les

_{profils génétiques}

des 7

_populations.

_Quelques

allèles

_(d,

S,

W et

_t

_b

₎

manifestent

même une tendance clinale.

_L’analyse

de la contribution de

chaque

locus à la diversité

génétique

observée entre les chats de

l’Espagne

et des Baléares

montre que la

plus grande

part de cette diversité est due à l’allèle

t

b

.

D’une manière

générale,

tous les

_{profils génétiques analysés}

montrent des

_{influences génétiques plus fortes}

des

populations

de chats de Méditerranée orientale et

d’Afrique

du Nord que de celles

d’Europe

occidentale. Mais _parmi les 7

_populations

de chats

étudiées,

celle de Palma

montre une

influence légèrement plus forte

des

_{populations d’Europe occidentale,}

alors _que

les

_populations

centrales et orientales de

_Minorque

_(Mahon,

Villacarlos et

_{particulièrement}

Mercadal et

_Alayor)

semblent avoir suivi une évolution

différente marquée

_par un

effet

fondateur,

une dérive

génétique

et/ou

des

flux géniques différentiels

à _partir d’autres localités autour de la Méditerranée qui n’ont _pas encore été étudiés d’une manière

_précise.

Les

_{origines possibles}

d’autres

espèces

de

_Mammifères

et les mouvements humains dans

ces îles

_pourraient

être

_parallèles

au modèle

proposé

pour les chats des îles Baléares. chat

_/

génétique des populations / gène de coloration / hétérogénéité génétique /

flux _génique

INTRODUCTION

More than 100 studies on the

frequencies

of alleles at loci that affect the fur of cats in more than 300

_populations

_throughout

the world have been carried out

of facts about the Iberian Peninsula and the Balearic islands has been remarkable

until the last 3 or 4 yr. This

_study

is an effort to

_provide

these

_genetic

data for

the Balearic cat

_populations.

In this

_work,

we use the

following

plan:

a)

observe

the individual existence of

_genetic

_{heterogeneity}

at each locus

and,

globally,

in the

_genetic

_profiles

between the 7 Balearic cat

_populations

taken into

_account;

b)

find out if this

_{heterogeneity}

found

_individually

at each locus and

_globally

is in any way

spatially organized

in Minorca and in the whole of Balearic

_islands;

and

_c)

_investigate

the

_possible

_origins

of the 7 Balearic cat

_populations.

Ruiz-Garcia

_{(1988, 1990b)}

stated that there were 2 areas on the

_Spanish

Mediterranean

coast with differentiated

_genetic

_pools

in their cat

_populations.

One of these is

Catalonia,

where we found

_genetic

profiles

similar to Greek and North-African

cat

_populations,

and the other is

_Spanish

_Levante,

where the western

_European

influence is

_{substantially}

clearer. It would be

_interesting

to find out to which of the 2 areas the Balearic cat

_{populations belong. Previously, Dyte (unpublished data)}

and Robinson

_{(unpublished data) (both}

of these references can be found in

Lloyd

and

_Todd,

₁₉₈₉₎

obtained small

_samples

of cats in

_unspecified

areas of the Balearic

Islands. These were

_probably

not

_{representative}

of all of the islands and could not

answer the

_questions

that we will

study

here

(for example,

Robinson’s

sample

in

Majorca

consisted of 45

_cats).

MATERIAL AND METHODS

Populations

and alleles studied

A total number of 2 096 cats was observed in

Minorca,

Majorca

and Ibiza

(Balearic

islands)

between March 1989 and March 1990. In

_Minorca,

1348 cats were

seen

(Mahon, n

= 474

_cats;

Villacarlos,

n = 226

_cats;

Mercadal and

Alayor,

n = 104

_cats;

Ciudadela, n

= 510 _{cats; the}

remaining

34 _cats were seen in other

parts

of Minorca:

_principally

Fornells,

Cala en

’Porter,

Punta Prima and

Binibeca).

In

_{Majorca (Palma}

_Majorca

and

_{nearby populations),}

475’cats

were observed. In

Ibiza,

273 cats were

_sampled

(Ibiza,

_city,

n = 210 and San

Antonio,

_n =

63).

Each

of these

_populations

was

_{extensively sampled}

to minimise whatever effects there

might

be of local deviations in allele

_frequencies.

Each cat

_sampled

was a

_stray,

an

alley-cat,

a feral cat or

&dquo;pseudo-wild&dquo;.

Careful measures were taken in order not to

_repeat

the observation of a cat

_previously

examined in the different incursions

made into these Balearic localities

_{(fig 1).}

The

_phenotypes

of the cats were recorded

directly

from observation of the

animals and the

_genetic

nomenclature used is in accordance with the Committee on Standardized Genetic Nomenclature for Cats

_(1968).

The

_genetic

characteristics studied here included

_{(table I):}

sex-linked

_(0,

o;

Orange vs

non-orange);

the

autosomial loci,

A

_(A,

a;

Agouti

vs

Non-agouti) ;

T

,

b

(t

t

+

,

T

a

;

Blotched vs Mackerel vs

_{Abyssinian tabby);}

D

_(D,

_{d ;}

Intense colour vs Dilute

_colour);

L

_(L,

_{l ;}

Short

hair vs

_{Long hair);}

S

_(S,

_s; White

_{spotting vs}

Non-white

_spotting);

W

_(W,

w; Dominant white vs Normal

colour);

C

(C,

_c!;

Full colour vs

Siamese);

M

(M,

_m;

approximation, assuming

a 1:1 sex ratio

_(a

fraction of the

_sample

was sexed and did

not

_{significantly}

differ from a 1:1 sex

_ratio),

was used to estimate the

_frequency

of

Orange

(Robinson, 1972), p(O) = (2a+6)/2N,

where a = number of

Orange (0/0

and

_0/-)

_phenotypes,

b = number of tortoiseshell

(0/+) phenotypes,

N = total

sample

size and _{p is} the

_frequency

of

_Orange.

The standard error for the estimate of

_Orange

was obtained

_by

the formula used

_by

Robinson and Machenko

(1981):

A test for random

_mating

at the O locus was

performed

_using

a G test

_(Sokal

and

_Rohlf,

₁₉₈₁₎

that

compared

observed

phenotypes

to those

predicted

from the

estimated mutant allele

_frequency.

Recessive mutant

_frequencies

_(q)

are taken as the _square roots of observed

phenotypic

frequencies,

while dominant mutant

_frequencies

_(p)

are taken as _{1 — q.} Standard errors are

_given

_by

the formulae:

for recessive and dominant

alleles,

respectively.

Sample

sizes for the various loci are different because

Orange

is

_epistatic

to

Agouti, Non-agouti

is

_epistatic

to

_Tabby

and Dominant white is

_epistatic

to all other coat colours.

Futher,

some

_diagnoses

are difficult or

_impossible

due to

_high

Genetic

_{heterogeneity}

and theoretical gene flow

To estimate the

_genetic

_{heterogeneity}

due to these _genes between the Minorcan cat

_populations

and between all the Balearic cat

_populations

_studied,

the

_Wright’s

F

st

statistic

_(Wright,

_1969,

₁₉₇₈₎

was used. In this

_work,

the

_F

_st

estimates were corrected for

_sampling

error

_using

the

_expression

q(1 - q)/2N (q

is the allele

frequency

studied and N is the number of

_{individuals) (Nei}

and

_{Imazumi, 1966;}

Wright,

1978).

Seven loci

_(0,

A,T, D, L, S,

W)

were used to

_compute

the

_F

_st

statistics. To test for

_genetic

_{heterogeneity,}

the

_chi-square

statistic for an

_M

_X

_N

contingency

table with

_{(M — 1)(N - 1) degrees}

of freedom where M is the number

of

_populations

and N the number of

alleles,

was used as introduced

by

Workman and

Niswander

_(1970).

Indirect

_(Nm)

_gene-flow

estimates were obtained from these

_F

_st

values. This can be estimated

_assuming

an n-dimensional island model

(Takahata,

1983;

Crow and

Aoki,

1984)

by

the

_expression:

Nm =

_{[(l/!)-l]/{4[n/(n-l)J!},}

In this

_model,

it is assumed that the efFects of

_migration

and

_genetic

drift are balanced in a subdivided

population.

These

gene-flow

values are

probably

underestimate of the real

_gene-flow

values, overall,

if there is a

_strong

_geometric

component

between the

_{populations (Kimura}

and

_Weiss,

_{1964) (eg,}

Slatkin

₍₁₉₈₅₎

stated that the infinite island model underestimates Nm for a 1-dimensional

stepping-stone

model).

The

_{phenotypic frequencies}

at each

locus

of each cat

_population

were also

compared

to other cat

_populations

_using

a 2 x 2

_chi-square

_contingency

test

(Simpson

et

_al,

₁₉₆₀₎

with Yates’ correction for

_continuity.

Spatial

autocorrelation

analysis

To

_study

whether the

_genetic

_{heterogeneity}

between the Balearic cat

_population

has

a

_significant

_{spatial trend,}

a

_spatial

autocorrelation

_analysis

was

employed (Sokal

and

_{Oden, 1978ab;}

Sokal and

_{Wartemberg, 1983).}

_Spatial

autocorrelation is the

dependence

of the value of a

particular

variable at 1 location on the value of that

same variable at other

_nearby

locations or at determined

_geographic

distance. The

spatial

autocorrelation statistic

_employed

was Moran’s I index

_(Sokal

and

Oden,

1978a).

To _carry out this

spatial

analysis,

4 distance classes were defined

(1

DC =

0-29

_km;

2 DC = 29-162

km;

3 DC = 162-303

km;

4 DC = 303-339

km)

where each

particular

distance class was chosen to

_optimize

the allocation of

_locality

_pairs

_(an

equal

number of

_point

_pairs)

among distance classes. A

binary

connection matrix

was formed

_according

to Sokal and Oden

_(1978b)

and to determine statistical

significance

for autocorrelation

coefficients,

the Bonferroni

_procedure

was used

(Oden, 1984).

Genetic distances

Three measures of

_{genetic relationships}

were

employed.

The Nei

_genetic

distance

(Nei, 1972)

was one of these. The values DNei < 20.00

_(multiplied

_by

₁₀₀₀₎

will

be

taken to indicate a close

_{genetic relationship}

between the different cat

_populations

analyzed (Ahmad

et

_{al, 1980; Ruiz-Garcia,}

_1990c).

Values 20.00 < DNei < 40.00 will be taken as intermediates in the

_genetic

relationships

between

populations

(Klein

et

_al,

_1988).

The Nei

_genetic

distance is a

_good

index when it measures

the

_genetic

_divergence

in accordance with the neutralist evolution

_theory

_(Kimura,

1983).

Nevertheless,

some

_polymorphic

loci in the cat

_populations

could be under the action of

_diversifying

natural selection

_(Blumenberg,

_1977;

_Blumenberg

and

Lloyd,

1980;

Lloyd, 1985).

For this reason, we have also used the Prevosti

_genetic

distance

_(Prevosti,

_1974;

Prevosti et

_al,

_1975).

This

_genetic

index is

_independent

of selective or neutral _processes and recurrent or non-recurrent _processes. In

addition,

the Cavalli-Sforza and Edwards

₍₁₉₆₇₎

chord distance was

_used,

as it has mathematical

_properties

different from the 2

_genetic

distances mentioned above.

Additionally

Nei et al

₍₁₉₈₃₎

showed that

_assuming

a constant evolution

_rate,

the

dendrograms

produced

when

_using

the UPGMA

_algorithm

and the

_Wagner

method with the Cavalli-Sforza and Edwards

₍₁₉₆₇₎

distance are those which

_produce

the

most

_precise

_topology

of the branches.

In this

_study,

7 loci

_(0,

A, T, D, L, S,

W)

were taken into account to obtain the

populations

and 70 selected

_European

and North-African cat

_populations.

The

genetic

profiles

of all of these cat

_populations

can be found in Ruiz-Garcia

(1988,

1990abc)

and

_Lloyd

and Todd

_(1989).

Manx

_(M)

and Siamese

_(c

_s

₎

are not included

in this

_analysis

because

_they

are

rarely

found above trace levels or are exotic characters.

In order to _compare the

_genetic

_{relationships}

of a fixed

_pair

of Balearic

pop-ulations to the

_{relationships}

between another

_pair

of Balearic

_{populations (using}

the 7 mentioned

_loci),

we have used Nei’s

(1978)

_genetic

identity

I coefficient with

variance

SDl

2

=

!(1 -

I)/In]

2

where n is the number of loci

_analyzed.

Mantel’s test

The Mantel’s test

_(Mantel,

_1967;

Hubert et

_{al, 1981;}

Hubert and

_Golledge,

1982)

has been used to detect for

_{possible relationships}

between the

_genetic

distance matrices obtained between the Minorcan cat

_populations

and Balearic cat

populations

and the

_geographic

distance matrices. In this

_work,

Mantel’s statistic

was normalized

_using

the Smouse et al

_{(1986) technique,}

which converts Mantel’s statistic into a correlation coefficient. In order to observe whether the

_type

of data may have some

_repercussion

on the

_{correlations, linear,}

_{logarithmic, exponential}

and

power functions were used.

Using

a Monte-Carlo simulation

(2 000 permutations)

or

using

an

_approximate

Mantel

_t-test,

we can test the

_significance

of the correlations

obtained.

Statistical studies of the 4 main Balearic

_populations

and the

_large

geographical

clusters

The 4 main Balearic

populations

studied here

_(Mahon,

Ciudadela,

Palma

Majorca

and

_Ibiza)

were related to the 70

_European

and North-African

populations

selected

using

geographical

clusters for each

_country

to which these

_{populations belong.}

To

find out whether there are

_significant

differences between _average Nei

_genetic

dis-tances between the different

_geographical

clusters for the same Balearic

_population

or to see whether there are

significant

differences between the different Balearic

populations

in relation to a fixed

geographical

cluster,

we used different

statis-tical

_techniques.

When the

_possible

existence of

_significant

statistical differences between the _average values of the Nei distance of the different

_geographical

clusters to a fixed Balearic

population

was

suspected,

an

_analysis

of the variances of the Nei _average distances was carried out. All the F-tests for the

_comparison

between

eastern Mediterranean and North-African

_(Greek

and

_{North-African)}

clusters and

western

_European

clusters

_(France

and Great

_{Britain) proved}

to be

_significant.

Be-cause of

this,

these

_comparisons

of means were carried out with a

_{non-parametric}

test

_{(Mann-Whitney}

_U-test;

Hollander and

Wolfe,

1973).

For the second case, in

which the

_{possible significant}

differences between the Nei _average distance between the different Balearic

_populations

to the same

_geographical

cluster were

_studied,

we were able to observe the existence of

_normality

on most occasions

_by

means of

the

Kolmogorov-Smirnov

test

_using

Lilliefors’ tables

_{(Lilliefors, 1967).}

We did not

observe any

significant

differences between the variances on most

_occasions,

so we

Phenograms

and

_cladograms

Different kinds of

_dendrograms

were constructed to

_explain

the

_{genetic relationships}

between the cat

_populations

of

_Minorca,

between the cat

_populations

in the Balearic islands and between these

_populations

and other

European

and

North-African

_populations.

To do

this,

we carried out a

phenetic approach

_using

different

algorithms.

These

_algorithms

used were the UPGMA

_{procedure (unweighted}

pair-group

method),

the SINGLE

procedure (single-linkage clustering).

The

description

of these

_algorithms

can be found in Sneath and Sokal

(1973)

and Dunn and Everitt

(1982).

To the different

_dendrograms

which were

_obtained,

goodness-of-fit

statistics

were

applied

to find the differences between the

_original

_genetic

distance matrices

(input)

and the

_patristic

distances

_(output).

These

_{goodness-of-fit}

statistics are as

follows: Farris’s F

_{(1972), Prager}

and Wilson’s F

_(1976),

Fitch and

_{Margoliash’s}

standard deviation

₍₁₉₆₇₎

and the

cophenetic

correlation coefficient

_(Sneath

and

Sokal, 1973).

In

addition,

some strict consensus trees

_{(Rohlf, 1982)}

were constructed between the

_{dendrograms by}

means of different

_algorithms

and different

_genetic

distances,

but

_they

are not shown in this article. To the

_populations

in Minorca and the whole of the Balearic

populations,

a

cladogenetic

_analysis

by

means of

_Wagner’s

method

_{(Farris, 1972)}

was

_applied

to find out whether the results obtained

_through

this method are

highly

similar to those obtained

_through

a

phenetic

analysis.

This

analysis

was carried out

_using

the Sforza and Edwards

₍₁₉₆₇₎

distance. For the

development

of this method we used the OTUS addition _sequence

_by

means of

the

_multiple

addition criterion

_{(MAC) algorithm}

_{(Swofford, 1981)}

and the tree

was rotated in order to

_produce

a tree conducted

_by

the

_midpoint

_rooting

method

(Farris, 1972).

Some

_cladograms

were also constructed for the Balearic

_populations

in which the Mahon

_population

was

_regarded

as the root of the tree in relation

to the rest of the

_populations

_{(outgroup method).}

This will

_help

us to ascertain how the other

_populations

have differed from the

_population

of Mahon

_(one

of the assumed

_points

of introduction of cats into

_Minorca).

Percentage

of genetic heterogeneity

attributed to each locus and to each

population

with the method of Adalsteinsson et al

₍₁₉₇₉₎

In order to calculate the

_genetic

_{heterogeneity}

_percentage

which each locus

con-tributes to the total

_genetic

_{heterogeneity}

of the loci

_studied,

and calculate the

genetic

heterogeneity

that can be attributed to each

_{population, pairs}

of

_genetic

differences between

populations

using

Kidd and Cavalli-Sforza’s

₍₁₉₇₄₎

_genetic

dis-tance have been used in the same _way as was done

by

Adalsteinsson et al

_(1979),

where :

where _{Pik is} the

_frequency

of the k allele in

_{the j sample,}

_Pik is the

_frequency

of the k allele in

_{the j sample}

and n is the number of the loci taken into account. This

analysis

was

_applied

to the Balearic

_populations

and to some Iberian

_populations.

This

_analysis

allows us to find out which loci introduce

_{heterogeneity}

and which

RESULTS .

Phenotypic frequencies,

gene

frequencies

and

Hardy-Weinberg

equi-librium

In table

II,

we

_give

the _gene

frequencies

for the 7 cat

_populations

studied in the

Balearic islands. In table

_III,

the results of the

_application

of a G test to the

0

locus are shown in order to check

_{Hardy-Weinberg equilibrium}

in these

_populations.

There was no

_significant

statistical deviation between the observed

_proportions

and those

_expected

for the

_populations

of Ibiza

_city,

San Antonio

_(Ibiza),

Palma

Majorca (Majorca),

Mahon and Villacarlos

_(Minorca).

So we can conclude that in

these

_populations

there are no

evolutionary

_agents

able to deviate the

_proportions

of

_homozygotes

and

_{heterozygotes}

from

_{Hardy-Weinberg}

_{equilibrium. However,}

it

turned out that the

_{Hardy-Weinberg equilibrium}

did not

_apply

at the 0 locus for the Minorca

_sample

as a

_whole,

and for the

_samples

from Ciudadela and Mercadal

and

_{Alayor (Minorca)}

which have an excess of

homozygotes.

In

_spite

of

this,

the

factor

_{(or factors)}

that increases the

_proportion

of

_{homozygotes significantly}

does

not affect allele

_{frequencies (Scribner}

et

_al,

_1991).

Genetic differentiation and theoretical _gene flow

The

_global

_genetic

differentiation between the cat

_populations

of Minorca

_(F

_st

=

0.0151)

and between all of the Balearic

populations

studied here

_(F

_st

=

0.0299)

are small

(table

IV).

This means that _any

_population

has an _average value of

98.49%

and

97.01%

of the total

_genetic

_diversity

found in the total

_population

of Minorca and the Balearic

_population

as a

_whole,

_{respectively.}

The values of theoretical gene flow in an n-dimensional island model for the cat

_populations

of Minorca were 9.16 cats

_entering

_per

_generation

and

_population

as an on _average

value and 5.94 for the islands as a whole. These values are much

_higher

than those found for other

_organisms

studied.

_{Nevertheless,}

the existence of

_{statistically}

significant

heterogeneity

can be observed. In Minorca and in the Balearics as a

whole,

all the alleles

_(except

the W

_allele)

showed the existence of

_significant

heterogeneity.

Another

_aspect

that can be observed is that the relative

_quantity

of

genetic

heterogeneity

introduced

_by

each locus is

_highly

different. For

_Minorca,

the

t

b

allele

_(F

_st

=

0.0567)

is the one which introduces the most

_genetic

_{heterogeneity}

and the W

_(F

_st

=

0.0000)

and 0

(F

st

=

0.0042)

alleles _are those which introduce the least

_{heterogeneity.}

When we consider the Balearic

_populations

as a

whole,

the

t

b

_(F

_st

=

0.0693)

and I

(F

st

=

0.0526)

alleles are those which introduce the most

genetic

heterogeneity,

while W

_(F

_st

=

0.0008)

and 0

(F

st

=

0.0065)

_are the alleles which introduce the least

_{heterogeneity.}

When we considered each allele

individually

between

pairs

of

populations

(table V)

we also observed a

_great

number of

significantly differentiating

alleles.

For

_example,

out of 9 alleles

_studied,

the

_population

of Mahon differs in 5 alleles

from the

population

of Palma

Majorca

and in 7 alleles from the

_population

of

Ibiza,

Spatial

autocorrelation

The

_0,

a, I alleles do not show _any kind of

_{significant spatial}

structure.

The t

b

allele,

on the other

hand,

has 3

statistically significant

Moran’s I

coefficients,

though

it does not reach a

_{significant global correlogram}

_{(table VI).}

Between

0-29.2 km and 29.2-162.1

km,

the values are

significantly

_positive

(high

_similarity

for

the t

b

allele

_{frequencies).}

On the

_contrary,

between 302.8 and 338.8

_km,

the value is

_{significantly}

_negative

_(highly

different tb

allele

_{frequencies).}

The

_{d, S,}

and

W alleles have

_{significant spatial}

_patterns

_(P

=

0.022,

P =

_0.001,

P =

_0.001,

respectively).

In the 3 cases there are

significantly

_positive

Moran’s I values for the first distance class and Moran’s I values are

_{significantly}

_negative

for the fourth

distance class

_{(302.8-338.8 km) (genetic}

differentiation at

_{long distance).}

The d

and W alleles showed a

_stronger

monotonic clinal

_tendency

than the S allele which

rather showed

_genetic

differentiation at

_long

_{distance. The average}

_correlogram

shows a clear clinal monotonic

tendency

for the 7 alleles studied as a whole with a

progressive

diminution of

_genetic

_similarity

as

_geographical

distances increases.

Mantel’s test

Mantel’s tests to _{prove associations} between

_geographical

and the Nei and Prevosti

genetic

distances for the cat

_populations

of Minorca and for the Balearic cat

populations

as a whole were

_analyzed.

For the Nei distance in

_Minorca,

_geographical

separation explains

between

2.25%

_(linear

regression;

r =

0.15023,

t =

0.315,

P =

_0.3762;

Monte-Carlo simulation

(2 000

_permutations

_at

random)

P =

0.484)

and

42.68%

_(logarithmic

transformation;

r =

0.65332,

t =

1.452,

P =

0.0732;

Monte-Carlo: P =

0.1785)

for

_genetic

variability.

For the Prevosti

distance,

t =

_0.454,

P =

_0.3249;

Monte-Carlo: P =

0.497)

and

36.87%

(logarithmic

transformation;

r =

_0.60721,

_t =

1.350,

P =

0.0855;

Monte-Carlo: P =

0.1620)

of

genetic

variability.

In no case were these values

significant.

Thus we can state that

geographical

distances between

_populations

of Minorca do not have an observable

significant

effect on the constitution of the

_genetic

profiles

of the cat

_populations

on this island.

_However,

when we consider all the Balearic cat

_populations

studied

(in

this case those of San Antonio and Ibiza

_City

were considered as one

_sample)

geographical

distance

_explains

between

31.57%

_(power

transformation;

r =

0.56187,

t =

1.959,

P =

0.0251;

Monte-Carlo: P =

0.0067)

and

46.20%

(logarithmic

transformation;

r =

_0.67975,

_t =

_2.414,

P =

_0.0079;

Monte-Carlo: P =

0.0123)

of the

_genetic

_{heterogeneity (in}

both cases, these values were

_{significant).}

Unlike what was observed in

Minorca,

for the Balearic

populations

as a

whole,

geographical

distance

_{significantly explains}

between a third and a half of the total

genetic

heterogeneity

found for these

_populations.

Genetic identities between the Balearic

_populations

studied

A

_question

which has been studied here is which of the 2 most

_important

popu-lations in Minorca

_(Mahon

and

Ciudadela,

the 2

_harbours)

has most

_decisively

influenced the other 2 small

_populations

that have been studied on this island

(Villa-carlos and Mercadal and

_{Alayor) (table VII).}

We can _prove that the

population

of Mahon is

_{significantly}

more similar to the

populations

of Villacarlos and Mercadal and

_Alayor

than the

_population

of Ciudadela

_(t

=

_11.64,

_12df,

P _<

0.001;

t =

_7.524,

12 df ,

P _<

0.001).

We also observe that the

_population

of Mahon is

_{significantly}

more similar to the 2 mentioned

_populations

on the same island than the rest of Balearic cat

_populations

studied

_(for

Villacarlos: Mahon-Palma: t =

_15.50,

12 df ,

P _<

_0.001;

Mahon-Ibiza: _t =

_15.68,

12 df ,

P <

_0.001;

Mahon-San Antonio: t =

16.69,

12 df,

P _<

0.001;

For Mercadal and

Alayor:

Mahon-Palma: t =

12.573,

12df,

P _<

0.001;

Mahon-Ibiza: t =

10.52,

12df,

P <

_0.001;

Mahon-San Antonio: t =

_12.57,

_12df,

P <

_0.001).

We also wanted to find out which of the 2 most

_important

_populations

in Minorca is

most similar to the rest of Balearic

_populations.

Ciudadela turned out to be

significantly

more similar to the

_populations

of Palma

_Majorca

and those of

Ibiza and San

_Antonio,

than what was observed for the

_population

of Mahon

(t

=

_3.93,

12 df ,

P _<

_0.05;

t =

_6.51,

12df,

P _<

_0.001;

_t =

_11.17,

12df,

P <

_0.001,

_{respectively).}

This shows that the

_populations

in central-eastern Minorca

_(Mahon,

_{Villacarlos, Mercadal,}

_Alayor)

differ

_slightly

but

_{significantly}

from the rest of Balearic

_populations

studied here.

Differences between the

_{large geographical}

clusters and the 4 most

important

Balearic

_populations

studied

When we _compare the Nei _average

_genetic

distances between 2

_{large geographical}

groups

(Greece

and North Africa

(n

=

14)

and Great Britain and France

(n

=

25))

and the 4 most

_important

Balearic

_populations

_(tables

VIII and

_IX),

we observe

significantly

lower mean values for the _group

_containing

Greece and North Africa

12.12 ! 5.79-29.35 ± 9.34 while that for Great Britain and France is 46.39 ± 18.44-106.51 !

_26.89).

We also observe that the

_populations

of Ciudadela and Ibiza show

significantly

lower mean values of the Nei distance with the _group of Greece and

North Africa than those of Palma

_Majorca

and Mahon

_(Ibiza

vs Palma: t =

5.11,

P <

_0.001;

Ibiza vs Mahon: t =

_3.83,

P _<

0.001;

Ciudadela vs Mahon: t

= 10.51,

P < 0.001 and Ciudadela vs Palma: t =

4.54,

P _<

0.001).

These

_comparisons

show that all the Balearic cat

_populations

are

genetically

more similar to the east

Mediterranean and North-African cat

_populations

than to the western

_European

ones with

regard

to the coat _genes. We also observe that the

_population

of Palma

Majorca

is

the

one that

_presents

the least difference between both _groups of

clusters,

being

the Balearic

_population

which seems the most

_closely

related to

the

_genetic

_profiles

of some western

_European

cat

_populations

_(especially

some

French and Italian

_{populations).}

With,

for

_example,

the Prevosti _average

_distance,

the value

_of

Palma

_Majorca

for the eastern Mediterranean and North-African group

(n

=

25)(D

= 10.01 ±

2.29)

is

practically

identical to the western

European

_group

(n = 33)(D = 10.99::1:: 2.44).

Phenetic and

_cladogenic

_study

All the

_phenetic

and

_{cladogenetic analyses}

of the 4

_populations

of Minorca offer the same

_groupings

between the

populations regardless

of the

algorithms

and

genetic

distances used

_{(fig 2).}

Mercadal and

_Alayor

is the

_population

which most

_clearly

statistics for the

_phenograms

and

_cladograms

corresponds

to the Cavalli-Sforza and

Edwards distance

_(eg,

the

_cophenetic

correlation coefficient for the Cavalli-Sforza and Edwards distance is

_0.966,

while for the Nei distance it is

_0.796).

With

_regard

to the Balearic

_populations

as a

_whole,

there are 2 clusters which

remain immutable: Mahon and Villacarlos

_{(eastern Minorca),}

Palma

_Majorca

and

Ibiza

_City.

For

_example,

in the

_{phenetic analyses}

_using

the UPGMA

_algorithm

with the Nei distance and the strict consensus tree with the

_UPGMA,

and SINGLE

(especially

with the Cavalli-Sforza and Edwards

_distance)

show trees with much

better

_{goodness-of-fit}

statistics than the trees obtained from a

phenetic analysis.

In

all these

_analyses

_using

the method of

_midpoint

_rooting

of the

_{longest path}

with either Cavalli-Sforza and Edwards or Prevosti distances the

populations

of

Mahon,

Villacarlos and Mercadal and

_{Alayor (eastern}

and Central

_Minorca)

differ from the

rest of the Balearic

_populations.

Ciudadela

_{(western Minorca)}

clusters with the

populations

of San Antonio

_(Ibiza)

while Palma

_Majorca

and Ibiza

_City

maintain their

_genetic

_similarity.

Phenetic

_analysis

of the 7 Balearic cat

_populations

studied and 70

European

and North-African cat

_populations

The 7 Balearic cat

_populations

on are

_clearly

and

_{significantly}

more related to the

North-African and eastern Mediterranean cat

_populations,

and to the Catalonian

cat

_populations

with

_possible

eastern Mediterranean and North-African

_origin,

than to western

_European

ones

_{(fig 3).}

All the

_{phenetic analyses}

show the same

relationships.

For

_instance,

the UPGMA

_{phenetic analysis}

with the Nei distance

shows that all 4 cat

_populations

in Minorca were

_closely

related with some

North-African

_populations

_(like

Constantine

_(Argelia),

and

_Tunis).

In all cases,

Palma

_Majorca

and Ibiza show a

_strong

_genetic

_similarity

to certain Catalonian

populations (like

Barcelona and

_Sitges)

and also with Rabat

_(Morocco),

Athens

or Samos

(Greece).

San Antonio

(Ibiza)

shows the most marked

_similarity

with

Tarragona

(Catalonia)

and

_{Argolis (Greece).}

All the Catalonian

_populations

also appear

together

with eastern Mediterranean and North-African

populations.

On the other

_hand,

the

_Spanish

Levante

_populations

_(like

Alicante,

Benidorm or

Murcia)

are in the western

_European

cluster. A

_principal

coordinates

analysis

and a

_principal

_component

analysis (not

shown

here)

also show the same kind of

_genetic

relationships.

Percentage

contribution to the

_genetic

differences classified

_by

loci between Balearic and

Spanish

cat

_populations

The

_percentage

contribution to the

_pairwise

_genetic

differences classified

_by

loci and locations is shown in table X. The

following

characteristics can be seen:

a)

the

greatest

contribution to the variation in the Balearic

_populations

comes from the

t

b

allele

_(31.86%)

with the 1 allele in second

_place

_(26.16%).

The a allele

(4.23%)

is the lower contribution to the variation between Balearic

populations

(W,

M and c’

were not

_{included); b)}

with the

_Spanish

_populations

taken as a

whole, the t

b

allele

(44.9%)

is the most

_{heterogeneous}

and the a allele distribution

(5.92%)

is the most

homogeneous. Undoubtedly,

q(t

b

)

frequency

is the most

_outstanding

factor in the differentiation of these cat

_populations.

DISCUSSION

Hardy-Weinberg

equilibrium

Generally,

when u and S loci

(Dreux, 1975)

are

analyzed

to

_{study Hardy-Weinberg}

of

_{electrophoretic}

characters in cat

_populations

have also confirmed the

_finding

of

panmixia

(Hardy-Weinberg equilibrium) (Spencer,

1979; O’Brien, 1980;

Ritte et

al, 1980;

Weghe

et

_{al, 1981;}

Brown and

_{Brisbin, 1983);}

Futher,

other felid

species

(like

Panthera

_pardus,

Panthera

_{leo, Leptailurus}

_serval,

Caracal

caracal,

Neofelis

nebulosa,

Leopardus (= Felis)

pardalis

and

Leopardus

wiedi)

conform also

_apparently

to

_{Hardy-Weinberg equilibrium (Newman}

et

al,

1985).

However,

as has

previously

been

_shown,

the

_{Hardy-Weinberg equilibrium}

at the 0 locus was not confirmed for the Minorca

_sample

as a

_whole,

nor for Ciudadela and Mercadal and

_Alayor

because these

_{populations apparently}

exhibited a

significant

excess of

_homozygotes.

If these

_samples

contained an excess of males this could

provoke

an

_apparent

excess

of

_homozygotes.

Nevertheless,

some of the

_samples

were sexed and there was no

significant departure

from a 1:1 sex-ratio. If the

disproportion

of sex could be excluded as an effective

_explanation,

the

_{consanguinity}

and/or

Wahlund’s effect

situations have been

_reported

for other

_mammals,

for

_{example, Procyon}

_lotor,

Beck

and

_Kennedy,

_1980;

_Oryctolagus

cuniculus,

Arana et

al, 1989;

Thomomys

bottae,

Daly

and

_Patton,

_1990;

_Cynomys

_{ludovicianus,}

_Chesser,

_1983;

_Calorriys

_laucha,

Garcia et

al,

1990).

Selective

_agents

are

_possibly

not the causes of the observed

situation.

Genetic differentiation of the Balearic cat

_populations

and

_spatial

pat-terns of some variables

-The amount of

_genetic

_{heterogeneity}

introduced

_by

each allele is different

_(for

example, t

b

introduces much more

_{heterogeneity}

than

_W,

0 or

_a),

which indicates

that the

_{evolutionary history}

of each has been

_notably

different. For

_example,

an allele which was introduced

_long

_{ago may} have become

highly homogeneized

throughout

the area in

_question

whereas an allele which arrived more

_recently

_may

present

a

_stronger

_genetic

heterogeneity.

In

short,

each allele _may have suffered different stochastic _processes

_depending

on the

_existing

_demographic

_population

parameters

at _any historical moment. Neither can we

_completely

rule out that the influence of

_diversifying

or

unifying

selective _processes

(depending

on the different

loci which have been

_studied)

is not _necessary to

_explain

the different amount of

genetic

heterogeneity

introduced

_by

each allele. The _average

_F

_St

values and the gene-flow estimates

_(Nm)

obtained for these Balearic cat

_populations

are

_{extraordinarily}

different from those observed for other island mammals. For

_example,

Navajas-Navarro and Britton-Davidian

₍₁₉₈₉₎

showed for Mus

_musculus,

an

_F

_st

value of

0.278 and an Nm value of 0.65 for this

_species

on the western Mediterranean islands. We may conclude that the

genetic

differentiation of the Balearic cat

_populations

is

relatively

limited and that gene flow is

_{substantially}

_greater

that what is observed

for other island

_species.

This

_strong

_gene flow _may be due to the intrinsic

_ethological

characteristics of the cat

_(Ruiz-Garcia

and

_Klein,

_1993),

_but,

above

_all,

this

_high

level of _gene flow is due to the association between man and cat, where the latter

depends

on the

high mobility

of the former.

Nevertheless,

in

_spite

of all

this,

we can observe the existence of

_significant

_genetic

_{heterogeneity}

for most of the studied loci.

_{Wright (1931)}

stated that if Nm > 1 then gene flow is

important

enough

to erase the

_genetic

_{heterogeneity}

between the

_populations

in

_equilibrium.

In this

study,

we obtained Nm estimates of

_{approximately}

9 cats in Minorca and 6 cats

in the Balearic islands as a whole.

_However,

a

significant

_genetic

_{heterogeneity}

was

observed. This

_{might perhaps}

be

_explained

_by

Allendorf and

_Phelps

₍₁₉₈₁₎

who

argued

that the most correct

_{interpretation}

of Nm > 1 is that the

_populations

share the same alleles

though

not

_necessarily

with the same allele

frequencies. By

means of simulation models

_they

showed that

_significant

allele

_divergence

occurred

in

50%

of the

_generations

with a _very

high

_gene flow of Nm = 50 and that

significant

allele differentiation occurred on most ocassions when Nm = 10.

Cats introduced at different

_points

of the islands _may

_originate

from diverse

places (though predominantly

from the Mediterranean

_world),

with different

fre-quencies

for the alleles introduced at different

_points.

This can be

_proved

_by

the

existence of

_{significant spatial}

autocorrelation of a clinal kind and of differentation

Genetic

_{relationships}

between the Balearic cat

_populations

and other

European

and North-African cat

_populations

Although

some

_significant

_genetic

differentiation is observable between the Balearic

cat

_populations,

we can state that the Balearic

_populations

which have been studied

are of a clear eastern Mediterranean and North-African

_origin.

All this is in

agree-ment with the

_history

of the inhabitants of Balearics.

_{Phoenicians, Greeks,}

Romans

and,

especially,

the

_{Carthagenian hegemony}

from

_{North-Africa,}

were

_present

in the Balearics. The Arabian _presence for 500 _{years in} Balearics

_{(700-1 200 BC)}

was also

very

important.

It is

probable

that these human movements in the Balearics were

responsible

for this

_genetic

_similarity

between the Balearic cat

_populations

and the

North-African and eastern Mediterranean ones.

_Moreover,

other historical events

can

_help

to understand the close

_genetic

_{relationships}

between these cat

popula-tions,

such as the

_{extraordinarily}

_strong

historical and commercial

_{relationships}

between Catalonia and the eastern Mediterranean

_{(Greece, especially)}

and North

Africa

_during

13th and 16th centuries. Catalonia became most

_important

in the

western Mediterranean area with direct contact with eastern Mediterranean and

North-African harbours in 14th-15th centuries. Catalonia first

_conquered

_Majorca,

Ibiza and Minorca.

_Later,

Catalonia

_conquered

_Athens,

_Arta,

_{Morea, Neopatria}

_(all

Greek

_cities)

and other areas in

Turkey

and established consulates in

_Syria,

North

Africa,

Malta and

_Cyprus.

Important

Greek cities were dominated

_by

Catalans for 1

_century

_(in

the Attica

_region,

the Catalans had

_possession

until 1456

_AD).

The Catalans

_probably

introduced an

_important

number of cats into Balearics on

their

_journeys

to eastern Mediterranean and North-African areas

_during

the

13th-16th centuries. If these Balearic cat

_populations

were much _younger

_(for

_example,

if

_they

were founded

_during

the last 2

_centuries)

_{they might}

have these eastern

Mediterranean and North-African

_genetic

characteristics because

_they

_might

stem

indirectly

from

_populations

of eastern Mediterranean

_origin

like the

_present

Catalan

populations.

Might

there be _any correlation between the introduction of

the

cat on

the Balearic islands and the

_origin

of other mammals on these islands ?

Some Balearic

_species

of mammals like the

_garden

dormouse

_{(Eliomys qvercinus)}

(at

least the

_population

of Minorca and a

_part

of the

population

of

_Majorca;

Kahmann and

_{Tiefenbacher, 1969;}

Kahmann and

_Thoms,

_1973;

Kahmann and

Alcover,

1974),

and the

_pine

marten

_{(Martes martes)}

or Mus muscul!s domesticus

in

_{Majorca (Navajas}

_y Navarro and

_{Britton-Davidian,}

_{1989) might}

be of western

European origin. However,

the introduction of other mammals to these islands

might

be in correlation with the introduction of the cat. Some Balearic

_species

of mammals which

_might

turn out to be of North-African and Eastern

_origin

are:

the Balearic

_hedgehog

_{(Aetechinus algirus)}

classified

_by

Thomas

₍₁₉₀₁₎

as a North-African

_species;

the rabbit

_{(Oryctolagv,s cuniculus)}

introduced into North-Africa

_by

the Phoenicians

_according

to Petter and Saint-Girons

₍₁₉₇₂₎

and

_possibly

also into

the Balearic

_islands;

and the black rat

_(Rattus

rattus

_frugivorus)

introduced

_by

the

Catalans or

by

the Arabs

according

to Alcover

_(1979).

Boursot et al

₍₁₉₈₅₎

showed