Polygenic control of an all-or-none morphological trait in Euplotes (Ciliata, Hypotrichea). Evolutionary significance of naturally occurring morphological variation in Ciliates

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Polygenic control of an all-or-none morphological trait

in Euplotes (Ciliata, Hypotrichea). Evolutionary

significance of naturally occurring morphological

variation in Ciliates

J Génermont, C Demar, G Fryd-Versavel, H Tuffrau, M Tuffrau

To cite this version:

Original

article

Polygenic

control

of

an

all-or-none

morphological

trait in

_Euplotes

_{(Ciliata, Hypotrichea).}

Evolutionary

significance

of

_naturally

occurring

morphological

variation

in

Ciliates

J

Génermont

C

_Demar,

G

_{Fryd-Versavel,}

H

_Tuffrau,

M Taffau

Université de _Paris-Sud, Laboratoire de _Biologie Evolutive et _Dynamique des

Populations, Bât _{446, 91405}

_{Orsay Cedex,}

France

(Received

7 October _{1991; accepted} 30 _January

₁₉₉₂₎

Summary - A

_{hereditary abnormality}

is described in

_Euplotes

crassus, consisting of disturbances of the ventral somatic

_ciliary

_pattern, as a result of alterations of the

development

of _primordia of frontoventral and transverse cirri. A

_{breeding analysis}

was _performed, the results of which are not consistent with monofactorial Mendelian

determination,

thus _permitting the consideration of an alternative hypothesis of

_polygenic

control. From a _survey of _presently known

_{spontaneously}

_occurring variants in

_Ciliates,

it is inferred that some _apparently minor

_changes

which take

_place

in the course of

morphological

divergence between related _{species probably} cannot be accounted for

_by

fixation of a low number of _mutations, but require some more _{complex genetic repatterning.}

ciliary pattern / Euplotes / polygenic inheritance _{/ speciation /} threshold character Résumé - Déterminisme _polygénique d’un caractère _{morphologique tout-ou-rien} chez

Euplotes (Ciliés Hypotriches). Signification évolutive de la variabilité morphologique

naturelle chez les Ciliés. Un clone présentant une anomalie _{morphologique} a été isolé

d’une _population de laboratoire constituée en vue de _perpétuer une _importante variabilité

génétique.

Sur les cellules _{végétatives} le nombre de cirres _{occupant l’emplacement} normal des transverses est _inférieur au nombre standard de _5, tandis _que des cirres sont disposés

de _façon désordonnée dans la partie

postérieure

de la zone

_{fronto-ventrale.}

L’étude de la

morphogenèse

de division _{montre que} les _primordiurw communs aux cirres transverses

et _{fronto-ventraux} se _{forment normalement,} mais

_qu’ils

_présentent des altérations lors de

*

leur

_fmgmentation

en ébauches de cirres et surtout que le positionnement des ébauches

postérieures

est

_anarchique.

Le croisement du clone anormal avec un clone normal issu de la même _population

fournissant

une Fl normale, l’hypothèse d’un déterminisme _{monofactoriel récessif} de l’anomalie _pouvait être émise. La descendance d’un bnck-c!sa, puis celles de _plusieurs croisements n’ont pas les compositions attendues sous cette _hypothèse, même en admettant

que des écarts aux _pmportions mendéliennes _puissent être introduits soit du _fait de la

mortalité à _laquelle sont _{sujets les} clones anormaux à l’issue de la _conjugaison, soit du fait de la mise en _jeu de _phénomènes d’hérédité _caryonidale comme on en connaît par

ailleurs chez les Ciliés. En _{conclusion, l’anomalie, qui,} it l’échelle du _clone, obéit à la

règle du tout-ou-rien, appamît comme un camctére à seuil, à déterminisme

polygénique.

D’un survol des _{différents types} de variants _{:morphologiques} connus chez les _Ciliés, et notamment de ceux _qui ont été obtenus sans _application d’un ttn,itement _mutagène, il

ressort que ces variants diffèrent en général bien plus du « standard» de leur

_espèce

que

ne

diffèrent

de celui-ci d’autres _espèces du même genre. En particulier, la variation sur

des caractères _apparemment aussi mineurs que la présence ou l’absence d’un cirre est

strictement

_{interspécifique.}

Elle met en _jeu, contrairement à ce qu’on aumit pu

prévoir,

d’importants remaniements _{génétiques,} sans commune mesure avec la _{très faible}

divergence

observée entre les _{espèces jumelles.}

caractère à seuil _/ ciliature _{/ Euplotes /} hérédité _{polygénique / spéciation}

INTRODUCTION

In

_Euplotes,

some

_{morphological}

features are

highly

conserved

throughout

the

evolutionary

diversification of the genus. Such is the case for the

general

_pattern

of the somatic ventral ciliature. It is

_composed

of cirri which are

_usually

classified

in 3 groups on the basis of their

morphological

_arrangement

in

_vegetative

cells

(fig 1).

The frontoventral cirri

_(FV)

are distributed on the anterior

_right

quarter of the ventral _area; their number and their

_{topological relationships}

are _very stable

within a

_species.

The transverse cirri

(T)

are attached to the ventral surface

_along

a

_{V-shaped subequatorial}

_line;

their number is 5 in all

_species

but one

(E

stmlkot4

Agamaliev).

The caudal cirri

_(usually

4 to

₆₎

lie very close to the

_posterior

_margin

of the cell.

This classification is no

_longer

valid when

_{morphogenesis}

is taken into consider-ation

_{(fig 2).}

One of the FVs is

_obviously

related to the oral ciliature: it may be

referred to either as the buccal

(Borror, 1972)

or as the

paroral

(

T

unrau

et

_al,

₁₉₇₆₎

cirrus. All other FVs and Ts

_originate

from a set of 5

_primordia,

each one

_consisting

of a

longitudinal

streak of

kinetosomes

which

_elongates,

then

_fragments

into 2 or

3 cirral

_anlagen:

the _{Ts emerge} from the 5

_posterior

_anlagen

and the FVs

_(except

for the buccal

_cirrus)

from the others. In those

_species,

such as E _crassus, which

possess 10

FVs,

3 cirral

anlagen

are derived from each

primordium,

_except for the most

_marginal

one

(at

the

_right

end of the

set)

which

_generates

only

2

anlagen.

All

cirri

_developed

from this set of 5

_primordia

make _up a

morphogenetical

unit which

will be referred to in the _present paper as the FVT

_complex,

a denomination which

is not a _synonym of the FVT _{system introduced}

_by

Gates

(1987b),

since the latter

since 2 of them

_originate

from

_{a primordium}

located on the left side of the ventral

area whereas the others are related to the dorsal kineties.

By mixing

many

conspecific

clones collected from a

large

_array of

stations,

a

synthetic population

was

established,

then maintained in the

laboratory in

such a

way that sexual processes were

_expected

to occur

repeatedly.

Later,

a few new clones were

occasionally

added,

so that a

significant

level of

genetic diversity

was

likely

to be _present in the

_population

some _years after its foundation.

_Many

clones were

isolated from this

_{synthetic population}

in order to _carry out an

_{experimental study}

of heritable variation for some

morphological

traits. Under

cytological

examination, one of these

_clones,

here referred to as

Go,

proved

to include a rather

_{high proportion}

(about 50%)

of cells in which the FVT _pattern was

severely

disturbed: such cells are referred to as fvtd cells.

A

_study

of such a variant trait is

expected

to

provide

some

insight

into

understanding

the mechanisms

_by

which cirral

_{anlagen positioning}

is

_achieved,

especially

if it is

_single-gene

determined. On the other

hand,

from an

evolutionary

point

of

view,

it is of some interest to

_appreciate

to what extent

_naturally

occurring

intraspecific

morphological

variation is related to

_{interspecific}

variation. Thus an

experimental study

was

designed

in order to

_elucidate

the

_genetic

control of the fvtd

_phenotype.

We report here the main results of this

_{study, together}

with a

brief

_description

of the variant

_phenotype

itself. A more detailed account of the

MATERIAL AND METHODS

The &dquo;abnormal&dquo; clone

_belongs

to a

_species

which in

_previous

_publications

has been

referred

_{to as}

_{&dquo;species}

No 2 of the

_Euplotes

vannus

complex&dquo;

(G6nermont

et

_al,

1976,

1985;

Machelon et

_al,

_1984).

_However,

the _proper name is E crassus rather

than E vannus

(see Discussion).

The cultures are _grown at room _temperature on dried lettuce infusion

_prepared

with

natural sea water

_(obtained

from the Laboratoire

_Maritime,

Luc sur

_Mer,

France),

sterilized,

then inoculated with Enterobacter _aerogenes.

Crosses were achieved

_{by mixing samples}

of 2

_{complementary}

_clones,

in the

maturity period

of the

_life-cycle,

_grown in test tubes _up to exhaustion of culture medium in order to induce sexual

_reactivity.

A few hours

_{later, pairs}

of

_presumptive

conjugants

could be

_isolated,

then next

_{day presumptive}

_{exconjugants.}

We retained

only

the cells which satisfied the

_following

3 criteria:

_i),

_regression

of buccal

true sexual _process is

_{completed. Cytogamy}

or

selfing

would be excluded

only

on the

basis of the

_hereditary

transmission of marker genes. No such genes were identified

in our

material,

_except for the

_mating

_type

_determining

series of alleles

_(Heckmann,

1964)

which

_provided

rather _poor

_information,

since in the whole course of our

breeding analysis only

2

_mating

_types were _present,

_segregating

in a 1:1 fashion in

any cross. No

_{significant departure}

from this ratio was ever

_observed,

and no clone was detected

_exhibiting

a third

_mating

_type, so that there is no evidence

_against

the idea that the isolated cells were true

_{exconjugants. Moreover,}

we know from

previous

extensive work that in our

experimental

conditions

selfing

and cytogamy

are

_highly

_exceptional

in

intraspecific

sexual reactions in the E crnssus

complex.

For

_cytological

examination under

_light

_microscope,

silver

_impregnation

was

performed

as

adapted

from Chatton and Lwoff

₍₁₉₃₀₎

_by

Frankel and Heckman

(1968)

and

_protargol

_staining

was carried out

_according

to the

_procedure

devised

_by

Tuffrau

_{(1967), slightly}

modified

_{by using}

the

_{slowly acting developer}

recommended

by Foley

(1943).

For

_rapid

_screening

of clones in the course of

_breeding

_analysis,

_{&dquo;ghosts&dquo;}

were

obtained

_by

means of

_detergent

treatment. Cells from a culture

sample

were

collected

_by

_low-speed

_{centrifugation,}

then

_suspended

for 10 min in a 2% Nonidet

P

40

@

solution in PHEM buffer

_(Schliwa

and Van

_Blerkom,

_1981).

After

_washing

with PHEM and

_{centrifugation,}

the

_ghost

_pellet

was

deposited

on a

_microscope

slide,

gently compressed

with a

_cover-glass

and observed with

_{phase optics.}

The

cirral _pattern of about 20

_ghosts

was ascertained. Most clones fell

clearly

into one or other of 2 discrete classes: those in which the

frequency

of fvtd was close to

100%,

referred to as fvtd

clones,

and those in which all cells were

_normal,

referred to as wt

_(wild-type).

A few clones remained unclassified since the

_sample

included

a mixture of fvtd and normal

cells; they

were further characterized on the basis of

standard

_{protargol staining;}

_they

were _{proven to} include at least

30-40%

fvtd

_cells,

and were therefore classified as fvtd clones.

RESULTS

The fvtd

_phenotype

The most

_prominent

and constant feature of the fvtd

_phenotype

is the presence

of fewer than 5 cirri in the usual

_place

of the Ts.

However,

it cannot be described

as the loss of some transverse

_cirri,

as revealed

_by

the _sequence of

_{morphogenetic}

events

_resulting

in the abnormal _pattern

_{(fig 3).}

During prefission

morphogenesis,

the 5

_primordia

which are

_expected

to

_yield

the FVT

_complex

seem to be

_quite

normal in both location of initial sites and

_early

stages of

_elongation

of kinetosome streaks.

_Later,

in the course of further

elongation

and

_thickening

of the

_streaks,

some

_{irregularities}

are

_observed,

such as breaks or

branching

resulting

in extra

_primordia.

Still

_later,

some cirral

anlagen, especially

the

_posterior

ones in each

_daughter

cell

_migrate

in a more or less random fashion

and a few

eventually disintegrate.

Finally

the number of FVT cirri may be either

higher

or lower than the standard number of 14 and some of

them,

including

one or

long

ascendant

_{subpellicular}

_fibres,

a feature which is known as

_{strictly T-specific}

in

_wild-type.

Breeding analysis

Preliminary

observations

The fvtd condition is inherited

_through

asexual

_{multiplication:}

when cells were

reisolated from the

_{Go clone,}

at clonal _ages

_exceeding

100

_fissions,

_they

_yielded

subclones all of which exhibited the fvtd condition at the same

_level,

ie the

_frequency

of fvtd cells was close to 50%.

First

_{crossing experiment}

The

_Go

clone was crossed to another

_clone,

isolated from the same

_synthetic

laboratory

population

and selected on the basis of

_exhibiting

the wt condition and sexual

_{compatibility}

to

_Go.

From this _cross, 24

_{conjugating pairs}

were isolated and the 48

_exconjugants

were

separated.

Out of these 48

cells,

35

_yielded

viable

clones,

making

up the

G

l

generation.

The

mortality

rate

_following

_conjugation

is

thus

_13/48

=

0.27,

a

figure

which falls within the _range of

_usually

observed values

in this

_species.

All

_G

_l

clones were

_undoubtedly

wt.

Second

_{crossing experiment}

Among

the

_G

_l

_clones,

at clonal _ages estimated at about 60

_fissions,

9 were found

to be

_{sexually compatible}

with

_Go,

so that

_they

could be backcrossed to their

ap-parently

recessive _parent. From each of the 9

_backcrosses,

12

_exconjugants

_(derived

from 12 different

_mating

_pairs)

were isolated. From a total of 108

_{exconjugants,}

46 viable clones

_(G

₂

_generation)

were recovered

_{(table I).}

_Among

_them,

7 are

un-ambiguously

classified as fvtd and 39 as wt. The viable clones and the fvtd clones

The

_fvtd/wt

_segregation

is

_apparently

_quite

different from the 1:1 ratio

_expected

if the fvtd condition is determined

_by

a

single

recessive allele.

_However,

some

attention must be

_paid

to the rather

_{high mortality}

rate:

_0.57,

_{significantly}

_higher

than the

_G

_l

_figure.

Two different

_phenomena,

which are not

_mutually

_exclusive,

may account for this:

_inbreeding

_depression

_(under

the

_assumption

that the 2

_parental

clones were neither inbred nor

related,

the

G

l

clones are not

inbred,

but in _any

G

2

clone the

_inbreeding

coefficient is

_1/4)

and

_positive

association between the fvtd condition and lower

_ability

to overcome the critical

early

stages of clonal life. The observed results are

_actually

not _very different from those that would be

_expected

on the basis of a 1:1 ratio of fvtd to wt

_clones,

combined with

_mortality

rates close

to _{0.15 among} wt clones and _{0.85 among} fvtd ones.

Third

_{crossing experiment}

If the

_&dquo;single

gene

hypothesis&dquo;

is true, all fvtd

_G

₂

clones are

homozygous

for the

recessive allele which determines the fvtd

_condition,

so that _any cross between 2

of these clones must

_yield

_{similarly homozygous, uniformly}

_fvtd,

progeny. It was

thus

_planned

to cross the fvtd

_G

₂

clones in all

_possible

combinations.

_Actually,

among these 7

clones,

one

(belonging

to the progeny of backcross No

7)

could not

be

_brought

to

_{mating reactivity.}

_Among

the 6 reactive ones, 2

mating-types

were

found,

allowing

8 crosses.

Unfortunately,

the

_reactivity

of one of the clones was very

weak,

as a consequence of

premature

senescence

(at

the time of the

_experiment,

the clonal _age was about 100

_fissions,

within the usual

_maturity

_period)

so that the effective number of

_progenies

_making

up the

G

3

generation

was 6. The results are

given

in table II. Two sets of conclusions can be drawn.

First,

the overall

_mortality

is

_{impressively high:}

about

90%.

It does not seem

possible

that

_inbreeding

could account for this: the

_inbreeding

coefficient of

G

3

clones is

_5/16,

not

_{substantially larger}

than in

_G

₂

_.

_Moreover,

the

_mortality

rates are _very different among separate

progenies:

from 0.6 in cross 3 x 4 to 1.0 in cross

7 x 8,

despite strictly

identical levels of

_inbreeding.

On the other

_hand,

the 5 clones which

_yielded

_G

₃

were

_obviously

_fully

_mature, so that the

_{postconjugation mortality}

is not attributable to

_parental

senescence. These results _support the

_idea,

expressed

above,

of an association between the fvtd condition and

_mortality

The second set of conclusions concerns the fvtd condition itself. It is

expressed

in 16 out of 32 viable

_G

₃

clones. This result seems to be in

_strong

_disagreement

with the

_{single-gene hypothesis.}

_However,

it cannot be excluded that so-called

to such a

hypothesis,

all of the 32

G

3

clones would be

homozygous

for the fvtd

determining

recessive

_allele,

but in 16 of them the macronuclei would have lost the

ability

to _{express it.} It is

_actually

known that in Ciliates the

_expression

of some

characters is conditioned

_by

some switch which occurs

_when,

in late

_stages

of sexual

phenomena,

the macronucleus

_develops

from a micronucleus: these characters have

been reviewed

_by

Sonneborn

_(1977).

Fourth

_{crossing experiment}

If all

G

3

clones are

_homozygous

for a fvtd

_determining

_allele,

_they

must transmit it

_{through conjugation, irrespective}

of their

_{expressed phenotype.}

All

_progenies

of

G

3

x

_G

₃

crosses

(either

fvtd x

_fvtd,

or fvtd x wt or wt x

_wt)

are thus

expected

to be similar.

Many

crosses between

_{sexually compatible}

_G

₃

clones were carried out. The

_G

₄

clones were classified

_according

to the _types

_{expressed by}

the 2 _parents. The overall results are

_given

in table III. From these results the

_probability

of

_displaying

the fvtd condition may be estimated for a clone when both _parents are fvtd

(13/29

=

0.45),

when

only

_{one parent} is fvtd

(37/156

=

0.24),

_or when both

parents are wt

_(4/87

=

0.05).

These 3

figures

are _very

_different,

so that the results

do not _support the

_{single-gene hypothesis.}

It is not

_strictly

excluded,

since some

situations are known in which

caryonidal

inheritance allows some

parent-offspring

correlation,

for instance the B _system of

_mating

_type

determination

_(Sonneborn,

1937)

and resistance to

CaCl

2

(G6nermont,

1961)

in Paramecium.

However,

it is

highly unlikely,

so that it is

_strongly

_suspected

that some alternative

_hypothesis

should fit the whole set of

_experimental

data.

DISCUSSION

Genetic control of the fvtd trait and associated

_mortality

According

to the above

_results,

_{it is very}

_unlikely

that the fvtd condition

_depends

on a

single

recessive allele. That it

depends

on recessive alleles at 2 different loci is still more

_unlikely.

Other models

_involving

2 loci do not fit our data

_{satisfactorily.}

It is thus

_proposed

that the

_genetic

control of the fvtd vs wt

_binary

variate is

polygenic. Though

the model of

_polygenic

inheritance was

_{initially developed}

with

categorical

data

_(Wright,

_1934a,

_1934b).

This model seems to fit _very well to the

results obtained in the _present

_{study, especially}

for the

G

4

generation

in which a

good positive

correlation appears between the

probability

for a clone to

_display

the fvtd condition and the number of its fvtd parents.

It can be

_questioned

if the fvtd condition is a true all-or-none character.

_Actually

it is clear that some of the clones which have been classified as fvtd are somewhat

intermediate,

since

_they

include both fvtd and normal cells: such was the case

of the

_Go

clone. These observations could

_suggest

a

study

of the

frequency

of

fvtd cells within a clone as a

quantitative

continuous character.

Though

we did not collect extensive

_data,

it became obvious that this character was not suitable for

_{quantitative analysis,}

since most clones are

_{phenotypically}

uniform;

_so, it was

arbitrarily

chosen to

_classify

the few intermediate

clones,

the

_frequency

of which is very low

(less

than

5%),

as fvtd.

Concerning mortality

rates, the

_experimental

data which have been accumulated

for some 15 years support the idea that the

_species

of the

_Euplotes

crassus

_complex

are outbreeders in natural conditions and that

inbreeding

often results in an increase

of the

_frequency

of inviable

_{exconjugants.}

It is thus

_likely

that the

_high

_mortality

rates which have been observed in the course of the _present work are to some extent

accounted for

_{by inbreeding depression.}

However,

the very

high mortality

rate of

the

_G

₃

_generation

needs some further

explanation.

As

suggested

above,

it may

be assumed that the fvtd condition favours

_exconjugant

_inviability,

more or less

irrespective

of the

_inbreeding

level.

_Thus,

in most

_experiments

the overall

_mortality

rate is the

_joint

effect of the 2 causes.

If this

_{interpretation}

is _correct, at a

given

inbreeding

level,

a

_positive

correlation is

expected

between

_mortality

rate and

_frequency

of fvtd clones. The results obtained

in

G

4

are not inconsistent with this

_idea,

since

G

4

includes clones of very different

inbreeding

levels. On the other

_hand,

it must be

_pointed

out that at any

generation

the fvtd vs wt ratios are biased: fvtd

_frequencies

are

systematically

underestimated.

In _any

_{breeding experiment,}

the observed ratios express the interactions of

at least 3 kinds of

_phenomena:

_segregation

of a

polygenic

_system,

inbreeding

associated

_mortality,

and fvtd associated

_mortality.

Because of the

_complexity

of such

interactions,

any attempt to estimate

_genetic

parameters

(heritability,

for

instance)

would be unfounded.

Genetic control of all-or-none traits

As far as we

know,

no all-or-none

qualitative

trait has been

reported

to date as

polygenically

determined in

Ciliates,

since the

only suggestion

of

polygenic

inheritance of

_{morphological}

characters deals with the number of dorsal

_ciliary

rows

_{(Frankel, 1973b).}

It must be

_pointed

out that in some instances erroneous

conclusions

_might

be drawn from

_segregation

data. This is well

_exemplified

_by

results obtained

_by

_Wright

_(1934b)

in the course of his

study

of

polydactyly

in

the

_{guinea pig.}

When a

_polydactyl

and a normal line were

crossed,

the

F

l

was

uniformly

normal. The backcross and

_F

₂

results show no

_{significant departure}

from

the classical Mendelian ratios of 1:1 and

_3:1,

_{respectively.}

Thus the

_assumption

that

_polydactyly

was controlled

_by

a

single

recessive allele seemed to be

strongly

generation,

since

_{relatively large}

numbers of normal animals were recovered from

crosses between

presumed homozygous

recessives. The final conclusion was that

polydactyly

was a so-called threshold character under

_polygenic

control.

From a _survey of the literature

_dealing

with

_genetics

of

_{morphological}

traits in

Ciliates,

it _appears that 3

_previously

described variants

_display

some similarities to fvtd: not induced

_by

_any

_mutagenic

treatment and

_including

alterations of the

_assembly

and

_positioning

of kinetosomes in the course of

completion

of the

overall

_ciliary

_pattern of the cell. These 3 are the mlm

(multileft-marginal)

variant

of

_{Paraurostyla weissei,}

the _mp

_{(membranellar pattern)}

variant of

_Tetrahymena

thermophila,

and the bbd

_(basal

_body

_deficient)

variant of

_Euplotes

minuta. Since

they

had been claimed to be

_single-gene

determined,

we reexamined the

_segregation

data on which these

_assumptions

had been founded.

In mlm

_clones,

most cells _possess 2 or 3 left

_marginal

rows of

_cirri,

instead of 1

in the

_wild-type

condition. The

_early

_stages of

_{prefission morphogenesis}

look

quite

normal,

such as in the fvtd situation. The alteration is initiated

by

the

_segmentation

of the

presumptive

primordium

of the left

_marginal

_row, a

_single

kinetosomal

streak;

the _segments become

_parallel

to each other and each one

develops

into a row of

cirri

_{(Jerka-Dziadosz}

and

_Banaczyk,

_1983;

Dubielecka and

_{Jerka-Dziadosz,}

_1989).

All known mlm lines are inbred clones derived from 2

_exconjugants

isolated from a

sample

collected from a

_{single pond. They}

have been

_pedigreed

over 10

_generations.

The

_progenies

of 3 crosses have been screened for

_segregation

of the mlm trait.

Both

_pedigree

and

_segregation

data

_strongly

_suggest a

_single-gene

control

(Jerka-Dziadosz and

_Dubielecka,

_1985),

so that this

hypothesis

_may be held as rather

firmly established,

in

_spite

of lack of direct evidence of

_homozygosity

of mlm

_clones,

since no viable _progeny could be recovered from mlm x mlm crosses. It must be

added that some clones have been found which exhibited a somewhat enhanced mlm

phenotype;

it is

_likely

that

_they

are

homozygous

not

only

for the mlm mutation

itself,

but also for a recessive allele at another locus

_{(Jerka-Dziadosz,}

_1989;

Jerka-Dziadosz et

al,

1989).

The mp

phenotype

was discovered in a

laboratory

inbred line. It consists of some alterations in the

_development

of the oral _apparatus,

_{especially during}

oral

replacement,

a _process which

_usually

occurs in starved

_cells,

so that the

_frequency

of abnormal cells in a

_growing

culture is rather low and increases up to 80% when the

_stationary

_phase

is reached.

_During

oral

_replacement

in a

_wild-type

cell,

an

oral

_primordium

arises from the fusion of 2 kinetosome

_fields,

one of which is

associated with the old

_{disintegrating}

oral _apparatus and the other related to

the so-called

_stomatogenic

_ciliary

row.

Later,

the

patterning

of the

single

anarchic

field results in the differentiation of the _components of the new oral _apparatus,

which includes 3 membranelles.

_Conversely,

in _mp cells the fusion of the 2 initial fields is

_delayed

and each one differentiates 3 membranellar

anlagen. Later,

the 6

anlagen

are

_incorporated

into a

_single

oral _apparatus, and some of them fuse in

such a _way that the final number of membranelles is

_usually

4 or 5

_{(Kaczanowski,}

1975).

The mp line was crossed to a

wild-type

line. The observed

_segregation

fit the

expected

ratios,

1:1 for back-cross and 1:2:1 in

_F

₂

_,

under the

_assumption

that the

mp

phenotype

is controlled

by

a

single

recessive allele. The

_F

₂

clones which were

classified as

_homozygous

recessive included in

_stationary

_phase

40 to 60% mp

cells,

About 50% of the

_F

₂

clones

_appeared

to be

_{phenotypically}

normal

_{during early}

stages of clonal

_life,

but later

_produced

_mp cells at low

_frequencies

₍₁

to

_10%):

_they

were

_interpreted

as

heterozygous,

_segregating

_{phenotypically}

_mp sublines

_by

means

of phenotypic

assortment, a

_phenomenon

which is well documented in

_{Tetrahyrreena}

thermo

P

hila

(Sonneborn,

1974;

Orias and

_Flacks,

_1975),

but which was not checked

in this

_particular

situation. The main difficulties for the

single

gene

hypothesis

arise from the occurrence of 2

F

l

clones which

_proved

to be

_{phenotypically}

_mp and not to _segregate

_wild-type

_progeny clones

_{by backcrossing}

or

_by

_{intercrossing.}

Kaczanowski

₍₁₉₇₅₎

_pointed

out that these

_unexpected

results

_might

deserve some

further

_attention,

but he nevertheless did not rule out the

_single

_gene

_hypothesis.

In our

_opinion,

these results indicate that the

_genetic

control of the mp

genotype

is

presumably

more

_complex

than first assumed.

_Among

a

large

range of

hypotheses,

a

_polygenic

_system is not excluded.

In the bbd variant of

_Euplotes

_minuta,

the

_primary

defect seems to be a

failure to

_produce

new basal bodies in

_prefission

_stages of

_{morphogenesis.}

Some

of the

_resulting

abnormalities of the

_ciliary

_pattern can be

interpreted

as direct

consequences of this defect: loss of dorsal

_ciliary

rows, lack of 1 or 2

right

caudal

cirri, presence of a few

incomplete

membranelles. Some others cannot be

_explained

in this

_simple

_way: for

_instance,

_{during proliferation}

of basal bodies within a

_given

ciliary

row, a few basal bodies _may become

_{mispositioned}

and then initiate a new row,

parallel

to the

_preexisting

one. Thus there are 2

_opposite

tendencies

_during

the

_growth

of a bbd

clone,

one to

_loss,

the other to addition of

_ciliary

rows, so that

the intraclonal

_variability

for the number of rows is much

higher

in bbd than in

normal clones. The rather

_complex

bbd

_phenotype

is _very

_likely

to be controlled

by

a

single

recessive allele

(Frankel,

_1973a).

Thus if our

_{interpretation}

is _correct, fvtd is the

_only

variant of

_ciliary

_pattern for which the

_breeding

data are

_{strongly suggestive}

of

_polygenic

inheritance.

Morphological

variants and evolution

If E balticus is

_excluded,

since the information available on this taxon _{is very} poor, the section A of the genus

Euplotes,

as defined

_by

Curds

_(1975),

is very

likely

to be

_monophyletic

on the basis of _many similarities in

_ciliary

_patterns and

arrangement of the dorsal argyrome

(Tuffrau,

1960; Curds, 1975; Gates, 1978a,

1978b),

mating

type systems

(Heckmann,

1963, 1964;

Nobili,

1966;

Wichterman,

1967),

electrophoretic

patterns

(Schlegel

et

_al,

_1988),

_habitat,

etc. Since the oldest

specific epithet

applying

to a _category included in this section is vannus

(Miiller,

1786),

this valid

_infrageneric

_{taxon may} be

_conveniently

referred to as &dquo;the vannus

group

_of

genus

Euplotes&dquo;

(G6nermont

et

al,

1985).

Within

_it,

3 entities can

be

_separated

on the basis of

_{morphometric,}

biochemical and

_ecological

criteria

(Machelon

et

_al,

_1984;

G6nermont et

_al,

_1985;

Valbonesi et

_al,

_1988;

Gianni and

Piras,

1990).

Since the 3 kinds of criteria

_provide

_{arguments in very}

_good

agreement with each

_other,

it may

reasonably

be assumed that these entities are

monophyletic

taxonomic units.

_They

are

obviously

rather

_closely

_related,

since

mating

reactions have been observed between members of different units

_(Nobili,

1964).

Genetic

_exchanges

are nevertheless

_impossible,

so that the taxonomic level of these entities is either

_specific

or

supraspecific. Morphometric

studies failed to

entities may be referred to as

_morphotypes.

It has been

_repeatedly

pointed

out

that these

_{morphotypes satisfactorily}

fit earlier

_diagnoses

of

Euplotes

vannus 0 F M

_(long

and

_{relatively straight}

_cells),

E crassus

Dujardin

(shorter

and

relatively

wider)

and E minuta

_{(definitely smaller):}

for a

_{thorough discussion,}

see for instance

Valbonesi et al

₍₁₉₈₈₎

and

_Schlegel

et al

_(1988).

One of these

_morphotypes

has been

_proved

to be a

complex

of several

(at

least

₅₎

_sibling

_species:

it was

_{&dquo;provisionally&dquo;}

named

_{&dquo;Euplotes}

vannus

_{complex&dquo;}

(G6nermont

et

_{al, 1976, 1985;}

Machelon et

al,

1984).

In the

_light

of _present

knowledge,

it is clear that this

_complex

is

_actually

identical to the E crassus

morphotype,

so that it must be renamed

_{&dquo;Euplotes}

crn.ssus&dquo;

_complex.

Thus the

species

in which the fvtd trait is

_reported

in the present paper is

conveniently

referred to as E crassus,

species

No 2.

The

_species

of the E crassus

complex

are

highly

similar at the

_{morphological}

level,

and also at the

_{electrophoretic}

and

_ecological

levels

_(Machelon

et

_al,

1984 and

unpublished

results),

so that it may be

suggested

that the

speciation

events which

promote an increase in the number of

_species

within the

_complex

result from a

more or less random accidental rise of

_mating

_barriers,

with _very little

_evolutionary

importance.

Some

_equilibrium

must be reached between

_speciation

and extinction

rates with minor

_ecological

_{consequences,} so that the effective

_evolutionary

unit is not the

_species,

but the

_complex

itself. Thus the

_evolutionary

_significance

of the E cmssus

complex

is _very different from that of some other

complexes

of

sibling

species,

for instance Pardmecium aurelim

_(Sonneborn,

₁₉₇₅₎

in which the

_ecological

niches are

only partly overlapping.

More

_important

from an

evolutionary point

of view is the differentiation of the

3

_morphotypes,

_cmssus, vannus and rrcinuta. At a

_{morphological}

_level,

crassus and vannus seem more similar to each other than either of them to minuta.

_Formally,

it seems that it would be

_possible

to transform crassus into vannus

_{by increasing}

cell

_length,

_decreasing

cell width and

_decreasing

the number of dorsal

_ciliary

rows.

Since

_{intraspecific genetically}

determined

_naturally

occurring

variation is known for

the number of

_ciliary

rows

(Heckmann

and

Frankel, 1968; Frankel,

1973b; Laloe,

1979),

and a

_positive

correlation has been shown between number of rows and cell

width

_(Machelon

et

_al,

_1984),

it could be

_expected

that some _progress towards

the vannus

phenotype

would be obtained

by

selecting

for a

high

length/width

ratio in a

_genetically

_{heterogeneous population}

of the crassus

_morphotype.

Such an

_experiment

was carried out over 5 sexual

_{generations, starting}

from the

_species

No 2

_synthetic

_population

described above.

_{Unexpectedly,}

_despite

a rather

_strong

selection _pressure, no _response was observed

(unpublished results).

On the other

_hand,

there are some indications that the loss of one of the

FV cirri

_might

have occurred several times

_{independently during}

the

_evolutionary

diversification of the genus

(Schlegel

et

_al,

_1988).

_{It may} be

_postulated

that such

a minor

_change

involves the

_fixation,

associated with some

_speciation

_event, of a

very low number of

mutations,

perhaps

a

single

one. If this is true, it should be

possible

to

_{find, by}

means of an extensive survey of

intraspecific

_variation,

some

exceptional

variants,

genetically determined, differing

from the

_species

standard

pattern

by

lacking

one cirrus. Such variants have not been

_found,

either

_naturally

More

_generally,

_however,

those variants which have been isolated

_usually

exhibit

very

large

deviations from the standard

ciliary

patterns, much

larger

than

inter-specific

differences within a _{genus. The} fvtd cells which are shown in

fig

2A and

2B

_display

a

non-Euplotes

cirral _pattern. The mlm mutant of

Paraurostyla

weissei

shows

_profound

alterations in some

morphogenetic

fields. The membranellar pat-tern of the _{mp variant in}

_{Tetrahymena thermophila}

is unstable. In the bbd variant

of

_Euplotes

minuta,

the

_phenotype

is

_by

no means reminiscent of the standard pat-tern _{of any} related

_species.

Most induced mutations determine

_highly

abnormal

patterns

(for

reviews,

see

_Frankel,

_1989;

Jerka-Dziadosz and

Beisson,

1990).

Moreover,

most _spontaneous variants are

likely

to be

_strongly

counterselected in

nature, since

_they

show either

_{exceptionally}

low fission rates or

exceptionally

high

postmating

mortality.

The fvtd variant does not seem to be altered in fission _rate, a

rather

_{unexpected result,}

since it _suggests that

_foraging

_activity,

a vital

function,

is

to a

large

extent uncorrelated with the FVT _pattern; it must be

admitted,

however,

that this conclusion _may not be valid in natural

conditions,

where food is much

scarcer than in

laboratory

cultures. The

capacity

for

uniting

with a

_mating

_partner

is also normal.

_However,

the

_experimental

results indicate that the fvtd trait is

associated with a _very

_{high mortality}

rate in

_{exconjugants.}

The

identification

of the critical

_stage

of the

_postmating

sequence will be the aim of a further

experimental

study.

The

_rarity

of

_genetically

determined

_{morphological}

variants in a natural

popu-lation is well accounted for

_by

such counterselection. When a variant

phenotype

is controlled

_by

a

single

recessive

allele,

the

frequency

of this allele is

necessarily

very low so that the

_homozygous

clones are

exceptional.

In

laboratory

conditions,

inbreeding

occurs, either

spontaneously

as a _consequence of small

population

size or

according

to some

_{experimental design, resulting}

in an increase of

homozygote

fre-quencies

and

_favouring

the detection of variants. The known

_{morphological}

mutants

of

_Pamurostyla

weissei were discovered in this manner: the recessive alleles were

present in a

pond population.

As for a

polygenically

determined trait the situation may be somewhat different. The fvtd variant has been isolated from a

_laboratory

population

in which

_{genetic exchanges}

took

_place

between

_previously

_separate gene

pools,

since the founder clones had been collected from

_{geographically}

distant areas;

possibly

it was

_generated

_by

recombination between these _gene

pools,

though

in a

given

locality

the full gene set able to determine the fvtd

_phenotype

would never

be _present. In the first situation some

_genetic

load is associated with the variant in

the

_populations

where the recessive allele is _present. In the second situation there is

no

_genetic

load in any

population,

and the

genetic

basis for the

expression

variant may be found

only

at the level of the whole

species.

It appears from this discussion that

nothing

is known of how some of the

characters which are

currently

used for

_species

diagnoses

are

_genetically

determined.

This conclusion _may be

_compared

to the ideas

_{expressed by}

Frankel

_(1983).

He

_suggested

a mode of

speciation

in which the

primary

event would be the

fixation in a

population

of a mutation

responsible

for a shift in life

_habits,

for

instance

_adaptation

to a new

_ecological

niche. Such a

population

is

expected

to

display

some

degree

of

reproductive

isolation from the bulk of the

species

and to be submitted to new selective _pressures

resulting

in

sequential

fixation of other

one

_by

several _genes. This model does not

_imply

a

_major

morphological

effect of

the

_primary

mutation

_and,

_according

to

_Frankel,

the

_{morphological divergence}

is

likely

to

_proceed

_slowly,

on a

_polygenic

basis. It

_implies,

_however,

that some

single-gene or

_{oligogenically}

determined variant

_{morphological}

traits are at least tolerated

by

natural selection. The variants which have been described or discussed in the

present paper are not tolerated.

_Presently

available data are far too restricted to

conclude that tolerated variants do not exist.

_They

nevertheless _suggest that a

given

morphological

pattern is controlled

_by

a rather

_large

_array of _genes with _very

complex intergenic

interactions,

in such a _way that a

change

even at the level of

a

single

gene is

likely

to

_severely

disturb these

_{interactions;}

it is thus

_suspected

that a new tolerated

_{morphological}

_pattern can be

_generated

only

_by

a dramatic

repatterning

of the whole

genic

system. Further research is

_undoubtedly

necessary in order to find new

naturally occurring

morphological

variants and assess their

fitness: this will allow an evaluation of the

_{evolutionary potentialities of intraspecific}

morphological diversity.

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