Download courtship and mating behavior as a reproductive isolating

AM. ZOOLOGIST, 4:147-153(1964).
COURTSHIP AND MATING BEHAVIOR AS A
REPRODUCTIVE ISOLATING MECHANISM IN
DROSOPHILA
LEE EHRMAN
The Rockefeller Institute, Neiu York
Species of sexually reproducing or*
ganisms are genetically closed systems.
They are closed systems because they do
not exchange genes or do so rarely enough
so that the species differences are not
swamped. Races are, on the contrary, genetically open systems. They do exchange
genes by peripheral gene flow, unless they
are isolated by extrinsic causes such as
spatial separation. The biological meaning
of the closure of a genetic system is simple
but important—it is evolutionary independence. Consider these four species—man,
chimpanzee, gorilla, orangutan. No mutation and no gene combination arising in
any one of them, no matter how favorable,
can benefit any of the others. It cannot do
so for the simple reason that no gene can
be transferred from the gene pool of one
species to that of another. On the contrary,
races composing a species are not independent in their evolution; a favorable
genetic change arising in one race is, at
least potentially, capable of becoming a
genetic characteristic of the species as a
whole.
Species are genetically closed systems because the gene exchange between them is
impeded or prevented by reproductive isolating mechanisms. The term "isolating
mechanism" was proposed by Dobzhansky
in 1937 as a common name for all genetically conditioned barriers to gene exchange
between sexually reproducing populations.
According to Mayr (1963), isolating mechanisms are ". . . perhaps the most important set of attributes a species has. . . ."
It is a remarkable fact that isolating mechanisms are physiologically and ecologically
a most heterogenous collection of phenomena. It is another remarkable fact that the
The work reported here has been carried out
under Contract No. AT-(30-l)-3096, U. S. Atomic
Energy Commission.
isolating mechanisms which maintain the
genetic separateness of species are quite
different not only in different groups of
organisms but even between different pairs
of species in the same genus.
Several classifications of the reproductive
isolating mechanisms have been proposed.
That of Mayr is a simple and convenient
one. The two major groups are the premating barriers which prevent the formation of hybrid zygotes, and the postmating
barriers which impede the survival or reproduction of these zygotes.
Three of the premating mechanisms are:
1) Potential mates do not meet (seasonal
and habitat isolation).
2) Potential mates meet but do not mate
(ethological or sexual isolation).
3) Copulation attempted but no transfer
of gametes takes place (mechanical isolation).
Four of the postmating barriers are:
4) Gametes transferred but no fertilization, and hence no zygote formation
takes place (gamete mortality).
5) Death of the zygotes (hybrid inviability).
6) The Fi zygotes are viable but partly
or completely sterile (hybrid sterility).
7) Fi hybrids are fertile but the fitness of
the F 2 or backcross hybrid is reduced
(hybrid breakdown).
Since our primary interest today centers
on the relations between genes and behavior, ethological isolation, sometimes
also termed sexual or psychological isolation, should be discussed here in more
detail. The phenomenon observed is usually that the mutual attraction between
conspecific females and males is greater
than the attraction between males and
females of different species. Successful
analysis of this form of isolation requires
(147)
148
LEE EHRMAN
a detailed description of the courtship rituals and the mating behavior in the pure
species concerned. Species of the genus
Drosophila offer abundant and favorable
material for such analysis; their courtship
and mating can be observed easily at any
season of the year and under reasonably
precisely controlled laboratory conditions.
My favorite materials are the incipient
species of the Drosophila paulistorum complex or superspecies. As the name "incipient species" implies, these forms are very
closely related and can be regarded either
as very similar sibling species or as very
distinct races standing on the brink of
full species separation. This close relationship is, of course, very favorable for
analytical purposes. What we wish to
study is speciation in the process and the
closeness permits a genetic analysis to be
carried further than would be possible
with full species.
The six incipient species of Drosophila paulistorum inhabit a part of the
Neotropical zoogeographic region, from
Guatemala and Trinidad in the north to
southern Brazil in the south. Each incipient species has a distribution area of its
own, but these are in part overlapping, so
that in some places two, three, or even
four incipient species live together, sympatrically, and apparently without producing any hybrids.
Thanks to the work of many authors,
especially H. T. Spieth (1952) the sexual
behavior in Drosophila is fairly well
known. Sexual recognition is a trial and
error affair among drosophilids. Males will
generally attempt to court females of any
Drosophila species, even distantly related
ones.
At least four distinct elements can be
distinguished in the courtship process in
Drosophila paulistorum. The first element
or stage is that of circling. The male approaches a female, attracts her attention,
and may limit her movements by running
around her. He never completes a full
circle however, turns around, reversing the
direction of his movement about every
330°.
Next he begins a tapping action—an important aspect of the courtship in this species (Fig. 1A) . The male lightly touches
the legs or the abdomen of the female with
his own legs. At first, only one leg of each
fly is involved. Since many taps may be
necessary before it is established that the
male has located a female of his own species, the male continues to circle between
contacts. Here, the question of the female's
receptivity is settled (this is probably mediated by samples taken by the great number
of chemoreceptive hairs on the body of
both insects); she will either remain still
so that circling is no longer necessary, and
then spread her wings to receive the male
between them; or she will vigorously kick
the courting male and do her best to
depart.
Licking and wing vibrating occur next
(Fig. IB), both as a prelude to mounting.
As Spieth (1952) has described it, the male
"goes to the rear of the female and assumes
a slightly crouched position with the tip
of his abdomen slightly curled. Having
positioned himself, he extends one wing
70° to 90° and vibrates it periodically,
twisting his body on the longitudinal axis
as he vibrates, taps (uppercuts), and occasionally licks or attempts to lick the
female." In licking, the male proboscis
contacts the female genitalia—and this is
a reliable sign that the male is about to
rush in for the mount. In D. paulistorum
wing vibrations are not so important a
part of courtship as in some of the
other species. The one wing that is
vibrated is used for leverage as the male
raises himself on the body of the female.
Mounting and insertion seem to be accomplished simultaneously. A portion of
the male reproductive organ is used in a
clasping manner and, after mounting, the
male secures his position on the female by
placing his forelegs on top of her slightlyspread wings. This additional support is
imperative because copulation lasts a fairly
long time in this species (an average of
17 minutes and 12 seconds). During the
copulatory period, the female may turn
about or walk around; and she may even
BEHAVIORAL ISOLATION IN
Drosophila
149
find herself having to fend off other males
(Fig- 2) •
When copulation is nearly over, the
female attempts to dislodge the male by
vigorously kicking and swinging her body
from side to side. The male loses his hold
on her wings when she snaps them together, and seconds later he falls off backwards. Thereafter, the female repels all
sexually excited males by raising the tip
of her abdomen, rendering her vaginal orifice inaccessible.
FIG. 2. Photograph of the full mount during which
time the female may move about freely and even
feed.
FIG. 1. These "stills" were taken from a 16 mm
color film prepared by Mr. Richard F. Carter of
the Rockefeller Institute and by the author; the
project was supported by a grant from The Society
of the Sigma Xi. This study of courtship be
havior in Drosophila paulistorum was shown, in
part, when this paper was delivered.
A. Tapping (the male is the smaller, active individual with the rumpled wings)—an initial stage
in courtship.
B. Licking—often seen just before mounting.
C. The formation of a chain wherein one re-
Notice that throughout this entire procedure, it is the female that at all stages,
is "discriminatingly passive" while the
male is "indiscriminately eager" (Bateman,
1948). D. paulistorum males are very active
and will court almost anything: a dead
fly, a mired fly, lumps of food, and often
other males. It is not unusual to observe
the formation of a chain initiated by a
male courting a female, and in turn being
himself courted by another male who is
being approached by yet another male.
These chains, of course, are of short duration (Fig. 1C).
jecting female (with extruded ovipositor) is being
courted by a male who is in turn being courted by
a male, etc., for a total of one female and three
males—these chains are necessarily of short duration but do emphasize the fact that D. paulistorum
males will certainly court the other males. See text
for detailed explanation.
150
LEE EHRMAN
Now that the normal courtship and
mating behavior of the species-complex
has been recorded, we are ready to consider the evolution of this superspecies
Drosophila paulistorum (Dobzhansky et
al., 1964).
One of the first of the many interesting
evolutionary phenomena exhibited by the
D. paulistorum complex of seven known
races or incipient species to be analyzed
genetically was the complete hybrid male
sterility discovered when crosses between
the races were successful. The male sterility was found to be genie in nature
(Ehrman, 1960a), and to be expressed via
the genotype of hybrid mothers. This kind
of hybrid sterility, unique in the genetic
literature, operates in the following manner. Suppose that we cross two races, A
and B, one or both of them having one
or more chromosomes marked with suitable mutant genes. The distribution in the
progeny of the chromosomes of different
racial origin can then be followed by inspection of the external morphology of the
Hies. The interracial hybrid males are
sterile, but the females are fertile and can
be backcrossed to either A or to B males.
After several backcrosses, flies are obtained
which carry all but one of the chromosomes from race A, or all but one from
race B. Females of this sort are, of course,
fertile and can again be backcrossed to
males of the recurrent race. Half of this
progeny, of either sex, will carry all chromosomes of the same race, and half will
contain one race-foreign chromosome.
(Crossingover is suppressed in these hybrids.) Now, the striking fact is that males
of both kinds are completely sterile. The
sterility of the males identical in chromosomal constitution with males of one of the
"pure" races can only mean that the sterility is in this case determined not by the
chromosomal constitution of the individual
itself but by that of his mother. The presence of at least one race-foreign chromosome in the developing egg cell before
meiosis somehow modifies the cytoplasm or
some other property of the egg, and makes
a male individual developing from this egg
sterile.
Once the above facts were established,
the question logically arose whether the
ethological isolation observed in races of
D. paulistorum has the same peculiar genetic basis as the sterility of the hybrid
males, that is, a genic-maternal effect. The
ethological isolation is in all probability
the mechanism which keeps the gene pools
of these races separate in nature where the
races share the same territory. Indeed, race
hybrids have never been found in nature,
and the cytological study of their chromosomes by Dobzhansky and Pavlovsky
(1962) shows that genetically effective
hybridization of the races is rare, if it
occurs at all.
The genetic basis of the ethological (sexual) isolation was studied by Ehrman
(1961). The method used was in principle the same as that applied for the
analysis of the hybrid sterility, i.e., making
crosses and backcrosses of strains of different races having some of their chromosomes
marked with suitable mutant genes. It was
here that the knowledge of the courtship
and mating behavior of D. paulistorum
became important.
Hybrid males of D. paulistorum transfer
no sperm into the body of the female with
which they copulate. F1 hybrid males produce no motile spermatozoa, because a
restitution nucleus is formed presumably
after the first meiotic division, and then all
the gametic material degenerates. Backcross hybrid males are even more profoundly sterile, since they frequently have
no testes at all, or only one testis, or no
gonial cells within the abnormally thick
testicular sheath. Yet these males are normal in external morphology and have normal genitalia and internal reproductive organs other than testes. Their patterns of
courtship and mating behavior are also
normal. Since dissections of the female
reproductive tract for the storage of transferred sperm (as is usually done in analyses
of this sort where fertile males are involved) would be out of the question here;
the entire analysis of the genetic architec-
BEHAVIORAL ISOLATION IN
ture of sexual isolation as a reproductive
isolating mechanism was made by using
the simple but informative, direct-observation technique.
One may conclude from these directobservation studies that the sexual isolation, which makes matings between the females and males of the D. paidistonim
races much less likely to occur than matings within the races, is due to polygenes
in every one of the three pairs of chromosomes which the species possesses. These
polygenes control the sexual preferences of
their carriers. Their effects seem to be
simply additive. A female of hybrid origin
which carries a majority of the chromosomal material derived from a given race is
most likely to accept a male of that race.
And conversely, a hybrid male is most
likely to be accepted by females whose
chromosomal constitution is closest to his.
The source of the cytoplasm or the genetic
constitution of the mother do not seem to
matter. This is clearly not at all comparable with the genetic basis of the hybrid
sterility, where the properties of an egg
are determined, as far as the sterility of
the backcross males is concerned, by the
genotype present in it before meiosis, and
not by that formed following fertilization.
We may surmise that this peculiar chromosomal-cytoplasmic mechanism of sterility arose first in the evolutionary history
of these races, and that it arose in allopatric races becoming adapted to different environments in their respective geographic
areas. Sexual isolation might then be built
by natural selection, as a much less wasteful and more efficient reinforcement of the
bar to gene exchange between the races.
When this sexual isolation became strong
or complete, the races became able to coexist sympatrically, as they have been
found to do in some localities in the northern part of the South American continent
(Dobzhansky et al, 1964) where as many
as four races have been found to live in
the same places.
How effective can sexual isolation be,
what with omnipresent variations in environmental influences and the undeniable
Drosophila
151
susceptibility of behavior patterns to these
influences? Consider the case of the rare
hybrids obtained in the laboratory between
the races of Drosophila paulistorum inhabiting northern and southern Brazil, respectively. These hybrid individuals are
most difficult to obtain and possess a genetic constitution discordant enough so
that the hybrid females repel the courtship
of all males, and will mate with none;
their sterile hybrid brothers will court and
will be rejected by almost all females, including their own hybrid siblings. There
is no question of gene flow through the
hybrid males, because they are absolutely
sterile. Their sisters, however, are potentially fertile. This has been verified not
only by dissection of their internal and
external reproductive apparatus, but more
conclusively by a direct experiment. Some
etherized hybrid females are exposed to
many mature, unanesthetized males; the
males approach, mount and inseminate the
females in question; the hybrid females
subsequently recover and deposit fertilized
eggs which develop normally (Ehrman,
1960b).
More recently, Carmody et al. (1962)
investigated a possible correlation between
the occurrence of hybrid sterility and sexual isolation within the D. paidistonim complex. In this massive study, more than sixteen thousand females from all of the then
known D. paulistorum races were dissected
and their sperm-storing organs checked.
Each Drosophila paulistorum female, hybrid or not, has three sperm-storing organs.
The "male choice" experimental method
was used to test mating preferences of
different strains comprising the races.
Briefly, this involves groups of ten virgin
females of each of two races ( a total of
twenty females), aged for three to four
days after hatching, marked for recognition by clipping a very small part of one
of the wings of one of two types of females,
and then confined with males of one of
the two races for twenty-four to fortyeight hours, i.e., A ^ X A ?? -)- B J J
or B ^ X B $? + A 5$. All the females
are then dissected in a physiological saline,
152
LEE EHRMAN
and their sperm receptacles are examined
for the presence or absence of sperm.
Strains of the same race but of different
geographic origin often show significant
preferences for homogamic matings, but
strains of different races show such preferences to a much greater extent. On the
average, sexual isolation is lower in interracial matings involving the transitional
race, the bridge to fertile hybrids between
all the other races. The degree of sexual
isolation shows only a weak positive correlation with the fertility or sterility of the
hybrids between the strains crossed.
More study is needed to unravel the interrelations of the sexual isolation and hybrid sterility. As indicated above, sexual
isolation is most effective in limiting or
preventing the appearance of interracial
hybrids with reduced reproductive fitness.
A problem of considerable interest is
whether isolating mechanisms which decrease the chances of production of hybrid
offspring are stronger between sympatric
than between allopatric populations of the
same pairs of races or subspecies or incipient species. When two or more Mendelian
populations of sexually reproducing and
cross-fertilizing organisms share the same
territory, these populations are exposed to
the risk of hybridization and gene exchange. If such a gene exchange leads to
production of adaptively inferior genotypes, natural selection may favor genetic
constitutions which hinder or prevent hybridization. On the other hand, the gene
exchange and introgression may weaken
the reproductive isolation, and may eventually lead to fusion of the previously separate populations. Experiments were made
to test whether the sexual isolation between sympatric strains of a given pair of
races is, on the average, greater or smaller
than that between allopatric strains of the
same races. They showed (Dobzhansky et
al., 1964) that natural selection had encouraged the spread and maintenance, in
sympatric populations of incipient species,
of those genes which limit or prevent the
reproductive wastage resulting from gene
ilow between these populations.
However, one may suppose as Muller
(1942) does that reproductive isolation
arises as an incidental by-product of genetic divergence. When populations become adapted to different environments
they are likely to become different in progressively more and more genes. Reproductive isolation then might arise because
the action of many genes is pleiotropic.
Some gene differences selected for different reasons, or resulting from random genetic drift, may thus have isolating sideeffects.
That selection can indeed produce, or
at least strengthen, reproductive isolation
has been demonstrated experimentally by
Koopman (1950) and by Knight, Robertson, and Waddington (1956). Koopman
set up experimental populations in laboratory population cages consisting of two
species, D. pseudoobscura and D. persimilis. Each species was homozygous for a
different recessive mutant gene with easily
visible external effects. The pure species
and the hybrids were thus made easily
distinguishable. In every generation the
adult flies were taken from the cages, etherized, classified, and counted. The hybrids
were then discarded and new population
cages were started with nonhybrid progenies. By these means Koopman was selecting the progenies of intraspecific, and excluding those of interspecific, matings. In
a surprisingly small number of generations
he obtained strains of D. pseudoobscura
and D. persimilis which showed a more
nearly complete sexual isolation than did
the original strains. The results of Knight,
Robertson, and Waddington are, in a way,
even more dramatic, since they obtained
by selection a significant, though of course
incomplete, sexual isolation of strains of
D. melanogaster which originally showed
no such isolation.
The incipient species of D. paulistorum
seem to furnish a good illustration of the
two processes postulated above. It is not
easy to imagine the sterility of male hybrids between these incipient species conferring am adapthe advantage upon them.
But once this sterility has arisen as a hy-
BEHAVIORAL ISOLATION IN
product of their genetic divergence, it is
probable that natural selection would favor
genetic constitutions which make the sterile hybrids rare. The sexual isolation between the incipient species may be a reproductive barrier contrived as such by
the action of natural selection.
Dobzhansky and Spassky (1959) first
suggested that Drosophila paulistorum is
a cluster of species in statu nascendi, a
borderline case of uncompleted speciation.
This suggestion is borne out by the work
since then. Drosophila paulistorum is a
superspecies composed of six races or
incipient species. The interest of the situation lies precisely in that these six may
be considered about equally legitimately
as very distinct races or as very closely
related species. Each race inhabits a geographic area different from the others, but
the areas of some of the races overlap.
Where two or more "races" share a common territory they apparently do not interbreed, and thus behave like full-fledged
species. The Transitional race and transitional strains yield, however, fertile hybrids with some other races. The possibility of gene flow between the incipient
species is, therefore not excluded, although
it is questionable whether it is actually
taking place.
Last year, in his Vice Presidential address on Genes and the Study of Behavior,
Caspari (1963) cautioned ". . . the analysis of Ft species hybrids cannot give us, in
principle, information about genetic units.
This will only be possible through further
crosses, F2 and backcrosses." The step-bystep, often tedious analysis of the D. paulistorum situation, as presented here today,
certainly indicates how correct he was. Understanding of the genetic basis of the sexual isolation among these races would have
been impossible without a systematic study
of the backcross individuals, in conjunction
with a survey of the courtship and mating
behavior in the Fj hybrids and in the
Drosophila
153
"pure" races. Surely "the genetic basis of
behavioral characters is important, because
it is the genes which are the basic units
transmitted and reshuffled in the evolutionary process and which are arranged by
selective forces into adaptive action patterns." (Caspari, 1963.)
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