The induction of sexual reproduction in Daphnia magna: genetic differences between

arctic and temperate populations

DENISEC. FERRARI' A N D PAULD. N. HEBERT

Department of Biology, Great Lakes Institute, University of Windsor, Windsor, Ont., Canada N9B 3P4
Received November 1 8, 1981
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FERRARI,
D. C., and P. D. N. HEBERT.
1982. The induction of sexual reproduction in Daphnia magna: genetic differences
between arctic and temperate populations. Can. J. Zool. 60: 2 143-2 148.
Even when provided with abundant food and grown in isolation, Daphnia magna from arctic Canada responds to declining
photoperiods by producing male and then ephippial offspring. In contrast, clones of English origin do not alter their reproductive
behaviour when exposed to photoperiod shifts. Hybrid clones show an intermediate response in terms of ephippial egg
production, but a variable response in male production. The results indicate that variation exists at gene loci controlling sexual
behaviour in local populations as well as between distant populations. 'The significance of this variation is discussed in relation to
the environmental heterogeneity encountered by D . magna over its extensive range.

FERRARI,
D. C., et P. D. N. HEBERT.
1982. The induction of sexual reproduction in Daphnia magna: genetic differences between
arctic and temperate populations. Can. J. Zool. 60: 2 143-2 148.
Meme gardees isolees en cultures, en presence d'une source abondante de nourriture, les daphnies Daphnia magna de
I'Arctique canadien reagissent au raccourcissement de la photoperiode en produisant d'abord des miles, puis des Cphippiums.
En revanche, des clones d'origine britannique ne changent pas leur comportement reproducteur lorsque la photoperiode est
modifiee. Les clones d'hybrides ont une reaction intermediaire en ce qui conceme la production d'oeufs tphippiaux, mais la
For personal use only.

production de miles est variable. Les rksultats indiquent qu'il y a des variations aux locus qui contrdlent le comportement sexuel
chez les populations et chez les populations Cloignees. La discussion porte sur l'importance de cette variation en fonction de
I'hCttrogCntite &cologiquerencontrke chez Daphnia magna dans toute son aire de repartition.
[Traduit par le journal]

Introduction northern Canada and Greenland to mesic habitats in

The cladoceran crustacean Daphnia magna repro- Europe and the southwestern United States. In northerly
duces by cyclic parthenogenesis. During the early locales population survival depends upon the formation
spring, populations often consist entirely of female of ephippial eggs, because they are the only life stage
individuals producing diploid parthenogenetic eggs resistant to freezing. In more temperate regions,
which may develop into either female or male offspring. ephippial eggs remain important in those habitats which
Sex is determined by environmental conditions and regularly dry up. However, in ponds which remain filled
broods are ordinarily single sexed. Photoperiod, year round, populations can reproduce parthenogenetic-
crowding, and food levels have all been implicated in ally for an extended period of time. Such populations do
the switch to producing male eggs (Banta and Brown not eliminate sexual reproduction entirely as ephippial
1929; Stross and Hill 1965, 1968; D'Abramo 1980). eggs remain important for colonizing new habitats.
Aside from producing parthenogenetic eggs, females However, in such permanent ponds there is no
can also produce haploid ephippial eggs which require requirement for sexuality to occur at a specific time of
fertilization. Ephippial egg formation in some cladocer- the year.
ans is induced by a rapid decline in food availability The aim of the present study was to compare the
(Slobodkin 1954; von Dehn 1956; D'Abramo 1980); in reproductive behaviour of six clones of Daphnia magna
others a strong photoperiod effect has been shown exposed to different photoperiods and culture densities.
(Stross 1969). It is clear from a number of studies that Two of the clones were collected from tundra ponds near
the reproductive behaviour of most cladoceran species is the northern range limit of the species in Canada, and
determined by a complex interaction of environmental two others were collected from a pond in England which
variables (Stross and Hill 1965; Stross 1969, 1971; permitted year-round parthenogenetic reproduction.
Stimpfl 1971). The remaining two clones were produced by hybridizing
Over its extensive geographic range Daphnia magna clones from the two geographic locales.
is found in a diversity of habitats from tundra ponds in Materials and methods
' ~ o r m e r D.
l ~ C. Woodrich. The Canadian clones, C l and C2, were collected from two
0008-4301 /82/092143-06$01 .OO/O
01982 Nati~nalResearch Council of Canada/Conseil national de recherches du Canada
 2144 CAN. J. ZOOL. VOL. 60, 1982
temporary ponds near Churchill, Manitoba. The English
clones, E l and E2, were collected from a permanent pond near
Hatley Hill, Cambridgeshire. The remaining two, H 1 and H4,
were hybrid clones produced by mating Canadian females with
English males. The maternal origin of HI was a C l female, for
H4 a C2 female. In both cases the Canadian females were
mated to E2 males. The hybrid offspring were produced by
placing E2 males with females in the early stages of ephippial
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formation. Ephippia were collected and stored in a dry state at
5C for 1-3 weeks. Thereafter, they were frequently flooded
with synthetic pond water at room temperature to induce
hatching. Electrophoretic analysis of several enzyme loci
verified the hybrid origin of these individuals (Crease 1981).
All stock and experimental animals were maintained in a
synthetic pondwater mixture (Hebert and Crease 1980).
Experimental animals were maintained in 100 mL of water and
fed Chlamydomonas sp. every 2 days. Chlamydomonas sp.
was cultured in an autoclaved medium containing 250mg
NaN03, 50 mg MgS04-7H20,100 mg K2HP04,25 mg NaCl,
33 mg CaC12.2H20, and 50 mL of soil extract in 1 L of distilled
water. The cultures were grown at room temperature under
continuous fluorescent illumination and used after 7- 10 days.
The optical density was adjusted to 0.15 at 620 nm. Dried liver
extract at a concentration of 2 mg/mL was mixed, allowed to
settle, and one part of the supernatant was added to five parts
For personal use only.
algae. One millilitre of a vitamin supplement (700 mg Ca
panthothenate, 60 mg thiamin, 40mg riboflavin, 130 mg
nicotinarnide, 330 mg folic acid, 500 mg choline, 30 mg
putrescine, and a trace amount of vitamin B ,2 in 1 L of distilled
water) was added weekly to each beaker.
All experiments were run at 20 + 1C; three photoperiods
(8, 16, and 24 h of light) were studied. Before beginning the
experiment, stocks of each clone were maintained for
several generations at 20C and 24 h of light. To initiate an
experiment, gravid females were placed individually in
beakers and shifted to the experimental photoperiod. Within
24 h of birth their offspring were removed and placed in cups
either individually or with nine other neonates of the same
clone. These individuals required about 10 days to mature;
thereafter their reproductive behaviour was monitored daily
and all offspring were removed and sexed.
At each feeding, cups containing a single animal were given
4, 3.5, and 3.0 mL of algal solution in the 8-h, 16-h, and 24-h
photoperiod groups, respectively. The animals in shorter
photoperiods were given more food in case the shorter periods
of photosynthesis reduced algal growth rates. The cultures
containing 10 animals were fed 10 mL at each feeding. To
prevent the accumulation of waste products one-half of the
culture medium was replaced each week.
Three experiments were carried out to study the effect of
shifts in photoperiod on the reproductive behaviour of isolated
individuals. Each experiment involved placing 10 individuals
of a clone in the three photoperiods (8, 16, 24 h). Each
individual was maintained until it had produced four broods
and the proportion of females producing male, female, or
ephippial eggs at each brood was calculated. Parthenogenetic
broods generally consisted entirely of male or female
offspring. Before statistical analysis, the data were arcsine
transformed.
Two experiments were carried out to determine if crowding
increased male or ephippial egg production. These studies
were run only at 24-h and 16-h photoperiods as difficulty was
experienced in maintaining crowded cultures in an 8-h
photoperiod. In each experiment, 4-6 replicate cups were set
up for each of the six clones. The parthenogenetic offspring
produced by the 10 females in each cup were sexed and the
number of ephippia was tallied daily. Cultures were
maintained for a period of 30 days, a length of time similar to
the experiments on isolated individuals. Females produced
offspring during the last 15 days of this period. The proportion
of male offspring produced by each clone was determined by
dividing the total number of males by the number of
parthenogenetic young produced during an experiment. The
absolute number of ephippial broods could be determined by
counting ephippia, but the proportion of ephippial broods had to
be estimated, as the number of parthenogenetic broods could
not be determined. The total number of broods produced
during an experiment was estimated by multiplying the
number of females (10) present by the length of reproductive
period (15 days) and dividing by the brood interval (2.5 days).
The total number of broods produced during an experiment
was calculated to be 60. Thus if 6 ephippia were produced
during an experiment, the proportion of ephippial broods was
estimated to be 10%.
Results
The experiments on isolated females (Table 1)
showed a very low incidence of ephippial egg
production in both 24- and 16-h photoperiods.
However, in an 8-h photoperiod, females of both arctic
clones produced a large proportion of ephippial eggs.
Both hybrid clones also produced ephippia in the short
day photoperiod, but to a lesser extent than their
Canadian parents. The English clones failed to produce
ephippia in any of the environments and produced only a
few males. The E2 clone produced more males than E 1,
but even it produced less than 10% males in 8-h days.
The Canadian clones were much less reluctant. In a 24-h
photoperiod only C2 produced males regularly but with
16 h of light both clones produced males in abundance.
An effect of brood number was evident. At 16-h
photoperiods C 1 females produced 57% female eggs in
brood 1, but less than 1% in brood 2. In 8-h days nearly
all of the parthenogenetic eggs produced by both arctic
clones developed into males. The two hybrid clones
showed remarkable differences in male production. H4
failed to produce any male offspring, while H 1 produced
males in all environments tested.
Figure 1 summarizes the clonal responses; the data
points were calculated by determining the average
proportion of female, male, and ephippial eggs for each
experiment over four broods and finally taking the
average of three experiments. Figure 1 clearly shows the
general similarity of response in the two clones from
each area and the intermediate response of the hybrids.
 FERRARI AND HEBERT

TABLE1 . The proportion of male offspring or ephippial eggs (E) during the first four broods for
six clones at three photoperiods. Each proportion is the average of three experimental runs. The
proportion of females producing female parthenogenetic eggs can be obtained by subtracting the
sum of the male and ephippial proportions from 1.00

Brood number

1 2 3 4
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Photoperiod Clone d E d E d E d E
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For this reason detailed statistical analysis of the data proportion of male offspring in crowded cultures at both
was carried out after pooling clones into three photoperiods although the increase was not striking.
categories (English, Canadian, and hybrid). The
three-way ANOVA in Table 2 shows that not only are Discussion
the main effects significant for all three dependent The present study has revealed clear evidence of
variables, but also for most of the interaction terms. intraspecific variation in the genes controlling reproduc-
Photoperiod and geographic origin are clearly very tive behaviour of D. magna. Clones from arctic Canada
important in determining a clone's reproductive respond to reduced photoperiod by producing males and
behaviour, but brood number also has a significant ephippial eggs, even when living in isolation and
influence. A Duncan's multiple range test documented provided with abundant food. In contrast, the English
that significant (5%)differences existed among the three clones show little change in reproductive behaviour in
categories, with the hybrid clones being intermediate response to shifts in crowding or photoperiod. Crease
between the two parent strains (Woodrich 1981). (198 1) has shown that these same clones can be induced
The results of the crowding experiments are to produce ephippia by rapidly reducing both food
graphically illustrated in Fig. 2. Again the six clones supply and culture volume. Clearly the factors
were pooled into three categories. Fewer than 1% of controlling reproductive behaviour of English and
their broods were ephippial when clones were kept Canadian clones differ qualitatively. The manner in
individually at 24- and 16-h photoperiods. However, in which these different inductive factors mediate control
crowded cultures the hybrid clones produced 8% of reproductive behaviour merits study.
ephippial eggs in 24-h photoperiods, while in a 16-h The two hybrid clones shared a common male parent
photoperiod the Canadian clones displayed a substantial and their female parents were from nearby ponds in
increase in ephippial egg production. The increased Canada. Nonetheless, the two hybrids showed a striking
production of ephippial eggs was particularly evident in difference in reproductive behaviour; .the HI clone
clone 100; clone 108 showed only a moderate increase. produced males abundantly while the H4 clone
All of the clones also showed a similar increase in the produced none. This difference indicates that either the
 2146 CAN. J. ZOOL. VOL. 60, 1982

EPHlPPIAL MALE FEMALE

TABLE2. Probability values (probability >F) from three-way
ANOVA of photoperiod, category, and brood on the three
dependent variables of proportion, female, male, and ephippial
(arcsine transformed) (n = number of degrees of freedom
for each source)

Proportion
- -
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Source n Male Female Ephippial

Photoperiod 2 0.0001 0.0001 0.0001

Category 2 0.0001 0.0001 0.0001
Brood 3 0.084 0.0006 0.0006
Photo x category 4 0.0001 0.0001 0.0001
Photo x brood 6 0.035 0.734 0.0001
Category X brood 6 0.618 0.173 0.078
PhotoXcategoryXbrood 12 0.290 0.817 0.031
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PHOTOPERIO D, HOURS

FIG. 1. The proportion of ephippial, male, or female

offspring produced by females of six clones in each of three
photoperiods.

English male was heterozygous at the locus (or loci)

controlling male production or that the Canadian clones
were variable at the same locus (or loci). Thus genetic
variation at the loci controlling reproductive behaviour
occurs within populations in at least one of the study
areas. Allozyme studies have revealed little genetic
variation at Churchill, but considerable polymorphism
in the English population (Crease 1981) suggesting that
variation in the locus controlling sexual response resided
in the English male parent. The present results are
compatible with the view that both male and ephippial
FIG.2. The effect of density on the production of males and
ephippial eggs by clones of English, Canadian and hybrid
origins grown in 16- and 24-h photoperiods.
egg production are under polygenic control. However,
analysis of a large number of F2 progeny is necessary to
firmly establish that several loci control each trait.
The response to shifts in photoperiod shown by the
arctic clones of D. magna &e similar to those seen in
Daphnia middendorfJiana from Barrow, Alaska (Stross
 FERRARI AND HEBERT
1969). This species is an obligate parthenogen and
produces no male offspring, but isolated females
produce only ephippial eggs when day lengths are less
than 20 h. Barrow is further north than Churchill (7 1 vs.
60N) and thus it is not surprising that the switch to
ephippial egg production occurs at longer daylengths.
Stross (1969) found that natural populations of D.
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rniddendor-ana began to produce ephippia while day
length was still 24 h. He argued that the photoperiod
response was overridden by reduced food abundance or
by diurnal temperature cycles in nature. It is apparent
from the present studies that the reproductive behaviour
of D. magna populations at Churchill also can not be
predicted from a photoperiod response alone. Crowd-
ing, for example, modified reproductive behaviour;
isolated arctic females produced few ephippia in 16-h
photoperiods, but produced more ephippia when they
were crowded.
It is logical from an adaptive standpoint that
photoperiod has such an important effect on the
reproductive behaviour of D. magna clones from arctic
Canada but not those from England. Populations at
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Churchill must produce ephippia to survive the period
when ponds are frozen. Photoperiod is an excellent
indicator of the approach of winter; day lengths become
shorter in a regular fashion as the season progresses.
Temperature is, by comparison, less useful, for low
temperatures not only occur in both spring and autumn,
but also sporadically during the summer. The
modification of the photoperiod response by density
cues is also reasonable. The production of a haploid egg
or a male offspring is maladaptive unless a mate is
present. Natural selection will favour those clones
which fail to produce sexual eggs until population
density is high enough to ensure that a mate will be
encountered. As the season advances, such considera-
tions become unimportant. Clearly, a clone which
produces an ephippial egg on the day before a pond
freezes has a higher fitness than a clone which produces
a parthenogenetic egg, even if the former has only a
small chance of being fertilized. At sufficiently short
day lengths, one expects then to see photoperiod act as a
master cue. While photoperiod provides a good
indication of environmental deterioration in the Arctic,
it is of much less value in England. Clones are able to
reproduce parthenogenetically for extended periods of
time. Indeed, in a permanent pond the production of
sexual offspring as opposed to female parthenogenetic
eggs directly reduces fitness. Thus a clone which
produces only males and ephippial eggs will be rapidly
displaced by clones which continue to produce female
offspring. AS in the Arctic, sexual reproduction will be
retained in situations where parthenogenetic survival is
uncertain. In England shifts in photoperiod are of little
value in predicting such situations. Instead, the shift to
2147
sexuality is precipitated by the unfavourable conditions
themselves, namely increased population density and
reduced food levels.
Over the past decade there has been a surge of interest
in the evolution of life history patterns (Steams 1977,
1980). The life history traits of related species (for
cladoceran examples see Allan 1976; Lynch 1980) have
often been compared and then adaptive significances
ascribed to observed differences in relation to the
environmental preferences of the species. Such
conclusions concerning the relationship between life
history traits and environmental conditions are in the end
untestable, because the life history differences represent
only a small proportion of the total genetic divergence
between the species. Untallied genetic differences may
be responsible for the habitat preferences. Critical
assessment of the adaptive significance of life history
variation ideally requires comparison of the fitness of
individuals which differ only at the loci controlling the
life history trait. Clearly this ideal can only be
approached when one is dealing with intraspecific
variation.
The present study has shown that variation not only
exists at the gene loci controlling reproductive
behaviour in D. magna, but also suggests that this trait
has been moulded by natural selection. Moreover, the
switch from parthenogenetic to sexual reproduction has
effects on both short-term and long-term fitness that can
be quantified and distinguished. Evidence of genetic
variation in local gene pools was also observed. It seems
likely that the extent of such variation may vary
regionally. In some locations, such as arctic Canada, the
physical environment places rather similar demands on
all populations. These may vary in timing from one year
to the next, but the qualitative demands (i.e., to
overwinter in the ephippial stage) remain unchanged.
By comparison, in mesic regions, qualitative differ-
ences exist among local habitats; some regularly impose
diapause, others do not. Successful occupation of such
habitats may well involve genetic diversification. It
seems likely that study of the reproductive patterns of
cladocerans in such habitats would be a profitable way to
extend our understanding of life history evolution.
Acknowledgements
We thank Drs. Harm and S- Schwartz and two
anonymous reviewers for their On the
manuscript. P. D. N. Hebert gratefully acknowledges
the suppofi of the Natural Sciences and Engineering
Research
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