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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 Two experiments were carried out to determine if crowding
clones, E l and E2, were collected from a permanent pond near increased male or ephippial egg production. These studies
Hatley Hill, Cambridgeshire. The remaining two, H 1 and H4, were run only at 24-h and 16-h photoperiods as difficulty was
were hybrid clones produced by mating Canadian females with experienced in maintaining crowded cultures in an 8-h
English males. The maternal origin of HI was a C l female, for photoperiod. In each experiment, 4-6 replicate cups were set
H4 a C2 female. In both cases the Canadian females were up for each of the six clones. The parthenogenetic offspring
mated to E2 males. The hybrid offspring were produced by produced by the 10 females in each cup were sexed and the
placing E2 males with females in the early stages of ephippial number of ephippia was tallied daily. Cultures were
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formation. Ephippia were collected and stored in a dry state at maintained for a period of 30 days, a length of time similar to
5C for 1-3 weeks. Thereafter, they were frequently flooded the experiments on isolated individuals. Females produced
with synthetic pond water at room temperature to induce offspring during the last 15 days of this period. The proportion
hatching. Electrophoretic analysis of several enzyme loci of male offspring produced by each clone was determined by
verified the hybrid origin of these individuals (Crease 1981). dividing the total number of males by the number of
All stock and experimental animals were maintained in a parthenogenetic young produced during an experiment. The
synthetic pondwater mixture (Hebert and Crease 1980). absolute number of ephippial broods could be determined by
Experimental animals were maintained in 100 mL of water and counting ephippia, but the proportion of ephippial broods had to
fed Chlamydomonas sp. every 2 days. Chlamydomonas sp. be estimated, as the number of parthenogenetic broods could
was cultured in an autoclaved medium containing 250mg not be determined. The total number of broods produced
NaN03, 50 mg MgS04-7H20,100 mg K2HP04,25 mg NaCl, during an experiment was estimated by multiplying the
33 mg CaC12.2H20, and 50 mL of soil extract in 1 L of distilled number of females (10) present by the length of reproductive
water. The cultures were grown at room temperature under period (15 days) and dividing by the brood interval (2.5 days).
continuous fluorescent illumination and used after 7- 10 days. The total number of broods produced during an experiment
The optical density was adjusted to 0.15 at 620 nm. Dried liver was calculated to be 60. Thus if 6 ephippia were produced
extract at a concentration of 2 mg/mL was mixed, allowed to during an experiment, the proportion of ephippial broods was
settle, and one part of the supernatant was added to five parts estimated to be 10%.
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algae. One millilitre of a vitamin supplement (700 mg Ca


panthothenate, 60 mg thiamin, 40mg riboflavin, 130 mg Results
nicotinarnide, 330 mg folic acid, 500 mg choline, 30 mg The experiments on isolated females (Table 1)
putrescine, and a trace amount of vitamin B ,2 in 1 L of distilled showed a very low incidence of ephippial egg
water) was added weekly to each beaker.
production in both 24- and 16-h photoperiods.
All experiments were run at 20 + 1C; three photoperiods
(8, 16, and 24 h of light) were studied. Before beginning the However, in an 8-h photoperiod, females of both arctic
experiment, stocks of each clone were maintained for clones produced a large proportion of ephippial eggs.
several generations at 20C and 24 h of light. To initiate an Both hybrid clones also produced ephippia in the short
experiment, gravid females were placed individually in day photoperiod, but to a lesser extent than their
beakers and shifted to the experimental photoperiod. Within Canadian parents. The English clones failed to produce
24 h of birth their offspring were removed and placed in cups ephippia in any of the environments and produced only a
either individually or with nine other neonates of the same few males. The E2 clone produced more males than E 1,
clone. These individuals required about 10 days to mature; but even it produced less than 10% males in 8-h days.
thereafter their reproductive behaviour was monitored daily The Canadian clones were much less reluctant. In a 24-h
and all offspring were removed and sexed. photoperiod only C2 produced males regularly but with
At each feeding, cups containing a single animal were given
16 h of light both clones produced males in abundance.
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 An effect of brood number was evident. At 16-h
photoperiods were given more food in case the shorter periods photoperiods C 1 females produced 57% female eggs in
of photosynthesis reduced algal growth rates. The cultures brood 1, but less than 1% in brood 2. In 8-h days nearly
containing 10 animals were fed 10 mL at each feeding. To all of the parthenogenetic eggs produced by both arctic
prevent the accumulation of waste products one-half of the clones developed into males. The two hybrid clones
culture medium was replaced each week. showed remarkable differences in male production. H4
Three experiments were carried out to study the effect of failed to produce any male offspring, while H 1 produced
shifts in photoperiod on the reproductive behaviour of isolated males in all environments tested.
individuals. Each experiment involved placing 10 individuals Figure 1 summarizes the clonal responses; the data
of a clone in the three photoperiods (8, 16, 24 h). Each
individual was maintained until it had produced four broods
points were calculated by determining the average
and the proportion of females producing male, female, or proportion of female, male, and ephippial eggs for each
ephippial eggs at each brood was calculated. Parthenogenetic experiment over four broods and finally taking the
broods generally consisted entirely of male or female average of three experiments. Figure 1 clearly shows the
offspring. Before statistical analysis, the data were arcsine general similarity of response in the two clones from
transformed. 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 FIG.2. The effect of density on the production of males and
were variable at the same locus (or loci). Thus genetic ephippial eggs by clones of English, Canadian and hybrid
variation at the loci controlling reproductive behaviour origins grown in 16- and 24-h photoperiods.
occurs within populations in at least one of the study
areas. Allozyme studies have revealed little genetic egg production are under polygenic control. However,
variation at Churchill, but considerable polymorphism analysis of a large number of F2 progeny is necessary to
in the English population (Crease 1981) suggesting that firmly establish that several loci control each trait.
variation in the locus controlling sexual response resided The response to shifts in photoperiod shown by the
in the English male parent. The present results are arctic clones of D. magna &e similar to those seen in
compatible with the view that both male and ephippial Daphnia middendorfJiana from Barrow, Alaska (Stross
FERRARI AND HEBERT 2147

1969). This species is an obligate parthenogen and sexuality is precipitated by the unfavourable conditions
produces no male offspring, but isolated females themselves, namely increased population density and
produce only ephippial eggs when day lengths are less reduced food levels.
than 20 h. Barrow is further north than Churchill (7 1 vs. Over the past decade there has been a surge of interest
60N) and thus it is not surprising that the switch to in the evolution of life history patterns (Steams 1977,
ephippial egg production occurs at longer daylengths. 1980). The life history traits of related species (for
Stross (1969) found that natural populations of D. cladoceran examples see Allan 1976; Lynch 1980) have
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rniddendor-ana began to produce ephippia while day often been compared and then adaptive significances
length was still 24 h. He argued that the photoperiod ascribed to observed differences in relation to the
response was overridden by reduced food abundance or environmental preferences of the species. Such
by diurnal temperature cycles in nature. It is apparent conclusions concerning the relationship between life
from the present studies that the reproductive behaviour history traits and environmental conditions are in the end
of D. magna populations at Churchill also can not be untestable, because the life history differences represent
predicted from a photoperiod response alone. Crowd- only a small proportion of the total genetic divergence
ing, for example, modified reproductive behaviour; between the species. Untallied genetic differences may
isolated arctic females produced few ephippia in 16-h be responsible for the habitat preferences. Critical
photoperiods, but produced more ephippia when they assessment of the adaptive significance of life history
were crowded. variation ideally requires comparison of the fitness of
It is logical from an adaptive standpoint that individuals which differ only at the loci controlling the
photoperiod has such an important effect on the life history trait. Clearly this ideal can only be
reproductive behaviour of D. magna clones from arctic approached when one is dealing with intraspecific
Canada but not those from England. Populations at variation.
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Churchill must produce ephippia to survive the period The present study has shown that variation not only
when ponds are frozen. Photoperiod is an excellent exists at the gene loci controlling reproductive
indicator of the approach of winter; day lengths become behaviour in D. magna, but also suggests that this trait
shorter in a regular fashion as the season progresses. has been moulded by natural selection. Moreover, the
Temperature is, by comparison, less useful, for low switch from parthenogenetic to sexual reproduction has
temperatures not only occur in both spring and autumn, effects on both short-term and long-term fitness that can
but also sporadically during the summer. The be quantified and distinguished. Evidence of genetic
modification of the photoperiod response by density variation in local gene pools was also observed. It seems
cues is also reasonable. The production of a haploid egg likely that the extent of such variation may vary
or a male offspring is maladaptive unless a mate is regionally. In some locations, such as arctic Canada, the
present. Natural selection will favour those clones physical environment places rather similar demands on
which fail to produce sexual eggs until population all populations. These may vary in timing from one year
density is high enough to ensure that a mate will be to the next, but the qualitative demands (i.e., to
encountered. As the season advances, such considera- overwinter in the ephippial stage) remain unchanged.
tions become unimportant. Clearly, a clone which By comparison, in mesic regions, qualitative differ-
produces an ephippial egg on the day before a pond ences exist among local habitats; some regularly impose
freezes has a higher fitness than a clone which produces diapause, others do not. Successful occupation of such
a parthenogenetic egg, even if the former has only a habitats may well involve genetic diversification. It
small chance of being fertilized. At sufficiently short seems likely that study of the reproductive patterns of
day lengths, one expects then to see photoperiod act as a cladocerans in such habitats would be a profitable way to
master cue. While photoperiod provides a good extend our understanding of life history evolution.
indication of environmental deterioration in the Arctic,
it is of much less value in England. Clones are able to Acknowledgements
reproduce parthenogenetically for extended periods of
time. Indeed, in a permanent pond the production of We thank Drs. Harm and S- Schwartz and two
sexual offspring as opposed to female parthenogenetic anonymous reviewers for their On the
eggs directly reduces fitness. Thus a clone which manuscript. P. D. N. Hebert gratefully acknowledges
produces only males and ephippial eggs will be rapidly the suppofi of the Natural Sciences and Engineering
displaced by clones which continue to produce female Research
offspring. AS in the Arctic, sexual reproduction will be
retained in situations where parthenogenetic survival is J . D.1976. Life history patterns in zoop]ankton. Am.
ALLAN,
uncertain. In England shifts in photoperiod are of little Nat. 110: 165- 180.
value in predicting such situations. Instead, the shift to BANTA,
A. M., and L. A. BROWN.
1929. Control of sex in
2 148 CAN. J. ZOOL. VOL. 60, 1982

Cladocerans. I. Crowding the mothers as a means of Leydig . Ph.D. thesis, Indiana University, Bloomington,
controlling male production. Physiol . Zool . 2: 80-92. IN.
CREASE, T. J. 1981. Genetic variation in natural populations of STROSS,R. G. 1969. Photoperiod control of diapause in
Daphnia. M.Sc. thesis, University of Windsor, Windsor, Daphnia. 11. Induction of winter diapause in the arctic.
Ont. Biol. Bull. (Woods Hole), 136: 264-273.
D'ABRAMO, L. R. 1980. Ingestion rate decrease as a stimulus 1971. Photoperiodism and diapause in Daphnia: a
for sexuality in populations of Moina macrocopa. Limnol. strategy for all seasons. Trans. Am. Microsc. Soc. 90:
Oceanogr. 25: 422-429. 110-112.
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HEBERT,P. D. N., and T. J. CREASE.1980. Another STROSS, R. G., and J. C. HILL.1965. Diapause induction in
planktonic paradox: coexisting clones of Daphnia pulex Daphnia requires two stimuli. Science (Washington,
Leydig. Science (Washington, D.C.), 207: 1363- 1365. D.C.), 150: 1462-1464.
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Q. Rev. Biol. 55: 23-42. fresh water crustacean Daphnia. Biol. Bull. (Woods Hole),
SLOBODKIN, L. B. 1954. Population dynamics in Daphnia 134: 176-198.
obtusa Kurz. Ecol. Monogr. 24: 69-88. VON DEHN, M. 1956. Die Geschlechtsbestimmung der
STEARNS, S. C. 1980. A new view of life history evolution. Daphniden. Die Bedeutung der Fettstoffe untersucht an
Oikos, 35: 266-281. Moina rectirostris. Zool. Jahrb. Abt. Allg . Zool. Physiol.
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the theory and a review of the data. Annu. Rev. Ecol. Syst . WOODRICH, D.C. 1981 . Ecological differences among clones
8: 145-171. of Daphnia. M. Sc. thesis, University of Windsor, Windsor,
STIMPFL,K. J. 1971. The effects of four environmental Ont .
variables on the induction of gamogenesis in Daphnia pulex
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