Molecular Ecology (2002) 11, 1209 –1217

New insights into the distribution of polyploid Daphnia:

Blackwell Science, Ltd

the Holarctic revisited and Argentina explored

S A R A H J . A D A M O W I C Z ,* T . R Y A N G R E G O R Y ,* M A R Í A C R I S T I N A M A R I N O N E ,† and
PAUL D. N. HEBERT*
*Department of Zoology, University of Guelph, Guelph, ON, Canada, N1G 2W1, †Departamento de Ciencias Biológicas,
Universidad de Buenos Aires, Pabellón II, 4to. Piso, 1428 Buenos Aires, Argentina

Abstract
It has long been known that polyploid organisms are more prevalent in arctic than in tem-
perate environments. Past explanations for this geographical trend have focused on the role
of glacial cycles in generating polyploids and the influence of abiotic factors in favouring
polyploidy in the north. In combination, these mechanisms probably suffice to explain the
observed geographical cline in ploidy levels in members of the Daphnia pulex complex in
the Holarctic. While only diploid members of the D. pulex complex are found in the tem-
perate regions of North America and Europe, allozyme and DNA quantification analyses
indicate that the southern Argentine pulex-complex fauna is dominated by polyploids.
Indeed, the present study is the first to document the presence of polyploid members of the
D. pulex complex in any temperate climate. The results of phylogeographic analyses suggest
that this difference in polyploid distribution between the northern and southern hemispheres
is based more on ecological and historical contingencies than direct selection for polyploidy.
Specifically, competition with diploid relatives probably limits the lower latitudinal range
of polyploids in the north, but appears not to have occurred in Argentina. Because of these
differences, the present study provides important insights into the diverse factors that
determine the distributions and evolutionary fates of polyploid organisms.
Keywords: DNA content, evolution, hybridization, parthenogenesis, zooplankton

Received 27 November 2001; revision received 12 March 2002; accepted 12 March 2002

Although several hypotheses have been advanced to

Introduction
Polyploidy is a phenomenon of particular interest in
evolutionary biology as it provides an opportunity for
instantaneous genome reconfiguration and speciation.
Not surprisingly, considerable attention has been paid to
the observation that arctic and alpine environments
harbour a substantially higher proportion of polyploid
organisms than temperate regions. Such an elevated
incidence of polyploidy in the north has long been
documented in plants (Stebbins 1950), and is also apparent
in animals such as insects (Suomalainen et al. 1987) and
zooplankton (Beaton & Hebert 1988; Little et al. 1997).
Notably, the dominant freshwater fish family in arctic
Canada, the Salmonidae, is also polyploid (Gregory &
Hebert 1999).
Correspondence: Sarah J. Adamowicz. E-mail: sadamowi@uoguelph.ca
account for these patterns, a comprehensive understanding
of the relationship between ploidy level and environmental
conditions has remained elusive, largely due to the fact
that polyploidy and parthenogenesis tend to be acquired
together (Glesener & Tilman 1978; Bell 1982; Suomalainen
et al. 1987; Beaton & Hebert 1988). However, studies on
plants have been particularly useful in demonstrating
the adaptive significance of polyploidy in the Arctic, as
comparisons of closely related diploid and polyploid
sexual species have permitted the effects of ploidy level
to be examined independently of variation in breeding
system (reviewed in Bierzychudek 1985). It is a freshwater
zooplankton species complex that has provided the
converse study system, in which geographical variation in
ploidy level has also been examined under a constant, this
time asexual, breeding system.
The Daphnia pulex group in North America contains
obligately parthenogenetic diploids and polyploids, as

© 2002 Blackwell Science Ltd

1210 S . J . A D A M O W I C Z E T A L .

well as sexual diploids. Surveys of clonal Daphnia popula-

tions have revealed that high Arctic environments are
dominated by polyploids, while both diploids and poly-
ploids occur at low Arctic sites, but only diploids are found
in temperate zones (Beaton & Hebert 1988). Studies on
Daphnia from other geographical regions (Ward et al. 1994)
and on other cladoceran genera (DeMelo & Hebert 1994;
Little et al. 1997) further suggest that polyploidy is pre-
valent among zooplankton inhabiting the Arctic, but is
absent in temperate regions. Thus, geographical patterns
of ploidy level in zooplankton suggest that polyploidy is
adaptive in the Arctic.
Members of the D. pulex complex have a broad Holarctic
distribution, and while they are absent from the tropical
regions of both North and South America (Paggi 1998;
Hebert and Finston 2001), they have been reported from
southern and alpine regions of the latter continent (e.g.
Villalobos 1994). Argentina, which boasts a diverse land-
scape and extends into the southernmost reaches of South
America, provides an excellent opportunity to explore
breeding system diversity and ploidy levels in the D. pulex
complex along a latitudinal gradient in the south.
This study presents the first genetic investigation of
members of the D. pulex complex in South America. Breed-
ing system, clonal diversity, ploidy levels and the origin of
Argentine populations were investigated using allozyme,
mitochondrial DNA (mtDNA) and DNA quantification
techniques. Previous explanations for the geographical
patterns of polyploidy and asexuality in the D. pulex com-
plex are re-evaluated in light of the present results, and a
new synthesis involving the roles of abiotic, ecological and
historical factors in the generation and maintenance of
polyploids is proposed.
Fig. 1 Map of collection sites for South American populations of
the Daphnia pulex complex.
Materials and methods
margin of the carapace (Schwartz et al. 1985). Populations
Specimen collection and identification
belonging to the D. pulex complex were encountered in
During sampling campaigns in November–December 1999 16 ponds in extra-Andean regions of the southernmost
and January–February 2001, zooplankton collections were provinces of Argentina (Santa Cruz and Tierra del Fuego),
made from more than 250 water bodies throughout numbered ARG 1–16 (Fig. 1; Appendix I).
Argentina, 160 of which contained Daphnia. Habitats
sampled included ponds, roadside ditches, playa lakes,
Allozyme analysis
alpine lakes, rivers, reservoirs and salt lakes. Initial species
assignments were made in the field based on morphology. Allozyme surveys were conducted on all Argentine
Animals were sorted alive and either flash-frozen in liquid populations by cellulose acetate electrophoresis using a
nitrogen for allozyme surveys or preserved in 95% ethanol Tris–glycine buffer (pH = 8.5) (Hebert & Beaton 1993).
for DNA analysis. Taxonomic assignments were later Populations were screened for variation at seven com-
revised based on the results of mtDNA analysis and more monly polymorphic loci: aspartate amino transferase
detailed morphological investigations. (AAT; EC 3.2.1.1), fumarate hydratase (FUM; EC 4.2.1.2),
Species of the subgenus Daphnia were identified by their glucose-6-phosphate isomerase (GPI; EC 5.3.1.9),
prominent medial pecten, and members of the D. pulex glyceraldehyde-3-phosphate dehydrogenase (G3PDH; EC
complex were separated from those of the D. obtusa 1.2.1.12), lactate dehydrogenase (LDH; EC 1.1.1.27),
complex by their lack of elongate setae along the internal mannose-6-phosphate isomerase (MPI; EC 5.3.1.8), and

© 2002 Blackwell Science Ltd, Molecular Ecology, 11, 1209 –1217


phosphoglucomutase (PGM; EC 5.4.2.2). All gels were
electrophoresed for 15 min, as pilot trials revealed that this
run time maximized allele separation while maintaining
crisp bands. From most populations, 18–44 individuals
were analysed, but only two individuals were available
from ARG 13. During each staining run, two individuals
from a clonal stock of North American Daphnia pulicaria
(from Lake Washington, USA) were used to standardize
scoring. A population of D. pulex (from a pond in Guelph,
Ontario, Canada) was also used in several runs for com-
parison. Particular attention was paid to the comparison of
alleles at LDH, as this locus is useful for diagnosing species
in the D. pulex complex (Hebert et al. 1989).
Allozyme surveys were used to estimate levels of genetic
diversity in Argentine populations and also to determine
their breeding systems, as this approach has been shown to
provide reliable diagnoses in studies that coupled alloz-
yme analysis with subsequent breeding experiments (e.g.
Hebert & Crease 1983). In addition, allozyme phenotypes
were used to gain an initial indication of ploidy levels.
Evidence of polyploidy includes the detection of three or
more alleles at a locus or asymmetrical banding patterns,
which indicate uneven (and hence multiple) allelic copy
numbers (e.g. Ward et al. 1994; Dufresne & Hebert 1995).
Mitochondrial DNA analysis
Total DNA was extracted from several individuals from
each population in 50 µL of proteinase K extraction buffer,
according to the protocol of Schwenk et al. (1998). A 710-
base-pair (bp) fragment of the cytochrome c oxidase
subunit I (COI) mitochondrial gene was amplified via the
polymerase chain reaction (PCR) (Saiki et al. 1988) from a
single individual from seven populations (ARG 1–4, 8, 9
and 15) using universal primers LCOI490 and HCO2918
(Folmer et al. 1994). A 550-bp fragment of the NADH
dehydrogenase subunit 5 gene (ND5) was amplified for
one individual from ARG 9 and ARG 11 using primers
designed for Daphnia (Colbourne et al. 1998). Each 50-µL
reaction consisted of 3–5 µL of DNA template, 5 µL of 10×
PCR buffer (10 mm Tris–HCl, pH 8.3; 50 mm KCl), 0.2 µm
of each primer, 1.5 mm MgCl2, 0.2 mm of each dNTP, and
1 unit of Taq DNA polymerase. The PCR thermal regime
for both genes was as follows: one cycle of 1 min at 94 °C;
five cycles of 1 min at 94 °C, 1.5 min at 45 °C, and 1.5 min
at 72 °C; 30 cycles of 1 min at 94 °C, 1.5 min at 50 °C,
and 1.5 min at 72 °C; and finishing with a final extension
at 72 °C for 5 min. PCR products were gel-purified
using the Qiaex II (Qiagen) kit and sequenced in one
direction (with primer LCOI490 for COI or ND5-A) using
the Big Dye Terminator (version 3) sequencing kit (ABI
Prism). Sequencing-reaction products were electro-
phoresed on an ABI 377 automated sequencer (Applied
Biosystems).
© 2002 Blackwell Science Ltd, Molecular Ecology, 11, 1209–1217
P O L Y P L O I D D A P H N I A I N A R G E N T I N A 1211
Sequence electropherograms were inspected and aligned
using the seqapp 1.9 sequence editor (Gilbert 1992), result-
ing in a final alignment of 630 bp for COI and 499 bp for
ND5. All unique sequences are available from GenBank
(accession numbers: AF489523–AF489527). Pairwise
genetic distances were calculated using Kimura’s (1980)
two-parameter model (K2P) in mega 2.1 (Kumar et al.
2001) and used to construct phenograms by the neighbour-
joining (NJ) algorithm (Saitou & Nei 1987). In all cases,
trees were constructed using pairwise deletion of miss-
ing sites, and bootstrap values were based on 1000
pseudoreplicates.
COI sequences for North American members of the
D. pulex complex were also included in the analysis.
Unpublished D. pulicaria and D. tenebrosa sequences were
contributed by J. Colbourne, while the D. pulex sequence
was from Crease (1999). Much of the past work on the
Holarctic members of the D. pulex complex also employed
the ND5 gene. Consequently, the two ND5 sequences
obtained in the present study were incorporated into the
data set of Colbourne et al. (1998) to examine the relation-
ship between North and South American members of the
D. pulex complex.
DNA quantification
Whole bodies of ethanol-preserved Daphnia from Argentina,
along with a diploid laboratory culture of D. pulicaria, were
stained in a standard Feulgen protocol (Beaton & Hebert
1988). Briefly, the animals were placed in glass tubes with
perforated caps which allowed their simultaneous
submersion in the various solutions. Daphnia from the live
culture were first placed in 95% ethanol and allowed to
dehydrate, and then all specimens were postfixed in an
MFA solution (85 methanol : 10 formalin : 5 glacial acetic
acid, v : v : v) for 30 min. A 30-min hydrolysis in 5 m HCl
was preceded by a 10-min rinse in running tapwater and
followed by a dip in 0.1 m HCl to prevent the carry-over of
strong acid. Hydrolysed specimens were then stained in
freshly prepared Schiff reagent for 60 min before being
passed through three rinses in fresh bisulphite solution.
A second 10-min tapwater rinse was followed by three
changes in distilled water, and the animals were then
transferred to 20% ethanol for storage.
Daphnia species display pronounced endopolyploidy in
most tissues, but the exopodites of the thoracic limbs show
no evidence of this phenomenon, and were therefore
selected for analysis (Beaton & Hebert 1988, 1989). Within
48 h of staining, exopodites were removed by dissection in
distilled water and allowed to air-dry onto microscope
slides. Relative nuclear DNA contents were determined
using the bioquant true color windows 98 v3.50.6
image analysis software package (R & M Biometrics Inc.)
along with an Optronics DEI-750 CE three-chip CCD
1212 S . J . A D A M O W I C Z E T A L .
colour camera connected via a BQ6000 frame-grabber
board to a Pentium II® 300 MHz PC (Hardie et al. 2002).
Specimens were visualized under a 100× oil-immersion
objective (ND = 1.515) on a Leica DM/LS microscope. The
exopodites were mounted in immersion oil with a refrac-
tive index of 1.520 to reduce glare caused by exoskeletal
chitin (Beaton & Hebert 1988). A 546-nm interference filter
was used to increase the contrast between the stained
nuclei and the background.
Integrated optical densities (IODs) were measured from
six individuals from ARG 11, for a total of 65 nuclei. In
addition to this, 70 nuclei were measured from individuals
of the diploid laboratory culture. Similar, though less
intensive, measurements were performed on individuals
from three other populations (ARG 2, 4 and 6). IOD meas-
urements were consistent across individuals of the same
population, generally showing coefficients of variation of
less than 10% when the IOD data were pooled. The DNA
contents of the purported polyploid clones were evaluated
by comparing the ratios of the mean IOD of each Argentine
population to that of the diploid D. pulicaria culture.
Results
Clonal diversity
Allozyme surveys revealed only two multilocus genotypes
in the 16 Argentine populations, which are subsequently
referred to as clones A and B (Table 1). Only a single clone
was detected in each habitat. Clone B was found at two
sites (ARG 11 and 13), while clone A occupied the
remaining 14 Argentine habitats. Both of these clones were
heterozygous for the same alleles at GPI, FUM and LDH,
and were homozygous for the same allele at PGM, AAT
and G3PDH. While FUM and LDH heterozygotes
exhibited normal phenotypes, the staining pattern of all
Table 1 Genotypes at seven allozyme loci of the two clones of
Daphnia pulicaria detected in Argentine habitats
Locus Genotype of Argentine populations
AAT 1.00/1.00
G3PDH 1.00/1.00
FUM 1.00/1.14
GPI* 0.88/1.00
LDH 0.83/1.00
MPI-Clone A 0.86/1.00
MPI-Clone B 0.86/0.92/1.00/1.09
PGM 1.00/1.00
Clones A and B differed only at MPI. The relative mobilities of
Argentine alleles are as compared with a laboratory clone of North
American D. pulicaria.
*Unbalanced staining (GPI1·00 heavier).
GPI heterozygotes was unbalanced, suggesting that both
clones were polyploid. Further evidence for the polyploid
state of clone B was observed at the final locus, MPI, which
is a monomer. Although clone A was a heterozygote with
two roughly equally staining alleles, clone B displayed a
phenotype suggesting that it possessed four different
alleles. Given the observed fixed heterozygosity, all 16
populations were assumed to reproduce by obligate
parthenogenesis.
COI and ND5 sequence diversity
There was very limited sequence divergence among the
seven COI sequences examined from the Argentine
populations (all containing allozyme clone A). Only three
nucleotide positions were variable among the sequences,
and the maximum K2P pairwise distance was 0.32%.
Analysis of ND5 sequences from a single individual of
each clone indicated that they were also closely allied, as
their sequence divergence was only 0.20%.
Phylogenetic relationships
Mitochondrial DNA analyses indicated that Argentine
populations assigned to the Daphnia pulex complex were
closely related to North American members of this
complex, specifically Daphnia pulicaria (Figs 2, 3). Daphnia
pulicaria consists of at least three distinct clades in the
Holarctic (Dufresne & Hebert 1997; Colbourne et al. 1998).
ND5 sequence variation indicates that the Argentine
populations of D. pulicaria form a group distinct from all
of the known Holarctic clusters (Fig. 3). Its average
ND5 sequence divergence from all members of the three
D. pulicaria clades was 2.94%.
Allozymic profiles were consistent with the mtDNA
results. In tetrameric enzymes such as LDH, the precise
relative mobilities of individual alleles are difficult to
measure when in heterozygous condition, due to the
complex banding pattern. However, the faster allele at
Fig. 2 Neighbour-joining tree based on COI sequences for
members of the Daphnia pulex complex. All Argentine sequences
are from one allozymic genotype (clone A). Daphnia pulex, D.
pulicaria and D. tenebrosa sequences are from North America
(sequences provided by J. K. Colbourne). The D. pulicaria sequence
belongs to the eastern D. pulicaria clade (see text and Fig. 3). Scale
bar indicates Kimura two-parameter genetic distance. Bootstrap
values are based on 1000 pseudoreplicates.
© 2002 Blackwell Science Ltd, Molecular Ecology, 11, 1209 –1217

Fig. 3 Neighbour-joining tree of ND5 sequence variation in the
Daphnia pulex complex. The Holarctic dataset with clade
designations is from Colbourne et al. (1998). South American
sequences from the present study are indicated in bold. ARG 9 is
from a population with allozyme genotype A, while ARG 11
displays genotype B. Scale bar indicates Kimura two-parameter
genetic distance. Bootstrap values (based on 1000 pseudo-
replicates) are presented for major clusters.
LDH in the Argentine populations appeared to have the
same mobility as the ‘F’ allele considered diagnostic for
D. pulicaria in North America, while the slower LDH allele
in Argentine populations appeared to have the same
mobility as the ‘S’ allele that is diagnostic for D. pulex
(Hebert et al. 1989).
Relative DNA content
The mean IOD of exopodite nuclei from individuals of
clone B (site ARG 11) was 163.7 (standard error ± 1.4), while
the laboratory culture of D. pulicaria showed a mean IOD of
83.8 (±2.0). Thus, the ratio of DNA contents in exopodite
nuclei from the Argentine clones and diploid D. pulicaria
was approximately 1.9. Individuals from allozyme clone A
© 2002 Blackwell Science Ltd, Molecular Ecology, 11, 1209–1217
P O L Y P L O I D D A P H N I A I N A R G E N T I N A 1213
(sites ARG 2, 4 and 6) had somewhat dispersed DNA in
their exopodite nuclei which complicated measurements,
but they showed a similarly elevated DNA content relative
to the diploid D. pulicaria (ratio ∼1:7).
Discussion
The first case of temperate polyploid Daphnia
The present study confirms that the Daphnia pulex complex
is represented in South America. Moreover, all Argentine
populations were asexual tetraploids, as shown by their
allozymic phenotypes and nuclear DNA contents. Given
their high heterozygosities, these clones are likely to be of
hybrid origin. Hybrid polyploid clones of the D. pulex
complex also dominate the most northerly portions of the
range of this group (Weider et al. 1987; Beaton & Hebert
1988; Dufresne & Hebert 1994), suggesting that similar
processes may have produced this apparent geographical
symmetry. However, there are important distinctions
between the environments occupied by polyploids in the
northern and southern hemispheres.
In North America and Europe, polyploids are found at a
minimum of 58° N and only achieve dominance at about
70° N (Beaton & Hebert 1988; Ward et al. 1994). By contrast,
Argentine polyploids were found within the much lower
latitudinal range of 46–54° S, where they experience longer
and warmer growing seasons than their Holarctic counter-
parts. The southern polyploids occupy habitats in a region
with a ‘temperate or cool-temperate’ climate (Paruelo et al.
1998), having mean January (i.e. summer) temperatures
between 9 °C and 16 °C (National Meteorological Ser-
vice of Argentina). On the other hand, arctic polyploids
only achieve dominance north of the 10 °C July isotherm
(Beaton & Hebert 1988; Ward et al. 1994). In fact, and
despite exhaustive sampling efforts, polyploids have never
been detected in any temperate region of North America or
Europe (e.g. 3erny & Hebert 1993; Hebert et al. 1993; Ward
et al. 1994; Hebert and Finston 2001). Thus, the detection
of polyploids in the relatively warm environments in
Argentina challenges the existing theories regarding the
relationship between polyploidy and environment and
may provide insight into the processes responsible for
the generation and maintenance of polyploids.
Polyploids in the north: production and persistence
Some authors have suggested that the elevated incidence
of polyploidy in the far north is attributable to the indirect
action of the Pleistocene glaciations in generating
polyploid lineages (Stebbins 1984; Dufresne & Hebert
1997). Specifically, it is argued that during glacial
advances, species’ ranges are reduced to isolated refugia
where gene pool divergence occurs. Hybridization of
1214 S . J . A D A M O W I C Z E T A L .
divergent genomes during secondary contact following
glacial retreat is thought to promote (allo)polyploidization.
However, while this glacial cycling mechanism may
promote the production of polyploids via hybridization, it
does not explain their persistence in arctic environments.
In Daphnia in particular, it is clear that hybridization alone
is not sufficient to account for the presence of polyploid
lineages, as interspecific hybridization is common between
many Daphnia species inhabiting temperate climates
where polyploids are never found (Taylor & Hebert 1993;
Colbourne & Hebert 1996; Hebert and Finston 2001).
Consequently, other authors have emphasized the role
of selection to explain the differential maintenance of
polyploids in arctic vs. temperate environments (Beaton &
Hebert 1988; Ward et al. 1994).
Some authors have proposed that the success of
polyploids in the north is due to the adaptive value of
parthenogenesis (e.g. Bell 1982), primarily since asexuals
are considered superior colonizers (Stebbins 1950) and
are also expected to perform well under conditions of low
biotic complexity (Glesener & Tilman 1978). However, in
the case of the D. pulex complex, the fact that diploid asex-
ual populations are found as far south as Mexico (Hebert
and Finston 2001) suggests that it is polyploidy itself that is
especially relevant in the Arctic. The proposed adaptive
significance of polyploidy in cold conditions relates prim-
arily to the effects of increased DNA content on cell size
(Gregory 2001). In plants, larger DNA contents in northern
species may reflect the necessary shift in emphasis from
cell division to cell enlargement as the former is impeded
in the cold (Grime & Mowforth 1982; Mowforth & Grime
1989). Similarly, a comparison of laboratory-reared clones
revealed that polyploid members of the D. pulex complex
mature faster than their diploid counterparts under cold
conditions (Dufresne & Hebert 1998). Because of their
increased DNA content, polyploid Daphnia also produce
larger (but fewer) eggs and offspring than diploids
(Dufresne & Hebert 1998) — a fact directly relevant to their
fitness at low temperatures where decreased predation
and competition favour the ‘few large offspring strategy’
(Yampolsky & Scheiner 1996).
Limits to polyploid distribution: competitive exclusion?
A combination of the production/selection mechanisms
outlined above is probably responsible for the
predominance of polyploids in the Arctic, but these do not
directly address the reasons for their exclusion from
temperate regions. Clearly, the occurrence — and even
dominance — of polyploid Daphnia in temperate South
American habitats indicates that polyploidy is not
‘forbidden’ in warmer climates. Instead, the relevant
difference appears to be the presence of closely related
diploid populations in North America, and their absence in
Argentina. In the north, competition with diploid relatives
may restrict the southerly distribution of polyploids, while
in Argentina polyploid populations are free to exploit
habitats in warmer regions. The observation that Argen-
tine D. pulex-group populations frequently co-occur with
distantly related sexually reproducing species (e.g. members
of the D. obtusa complex and subgenus Ctenodaphnia)
further suggests that polyploids can hold their own in
temperate regions so long as they are not in competition
with close diploid relatives.
Polyploids in the south: fortunate founders?
The proposed ‘competitive-release’ explanation for the
presence of polyploid Daphnia in temperate Argentina is
largely dependent on negative evidence: the lack of diploid
and/or sexual members of the D. pulex complex. Although
the broader survey was extensive, including genetic
characterization of 160 Daphnia populations throughout
the country, the possibility that such diploid pulex-group
populations simply eluded detection cannot be dismissed.
However, this (apparent) absence of diploids in Argentina
is consistent with other phylogeographic evidence
suggesting that the Argentine polyploids were not
generated in situ, but in the Holarctic. Northern polyploid
members of the D. pulex complex typically consist of
hundreds of distinct clones (Weider et al. 1999) and
numerous mitochondrial lineages (Dufresne & Hebert
1997; Colbourne et al. 1998), while in Argentina levels of
clonal and mtDNA diversity were severely diminished.
This unbalanced distribution of variation between the
Holarctic and Argentina strongly supports the conclusion
that the northern hemisphere is the birthplace of the D.
pulex complex. One plausible scenario for their arrival in
Argentina involves a sweepstake dispersal event of one or
a few hybrid, polyploid propagules from North America.
In this case, the observed mitochondrial genotypes would
indicate that D. pulicaria must be the maternal parent, and
while the identity of the paternal parent is not entirely
clear, D. pulex is a likely candidate, as indicated by
allozyme evidence. Indeed, interspecific hybridization
events involving maternal D. pulicaria and paternal D.
pulex always produce polyploids, while the reverse cross
results in diploid asexuals (Dufresne & Hebert 1994).
This demonstrated capacity for intercontinental ex-
change, however, raises the question of why neither sexual
nor asexual diploid populations of D. pulex or D. pulicaria
from North America have managed to invade southern
Argentina. Based on their life history traits (Dufresne &
Hebert 1998) and biogeographic patterns in North America
(Beaton & Hebert 1988), it appears that the selective advant-
age of diploids is more than sufficient to expect such a take-
over upon their arrival in Argentina. It may be simply by
chance that it was polyploid rather than diploid individuals
© 2002 Blackwell Science Ltd, Molecular Ecology, 11, 1209 –1217

that first (and perhaps exclusively) colonized southern
Argentina. An examination of bird migration routes
indicates that this is plausible, as it is primarily Arctic birds
that make transoceanic migrations to the southernmost
reaches of South America (Alerstam 1982; Mead 1993).
Such migrants would be expected to transport ephippia
from the dominant type of Daphnia in the arctic — namely
polyploids. Thus, whereas mechanisms of polyploid
generation, in combination with various selective pressures,
may explain the prominence of polyploids in the Arctic (as
well as their absence in temperate North America), these
are not applicable in South America. Instead, polyploids
may dominate the temperate regions of Argentina solely
because of a fortuitous priority of arrival.
The study of arctic organisms has revealed much regard-
ing the importance of environmental factors in producing
and maintaining polyploids. The present comparison
involving North and South American populations of
Daphnia has instead highlighted the influence of contin-
gencies of both history and ecology in determining the
distribution and evolution of polyploids. Of course,
detailed studies of other closely related taxa with diploid
and polyploid members will be required to assess the
generality of the trend reported here. However, it is clear
from the present study that the evolutionary fate of
polyploids is determined by a complex and intriguing
interaction between the biotic and abiotic environments.
Acknowledgements
Silvina Menu Marque and Agustín Bachmann provided extensive
assistance during the field collections in Argentina. We also thank
John Colbourne for providing sequence data, Ian Smith for aiding
with figures, Angela Holliss for DNA sequencing, and Teri Crease
and two anonymous reviewers for providing helpful comments
on an earlier draft. This research was supported by NSERC post-
graduate scholarships to S.J.A. and T.R.G., a University of Guelph
Alumni Doctoral scholarship to T.R.G., and by grants from
NSERC and the Canada Research Chairs Program to P.D.N.H.
References
Alerstam T (1982). Bird Migration. Cambridge University Press,
Cambridge.
Beaton MJ, Hebert PDN (1988) Geographical parthenogenesis and
polypoidy in Daphnia pulex. American Naturalist, 132, 837–845.
Beaton MJ, Hebert PDN (1989) Miniature genomes and endo-
polyploidy in cladoceran crustaceans. Genome, 32, 1048–1053.
Bell G (1982) The Masterpiece of Nature: the Evolution and Genetics of
Sexuality. Croom-Helm, London.
Bierzychudek P (1985) Patterns in plant parthenogenesis. Experi-
entia, 41, 1255 –1264.
3erny M, Hebert PDN (1993) Genetic diversity and breeding sys-
tem variation in Daphnia pulicaria from North American lakes.
Heredity, 71, 497–507.
Colbourne JK, Hebert PDN (1996) The systematics of North Amer-
ican Daphnia (Crustacea: Cladocera): a molecular phylogenetic
© 2002 Blackwell Science Ltd, Molecular Ecology, 11, 1209–1217
P O L Y P L O I D D A P H N I A I N A R G E N T I N A 1215
approach. Philosophical Transactions of the Royal Society of London
B, 351, 349–360.
Colbourne JK, Crease TJ, Weider LJ, Hebert PDN, Dufresne F,
Hobaek A (1998) Phylogenetics and evolution of a circumarctic
species complex (Cladocera: Daphnia pulex). Biological Journal of
the Linnean Society, 65, 347–365.
Crease TJ (1999) The complete sequence of the mitochondrial
genome of Daphnia pulex (Cladocera: Crustacea). Gene, 233, 89 –
99.
DeMelo R, Hebert PDN (1994) Allozymic variation and species
diversity in North American Bosminidae. Canadian Journal of
Fisheries and Aquatic Sciences, 51, 873–880.
Dufresne F, Hebert PDN (1994) Hybridization and origins of
polyploidy. Proceedings of the Royal Society of London B, 258, 141–
146.
Dufresne F, Hebert PDN (1995) Polyploidy and clonal diversity in
an arctic cladoceran. Heredity, 75, 45–53.
Dufresne F, Hebert PDN (1997) Pleistocene glaciations and
polyphyletic origins of polyploidy in an arctic cladoceran. Pro-
ceedings of the Royal Society of London B, 264, 201–206.
Dufresne F, Hebert PDN (1998) Temperature-related differences
in life-history characteristics between diploid and polyploid
clones of the Daphnia pulex complex. Écoscience, 5, 433 – 437.
Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R (1994) DNA
primers for amplification of mitochondrial cytochrome c oxi-
dase subunit I from diverse metazoan invertebrates. Molecular
Marine Biology and Biotechnology, 3, 294 –299.
Gilbert D (1992) SeqApp, Version 1.9a. A multiple sequence editor for
Macintosh computers, distributed over the Internet by the Author
at http://iubio.bio.indiana.edu/.
Glesener RR, Tilman D (1978) Sexuality and the components of
environmental uncertainty: clues from geographic partheno-
genesis in terrestrial animals. American Naturalist, 112, 659 – 673.
Gregory TR (2001) Coincidence, coevolution, or causation? DNA
content, cell size, and the C-value enigma. Biological Reviews, 76,
65–101.
Gregory TR, Hebert PDN (1999) The modulation of DNA content:
proximate causes and ultimate consequences. Genome Research,
9, 317–324.
Grime JP, Mowforth MA (1982) Variation in genome size — an
ecological interpretation. Nature, 299, 151–153.
Hardie DC, Gregory TR, Hebert PDN (2002) From pixels to
picograms: a beginners’ guide to genome quantification by
Feulgen image analysis densitometry. Journal of Histochemistry
and Cytochemistry, 50, 735–749.
Hebert PDN, Beaton MJ (1993) Methodologies for Allozyme Analysis
Using Cellulose Acetate Electrophoresis. Helena Laboratories,
Beaumont, TX, USA.
Hebert PDN, Crease T (1983) Clonal diversity in populations
of Daphnia pulex reproducing by obligate parthenogenesis.
Heredity, 51, 353–369.
Hebert PDN, Finston TL (2001) Macrogeographic patterns of
breeding system diversity in the Daphnia pulex group from the
United States and Mexico. Heredity, 86, 1–9.
Hebert PDN, Beaton MJ, Schwartz SS, Stanton DJ (1989)
Polyphyletic origins of asexuality in Daphnia pulex. I. Breeding
system variation and levels of clonal diversity. Evolution, 43,
1004–1015.
Hebert PDN, Schwartz SS, Ward RD, Finston TL (1993) Macro-
geographic patterns of breeding system diversity in the Daphnia
pulex group. I. Breeding systems of Canadian populations.
Heredity, 70, 148–161.
1216 S . J . A D A M O W I C Z E T A L .
Kimura M (1980) A simple method for estimating evolutionary
rates of base substitutions through comparative studies of
nucleotide sequences. Journal of Molecular Evolution, 16, 11–120.
Kumar S, Tamura K, Jakobsen IB, Nei M (2001) MEGA2: Molecular
Evolutionary Genetics Analysis software, Version 2.1. Arizona State
University, Tempe, Arizona, USA. Distributed over the Internet
by the authors at www.megasoftware.net.
Little TJ, DeMelo R, Taylor DJ, Hebert PDN (1997) Genetic charac-
terization of an arctic zooplankter: insights into geographic
polyploidy. Proceedings of the Royal Society of London B, 264,
1363 –1370.
Mead C (1993). Bird Migration. Newnes Books, The Hamlyn Pub-
lishing Group Ltd, New York.
Mowforth MA, Grime JP (1989) Intra-population variation in
nuclear DNA amount, cell size and growth rate in Poa annua L.
Functional Ecology, 3, 289 –295.
Paggi JC (1998) ‘Cladocera’ (Anomopoda y Ctenopoda). In: Bio-
diversidad de Artrópodos Argentinos: Una Perspectiva Biotaxonómica
(eds Morrone JJ, Coscarón S), pp. 507–518. Ediciones Sur, La
Plata, Argentina.
Paruelo JM, Beltrán A, Jobbágy Sala OE, Golluscio RA (1998) The
climate of Patagonia: general patterns and controls on biotic
processes. Ecología Austral, 8, 85 –101.
Saiki RK, Gelfand DH, Stoffel S (1988) Primer-directed enzymatic
amplification of DNA with a thermostable DNA polymerase.
Science, 239, 487– 491.
Saitou N, Nei M (1987) The neighbor-joining method: a new
method for reconstructing phylogenetic trees. Molecular Biology
and Evolution, 4, 406 – 425.
Schwartz SS, Innes DJ, Hebert PDN (1985) Morphological
separation of Daphnia pulex and Daphnia obtusa in North America.
Limnology and Oceanography, 30, 189–197.
Schwenk K, Sand A, Boersma M et al. (1998) Genetic markers,
genealogies and biogeographic patterns in the cladocera.
Aquatic Ecology, 32, 37–51.
Stebbins GL (1950) Variation and Evolution in Plants. Columbia
University Press, New York, NY.
Stebbins GL (1984) Polyploidy and distribution of arctic-alpine
flora: new evidence and a new approach. Botanica Helvetica, 94,
1–13.
Suomalainen E, Saura A, Lokki J (1987) Cytology and Evolution in
Parthenogenesis. CRC Press, Boca Raton, FL.
Taylor DJ, Hebert PDN (1993) Habitat dependent hybrid parent-
age and differential introgression between neighboringly sym-
patric Daphnia species. Proceedings of the National Acadademy of
Sciences of the USA, 90, 7079–7083.
Villalobos L (1994) Distribution of Daphnia in high mountain and
temperate lakes of South America. Verhandlungen der Inter-
nationalen Vereinigung Fuer Theoretische und Angewandte Limnologie,
25, 2400–2404.
Ward RD, Bickerton MA, Finston T, Hebert PDN (1994) Geo-
graphical cline in breeding systems and ploidy levels in
European populations of Daphnia pulex. Heredity, 73, 532–543.
Weider LJ, Beaton MJ, Hebert PDN (1987) Clonal diversity in high-
arctic populations of Daphnia pulex: a polyploid apomictic
complex. Evolution 41, 1335–1346.
Weider LJ, Hobaek A, Hebert PDN, Crease TJ (1999) Circumarctic
phylogeography of an asexual species complex II: allozymic
variation and clonal structure in arctic Daphnia. Molecular Eco-
logy, 8, 1–13.
Yampolsky LY, Scheiner SM (1996) Why larger offspring at lower
temperatures: a demographic approach. American Naturalist,
147, 86–100.
Sarah Adamowicz’s research has examined the daphniid fauna of
Argentina in order to address patterns of biogeography and
speciation in the genus. She recently completed her MSc thesis in
the laboratory of Paul Hebert, whose research interests span many
aspects of the evolution of freshwater organisms. Ryan Gregory’s
research focus is on the evolution of genome size, particularly in
invertebrates, while María Cristina Marinone works on the
ecology of freshwater invertebrates in Argentina.
© 2002 Blackwell Science Ltd, Molecular Ecology, 11, 1209 –1217
 P O L Y P L O I D D A P H N I A I N A R G E N T I N A 1217

Appendix I
Collection sites and codes for populations belonging to the Daphnia pulex complex from Argentina. (SC = Province of Santa Cruz; TF = Tierra
del Fuego). The clonal identity of each population is designated by letter (see Table 1 for clones), with sample size from each population
indicated. Populations for which mtDNA sequence data were obtained are designated by the name of the gene, followed by the GenBank
accession code for each unique sequence

Population Gene sequence

code Sampling locality Habitat Latitude Longitude Clone (n) (GenBank code)

1 Las Heras, SC quarry pond 46°33’00″ 68°46′12″ A (22) COI (AF489523)

2 Las Heras, SC quarry pond 46°33′00″ 68°46′12″ A (22) COI (same as 3)
3 Las Heras, SC quarry pond 46°33′00″ 68°46′12″ A (22) COI (AF489525)
4* Puerto San Julian, SC pond 48°53′33″ 67°40′29″ A (22) COI (same as 3)
5** Provincial Route #5, SC pond 51°21′59″ 70°15′30″ A (18) —
6 Chorrillo de los Frailes, SC dried river pond 51°54′22″ 69°07′43″ A (22) —
7 Cabo Vírgenes, SC pond 51°54′22″ 69°07′43″ A (22) —
8 Río Grande, TF quarry 53°43′47″ 67°47′08″ A (22) COI (same as 3)
9** Río Grande, TF large pond 53°47′15″ 67°47′46″ A (32) COI (AF489524)
ND5 (AF489526)
10*** Río Grande, TF ditch 53°47′32″ 67°48′57″ A (22) –
11 near Laguna Bombilla, TF pond 54°36′29″ 67°49′31″ B (44) ND5 (AF489527)
12 near Laguna Bombilla, TF pond 54°36′20″ 67°51′46″ A (22) —
13** near Lago Fagnano, TF pond 54°35′38″ 67°35′13″ B (2) —
14 Estancia Harberton, TF floodplain pool 54°52′37″ 67°18′22″ A (22) —
15 Estancia Harberton, TF large pond 54°52′23″ 67°20′06″ A (22) COI (same as 3)
16** Estancia Harberton, TF pond 54°51′31″ 67°27′48″ A (22) —

*Population found co-occurring with D. menucoensis.

**Populations co-occurring with species belonging to the D. obtusa complex.
***Population co-occurring with D. dadayana.

© 2002 Blackwell Science Ltd, Molecular Ecology, 11, 1209–1217