Copyright Ó 2008 by the Genetics Society of America
DOI: 10.1534/genetics.107.084657
Localization of the Genetic Determinants of Meiosis Suppression
in Daphnia pulex
Michael Lynch,1 Amanda Seyfert, Brian Eads and Emily Williams
Department of Biology, Indiana University, Bloomington, Indiana 47405
Manuscript received November 16, 2007
Accepted for publication June 10, 2008
ABSTRACT
Although 1 in 10,000 animal species is capable of parthenogenetic reproduction, the evolutionary causes
and consequences of such transitions remain uncertain. The microcrustacean Daphnia pulex provides a
potentially powerful tool for investigating these issues because lineages that are obligately asexual in terms of
female function can nevertheless transmit meiosis-suppressing genes to sexual populations via haploid
sperm produced by environmentally induced males. The application of association mapping to a wide
geographic collection of D. pulex clones suggests that sex-limited meiosis suppression in D. pulex has spread
westward from a northeastern glacial refugium, conveyed by a dominant epistatic interaction among the
products of at least four unlinked loci, with one entire chromosome being inherited through males in a
nearly nonrecombining fashion. With the enormous set of genomic tools now available for D. pulex, these
results set the stage for the determination of the functional underpinnings of the conversion of meiosis to a
mitotic-like mode of inheritance.
S
EVERAL of the central unresolved issues in evolutionary genetics concern the evolution of sexual vs.
asexual reproduction. Although substantial attention
has been given to the selective advantages and disadvantages associated with meiotic recombination (e.g., Peck
1994; Barton and Charlesworth 1998; Otto and
Barton 2001), less clear are the molecular/cytogenetic
mechanisms that lead to the conversion of an ancestrally
meiotic condition to a derived form of obligate asexuality,
in particular, reproduction via the use of unfertilized eggs
(obligate parthenogenesis) (Suomalainen et al. 1987). A
major impediment to resolving these matters is the general difficulty in performing genetic analysis with purely
asexual eukaryotes. However, a few asexual species exhibit
biological features that provide a potential entrée into the
mechanisms underlying sexual-to-asexual transitions.
Such species include asexual lineages that retain the
capability of producing fully functional males (via an
environmental sex-determination pathway). One such
species, the aquatic microcrustacean Daphnia pulex, is the
subject of the following investigation.
Nearly all members of the Cladocera are capable of
both sexual and asexual reproduction, with the former
being triggered by environmental induction of male
and haploid resting-egg production. However, the
D. pulex complex is unique in harboring numerous
lineages in which females have lost the ability to engage
in meiosis and instead produce diploid resting eggs by a
mechanism that is genetically equivalent to mitosis
(Hebert and Crease 1980). Moreover, many lineages
of this type retain the ability to produce males, at least
1
Corresponding author: Department of Biology, Indiana University,
Bloomington, IN 47405. E-mail: [email protected]
Genetics 180: 317–327 (September 2008)
some of which are capable of meiotic production of
haploid sperm (Hebert and Crease 1983; Innes and
Hebert 1988). Such sex-limited meiosis suppression
provides a potentially powerful mechanism for the
transformation of an entire sexual population to asexuality, as males from otherwise obligately asexual lineages backcross to sexual females and in doing so convert
a proportion of the hybrid progeny to obligate asexuality. Because this process could eventually assimilate
much of the diversity of a sexually reproducing species
into a diverse clonal assemblage, such a drive mechanism might explain the widespread geographic distribution of numerous obligately asexual lineages of D.
pulex throughout eastern Canada into the midwestern
United States (Hebert et al. 1988).
Under the simplest model envisioned for this system,
sex-limited meiosis suppression is conferred by a dominant allele at a single locus, such that a cross between a
sexual female and an adventitious male from an asexual
lineage is expected to yield a 1:1 ratio of obligately asexual and cyclically parthenogenetic progeny (Richards
1973; Jaenike and Selander 1979; Hebert 1981).
Crosses between the two breeding systems have led to
the synthesis of novel obligate parthenogens, demonstrating that successful transmission of meiosis-suppressing factors is indeed possible, although the number of
observations is too small to either confidently accept or
reject the ‘‘dominant sex-limited meiosis suppressor’’
model (Hebert et al. 1988). Here we apply a large battery
of molecular markers to a broad geographic survey of D.
pulex to gain insight into the likely mechanism of
meiosis suppression (monogenic vs. polygenic), the
approximate ages of individual asexual lineages, and
318
M. Lynch et al.
the potential source of origin of meiosis suppression in
the system. The recent development of a wide array of
genomic tools, including a complete genome sequence,
a genetic map, and a well-characterized, high-coverage
cDNA library, makes this a powerful system for understanding the genetic basis of obligate parthenogenesis.
MATERIALS AND METHODS
The work described herein was based on the analysis of
clones collected from broad geographic surveys ranging from
the midwestern United States to eastern Canada, made in 2003
to 2004 and subsequently maintained in the laboratory as
clonal isolates (supplemental Table 1). In accordance with prior
observations, clones were deemed to be obligately asexual if
they were able to produce resting eggs in the absence of males,
contrary to the situation in cyclical parthenogens, where
fertilization is required before release of an egg into an
ephippium (Paland et al. 2005).
DNA extraction and amplification: In an effort to find allelespecific associations between breeding systems and molecular
markers, we initially relied on genetically mapped microsatellite loci reported on in Cristescu et al. (2006) and then
supplemented such analyses with additional physically mapped markers derived from scaffolds associated with the fully
sequenced D. pulex genome (supplemental Table 2). Primers
for genetically mapped microsatellite loci were obtained from
Cristescu et al. (2006). Those for additional markers, which
were added to further saturate the physical map prior to
analysis (but with a few added post hoc in regions identified as
highly informative in the first pass), were created using the
program Primer3 (Rozen and Skaletsky 2000), following
BLASTN searches of the D. pulex genome sequence on
WFleaBase (http://wfleabase.org), using scaffolds with known
chromosomal affinities. Locus-specific amplifications were carried out using the 12-ml polymerase chain reaction described in
Cristescu et al. (2006). The touchdown thermal cycle program
began with a 3-min denaturation at 95°, followed by 15 cycles of
35-sec denaturation at 95°, 15 cycles of 35-sec annealing at 56°
(the temperature was reduced by 1° for each of the 14 remaining
cycles), and 15 cycles of 45-sec extension at 72°. The latter step was
followed by 45 cycles of 95° for 35 sec, 48° for 35 sec, and 72° for
45 sec and one final extension cycle at 72° for 10 min.
Primer sequences for mitochondrial genes were found,
using the complete mitochondrial genome sequence (Crease
1999) and the program Primer3. Mitochondrial genes were
PCR amplified, using the same 12-ml polymerase chain reaction noted above with a thermal cycle program beginning
with 2 min of denaturation at 94° followed by 30 cycles of 30sec denaturation at 94°, 30 cycles of 30-sec annealing at 51°–
61°, 30 cycles of 2-min extension at 72°, and a final cycle of
5-min extension at 72°. Different annealing temperatures were
used to optimize amplification of the five genes: 61° for Cox I,
58° for Cox II, 51° for Cox III, 61° for CytB, and 51° for ND5.
Genomic DNA was extracted using a modified cetyltrimethylammonium bromide (CTAB) extraction method as in
Cristescu et al. (2006). Eight to 10 adult individuals were
extracted for each sample.
Genotyping and sequencing: Microsatellite loci were
labeled using the M13(-21) fluorescent labeling system and
genotyped on an ABI 3730 genotyper. The products of four
loci (each labeled with a different fluorescent dye) were
combined and diluted 60- to 100-fold. One microliter of this
dilution was then added to 8.95 ml of water and 0.05 ml of
GeneScan-500 Liz size standard. This solution was denatured
for 5 min at 93° and then cooled immediately on ice.
Genotyping data were analyzed using Genemapper v.4.0
software (Applied Biosystems, Foster City, CA). In the first
pass of such analyses, any potential triploids were reextracted
and regenotyped to eliminate the possibility of contamination.
Mitochondrial genes were sequenced using a 10-ml reaction
mixture with 1 pmol primer, 0.5 ml of BigDye Terminator v3.1
(Applied Biosystems), and 1 ml (10–20 ng) of amplified PCR
product. A two-step thermal cycle program was used for the
sequencing reaction, beginning with a 2-min 96° denaturing
step, followed by 27 cycles of 96° for 30 sec, 60° for 4 min, and
then one cycle of 10° for 3 min. Sequence data were analyzed
using CodonCode Aligner.
Statistical methods for sequence and microsatellite analysis
follow the procedures outlined in Lynch et al. (1999) and
utilize software developed by the first author.
Sexual 3 asexual crosses: In an effort to test the ability of
sons of obligately asexual females to mate with sexual females
and to evaluate the genetic consequences of such events,
hybrid crosses were carried out by combining males from an
obligately asexual clone with females from a cyclically parthenogenetic clone in a 1:5 ratio. To ensure that crosses could
proceed in only one direction, the sexual clones chosen for
use were usually incapable of male production, as such variants
are frequent in natural populations, independent of the
sexual/asexual breeding system. Crosses were performed at
20° on a 12-hr light:12-hr dark cycle and checked every 2 days
for the removal of any live young. Resting eggs were collected
and placed in the dark at 4° for 2 weeks, after which the eggs
were removed from their ephippium and then exposed to
high light at 15° with a 12-hr light:12-hr dark cycle.
To assist in the interpretation of the following results by
those unfamiliar with the Daphnia system, an overview of the
alternative breeding systems is provided in Figure 1.
RESULTS
Phylogenetic distribution of meiosis suppression:
Under the assumption that obligate asexuality arises
either by de novo cytogenetic events within sexual lineages
or by transmission of sex-limited meiosis-suppressing
genes via asexually produced males, the maternally inherited mitochondrial genome is expected to provide an
unbiased view of the phylogenetic background within
which asexuality has arisen. Like several preceding
studies (e.g., Crease et al. 1989; Paland et al. 2005),
our results indicate that obligate asexuality is widely
distributed throughout the entire D. pulex lineage, with
individual asexual lineages more or less randomly
distributed among those reproducing by cyclical parthenogenesis (Figure 2). All lineages of both types
from the midwestern United States, Maine, and Ontario are contained within a single clade, with two
other distinct clades appearing at the base of the tree,
one from Oregon and the other from northeastern
Canada (Quebec and New Brunswick). On the basis
of the reproductive modes of outgroup species, the
ancestral state of D. pulex is inferred to be cyclically
parthenogenetic, although the northeastern Canadian
lineage appears to be nearly entirely asexual, with the
possibility of a single reversion to sexuality appearing
in a clone from Michigan.
Meiosis Suppression in Daphnia
319
Figure 1.—Schematic of the breeding systems of the alternative forms of
Daphnia pulex. Note that (1) cyclical parthenogens are not truly ‘‘cyclical,’’ as
they are capable of switching to sexual
reproduction at any time, and (2) not
all cyclical or obligate parthenogens
produce males, although males are essential for resting-egg production by cyclical parthenogens and nonessential in
obligate asexuals.
To estimate the times of origin of the various asexual
clades, we employed as a molecular clock the only direct
estimate of the rate of nucleotide substitutional mutation for the mitochondrial genome, 9.7 3 108/site/
generation, derived from the nematode Caenorhabditis
elegans (Denver et al. 2000). Assuming neutrality, substitutions at silent sites in protein-coding genes are
expected to accumulate at this rate, providing a basis for
estimating the timing of internal nodes of a phylogenetic tree on the basis of silent sites alone. Such analyses
suggest that all but the New Brunswick/Quebec clade
have depths in the range of 12,000–58,000 generations,
whereas the northeastern Canada clade extends back
172,000 generations (Figure 2). These estimates may
be downwardly biased, as indirect estimates of the
mitochondrial silent-substitution rate derived from interspecific comparisons and assumed divergence dates
based on the fossil record average 4.7 3 108/site/year
for four groups of invertebrates (Lynch et al. 2006).
Assuming 10 D. pulex generations per year, the latter rate
leads to estimates 21 times greater than those given in
Figure 2. Even at this extreme, however, the divergence
times of most asexual clades are still in the range of
25,000–122,000 years. Moreover, regardless of which set
of estimates is deemed most appropriate, they are both
upwardly biased because the precise timing of the
transition to asexuality within individual clades must
postdate the base of the clades, and more intensive
clonal sampling could break up apparently monophyletic lineages of asexuals. Thus, taken together, these
results imply a relatively short evolutionary longevity for
individual asexual lineages, while not ruling out an
ancient origin for the meiosis suppressor itself.
Further inferences from divergence of nuclear
markers: One of the central goals of this study is to
identify markers associated with obligate asexuality. However, a concern with any attempt to identify marker–
phenotype associations over a broad geographic range is
the possibility of spurious correlations resulting from
geographic divergence of phenotypes, in this case of
alternative breeding systems. This appears not to be a
significant issue in this study, as chromosomewide esti-
mates of Gst (a measure of population subdivision, on a 0–
1 scale; Nei 1987), contrasting all obligate asexuals with all
cyclical parthenogens, are all ,0.08 for nuclear microsatellite loci after a few very strong associations (below)
are eliminated from the analysis. The average Gst is just
0.023 (SE ¼ 0.004) across the entire genome (Figure 3).
In other words, with the exception of a few candidate
regions discussed below, only 2% of the total variation at
microsatellite loci in D. pulex is associated with breedingsystem differences. Thus, while prior Gst estimates for
populations within breeding systems are large for this
species, typically on the order of 0.3–0.5 for a region the
size of a few U.S. states (Lynch et al. 1999), the additional
subdivision between breeding systems is negligible.
Once locked into a regimen of obligate asexuality, a
diploid locus is expected to experience an increased
level of allelic divergence owing to the accumulation of
nonsegregating mutations, unless gene conversion is of
overwhelming importance. Consistent with this expectation, the average microsatellite heterozygosity in
asexual derivatives of D. pulex exceeds that in sexual
clones on 11 of 12 chromosomes, although not greatly
so, with an average elevation of 31.21 (0.04) or, in
absolute terms, of 0.092 (0.020) (Figure 4). The divergence of allele sizes within individuals is also greater
in asexuals than in sexuals on 11 of 12 chromosomes,
but again only to a small extent, with an average of 0.97
(0.30) additional nucleotides separating any two alleles
in clones of the former (Figure 4). Such differences are
most notable on chromosome IX.
These low levels of microsatellite divergence both
within asexual lineages and across the two breeding
systems are qualitatively consistent with our conclusion
that individual asexual lineages are relatively young, but
also yield to more quantitative perspective. Given an
initial level of heterozygosity H0, and per-generation
rates of mutation and gene conversion, u and c, respectively, the expected heterozygosity within an asexual
line of descent can be described as
Ht11 ¼ ð1 cÞHt 1 2uð1 Ht Þ;
which generalizes to
320
M. Lynch et al.
Figure 2.—A phylogeny of cyclically parthenogenetic (dotted
lines, regular type) vs. obligately
asexual (solid lines, boldface
type) lineages based on mitochondrial sequence (including
both silent and replacement
sites). The scale is in units of substitutions per nucleotide site, and
the bootstrap support for individual nodes is given on internal
branches (in percentage). Tree
construction used the neighborjoining method (Saitou and
Nei 1987), as implemented in
MEGA (Tamura et al. 2007). Ages
of various clades are determined
from silent-site divergence alone,
as described in the text. The base
of the tree is rooted with sequences from two clones of Daphnia
melanica. Clone notation: state/
province, clone number, breeding system (A, obligately asexual;
S, cyclical parthenogen).
Meiosis Suppression in Daphnia
321
Figure 3.—Estimates of the degree of average marker subdivision between the aggregated sets of sexual and asexual lineages.
Ht ¼ H * 1 ð1 c 2uÞt ðH0 H *Þ;
where H * ¼ 2u/(c 1 2u) is the expected level of
heterozygosity at conversion–mutation equilibrium.
Although this expression ignores the possibility of loss
of heterozygosity by back mutations, this assumption
appears reasonable for moderate timescales given that
most microsatellite mutations in Daphnia involve multiple-step changes and that almost all single-step changes
involve single-repeat insertions to the exclusion of deletions (Seyfert et al. 2008). Results from long-term
mutation-accumulation experiments indicate that, for
the loci involved in this study, average u ¼ 7.1 (2.6) 3
105/allele/generation (Seyfert et al. 2008) and that
the average rate of loss of heterozygosity by gene
conversion (c) ¼ 1.2 (0.6) 3 104/allele/generation
(Omilian et al. 2006).
Under this model, the expected equilibrium level of
microsatellite heterozygosity within an obligately asexual clone is 0.523, whereas the observed average is 0.508
excluding the highly divergent chromosome IX (discussed below). Assuming that newly arisen asexual
lineages have an average level of heterozygosity equal
to that in the sexual subdivision of the species (0.431),
the time to achieve the observed average level in asexual
lineages is just 4487 generations. Because there is a
slight level of divergence among sexuals and asexuals, as
reflected in an average Gst of 0.023, a newly arisen
obligate asexual resulting from a backcross to a sexual
individual is expected to have a level of heterozygosity
elevated above that in the sexual species by 0.011, but
this reduces the prior estimate only to 4090 generations.
Although the standing levels of heterozygosity for
most chromosomes in the sexual species are somewhat
smaller than the equilibrium expectations given above
(Figure 4), this is at least in part because random genetic
drift among segregating alleles constitutes an additional
Figure 4.—Estimates of the average degrees of marker heterozygosity and allelic-size divergence for each chromosome with respect to the alternative breeding systems. The dashed line in the
top denotes the level of heterozygosity expected in a purely asexual lineage under mutation–gene conversion equilibrium.
mechanism of loss of variation from sexual populations
(which does not apply to the heterozygosity within a
nonsegregating asexual lineage). An average additional
loss of heterozygosity of 6.8 3 105/locus/generation is
sufficient to explain the observed levels of variation
within sexual individuals, which in turn implies average
effective population sizes of 7400 clones.
Association mapping of meiosis-suppressing loci:
Given the results noted above, any marker exhibiting a
Gst significantly .0.08 can be viewed confidently as
residing in a candidate-gene region. Relying on this
conservative criterion, several potential regions for the
determinants of meiosis suppression are apparent,
including small sections of chromosomes V, VIII, and
X (Figure 5). Most notable, however, is chromosome IX,
which exhibits highly significant associations over its full
length, with only three exceptions appearing near the
tips. For the most significant loci on all of these
chromosomes, one highly informative marker allele is
present in all to nearly all obligate asexuals and absent
to nearly absent from all sexual members of the species
(Table 1).
For each of the diagnostic marker alleles, a 200-kb
window around the marker was searched for highly
conserved proteins with known involvement in reproduc-
322
M. Lynch et al.
Figure 5.—Chromosomal distribution of marker significance associated with obligate asexuality vs. cyclical parthenogenesis.
Bars on the left denote regions containing markers with asexual–sexual Gst values significant at the level P , 0.001 and extend to
the midpoints with the first flanking markers on both sides deemed as insignificant. The statistic D is a more conservative measure
of significant phenotypic association, the number of standard errors by which the asexual–sexual measure of Gst exceeds the upper
bound on the baseline value (0.08).
tion, using gene predictions and their imputed functions
derived from the full-genome sequence of this species.
For each marker, we found putative homologs of proteins
involved in a variety of processes, including meiosis,
gametogenesis, DNA repair, and sister-chromatid cohesion (supplemental Table 3). For example, contained
within the significant region on chromosome V is an
ortholog of Cdc6, a master regulator of DNA replication
known to have a critical role in meiotic spindle formation
in the mouse (Anger et al. 2005). Putative orthologs of a
number of proteins with characterized meiotic functions,
including polo kinase, pelota, and Esco1, are located on
chromosome IX.
Residing within the significant region on chromosome VIII are two very relevant proteins, Brca1 and
Rec8. Extensive studies have revealed the involvement
of Brca1 in DNA repair and in the silencing of unsynapsed meiotic chromosomes (Turner et al. 2004),
and equally intriguing is the role of Rec8 in sisterchromatid cohesion during meiosis. Rec8 is conserved
from yeast to mammals, where it replaces the mitotic
Rad21 in the multiprotein cohesin complex (Revenkova
and Jessberger 2005). The D. pulex genome harbors
three copies of Rec8, including two highly similar copies
in a head-to-head arrangement, whereas only one copy is
found in other well-studied eukaryotes (Revenkova and
Jessberger 2005).
This unusual situation prompted us to begin a
molecular analysis of the Rec8 loci in cyclical and
obligate parthenogens. Application of PCR primers
flanking the highly similar duplicated regions in the
two Rec8 copies on scaffold 77 revealed a striking length
polymorphism 650 bp upstream of one of the copies.
In every cyclical parthenogen tested (n ¼ 38), a band of
1.4 kb was amplified in this region, while all obligate
asexuals tested (n ¼ 42) revealed the 1.4-kb band as well
as an additional 2.7-kb band. Preliminary sequence
analysis of a small number of genotypes demonstrates
that the polymorphism results from a 1.3-kb insertion,
and work is ongoing to characterize the expression
differences associated with this polymorphism.
Asexual 3 sexual crosses: We were able to produce 51
hybrid progeny through several different crosses involving males produced by otherwise obligate asexuals
and females from cyclically parthenogenetic lineages.
Because a large fraction of the hybrids were the products
Meiosis Suppression in Daphnia
TABLE 1
Highly diagnostic microsatellite markers, with the fractional
representation among sampled alleles given for each of
the two reproductive systems
Locus
Allele
Asexuals
Sexuals
d160
265
Chromosome V
20/35
1/39
d165
Chromosome VIII
184
32/34
2/40
d178
d171
d043
d099
d149
d011
9-3
9-8
9-10
9-41
9-42
13-5
13-14
13-48
99-50
99-52
240
221
163
300
181
261
157
268
141
218
254
151
110
205
162
165
Chromosome IX
13/31
26/31
26/34
24/24
31/33
24/28
32/34
37/37
33/33
38/39
30/35
30/36
30/30
23/37
33/37
39/39
1/38
0/30
1/28
2/50
0/40
0/28
0/39
0/40
2/37
0/40
2/27
3/37
2/26
7/36
0/35
6/40
d005
302
Chromosome X
31/37
0/37
Allele designations are based on the estimated lengths of
the PCR products.
of unreduced male gametes, these numbers are far too
small for reliable analyses of segregation ratios, but the
resultant observations do resolve several issues. First, all
progeny resulting from these crosses inherited malespecific markers, ruling out the possibility of gynogenesis. Second, there is no evidence that any marker
strongly associated with obligate asexuality is subject to
a strong drive process. In all informative cases, both
alternative alleles within males were observed to be
transmitted into at least some progeny. This tentatively
suggests that the asexual-specific markers do indeed
reflect their adjacency to factors causally involved in
meiosis suppression, rather than simply being residents
of chromosomal segments that happen to have elevated
delivery rates to successful sperm (e.g., chromosomes
experiencing male-specific meiotic drive). Third, although the data are limited, the diagnostic markers
associated with chromosome IX (as mapped in sexual
crosses) appear to be strongly linked on the same
chromosome, rather than being distributed over both
members of homologous pairs, and are therefore largely
heritable as a unit, albeit with some recombination
(Table 2). Prior work indicates that chromosome IX
recombines normally in cyclical parthenogens, as demonstrated by our ability to discriminate map positions
(Cristescu et al. 2006). Fourth, as noted above, some
323
males experience aberrant gametogenesis, as reflected
in the presence of allelic pairs of unambiguous paternal
markers in hybrid progeny, indicative of triploidy for
one or more chromosomes. Of the 2 progeny from the
cross Tre1 # 3 Pa32 $, one had a genotype consistent
with complete diploidy, whereas the other was consistent with complete triploidy; and all 32 of the progeny
from the Sed2 # 3 Pa33 $ cross had unbalanced
genotypes (i.e., an apparent combination of disomy
and trisomy across different loci).
One caveat in these analyses is that the assignment of
chromosomal locations is based on a prior mapping
exercise derived entirely from a sexual 3 sexual cross. In
principle, the asexuals might have experienced some
form(s) of chromosomal rearrangement that placed all
of the strong markers that we identified into a single
linkage group, in which case our inference of at least
four independent factors associated with meiosis suppression would be in error. Although the data for
informative markers off of chromosome IX in these
crosses are limited, they are sufficient to demonstrate
that they are unlinked to chromosome IX (Table 2).
Moreover, our observations on chromosome IX itself
reveal eight apparent crossover events in 15 meiotic
products (Table 2), arguing against the presence of a
strong crossover-suppressing rearrangement on this
chromosome in asexuals. Thus, we tentatively conclude
that the asexuals have not experienced major chromosomal rearrangements.
DISCUSSION
Our results are qualitatively consistent with the hypothesis that the geographical spread of a meiosissuppressing mechanism in D. pulex initiated from a
northeastern glacial refugium, proceeding westward in
a stepping-stone fashion by gradual backcrossing and
conversion of sexual populations, but with populations in
the far western United States remaining isolated from
such events to this day (Paland et al. 2005). Consistent
with such an origin, the northeastern asexual clade
identified in the mitochondrial phylogeny exhibits an
average heterozygosity at microsatellite loci of 0.564
(0.036), which is substantially greater than that for the
entire collection of asexuals (0.508) and not significantly
different from the expectation under long-term mutation–gene conversion equilibrium (0.523). Also consistent with the contagious spread of obligate asexuality by
backcrossing to sexual lineages is the fact that all of the
highly diagnostic microsatellite markers for asexuality
(Table 1) are generally present as unique, single alleles
within asexual isolates, with the aggregate pool of nondiagnostic alleles at the same loci being consistent with
those segregating among sexual individuals.
Although the precise nature of the mechanism of
meiosis suppression in D. pulex is not yet clear, two
conclusions now appear possible. First, as postulated by
324
M. Lynch et al.
TABLE 2
Genotypes and ploidy levels of sperm contributing to progeny of crosses between males derived from obligate asexuals
and females derived from cyclical parthenogens
Linkage group
Asexual male
IX
V:
VIII:
X
Sexual female
Sperm ploidy
41
d171
8
50
d149
d11
14
d160
d165
d005
n
Lau1
Rw1
Stm2
Stm2
Rng1
Cc6
Op1
Op1
Tre1
Pa32
Nos1
Nfl3
Sed2
Pa33
Haploid
Haploid
Haploid
Haploid
Haploid
Haploid
Haploid
Haploid
Diploid
Unbalanced
Unbalanced
Unbalanced
Unbalanced
Unbalanced
Unbalanced
Unbalanced
Unbalanced
Unbalanced
Unbalanced
Unbalanced
Unbalanced
Unbalanced
Unbalanced
Unbalanced
Unbalanced
Unbalanced
Unbalanced
Unbalanced
M
M
m
m
m
M
m
M
Mm
Mm
Mm
Mm
Mm
Mm
Mm
Mm
Mm
Mm
Mm
Mm
Mm
Mm
Mm
Mm
Mm
M
M
M
M
m
m
m
m
M
M
M
Mm
Mm
Mm
M?
M?
M?
M?
M?
M?
M?
M?
M?
M?
Mm
Mm
Mm
Mm
Mm
M?
M?
M
M
m
m
m
M
m
M
Mm
m
m
M
m
m
M
M
M
Mm
Mm
Mm
Mm
M
M
Mm
m
Mm
Mm
M
M
M
m
m
m
M
m
M
Mm
–
–
M
Mm
Mm
Mm
Mm
Mm
Mm
Mm
Mm
Mm
Mm
Mm
Mm
Mm
Mm
Mm
Mm
M
M
m
m
m
M
M
M
Mm
M?
M
m
m
M?
m
M?
M?
M?
M?
m
m
M?
Mm
M?
m
m
M?
M?
M
M
m
m
m
M
M
M
Mm
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
M
M
m
m
m
M
m
M
Mm
M?
M?
Mm
Mm
Mm
Mm
Mm
M?
Mm
M?
M?
Mm
Mm
Mm
Mm
Mm
Mm
Mm
M?
4/1
0/1
2/0
1/1
1/1
0/1
1/0
/
/
1/
1/
/
1/0
1/0
2/0
7/0
1/1
1/0
1/0
1/0
1/0
1/
/
1/0
1/0
/
/
2/0
2/3
1/0
1/1
1/1
/
/
/
/
/
1/
1/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
2/3
1/0
2/0
2/0
1/1
1/0
0/1
1/0
1/1
/
/
/
1/0
1/0
2/0
7/0
2/0
1/0
1/0
1/0
1/0
2/0
1/0
1/0
1/0
0/1
1/0
2/0
5
1
2
2
2
1
1
1
1
1
1
1
1
1
2
7
2
1
1
1
1
2
1
1
1
1
1
2
The seven designated loci extend over the full length of chromosome IX (Figure 5). For the two codominant markers, M
denotes a marker allele diagnostic of obligate asexuality, m denotes any alternative allele, – denotes an uninformative parental
genotype, and a question mark denotes uncertainty regarding a second allele caused by shared parental markers. Note that the
presence of two markers from an asexual parent (Mm) implies transmission via a diploid sperm. The number of times each presumptive gamete was observed in a cross is denoted by n.
previous authors (Hebert 1981; Hebert and Crease
1983; Innes and Hebert 1988), meiosis suppression in
D. pulex is largely sex limited. Although males produced
by some asexual lineages generate chromosomally
aberrant gametes, others appear to be meiotically competent, producing only diploid progeny when backcrossed to the sexual species. Second, the fact that
highly significant marker–breeding system associations
reside on four chromosomes implies that the capacity
for meiosis suppression may be conferred by the joint
action of multiple loci, rather than by the single
dominant mutation envisioned by Hebert (1981).
In principle, at least one of the markers for asexuality
could simply be associated with male production, as
such a capacity is essential for the spread of a meiosis
suppressor into the sexual segment of a species. Only a
fraction of D. pulex are capable of male production, and
although the genetic details remain to be worked out,
the limited crosses that have been made between male-
and nonmale-producing clones appear to be qualitatively consistent with a single-locus model in which
individuals with male/female function are either MM or
Mm and those incapable of male production are mm,
with a dominant allele (or tight linkage group) M
conferring the ability to produce males (Innes and
Dunbrack 1993; Innes 1997; Tessier and Cáceres
2004). However, none of the significant markers that
we identified in obligate asexuals were associated with
the ability to produce males (revealed by application
of male-inducing methyl farnesoate; Olmstead and
Leblanc 2002). In addition, our broad geographic
survey provides little support for the hypothesis that
obligate asexuals tend to lose the ability to produce
males (Innes et al. 2000), as 59% (n ¼ 39) of the sexual
clones and 52% (n ¼ 40) of the asexual clones in our
survey produce males. However, the possibility that
newly arisen asexual lineages have a frequency of male
production in excess of 52% cannot be ruled out.
Meiosis Suppression in Daphnia
Thus, our results support the idea that as many as four
epistatically interacting factors, likely operating together in a dominant fashion, yield a system of sexlimited meiosis suppression in D. pulex. Assuming these
factors are located on different chromosomes, independently assorting as they do in cyclical parthenogens, just
one-sixteenth of the male gametes produced by asexual
lineages would be expected to convey the capacity for
meiosis suppression, in contrast to the 50% expected
under the single-factor hypothesis. Consistent with this
low expected yield, of the 31 hybrid progeny that we
have been able to produce, only 2 have produced viable
resting eggs, and both such clones were triploid (from
the cross Sed2 # 3 Pa33 $; Table 2), having inherited
the full suite of genes from the asexual parent (none of
the 15 diploid hybrids have initiated the production of
resting eggs, and hence their reproductive status remains unknown). Innes and Hebert (1988) report a
similarly low success rate for asexual–sexual crosses, but
were able to produce 10 putatively diploid hybrid clones
capable of viable resting egg production, of which 4
were obligately asexual and 6 were sexual. Although the
latter ratio is close to the 1:1 expectation under the
single-factor model, it would also be consistent with a
four-factor model if successful resting egg production
(either haploid or diploid) requires that a hybrid procure a full complement of the four genes of a specific
mating type from the male (asexual) parent.
Because a polar body is extruded during the process
of cell division during even normal parthenogenetic
reproduction in Daphnia, it is highly likely that the
transition to obligate asexuality in Daphnia does not
simply involve a switch to mitosis (Weismann 1886;
Schrader 1926; Lumer 1937; Zaffagnini and Sabelli
1972), and this simple observation helps reveal why the
emergence of a system endowed with the capacity for
the contagious spread of asexuality may require several
cytological modifications. First, the production of a
diploid, genetically clonal egg without fertilization
requires a normal meiosis followed by the fusion of
nonsister nuclei or a single equational division (as in
mitosis). Thus, as there is no evidence for segregation or
recombination in obligately asexual D. pulex (beyond
background mitotic levels; Omilian et al. 2006), it is
likely that sister kinetochores are oppositely oriented
(as in mitosis) rather than co-oriented (as in normal
meiosis), and that an equational division takes place.
Second, because all sister-chromatid cohesion must
be lost at or before anaphase I if a single equational
division is to occur, the switch to diploid, asexual resting
egg production must require a modification of the
mode of cohesion from its meiotic form. Thus, it is
notable that two copies of the meiosis-specific cohesin,
Rec8, are localized near one marker for meiosis suppression, while a putative ortholog of Polo kinase, a
protein with known roles in mitotic and meiotic
kinetochore function (Kamieniecki et al. 2005; Qi
325
et al. 2006), is found on chromosome IX. In the fission
yeast Schizosaccharomyces pombe, a knockout of the Rec8
locus results in the separation of sister chromatids
during meiosis I, thereby eliminating the reductional
phase of chromosome segregation (Watanabe and
Nurse 1999), in accordance with the scenario outlined
above. Such an alteration would result in a heterozygosity-maintaining mechanism of diploid egg production
like that seen in obligately asexual Daphnia.
Third, an alteration that enables development to
initiate spontaneously, without fertilization, is required.
In principle, diploid-egg production may be sufficient
to trigger downstream development, as cyclically parthenogenetic Daphnia are already predisposed to the
immediate development of diploid eggs during the
clonal phase of propagation.
Fourth, as noted above, if a meiosis-suppressing mechanism is to spread via backcrossing to a sexual species,
obligately asexual lineages must retain the capacity for
male production and such males must be capable of the
meiotic production of functional haploid sperm. Because
sex-specific meiosis genes are known to exist in Drosophila (Baker 1975; Bopp et al. 1999; Tomkiel et al. 2001;
Hirai et al. 2004; Thomas et al. 2005), and sex-specific
differences exist in meiotic progression and dysfunction
in mammals (Morelli and Cohen 2005), the restriction
of a meiosis-suppressing gene to one sex may entail no
special requirements.
Even with as many as four independently segregating
factors critical to meiosis suppression, the spread of
obligate asexuality into a sexual population by repeated
backcrossing can be extremely rapid, provided any
immediate fitness costs to asexuality are small relative
to the fraction of hybrid progeny converted to asexuality. Assuming random mating of males produced by
asexuals with females of the sexual population, the
change in frequency of asexuals over a generation of
mating is
pt11 ¼ pt 1 bpt ð1 pt Þ;
where b is the fraction of hybrid progeny converted to
obligate asexuality, the solution of which is
pt 1=f1 1 ½ð1 p0 Þ=p0 e bt g
for small b. Under this model, starting from an undetectable frequency of asexuals, essentially complete loss of
sexual reproduction is expected to occur within 50 sexual
generations under a one- or a two-locus model (b ¼ 0.5
and 0.25, respectively) and within 300 generations under
a four-locus model (b ¼ 0.0625) (Figure 6). Thus,
because Daphnia populations typically experience at
least one bout of sexual reproduction per year, even
under a four-factor mechanism, complete displacement/
assimilation of a sexual population by a single asexual
invader can be accomplished in just a few centuries.
Finally, although our results suggest that individual
lineages of obligately asexual D. pulex typically survive
326
M. Lynch et al.
the rapidly emerging field of functional genomics,
bodes well for the future elucidation of the molecular
mechanisms responsible for the conversion of sexual
populations to effectively clonal lineages.
We thank J. Colbourne, J. Dudycha, S. Paland, and S. Schaack for
providing many of the clones included in this study and T. Crease, M.
Cristescu, D. Innes, and M. Zolan for helpful comments. We are also
grateful to Alice Prickett for the artwork. This project was supported by
a National Science Foundation Frontiers in Integrative Biological
Research grant to M.L. and colleagues.
LITERATURE CITED
Figure 6.—Dynamic takeover of a sexual population by the
invasion of a male-producing obligate asexual, starting with
an initial asexual frequency of 0.0001. n denotes the number
of freely segregating loci containing dominant alleles essential to sex-limited meiosis suppression, with obligate asexuality
being conferred only on individuals harboring such alleles at
all n loci.
for no more than a few thousand years, a likely
consequence of deleterious-mutation accumulation in
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and Lynch 2006), this does not rule out a much deeper
evolutionary origin of the meiosis-suppressing mechanism itself, which may survive in the long run via
periodic transfers into fresh sexual backgrounds with
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enriched in the close chromosomal vicinity of the
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(except during rare phases of transmission through
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Communicating editor: D. Charlesworth