Genetic Differentiation Among Oregon Lake
Populations of the Daphnia pulex Species
Complex
D. J. Straughan and N. Lehman
Gene flow among invertebrate populations inhabiting bodies of nonflowing freshwater such as ponds or lakes must at some stage involve transport across habitat
unsuitable for adult stages. Consequently the potential for interpopulational differentiation is high in these species, yet empirical studies of lake populations of Cladocerans such as Daphnia have failed to reveal high levels of genetic distinctiveness among populations and have led to much speculation about how these
populations exchange genes and remain cohesive evolutionary units. In this study
we surveyed 42 Oregon lake populations of Daphnia from the D. pulex species
complex for genetic variation within the mitochondrial DNA control region. We have
used this data to test the relative abilities of various ecological factors to explain
the observed patterns in genetic differentiation among lakes. Despite limited genetic variation detected among our samples—11 very similar RFLP-defined mtDNA
genotypes from 388 individuals—analyses of nucleotide variance using analogs to
Wright’s F statistics indicate that when multilake populations are defined in terms
of the river drainage basin to which they belong, strong and significant amounts
of among-population genetic variation can be detected at this locus (FST estimates
between 0.5 and 0.6). In contrast, we fail to detect consistent significant amongpopulation variation when populations are defined on the basis of regional physical
geography, bird migratory flyways, or lake trophic status. The manner in which the
data are compiled, that is, whether RFLPs or nucleotide sequences are used, has
little effect on the overall conclusions, yet it is clear that nucleotide sequence data
would lower the standard errors of FST estimates. We propose that periodic widescale flooding during the late Pleistocene may be an important mechanism to homogenize genetic differences among lake Daphnia continent-wide south of the
southern-most extent of Pleistocene glaciation.
From the Department of Biological Sciences, California
State University, Long Beach, Long Beach, California
(Straughan) and the Department of Biological Sciences,
University at Albany, SUNY, 1400 Washington Ave., Albany, NY 12222 ( Lehman). We thank D. Decker for valuable discussions and help in all phases of this project;
M. Joshi, S. Smith, D. Fera, and D. Tieman for technical
assistance; and J. Hicks, M. Pfrender, and S. Straughan
for help in obtaining lake samples. This work was supported by California State University, Long Beach and
the University at Albany, SUNY. Address correspondence to Niles Lehman at the address above or e-mail:
[email protected].
q 2000 The American Genetic Association 91:8–17
8
The freshwater microcrustacean Daphnia
(water fleas) is a common and abundant
inhabitant of North American lakes and
freshwater ponds. Although population
densities in these lentic environments can
reach very high values, free-swimming
Daphnia are rare or absent from most lotic
environments such as rivers and streams.
Consequently the mechanisms of gene
flow among lake or pond populations, and
invasion of newly created habitats are
largely unknown. It has often been postulated that the desiccation-resistant resting
stage of Daphnia, hard dizygotic encasings
known as ephippia, can be transported
from waterbody to waterbody either by
wind, moving water, or on the feet of migratory birds such as waterfowl (Crease et
al. 1997; Dodson and Frey 1991; Talling
1951; Weider et al. 1996). Yet many hypotheses concerning gene flow among
populations remain untested with highresolution molecular studies.
The waterbodies of Oregon provide a
perfect milieu in which to examine proposals concerning the population-genetic
(and phylogenetic) patterns of Daphnia.
Although the state may appear to be an
arbitrary political construction, it is actually well defined geographically. It is
bounded on the west by the Pacific Ocean.
It is bounded on the north and east in
large part by the Columbia and Snake Rivers, which are major hydrologic features
of the Pacific Northwest. Its southern
boundary is less well defined, but does include the Siskiyou mountain range, which
partitions the weather patterns and flora
sharply, with wetter forms to the north
and drier forms to the south, in California.
Within the boundaries of the state lie an
extreme range of geologic features and
ecotones. High alpine, marsh, fertile low-
land plains, sand dune, volcanic flow
channel, glacial valley, high desert plain,
low desert basin, and subalpine forest
habitats are all present and abundantly
represented. Within the state are some of
the driest as well as some of the wettest
places in the United States. Accordingly,
Oregon can be divided into 10 distinct
geomorphic regions ( Loy et al. 1976),
each of which contains many permanent
water bodies ( lakes and reservoirs) and
numerous temporary ponds. The lakes
that are found in Oregon have been
formed by a wide variety of processes,
from man-made (i.e., artificial) reservoirs,
to tectonic basins, to lakes formed by
landslides and river activity (Johnson et
al. 1985). In addition to these geomorphic
regions, Oregon has many rivers, and 18
major river drainage basins can be delineated (Johnson et al. 1985). Migratory flyways of birds tend to transect this region
in a north/south fashion following general
geographic features ( Evanich 1990). The
boundaries of the geomorphic regions, river drainage basins, and migratory flyways
as defined by Evanich (1990) and Johnson
et al. (1985) are indicated in Figure 1.
The control region of the mitochondrial
DNA (mtDNA) genome has become the locus of preference for a wide range of population- and species-level evolutionary
studies in animals. The rapid rate of nucleotide substitution in this region, combined with desirable characteristics of animal mtDNA in general ( high copy number
in the cell, uniparental inheritance, lack of
recombination, strong conservation of
gene order, and amino acid sequence over
many taxa) has made it amenable to investigations that require a significant
amount of polymorphism unconfounded
by allelic heterogeneity (Moritz et al.
1987). Availability of the polymerase chain
reaction (PCR) with ‘‘universal’’ oligonucleotide primers anchored in flanking RNA
regions that are highly conserved has allowed many investigators to generate sequence and/or RFLP data quickly with
very little prior knowledge of the target
species’ evolutionary history ( Hillis et al.
1996). And despite potential pitfalls of
mtDNA analysis such as interspecific introgression (e.g., Lehman et al. 1991, Spolsky and Uzzell 1984), nuclear integration
(e.g., Zhang and Hewitt 1996), occasional
heteroplasmy (e.g., Bermingham et al.
1986; Rand and Harrison 1989 and references therein), and lack of concordance of
mtDNA gene trees with species trees (e.g.,
Avise 1989; Takahata 1989), the control region promises to remain an important
Figure 1. Population delineations in this study. Dots
indicate the cities of Portland, Eugene, and Medford,
from north (top) to south ( bottom). (A) The ten geomorphic regions of Oregon ( Loy et al. 1976): CR 5
Coast Range, KL 5 Klamath, WM 5 Willamette, WC 5
Western Cascades, HC 5 High Cascades, DU 5 Deschutes-Umatilla, BM 5 Blue Mountains, HL 5 High Lava
Plains, OW 5 Owyhee, BR 5 Basin and Range. (B) The
eighteen major river drainage basins in Oregon (Johnson et al. 1985): NC 5 North Coast, MC 5 Mid Coast,
SC 5 South Coast, WM 5 Willamette, UM 5 Umpqua,
RG 5 Rogue, SN 5 Sandy, HD 5 Hood, DE 5 Deschutes,
KL 5 Klamath, JD 5 John Day, GS 5 Goose and Summer, UT 5 Umatilla, GR 5 Grande Range, PO 5 Powder,
MR 5 Malheur River, ML 5 Malheur Lake, OW 5 Owyhee. The shaded region drains into California. (C) The
four north-south bird migratory flyways ( Evanich
1990): CO 5 Coast, VA 5 Valley, CE 5 Central, EA 5
East.
source of fine-scale evolutionary information for many laboratories.
The control region encompasses less
than 5% of most animal mtDNA genomes
( Brown 1985) and represents the predominant fraction of untranslated mtDNA in
multicellular animals. Thus, in contrast to
the balance of the mtDNA, which is partially subject to strong stabilizing selection imposed by the critical nature of the
genes that are encoded therein, the control region is relatively unconstrained in
terms of nucleotide sequence or length. A
few promoter-like elements, such as the
origin of replication and the D-loop, are
known to be embedded in the control region, but even these show poor phylogenetic conservation even among related
taxa. Surveys of a diversity of species
have shown that the control region accumulates mutations at very high, but variable, rates ranging from 1% to 20% per million years (Stanley et al. 1996) and that
small insertions and deletions (‘‘indels’’)
are common mutational events. Consequently the control region varies greatly in
total size among animals and contributes
greatly to overall mtDNA length variation
in animal taxa (Moritz et al. 1987).
Previous analyses of mtDNA variation in
the Daphnia pulex species complex (Colbourne et al. 1998; Crease et al. 1997; Dufresne and Hebert 1997; Lehman et al. 1995;
Van Raay and Crease 1994; Weider and
Hobaek 1997; Weider et al. 1996) suggest
that many Daphnia ‘‘species’’ may have
polyphyletic or paraphyletic origins of
their mtDNA lineages. Accordingly, few
consistent phylogeographic patterns have
emerged that would prove useful in the
study of gene flow across large physical
distances (Crease et al. 1997). In particular, lake populations of members of the pulex complex, often classified as a distinct
species D. pulicaria, have exhibited poorly
differentiated populations at many geographic scales, as assayed by either allozyme electrophoresis (Cerny and Hebert
1993) or by total mtDNA genome RFLP
analysis (Crease et al. 1997). The purpose
of the current study is twofold. The first is
to examine whether the mtDNA control region has utility in a fine-scale examination
of population-genetic variation for testing
of specific gene-flow mechanism hypotheses among Oregon lake populations. Specifically, the significance of among-population variation in mtDNA genotypes will
be estimated when populations are defined on the basis of river drainage basin,
geomorphic region, bird migratory flyway,
or lake trophic status. The second is to
compare various levels of genetic resolution for their efficacies in such hypotheses
testing. Specifically, it will be examined
whether different conclusions are reached
depending on the manner in which the genetic data are obtained: RFLP data, direct
nucleotide sequence data, or a hybrid of
the two.
Materials and Methods
Sample Collection
Daphnia samples were collected in the
summers of 1996 and 1997. In total, 90
Oregon lakes were successfully sampled
Straughan and Lehman • Daphnia Population Genetics 9
with the goal of an even geographic distribution about the state. Eighty-four of
these lakes were sampled in 1996, eight of
which were resampled in 1997 along with
six that had not been sampled previously.
Sampling was performed so as to cover all
defined geomorphic regions and river
drainage basins ( Figure 1), although lake
accessibility did restrict optimal sampling
uniformity. At the time of sampling, all collected Daphnia were treated as though
they were from the pulex complex; no
samples were excluded from subsequent
genetic analysis despite morphological
variation observed within and among
lakes. Samples were collected using a
small-mouthed plankton net ( BioQuip,
Gardena, CA) either from a kayak or from
shore. In general, sampling was attempted
from the deepest regions of the lake, following the bathymetric maps of Johnson
et al. (1985). In the majority of cases sampling was performed from only one location, but each involved multiple net pulls
across several meters of lake depth so
that individuals were unlikely to be clutchmates. Daphnia were concentrated on
mesh screens and preserved in 70% ethanol at 48C until genetic analysis.
DNA Extraction, Amplification, and
Sequencing
Genomic DNA was liberated from preserved Daphnia using the Chelex method
(Morin et al. 1993; Walsh et al. 1991). Single individuals were placed in 600 ml microcentrifuge tubes containing 300 ml 10%
Chelex 100 resin ( BioRad Laboratories) in
ultrapure water. Lid locks were secured to
the microcentrifuge tubes and the tubes
were autoclaved at 1218C at 10 psi for exactly 20 min. Upon completion of the sterilization cycle, the autoclave chamber was
fast exhausted and the microcentrifuge
tubes were placed on wet ice. These samples were stored indefinitely (up to 1 year)
at 48C before amplification.
Immediately prior to amplification via
the PCR (Mullis et al. 1986), the tubes were
vortexed gently and quickly spun in a microcentrifuge. To amplify high-quality DNA
from the mtDNA control region, a nested
PCR approach was taken. Initial amplification was achieved using the 12S primer (59TAACCGCGACGGCTGGCAC-39), which anneals in the 12S rRNA gene immediately
adjacent to the control region, and the fMet
primer (59-GGGCATGAACCCACTAGCTT-39),
which anneals to the fMet tRNA immediately adjacent to the control region on the opposite side ( Lehman et al. 1995). These reactions utilized 10 ml Chelex supernatant in
10 The Journal of Heredity 2000:91(1)
50 ml reaction volumes. Forty amplification
cycles were performed with an annealing
temperature of 508C to generate products
approximately 1000 nucleotide pairs in
length. Following successful external amplification, the PCR products were diluted
1:1000 in water and 1 ml was used as a template for amplification with the DPUDL-1
(59-CAATCTAGAGCCAAAGCCAGATTCA-39)
and DPUDL-2 (59-CCTCTGCAGGTAGCCCTTTAATCAGGCATC-39) primers. Twenty-five
amplification cycles were performed with
an annealing temperature of 538C to generate products approximately 767 nucleotide pairs in length. All amplifications were
accomplished in 50 ml reaction volumes using 8.75 pmol of each primer and 1.25 units
AmpliTaq DNA polymerase (Perkin-Elmer)
in a PTC-100 thermocycler (MJ Research).
For subsequent RFLP analysis, no post-PCR
purification was necessary. For subsequent
sequence determination, the PCR products
were subject to the GeneClean ( Bio 101)
process for desalting and were rehydrated
in 25 ml of water prior to sequence analysis.
Successful DPUDL-1/2 PCR products
were subjected to digestion with a panel of
seven restriction endonucleases (BamHI,
DraI, HhaI, MseI, PalI, RsaI, and Sau96I;
New England BioLabs) and the resulting
fragments were then separated on either
2% or 3% agarose gels, and bands were visualized by ethidium bromide staining and
UV transillumination. Digests were performed using 6–10 ml of unpurified PCR
product, 2 ml of manufacturer’s supplied
103 buffer, and 2–8 units of enzyme in 20
ml total volume at 378C for at least 2 h. The
seven-enzyme fragment profiles were compiled to generate composite mtDNA genotypes for 2–20 individual Daphnia per
lake. After all individuals were unambiguously genotyped with this panel of enzymes, and nucleotide sequences were obtained for representative individuals (see
below), a subset of all individuals in the
study were genotyped with an eighth restriction enzyme, Tsp509I. This was done
to probe for additional variation within
one of the defined genotypes (see Results).
For each of the composite seven-enzyme RFLP genotypes, a nearly complete
nucleotide sequence of the 767 bp DPUDL1/2 PCR product was obtained by direct
sequencing of this product from at least
one individual. Nucleotide sequencing was
performed manually on the double-stranded PCR products as follows. Four to 7 ml
of GeneCleaned DNA were mixed with 100
pmol primer, boiled 5 min and snapcooled in a dry-ice/ethanol slurry. Se-
quencing was then carried out utilizing
[35S]dATP, the modified T7 DNA polymerase (Sequenase, version 2.0, US Biochemical Corp.), and dideoxynucleotide triphosphates. Reaction products were
separated on 6% polyacrylamide 0.4 mm
gels containing 7 M urea and 0.53 TBE
buffer in 38 cm 3 50 cm Sequi-Gen apparatuses ( BioRad Laboratories) and visualized by autoradiography. Five primers were
used to completely span the control region: DPUDL-1, DPUDL-2, DCRI-1 (59-TCGGAACCACTTTAGCGCAAG-39) and DCRI-2 (59GTTTAAACCCCTTTTCTAATTTTTGGACC-39)
which anneal approximately 100 nucleotides
internal to the DPUDL-1 and DPUDL-2 primers, respectively, and DSP-2 (59-GAAAATTAGTATTATAATC-39), which anneals in the middle of the control region and is necessary to
resolve precisely the homopolymer runs of
cytosine (see Results). These sequences
were compared to the published D. pulex
control-region sequence from Amana Pond,
Iowa (Van Raay and Crease 1994).
Sequences were aligned first by use of
the Jotun-Hein method contained within
the MegAlign module of the Lasergene
DNA software package ( DNASTAR, Inc.)
and then refined by eye. Observed transitions, transversions, and insertions/deletions (indels) among the aligned sequences, along with percent A 1 T content of
the sequences and absolute (uncorrected)
numbers of pairwise nucleotide differences, were computed manually.
Population Genetic Analyses
The nucleotide sequences were used to
construct restriction maps with high certainty and these maps could then be used
to produce a presence-absence matrix of
restriction sites for all individuals genotyped through the RFLP technique. This
matrix was input into the HAPLO2 computer program ( Lynch and Crease 1990) in
order to calculate estimated pairwise nucleotide substitutions per site among genotypes [d̂xy, corrected from observed values using the method of Jukes and Cantor
(1969)], and to estimate the proportion of
total mtDNA genetic variance at the nucleotide level that was due to among-population differentiation (NST). In addition, the
traditional GST statistic, which unlike NST,
treats all genotypes as being equally distinct from one another, was calculated by
hand using the weighting method of Nei
(1987). Populations were defined in these
computer analyses in several different
fashions: all lakes within a geomorphic region, all lakes within a river drainage basin, and all lakes within a north-south fly-
way ( Figure 1). As a control for spurious
results from pooling individuals from
many different lakes in a single population, an additional analysis was performed
in which populations were constructed by
pooling lakes of a given trophic status (oligotrophic, mesotrophic, eutrophic, and
hypereutrophic; status as of 1985, Johnson et al.) regardless of geographic location.
Genetic-structure analysis using the NST
statistic was performed by treating the
raw data in three ways. First, the presence-absence matrix of restriction sites
was used to estimate d̂xy values among genotypes. This approach, indicated by the
label ‘‘RFLP’’ in the Results, uses the maximum-likelihood estimates of Nei and Tajima (1983) to compute the expected pairwise sequence divergences of two
genotypes based on the number and type
(‘‘4-cutter,’’ ‘‘6-cutter,’’ etc.) of restriction
enzymes employed to genotype individuals. As such, this approach does not utilize
all the sequence information present in
each PCR product. However, because all
the individuals in the study were typed
with all seven restriction enzymes, but
only a small fraction of these individuals
had the nucleotide sequences of their PCR
products determined completely, the
RFLP approach will detect any sequence
variation within those individuals at the
relevant restriction sites. In contrast, a
second approach to data treatment was
the utilization of the complete nucleotide
sequence data for each genotype, based
on the sequences obtained from representative individuals from each genotype.
Here, all nucleotide sequence data within
the PCR product is utilized, but an assumption is made that all individuals with
a given RFLP-based genotype have identical nucleotide sequences, even at nucleotides not assayed by the restriction enzymes. This approach is indicated by the
label ‘‘Full Seqs’’ in the Results. A third approach, a hybrid of the first two and indicated by the label ‘‘RFLP/Seq’’ in the Results, was to input the actual nucleotide
sequences at the restriction sites into the
matrix used to calculate d̂xy values. For example, if genotype 1 had an RFLP presence-absence description for three MseI
restriction sites of ‘‘111’’, this would be
converted into an RFLP/Seq genotype of
‘‘TTAATTAATTAA’’, because the recognition sequence of MseI is 59-TTAA-39. Similarly, if genotype 2 had the RFLP genotype
of ‘‘1–1’’, its RFLP/Seq genotype might be
‘‘TTAATTAGTTAA’’; the actual no-cut sequences were determined by the represen-
tative sequences of each RFLP genotype.
This hybrid approach potentially improves the estimates of d̂xy values among
genotypes over the straight RFLP approach without making assumptions
about sequences not covered by restriction sites. For each method of analysis, a
subset of the data was analyzed in which
artificial lakes (reservoirs) were excluded,
with only naturally formed lakes being included.
Results
Of the 90 lakes sampled, successful
DPUDL-1/2 PCR amplifications were obtained from individual Daphnia from 42 of
these lakes ( Table 1). These 42 lakes included representatives from 9 of the 10
geomorphic regions, 14 of the 18 river
drainage basins, and all of the four defined
migratory flyways and four trophic status
levels. A roughly uniform distribution of
success was achieved geographically, with
fewer successes in the southeastern portion of the state, which is drier and supports fewer permanent waterbodies, and
more successes along the coast and in the
Cascade Range ( Figure 2). Among the unsuccessful lakes, failure was approximately evenly split between lakes in which no
Daphnia could be found, and in which
Daphnia that did not consistently generate DPUDL-1/2 PCR products. The latter
situation could be a consequence of either
sample degradation prior to genetic analysis or the predominance of Daphnia not
in the pulex group. The DPUDL PCR primers work much less efficiently when nonpulex-group Daphnia are used as templates ( Lehman et al. 1995). For example,
amplification with these primers is not reliable from D. middendorffiana, D. tenebrosa, and most notably D. galeata, which is
a common lake inhabitant in other portions of North America.
From 2 to 20 individuals from each successful lake were genotyped with the seven restriction enzyme panel (mean 9.2 individuals per lake); a total of 388
individual Daphnia were typed. Among
these individuals, a total of 11 distinct
RFLP genotypes were detected ( Table 2).
These genotypes were all relatively similar
to one another, with no more than nine
restriction site differences separating the
most divergent types (15- and 17; Table 3).
Three of the genotypes (11-, 12-, and 15-)
exhibited a deletion in the DPUDL-1/2 region that was detectable in most RFLP gel
electrophoreses, and later demonstrated
by sequence analysis (see below) to be a
synapomorphic 50 bp deletion in the middle of the control region. Scoring this deletion as the loss of two restriction sites,
a parsimony network of site differences
among all genotypes was constructed by
hand ( Figure 3). The fact that no single
most-parsimonious network could be constructed (genotypes 12- and 17 can articulate in more than one equiparsimonious
way), combined with the small numbers of
restriction-site differences separating
most genotypes, indicates that these genotypes are all closely related, perhaps
being relatively recent derivatives of one
another. The strong interconnectivity of
the network suggests further that some of
the site gains and losses may be homoplaseous.
Three of these genotypes (7, 8, and 9)
were relatively widespread ( Figure 2), being detected in all populations, regardless
of how the populations were defined. The
remaining eight genotypes were much rarer, being present in at most 11 of the 388
genotyped individuals and being restricted to single populations ( Table 1). A rankorder frequency histogram of all 11 genotypes shows the discontinuity in
frequencies between the common and
rare genotypes ( Figure 4). Notably, the
three common genotypes represent the
‘‘core’’ of the connectivity network, with
the most common genotype by far (genotype 7) being at the center, at least as depicted in Figure 3. A haplotype diversity of
0.641 was calculated from all genotype frequencies; this figure represents the
mtDNA equivalent of heterozygosity
among all individuals ( Nei 1987). With the
exception of one lake (MER), no apparent
variation in genotype frequencies was observed when the same lake was sampled
in successive years ( Table 1). This was
confirmed by R 3 C G-tests (with the Williams correction) of genotype frequency
independences year to year for each individual lake. Only Mercer Lake revealed a
significant (P , .05) frequency shift between the 1996 and 1997 samplings. This
shift could represent a selection-driven
temporal genotypic turnover as detected
in some pond Daphnia ( Lynch 1987), or
an extremely rapid colonization of this
lake by an invading genotype, but the
small sample sizes (n # 8 in each year)
allow for sampling error effects to occur
in individual lakes and preclude any definitive conclusions to be drawn.
Nucleotide sequences of approximately
679 bp of the DPUDL-1/2 PCR product from
the eleven genotypes (GenBank accession
numbers AF165861-AF165872) were aligned
Straughan and Lehman • Daphnia Population Genetics 11
Table 1. Summary of lake and RFLP data in this study
Lake
GeoDrainage morphic Fly
basin
region way
Trophic
status
Source
Sunset (SUL)
Devil’s ( DVL)
NC
MC
CR
CR
CO
CO
Eu
Eu
Natural
Natural
Elbow ( ELB)
Mercer (MER)
MC
MC
CR
CR
CO
CO
Unknown
Meso
Natural
Natural
Munsel (MUL)
Sutton (STL)
Triangle ( TRL)
North Tenmile ( NTL)
Tenmile ( TML)
Eel ( EEL)
Saunders (SAL)
Marie (MAL)
Hills Creek ( HCR)
Scott (SCL)
Gold (GOL)
Lemolo ( LEM)
Applegate (AGR)
Lost Creek ( LCL)
Emigrant ( EMR)
Upper Squaw ( USL)
Laurance ( LAL)
Wickiup (WKR)
Pine Hollow (PHR)
Lava ( LAV)
MC
MC
MC
SC
SC
SC
SC
SC
WM
WM
WM
UM
RG
RG
RG
RG
HD
DE
DE
DE
CR
CR
CR
CR
CR
CR
CR
CR
WC
HC
HC
WC
KL
KL
KL
KL
HC
HC
DU
HC
CO
CO
CO
CO
CO
CO
CO
CO
VA
CE
CE
VA
VA
VA
VA
VA
CE
CE
CE
CE
Meso
Eu
Meso
Eu
Eu
Meso
Meso
Unknown
Meso
Oligo
Meso
Meso
Meso
Meso
Eu
Meso
Meso
Meso
Meso
Oligo
Natural
Natural
Natural
Natural
Natural
Natural
Natural
Natural
Artificial
Natural
Natural
Artificial
Artificial
Natural
Artificial
Natural
Natural
Artificial
Artificial
Natural
Ochoco (OCR)
Prineville (PRR)
Paulina (PAL)
DE
DE
DE
HL
BM
HL
CE
CE
CE
Eu
Eu
Meso
Artificial
Artificial
Natural
East ( EAL)
Hosmer ( HOL)
DE
DE
HL
HC
CE
CE
Meso
Meso
Natural
Natural
Little Cultus ( LTL)
Hyatt ( HYR)
Upper Klamath ( KLL)
DE
KL
KL
HC
KL
BR
CE
VA
CE
Oligo
Eu
Hyper
Natural
Artificial
Natural
Gerber (GRR)
Howard Prairie ( HPR)
Willow Creek (WIR)
McKay (MCR)
Mangone (MGL)
Morgan (MRL)
Phillips (PIR)
Wolf Creek (WCR)
Unity ( UNR)
Antelope (ANR)
Totals
KL
KL
UT
UT
JD
GR
PO
PO
PO
OW
BR
KL
DU
DU
BM
BM
BM
BM
BM
OW
EA
VA
EA
EA
EA
EA
EA
EA
EA
EA
Eu
Meso
Meso
Eu
Meso
Eu
Meso
Eu
Eu
Eu
Artificial
Artificial
Artificial
Artificial
Natural
Natural
Artificial
Artificial
Artificial
Artificial
Year
sampled N
1996
1996
1997
1996
1996
1997
1996
1996
1997
1996
1996
1997
1997
1997
1996
1997
1997
1996
1996
1996
1996
1996
1996
1996
1996
1996
1997
1996
1996
1996
1997
1996
1996
1997
1997
1996
1996
1997
1996
1996
1996
1996
1996
1996
1996
1996
1996
1996
7
7
12
5
8
7
8
8
8
8
8
12
12
9
6
9
10
4
8
8
8
6
8
8
8
8
12
9
8
8
10
6
8
8
8
10
8
9
8
6
8
2
10
8
9
9
7
7
388
7
8
7
3
4
5
8
1
8
7
9
2
3
12–
13
15–
16
17
1
8
8
7
1
9
1
4
5
7
1
5
3
2
3
2
7
6
2
1
8
4
6
12
1
5
8
8
8
6
8
3
1
8
12
8
3
1
5
11–
6
8
12
9
5
9
10
3
1
2
6
3
5
6
6
5
2
1
3
5
5
1
1
1
1
10
8
9
9
7
7
207
55
88
1
8
8
6
11
2
Abbreviations for drainage basins, geomorphic regions, and flyways correspond to those in Figure 1. Abbreviations for trophic status are as follows: Oligo 5 oligotrophic,
Meso 5 mesotrophic, Eu 5 eutrophic, Hyper 5 hypereutrophic. Because the statuses for ELB and MAL could not be unambiguously determined, these two lakes were left
out of the analyses involving trophic status. The numbers in the 11 right columns indicate the numbers of each genotype found in each lake by a seven-enzyme RFLP screen
of the mtDNA control region.
in comparison to the published Iowa D. pulex sequence of the same nucleotide span
( Van Raay and Crease 1994). This ,680 bp
region begins 12 bp downstream of the 39
end of the DPUDL-1 primer-binding site, at
the first nucleotide of the control region immediately adjacent to the 39 end of the 12S
rRNA gene, and this region ends approximately 24 bp upstream of the 39 end of the
DPUDL-2 primer-binding site, at the last nucleotide of the control region ( Van Raay
and Crease 1994). The general features of
this region in any of these sequences do
not differ radically from the D. pulex sequence. The A 1 T content of the D. pulex
sequence is 66.6%, while the A 1 T con-
12 The Journal of Heredity 2000:91(1)
tents of these sequences ranges from 63.0%
(genotype 17) to 66.9% (genotype 8). Distinct runs of homopyrimidines are evident
near the 12S rRNA bordering side of the sequences. There is a pair of direct repeats
of 29 nucleotide pairs in length in every sequence and a putative stem-loop structure
located between nucleotide positions 542
and 609 from the 59 end of the control region. Pairwise sequence divergences for
the DPUDL-1/2 fragment as a whole range
from 0.29% to 3.5% (counting each indel,
regardless of length, as a single nucleotide
change), and average pairwise nucleotide
substitutions per site (d̂xy) values range
from 0.0032 to 0.0451 ( Table 3). These val-
ues are average or even below average for
typical within-species comparisons of the
mtDNA control region in animals (Stanley
et al. 1996), and support the assessment
that all samples in this study are of the
same species. The most divergent sequence is genotype 17, which was detected
in only two individuals from Gerber Reservoir. Notably this genotype is also a rare
inhabitant of some coastal ponds [genotype S82 of Lehman et al. (1995)] and may
represent a ‘‘pond’’ genotype found in a
lake (c.f. Cerny and Hebert 1993; Crease et
al. 1997; Pfrender ME, Spitze K, and Lehman
N, manuscript in review). Failure of a phylogenetic analysis by PAUP to recover a sin-
Figure 2. Map of the 42 lakes included in the genetic survey and geographic distributions of the major mtDNA
RFLP-based genotypes. Genotypes 10–17 are lumped as ‘‘other.’’
ferences. When all genotypes are included,
all of these NST values are significantly different from zero using the D statistic (D 5
[NST/SE]2, where SE is the standard error
in NST), which is expected to have a chisquare distribution with one degree of
freedom ( Lynch and Crease 1990). By
comparison, the GST value for drainage basins is 0.567, indicating that nucleotide differences among the genotypes have a minimal effect on the degree of genetic
partitioning as would be predicted from
the high sequence similarity of all genotypes.
To test whether this differentiation was
a consequence of the rare genotypes that
are typically found in only one drainage
basin, the analysis was rerun with all genotypes removed except the common
ones (7, 8, and 9). Of interest, this operation actually increased the NST value to
gle most parsimonious tree of the 11 genotypes or to give a significant phylogenetic signal by the g1 statistic ( Hillis and
Huelsenbeck 1992) further supports the
homoplaseous nature of many of the observed nucleotide differences (data not
shown).
Analyses of population-genetic partitioning using the NST statistic are summarized in Table 4. The most obvious conclusion from this summary is that when
populations are defined by the river drainage basin, genetic subdivision is the greatest and most significant. When full datasets are used, values of NST range from
0.484 to 0.592 when populations are defined this way, meaning that between 48%
and 59% of all genetic differentiation at the
nucleotide level is attributable to amongpopulation (among drainage basin) differences as opposed to within-population dif-
Table 2. Mitochondrial DNA control region RFLP genotypes detected among Oregon lake Daphnia
Genotype
Type
clone
BamHI
GGATCC
DraI
TTTAAA
HhaI
GCGC
MseI
TTAA
PalI
GGCC
RsaI
GTAC
Sau96I
GGNCC
7
8
9
10
11–
12
12–
13
15–
16
17
USL A-1
PHR A-2
TML A-1
DVL A-9
HPR C-4
PAL A-2
HYR A-2
PAL A-4
MCR A-6
MCR A-3
SCP-82
A
A
A
A
F–
F
F–
F
F–
A
A
B
B
B
B
B–
B
B–
B
B–
B
D
A
A
A
A
A–
A
A–
A
A–
B
A
A
A
L
A
A–
A
A–
A
L–
A
E
F
F
F
F
F–
F
F–
A
F–
F
A
F
B
F
F
F–
F
F–
F
F–
F
F
C
C
C
B
B–
C
C–
C
B–
C
C
Restriction fragment patterns are assigned an arbitrary letter, with the exception that patterns exhibiting no cuts
within the 767 bp DPUDL-1/2 PCR product are designated ‘‘F.’’ The recognition sequences for the enzymes used
are indicated below their names. The negative signs indicate the presence of a 50 bp deletion in the middle of the
control region for three of the genotypes. Actual RFLP patterns are available upon request from the authors.
0.715 or 0.665, depending on whether
RFLPs or full sequences were used as the
raw data. However, if genotype 9 is removed from the dataset instead, the NST
value drops to 0.327 and becomes not significantly different from zero (P . .05).
These manipulations suggest that genotype 9 is the single greatest contributor to
population differentiation among drainage
basins. Examination of the geographic distribution of genotypes ( Table 1 and Figure
2) reveals that genotype 9 is common
along the Pacific Coast and in the northeastern portion of the state, but rare inbetween. To help ensure that a subtle difference between ‘‘western’’ and ‘‘eastern’’
forms of genotype 9 did not go undetected
by our seven-enzyme RFLP analysis (a difference that would actually increase the
magnitude and significance of the NST value), a subset of lakes containing high frequencies of genotype 9 (MUL, MER, DVL,
and EEL from the west, and MRL, WCR,
WIR, PIR, PRR, and UNR from the east) was
further assayed with the enzyme Tsp509I.
This enzyme has the recognition sequence
59-AATT-39 and would be expected to cut
genotype 9 at 10 sites, surveying approximately an additional 34 nucleotides for
variation. No variation among these lakes
was detected with this enzyme, however,
lending confidence that the NST values obtained with the original RFLP screen were
robust.
Removing all artificial lakes from the
analysis was performed to ensure that recent anthropomorphic influences were not
artificially influencing the results. All artificial lakes in this study were constructed
by human activity since 1900 and must
contain immigrant populations of Daphnia
from other waterbodies. Visual examination of Table 1 shows no obvious differences in genotype frequencies between
natural and artificial lakes. For example,
the combined frequency of the three common genotypes (7, 8, and 9) is only marginally significantly different between the
two types of lakes by a G test of independence (P 5 .04), but when the putative
‘‘pond’’ genotype 17 is removed from the
analysis, the difference becomes nonsignificant (P . .05). For the eight rare genotypes, each genotype was found only in
natural or only in artificial lakes, but three
genotypes were restricted to natural and
five to artificial lakes, so one might conclude that recently formed waterbodies
harbor a disproportionate amount of this
class of genetic variation (see Discussion).
Removal of artificial lakes from the NST calculations has little effect on genetic parti-
Straughan and Lehman • Daphnia Population Genetics 13
Table 3. Observed pairwise divergences among mtDNA genotypes
7
8
9
10
11–
12
12–
13
15–
16
17
7
8
9
10
11–
12
12–
13
15–
16
17
—
0.0192
0.0241
0.0241
0.0241
0.0258
0.0373
0.0258
0.0340
0.0290
0.0350
1
—
0.0241
0.0241
0.0225
0.0291
0.0356
0.0291
0.0356
0.0290
0.0383
1
2
—
0.0032
0.0225
0.0274
0.0323
0.0307
0.0340
0.0274
0.0417
1
2
2
—
0.0225
0.0274
0.0323
0.0307
0.0340
0.0274
0.0417
4
5
5
3
—
0.0307
0.0160
0.0307
0.0144
0.0274
0.0400
1
2
2
2
3
—
0.0242
0.0032
0.0209
0.0160
0.0367
3
4
4
4
1
2
—
0.0274
0.0080
0.0208
0.0451
2
3
3
3
4
1
3
—
0.0242
0.0193
0.0334
5
6
4
4
1
4
2
5
—
0.0208
0.0451
1
2
2
2
5
2
4
3
6
—
0.0383
6
7
7
7
8
7
7
6
9
7
—
Above the diagonal, number of observed pairwise restriction site differences between genotypes. Below the diagonal, average pairwise nucleotide substitutions per site between full-sequence genotypes (d̂xy values).
tioning by drainage basin. This manipulation lowers the NST value from 0.523 to
0.508 for the RFLP data, and from 0.592 to
0.533 in the Full Seqs data. By contrast, if
natural lakes are removed from the analysis and only the 17 artificial lakes are analyzed, the NST value is lowered to 0.391,
which is now only of marginal significance
(P 5 .05) as a consequence of the reduced
population size.
Among-population differentiation is
much weaker and less significant if populations are defined on the basis of geomorphic region, bird migratory flyways, or
lake trophic status ( Table 4). Defining
populations by geomorphic regions produces the highest NST values of the three,
with the RFLP-based value at 0.265, but
this is only marginally significantly different from zero (P 5 .046). The Full Seqs
value for geomorphic regions is slightly
greater at 0.283, and because of the lowered standard error values engendered by
using complete nucleotide sequences, this
value is significantly different from zero
based on the D statistic. However, it
should be recalled that using full sequences assumes that each RFLP-defined genotype is identical at all sites not surveyed
by restriction enzymes, and thus some
within-population variation could go undetected that could reduce the NST estimation. RFLP-based NST calculations for
flyway and trophic status are 0.201 and
0.141, respectively, and neither is significantly nonzero.
Figure 3. Parsimony network of mtDNA control region RFLP genotypes discovered in Oregon lake Daphnia. Each
cross-hatch indicates a single gain or loss of a restriction site. The synapomorphic 50 bp deletion detected in
genotypes 11-, 12-, and 13- is scored as the loss of two restriction sites. Multiple equiparsimonious paths to
genotypes 12- and 17 demonstrate the strong interconnectivity of this network and the homoplaseous nature of
some of these restriction site mutations. The Iowa D. pulex genotype (1, dashed circle) is included for reference,
but the restriction enzymes utilized in this study were chosen in part based on their ability to differentiate among
Oregon lake genotypes and hence underestimate the genetic distance between Iowa pond and Oregon lake samples.
14 The Journal of Heredity 2000:91(1)
Figure 4. Rank order frequency histogram of the 11
mtDNA control region genotypes.
Because the NST values tend to increase
as the geographic area of the populations
decreases, it is important to demonstrate
that the significance of the drainage basin
results is not an artifact of an isolation-bydistance phenomenon. Populations that
are not subdivided at larger geographic
scales can show (inconsequential) subdivision if the scale is made small enough to
ensure isolation of rarer genotypes (e.g.,
Lehman and Wayne 1991). For example, in
the present study, if each of the 42 lakes
is considered a separate population, NST
rises to 0.73. To test further the significance of drainage basin subdivision,
Oregon, being roughly rectangular, was artificially partitioned into rectangular sections by overlaying 3 3 6 and 6 3 3 grids.
The resulting 18 sections average in size
exactly as do the 18 river drainage basins,
but encompass different sets of lakes. Auspiciously, both the 3 3 6 grid, making tall,
thin sections, and the 6 3 3 grid, making
short, fat sections, produce 14 lake-containing populations to mirror the 14 drainage basins that contain lakes in this study.
Both grid-generated populations produce
markedly lower NST values (3 3 6: 0.383; 6
3 3: 0.299) with RFLP data than do the
drainage basin populations. Thus, despite
the fact that all three constructions are of
the same number and average size, and
even contain many of the same lake group-
Table 4. Population subdivision among mtDNA genotypes in Oregon lake Daphnia
Population
division
Data
format
Lakes
included
Genotypes
included
NST
SE in
NST
D
Significance
RFLPs
RFLPs
RFLPs
RFLPs
RFLPs
RFLP/seqs
Full seqs
Full seqs
Full seqs
Full seqs
Left CR seq
Mid CR seq
Rt CR seq
All
All
All
Natural
Artificial
All
All
All
All
Natural
All
All
All
All (11)
7, 8, 9 only
All except 9
All (6)
All (8)
All (11)
All (11)
7, 8, 9 only
All except 9
All (6)
All (11)
All (11)
All (11)
0.523
0.715
0.327
0.508
0.391
0.484
0.592
0.665
0.402
0.533
0.614
0.530
0.621
0.139
0.184
0.193
0.104
0.203
0.148
0.029
0.056
0.041
0.062
0.033
0.061
0.036
14.2
15.1
2.88
24.1
3.71
10.6
417
143
94.5
72.8
345
74.9
297
P,
P,
N.S.
P,
P5
P,
P,
P,
P,
P,
P,
P,
P,
.01
.01
RFLPs
RFLPs
RFLPs
RFLP/seqs
Full seqs
Full seqs
All
Natural
Artificial
All
All
Natural
All
All
All
All
All
All
(11)
(6)
(8)
(11)
(11)
(6)
0.265
0.426
0.302
0.230
0.283
0.395
0.132
0.070
0.169
0.040
0.055
0.060
4.05
37.5
3.20
32.4
26.4
43.0
P5
P,
N.S.
P,
P,
P,
.046
.01
RFLPs
RFLPs
RFLPs
RFLP/seqs
Full seqs
Full seqs
All
Natural
Artificial
All
All
Natural
All
All
All
All
All
All
(11)
(6)
(8)
(11)
(11)
(6)
0.201
0.223
0.274
0.173
0.152
0.219
0.120
0.161
0.143
0.049
0.041
0.049
2.82
1.92
3.68
12.3
14.1
20.3
N.S.
N.S.
P5
P,
P,
P,
RFLPs
RFLPs
RFLP/seqs
Full seqs
Full seqs
All
Natural
All
All
Natural
All
All
All
All
All
(11)
(6)
(11)
(11)
(6)
0.141
0.214
0.125
0.132
0.156
0.078
0.111
0.035
0.025
0.026
3.27
3.72
12.8
28.4
35.6
N.S.
N.S.
P , .01
P , .01
P , .01
Drainage Basins
Geomorphic regions
.01
.05
.01
.01
.01
.01
.01
.01
.01
.01
.01
.01
.01
Flyways
.05
.01
.01
.01
Trophic status
ings as a consequence of geographic proximity, the information contained in the
boundaries of river drainage basins has a
strong positive effect on genetic subdivision.
In all cases, including the drainage basin
case, the RFLP/Seqs datasets generate NST
values that are lower than those from the
RFLP datasets ( Table 4). The SE values for
the RFLP/Seqs analyses are also lower, as
expected. By contrast, use of the Full Seqs
dataset affects the NST value in no consistent fashion, raising it in the drainage basin and geomorphic region cases, lowering
it in the flyway case, and leaving it essentially unaffected in the trophic status case.
In the drainage basin case, focusing on the
left and right portions of the control region will increase the NST values over that
from the entire 679 bp span studied, while
focusing on the middle portion of the control region lowers the NST value in accordance with observations that the D-loop
region of the control region is more constrained than its flanking regions (c.f. Moritz et al. 1987).
Discussion
Genetic partitioning among mtDNA genotypes of lake populations of members of
the D. pulex species complex appears to
be the greatest among river drainage basins in Oregon. All other manners in which
the total population was subdivided
showed systematically lower among-population differentiation with analogues of
Wright’s F statistics. When geomorphic regions were used to construct populations,
the NST values were all significant, but typically one-half of the corresponding drainage basin values. The NST and GST values
for mtDNA genotypes among river drainage basins range from roughly 0.5 to 0.6,
indicating that about half of the total
amount of variation, as detected by a seven-enzyme RFLP screen of roughly 750 bp
of the control region, is a consequence of
variation among the river drainage basins
of the state. Gerrymandering the state into
regions of physical-geographical similarity
shows marginal levels of among-population differentiation, on the order of 20–
30%. However, comparison of Figure 1a
and b reveals a significant amount of covariation between lakes found in drainage
basins and geomorphic regions. Thus the
among-population variation detected in
the latter could be in fact an orthogonal
effect of the former.
It is not unreasonable to assume that
migration among ‘‘permanent’’ waterbodies such as lakes could be mediated by
transport of ephippia through lotic environments. With a low specific gravity and
a hydrophobic surface, ephippia usually
float on the surface of the water ( Dodson
and Frey 1991). Thus they would be subject to dispersal through the inlet and outlet streams of lakes, which the lakes of
Oregon typically possess. In fact Dobson
and Frey (1991) note that Cladocerans, the
group to which Daphnia belong, are occasionally found as adults in quiet moving
water and in marginal vegetation bordering streams. Notably, 2 of the 11 genotypes
detected in this study (12 and 13) were
confined to the twin caldera lakes of an
extinct volcano (Paulina Lake and East
Lake), to which there are no inlet streams,
a situation that would favor the in situ accumulation of mutations. While the present study does not rule out gene flow aided by waterfowl, fish, wind, or groundwater,
it identifies the interconnectivity of lakes
by surface water as the most evident
source of among-population migration.
The similarity of mtDNA genotypes and
low overall sequence divergences detected among lake mtDNA genotypes could be
the result of geologically recent events
that affected the region. Oregon lies below
the southern extent of Pleistocene glaciation (Pielou 1991) and thus its lakes were
not covered by the Cordilleran ice sheet.
However, the region, especially what is
now southern Washington and northern
Oregon, was subjected to repeated mass
floodings from the release of water from
the glacial lakes Columbia and Missoula.
These large lakes were present during the
Pleistocene and contained more than 2000
km3 of water to the north and east of modern Oregon, and would periodically flood
toward the Pacific Ocean with the sudden
failures of the ice dams that maintained
them (Pielou 1991). The last such event
occurred approximately 10,000 years before present and could readily explain the
genetic homogenization that is depicted in
the distributions of the common, and similar, mtDNA genotypes 7, 8, and 9. Furthermore, Lake Missoula spanned what is now
the continental divide and had a variable
drainage basin that probably periodically
included the modern Mississippi River Valley through predecessors of the Missouri
River. Thus if Daphnia migration was aided by river drainages, it is not surprising
that poor differentiation between Oregon
lake and Midwest lake genotypes was discovered using an RFLP analysis of whole
Straughan and Lehman • Daphnia Population Genetics 15
mtDNA genomes (NST 5 0.36 6 0.24;
Crease et al. 1997).
Periodic large-scale flooding and river
transport of cladoceran ephippia thus can
help to explain not only the populationgenetic patterns (or lack thereof ) reported in this study, but the commonality of
paraphyly in the D. pulex species complex
at both the population and species levels.
A companion study to this report where
Oregon pond populations were analyzed
for genetic differentiation suggested that
the D. pulex (i.e., pond) and D. pulicaria
(i.e., lake) populations in this region are
very closely related and that the lake populations may actually provide a genetic
source for the more ephemeral pond populations through flooding events (Pfrender ME, Spitze K, and Lehman N, manuscript in review). Mass floodings could
actually impede speciation in preexisting
lakes, while augmenting speciation overall, by creating new waterbodies that are
unoccupied habitats.
An alternate scenario to river transport
and flooding as sources for gene flow
would be that recent human activity has
disrupted the prehistoric patterns of genetic differentiation. Although this cannot
be ruled out by the existing data, it is numerically unlikely. The lakes in this study
each contain in excess of 1 million m3 of
water (average, 100 million m3), the smallest being Scott Lake at 1.2 million m3. Assuming an average density of 1 animal/cm3
for Daphnia in lakes where they exist in
moderate to high abundance (unpublished observations), as is the case in all
42 lakes reported here, an minimum estimate of 1.2 3 1012 animals/lake can be
made. Even if stratigraphic variation within lakes were to lower the average density
two to three orders of magnitude below 1/
cm3, these lakes would still support more
than 1 billion individuals. In the absence
of selection, such large population sizes
should be rather insensitive to drift-driven
genotypic turnover by innoculums on the
order of tens or hundreds as would be expected by lake-to-lake watercraft (or waterfowl) transport of live Daphnia. Selection has been demonstrated to occur
among nuclear genotypes in pond Daphnia as monitored by allozyme markers
( Lynch 1987), but no reports exist of selective differences among mtDNA genotypes in Daphnia.
In contrast, flooding and river transport
have the potential to affect Daphnia distributions over longer periods of time,
larger land areas, and continually over
time. Lakes are often thought of as ‘‘per-
16 The Journal of Heredity 2000:91(1)
manent’’ waterbodies, especially in comparison to temporary ponds, but even the
natural lakes in this study are of geologically recent origin, being formed by landslides, volcanic activity, tectonic activity,
river quiescence, and glacial scouring in
the high Cascades (Johnson et al. 1985).
Many have inlet and outlet streams that
connect them to other lakes. For example,
the nearby Hyatt and Howard Prairie Reservoirs are part of the same stream system that eventually drains into the Klamath River, and these two lakes were the
only ones to harbor the rare mtDNA RFLP
genotype 5. It would be of interest to compare more lakes found along the same specific waterway, as gene flow by river transport would be expected to have a strong
downstream component. In this study,
however, an attempt was made to sample
the state uniformly, and thus nearby lakes
along the same waterway were avoided
during sampling. Similarly, lakes in the
northern Rocky Mountains, such as Montana, Alberta, and British Columbia, which
may have been source populations for
Oregon lakes if the latter were indeed populated by Pleistocene floods, should possess a higher clonal diversity and greater
differentiation among mtDNA genotypes
than those detected in this study.
Also somewhat suggestive of river
transport is the diversity among artificial
lakes in this study. All of these lakes were
formed by the creation of dams along major Oregon rivers during the past century
and thus have been recently populated.
When only artificial lakes are analyzed, all
RFLP-based NST values become marginally
significant or nonsignificant ( Table 4).
This indicates that, to a certain extent, no
single factor tested is responsible for gene
flow into these lakes. Moreover, five of the
eight rarer genotypes are found among the
17 artificial lakes, while only three of the
rare genotypes are found among the 25
natural lakes. Yet a closer inspection of
the length of time these lakes have been
in existence reveals a striking pattern. Of
the four oldest artificial lakes in this study
(OCR, HYR, GRR, and MCR), all constructed during the 1920s, only OCR fails to harbor a unique rare genotype not found elsewhere. The other artificial lakes were
formed by dam projects that were completed after 1939. Newer reservoirs thus
are populated by common Oregon genotypes, and because they are all suddenly
created in the midst of a river flow, lotic
transport would be the most apparent
source of immigrants. The longer a reservoir exists, the more likely it becomes that
novel genotypes are brought in by human,
wind, and/or animal transport. The natural lakes on the other hand could represent an even longer-term stasis state that
is dominated by mass floodings which
tend to homogenize populations and reestablish the predominance of the common genotypes in the region, in this case
genotypes 7, 8, and 9. Again, testing of this
scenario awaits study of additional lakes.
Because of taxon-specific rate heterogeneities and the high frequencies of insertions and deletions, it is hard to calibrate a molecular clock for the mtDNA
control region ( Lynch and Jarrell 1993).
Thus it is difficult to estimate how long
ago the genotypes in this study diverged
from one another. In a comparison study,
Weider et al. (1996) determined nucleotide
sequences from a 254 bp region of the
mtDNA control region from Greenland and
Iceland pond isolates of D. pulex. Mean
pairwise sequence divergence values in
that study ranged from 2% to 10% from genotypes that likely diverged during the
Pleistocene, providing further indirect evidence that Oregon lake Daphnia genotypes diverged through mutational processes in the last 10,000–50,000 years. For
context, an analysis of mtDNA proteincoding gene ( ND5) evolution in Daphnia
places the split of the D. pulex group from
its closest relatives (D. melanica and D.
middendorffiana) at 1.4–1.6 million years
ago (Colbourne et al. 1998), and RFLP variation within this gene among 14 North
American lake populations of pulex group
members suggests the existence of two
distinct genotype clades that likely diverged during the mid-Pleistocene ( Dufresne and Hebert 1997).
From Table 4 it can be seen that the way
in which the data are formatted has little
effect on the general results. The NST values estimated from RFLPs, RFLP/Seqs, or
Full Seqs are similar in all cases, never differing by more than about 0.1. The main
distinction among these formats is the estimations of the standard errors, which
appear to drop when Full Seqs are used.
However, this increase in statistical accuracy must be weighed against uncertainties generated when full sequences are
predicted from RFLP-based population
surveys. Clearly the optimal approach
would be to obtain complete nucleotide
sequences from each individual in the survey—an increasingly plausible venture
with advances in rapid sequencing technologies. The similarities in NST values
across data formats generated in this
study may be a consequence of the low
overall divergences among genotypes and
it remains to be seen whether this situation will be observed in more diverse datasets.
The results of this study indicate that
even with low levels of nucleotide sequence divergence among a small number of genotypes, a fine-scale mtDNA
analysis can be used successfully to test
hypotheses about patterns of population
differentiation. Among-population variation reaches its highest proportion when
populations are defined in terms of river
drainage basins and its lowest when they
are defined in terms of lake trophic status, which has very little geographical
correlation. Taken together, these observations suggest that there is a pattern to
recent gene flow among Daphnia-inhabited waterbodies, but that without a detailed analysis of large numbers of samples, the pattern may go undetected.
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Received February 22, 1999
Accepted September 14, 1999
Corresponding Editor: Robert Wayne
Straughan and Lehman • Daphnia Population Genetics 17