Download Inferring natural selection in a fossil threespine stickleback

Paleobiology, 32(4), 2006, pp. 562–577
Inferring natural selection in a fossil threespine stickleback
Michael A. Bell, Matthew P. Travis, and D. Max Blouw
Abstract.—Inferring the causes for change in the fossil record has been a persistent problem in evolutionary biology. Three independent lines of evidence indicate that a lineage of the fossil stickleback fish Gasterosteus doryssus experienced directional natural selection for reduction of armor.
Nonetheless, application to this lineage of three methods to infer natural selection in the fossil
record could not exclude random process as the cause for armor change. Excluding stabilizing selection and genetic drift as the mechanisms for biostratigraphic patterns in the fossil record when
directional natural selection was the actual cause is very difficult. Biostratigraphic sequences with
extremely fine temporal resolution among samples and other favorable properties must be used to
infer directional selection in the fossil record.
Michael A. Bell and Matthew P. Travis. Department of Ecology and Evolution, Stony Brook University,
Stony Brook, New York 11794-5245. E-mail: [email protected]
D. Max Blouw. Biology Program and Office of the Vice President for Research, University of Northern
British Columbia, Prince George, British Columbia V2N4Z9, Canada
Accepted:
17 April 2006
Introduction
Attributing process to patterns in the fossil
record has been an enticing but elusive objective in paleontology since Darwin. This problem reflects the unavoidable fact that stratigraphic sequences with high completeness,
fine resolution, and large temporal scope are
rare (Schindel 1980; Sadler 1981). Defining a
pattern in the fossil record and applying appropriate methods to test for its existence pose
additional problems. When Darwin (1859)
proposed natural selection as the cause for biological evolution, he realized that the fossil
record would provide one of the most important sources of support for his theory. He
clearly believed that evolution would appear
as a slow, gradual trend in the fossil record.
Thus, in his original discussion of natural selection, Darwin (1859: p. 152) stated, ‘‘That
natural selection will always act with extreme
slowness, I fully admit.’’ Similarly, in reference to the evolution of complex structures,
Darwin (1859: pp. 217–218) supposed that it
might be necessary to ‘‘descend far beneath
the lowest known fossiliferous stratum to discover the earlier stages, by which the eye has
been perfected.’’ As a result, he believed that
evolutionary mechanisms apparent from extant
populations should cause gradual transitions
between ancestral and descendant species in
䉷 2006 The Paleontological Society. All rights reserved.
biostratigraphic sequences. In Chapter 9, ‘‘On
the Imperfection of the Geological Record,’’
Darwin (1859: pp. 297–309) went to great
lengths to explain why such transitions had
not been discovered, attributing their absence
to the incompleteness of the fossil record.
In the years since Darwin, several examples
of gradual transitions between fossil species
have been reported (reviewed in Erwin and
Anstey 1995). Although contributors to the
evolutionary synthesis varied in their views
on the rate and gradualness of evolution, the
consensus clearly favored Darwin’s expectation for gradual evolutionary change in the
fossil record, rather than more radical proposals, such as those of Goldschmidt or Waddington (Gould 2002). Simpson (1944) was responsible for incorporating paleontology into the
evolutionary synthesis. Even though some of
Simpson’s most important work concerned
variation of evolutionary rates in the fossil record, he never suggested that evolution should
not generally be slow and gradual. Darwin’s
expectation remained the conventional wisdom for the next three decades.
However, in their classic paper on punctuated
equilibrium, Eldredge and Gould (1972) contested this expectation. They proposed instead
that dominance of stasis in the fossil record accurately portrays the history of life and reflects
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NATURAL SELECTION IN THE FOSSIL RECORD
Mayr’s (1963) view that species represent wellintegrated genetic systems that become disrupted only during speciation. They concluded
that macroevolution occurs during brief intervals of genetic instability (punctuations) and
that long-term trends require species selection.
Their claims generated heated controversy for
two reasons: (1) the process they proposed had
limited empirical support, relegating population genetic mechanisms that are readily observed in extant populations to little more than
noise, and (2) there were no objective criteria to
distinguish stasis (equilibrium) from gradual
change. Thus, not only was there disagreement
about the processes responsible for macroevolution, but there could be disputes about whether a specific biostratigraphic sequence represented stasis, gradualism, or punctuated equilibrium. There is still no broad consensus on the
dominance of any particular set of processes for
patterns in the fossil record (Erwin and Anstey
1995), but this controversy inspired attempts to
develop quantitative methods to distinguish
stasis from gradual change.
Methods to distinguish stasis from either
directional or random change depend either
on consistency in the direction of change for a
trait among several samples in a time series or
on the rate of change between consecutive
samples. Four criteria can be used to compare
change among samples within biostratigraphic sequences with expectations from a random
process: (1) rates of change between consecutive samples (Lynch 1988, 1990), (2) the rate of
change between consecutive samples compared with those on longer time scales (Gingerich 1993, 2001), (3) magnitude (Hurst 1951;
Bookstein 1987, 1988; Roopnarine et al. 1999;
Roopnarine 2001) of change from the beginning to the end of a sequence compared with
that expected from a random walk with appropriate statistical properties, and (4) the
number of sets of consecutive increases or decreases of trait values without reversal (i.e.,
runs) (Raup and Crick 1981). Some authors
(Roopnarine et al. 1999; Sheets and Mitchell
2001) have advocated comparing the result
from method (2) above with the expectation
from a random walk generated from the data
in similar fashion to method (3). Doing this
should mean that methods (2) and (3) give
similar results (Sheets and Mitchell 2001), and
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we therefore use only method (3) in this paper.
Directional selection is implied by higher
rates, greater change, or fewer runs than expected by chance, and the converse of these results indicates stabilizing selection. Unfortunately, a wide range of intermediate values for
these criteria is indistinguishable from random processes.
Previous studies using these methods have
generally failed to falsify the hypotheses that
samples in a biostratigraphic sequence are
static or that change can be explained by random processes, implicating stabilizing selection or genetic drift, respectively (Raup and
Crick 1981; Charlesworth 1984; Bookstein
1987, 1988; Lynch 1990; Cheetham et al. 1993;
Cheetham and Jackson 1995; Sheets and
Mitchell 2001). Exceptions to this generalization were findings of directional change by
Clyde and Gingerich (1994) and Gingerich
and Gunnell (1995), but Sheets and Mitchell
(2001) have criticized their test criteria. Failure
to infer directional natural selection in biostratigraphic sequences has been interpreted
to support the dominance of stasis in macroevolution (Cheetham et al. 1993; Cheetham
and Jackson 1995) in accordance with the
punctuated equilibrium model (Eldredge and
Gould 1972).
The potential for any of these methods to
detect directional change when it actually occurred is reduced when fossil preservation is
poorer (Martin 1999), the rate of sediment deposition is irregular (Schindel 1980; Sadler
1981), and the amount of time between samples is greater (Gingerich 1983, 1993). Less
than ideal quality of any of these factors will
not simply obscure the details of stratigraphic
variation, but will bias analyses toward a result of no directional change within the sequence (Sheets and Mitchell 2001). Without independent evidence for the cause of change in
a lineage, it is impossible to know whether
failure to falsify the null hypotheses of random change indicates that directional selection was not involved or only that the effects
of directional selection were obscured by the
poor quality of the samples or sequence.
A biostratigraphic sequence of the stickleback fish Gasterosteus doryssus from a late middle Miocene lake deposit (10 Ma; Perkins et al.
1998) in the Truckee Formation, Nevada, offers
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MICHAEL A. BELL ET AL.
FIGURE 1. Gasterosteus doryssus with three dorsal spines (D1–D3), seven predorsal pterygiophores (R1–R7, with asterisks indicating intervening pterygiophores) with five contact points between them, and a fully expressed pelvis
(P). Diagrams below the specimen indicate the range of pelvic phenotypes and their scores.
an excellent opportunity to infer evolutionary
causes from a fossil sequence. G. doryssus is
abundant and well preserved (Fig. 1), the sequence comprises numerous samples, and uninterrupted annual laminations throughout
the exposure allow temporal precision of decades to years (Bell et al. 1989; Bell 1994). G.
doryssus belongs to the well-known threespine
stickleback (Gasterosteus aculeatus) complex
(Bell and Foster 1994), providing an excellent
context to interpret paleoecology and patterns
of variation among samples within biostratigraphic sequences. In both modern and fossil
threespine stickleback, armor is highly variable (e.g., Bell 1976; Reimchen 1994) and can
evolve rapidly (Bell 2001; Bell et al. 2004). Research using modern populations has shown
that at least two factors select for armor reduction; the absence or rareness of predatory
fishes and low concentration of the ions necessary for bone formation (e.g., Giles 1983;
Reimchen 1983, 1994; Bell et al. 1993).
In this deposit, an initially low-armor (i.e.,
one dorsal spine, vestigial pelvis) G. doryssus
lineage experienced further reduction of pelvic structure during the first 93,000 years exposed in the study section (Bell et al. 1985).
Although the frequency of occurrence of the
small, third dorsal spine increased over the
course of the sequence for this lineage, sample
means were always less than one spine per
fish, which is an extremely low-armor state.
The first lineage was replaced by a high-armor
(i.e., three dorsal spines, two pelvic spines)
lineage that evolved reduced armor during
the next 17,000 years, before it disappeared
from the sequence.
We believe that the transition from low- to
high-armor forms represents species replacement. The later samples from the original lineage had such severely reduced armor that the
population probably lacked the genetic variation necessary for rapid evolution of increased
armor (see Foster et al. 2003). The low-armor
and high-armor forms co-occur for about a
century, during which few intermediate phenotypes are present, and then the high-armor
form continues alone up section for several
thousand years (Fig. 2). Unlike this replacement event, even rapid contemporary evolution of armor reduction involves a progression
of intermediate phenotypes (Bell 2001; Bell et
al. 1985, 2004). Presence of high-armor stickleback in sample 4 of Bell et al. (1985) suggests
that a high-armor lineage had long occupied
an adjacent habitat. Co-occurrence with few
intermediates indicates that the low- and
high-armor forms represented separate biological species but does not preclude gene
flow between them. Sympatric species pairs of
threespine sticklebacks, between which low
levels of gene flow take place, occur rarely
within modern lakes and commonly in modern lakes plus their associated streams (McPhail 1994; McKinnon and Rundle 2002).
Thus, presence of two biological species of
Gasterosteus within this deposit has modern
analogues.
Three armor traits—pelvic structure and
number of dorsal spines and predorsal pter-
NATURAL SELECTION IN THE FOSSIL RECORD
565
FIGURE 2. High-resolution plots for three armor traits between 4250 and 4500 years after the beginning of the
sequence. The plots on the right represent phenotypic change throughout the sequence (see Fig. 3), and those on
the left show the three armor phenotypes for individual specimens within the interval between 4250 and 4500 years.
ygiophores (Fig. 1)—vary conspicuously
within and among G. doryssus samples (Bell et
al. 1985). Armor is important for defense of
threespine stickleback against piscivorous
fishes (e.g., Hagen and Gilbertson 1972; Bell et
al. 1993; Reimchen 1983, 1994). Extreme pelvic
reduction and loss of spines occur in extant
stickleback populations from lakes that lack
predatory fishes (Bell 1974; Reimchen 1983;
Bell et al. 1993), and the number of contacts
between pterygiophores is reduced in populations with pelvic reduction (Bell and Nagappan unpublished data). Predorsal pterygiophores lie within the musculature along the
dorsal midline anterior to the dorsal fin, and
some support dorsal spines (Bowne 1994).
Overlap of armor bones in modern stickleback
stabilizes the dorsal spines (Reimchen 1983,
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MICHAEL A. BELL ET AL.
Bergstrom and Reimchen 2000, 2003), and
overlap of adjacent pterygiophores in G. doryssus presumably had the same function.
The genetics of armor traits in extant G. aculeatus has been studied extensively. The size
and complexity of the pelvis are determined
by one major and at least four modifier genes
(Shapiro et al. 2004; Cresko et al. 2004). Spine
number is moderately to highly heritable in
the fourspine stickleback, Apeltes quadracus
(Hagen and Blouw 1983). There is very limited
evidence that dorsal spine number is heritable
in G. aculeatus (Lindsey 1962), and two unlinked dorsal spine-length genes have been
identified (Peichel et al. 2001). There are no genetic data concerning the number of touching
predorsal pterygiophores, but the number of
pterygiophores varies among modern populations (Penczak 1962, 1965; Bell 1974) and
probably has a genetic basis, as well (Lindsey
1962). Consequently, these three armor traits
probably had a strong genetic basis in fossil G.
doryssus, and stratigraphic variation of armor
traits in this sequence represents evolution.
Three lines of evidence that are independent
of the pattern of change in the stickleback biostratigraphic sequence itself indicate that armor reduction during the last 17,000 years of
the sequence reported by Bell et al. (1985) was
due to directional natural selection. (1) Predatory fishes are extremely rare in the deposit
from which G. doryssus comes (Bell 1994), and
such absence is strongly associated with armor reduction in extant G. aculeatus populations (e.g., Hagen and Gilbertson 1972; Bell et
al. 1993; Reimchen 1994). (2) Multiple armor
traits, including the three reported in this paper and others not reported here (Travis et al.
unpublished data), simultaneously underwent reduction in size or number during the
period that the second lineage occurred. Dorsal spine number and pelvic structure vary independently among individuals within mass
mortality layers of G. doryssus and samples
from modern G. aculeatus populations (Bell et
al. 1989). Moreover, linkage mapping using
several extant threespine stickleback populations suggests that genes for armor traits are
distributed among several linkage groups and
are rarely closely linked on a chromosome
(Peichel et al. 2001; Shapiro et al. 2004; Colosimo et al. 2004, 2005; Cresko et al. 2004).
Thus, three armor traits that are free to vary
independently among individuals all exhibit
evolutionary reduction, suggesting that they
responded independently to directional selection favoring armor reduction. (3) The earlier
stickleback lineage during the first 93,000
years in this deposit also had extremely reduced armor, indicating that low armor was
usually favored by natural selection in the lake
inhabited by G. doryssus. Despite the high
quality of our material and this independent
evidence that change of genetically determined traits in this fossil sequence was caused
by directional selection, analysis of biostratigraphic sequence data alone did not falsify the
hypothesis that random processes caused dramatic morphological change in this time series.
Methods
Fossil Sampling and Preparation. The last
21,500 years of the stratigraphic section sampled by Bell et al. (1985) was resampled much
more intensively for this study. Specimens
were collected in an open pit mine at
39.526⬚N, 119.094⬚W (Two Tips, Nevada, 15minute series, topographic, U.S.G.S.). The fossil G. doryssus came from the 21,500-year
stratigraphic interval, beginning about 4500
years before the appearance of the high-armor
lineage (Bell et al. 1985) (Fig. 3). Sharpened
putty knives were used to split slabs of rock
at arbitrary points along bedding planes to expose the stickleback. The approximate stratigraphic position (⫾2 cm; about 60–100 years)
of each specimen was recorded. Most specimens lie on their sides on a bedding plane
with individual bones approximating life positions, but structures may be covered by rock,
which was removed under a dissecting microscope using probes.
Chronology. Each specimen was dated by
converting stratigraphic distance to years. The
7-m stratigraphic section from which specimens came was measured along two transects
about 6 m apart, and the mean of the two measurements was marked every 10–20 cm on exposed bedding planes adjacent to the sampling pit (⫾2 cm). The stratigraphic distance
of each specimen from a marked bedding
plane was determined during collection by
measuring distances of the fossil fish from this
NATURAL SELECTION IN THE FOSSIL RECORD
FIGURE 3. Mean values for three armor traits of fossil
Gasterosteus doryssus during about 21,500 years. The
time interval included corresponds to the period between samples 19 and 26 of Bell et al. (1985). Each point
represents the mean from specimens within a 250-year
interval plotted at the beginning of that interval. Square
symbols represent the original low-armor lineage before
the species replacement event, and diamonds represent
the second, highly armored lineage. Closed symbols
mark the start of intervals with high enough rates (⌬) to
indicate directional selection. Analyses using the x-statistic and runs tests began with sample 18 (arrow) and
include all subsequent samples in each time series. Intermediate values within the ascending phase of each
plot (represented by the dotted lines in the figure) at
4250 years represent mixtures of the low-armor and initially high-armor lineages. The horizontal line represents the approximate mean for the armor characters in
the original lineage.
series of mean measurements recorded on the
rock.
A lithological sample representing the entire 7-m stratigraphic section was divided into
1–3 cm segments and mounted on microscope
slides. The deposit is a diatomaceous shale
567
with nearly continuous couplets of light and
dark laminations interrupted only by conformably deposited volcanic ash (F. Brown
personal communication 1987). The couplets
have been interpreted as annual layers
(varves), representing deposition of diatoms
during the summer and terrigenous silt during the winter (Bell and Haglund 1982; reviewed in Bell 1994). The number of varves
per lithological segment was counted three
times by each of three observers. The weighted grand mean number of years per segment
for all three observers was computed by using
the mean from each observer’s three counts
weighted by the inverse of the variance of that
observer’s counts for that segment. The year of
deposition of each specimen was estimated by
using the cumulative number of years prior to
the segment in which it occurs plus the number of years within the segment prior to the
position of its occurrence, using a linear approximation for years during the segment
within which the specimen occurs.
Morphology. We scored three armor phenotypes—pelvic score, number of dorsal
spines, and number of predorsal pterygiophores touching along the dorsal midline.
Dorsal spines and the number of touching
predorsal pterygiophores were counted (Fig.
1). Pelvic phenotypes were placed into numerical categories by comparison to drawings
from Bell (1987: Figs. 7–10): 0, absent; 1, vestige of only the anterior portion of the pelvis;
1.2–2.8, anterior vestige plus vestige of posterior portion of the pelvis, increasing in increments of 0.2, according to size and complexity of the posterior portion; 3, all major elements of the ancestral pelvic structure (i.e.,
anterior process, ascending branch, posterior
process, and spine) present, regardless of size
(Fig. 1). Regression of the digitized area of
pelvic girdles (n ⫽ 78) with scores of 1 to 3 on
pelvic score is significant (F1,76 ⫽ 341.57, p ⬍
0.0001) and explains 82% of the variance in
pelvic girdle area. Thus, pelvic score takes
morphological complexity into account but is
also a good estimate of pelvic size.
Sample Formation. Sample means were calculated by using data for each trait from specimens within each of 83 contiguous 250-year
intervals (Appendix). Most but not all specimens were scored for all three traits. Thus,
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MICHAEL A. BELL ET AL.
means for different traits in the same interval
were based on somewhat different sets of
specimens. We scored 4720 specimens, and
the mean and standard deviation of sample
sizes per 250-year interval were 44.7 ⫾ 28.0 for
pelvic score, 41.4 ⫾ 26.0 for dorsal spine number, and 36.4 ⫾ 23.5 for number of touching
pterygiophores. We assessed the potential for
stratigraphic measurement error to cause assignment of specimens to the wrong 250-year
interval, which could artificially reduce evolutionary rate estimates, by computing rates
between pairs of odd-numbered and evennumbered, non-consecutive intervals (i.e.,
250-year groups separated by 250-year gaps).
The gapped data resulted in fewer high rates
of change, indicating that doubling the time
interval between observations reduces evolutionary rate estimates (see Gingerich 1983,
1993) more than misassignment of specimens
between adjacent 250-year intervals does.
Comparison with Expectations from Random
Processes. We used three methods to compare biostratigraphic sequences of mean phenotypes with expectations for a random process. The computational details are described
in the papers in which these methods were developed and will not be repeated here. The
first method was developed by Lynch (1990).
In a previous paper, Lynch (1988) reported the
range of rates of neutral morphological evolution in a variety of metazoans by examining
rates of neutral mutation (Lynch 1988). Lynch
(1990) used the range of rates of phenotypically detectable neutral mutations as a standard against which to evaluate rates of evolution calculated from data for mammalian
fossils. If rates based on fossil samples were
lower than the lowest neutral mutation rates,
they indicate stasis. Those within the range of
neutral mutation rates are consistent with
neutral morphological evolution, though various mixtures of other processes are consistent
with this range of rates, as well. Rates that exceed the range for neutral mutation are attributed to directional natural selection. This is
obviously an inexact estimate of what the neutral rate of morphological evolution would be
for traits in G. doryssus, but the range of neutral rates in diverse extant species probably
encompasses the true neutral mutation rate of
the three armor characters we investigate.
Neutral mutation rates range from 10⫺4, separating stabilizing selection and genetic drift,
to 5 ⫻ 10⫺2, separating genetic drift from directional selection (Lynch 1988).
Lynch’s (1990) method measures the rate of
evolution between successive samples in variance units and this rate can be compared with
the range of neutral rates calculated by Lynch
(1988). The evolutionary rate between successive samples was calculated from equation (1):
⌬ ⫽ VarB(lnz)/[tVarW(lnz)]
(1)
where VarB(lnz) and VarW(lnz) are the observed between-sample and within-sample
components of the phenotypic variance for
log-transformed measures, z is the phenotypic
value, and t is the number of generations between samples. Within-sample and betweensample variances were obtained from mean
squares generated by performing analysis of
variance on the data from each set of two consecutive intervals (Lynch 1990). We used a
generation time of two years, which is common in extant threespine stickleback (Baker
1994), resulting in 125 generations between
consecutive samples. A one-year generation
time is also common in extant stickleback, but
populations with a one-year generation time
tend to be smaller bodied than G. doryssus. If
we had used a generation time of one year for
G. doryssus, we would have obtained lower
rates; thus, we have biased our analysis in favor of finding evidence for directional selection.
The second method, the x-statistic from the
theorem of the scaled maximum (Bookstein
1987), estimates the probability of observing a
maximum deviation from the mean of the first
sample, which exceeds the deviation produced
by a random walk using the number of steps,
mean step distance, and the variance of the
step distances in the observed data. We did
not use two other methods, the Log-Rate-Interval (LRI) method (Gingerich 1993) and the
Hurst exponent (Hurst 1951), which use a premise similar to that of the theorem of the
scaled maximum and should produce similar
results (Sheets and Mitchell 2001).
The third method (Raup and Crick 1981)
uses two types of runs tests, one assuming
that steps are independent events, and a more
conservative one that does not assume inde-
NATURAL SELECTION IN THE FOSSIL RECORD
pendence. If the data include too few runs
based on the null expectation for both tests,
directional selection is indicated. If the data
include too many runs for both tests, stabilizing selection is indicated. If the number of
runs does not deviate from the null expectation for either test, drift is indicated, and if the
null hypothesis for the first and not the second
test is falsified, the data are too noisy to interpret (Raup and Crick 1981). Sheets and Mitchell (2001) recommended testing sequences for
a deviation in the frequency of positive and
negative steps from 0.5 in conjunction with
these runs tests, because the runs tests will
have little power to detect a deviation from a
random walk if there are too few steps in one
direction. A significant deviation from 0.5
may provide some evidence of an evolutionary trend different from a random walk, even
if runs tests do not indicate such a trend. We
used the x-statistic and both runs tests for all
three traits beginning with sample 18 and
continuing to the end of the time series (Fig.
3).
Natural logarithms were used except in the
runs tests. One was added to each specimen
score before taking natural logarithms to
avoid loss of information on specimens with a
trait value of 0, and means were computed
from the log-transformed observations. However, for clarity, means of untransformed data
were used in the figures and text.
Results
Temporal variation of mean phenotypes for
all three armor traits form similar patterns
(Fig. 3). Low values occur at the outset and simultaneously reach their maxima at about
4500 years, apparently reflecting replacement
of the low-armor lineage by the high-armor
one (Fig. 2). Evolution of reduced numbers of
dorsal spines and touching pterygiophores
begins almost immediately after the replacement event, but there is a 2750-year delay before pelvic score declines. All three traits
reach mean values resembling those of the
first low-armor lineage about 2000 years after
they begin to decline and vary irregularly
near these values thereafter.
A total of 235 evolutionary rates between
successive samples was calculated for the
three traits and compared with rates expected
569
FIGURE 4. Evolutionary rates (⌬) for three armor traits
of fossil Gasterosteus doryssus. Most rates fall within the
range expected for genetic drift (black), and few are
within the range expected for stabilizing (hatched) or
directional selection (open). See text for calculation of ⌬.
by neutral mutation (Fig. 4). Only five rates for
pelvic structure, three for dorsal spine number, and five for the number of touching predorsal pterygiophores (closed points, Fig. 3)
are within the range of high rates that indicate
directional selection. Four of the five high
rates for pelvic score and number of touching
pterygiophores occur during the interval of
pronounced armor reduction, and the direction of change accompanying these high rates
is always consistent with the declining trend.
However, none of the rates between samples
during the interval in which spine number
evolved from 3 to 1 are within the range of
rates for directional selection. Only 5.5% of the
rates for all three traits exceed that expected
from neutral mutation, implying that directional selection was inconsequential or rarely
important for the observed change. Although
few rates for each trait are low enough to indicate stabilizing selection, there are about
twice as many rates within this low range as
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MICHAEL A. BELL ET AL.
TABLE 1. Tests for random walks in sequences of mean phenotypic values using the x-statistic from the theorem
of the scaled maximum. p is the probability that random processes produced the observed change under the assumptions of the test.
Phenotype
x-statistic
p
Interpretation
Pelvic score
No. of dorsal spines
No. of touching pterygiophores
1.577
1.301
1.327
0.75
0.68
0.63
Random walk
Random walk
Random walk
there are within the range for directional selection.
Using the x-statistic from the theorem of the
scaled maximum, none of the sequences for
three traits differs significantly from the expectation for a random walk (Table 1). Neither
pelvic score nor number of touching pterygiophores deviated from 0.5 for the proportion of positive to negative steps in the sequence (pelvic score: 33 decreasing steps, 24
increasing steps, p ⬎ 0.05; number of touching
pterygiophores: 36 decreasing steps, 32 increasing steps, p ⬎ 0.05) . However there were
significantly more decreasing (42) than increasing (26) steps (p ⫽ 0.034) for dorsal spine
number. The excess of downward steps in the
number of dorsal spines may indicate directional selection, but the subsequent runs test
may not have the power to detect a deviation
from a random walk. However, this test was
so close to the critical significance level (one
less downward step would have resulted in a
finding of no significant deviation from 0.5)
that we also present the results of the runs test
for this character. Runs tests indicate that pelvic score and spine number are too noisy to
interpret, and the number of touching pterygiophores is consistent with a random walk
(Table 2). Thus, the three methods we applied
to this biostratigraphic sequence usually
failed to implicate directional selection as the
cause for temporal change in this sequence of
G. doryssus.
Discussion
We report an unusually fine-scale biostratigraphic sequence in which a threespine stickleback evolves dramatically reduced armor.
The number and size of individual samples,
the quality of the fossil specimens, and the accompanying chronology are all excellent relative to comparable studies, maximizing our
chances to detect natural selection from rates
and patterns of change alone (see Martin 1999;
Gould 2002). In particular, the short time intervals separating samples should capture
high rates of phenotypic change that would be
obscured using longer time intervals (Gingerich 1983). The ecology, evolution, and genetics
of the threespine stickleback are also unusually well known (Bell and Foster 1994), providing a rich backdrop against which to interpret this fossil sequence.
When the high-armor lineage first appeared, all specimens possessed three long
dorsal spines, a robust pelvic structure, with a
long spine on each side, and numerous overlaps between adjacent predorsal pterygiophores (Fig. 1). It is unclear why the high-armor lineage replaced the original low-armor
one, but its abrupt appearance is unlikely to
represent evolutionary change (see ‘‘Introduction’’).
The numbers of dorsal spines and contacts
between pterygiophores declined soon after
the armored lineage appeared, but pelvic re-
TABLE 2. Runs tests for random walks in sequences of mean phenotypic values, assuming that events are independent or non-independent. Obs., observed number of runs; Exp., expected number of runs; p, probability that
the observed number of runs differs from a random walk; and n.s., nonsignificant.
Independent events
Non-independent events
Phenotype
Obs.
Exp.
p
Obs.
Exp.
p
Interpretation
Pelvic score
No. of dorsal spines
No. of touching pterygiophores
38
41
41
37.7
44.3
44.3
n.s.
n.s.
n.s.
38
41
41
28.8
33.1
34.9
⬍0.01
⬍0.025
Noise
Noise
Random walk
n.s.
NATURAL SELECTION IN THE FOSSIL RECORD
duction was delayed by more than a thousand
generations. Armor reduction in the second G.
doryssus lineage conforms to expectations for
the evolutionary response of inherited traits to
directional selection, which favors armor reduction in the absence of fish predation in extant populations (e.g., Bell 1974, 1987, 1988;
Reimchen 1983; Bell et al. 1993).
The high-armor G. doryssus lineage appears
to have experienced the ecological conditions
and to have possessed the genetic properties
necessary for a strong evolutionary response
to selection for reduced armor (see ‘‘Introduction’’). Absence of predatory fishes from the
deposit and repeated evolutionary reduction
of multiple armor traits in both lineages in the
same deposit indicate that directional selection favored armor reduction. The genetics of
armor in several extant threespine stickleback
populations indicate that variation of the
length of spines and the strength of the pelvis
includes a major component of genetic variation. The genetics of dorsal spine number and
the number of touching pterygiophores have
not been studied in modern G. aculeatus, but it
would be a radical departure from the genetics of the same traits in other stickleback genera and of other armor traits in extant G. aculeatus if they were not heritable. Thus, the
high-armor lineage probably possessed genetic variation to respond to selection for reduced armor, it inhabited a lake in which natural selection favored armor reduction, and,
as expected, it evolved armor reduction.
The first problem to address is the delay of
pelvic reduction for 2750 years. Presumably,
the decline in the number of dorsal spines and
touching pterygiophores indicates directional
selection for armor reduction when the higharmor lineage appeared in the sequence.
There may be a genetic cause for this difference in evolutionary response. Although there
is limited information on dorsal-spine number
genetics (Hagen and Blouw 1983), the distribution of dorsal-spine and touching-pterygiophore numbers within samples is generally
unimodal (Travis unpublished data), suggesting that they are controlled by multiple genes
with additive interactions and should respond
readily to directional selection (Futuyma
2005). In contrast, pelvic reduction in most extant G. aculeatus populations results from si-
571
lencing of the transcription factor, Pitx1,
which also tends to produce a larger pelvic
vestige on the left side than the right (Cole et
al. 2003; Cresko et al. 2004; Shapiro et al.
2004). Although the structure of pelvic vestiges in G. doryssus differs from that of extant
threespine stickleback with pelvic reduction
(Bell 1987), pelvic vestiges in G. doryssus also
tend to be left biased, implicating a null Pitx1
allele in pelvic reduction in the fossil form
(Bell et al. unpublished data). Null alleles of
Pitx1 in extant G. aculeatus are recessive to
functional alleles associated with full pelvic
expression (Cresko et al. 2004; Shapiro et al.
2004). Because rare recessive alleles are unlikely to be expressed phenotypically, directional natural selection favoring their increase
will be inefficient until they rise to higher frequencies (Roughgarden 1996). Thus, pelvic reduction in G. aculeatus may have been delayed
because it depended on selection for a rare recessive allele.
The more general problem, however, is the
failure of our analyses of biostratigraphic rates
and patterns in G. doryssus to exclude genetic
drift as the mechanism for evolution of armor
reduction when extrinsic evidence indicates
that directional selection caused it. Although
we did find a significant deviation from 0.5 in
the proportion of downward steps for one of
the three armor traits, the subsequent runs
tests for this character did not indicate directional selection. Likewise, a small fraction of
the rates of evolution we calculated are high
enough, are in the right direction (i.e., reduction), and in the right portion of the sequence
for directional selection to be indicated, but
the vast majority of the rates, even in the part
of the sequence that should be experiencing
the strongest and most consistent selection
pressures, are not high enough to indicate directional selection. There is remarkably little
evidence of directional selection, given the
quality of our biostratigraphic sequence and
our extrinsic evidence for directional selection, indicating that the methods to detect directional selection in biostratigraphic sequences are unreliable.
We believe that there are two general explanations that apply to all methods and account
for failure to detect deviations from random
change. They assume that natural selection
572
MICHAEL A. BELL ET AL.
will consistently favor evolution in the same
direction. In most sequences, however, random or pseudo-random effects that obscure
the effects of directional selection can be
caused by phenotypic plasticity, short-term
variation in the intensity or direction of selection (Gingerich 1983; Reznick and Ghalambor
2001), natural selection tracking a random or
cyclical environmental variable (Hendry and
Kinnison 1999), poor preservation, sample error due to the limited size of fossil samples,
and mixing of lineages within samples (Bell
and Haglund 1982). Even at 250-year intervals, evolutionary reversals and pooling of
high and low rates of evolution average to low
mean values that are consistent with genetic
drift and even stabilizing selection.
Lynch’s (1990) method has the added potential liability of depending on a specific genetic
model and empirical values from other traits
in other species. His estimates of the rates of
neutral evolution of morphological traits apparently were based on traits that did not differ greatly from additivity, but he noted that
such deviations should have little effect on
rates of neutral evolution. The wide range of
characters and taxa used to derive Lynch’s
(1988) estimates should be applicable to evolution for all three armor traits in G. doryssus.
However, as explained above, dependence of
pelvic reduction on a recessive allele of a gene
with a major phenotypic effect would retard
the response to selection for pelvic reduction
when that allele is rare.
Successive samples in our study were 250
years apart, and random factors are less likely
within these short time intervals than the entire 17,000-year series to incorporate random
or pseudo-random effects that obscure evolutionary trends. Nevertheless, Lynch’s (1990)
method detected very few rates exceeding
those expected from genetic drift. It is encouraging that eight of the high rates indicating directional selection are within the part of
the sequence when there appeared to be a longer-term trend for armor reduction, and that
these changes were in the direction (i.e., lower
values) expected, but most rates from this part
of the sequence are within the range expected
for genetic drift, and many imply stabilizing
selection. The genetic assumptions of Lynch’s
(1990) method do not appear to be a problem,
but the assumption that the direction of natural selection will be consistent within the
time interval between successive samples may
be violated frequently at even 250-year intervals.
The problems inherent in averaging adjacent high and low rates and reversals in the
direction of natural selection increase as the
time interval spanned by a series of samples
increases. Results of our x-statistic and runs
tests incorporate data spanning the entire
17,000 years after the high-armor lineage appeared and should have been even more
strongly affected by temporal pooling than
the shorter-term rate measurements using
Lynch’s (1990) method. Thus, these methods
are even more sensitive to the assumption that
directional selection will be consistent from
generation to generation over the entire period
of time sampled and other random factors will
not conceal the effects of directional selection.
Despite the favorable properties of our material to detect directional natural selection in a
biostratigraphic sequence, random or pseudorandom effects apparently obscured it.
Bookstein (1987, 1988) and Raup and Crick
(1981) also failed to find evidence for directional selection from the sequences they analyzed using the methods they developed. This
is not surprising in light of our results. Rates
between pairs of odd- or even-numbered samples (i.e., at 500-year intervals) in the high-armor G. doryssus lineage produced fewer rates
(i.e., eight rates) within the range of directional selection than rates between consecutive
samples (i.e., 13 rates). Lynch’s (1990) original
comparison of rates in fossil sequences used
longer time intervals than ours, and all but
one of his rates were even lower than ours, and
fell within the range expected for stabilizing
selection. Notably, his only rate high enough
to be within the range expected for genetic
drift came from the shortest time interval he
used. Clegg et al. (2002) used Lynch’s (1990)
method to infer whether directional selection
had caused evolutionary diversification of the
silvereye (bird) species complex on southern
Pacific islands. These birds diverged and speciated as they dispersed to islands around
Australia and New Zealand. Clegg et al.
(2002) estimated that the time separating speciation events in this clade ranges from 1000
NATURAL SELECTION IN THE FOSSIL RECORD
to 2000 generations for the youngest divergence to 100,000 to 1,000,000 generations for
the oldest divergences. They presented evidence that directional selection caused the divergence of these bird species, but all of the
rates they calculated were within the range of
drift or stabilizing selection. However, the
shortest time intervals separating divergent
species produced the highest rates of evolution. The message that emerges from these
studies is that current methods based on rates
and patterns of evolution cannot detect directional selection even when there is strong evidence that it occurred and that the effectiveness of these methods declines as the time between samples increases.
Nevertheless, the failure to detect directional selection with these methods has been interpreted to indicate that it was inconsequential in evolution. Lynch (1990) concluded that
stabilizing selection has dominated the evolution of cranial morphology in mammals.
Similarly, Cheetham and Jackson (1995) concluded that the low rates of evolution they calculated with Lynch’s (1990) method indicated
that directional selection was not likely to be
important to explain the patterns of diversification within a fossil bryozoan clade. The
time intervals over which they calculated their
rates, however, were on the order of millions
of years. They concluded that their failure to
detect a signature of directional selection in
their data indicates punctuated equilibrium in
which evolution is precluded between punctuations. Our results combined with others at
longer time intervals suggest instead that failure to detect directional selection in biostratigraphic sequences is likely to be an artifact of
long time interval between samples, even
when those intervals are as short as 250 years
(i.e., 125 generations).
Temporal resolution in biostratigraphic sequences is usually much coarser than 5000
years (Schindel 1980; Sadler 1981; Martin
1999), and recent analyses of extant populations indicate that major evolutionary changes
can occur within much less time (Reznick and
Ghalambor 2001; Bell 2001; Bell et al. 2004).
Thus, failure to reject the hypothesis that
change in biostratigraphic sequences is due to
random process, using the methods we employed, does not justify the conclusion (see
573
Lynch 1990; Cheetham et al. 1993; Cheetham
and Jackson 1995) that directional selection
was inconsequential for change in the fossil
record.
The fossil record contains unique evolutionary information. However, we are forced to
conclude that attempts to infer evolutionary
processes exclusively from patterns of change
within individual biostratigraphic sequences
using available analytical methods are unlikely to provide robust evidence for directional
natural selection, even when it actually caused
the change. Evolutionary reversals and rate
averaging over shorter time intervals than can
be resolved in most geological sequences will
depress rates from which directional selection
could be inferred (Gingerich 1983). Existing
methods to infer directional selection from
biostratigraphic pattern (e.g., x-statistic, runs
tests) are limited by unrealistic assumptions,
but increasing the reality of these assumptions
(e.g., allowing for evolutionary reversal)
would produce such lenient criteria to reject
the null hypothesis of drift that any biostratigraphic sequence could be erroneously attributed to directional selection. Our confidence
that directional selection was a major factor in
observed patterns of temporal change in our
G. doryssus sequence is based on extensive
knowledge of the ecology and evolution of
modern stickleback populations, not on incompatibility of the pattern of change with
drift. Indeed, if we had analyzed the same
pattern of change in a less well known species
or less easily interpretable set of traits, we
would have no way of justifying our view that
natural selection was involved in the evolution
of armor reduction in these fish (see also
Bookstein 1988). What is unique about our
biostratigraphic sequence is that the problems
associated with inferring process from pattern
in the fossil record are minimized compared
to other sequences previously reported. Nevertheless, we were unable to find evidence of
the action of directional selection without using evidence extrinsic to the biostratigraphic
pattern.
The longer the time intervals are between
successive fossil samples, the more likely it is
that the effects of directional selection will be
obscured by random processes. The fine temporal resolution required to detect natural se-
574
MICHAEL A. BELL ET AL.
lection is well beyond that possible for land
mammals, marine macroinvertebrates, and
most marine microfossils (Schindel 1980; Sadler 1981), the groups that have attracted the
most attention from paleontologists (Erwin
and Anstey 1995). Given the difficulties of interpreting the results from these methods, we
suggest that studies of evolutionary processes
based on biostratigraphic sequences focus on
cases with extremely fine temporal resolution
and complementary sources of mechanistic
explanations (e.g., see Goodfriend and Gould
1996). Comparison of patterns in fossil lineages with expectations based on modern populations of similar species, as we have done, is
one possible source of complementary evidence. Other possibilities include comparison
of multiple biostratigraphic patterns in independent sequences or functional analysis of
traits of interest. Unfortunately, much of the
fossil record cannot be resolved finely enough
to infer evolutionary mechanisms responsible
for change in biostratigraphic sequences. Current methods to study rates or patterns of phenotypic evolution in the fossil record are
strongly biased against detecting directional
selection, and failure to obtain such evidence
must be interpreted with extreme caution.
Acknowledgments
We thank J. V. Baumgartner, E. C. Olson, L.
Olson, R. Tintle, and several Earthwatch volunteers for helping collect fossils; N. J. Buck,
W. J. Caldecutt, J. Qiao, F. J. Rohlf, and T. T.
Zhang for assistance with the chronology; R.
Banks, and S. Oomen for rechecking phenotypes; J. M. O’Shea for data entry and editing;
and I. D. Chase, D. E. Dykhuizen, A. P. Hendry, J. P. Hunter, and H. D. Sheets for comments on earlier versions of the manuscript.
Thoughtful comments by P. D. Gingerich and
two reviewers also improved the final manuscript. Cyprus Industrial Minerals and CR
Minerals permitted us to collect fossils in their
mine. This research was supported by grants
from the National Science Foundation
(BSR8111013, EAR9870337, and DEB0322818),
Center for Field Research (Earthwatch), and
National Geographic Society (2869-84) grants
and appointment of M.A.B. as W. F. James Professor of Pure and Applied Sciences (visiting)
in the Department of Biology, St. Francis Xa-
vier University. This is contribution 1147 from
Ecology and Evolution at Stony Brook University.
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Appendix
Phenotypic means, variances, and sample sizes for three armor traits of Gasterosteus doryssus. The interval is the
beginning of the 250-year time interval since the first year of the 21,500-year stratigraphic section. Abbreviations:
PS, mean of the natural log of pelvic score; DS, mean of the natural log of the number of dorsal spines; TP, mean
of the natural log of the number of touching predorsal pterygiophores; Var. PS, Var. DS, and Var. TP are the variances
of the natural log of each character. n is sample size. Raw trait values have been adjusted by adding 1 to every
observation before taking the natural logarithm of the value to avoid losing data by throwing out observations that
would require taking the natural logarithm of 0. See methods for definition of traits.
NATURAL SELECTION IN THE FOSSIL RECORD
Appendix. Continued.
577