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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 0094-8373/06/3204-0003/$1.00 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 563 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 564 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, 566 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, 568 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 570 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. 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Genetica 112– 113:105–125. Simpson, G. G. 1944. Tempo and mode in evolution. Columbia University Press, New York. 576 MICHAEL A. BELL ET AL. 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