Intraspecific Heterochrony and Life History Evolution: Decoupling Somatic and Sexual Development in a Facultatively Paedomorphic Salamander

Proc. Natl. Acad. Sci. USA Vol. 95, pp. 5643–5648, May 1998 Evolution

Intraspecific heterochrony and life history evolution: Decoupling somatic and sexual development in a facultatively paedomorphic salamander

TRAVIS J. RYAN*† AND RAYMOND D. SEMLITSCH†

*Savannah River Ecology Laboratory, Drawer E, Aiken, SC 29802; †Division of Biological Sciences, University of Missouri, Columbia, MO 65211

Communicated by George C. Williams, State University of New York at Stony Brook, Stony Brook, NY, March 9, 1998 (received for review June 17, 1997)

ABSTRACT Morphological features such as size and phase ends at metamorphosis when individuals lose features shape are the most common focus in studies of heterochronic associated with aquatic life (e.g., external gills and extensive change. Frequently, these easily observed and measured fea- tail fins). Usually, metamorphosed juveniles attain maturity on tures are treated as a major target of selection, potentially land and return to aquatic habitats seasonally to reproduce. ignoring traits more closely related to fitness. We question the The duration of the larval period varies widely within the primacy of morphological data in studies of heterochrony, and family: most species have a brief larval period (e.g., Ͻ3 months instead suggest that principal sources of fitness, such as life in Ambystoma maculatum; ref. 13) although some larvae history characteristics, are not only the chief targets of overwinter and do not metamorphose until Ͼ12 months selection, but changes in them may necessitate changes in posthatching (e.g., some A. tigrinum, ref. 14). Other species other (subordinate) elements of the organism. We use an have abandoned the complex life cycle and bypass metamor- experimental approach to investigate the timing of metamorphosis altogether (e.g., the Mexican axolotl, A. mexicanum, ref. phosis and maturation in a facultatively paedomorphic 15). These individuals mature while retaining the larval mor- salamander, Ambystoma talpoideum. We determine that indi- phology, a derived state termed complete or larval paedomor- viduals possessing the well-known paedomorphic phenotype phosis (meaning ‘‘child-shape’’ or ‘‘underdeveloped,’’ ref. 1, 4). are peramorphic with regard to maturation, through the Paedomorphosis evolves by modifications of the timing process of predisplacement (an earlier onset of maturation). and͞or rate of key developmental events (reviewed in refs. 2, Combining the well studied ecology of dimorphic A. talpoideum 4, 16). The most sophisticated models predict that larval populations with theories of heterochronic mechanisms and paedomorphosis in salamanders may be achieved through life history evolution, we conclude that age at maturation is the three processes (Fig. 1) operating either singly or in consort: principal target of selection and that morphological changes neoteny (also referred to as ‘‘deceleration,’’ ref. 4), the decel- are secondary effects. Increased attention to the intimate erated rate of somatic development; postdisplacement, the connection between life history evolution and heterochrony is delayed onset of metamorphic change or somatic differentia- the most promising route to a better understanding of both. tion; or hypomorphosis, the precocious cessation (or negative offset) of somatic differentiation. Each of these processes has a corollary that shares a common morphological result termed Heterochrony, evolutionary changes in patterns of develop- peramorphosis (meaning overdeveloped): for example, the ment, has experienced a rebirth since Gould’s magnum opus opposite of postdisplacement is predisplacement, the earlier (1), and recently several synthetic reviews of the topic have onset of metamorphosis. Gould (1) has asserted that paedo- been published (2–4). To understand how patterns of growth morphosis in salamanders is the result of neoteny, although he and development evolve, researchers have used a wide array of was mindful to note that a comparison of ontogenies of groups techniques and conceptual approaches, including quantitative with a well established ancestor-descendant relationship—the genetics (5), paleontology (6), comparative developmental data necessary for determining heterochronic changes—are biology (7), and experimental ecology (8). However, McKin- often conspicuously absent from the literature. It is important ney and Gittleman (9) carefully note a major deficiency in the to note that all paedomorphic Ambystoma are derived from modern approach to heterochrony since Gould (1): there has naturally metamorphosing ancestors (17); therefore we must been surprisingly little attention paid to the close relationship focus on the developmental changes exhibited in derived between life history evolution and heterochrony. Within each paedomorphic forms relative to their closest metamorphosing of these broad frameworks of evolutionary biology the timing ancestor (2). For the axolotl, this is most likely A. tigrinum (18). of maturation is a critical event influencing both the morphol- However, once cladogenesis (speciation) occurs, each lineage ogy and fitness (i.e., lifetime reproductive success) of the evolves independently, and subsequent changes in the devel- individual (1, 2, 10) as well as the demographic structure of opmental programs of both lineages are likely to confound populations and species (11, 12). With this in mind, we turn to inference of heterochronic patterns. Populations where both a classic example of heterochrony, paedomorphosis in amby- metamorphic and paedomorphic life cycles are manifested stomatid salamanders, and experimentally investigate the evo- simultaneously are termed facultatively paedomorphic, and we lution of derived developmental and life history patterns. believe it is in these populations where we can elucidate the Members of the family Ambystomatidae (Amphibia: Cau- selective pressure(s) and distinguish the heterochronic pro- data) are moderately sized, morphologically conservative cess(es) that result in paedomorphosis. salamanders widely distributed throughout North America. We focus on the timing of metamorphosis and maturation as All species have aquatic eggs and larval stages. The larval the key developmental landmarks in experimental populations of Ambystoma talpoideum, a facultatively paedomorphic The publication costs of this article were defrayed in part by page charge salamander from the southeastern United States. Our reasons payment. This article must therefore be hereby marked ‘‘advertisement’’ in for concentrating on these events are straightforward. Larval accordance with 18 U.S.C. §1734 solely to indicate this fact. paedomorphosis is a radical departure from the ancestral life © 1998 by The National Academy of Sciences 0027-8424͞98͞955643-6$2.00͞0 cycle, one that is adaptive and thus shaped by natural selection PNAS is available online at http:͞͞www.pnas.org. (8, 19, 20). Selection is most likely to act on those characters

5643 Downloaded by guest on October 1, 2021 5644 Evolution: Ryan and Semlitsch Proc. Natl. Acad. Sci. USA 95 (1998)

FIG. 1. Heterochronies that may result in paedomorphosis in salamanders. Lines (A–D) represent hypothetical developmental trajectories. The ancestral pattern is represented by metamorphic adults (A). Derived patterns resulting in paedomorphic adults include (B) hypomorphosis, where somatic development ceases before the metamorphic threshold (dashed horizontal line); (C) deceleration, where somatic development proceeds at a slower rate than A with the maturation threshold (dashed vertical line) being reached before the metamorphic threshold; (D) postdisplacement, where somatic development is initiated at a later ontogenetic age than A; or some combination of B–D (e.g., see ref. 4). Images modified from refs. 4 and 47.

that are important to organismal fitness, such as age at randomized design. These experimental ponds have become a metamorphosis (21) and age at maturation (22). Because stalwart of amphibian ecological and evolutionary studies, and morphological reorganization consumes considerable time offer a practical mixture of ecological realism and experimen- and energy (17, 23), we hypothesize that, all other things being tal rigor (26, 27). We reared larvae at two densities (18 larvae held equal, salamanders foregoing metamorphosis may have per pond ϭ low density; 36 larvae per pond ϭ high density) more energy available for growth and (or) sexual development. within the range of natural densities (28), and collected whole For example, we expect that those individuals bypassing meta- populations at four times (September, October, November, morphosis should either attain maturation earlier than meta- and December). Each treatment combination was replicated morphic salamanders, or attain maturation at the same time as four times, for a total of 32 experimental populations. metamorphs but at a larger body size. In the first case, energy Procedures. Experimental populations were derived from and available resources can be channeled to the rapid devel- the progeny of metamorphic adult A. talpoideum intercepted opment of gonads and gametes rather than extensive morpho- during breeding migrations at Rainbow Bay, a temporary pond logical rearrangement (24, 25). Thus we expect if paedomorphs in Barnwell County, South Carolina. Because Rainbow Bay and metamorphs mature at the same body size, paedomorphs dries annually (29, 30), all members of the breeding population should mature at a younger age. Alternatively, if the two adult are necessarily metamorphic, indicating this as the life history morphs mature at the same point in time, we might predict pattern ancestral to the population. The members of this paedomorphic individuals to have increased body size relative population, nonetheless retain the ability to become paedo- to metamorphic individuals, with the energy and resources morphic when released from the pressure exerted by pond required for metamorphosis having been committed to overall drying, both under natural (31) and experiment (29) situations. growth in paedomorphs. Other complex trade-offs between Under constant water conditions, expression of the adult life history pathways and components of fitness (e.g., morph- morphs is near parity (29). specific fecundity, mating success) are also likely to be impor- Thirty-two experimental ponds (polyethylene cattle tanks, tant in determining the evolution and maintenance of facul- 1.83 m diameter, Ϸ1,300 l volume) were randomly assigned tative paedomorphosis. Herein we present the results of an treatment combinations and positions in an array at the experiment investigating the timing of metamorphosis and Reactor Research Park (University of Missouri, Columbia), differences in the timing of maturation between metamorphic and were filled with Ϸ1,000 l tap water on 4–5 March 1996. and paedomorphic A. talpoideum, and discuss the selective Each pond was equipped with an internal adjustable standpipe pressures that likely account for the heterochronic patterns to standardize and limit water depth. We added 1 kg of observed. homogenized leaf litter to each pond on 12 March 1996 and added equal-sized aliquots of a concentrated zooplankton METHODS solution to each pond several times before the addition of larvae (8, 29). Ponds were covered during the day to prevent Experimental Design. We raised populations of larval A. colonization of predacious insect larvae (e.g., odonates) and talpoideum in experimental artificial ponds in a two-factor uncovered at night to allow for natural colonization of other Downloaded by guest on October 1, 2021 Evolution: Ryan and Semlitsch Proc. Natl. Acad. Sci. USA 95 (1998) 5645

insects (e.g., chironomids) that serve as a food source. On 12 domorphs and metamorphs differed in each of the three March 1996, 10 males and 18 females were introduced to a treatment combinations where both morphs were present. In 1,500-l experimental pond, containing a leaf litter mixture to these tests, the null hypothesis is that there is a significant serve as a site for oviposition, and allowed to mate at random. difference in body size of the two morphs, and if the null Adults were removed from the breeding pond once oviposition hypothesis is rejected the morphs are considered indistinguish- was observed (14–20 March 1996). Eggs were allowed to able with regard to body size. In all comparisons, the null develop and hatch in the breeding pond. Hatchling A. talpoi- hypothesis was clearly rejected (t values ϭ 11.23–52.31; all P deum larvae were randomly assigned to experimental ponds values Ͻ 0.001). We thus regarded these adult morphs as according to density treatments on 3 May 1996 (day 1). representing a single class in a three-way ANOVA testing for We monitored the ponds daily and searched for metamorphs differences in body size, with morph (adult morph or immature every 2–3 nights beginning 1 June 1996 until the end of the larvae), month of collection, and density as main effects. Body experiment on 2 December 1996. Metamorphs were collected size was log-transformed for all statistical analyses. via dipnet, measured for snout-vent length (the distance from the tip of the snout to the posterior margin of the cloacal aperture) in mm, and given a unique mark (toe-clip). We RESULTS released metamorphs into polyethylene tanks filled with Ϸ10 Survival and Size. There were no significant differences in cm topsoil and Ϸ10 cm leaf litter and a 30 cm ϫ 30 cm survival among the treatment combinations (density effect: ϭ ϭ ϭ coverboard for refuge (referred to hereafter as ‘‘pens’’). The F1,24 2.01, P 0.1686; month of collection effect: F3,24 ϭ ϭ ϭ natural soil and leaf litter substrate contained an ad libitum 1.87, P 0.1613; interaction effect: F3,24 1.96, P 0.1473) food supply; arthropods, annelids, and other invertebrates as survival was high among all treatments (92–100%). Mean were observed during pen construction and dismantling. One body size varied significantly with respect to both density (F1,45 ϭ Ͻ ϭ pen was available for each collection month, and density in the 312.37; P 0.0001) and month of collection (F3,45 26.14; pens did not exceed natural densities of juveniles (32). P Ͻ 0.0001), but not between immature larvae and adult ϭ ϭ We harvested the contents of four replicate low density and morphs (F1,45 0.24; P 0.6247), and there were no signif- four replicate high density ponds on 8 September (day 129), 6 icant interactions among main effects. At each of the four October (day 157), 3 November (day 185), and 2 December collections, the low density survivors were significantly larger (day 224). This period, from 4–8 months posthatching, cov- than their high density counterparts. Within each density ered the time during which nearly all metamorphosis takes survivors collected in September were significantly smaller place in natural populations (22, 33). Past this point, most than those in the remaining collections, and survivors collected immature larvae have been found to remain as immature December were significantly larger than those collected in larvae until the following spring when they metamorphose October. These results demonstrate that initial larval density with the first of the new cohort. During each harvest, the influenced the body size of individuals, but that growth was appropriate ponds were drained and all leaf litter was thor- similar among the morphs at both densities. oughly searched for nonmetamorphosed individuals. Similarly, Metamorphosis and Maturation. At low density, paedo- the corresponding terrestrial pens were emptied each month morphs were present in the collections (September) before the and all metamorphic individuals were recovered. All individ- appearance of any metamorphs (October) (Fig. 2). Further- uals were sacrificed in a dilute ethanol solution and preserved more, none of the metamorphs from any of the collections in 7% formalin. Each individual was measured for snout-vent length and dissected to determine reproductive status. Mature or maturing males were identified by the presence of enlarged testes and͞or pigmented vasa deferentia. Mature or maturing females were identified by the presence of enlarged yolky ovarian follicles and͞or enlarged convoluted oviducts. We regarded all individuals exhibiting both the larval morphology (external gills and large tail fins) and sexual maturation as paedomorphs, as these individuals have ‘‘committed’’ to that life history pathway (29, 33); individuals that metamorphose Ͻ1 yr posthatching do not emerge from natural or experimental ponds sexually mature (unpublished data from 8, 22, 29, 33). All other nonmetamorphic individuals were regarded as immature larvae. Analysis. We performed a two-way ANOVA to detect differences in survival among the eight treatment combinations, using the arcsine-square root of the proportion of individuals surviving to the time of collection. We recorded size (snout-vent length in mm) and morph (metamorph, paedomorph, or immature larvae) of each individual, and calculated the proportion of each population (i.e., experimental pond) exhibiting each adult morph (metamorph or paedomorph). Preliminary analyses indicated that the proportion of paedomorphs and metamorphs in low density pons did not differ significantly over time, thus we used a two-way ANOVA to determine whether the (arcsine-square root transformed) proportion of paedomorphs differed with regard to density and month of collection. The proportion comprising the immature larvae was uniquely determined by the proportion of paedomorphs in all high density ponds and was therefore excluded from analyses of population composition. FIG. 2. Mean proportion of each adult morph (o, paedomorphs; ■, We performed equivalence tests (tests-of-no-effect, see ref. metamorphs) present in each collection under (A) low density and (B) 34 for review) to determine whether the mean sizes of pae- high density. Bar ϭ 1 SEM. Downloaded by guest on October 1, 2021 5646 Evolution: Ryan and Semlitsch Proc. Natl. Acad. Sci. USA 95 (1998)

showed any signs of sexual maturation. The expression of the summer of 1992 (39). Released from the ecological pres- paedomorphosis varied significantly with regard to density sure of pond drying in the summer and fall of 1991, many ϭ Ͻ (F1,28 35.04; P 0.0001) but not with month of collection larvae became paedomorphic in this population where the ϭ ϭ (F3,28 0.32; P 0.7301), and the interaction between these metamorphic pathway is the norm. In the winter of 1991 ϭ ϭ two factors was not significant (F3,28 1.24; P 0.3212). The courtship, sperm transfer, insemination, and oviposition were proportion of paedomorphs in low density ponds was greater completed by paedomorphic adults and hatching of some of than that of low density ponds at each of the collections, and their progeny occurred all prior to the arrival of metamorphic did not appear to increase or decrease appreciably throughout adults. All breeding paedomorphs in the population were Ͻ1 the experiment (Fig. 2). No metamorphs emerged from any of yr posthatching at the time reproductive activity was docu- the high density ponds by the termination of the experiment. mented (39). Although metamorphs may reproduce initially at Ϸ1 yr posthatching, reproduction is often delayed an addi- DISCUSSION tional year (or more), with the size and age at metamorphosis acting as a primary influence on reproductive attributes (21). We collected paedomorphic individuals from our experimen- At Ellenton Bay (a permanent pond about 15 km from tal populations before collecting any metamorphic individuals, Rainbow Bay), Krenz and Sever (40) found that 50% of in accordance with our predictions. Furthermore, no meta- paedomorphic females had been inseminated and begun ovi- morphs emerged from any of the high density ponds, although position about 1.5 months before the climax of migration by paedomorphs were present in each of the four collections, metamorphs. Although the ages of both morphs were un- albeit at relatively low frequency. Mean body size within a known in this study, it again demonstrates that migratory given density over time and between densities at any point in activity of metamorphs is constrained by local weather con- time differed significantly in a predictable manner consistent ditions, and thus may result in delayed reproduction relative to with growth rates in natural populations (21, 33). There was no paedomorphs. Differences in maturation among the morphs of evidence that growth rate influenced the probability of be- facultatively paedomorphic salamander species have largely coming paedomorphic or metamorphic as metamorphs, pae- been neglected, leading several authors to assume that matu- domorphs, and immature larvae were of similar size within any ration is synchronous (1, 4, 22, 42) and that breeding activity given treatment combination (see also 33). In general, the sizes is concomitant with the arrival of terrestrial metamorphic of metamorphs and paedomorphs are comparable to those adults. collected in other experiments (e.g., refs. 28 and 33) and in We suggest age at maturation as the principal target of natural populations (e.g., refs. 21 and 22). natural selection in the evolution of facultative paedomorpho- The most conspicuous difference in the developmental sis, with the retention of larval morphology following as patterns of the metamorphic (ancestral) and paedomorphic secondary effect. Age at maturation is a major factor in an (derived) life histories pathways of A. talpoideum in our organism’s life history, both in theory and in practice (11, 12, experimental populations is the presence of mature paedo- 42). The primary advantages of earlier maturation are in- morphs before any detectable metamorphic activity. Although creased survival to first reproduction and shortened genera- the pattern of somatic development may be paedomorphic, the tion times (11). Additionally, and perhaps more importantly, pattern of sexual development is clearly peramorphic via the when competition and other density-dependent factors influ- process of predisplacement, the earlier onset or initiation of ence growth and survival as they do in A. talpoideum (28) and the development of a particular trait (2, 4), in this case other amphibians (36, 43, 44), precocious maturation and early maturation. It appears that at least two separate processes reproduction of paedomorphs provides growth and survival involving the development of somatic and reproductive organs advantages to their offspring in extremely low density condi- operate to produce the paedomorphic phenotype. tions. These early growth and͞or survival benefits increase the In organisms with complex life cycles, metamorphosis is a overall quality of the offspring, which later translate into necessary threshold that must be crossed to reach the next increased adult performance or fitness (21). The advantages of stage (35). For most amphibians, the transition from the larval early growth are, of course, not exclusive to salamander or to the postmetamorphic form must occur before the initiation other amphibian larvae; e.g., the fitness benefits of early of maturation (17): few caudate and fewer anuran amphibians emergence in most plant communities are well documented are sexually mature at the completion of metamorphosis. In (45). this context metamorphosis is largely a means to an end. When The benefits of early maturation are sacrificed, however, if new metamorphosis and maturation (i.e., the ‘‘means’’ to the the age at first reproduction is the same as those that mature ‘‘end’’) are decoupled a developmental and life history pat- later; in fact, early maturation then may carry with it significant terns, such as larval paedomorphosis, may evolve (1). To costs (ref. 11; e.g., reduced body size, reduced parental invest- understand the selective pressures resulting in decoupling ment). Thus, if an individual matures early and then undergoes these developmental landmarks, the reproductive ecology of metamorphosis, the benefits of increased survival and higher the organisms must be considered. quality offspring are lost, because the age at first reproduction For A. talpoideum, rainfall and temperature determine the then becomes dependent on the abiotic conditions that permit initiation of breeding migrations, when mature metamorphic the breeding migrations to occur. Rather, to reap the benefits adults leave their subterranean refugia and move to aquatic of early maturation the individual must bypass metamorphosis breeding sites (37). Variation in these abiotic parameters to retain the larval morphological characters that facilitate accounts for a large proportion of the variation in the timing survival in the aquatic habitat. This completed, the age at first of migrations (37, 38); paedomorphic adults, however, are reproduction is constrained less by abiotic factors than by present at the breeding site as soon as they attain maturation. availability of mates and other considerations (e.g., mate Although breeding migrations have been shown to be heavily choice). Therefore, selection favors the retention of the larval dependent on ecological factors, breeding per se has not. morphology in adult salamanders indirectly to allow for the Breeding begins in late September and lasts as late as March potential fitness benefits of an early age at maturation to be (21, 22). When climatic conditions do not permit migration to realized through an early age at first reproduction (Fig. 3). occur until late in the breeding season, paedomorphic adults Because most A. talpoideum retain the ability to metamor- may complete reproduction before the arrival of metamorphic phose after initially reproducing as a paedomorphs (even after adults. For example, Ginger’s Bay (within 10 km of Rainbow several years), we regard the paedomorphic morphology to Bay) usually dries annually but, due to an exceptionally wet result primarily from the indefinite postdisplacement of meta- year, held water continuously from the winter of 1990 through morphosis (Fig. 3). When it occurs, the transition from pae- Downloaded by guest on October 1, 2021 Evolution: Ryan and Semlitsch Proc. Natl. Acad. Sci. USA 95 (1998) 5647

FIG. 3. Reproductive and somatic heterochronies that result in paedomorphosis in Ambystoma talpoideum. A significant decrease in the age at maturation, indicated by shifting the maturation threshold (dashed vertical line) to the left, requires the retention of the larval morphology, regardless of the developmental trajectory (A–D as in Fig. 1). When larvae become paedomorphic through predisplacement of maturation, metamorphosis becomes indefinitely postdisplaced (see text). Images modified from refs. 4 and 47.

domorphic to metamorphic adult does not happen until the assistance in setting up and maintaining the experimental ponds; spring, after the breeding season when other metamorphs (i.e., Amanda Ryan for invaluable assistance in draining the ponds and young-of-the-year) also leave the pond. This allows paedo- collecting the survivors; and Emma Ryan for her patience during the morphs to react adaptively to drastic changes in the usually preparation of the manuscript. The comments and opinions commu- benign and often favorable aquatic habitat (19, 22). nicated by S. L. Ball, J. R. Bodie, J. P. Collins, P. M. Dixon, R. N. Harris, M. L. McKinney, D. Nolte, S. R. Voss, A. M. Welch, H. H. Ambystomatid salamanders possess a considerable amount Whiteman, and three anonymous reviewers improved the clarity of the of developmental plasticity, and the retention of larval features manuscript. This research was made possible through a Grant-in-Aid in reproductive adults may arise through multiple pathways, from Sigma Xi (T.J.R.), Oak Ridge Associated Universities Travel selected by entirely different suites of ecological pressures Contracts (T.J.R. and R.D.S.), and University Research Board Grant (19). The value in our study is that we have compared the 94–074 (R.D.S.). Manuscript preparation was supported by Financial ontogenetic trajectories of organisms whose evolutionary his- Assistance Award Number DE-FC09–96SR18546 from the U.S. De- tory is known, and interpreted them in the context of ecolog- partment of Energy to the University of Georgia Research Foundation ical conditions and fitness benefits that have been vigorously (T.J.R.). studied. A comparative study of this sort is critical to expli- cating the evolutionary processes accounting for the apparent 1. Gould, S. J. (1977) Ontogeny and Phylogeny (Harvard Univ. Press, (morphological) results (1) and has rarely been accomplished Cambridge). 2. McKinney, M. L. & McNamara, K. J. (1991) Heterochrony: The (2). Instead of describing only changes in morphology (e.g., ref. Evolution of Ontogeny (Plenum, New York). 46), though informative, we have evoked an explanation 3. McNamara, K. J. (1995) Evolutionary Change and Heterochrony congruent with heterochronic and life history theories as well (Wiley, New York). as the ecology of the organisms under study. This holistic 4. Reilly, S. M., Wiley, E. O. & Meinhardt, D. J. (1997) Biol. J. Linn. approach leads us to identify age at maturation, a principal Soc. 60, 119–143. component of individual- and population-level fitness, as the 5. Slatkin, M. (1987) Evolution (Lawrence, Kans.) 41, 799–811. most likely target of selection, with retention of the larval 6. Schoch, R. (1995) in Evolutionary Change and Heterochrony, ed. morphology as a secondary effect. The result is a paedomor- McNamara, K. J. (Wiley, New York), pp. 107–124. phic appearance, although the selective forces have been acting 7. Hall, B. K., Miyake, T. (1995) in Evolutionary Change and on life history characters in a manner consistent with per- Heterochrony, ed. McNamara, K. J. (Wiley, New York), pp. 107–124. amorphic development. The error in diagnosing evolutionary 8. Semlitsch, R. D. & Wilbur, H. M. (1989) Evolution (Lawrence, changes in patterns of development based solely on morphol- Kans.) 43, 105–112. ogy is that the primary target of selection, the very character 9. McKinney, M. L. & Gittleman, J. L. (1995) in Evolutionary driving the evolution of ontogeny, may be ignored. Change and Heterochrony, ed. McNamara, K. J. (Wiley, New York), pp. 21–47. We thank Whit Gibbons and Joe Pechmann for supplying the 10. DeBeer, G. (1958) Embryos and Ancestors (Oxford Univ. Press, breeding stock for this experiment; Shelley Ball for much needed London), 3rd Ed. Downloaded by guest on October 1, 2021 5648 Evolution: Ryan and Semlitsch Proc. Natl. Acad. Sci. USA 95 (1998)

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