describe heterochrony, and suggest a more general means of describing hete- ... Events during ontogeny occur in a temporal order, often called a ... A third kind of developmental sequence occurs when an early event is causally required .... many cellular and molecular processes, such as transcription and induction, do not.
J. evol.
Biol.
2: 409-434
(1989)
1010 -061X/89/06409 ~26 $ 1.50 +0.20/O :(; 1989 Birkhiuser Verlag, Base1
Heterochrony: Developmental and evolutionary results Rudolf A. Raff and Gregory
mechanisms
A. Wray
Institute for Molecular and Cellular Biology, University Bloomington, IN 47405, U.S.A. Key words:
Heterochrony;
developmental
and Department
sequence; evolution
of Biology,
Indiana
of development.
Abstract The concept of heterochrony, that the relative timing of ontogenetic events can shift during evolution, has been a major paradigm for understanding the role of developmental processes in evolution. In this paper we consider heterochrony from the perspective of developmental biology. Our objective is to redefine heterochrony more broadly so that the concept becomes readily applicable to the evolution of the full range of ontogenetic processes, from embryogenesis through the adult. Throughout, we stress the importance of considering heterochrony from a hierarchical perspective. Thus, we recognize that a heterochronic change at one level of organization may be the result of non-heterochronic events at an underlying level. As such, heterochrony must be studied using a combination of genetic, molecular, cellular, and morphological approaches.
Introduction As phenotype changes during the course of evolution. so must the developmental processes which give rise to phenotype, and so too the genes which underly development. There are a variety of ways a descendant ontogeny can come to differ from that of its ancestor. These include temporal and spatial changes in development processes, as well as excisions of existing programs and intercalations of novel ones. Any such change, regardless of its nature, depends upon dissociation between developmental process (Needham, 1933). Although ontogeny appears to unfold in a precisely orchestrated fashion, individual processes are often loosely linked to one another. Many developmental events, especially during late development, occur through mechanistically distinct and causally independent pathways. Dissociability of developmental processes is evolutionarily important, because it allows 409
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functionally adaptive modifications of particular developmental programs without macromutational changes of an entire complex and integrated genetic system. Heterochrony, a shift in the relative timing between two developmental processes in a descendant ontogeny, has long been recognized as a major class of developmental dissociations during evolution (Garstang, 1922; Haldane, 1932; DeBeer, 1958; Gould, 1977; Raff and Kaufman, 1983). The concept of heterochrony has been of particular importance in theoretical treatments of the relationships between development and evolution, both because of its utility in evaluating morphological trends in the fossil record, and because heterochronies can be readily recognized and categorized. Numerous studies have documented changes in the relative timing of appearance of morphological features in a wide variety of organisms. In fact, morphological heterochronies are so prevalent, it has become commonplace to regard heterochrony as the most important mechanism for phenotypic change (deBeer, 1958; Gould, 1977; Buss, 1987). Although this may ultimately prove to be the case, the absence of mechanistic information for the overwhelming majority of morphological heterochronies renders such generalizations questionable. The developmental and genetic bases for heterochrony have thus far received scant attention, and have proven more difficult to define than morphological heterochronies. In this paper we examine current concepts of heterochrony from the perspective of developmental biology, and attempt to disentangle heterochronic result from developmental mechanism by stressing the importance of hierarchy in considering heterochronic processes and patterns. We begin by reviewing the distinctions between temporal sequences and causal sequences during development. Next we consider constraints inherent in the traditional nomenclature commonly used to describe heterochrony, and suggest a more general means of describing heterochronic process. We discuss the problem of practical reference points against which to measure timing changes, and then consider the kinds of heterochrony most likely to occur during successive phases of development. Finally, we discuss cases where heterochronic patterns derive from heterochronic as well as from non-heterochronic processes, and briefly review systems where the genetic basis for heterochrony is beginning to emerge.
Temporal series and temporal dissociations Events during ontogeny occur in a temporal order, often called a developmental sequence, but such sequences in development do not necessarily mirror causal sequences (Alberch, 1985). In the hypothetical developmental sequence A-B-C (Fig. I), event C may or may not depend causally upon event B. It could alternatively depend on A or even some other preceding event. The failure to distinguish temporal and causal sequences has led to two widely held, but highly questionable, generalizations which have their roots in the 19th Century ideas of Ernst Haeckel on recapitulation. The first generalization holds that evolutionary modifications of developmental sequences result primarily from terminal additions, deletions, or alterations (Nelson, 1978; Patterson, 1982; Fink, 1982).
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Fig. 1. Temporal sequences and causal sequences, a. Three events, A, B, and C occur in a hypothetical ontogeny in the order given. From this data alone, nothing can be deduced regarding causal relationships There are four formal classes of causal relationships that may exist between these events, as illustrated in the following panels. b. Direct causal sequence, where event A is required for B, which in turn is required for C. c. Indirect causal sequence, where event A causes B, but another event, A’ may substitute for A. d. Bifurcating causal sequences, where event A is required for both B and C, but event B is not required for C. e. Independent causal sequences, where all three events require other, preceeding events, but do not have any influence upon each other. See text for examples and full discussion.
The second, related generalization is that modifications in early development are very uncommon because of their supposedly profound effects on subsequent events (Gould, 1977; Raff and Kaufman, 1983; Eldredge, 1985; Buss, 1987; Arthur, 1988). Empirical evidence demonstrates that the first of these views is simply incorrect, and the second subject to numerous exceptions (Raff et al., 1990). Evolutionary modifications are possible at any point in ontogeny, including the very earliest stages (Sedgewick, 1894; Lillie, 1898; Garstang, 1922; Haldane, 1932; deBeer, 1958; Raff and Kaufman, 1983; Roth, 1984; Alberch, 1985; Wray and McClay, 1989). By definition, any change in the relative order of events in a developmental sequence consititues a heterochrony. Yet the kinds of heterochronies that can arise are constrained by the causal relationships between processes that constitute the sequence. For the temporal sequence of events A-B-C shown in Fig. 1, four broad classes of causal relationships are possible. The nature of causal relationships in a developmental system will determine the relative ease of dissociations of events, and hence the range of possible heterochronies. The first case is a true causal sequence, A-+ B-+C, where each process is necessary for the next (Fig. lb). An example of this kind is found in the development of sea urchin primary mesenchyme cells, which ingress, migrate, and finally synthesize calcareous spicules. The latter two processes cannot occur until its precedecessor is complete. Although the relative order of events cannot be shifted, heterochronies are still possible in rates of execution. Thus event C will always follow event B, but the elapsed time between them can change. A second class of relationships arises in cases of induction (Fig. lc). Here event
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A induces B, which in turn directly causes C. Although B, and therefore C, are mechanistically dependent upon A, dissociation is readily achieved if A is only one element of an inductive network. In such cases, other inducers may be sufficient to trigger event B, and dissociation can occur by a shift to a different major inducer. Event A is thus sufficient, but not necessary, for induction of event B. An example is found in vertebrate eye lens induction, where several tissues contribute to induction of the lens placode. If one inducing tissue is removed experimentally, induction still occurs (Jacobsen, 1966). Here relatively weak constraints maintain the temporal sequence. Another inducing event, A’, can substitute for A, thereby allowing a shift in order as well as elapsed time between A and B-C. A third kind of developmental sequence occurs when an early event is causally required for two separate later events (Fig. Id). In this case, the temporal sequence is still A-B-C, but event C does not depend on event B, it simply follows it in time. Because A -+ B and A + C are independent processes, heterochronies inverting the order of events B and C are possible. The only constraint is that, since B and C are both dependent upon A, neither can precede A. This situation is illustrated by the early development of sea urchins, in which both mesenchyme cell ingression and gastrulation require prior formation of the vegetal plate in the blastula. The two later events are not causally linked to each other: mesenchyme cell ingression occurs before gastrulation in the Euechinoidea, and after it in the Cidaroidea (Tennent, 1922; Wray and McClay, 1988). A final class of temporal sequences occurs when events A, B, and C are all causally unrelated (Fig. le). Since the temporal sequence A-B-C has no causal basis, there are no constraints to inversions of order among the events. Dissociability between processes not causally linked during development provides the basis for two important kinds of evolutionary changes. The first is radical change in early development, in which the larval developmental program is unlinked from that of the adult (Raff et al., 1989, 1990). The second is mosaic evolution, the apparently independent evolution of body parts. Mosaic evolution has long been known from the fossil record (Mayr, 1982). From the perspective of functional morphology, mosaic changes are interpreted as resulting from different selection pressures on various parts of the body. From a developmental perspective, mosaic evolution is possible because development of these features are not mechanistically coupled.
Heterochrony
and the constraints
of nomenclature
Terminology has profoundly influenced perceptions of heterochrony (Gould, 1977; Mayr, 1982). DeBeer’s (1958) classic treatment of heterochrony remains the basis for modern systems of nomenclature (Gould, 1977; Alberch et al., 1979; McNamara, 1986). The central component of what we will refer to as deBeerian heterochrony is the explicit comparison of somatic growth or morphogenesis with the onset of sexual maturity. This makes deBeerian classification particularly useful for analyzing global heterochronies which affect parameters such as overall body size or extent of somatic development at reproductive maturity. Such heterochronies
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may alter life history strategies, and are thus of ecological significance (Haldane, 1932; Wilbur and Collins, 1973; Gould, 1977; Calder, 1984; McKinney, 1986; McNamara, 1986). As will be elaborated in this section, deBeerian definitions nonetheless impose significant conceptual constraints on a more general study of heterochrony. Gould’s ( 1977) revised system of nomenclature considerably clarified deBeer’s (1958) terminology for classes of heterochrony. It has since been elaborated upon by Alberch et al. ( 1979) and McNamara ( 1986) and is in widespread use today. The detection of heterochrony in an altered ontogeny requires that the timing of one event be compared to a second, temporally unchanged event, which acts as a reference point. The onset of sexual maturity is used as the time reference point for deBeerian heterochronies, and the terminology explicitly compares timing rates of growth or appearance of somatic structures relative to the time of sexual maturity. DeBeerian heterochronies fall into eight categories (Table 1) (Alberch et al., 1979; McNamara, 1986). Six of these, the so-called “pure” forms of heterochrony, can affect both size and shape. Three result in paedomorphosis: adults which resemble juveniles of their ancestors. The other three “pure” heterochronies have the opposite result, paramorphosis: adults whose morphology progresses “beyond” that of their ancestors. The remaining two categories of deBeerian heterochrony affect only size, and do not yield paedomorphic or peramorphic results. McNamara ( 1986) reviews diagnostic patterns for classes of deBeerian heterochrony, and provides a useful list of synonyms for the often confusing names by which these catagories have been known. Heterochrony is defined as any change in relative time of appearance of features during ontogeny. Most theoretical and empirical treatments, however, have described heterochrony exclusively in deBeerian terms, as ocurring relative to the time Table 1. The eight classes of deBeerian heterochrony. deBeerian changes in size or shape in a descendent ontogeny. The so-called “pure” these are the first six classes listed. Two additional classes affect only and Alberch et al. (1979).
heterochronies involve temporal forms affect both size and shape; size. Adapted from Gould (1977)
Alteration
Pattern heterochrony
Morphological expression
Gonadal maturation
Somatic maturation
Acceleration Hypermorphosis
same delayed
accelerated same
peramorphosis peramorphosis
Neoteny Progenesis
same accelerated
delayed same
paedomorphisis paedomorphosis
Predisplacement Postdisplacement Proportioned Proportioned
dwarfism giantism
Growth rate
Onset of growth
same same
earlier later
slower faster
same same
peramorphosis paedomorphosis
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of sexual maturity. Definitions which derive exclusively from considerations of temporal alterations relative to sexual maturity impose conceptual and practical constraints on studies of heterochrony. We suggest that a more general concept of heterochrony be adopted, in which “somatic maturation” and “gonadal maturation” of deBeerian definitions are replaced by any two developmental events. This broader perspective is more representative of the spectrum of heterochronic phenomena actually encountered. Because of the way they are defined, deBeerian heterochronies deal primarily with global morphological events late in ontogeny. In the remainder of this section, we consider heterochronies that are not necessarily global, morphological, or late events (see also Fig. 2).
I. Spatial domains DeBeerian heterochronies represent an analytically useful set of definitions for dealing with global heterochronies affecting overall body size and shape. Local heterochronies also occur, however, involving shifts in relative timing between spatially delimited somatic features. Limb morphogenesis in arboreal salamanders of the genus Bolitoglossa provides examples (Larson, 1983). These heterochronies are best considered exclusively within the context of limb development, without reference to morphology beyond the feet. Indeed, most heterochronies are likely to be local rather than global in mechanisms and effects, particularly during late development (Raff et al., 1990). Local heterochronies may be more likely to produce novel morphological features than global heterochronies that preserve ancestral growth patterns. The preponderance of published examples of global rather than local heterochronies may be due in part to the constraints of deBeerian nomenclature: concepts like neoteny are so tied to global effects that is is hard to separate them from it. Even in classic cases of global deBeerian heterochrony, local heterochronies can result from the differential responses of somatic tissues to the dissociation. Thus, in the axolotl, although most global features of the adult retain their larval morphology, some somatic features undergo cryptic metamorphosis: the hematopoetic system, for example, has adult characteristics (Dulcibella, 1974).
2. Temporal domains Heterochronies occur at all stages of development. Although the best known examples occur late in development and are subsumed under deBeerian terminology, heterochronies are also common during early development (Berrill, 1955; Elinson, 1987; Raff, 1987; Wray and McClay, 1989; Raff et al., 1990). Examples of embryonic heterochronies include changes in the number of mitoses preceeding gastrulation in ascidians and sea urchins (Berrill, 1955; Parks et al., 1988) very early initiation of limb bud differentiation in direct developing frogs (Lynn, 1942) and inversions in the order of morphogenetic events in sea urchins (Raff, 1987;
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Fig. 2. Domains of heterochrony. The distribution of theoretical and empirical studies of heterochrony is both uneven and unrepresentative. Here we plot these studies in three dimensional space according to the point in the life cycle at which the heterochrony occurs (eg: fertilization through adult), the space over which the heterochrony is documented (local through global), and the level of biological organization at which the heterochrony occurs (molecular through morphological). Despite the broad definition of heterochrony often quoted, most studies have dealt with global changes in morphology relatively late in development (a). A number of recent studies have begun to expand our knowledge of other types of heterochrony (b-h; see text). Open circles represent l-3 papers, closed circles represent 10 papers; numbers and positions of studies diagrammed are not intended to be exact. a = 251 studies, an estimated compiled from totalling relevant references in Gould (1977) [pre-19771 and papers subsequently published in the journals Paleobiology and Evolution [ 19777mid 19881. b = Raff et al., 1984; Regier and Vlahos, 1988. c = Parks et al., 1988; Wray and McClay, 1989. d = Berrill, 1935; Wray and McClay, 1988. e = Lillie, 1898; Ambros and Horvitz, 1984; Wray and Raff, 1989. f = Raff, 1987; Elinson, 1987. g = Larson, 1983.
Wray and McClay, 1988). Many of these heterochronies do not appear to affect adult morphology, although some proufoundly influence life history strategy. It is clearly impractical to measure timing changes of hours or even minutes in early development relative to sexual maturity, which may occur years later. In addition, such measurements may be meaningless, since reproductive maturity can be triggered by environmental or behavioral factors rather than elapsed time or explicit
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developmental timing cues. Heterochronies measured relative to other events occurring
3. Organizational
in early development in early development.
must instead be
domains
Becausethey are most apparent, the large majority of documented heterochronies involve morphological features. A rapidly growing list of non-morphological heterochronies reflects the fact that, although size and shape are the most commonly studied independent variables, heterochronies occur at all levels of organization in development, including molecular and cellular processes(Cohen and Brown, 1963; Ambros and Horvitz, 1984; Raff et al., 1984; Parks et al., 1988; Regier and Vlahos, 1988; Wray and McClay, 1988, 1989; Wray and Raff, 1989). Temporal changes in many cellular and molecular processes,such as transcription and induction, do not readily admit to classification in deBeerian terms. Instead, treatment of nonmorphological heterochronies requires terms which describe temporal alterations of developmental processesin an appropriate mechanistic framework.
Pattern and process The existence of non-morphological heterochronies raises an important question: what constitutes heterochronic mechanism, molecular and cellular change or morphological change? The answer lies in understanding that distinctions between pattern and process in biology are always relative (Mayr, 1982; Fisher, 1985). As such, the mechanismsof heterochrony do not reside at any single level of processes operative during development. Analyses at all levels of biological organization are thus pertinent to understanding heterochronic mechanism. Given the strong historical bias towards analysis of heterochrony at the level of gross morphology, however, we feel that much remains to be learned from mechanistic approaches centered on other levels of organization. In particular, we expect genetic, molecular, and cellular techniques to complement existing morphological analyses of heterochrony. Studies of this nature often require a perspective of heterochrony more general than the deBeerian one. This section presents a description of heterochrony from the perspective of developmental mechanism. It builds upon existing notions of heterochrony, in particular concepts presented by Alberch et al. (1979). This description is quite general, and may be applied to processesas varied as gene transcription and limb growth. Fundamentally, there are three aspects of a developmental process subject to temporal change: initiation, termination, and rate (Alberch et al., 1979). Initiation and termination may appear either earlier or later in a descendant ontogeny, and rate may increase or decrease. The results will depend upon both the kind of heterochrony which has occurred and the nature of the developmental process in question: either or both the duration of the processand the level of its product may change. The units appropriate for measuring product level will depend upon the
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developmental process in question, and may include size (length, width, volume), shape, concentration (per unit volume or area), number, and so forth. The twelve classes of process heterochrony are represented graphically in Figs. 3-5, and their results are summarized in Table 2. Initiation of a developmental process may occur earlier or later in a descendant than in its ancestor (Fig. 3). Heterochronies in the initiation of a developmental process will alter either both its duration and level, or neither. The duration of a process may be under explicit control, in which case both the times of initiation and termination will both be shifted an equal amount. Neither duration nor level will be affected (Fig. 3A, C). Mechanistically, this situation could arise where a process ends in response to its product reaching a threshold amount (feedback inhibition). Alternately, the time of termination might be under explicit control, and only the time of initiation will be shifted. In this case, both duration and level will change (Fig. 3B, D). Whether process duration and level change as a result of a heterochrony in initiation will determine its outcome. Early initiation of somatic growth provides an illustration: in organisms with determinate growth (duration unchanged), predisplacement or acceleration will result, whereas organisms with indeterminate growth (duration changed) will exhibit proportioned gigantism. The second class of process heterochronies affects the time when a developmental process ends. It may end earlier or later in the descendant ontogeny (Fig. 4). Heterochronies in the termination of a developmental process necessarily alter both its duration and level (Fig. 4A, B). The nature of the developmental process itself Table 2. The ten classes of process heterochrony. Process heterochronies are changes in the time of initiation, time of termination, or rate of any developmental process (Figs. 335). The level of a developmental process (right column) is a relative index of its product or outcome. Two classes of initiation and rate heterochronies can be distinguished (a and b) on the basis of their effects; which situation obtains for any particular developmental process must be determined empirically. Note that different kinds of process heterochronies can produce similar changes in the duration and level of a developmental process. Process heterochrony
Effect Beginning
Early initiation Early initiation Late initiation Late initiation Early termination Late termination
(a) (b) (a) (b)
End
Duration
Level
early early late late same same
same early same late early late
longer same shorter same shorter longer
higher same lower same lower higher
Faster Faster
rate (a) rate (b)
same
same earlier
same shorter
higher same
Slower Slower
rate (a) rate (b)
same same
same later
same longer
lower
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Fig. 3. Heterochronies in process initiation, A hypothetical developmental process in an ancestor (a) and a descendant (d) are compared with regard to developmental time (x-axis) and progress of the particular developmental process (y-axis). For the sake of simplicity, this is diagrammed as a linear relationship; more complex situations often exist in actual developmental systems. A, B. Early initiation. C, D. Late initiation. Whether process duration changes in the descendant depends upon how process termination is regulated: in response to product level (A, C) or at an explicit time (B, D). See text for further details.
Fig. 4. Heterochronies in process termination. A. Early termination. B. Late termination. In both cases, both the duration of the process and its level are altered. See text for details. The format of this figure follows that of Fig. 3.
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Fig. 5. Heterochronies in process rate. A, B. Faster rate. C, D. Slower rate. Either duration or product level are altered in the descendant; the outcome depends upon how process termination is regulated, in response to product level (A, C) or at an explicit time (B, D). See text for details. The format of this figure follows that of Fig. 3.
will determine the effects on pattern. If the process is cell proliferation underlying growth, early termination might result in proportioned dwarfism and late termination in proportioned giantism. If, on the other hand, the process is cell death, the effects will be reversed. Finally, a development process may progress at a different rate. The new rate may be either faster of slower than that of the ancestor (Fig. 5), and will alter either the duration of a process or its level. Thus a faster rate may result in either a shorter duration (Fig. 5A) or a higher level (Fig. 5B). As with heterochronies in initiation, which situation occurs will depend upon the mechanisms controlling the time of termination. It is important to note that alterations in rate can mechanistically derive either from timing changes or from changes in amount, since rate = amount/ time. Evolutionary change in amount has been termed heteroposy (Regier and Vlahos, 1988). The relationship between a heterochrony in developmental process and its effect on phenotype is often indirect. One type of morphological heterochrony can be caused by more than one kind of heterochrony in developmental process (Table 2).
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For example, a shortened period of growth can be the result of late initiation, early termination, or faster rate (compare Figs. 3A, 4A, and SA). In many cases, careful comparison of both duration and sequence patterns in ancestor and descendant ontogenies may provide a clue to the underlying process alterations (Table 2). Thus, if the duration of an event is unchanged, but it both starts and ends earlier, it is likely due to early initiation with duration explicitly defined (i.e., Fig. 3A). It is important to note, however, that inferences about process based on growth patterns alone are valid only in cases where a single process heterochrony has occurred, and should therefore be made with caution. Finally, as will be amplified below, not all morphological heterochronies result from heterochronies in developmental process. Similarly, not all heterochronies in developmental process produce morphological heterochronies. Classifying heterochronic change in terms of alterations in developmental process offers several heuristic and practical advantages. First, by focusing on initiation, rate, and termination, it allows a more concise description of the nature of timing changes in development (Alberch et al., 1979). Second, because it is based on a broader concept of heterochrony, this description has broader utility. It can be applied to heterochronies of any spatial scope and at any level of organisation, and because it does not rely on gonadal maturation as a temporal reference point, it is readily applicable to heterochronies at any point in the life cycle. And finally, heterochronic change is categorized in mechanistically descriptive, semantically neutral terms.
The problem of practical reference points In practical terms, documenting a heterochrony requires that a change in relative timing of two events be shown between ancestor and descendant ontogenies. Given a timing change, the next question concerns the polarity of the heterochrony, whether the event occurs earlier or later in the ontogeny of the descendant relative to that of the ancestor. Were the ontogeny of both ancestor and descendant known, the polarity of a pattern heterochrony could be convincingly argued. This ideal is, however, never fully realized: the ancestor is rarely known, and its ontogeny is never sufficiently well characterized. Standard phylogenetic tools of cladistic analysis can often be brought to bear in determining the ancestral condition for any evolutionary change in a developmental event or process. As with phylogenetic analysis, accurate choice of the outgroup is vital, but often problematic. The use of closely related groups reduces irrelevant noise, offers a better chance of finding appropriate reference events, and maximizes confidence that homologous processes are being compared (Raff et al., 1989). A particularly nice example is seen in Lord and Hill’s (1987) analysis of heterochronies in angiosperms, where two flower forms occur in the same plant. Assuming that appropriate phylogenetic information is available for the ontogenies being compared, determining the polarity of a heterochrony requires some sort of developmental time frame. Unfortunately, there exists no objective frame of
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reference against which to measure timing changes, since all events during development are subject to evolutionary modification. The time of gonadal maturation is commonly used as temporal reference point 1986). Yet, as discussed above, this (deBeer, 1958; Gould, 1977; McNamara, imposes severe practical problems for measuring small shifts in timing during early development; most heterochronies in the embryo would remain undetected if timed relative to sexual maturity. The usefulness of this referent is further lessened in organisms where the attainment of sexual maturity is influenced by environmental or behavioral cues. Buss (1987) has suggested that the time of germ line segregation be substituted for gonadal maturation as a reference point early in development. However, this event is often difficult to determine in extant species, and is clearly impractical from fossils. Other temporal frameworks for ontogeny have their own disadvantages for assessing the polarity of a heterochrony. Using overall developmental stage removes the reliance upon a single event whose timing might itself have changed. Thus, one might frame heterochronies in relation to gastrulation, which is a key early developmental process. This formulation, however, introduces several other problems. The most obvious is that embryos often evolve in a mosaic manner (Garstang, 1928; Needham, 1933; Alberch, 1985; Raff et al., 1989) making it difficult to identify truly equivalent developmental stages. For example, embryos of related species may be at the “same” stage with regard to number of somities, but not with regard to extent of neural tube closure. Even embryos which appear to be very similar can differ significantly in non-morphological aspects (Wray and McClay, 1989) making direct comparisons between morphologically similar stages problematic. A further difficulty arises when entire developmental stages are missing or out of sequence in the descendant (Raff, 1987; Wray and McClay, 1988). Another temporal framework, absolute time, is also of limited utility, because few embryos develop at the same overall rate. Rearing embryos at the same temperature may provide equivalent overall rates in closely related species, but the rates of individual developmental processes may not be uniform. Another possible temporal framework is provided by measuring ratios of absolute times of developmental events. Thus, an event which occurs at 25 % of the time interval between gastrulation and hatching in the ancestor, but at 30 % of the same interval in a descendant would constitute a retardation. The drawback, of course, is that the timing of the endpoints which define the interval also may undergo evolutionary change. The fact that all events in ontogeny are subject to heterochronic change presents a fundamental and inescapable problem in interpreting empirical information. The only event in development not subject to heterochrony appears to be fertilization. However, fertilization is hardly an ideal referent against which to measure most heterochronies. Although all developmental events are subject to temporal change in evolution, every system for classifying and analyzing heterochronies assumes that polarities of individual heterochronies can be determined. The most convincing support for polarity is to show a consistent offset in the timing of the altered developmental event relative to the timing of several other reference events. In general, the more
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such reference events, the more strongly corroborated the polarity of the heterochrony becomes. The most useful reference events are those occurring at about the same time during ontogeny as the altered event, as these are most capable of revealing subtleties in timing. Referent choice is as important in analyzing heterochrony as character choice is in systematics, and for much the same reason. Reference events are only useful if they are not themselves causally linked to the displaced event. Further, where multiple reference events are used, it is also important that they be causally independent of each other. If instead they form a linked class of events, such as a series of events triggered by a single hormonal cue, they effectively comprise a single referent.
Temporal domains As mentioned earlier, there are convincing examples of heterochronies throughout ontogeny. In this section, we discuss how the mechanistic bases for heterochronies at different phases of animal development may differ.
I. Early development During early development, the major events revolve around rapid partitioning of the egg cytoplasm into a large number of cells, and commitment of newly established cell lineages to specific and regionalized pathways of differentiation in the embryo. Heterochronies are extremely common during early development in numerous groups, including coelenterates, annelids, mollusks, crustaceans, echinoderms, ascidians, and amphibians (Berrill, 1935; Raff and Kaufman, 1983; Elinson, 1987; Raff, 1987; Matsuda, 1987; Wray and McClay, 1989). The most widely studied heterochronies in embryogenesis are those associated with changes in developmental mode. The evolution of larval development entails intercalations of novel structures and developmental processes to aid feeding and dispersal (Garstang, 1929) whereas abbreviation to more direct development results in loss or modification of larval features (Elinson, 1987; Matsuda, 1987; Raff, 1987). Such cases have been analyzed in amphibians (Wake and Larson, 1987; Lynn, 1942; Elinson, 1987) and in many invertebrate groups (Mileikovsky, 1971; Whittaker, 1979; Raff, 1987; Matsuda, 1987). Heterchronies may in part be possible in early embryos because distinct genetic compartments underly production of the larva and adult. The development of a juvenile may not require expression of all genes of larval development, nor development of the typical suite of feeding larval structures (Raff et al., 1989). Heterochronies are also possible in early ontogeny in instances where a lack of cell interactions allows dissociation of developmental processes. Cell lineages often differentiate autonomously during early development. As development proceeds, interactive controls, such as induction, become important and cell lineage autonomy decreases. The relative degree of autonomy and interaction among cell lineages
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varies greatly between groups: in nematodes, relatively few inductive interactions occur, whereas in vertebrates, there are extensive inductive interactions. Temporal dissociations can be readily achieved under circumstances of cell lineage autonomy, as long as developmental processes eventually produce a defined set of cells capable of coherent interaction later in development.
2. Organogenesis Gastrulation, which produces the first juxtapositions of cell sheets, marks the onset of organogenesis. Inductive interactions typically begin to assume major roles in specifying tissue identities, and in localizing organs. The first inductions often occur during gastrulation, and block out major regional identities (Mangold, 1933; Holtfreter, 1936); specification of individual structures becomes more precise with additional inductions. Processes involving inductive interactions are very rich in potential heterochronies. Changes in timing can occur if production of an inducer begins at a different time or occurs at different rate. Dissociation can also occur if the duration of competence to respond changes. Together, production of inducer and competence to respond creates a window within which induction is constrained. Further possibilities arise from situations in which the inducing tissues are moving relative to the induced tissue. In these cases the dwell time of the inducer in the vicinity of the induced tissue can be critical in determining both the size and placement of structures. Both induction and morphogenetic movements of cells and cell sheets relative to one another are prevalent in animal development (Nieuwkoop et al., 1985). Induction of the amphibian eye lens depends not upon a single induction by the retina, but by a continuing series of inductions by underlying sheets of endoderm and mesoderm as they move past the prospective eye region (Jacobson, 1966; Henry and Grainger, 1987). The retina, which arises from neural tissue, serves as the last inducer. Unfortunately, no studies of heterochronies involving induction are available. An example of the kind of heterochronies that should be sought is specification of limb bud formation in vertebrates. Limb buds are induced by the underlying somites. The site of limb bud formation, which varies, has been demonstrated by grafting experiments to be controlled by the inducing, and not the responding tissue (Kieny, 1971; Raymond, 1977). Timing of limb bud formation varies as well, and in the direct developing frog Eleutherodactylus is initiated very early (Lynn, 1942).
3. Metamorphosis Most animals undergo some form of metamorphosis, and evolutionary modifications of adult body form can result from changes in metamorphic controls. Numerous examples are known from the amphibians, which provide some of the best studied examples of heterochrony. One class of heterochronies affecting amphibian metamorphosis involves retention of ancestral larval features by a
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reproductively mature adult descendant. There presistent larval features of aquatic, gilled salamanders provided the first and most dramatic examples of neoteny (Kollman, 1885). The Mexican axolotl, then called Siredon mexicanum, had been observed by Dumeril (1865, 1867) to undergo metamorphosis into adults of Ambystoma, a genus of terrestrial salamanders. The axolotl became the prototype of neoteny.
4. Late development Dramatic shifts in final body proportions are achieved by heterochronies in the growth of individual body parts late in development. This has been especially well documented for domestic dogs, which constitute a single, morphologically diverse species (Wayne 1986a, b). All dog crania are similar in proportion and size at birth. Most small breeds are paedomorphic with respect to ratio of skull width to length, with a close correspondence of ontogenetic and intraspecific allometries. During postnatal growth of some breeds, relative growth rates deviate from the ontogenetic trajectory of the more wolf-like breeds. This alteration results in a relatively wider head. Dogs also differ significantly in limb proportions, but here the basis for heterochronic dissociation is somewhat different. Most of the differences in leg length result from differences in prenatal and perinatal growth rates. These results are significant because domestic dogs are genetically very closely related, and all have similar gestation times (60-63 days) and perinatal growth periods (0 to 40 days of age) (Asdell, 1966; Wayne, 1986b). Despite apparent constraints on cranial growth patterns and gestation time in domestic dogs, intraspecific heterochronies in late development have resulted in body forms as distinct as those seen between other canid genera. Changes in adult body proportion are often examples of hypermorphosis. This heterochronic result represents growth along the ancestral ontogeny, followed by further growth beyond the ancestral adult state. Although the result is recapitulation, a quite distinctive new body morphology can be achieved. An example is provided by the large extinct European deer called the Irish Elk (Gould, 1974). Its huge antlers are a direct result of larger body size rather than a novel growth pattern. Since antler growth has a strong positive allometry with body size, a large deer grows very large antlers. Heterochronies arise during all phases of development from dissociations between developmental processes. Dissociations occur at all stages, although by different mechanisms as ontogeny proceeds, and as different kinds processes come to dominate development. In the following sections, we consider the kinds of developmental mechanisms which produce heterochronic results.
Heterochronic
mechanisms
Whether one sees a particular heterochrony generally depend on the level of organization
as a result or as a mechanism will being examined. In some instances,
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such as paleontological studies, where developmental information is rare, or in dealing with ecological consequences of heterochrony, data will usually be sought at the morphological level of organization and late in development. Gould’s (1977) analysis of heterochrony and patterns of selection in ambystomid salamanders is an example. The precise molecular or cellular mechanisms may be irrelevant to understanding neoteny as a mechanism at this level, and deBeerian concepts and terminology are appropriate. If heterochrony is to be studied from a developmental perspective, however, morphological heterochronies become the result of mechanisms operating at cellular and molecular levels of organization, and it is these mechanisms which are of primary interest. As developmental biologists interested in relationships between ontogeny and phylogeny, we would like to consider two important questions about heterochronic mechanism. First, is heterochrony really the predominant mode of evolutionary change? If so, this tells us something profound about the logic of development and points to the sorts of phenomena upon which we should focus attention. And second, what kinds of alterations in developmental processes underly morphological heterochronies? The nature of these alterations reflects how genotypic changes are translated into phenotypic changes. Although the first question is likely to remain unanswered for some time, the second can be addressed with data at hand. It is clear that morphological heterochronies can sometimes result from developmental mechanisms that do not involve timing control per se. In this section, we consider examples of morphological heterochrony which stem from non-heterochronic mechanisms (Table 3A) as well as heterochronic mechanisms (Table 3B). The clearest example of non-heterochronic mechanism yielding a heterochronic result is amphibian neoteny, the classic case of deBeerian heterochrony. The hormonal basis for amphibian metamorphosis and neoteny has been extensively studied (Etkin, 1970; Dodd and Dodd, 1976; White and Nicoll, 1981; Duellman and Trueb, 1986). Metamorphosis in amphibians is primarily a responseof body tissues to a dramatic rise in thyroxine, a hormone produced by the thyroid gland in response to maturation of the hypothalamus. In casesof neoteny, metamorphosis does not occur. Some species are permanent neotenes; their target tissues are no longer responsive to exogenous thyroxine. Other species, such as the axolotl, are facultative neotenes. Their thyroxine levels do not normally rise high enough to trigger metamorphosis; however, artificial exposure of the animal to thyroxine will induce metamoprhosis. Neoteny is due to a failure in the hypothalamic release mechanism (Norris and Gern, 1976) in turn the result of a single gene change (Tompkins, 1978). The loss of thyroid function in this animal produces a classical global neoteny. Since gonadal maturation does not require thyroxine (Dodd and Dodd, 1976), it is dissociated from the events of metamorphosis. Additional cryptic metamorphic changes, such as in serum proteins and hemoglobins, also occur (Ducibella, 1974). These metamorphic changes may occur in tissuesrequiring very low levels of thyroxine. Whereas the morphological result is a heterochrony in which somatic growth is retarded and gonadal maturation is unchanged, the developmental mechanism does not involve timing of development processesat all. What has in fact occurred is that some tissues, including reproductive tissues, have achieved an adult state, while
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others have not. The descendant is a mosaic. Dissociation has indeed occurred, but at a mechanistic level it is more validly viewed as a dissociation in hormonal response than as a heterochrony. Its importance as a heterochrony resides in its consequencesfor life history strategy and ecology (Wilbur and Collins, 1973). The next example in Table 3 involves an apparent acceleration in cell division during early seaurchin development. The direct developing sea urchin Heliocidaris erythrogramma contains roughly ten times the number of cells as its typical developing congener H. tuberculata, at both the sameembryonic stage and the same absolute time (Parks et al., 1988). This results not from a change in rates of cell division, but instead from a change in the time division ends. This in turn is controlled by the ratio of nuclear to cytoplasmic volume (Newport and Kirschner, 1982a,b). The difference in egg volume between the two Heliocidaris species is approximately IOO-fold, resulting in a change in the time cleavage ends. The developmental mechanism underlying the morphological heterochrony thus lies in a Table
3. Developmental
mechanisms
underlying
Example A. Mechanisms Axolotl (adult larval body)
heterochrony.
mechanism
in timing. failure to activate hypothalamicpituitary-thyroid axis
neoteny
target tissues unable to respond to thyroxine by metamorphosing
More cells in gastrula of lecithotrophic sea urchin
acceleration
altered nucleo/cytoplasmic ratio during cleavage
Precocious vertical cleavage in sea urchin vegetal cells
predisplacement
imposition of novel mechanisms cleavage plane determination
larval
B. Mechanisms Sculpturing egg shell
in
in the text
neoteny
Permanently salamanders
gonad
involved
cited
Developmental
Result not directly
Sources
directly
involving
of silkmoth
volume
for
timing. hypermorphosis
late expression of duphcated chorion genes; higher level of filler protein
Direct development of adult rudiment in sea urchins
predisplacement acceleration
Retarded somatic development in nematode
neoteny
persistence
Smaller yeast
progenesis
mitotic shorten
changed
to
hypermorphosis
mitotic timing control changed lengthen growth phase
to
Larger yeast
cell size in fission
cell size in fission
and
excision of larval gene expression; early initiation of adult genes of /in- I4 protein
timing control growth phase
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change in nucleo-cytoplasmic ratio, not an explicit change in timing. Dramatic effects of egg size on cell number also occur in ascidians (Berrill, 1935). The last example from Table 3A involves a heterochrony which distinguishes sea urchin development from that of other echinoderm embryos. During the fourth cleavage of non-echinoid embryos, all mitotic spindles are oriented horizontally. During the next cleavage all mitotic spindles are oriented vertically. In sea urchin embryos at fourth cleavage, four cells have the expected horizontal mitotic spindles, but four have the vertical spindles expected of the next cleavage, a predisplacement. The mechanism, however, is not a predisplacement of the normal vertical fifth cleavage spindle. Instead, an elaborate cellular structure in the relevant cells removes the spindles from their normal horizontal fourth cleavage orientation and reorients them into a vertical plane (Dan, 1979). The mechanism is profoundly different than a simple predisplacement of a normal fifth cleavage. Because the regulation of timing in biological systems is still not well understood, it is difficult to pin down heterochronic mechanisms in developmental processes underlying morphology. In a few cases, however, significant progress has been made, and it is becoming possible to study timing controls in development experimentally. Table 3B presents examples of heterochronic results that can be attributed to mechanisms directly involving timing changes in development. Because studies of this nature have just begun, the depth of understanding of mechanism varies among these examples. In general, mechanisms of timing change are more amenable to experimental analysis earlier in development and at more simple levels of organization. The first example involves the evolution of sculpting on silkmoth egg shells (Hatzopoulos and Regier, 1987). These egg shells are assembled from chorion proteins produced from a large family of chorion genes. To a rough approximation, there are early, middle, and late expressed chorion genes which encode proteins specific to various layers of the egg shell. Antheraea polyphemus egg shells differ from those of Hyalophora cecropiu primarily in elaborate ornamentation of the terminally deposited, outermost chorion layer. The basis of this late ornamentation lies in a higher level of expression of a filler protein present in both species, as well as the expression of a devergent set of lamellar protein genes unique to A. polyphemus. These late expressed genes apparently represent modified descendants of a duplicated set of genes expressed earlier in silkmoth oogenesis. This “hypermorphosis” in eggshell structure thus appears to arise from changes in timing of expression of a specific set of genes during a specific phase of egg shell development. Heterochronic changes in chorion gene expression are apparently involved in differences in eggshell formation between silkmoths and other families of moths as well (Regier and Vlahos, 1988). Heterochronies affecting embryonic development are prevalent in the evolution of direct developing sea urchins (Raff, 1987; Parks et al., 1988; Wray and Raff, 1989). Acceleration of adult gene expression and morphogenesis appears to occur through two complementary processes: elimination of larval programs of cell differentiation and early differentiation of critical adult cell lineages. Thus larval expression of the gene msp130 appears to be eliminated in direct developers, while adult expression
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is accelerated in both absolute and relative developmental time (Parks et al., 1988). Recent studies demonstrate that gene expression is largely controlled by the presence of proteins which bind to upstream DNA sequences (Watson et al., 1987). Presumably the basis of the msp130 heterochrony lies in an alteration in the presence of such a regulatory protein. Mutations in the lin-14 gene of the nematode Caenorhabditis elegans produce heterochronies at several levels of developmental process (Ambros and Horvitz, 1984, 1987; Ruvkun and Guisto, 1989). The lin-14 locus regulates timing during nematode development, and two classesof mutations in this gene cause opposite heterochronic effects. Semidominant mutations produce a neotenous result: sexual maturity occurs on time, but larval cuticular structures and cell lineage patterns are retained. Recessive mutations in the same gene produce the opposite effect: adult cuticular structures and cell lineage patterns appear in larval stages, a progenetic result. Semidominant mutations causethe gene product, a nuclear protein, to persist well past its wild type time of degradation. The fin-14 gene product acts as a developmental switch between early and late cell lineage behaviors, and also participates in regulating the larval-to-adult developmental switch (Ambros, 1989). This system beautifully illustrates the hierarchical nature of heterochrony: morphological changes result from alterations in cell lineage patterns, which in turn are controlled by changes in gene activity. The fission yeast, Schizosaccharomyces pombe offers the simplest genetic system which, upon mutation, produces heterochronic effects (Nurse, 1985; Russell and Nurse, 1986, 1987a, b). Fission yeast are rod-shaped cells which normally undergo mitosis when they reach a length of 14 pm. Mutations have been recovered which result in initiation of mitosis at both shorter (7.8 pm) and longer (up to 34 pm) cell lengths. These mutations reveal a temporal control system composed of cdc2, whose product initiates mitosis, and wee1 and cdc2.5, which act respectively as repressor and activator of cdc2. The antagonism between repressor and activator regulator proteins allows isolation of mutant yeast that exhibit deBeerian heterochronies. Mitosis constitutes reproductive maturity in yeast, and cell length is an appropriate index of growth. If the ratio of activator protein to repressor protein is high, cell division is initiated at a small size, and is analogous to progenesis. If the ratio is low, cell division occurs in long cells. The individual recapitulates the normal course of development and continues beyond it, a situation corresponding to hypermorphosis. The analogy to the classic global heterochronies defined for more complex animal systems is evident. Genetic manipulations affect a regulatory system which governs the relationship between cell cycle events and growth, and illustrates how heterochrony can result form very simple regulatory interactions. A second important result has emerged from characterization of cell cycle control genes in fission yeast. DNA sequence comparisons have allowed identification of genesin animals homologous to cdc2 and cdc25, as well as to cdcl3, which acts in concert with cdc2. Mutational analysis demonstrates that these loci also control mitosis during animal development. A cdc2 protein homolog is a component of maturation-promoting factor (Dunphy et al., 1988; Gautier et al., 1988), which
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controls the initiation of mitosis in a wide variety of invertebrate and vertebrate embryos (Ford, 1985; Kirschner et al., 1985; Labbe et al., 1988). The animal homolog of yeast cdcl3 is known as cyclin (Evans et al., 1983; Solomon et al., 1988; Goebl et al., 1988). Following fertilization, cyclin is synthesized during each cell cycle, and is destroyed as cells enter mitosis. These oscillations regulate the activity of maturation-promoting factor and provide a cyclical timing mechanism controlling cell cleavage during early development (Swenson et al., 1986; Draetta et al., 1989). The srg locus of Drosophila, which is homologous to yeast cdc25, also regulates embryonic cell division (Edgar and O’Farrell, 1989); spatial and temporal patterns of stg protein expression control localized cell division in the early embryo. Embryologists have long recognized the existence of a “cleavage clock” operating during early development (Hiirstadius, 1973; Freeman, 1983). If cleavage is reversibly blocked during sea urchin development, a characteristic unequal mitosis occurs at the proper absolute time, but one cleavage cycle early. This and other experiments reveal dissociability between cleavage number and cleavage pattern. We have demonstrated heterochronies in early embryos of direct developing sea urchins which involve changes in both cleavage kinetics and timing of cell fate decisions (Parks et al., 1988; Wray and Raff, 1989). Whether cleavage clock mechanisms are involved in these heterochronies remains to be investigated. However, at least some aspects of the genetic control system governing mitosis now appear to be common to a wide variety of eukaryotes (Lee and Nurse, 1988). Loci which regulate cell division offer a great potential for mechanistic alterations which can produce both local and global morphological heterochronies. These genes clearly deserve further scrutiny, and provide useful experimental systems for investigating heterochrony.
Conclusions Heterochrony continues to be a major paradigm for considering the interplay between developmental and evolutionary processes. A rich diversity of examples demonstrates that morphological heterochronies are common, and may provide important evolutionary innovations. As appropriate experimental tools become available, it is important that the mechanistic changes in development which underlie these heterochronies be characterized. Not surprisingly, some of the conceptual bases employed for primarily morphological studies are inappropriate for cellular and molecular approaches to heterochrony. We have presented a system describing heterochronic changes which is suitable for mechanistic studies at these levels. By avoiding explicit comparisons of timing changes during development to gonadal maturation, it is possible for studies of heterochrony to transcend the realm of global morphological events occurring late in development. In presenting this broader description of heterochrony, we do not intend to displace existing teminology and approaches. The traditional deBeerian framework is appropriate for describing many morphological heterochronies. The new framework describes a broader set of both pattern and process heterochronies. For
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systems where developmental mechanism has not been, or cannot be explored, the traditional nomenclature is preferable. This includes the vast majority of documented cases of heterochrony: most of these are global in nature and many derive from the fossil record. Paleontologists, comparative morphologists, and systematists encounter and describe heterochronies as morphological change. Developmental biologists, on the other hand, now have the opportunity to analyze heterochrony at several organizational levels. In studying heterochrony, whether the particular case be test length in Eocene echiniods or gene expression patterns in living echinoids, it is helpful to keep several points in mind. First, developmental sequences can be, but are not necessarily, causal sequences. The nature of casual relationships within a developmental sequence can constrain the kinds of evolutionary changes that are likely to occur. Second, heterochrony can occur at any stage of the life cycle, and in any developmental process at any level of biological organization. Although most published examples describe deBeerian heterochronies, a growing literature has begun to document timing changes in early development, in spatially localized processes, and at the level of cells and molecules. Third, the selection of an appropriate temporal framework against which to measure heterochronic change is problematic, since all reference points are themselves subject to evolutionary shifts in timing. In so far as is possible, reference points should be independent of the development event being studied, and several reference points are better than one. Fourth, morphological heterochronies are not always the result of heterochronies in developmental processes; similarly, not all process heterochronies result in overt morphological heterochronies. The relationship between developmental process and pattern is sufficiently complex as to require empirical rather than theoretical explication, since similar morphological results can ensue from quite distinct underlying causes. Dissociation provides a productive concept upon which to build an understanding of the interrelationships between developmental and evolutionary processes. Heterochrony has proven the most readily demonstrable form of dissociation. Its continued utility as a conceptual paradigm depends both upon extending studies beyond the classical bounds of global morphological events late in development, and upon probing developmental mechanisms underlying timing changes. Experimental approaches to heterochrony are becoming feasible, allowing testable predicitons about evolutionary change to be made. It is important to caution, however, that the temporal nature of development guarantees almost any change in developmental process will produce some effect on timing. As we continue to study the role of development in evolution, heterochronies causing evolutionary change must therefore be distinguished from heterochronies arising as incidental results of other dissociations. If this is not done, we risk robbing the concept of heterochrony of explanatory power by uncritically ascribing all morphological change to it. Acknowledgements We thank Allan Larson, Susan Lawler, Brian Parr, Jerome Regier, and two anonymous reviewers for insightful comments and suggestions. This study was supported by NIH research grant HD21337 and a Guggenheim Fellowship to RAR, and by NIH postdoctoral fellowship GM12495 to GAW.
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