Analyzing developmental sequences within a phylogenetic framework

Heterochrony is important as a potential mechanism of evolutionary change. However, the analysis of developmental timing data within a phylogenetic framework to identify important shifts has proven difficult. In particular, analytical problems with sequence (event) heterochrony revolve around the lack of an absolute time frame in development to allow standardization of timing data across species. An important breakthrough in this regard is the method of "event-pairing," which compares the relative timing of developmental events in a pairwise fashion. The resulting event-pair-encoded data can be mapped onto a phylogeny, which can provide important biological information. However, event-paired data are cumbersome to work with and lack a rigorous quantitative framework under which to analyze them. Critically, the otherwise advantageous relativity of event-pairing prevents an assessment of whether one or both events in a single event-pair have changed position during evolutionary history. Building on the method of event-pairing, we describe a protocol whereby event-pair transformations along a given branch are analyzed en bloc. Our method of "event-pair cracking" thereby allows developmental timing data to be analyzed quantitatively within a phylogenetic framework to infer key heterochronic shifts. We demonstrate the utility of event-pair cracking through a worked example and show how it provides a set of desired features identified by previous authors.     

A genetic basis for intraspecific differences in developmental timing?

Heterochrony, altered developmental timing between ancestors and their descendents, has been proposed as a pervasive evolutionary feature and recent analytical approaches have confirmed its existence as an evolutionary pattern. Yet, the mechanistic basis for heterochrony remains unclear and, in particular, whether intraspecific variation in the timing of developmental events generates, or has the potential to generate, future between‐species differences. Here we make a key step in linking heterochrony at the inter‐ and intraspecific level by reporting an association between interindividual variation in both the absolute and relative timing (position within the sequence of developmental events) of key embryonic developmental events and genetic distance for the pond snail, Radix balthica. We report significant differences in the genetic distance of individuals exhibiting different levels of dissimilarity in their absolute and relative timing of developmental events such as spinning activity, eyespot formation, heart ontogeny, and hatching. This relationship between genetic and developmental dissimilarity is consistent with there being a genetic basis for variation in developmental timing and so suggests that intraspecific heterochrony could provide the raw material for natural selection to produce speciation.     

Polymorphism in developmental timing: intraspecific heterochrony in a Lake Victoria cichlid

SUMMARY Biologists measure developmental time by dividing development into arbitrary time blocks called "stages." This is a reasonable approach, provided that developmental timing is precisely controlled within a species. However, the degree of this precision is unknown. This is unfortunate because precision in developmental timing at the population level is a central issue to the whole research program of heterochrony. To examine this issue, we apply Ontogenetic Sequence Analysis to 261 embryos of the Lake Victoria cichlid Haplochromis piceatus . The result of our analysis can be mapped as a complex web of 26,880 equally parsimonious developmental sequences. This topology reflects timing polymorphism (intraspecific heterochrony) among embryos of this species. Because of this timing polymorphism, it is not possible to define discrete "stages" in this cichlid (although there is sufficient sequence signal to assess the maturity of embryos). More generally, we show that sequence polymorphism creates uncertainty about how a given embryo will develop implying that the mechanisms controlling developmental timing in embryos lack precision. For this reason, it is imperative to consider patterns of embryonic variability when measuring developmental time.     

Spatiotemporal reorganization of growth rates in the evolution of ontogeny

Abstract. Heterochrony, evolutionary changes in rate or timing of development producing parallelism between ontogeny and phylogeny, is viewed as the most common type of evolutionary change in development. Alternative hypotheses such as heterotopy, evolutionary change in the spatial patterning of development, are rarely entertained. We examine the evidence for heterochrony and heterotopy in the evolution of body shape in two clades of piranhas. One of these is the sole case of heterochrony previously reported in the group; the others were previously interpreted as cases of heterotopy. To compare ontogenies of shape, we computed ontogenetic trajectories of shape by multivariate regression of geometric shape variables (i.e., partial warp scores and shape coordinates) on centroid size. Rates of development relative to developmental age and angles between the trajectories were compared statistically. We found a significant difference in developmental rate between species of Serrasalmus , suggesting that heterochrony is a partial explanation for the evolution of body shape, but we also found a significant difference between their ontogenetic transformations; the direction of the difference between them suggests that heterotopy also plays a role in this group. In Pygocentrus we found no difference in developmental rate among species, but we did find a difference in the ontogenies, suggesting that heterotopy, but not heterochrony, is the developmental basis for shape diversification in this group. The prevalence of heterotopy as a source of evolutionary novelty remains largely unexplored and will not become clear until the search for developmental explanations looks beyond heterochrony.     

Using heterochrony plots to detect the dissociated coevolution of characters

The comparison of developmental sequences among species is notoriously difficult. Here, heterochrony plots are introduced as a new graphic method to detect temporal shifts in the development of characters in pair-wise species comparisons. Plotting the timing of character development in one species against the timing of character development in another species allows us to compare a principally unlimited number of characters simultaneously and can detect whether suites of characters are dissociated from one another or not. Such heterochrony plots can be embedded into a comparative phylogenetic analysis in order to establish whether observed patterns of character codissociation are indeed due to their dissociated coevolution. Comparative phylogenetic analysis may also reveal multiple independent events of dissociated coevolution of the same suite of characters in a certain lineage, suggesting that the characters of this suite reciprocally constrain their evolutionary modifiability, thereby forming a unit of evolution. This ability to identify units of evolution is a prerequisite for assessing the validity of recently proposed scenarios, suggesting that modules of development and/or function tend to act as units of evolution. Starting from a detailed heterochrony plot comparing development in the direct developing frog Eleutherodactylus coqui and in the biphasically developing frog Discoglossus pictus, this comparative approach is illustrated focusing on the evolution of development of limbs, the nervous system and the pharyngeal arches in amphibians.     

Time's arrow: heterochrony and the evolution of development

The concept of heterochrony, which denotes a change in the relative timing of developmental events and processes in evolution, has accompanied attempts to link evolution and development for well over a century. During this time the definition of heterochrony and the application of the concept have varied and by the late 1990's, many questioned the usefulness of the concept. However, in the past decade studies of heterochrony have been revitalized by a new focus on developmental sequence, an examination of heterochrony in explicit phylogenetic contexts and increasing tendencies to examine the heterochrony of many kinds of events, including cellular, molecular and genetic events. Examples of such studies are reviewed in this paper and it is argued that this new application of heterochrony provides an extraordinarily rich opportunity for understanding the developmental basis of evolutionary change.     

Inverting the hourglass: quantitative evidence against the phylotypic stage in vertebrate development

The concept of a phylotypic stage, when all vertebrate embryos show low phenotypic diversity, is an important cornerstone underlying modern developmental biology. Many theories involving patterns of development, developmental modules, mechanisms of development including developmental integration, and the action of natural selection on embryological stages have been proposed with reference to the phylotypic stage. However, the phylotypic stage has never been precisely defined, or conclusively supported or disproved by comparative quantitative data. We tested the predictions of the 'developmental hourglass' definition of the phylotypic stage quantitatively by looking at the pattern of developmental-timing variation across vertebrates as a whole and within mammals. For both datasets, the results using two different metrics were counter to the predictions of the definition: phenotypic variation between species was highest in the middle of the developmental sequence. This surprising degree of developmental character independence argues against the existence of a phylotypic stage in vertebrates. Instead, we hypothesize that numerous tightly delimited developmental modules exist during the mid-embryonic period. Further, the high level of timing changes (heterochrony) between these modules may be an important evolutionary mechanism giving rise to the diversity of vertebrates. The onus is now clearly on proponents of the phylotypic stage to present both a clear definition of it and quantitative data supporting its existence.     

Sequence heterochrony and the evolution of development

One of the most persistent questions in comparative developmental biology concerns whether there are general rules by which ontogeny and phylogeny are related. Answering this question requires conceptual and analytic approaches that allow biologists to examine a wide range of developmental events in well-structured phylogenetic contexts. For evolutionary biologists, one of the most dominant approaches to comparative developmental biology has centered around the concept of heterochrony. However, in recent years the focus of studies of heterochrony largely has been limited to one aspect, changes in size and shape. I argue that this focus has restricted the kinds of questions that have been asked about the patterns of developmental change in phylogeny, which has narrowed our ability to address some of the most fundamental questions about development and evolution. Here I contrast the approaches of growth heterochrony with a broader view of heterochrony that concentrates on changes in developmental sequence. I discuss a general approach to sequence heterochrony and summarize newly emerging methods to analyze a variety of kinds of developmental change in explicit phylogenetic contexts. Finally, I summarize a series of studies on the evolution of development in mammals that use these new approaches.     

Ontogeny and phylogeny of the trunk lateral line system in cichlid fishes

An examination of the ontogeny of the lateral line trunk canal and the diversity of adult trunk canal patterns among cichlids indicates that bidirectional canal formation is a general ontogenetic pattern in the Cichlidae with the exception of Cichla and those few species with a complete trunk canal pattern. In addition to the tubed scales which make up the trunk canal, some lateral line scales have pits containing superficial neuromasts. These are recognized as components of the lateral line system of the trunk in adult cichlids for the first time. Eight trunk canal patterns that are variations on a simple disjunct pattern are defined among the 17 cichlid genera examined. Using bidirectional canal formation as a developmental model, these patterns can be placed along an ontogenetic spectrum. This suggests that heterochrony (alterations in the timing of development) is an important mechanism of evolutionary change in the lateral line system of the trunk in cichlid fishes.     

Evo-Devo: evolutionary developmental mechanisms

Evolutionary developmental biology (Evo-Devo) as a discipline is concerned, among other things, with discovering and understanding the role of changes in developmental mechanisms in the evolutionary origin of aspects of the phenotype. In a very real sense, Evo-Devo opens the black box between genotype and phenotype, or more properly, phenotypes as multiple life history stages arise in many organisms from a single genotype. Changes in the timing or positioning of an aspect of development in a descendant relative to an ancestor (heterochrony and heterotopy) were two evolutionary developmental mechanisms identified by Ernst Haeckel in the 1870s. Many more have since been identified, in large part because of our enhanced understanding of development and because new mechanisms emerge as development proceeds: the transfer from maternal to zygotic genomic control; cell-to-cell interactions; cell differentiation and cell migration; embryonic inductions; functional interactions at the tissue and organ levels; growth. Within these emergent processes, gene networks and gene cascades (genetic modules) link the genotype with morphogenetic units (cellular modules, namely germ layers, embryonic fields or cellular condensations), while epigenetic processes such as embryonic inductions, tissue interactions and functional integration, link morphogenetic units to the phenotype. Evolutionary developmental mechanisms also include interactions between individuals of the same species, individuals of different species, and species and their biotic and/or abiotic environment. Such interactions link ecological communities. Importantly, there is little to distinguish the causality that underlies these interactions from that which underlies inductive interactions within embryos.     

Predator cues alter the timing of developmental events in gastropod embryos

Heterochrony, differences in the timing of developmental events between descendent species and their ancestors, is a pervasive evolutionary pattern. However, the origins of such timing changes are still not resolved. Here we show, using sequence analysis, that exposure to predator cues altered the timing of onset of several developmental events in embryos of two closely related gastropod species: Radix balthica and Radix auricularia. These timing alterations were limited to certain events and were species-specific. Compared with controls, over half (62%) of exposed R. auricularia embryos had a later onset of body flexing and an earlier occurrence of the eyes and the heart; in R. balthica, 67 per cent of exposed embryos showed a later occurrence of mantle muscle flexing and an earlier attachment to, and crawling on, the egg capsule wall. The resultant developmental sequences in treated embryos converged, and were more similar to one another than were the sequences of the controls for both species. We conclude that biotic agents can elicit altered event timing in developing gastropod embryos. These changes were species-specific, but did not occur in all individuals. Such developmental plasticity in the timing of developmental events could be an important step in generating interspecific heterochrony.     

Mammalian organogenesis in deep time: tools for teaching and outreach

Mammals constitute a rich subject of study on evolution and development and provide model organisms for experimental investigations. They can serve to illustrate how ontogeny and phylogeny can be studied together and how the reconstruction of ancestors of our own evolutionary lineage can be approached. Likewise, mammals can be used to promote 'tree thinking' and can provide an organismal appreciation of evolutionary changes. This subject is suitable for the classroom and to the public at large given the interest and familiarity of people with mammals and their closest relatives. We present a simple exercise in which embryonic development is presented as a transformative process that can be observed, compared, and analyzed. In addition, we provide and discuss a freely available animation on organogenesis and life history evolution in mammals. An evolutionary tree can be the best tool to order and understand those transformations for different species. A simple exercise introduces the subject of changes in developmental timing or heterochrony and its importance in evolution. The developmental perspective is relevant in teaching and outreach efforts for the understanding of evolutionary theory today.     

From Haeckel to event-pairing: the evolution of developmental sequences

Summary Development involves a series of developmental events, separated by transformations, that follow a particular order or developmental sequence. The sequence may in turn be arbitrarily subdivided into contiguous segments (developmental stages). We discuss the properties of developmental sequences. We also examine the differing analytical approaches that have been used to analyse developmental sequences in an evolutionary context. Ernst Haeckel was a pioneer in this field. His approach was evolutionary and he introduced the idea of sequence heterochrony (evolutionary changes in the sequence of developmental events). Despite the availability of detailed developmental data (e.g. Franz Keibel’s ‘Normal Tables’), Haeckel was unable to undertake a quantitative analysis of developmental data. This is now possible through computer-based analytical techniques such as event-pairing, which can extract important biological information from developmental sequences by mapping them onto established phylogenies. It may also yield data that can be used in phylogeny reconstruction, although the inherent ‘non-independence’ of the data may make this invalid. In future, the methods discussed here may be applied to the analysis of patterns of gene expression in embryos, or adapted to studying gene order on chromosomes.     

Cranial modularity and sequence heterochrony in mammals

Heterochrony, the temporal shifting of developmental events relative to each other, requires a degree of autonomy among those processes or structures. Modularity, the division of larger structures or processes into autonomous sets of internally integrated units, is often discussed in relation to the concept of heterochrony. However, the relationship between the developmental modules derived from studies of heterochrony and evolutionary modules, which should be of adaptive importance and relate to the genotype-phenotype map, has not been explicitly studied. I analyzed a series of sectioned and whole cleared-and-stained embryological and neonatal specimens, supplemented with published ontogenetic data, to test the hypothesis that bones within the same phenotypic modules, as determined by morphometric analysis, are developmentally integrated and will display coordinated heterochronic shifts across taxa. Modularity was analyzed in cranial bone ossification sequences of 12 therian mammals. A dataset of 12-18 developmental events was used to assess if modularity in developmental sequences corresponds to six phenotypic modules, derived from a recent morphometric analysis of cranial modularity in mammals. Kendall's tau was used to measure rank correlations, with randomization tests for significance. If modularity in developmental sequences corresponds to observed phenotypic modules, bones within a single phenotypic module should show integration of developmental timing, maintaining the same timing of ossification relative to each other, despite differences in overall ossification sequences across taxa. Analyses did not find any significant conservation of developmental timing within the six phenotypic modules, meaning that bones that are highly integrated in adult morphology are not significantly integrated in developmental timing.     

Heterochrony and allometry: the analysis of evolutionary change in ontogeny

The connection between development and evolution has become the focus of an increasing amount of research in recent years, and heterochrony has long been a key concept in this relation. Heterochrony is defined as evolutionary change in rates and timing of developmental processes; the dimension of time is therefore an essential part in studies of heterochrony. Over the past two decades, evolutionary biologists have used several methodological frameworks to analyse heterochrony, which differ substantially in the way they characterize evolutionary changes in ontogenies and in the resulting classification, although they mostly use the same terms. This review examines how these methods compare ancestral and descendant ontogenies, emphasizing their differences and the potential for contradictory results from analyses using different frameworks. One of the two principal methods uses a clock as a graphical display for comparisons of size, shape and age at a particular ontogenic stage, whereas the other characterizes a developmental process by its time of onset, rate, and time of cessation. The literature on human heterochrony provides particularly clear examples of how these differences produce apparent contradictions when applied to the same problem. Developmental biologists recently have extended the concept of heterochrony to the earliest stages of development and have applied it at the cellular and molecular scale. This extension brought considerations of developmental mechanisms and genetics into the study of heterochrony, which previously was based primarily on phenomenological characterizations of morphological change in ontogeny. Allometry is the pattern of covariation among several morphological traits or between measures of size and shape; unlike heterochrony, allometry does not deal with time explicitly. Two main approaches to the study of allometry are distinguished, which differ in the way they characterize organismal form. One approach defines shape as proportions among measurements, based on considerations of geometric similarity, whereas the other focuses on the covariation among measurements in ontogeny and evolution. Both are related conceptually and through the use of similar algebra. In addition, there are close connections between heterochrony and changes in allometric growth trajectories, although there is no one-to-one correspondence. These relationships and outline links between different analytical frameworks are discussed.     

Is Sequence Heterochrony an Important Evolutionary Mechanism in Mammals?

It is postulated widely that changes in developmental timing (i.e., heterochrony) represent a major mechanism of evolutionary change. However, it is only with recent methodological advances that changes in the order in which development proceeds (sequence heterochrony) can be identified and quantified. We apply these techniques to examine whether heterochrony in the early embryonic (organogenetic) period has played an important role in the diversification of mammals. Although we find clear instances of sequence heterochrony in mammals, particularly between eutherians and marsupials, the majority of mammalian lineages that we could examine (those within the major clades Euarchontoglires and Laurasiatheria) show few or no heterochronic changes in the 116 events examined (e.g., Artiodactyla, Euarchonta, Fereuungulata, Glires, Primates, Rodentia). This is in contrast with the timing shifts reported between and within other tetrapod clades. Our results suggest that sequence heterochrony in embryonic stages has not been a major feature of mammalian evolution. This might be because mammals, and perhaps amniotes in general, develop for an extended time in a protected environment, which could shield the embryos from strong diversifying selection. Our results are also consistent with the view that mammal embryos are subject to special developmental constraints. Therefore, other mechanisms explaining the diversity of extant mammals must be sought.