Methods for the analysis of developmental sequence data

SUMMARY Heterochrony, evolutionary changes in developmental timing, can be studied either by examining changes in growth or changes in the sequence of developmental events. Developmental sequence data has the potential to address many important questions in the field of developmental evolution, but methodological challenges remain due to the biological and logical dependence of events in a ranked sequence. In the past 10 years, the study of sequence heterochrony has undergone a rebirth, with the creation of several new methods for the analysis of this type of data. These methods can be divided into two broad categories: phenetic comparisons between terminal taxa that strive to uncover integrations within the developmental sequences and putative shared sequence heterochronies, and phylogeny-based methods that derive ancestor-descendent sequence heterochronies and establish statements of sequence evolution. In this review, we will discuss the strengths and weaknesses of the methodologies that have been proposed to quantitatively examine developmental sequence data, and studies that have attempted to implement them in an evolutionary context.     

Frequent nonrandom shifts in the temporal sequence of developmental landmark events during teleost evolutionary diversification

Morphological transformations can be generated by evolutionary changes in the sequence of developmental events. In this study, we examined the evolutionary dynamics of the developmental sequence on a macroevolutionary scale in teleosts. Using the information from previous reports describing the development of 31 species, we extracted the developmental sequences of 19 landmark events involving the formation of phylogenetically conserved body parts; we then inferred ancestral developmental sequences by two different parsimony‐based methods—event‐pairing and continuous analysis. The phylogenetic comparisons of these sequences revealed event‐dependent heterogeneity in the frequency of sequence changes. Most of the sequence changes occurred as exchanges of temporally neighboring events. These heterochronic changes in developmental sequences accumulated along evolutionary time, but the precise distribution of the changes over the teleostean phylogeny remains unclear due to technical limitations.     

Heterochrony revisited: the evolution of developmental sequences

The concept of heterochrony is a persistent component of discussions about the way that evolution and development interact. Since the late 1970s heterochrony has been defined largely as developmental changes in the relationship of size and shape. This approach to heterochrony, here termed growth heterochrony, is limited in the way it can analyse change in the relative timing of developmental events in a number of respects. In particular, analytical techniques do not readily allow the study of changes in developmental events not characterized by size and shape parameters, or of many kinds of events in many taxa. I discuss here an alternative approach to heterochrony, termed sequence heterochrony, in which a developmental trajectory is conceptualized as a series of discrete events. Heterochrony is demonstrated when the sequence position of an event changes relative to other events in that sequence. I summarize several analytical techniques that allow the investigation of sequence heterochrony in phylogenetic contexts and also quantitatively. Finally, several examples of how this approach may be used to test hypotheses on the way development evolves are summarized.     

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.     

An integrative approach identifies developmental sequence heterochronies in freshwater basommatophoran snails

Adopting an integrative approach to the study of sequence heterochrony, we compared the timing of developmental events encompassing a mixture of developmental stages and functional traits in the embryos of 12 species of basommatophoran snails in an explicit phylogenetic framework. PARSIMOV analysis demonstrated clear functional heterochronies associated both with basal branches within the phylogeny and with terminal speciation events. A consensus of changes inferred under both accelerated transformation and delayed transformation optimizations identified four heterochronies where the direction of movement was known plus six twin heterochronies where the relative movements of the two events could not be assigned. On average, 0.5 and 0.58 events were inferred to have changed their position in the developmental sequence on internal and terminal branches of the phylogeny, respectively; these values are comparable with frequencies of sequence heterochrony reported in mammals. Directional heterochronies such as the early occurrence of body flexing in relation to the ontogeny of the eye spots, heart beat, and free swimming events occurred convergently and/or at different levels (i.e., familial, generic, and species) within the phylogeny. Such a functional approach to the study of developmental sequences has highlighted the possibility that heterochrony may have played a prominent role in the evolution of this group of invertebrates.     

Can heterochrony help explain the high morphological diversity within the genus Niphargus (Crustacea: Amphipoda)?

Postembryonic development of nine species of Niphargus (Crustacea: Amphipoda) was studied, with spine development and shifts in allometric growth being combined in developmental sequences that were compared across species. The developmental sequences show high diversity with respect to the position of individual events in the sequence, as well as a high frequency of events being inapplicable in some species. Within Niphargus, the highest degree of independence between events occurs mainly in early mid-aged instars, where the shifts in position are both the largest in magnitude and also the most frequent in occurrence. Constructive troglomorphic features of subterranean species were inferred to develop more because of accelerated growth rather than a delayed offset of growth. Shifts in both relative timing and growth rate appeared to have played a role in the evolution of sexually dimorphic elongated appendages. Growth patterns differed greatly between species for individual sexually dimorphic and troglomorphic traits, hinting at their possible independent origin. The independence between developmental events that was generally apparent might indicate the existence of an important genetic basis for the extreme intrageneric morphological variation arising from numerous highly variable body parts being combined in a ‘mosaic’ manner. Moreover, we suggest that the overall diversity of the genus might actually be underestimated in light of possible convergent features accompanied by ‘cryptic’ speciation. We conclude that both sequential and growth heterochrony appear to have played a key role in the evolution of Niphargus, the most diverse genus of freshwater amphipods. A preliminary list of heterochronic characters is provided.     

DIFFERENCES IN THE TIMING OF PRECHONDROGENIC LIMB DEVELOPMENT IN MAMMALS: THE MARSUPIAL–PLACENTAL DICHOTOMY RESOLVED

In contrast to placentals, marsupials are born with forelimbs that are greatly developmentally advanced relative to their hind limbs. Despite significant interest, we still do not know why this is the case, or how this difference is achieved developmentally. Studies of prechondrogenic and chondrogenic limbs have supported the traditional hypothesis that marsupial forelimb development is accelerated in response to the functional requirements of the newborn's crawl to the teat. However, limb ossification studies have concluded that, rather than the forelimb being accelerated, hind limb development is delayed. By increasing the taxonomic coverage and number of prechondrogenic events relative to previous studies, and combining traditional phylogenetic analyses of event sequences with novel analyses of relative developmental rates, this study demonstrates that the timing of limb development in marsupials is more complex than commonly thought. The marsupial phenotype was derived through two independent evolutionary changes in developmental rate: (1) an acceleration of the forelimb's first appearance and (2) a delay of hind limb development from the bud stage onward. Surprisingly, this study also provides some support for an evolutionary acceleration of the marsupial hind limb's first appearance. Further study is needed on the developmental and genetic mechanisms driving these major evolutionary transitions.     

Evolution of ossification sequences in salamanders and urodele origins assessed through event-pairing and new methods

SUMMARY Ossification sequences of the skull in extant Urodela and in Permo‐Carboniferous Branchiosauridae have already been used to study the origin of lissamphibians. But most of these studies did not consider some recent methods developed to analyze the developmental sequences within a phylogenetic framework. Here, we analyze the ossification sequences of 24 cranial bones of 23 extant species of salamanders using the event‐pairing method. This reveals new developmental synapomorphies for several extant salamander taxa and ancestral sequences for Urodela under four alternative reference phylogenies. An analysis with the 12 bones for which ossification sequence data are available in urodeles and in the branchiosaurid Apateon is also performed in order to compare the ancestral condition of the crown‐group of Urodela to the sequence of Apateon. This reveals far more incompatibilities than previously suggested. The similarities observed between some extant salamanders and branchiosaurids may result from extensive homoplasy, as the extreme variation observed in extant Urodela suggests, or be plesiomorphic, as the conservation of some ossification patterns observed in other remotely related vertebrates like actinopterygians suggests. We propose a new, simpler method based on squared‐change optimization to estimate the relative timing of ossification of various bones of hypothetical ancestors, and use independent‐contrasts analysis to estimate the confidence intervals around these times. Our results show that the uncertainty of the ancestral ossification sequence of Urodela is much greater than event‐pairing suggests. The developmental data do not allow to conclude that branchiosaurids are closely related to salamanders and their limited taxonomic distribution in Paleozoic taxa precludes testing hypotheses about lissamphibian origins. This is true regardless of the analytical method used (event‐pairing or our new method based on squared‐change parsimony). Simulations show that the new analytical method is generally more powerful to detect evolutionary shifts in developmental timing, and has lower Type I error rate than event‐pairing. It also makes fewer errors in ancestral character value or state assignment than event‐pairing.     

Variation, plasticity and modularity in anuran development

Although anuran development is generally thought to be relatively conservative, a great deal of variation is evident when different species are compared. This report summarizes the results of comparative analyses of different aspects of anuran development. These include differences in sequence and timing of developmental events, the effects of genome size, and the effects of different life history strategies on anuran embryogenesis. The results show that anuran development is plastic at the evolutionary level, and many changes can occur in the developmental processes of anurans throughout their evolution. Changes are apparently rapid, and are as common as cladogenic events. This evolutionary plasticity can be attributed to the modular nature of anuran development. Different modules can shift relative to one another in time or in space, creating variations in the observed developmental patterns. However, shifts in modules can occur even without having a significant effect on the ultimate outcome of the process. I discuss the implications of the modular nature of development on the evolution of anuran development, and of the group in general.     

Estimating evolution of temporal sequence changes: a practical approach to inferring ancestral developmental sequences and sequence heterochrony

Developmental biology often yields data in a temporal context. Temporal data in phylogenetic systematics has important uses in the field of evolutionary developmental biology and, in general, comparative biology. The evolution of temporal sequences, specifically developmental sequences, has proven difficult to examine due to the highly variable temporal progression of development. Issues concerning the analysis of temporal sequences and problems with current methods of analysis are discussed. We present here an algorithm to infer ancestral temporal sequences, quantify sequence heterochronies, and estimate pseudoreplicate consensus support for sequence changes using Parsimov-based genetic inference [PGi]. Real temporal developmental sequence data sets are used to compare PGi with currently used approaches, and PGi is shown to be the most efficient, accurate, and practical method to examine biological data and infer ancestral states on a phylogeny. The method is also expandable to address further issues in developmental evolution, namely modularity.     

Variation in anuran embryogenesis: differences in sequence and timing of early developmental events

Comparative embryology of closely related species can shed light on the evolution of developmental processes. An important mechanism in the evolution of developmental processes, which can lead to significant changes in larval or adult form, is variation in the sequence and timing of developmental events. We compared the development of 12 species of anurans, including a wide taxonomic range as well as a number of congeneric species. The comparison consisted of monitoring a series of external morphological markers and histological markers. For each species we noted the timing of each of the markers, using a uniform parameter of normalized time. We compared the normalized time of each of these events among the species, as well as the sequence of the events. Our analysis revealed many differences in sequence and in timing of developmental events. We mapped these differences on a cladogram of the studied species, using sequence units as discrete characters. The differences do not seem to be connected to the phylogenetic relations between the species or to any obvious ecological factors. We suggest a hypothetical ancestral sequence of developmental events, and discuss the possible factors that could have caused the observed variations from the ancestral sequence.     

Assessing the phylogenetic utility of sequence heterochrony: evolution of avian ossification sequences as a case study

The evolution of developmental sequences, or sequence heterochrony, is an emerging field of study that addresses the temporal interplay between evolution and development. Some phylogenetic signal has been found in developmental sequence data, but sampling has generally been limited to small numbers of taxa and few developmental events. Here we present the largest ossification sequence dataset to date. The sequences are composed of ossification events throughout the avian skeleton, and are used to address the evolutionary signal of ossification sequence data within this clade. The results indicate that ossification sequences are conserved in birds, and show a stronger phylogenetic signal than previous studies, perhaps due to the volume of data. Phylogenetic signal is not strong enough, however, to consider ossification sequence data to be any better at resolving phylogenetic hypotheses than other morphological data and just as prone to evolutionary convergence. There is no one-to-one correlation between ossification sequence and developmental stage. We discuss some methodological implications of our findings, as well as commonalities in avian ossification sequences such as early ossification of the long bones relative to the dermatocranium, and of the hindlimb over the forelimb.     

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.     

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.     

Heterochrony in plant evolutionary studies through the twentieth century

The evolution of plant morphology is the result of changes in developmental processes. Heterochrony, the evolutionary change in developmental rate or timing, is a major cause of ontogenetic modification during evolution. It is responsible for both interspecific and intraspecific morphological differences. Other causes include heterotopy, the change of structural position, and homeosis, the replacement of a structure by another. This paper discusses and reviews the role of heterochrony in plant evolution at the organismal, organ, tissue, cellular, and molecular levels, as well as the relationships among heterochrony, heterotopy, and homeosis. An attempt has been made to include all published studies through late 1999. It is likely that most heterochronic change involves more than one of the six classic pure heterochronic processes. Of these processes, we found neoteny (decreased developmental rate in descendant), progenesis (earlier offset), and acceleration (increased rate) to be more commonly reported than hypermorphosis (delayed offset) or predisplacement (earlier onset). We found no reports of postdisplacement (delayed onset). Therefore, although rate changes are common (both neoteny and acceleration), shifts in timing most commonly involve earlier termination in the descendant (progenesis). These relative frequencies may change as more kinds of structures are analyzed. Phenotypic effects of evolutionary changes in onset or offset timing can be exaggerated, suppressed, or reversed by changes in rate. Because not all developmental changes responsible for evolution result from heterochrony, however, we propose that plant evolution be studied from a viewpoint that integrates these different developmental mechanisms.     

The power of zebrafish models for understanding the co‐occurrence of craniofacial and limb disorders

Craniofacial and limb defects are two of the most common congenital anomalies in the general population. Interestingly, these defects are not mutually exclusive. Many patients with craniofacial phenotypes, such as orofacial clefting and craniosynostosis, also present with limb defects, including polydactyly, syndactyly, brachydactyly, or ectrodactyly. The gene regulatory networks governing craniofacial and limb development initially seem distinct from one another, and yet these birth defects frequently occur together. Both developmental processes are highly conserved among vertebrates, and zebrafish have emerged as an advantageous model due to their high fecundity, relative ease of genetic manipulation, and transparency during development. Here we summarize studies that have used zebrafish models to study human syndromes that present with both craniofacial and limb phenotypes. We discuss the highly conserved processes of craniofacial and limb/fin development and describe recent zebrafish studies that have explored the function of genes associated with human syndromes with phenotypes in both structures. We attempt to identify commonalities between the two to help explain why craniofacial and limb anomalies often occur together.     

Development of the cranium and paired fins in Betta splendens (Teleostei: Percomorpha): Intraspecific variation and interspecific comparisons

The development of the chondrocranium and the relative timing of ossification of the osteocranium is described in the teleost fish Betta splendens from a large series of cleared and differentially stained specimens. General trends in ossification patterns are examined from developmental, phylogenetic, and functional contexts. As in many other vertebrates, dermal bones form before cartilage bones. Ossification sequence conforms to functional need in a very general way, but there are many inconsistencies in the details of order. For example, some bones that are directly involved in feeding ossify no earlier than bones more indirectly involved. Comparisons of ossification sequence within specific cranial regions are made among Betta splendens, Oryzias latipes (Atherinomorpha), and Barbus barbus (Ostariophysi) within a phylogenetic framework. Many evolutionary changes in relative sequence of ossification are evident within regions among these taxa, yet many other sequences are conserved. The logistic difficulty of comparing entire cranial ossification sequences (vs. regional sequences) makes evident the need for new methods for identifying and quantifying sequence changes. Intraspecific variation in order of ossification is described for the first time in teleost fishes. To the extent that ossification sequence varies intraspecifically, conclusions drawn from previous interspecific comparisons are compromised. Understanding the importance of changes in ossification order within and among taxa will require experimental, functional, and evolutionary work. © 1996 Wiley-Liss, Inc.     

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.     

Different cranial ontogeny in Europeans and Southern Africans

Modern human populations differ in developmental processes and in several phenotypic traits. However, the link between ontogenetic variation and human diversification has not been frequently addressed. Here, we analysed craniofacial ontogenies by means of geometric-morphometrics of Europeans and Southern Africans, according to dental and chronological ages. Results suggest that different adult cranial morphologies between Southern Africans and Europeans arise by a combination of processes that involve traits modified during the prenatal life and others that diverge during early postnatal ontogeny. Main craniofacial changes indicate that Europeans differ from Southern Africans by increasing facial developmental rates and extending the attainment of adult size and shape. Since other studies have suggested that native subsaharan populations attain adulthood earlier than Europeans, it is probable that facial ontogeny is linked with other developmental mechanisms that control the timing of maturation in other variables. Southern Africans appear as retaining young features in adulthood. Facial ontogeny in Europeans produces taller and narrower noses, which seems as an adaptation to colder environments. The lack of these morphological traits in Neanderthals, who lived in cold environments, seems a paradox, but it is probably the consequence of a warm-adapted faces together with precocious maturation. When modern Homo sapiens migrated into Asia and Europe, colder environments might establish pressures that constrained facial growth and development in order to depart from the warm-adapted morphology. Our results provide some answers about how cranial growth and development occur in two human populations and when developmental shifts take place providing a better adaptation to environmental constraints.