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.     

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.     

Mosaic heterochrony and evolutionary modularity: the trilobite genus Zacanthopsis as a case study

Logical connections exist between evolutionary modularity and heterochrony, two unifying and structuring themes in the expanding field of evolutionary developmental biology. The former sees complex phenotypes as being made up of semi-independent units of evolutionary transformation; the latter requires such a modular organization of phenotypes to occur in a localized or mosaic fashion. This conceptual relationship is illustrated here by analyzing the evolutionary changes in the cranidial ontogeny of two related species of Cambrian trilobites. With arguments from comparative developmental genetics and functional morphology, we delineate putative evolutionary modules within the cranidium and examine patterns of evolutionary changes in ontogeny at both global and local scales. Results support a case of mosaic heterochrony, that is, a combination of local heterochronies affecting the different parts individuated in the cranidium, leading to the complex pattern of allometric repatterning observed at the global scale. Through this example, we show that recasting morphological analyses of complex phenotypes with a priori knowledge or hypotheses about their organizational and variational properties can significantly improve our interpretation and understanding of evolutionary changes among related taxa, fossil and extant. Such considerations open avenues to investigate the large-scale dynamics of modularity and its role in phenotypic evolution.     

Cranial Suture Closure Patterns in Sciuridae: Heterochrony and Modularity

Studies of sequence heterochrony in mammalian evolution have revealed differences in bone ossification between and within major clades. Sequences of late stage developmental events have been less well studied, and the relation of modularity to sequence heterochrony at these stages has not been explicitly tested. Here, the first data on cranial suture closure are provided for members of Sciuridae. Sequence heterochrony is quantified using the recently developed Parsimov-based genetic inference (PGi) algorithm to identify shifts in suture closure sequence, and modularity in heterochronic shifts is tested. Results indicate that suture closure pattern was quite variable among sciurids, and interspecific correspondence in closure sequence was generally lower than has been found for other rodents, and for carnivorans. A number of sequence heterochronies were detected for inclusive clades, and these were not randomly distributed but mainly concentrated among sutures that exhibited high rank variability and belonged to the orbit and cranial vault modules, suggesting that some regions of the cranium exhibited a greater capacity for variation in suture closure. Heterchronies were not detected for sutures belonging to the basicranium, or anterior orbit-nasal modules, both of which are recognized as highly integrated modules based on landmark data. Modularity of suture closure sequence was not significant for any modules following multiple-comparison correction, which contrasts with modularity that has been recovered in early stage, ossification sequence events among other mammals.     

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.     

Parent–offspring similarity in the timing of developmental events: an origin of heterochrony?

Understanding the link between ontogeny (development) and phylogeny (evolution) remains a key aim of biology. Heterochrony, the altered timing of developmental events between ancestors and descendants, could be such a link although the processes responsible for producing heterochrony, widely viewed as an interspecific phenomenon, are still unclear. However, intraspecific variation in developmental event timing, if heritable, could provide the raw material from which heterochronies originate. To date, however, heritable developmental event timing has not been demonstrated, although recent work did suggest a genetic basis for intraspecific differences in event timing in the embryonic development of the pond snail, Radix balthica. Consequently, here we used high-resolution (temporal and spatial) imaging of the entire embryonic development of R. balthica to perform a parent–offspring comparison of the timing of twelve, physiological and morphological developmental events. Between-parent differences in the timing of all events were good predictors of such timing differences between their offspring, and heritability was demonstrated for two of these events (foot attachment and crawling). Such heritable intraspecific variation in developmental event timing could be the raw material for speciation events, providing a fundamental link between ontogeny and phylogeny, via heterochrony.     

Skeletogenesis and sequence heterochrony in rodent evolution, with particular emphasis on the African striped mouse, Rhabdomys pumilio (Mammalia)

Data documenting skeletal development in rodents, the most species-rich ‘order’ of mammals, are at present restricted to a few model species, a shortcoming that hinders exploration of the morphological and ecological diversification of the group. In this study we provide the most comprehensive sampling of rodent ossification sequences to date, with the aim of exploring whether heterochrony is ubiquitous in rodent evolution at the onset of skeletal formation. The onset of ossification in 17 cranial elements and 24 postcranial elements was examined for eight muroid and caviomorph rodent species. New data are provided for two non-model species. For one of these, the African striped mouse, Rhabdomys pumilio, sampling was extended by studying 53 autopodial elements and examining intraspecific variation. The Parsimov method of studying sequence heterochrony was used to explore the role that changes in developmental timing play in early skeletal formation. Few heterochronies were found to diagnose the muroid and caviomorph clades, suggesting conserved patterning in skeletal development. Mechanisms leading to the generation of the wide range of morphological diversity encapsulated within Rodentia may be restricted to later periods in development than those studied in this work. Documentation of skeletogenesis in Rhabdomys indicates that intraspecifc variation in ossification sequence pattern is present, though not extensive. Our study suggests that sequence heterochrony is neither pivotal nor prevalent during early skeletal formation in rodents.     

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.     

A new technique for identifying sequence heterochrony

Sequence heterochrony (changes in the order in which events occur) is a potentially important, but relatively poorly explored, mechanism for the evolution of development. In part, this is because of the inherent difficulties in inferring sequence heterochrony across species. The event-pairing method, developed independently by several workers in the mid-1990s, encodes sequences in a way that allows them to be examined in a phylogenetic framework, but the results can be difficult to interpret in terms of actual heterochronic changes. Here, we describe a new, parsimony-based method to interpret such results. For each branch of the tree, it identifies the least number of event movements (heterochronies) that will explain all the observed event-pair changes. It has the potential to find all alternative, equally parsimonious explanations, and generate a consensus, containing the movements that form part of every equally most parsimonious explanation. This new technique, which we call Parsimov, greatly increases the utility of the event-pair method for inferring instances of sequence heterochrony.     

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.     

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.     

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.     

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.     

Phylogenies of the Frigatebirds (Fregatidae) and Tropicbirds (Phaethonidae), two divergent groups of the traditional order Pelecaniformes, inferred from mitochondrial DNA sequences

The frigatebirds (Fregatidae) and Tropicbirds (Phaethonidae) represent the most morphologically and behaviorally distinct members of the traditional Order Pelecaniformes. Using 1756bp of mitochondrial DNA sequence consisting of the 12S, ATPase-6, ATPase-8, and COI genes obtained from all extant species, we derive a completely resolved phylogeny for both groups. The inferred relationships among these species are robust to the method of phylogenetic estimation used, and all branches are well supported, in spite of the relatively recent radiation within the frigatebirds. The two families are not closely related either to each other, or to any other putative relatives (e.g., pelicans; Pelecanidae).     

Experimental heterochrony in hydractiniid hydroids: Why mechanisms matter

As with most clonal organisms, hydractiniid hydroids display a range of morphological variation from sheet-like to runner-like forms. Life history differences correlate with these morphological traits, exhibiting patterns commonly found in studies of heterochrony. Experimental studies of two hydractiniid species show that both morphological and life history heterochronies correlate with patterns of gastrovascular circulation. Similar experimental perturbations of energy metabolism, however, have opposite heterochronic effects on the two species. Treatment with 2,4-dinitrophenol, an uncoupler of oxidative phosphorylation, produced diminished peripheral circulation and sheet-like peramorphic morphologies in the runner-like species, Podocoryne carnea. In contrast, similar manipulations of the sheet-like species, Hydractinia symbiolongicarpus, produced variable peripheral circulation and more runner-like, juvenilized morphologies. These results diverge at the level of morphological and life history pattern, but are consistent when viewed in terms of the physiology of the gastrovascular system. Interpretations of patterns of heterochrony must focus on the physiological and developmental mechanisms which mediate the expression of heterochronic traits.     

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.     

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, variability, and the origin of the avian endocranium: insights from the anatomy of Alioramus altai (Theropoda: Tyrannosauroidea)

The internal braincase anatomy of the holotype of Alioramus altai, a relatively small-bodied tyrannosauroid from the Late Cretaceous of Mongolia, was studied using high-resolution computed tomography. A number of derived characters strengthen the diagnosis of this taxon as both a tyrannosauroid and a unique, new species (e.g., endocranial position of the gasserian ganglion, internal ramification of the facial nerve). Also present are features intermediate between the basal theropod and avialan conditions that optimize as the ancestral condition for Coelurosauria--a diverse group of derived theropods that includes modern birds. The expression of several primitive theropod features as derived character states within Tyrannosauroidea establishes previously unrecognized evolutionary complexity and morphological plasticity at the base of Coelurosauria. It also demonstrates the critical role heterochrony may have played in driving patterns of endocranial variability within the group and potentially reveals stages in the evolution of neuroanatomical development that could not be inferred based solely on developmental observations of the major archosaurian crown clades. We discuss the integration of paleontology with variability studies, especially as applied to the nature of morphological transformations along the phylogenetically long branches that tend to separate the crown clades of major vertebrate groups.     

Reconciling gene and genome duplication events: using multiple nuclear gene families to infer the phylogeny of the aquatic plant family Pontederiaceae

Most plant phylogenetic inference has used DNA sequence data from the plastid genome. This genome represents a single genealogical sample with no recombination among genes, potentially limiting the resolution of evolutionary relationships in some contexts. In contrast, nuclear DNA is inherently more difficult to employ for phylogeny reconstruction because major mutational events in the genome, including polyploidization, gene duplication, and gene extinction can result in homologous gene copies that are difficult to identify as orthologs or paralogs. Gene tree parsimony (GTP) can be used to infer the rooted species tree by fitting gene genealogies to species trees while simultaneously minimizing the estimated number of duplications needed to reconcile conflicts among them. Here, we use GTP for five nuclear gene families and a previously published plastid data set to reconstruct the phylogenetic backbone of the aquatic plant family Pontederiaceae. Plastid-based phylogenetic studies strongly supported extensive paraphyly of Eichhornia (one of the four major genera) but also depicted considerable ambiguity concerning the true root placement for the family. Our results indicate that species trees inferred from the nuclear genes (alone and in combination with the plastid data) are highly congruent with gene trees inferred from plastid data alone. Consideration of optimal and suboptimal gene tree reconciliations place the root of the family at (or near) a branch leading to the rare and locally restricted E. meyeri. We also explore methods to incorporate uncertainty in individual gene trees during reconciliation by considering their individual bootstrap profiles and relate inferred excesses of gene duplication events on individual branches to whole-genome duplication events inferred for the same branches. Our study improves understanding of the phylogenetic history of Pontederiaceae and also demonstrates the utility of GTP for phylogenetic analysis.