Jonathan E. Jeffery

Fins to limbs: what the fossils say1

SUMMARY A broad phylogenetic review of fins, limbs, and girdles throughout the stem and base of the crown group is needed to get a comprehensive idea of transformations unique to the assembly of the tetrapod limb ground plan. In the lower part of the tetrapod stem, character state changes at the pectoral level dominate; comparable pelvic level data are limited. In more crownward taxa, pelvic level changes dominate and repeatedly precede similar changes at pectoral level. Concerted change at both levels appears to be the exception rather than the rule. These patterns of change are explored by using alternative treatments of data in phylogenetic analyses. Results highlight a large data gap in the stem group preceding the first appearance of limbs with digits. It is also noted that the record of morphological diversity among stem tetrapods is somewhat worse than that of basal crown group tetrapods. The pre‐limbed evolution of stem tetrapod paired fins is marked by a gradual reduction in axial segment numbers (mesomeres); pectoral fins of the sister group to limbed tetrapods include only three. This reduction in segment number is accompanied by increased regional specialization, and these changes are discussed with reference to the phylogenetic distribution of characteristics of the stylopod, zeugopod, and autopod.

Forelimb-hindlimb developmental timing changes across tetrapod phylogeny

BackgroundTetrapods exhibit great diversity in limb structures among species and also between forelimbs and hindlimbs within species, diversity which frequently correlates with locomotor modes and life history. We aim to examine the potential relation of changes in developmental timing (heterochrony) to the origin of limb morphological diversity in an explicit comparative and quantitative framework. In particular, we studied the relative time sequence of development of the forelimbs versus the hindlimbs in 138 embryos of 14 tetrapod species spanning a diverse taxonomic, ecomorphological and life-history breadth. Whole-mounts and histological sections were used to code the appearance of 10 developmental events comprising landmarks of development from the early bud stage to late chondrogenesis in the forelimb and the corresponding serial homologues in the hindlimb.ResultsAn overall pattern of change across tetrapods can be discerned and appears to be relatively clade-specific. In the primitive condition, as seen in Chondrichthyes and Osteichthyes, the forelimb/pectoral fin develops earlier than the hindlimb/pelvic fin. This pattern is either retained or re-evolved in eulipotyphlan insectivores (= shrews, moles, hedgehogs, and solenodons) and taken to its extreme in marsupials. Although exceptions are known, the two anurans we examined reversed the pattern and displayed a significant advance in hindlimb development. All other species examined, including a bat with its greatly enlarged forelimbs modified as wings in the adult, showed near synchrony in the development of the fore and hindlimbs.ConclusionMajor heterochronic changes in early limb development and chondrogenesis were absent within major clades except Lissamphibia, and their presence across vertebrate phylogeny are not easily correlated with adaptive phenomena related to morphological differences in the adult fore- and hindlimbs. The apparently conservative nature of this trait means that changes in chondrogenetic patterns may serve as useful phylogenetic characters at higher taxonomic levels in tetrapods. Our results highlight the more important role generally played by allometric heterochrony in this instance to shape adult morphology.

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.

Analyzing evolutionary patterns in amniote embryonic development

SUMMARY Heterochrony (differences in developmental timing between species) is a major mechanism of evolutionary change. However, the dynamic nature of development and the lack of a universal time frame makes heterochrony difficult to analyze. This has important repercussions in any developmental study that compares patterns of morphogenesis and gene expression across species. We describe a method that makes it possible to quantify timing shifts in embryonic development and to map their evolutionary history. By removing a direct dependence on traditional staging series, through the use of a relative time frame, it allows the analysis of developmental sequences across species boundaries. Applying our method to published data on vertebrate development, we identified clear patterns of heterochrony. For example, an early onset of various heart characters occurs throughout amniote evolution. This suggests that advanced (precocious) heart development arose in evolutionary history before endothermy. Our approach can be adapted to analyze other forms of comparative dynamic data, including patterns of developmental gene expression.

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

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 supertree of early tetrapods

A genus–level supertree for early tetrapods is built using a matrix representation of 50 source trees. The analysis of all combined trees delivers a long–stemmed topology in which most taxonomic groups are assigned to the tetrapod stem. A second analysis, which excludes source trees superseded by more comprehensive studies, supports a deep phylogenetic split between lissamphibian and amniote total groups. Instances of spurious groups are rare in both analyses. The results of the pruned second analysis are mostly comparable with those of a recent, character–based and large–scale phylogeny of Palaeozoic tetrapods. Outstanding areas of disagreement include the branching sequence of lepospondyls and the content of the amniote crown group, in particular the placement of diadectomorphs as stem diapsids. Supertrees are unsurpassed in their ability to summarize relationship patterns from multiple independent topologies. Therefore, they might be used as a simple test of the degree of corroboration of nodes in the contributory analyses. However, we urge caution in using them as a replacement for character–based cladograms and for inferring macroevolutionary patterns.

Proximodistal patterning of the limb: insights from evolutionary morphology

There is an active debate about how skeletal elements are encoded along the proximodistal (PD) axis of the developing limb. Our aim here is to see whether consideration of the evolutionary morphology of the limb can contribute to our understanding of patterning mechanisms. Of special interest in this context are animals showing reiterated skeletal elements along the PD axis (e.g., dolphins and plesiosaurs with hyperphalangy). We build on previous hypotheses to propose a two‐step model of PD patterning in which specification of broad domains in the early limb bud is distinct from subsequent processes that divides an initial anlage into a segmental pattern to yield individual skeletal elements. This model overcomes a major evolutionary problem with the progress zone model, which has not previously been noted: pleiotropy. Parallels with other developmental systems are briefly discussed.

Genomic annotation and transcriptome analysis of the zebrafish (Danio rerio) hox complex with description of a novel member, hoxb13a

Summary The zebrafish (Danio rerio) is an important model in evolutionary developmental biology, and its study is being revolutionized by the zebrafish genome project. Sequencing is at an advanced stage, but annotation is largely the result of in silico analyses. We have performed genomic annotation, comparative genomics, and transcriptional analysis using microarrays of the hox homeobox‐containing transcription factors. These genes have important roles in specifying the body plan. Candidate sequences were located in version Zv4 of the Ensembl genome database by TBLASTN searching with Danio and other vertebrate published Hox protein sequences. Homologies were confirmed by alignment with reference sequences, and by the relative position of genes along each cluster. RT‐PCR using adult Tübingen cDNA was used to confirm annotations, to check the genomic sequence and to confirm expression in vivo. Our RT‐PCR and microarray data show that all 49 hox genes are expressed in adult zebrafish. Significant expression for all known hox genes could be detected in our microarray analysis. We also find significant expression of hox8 paralogs and hoxb7a in the anti‐sense direction. A novel gene, D. rerio hoxb13a, was identified, and a preliminary characterization by in situ hybridization showed expression at 24 hpf at the tip of the developing tail. We are currently characterizing this gene at the functional level. We argue that the oligo design for microarrays can be greatly enhanced by the availability of genomic sequences.