title: High-precision morphology: bifocal 4D-microscopy enables the comparison of detailed cell lineages of two chordate species separated for more than 525 million years.
abstract: BACKGROUND
  Understanding the evolution of divergent developmental trajectories requires detailed comparisons of embryologies at appropriate levels. Cell lineages, the accurate visualization of cleavage patterns, tissue fate restrictions, and morphogenetic movements that occur during the development of individual embryos are currently available for few disparate animal taxa, encumbering evolutionarily meaningful comparisons. Tunicates, considered to be close relatives of vertebrates, are marine invertebrates whose fossil record dates back to 525 million years ago. Life-history strategies across this subphylum are radically different, and include biphasic ascidians with free swimming larvae and a sessile adult stage, and the holoplanktonic larvaceans. Despite considerable progress, notably on the molecular level, the exact extent of evolutionary conservation and innovation during embryology remain obscure.
  
  
  RESULTS
  Here, using the innovative technique of bifocal 4D-microscopy, we demonstrate exactly which characteristics in the cell lineages of the ascidian Phallusia mammillata and the larvacean Oikopleura dioica were conserved and which were altered during evolution. Our accurate cell lineage trees in combination with detailed three-dimensional representations clearly identify conserved correspondence in relative cell position, cell identity, and fate restriction in several lines from all prospective larval tissues. At the same time, we precisely pinpoint differences observable at all levels of development. These differences comprise fate restrictions, tissue types, complex morphogenetic movement patterns, numerous cases of heterochronous acceleration in the larvacean embryo, and differences in bilateral symmetry.
  
  
  CONCLUSIONS
  Our results demonstrate in extraordinary detail the multitude of developmental levels amenable to evolutionary innovation, including subtle changes in the timing of fate restrictions as well as dramatic alterations in complex morphogenetic movements. We anticipate that the precise spatial and temporal cell lineage data will moreover serve as a high-precision guide to devise experimental investigations of other levels, such as molecular interactions between cells or changes in gene expression underlying the documented structural evolutionary changes. Finally, the quantitative amount of digital high-precision morphological data will enable and necessitate software-based similarity assessments as the basis of homology hypotheses.
authors.affiliation: Institut für Biologie, Kompetenzzentrum Elektronenmikroskopie, Humboldt-Universität zu Berlin, Philippstrasse 13, Haus 14, 10115, Berlin, Germany. thomas.stach@hu-berlin.de.
authors.forename: Thomas
authors.initials: T
authors.lastname: Stach
authors.affiliation: Dipartimento di Biologia, Università degli Studi di Padova, Via Ugo Bassi 58/B, 35131, Padova, Italy. anselmi-chiara@libero.it.
authors.forename: Chiara
authors.initials: C
authors.lastname: Anselmi
mesh_terms: D000818:Animals; D005075:Biological Evolution; D019070:Cell Lineage; D008853:Microscopy; D013045:Species Specificity; D014561:Urochordata / Q000033:anatomy & histology / Q000196:embryology*