Assessing the phylogenetic utility of sequence heterochrony: evolution of avian ossification sequences as a case study Erin E. Maxwell a,n , Luke B. Harrison b , Hans C.E. Larsson b a Department of Biological Sciences, University of Alberta, 11455 Saskatchewan Drive, Edmonton, Canada, AB T6G 2E9 b Redpath Museum, McGill University, 859 Rue Sherbrooke Ouest, Montreal, Canada, QC H3A 2K6 article info Article history: Received 26 April 2009 Received in revised form 26 June 2009 Accepted 28 June 2009 Keywords: Aves Avian skeletal development Event-pairing Phylogenetic signal Parsimov-based genetic inference abstract 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. & 2009 Elsevier GmbH. All rights reserved. Introduction The evolution of organismal morphology is a direct result of changes in organismal development (Haeckel, 1902; Gould, 1977; Hall, 1999). Heritable changes are directed by genetic changes while non-heritable changes are the result of alterations of epigenetic influences, such as climate. Heterochrony has been an important concept for the examina- tion of large-scale evolutionary and developmental changes, both as a system of measurement and description, and as a framework for permitting the discussion of evolutionary processes acting through development. Heterochrony encompasses the entire suite of possible changes in developmental timing that result in evolutionary change. This relatively straightforward evolutionary mechanism has been found to act across invertebrate and vertebrate animals and through all stages of development (Gould, 1977; Alberch et al., 1979; McKinney, 1988), and is considered to be an important pattern underlying morphological change. More recent work on heterochrony has divided it into two main components based on the type of developmental change (Smith, 2001). Growth heterochrony describes the differential growth rates and differential timing of onset and offset of somatic growth, generally compared to the onset of sexual maturity. The comparisons are usually bivariate. Growth heterochronies result in changes in size and shape of morphological structures. Sequence heterochrony describes evolutionary changes in the temporal order of developmental events (Smith, 2001). Sequence heterochronies are currently recognised as changes in a rank- ordered sequence of developmental events, but the utility of defining sequence heterochronies based upon absolute or stage- based temporal information has also been discussed (Smith, 2001). Ossification sequences are species-specific patterns of bone formation that appear to exhibit some degree of conservation within species (Sheil, 2003). The sequences are not invariant, however, and differences occur within species (intraspecific polymorphism) (Sheil and Greenbaum, 2005), between closely related species (Prochel et al., 2008), and between higher taxonomic groups (Sa ´ nchez-Villagra, 2002). Ossification se- quences of primary centres of ossification have not been found to exhibit strong phylogenetic signal when tested in an explicit context using objective methodologies (Sa ´ nchez-Villagra, 2002; Schoch, 2006; Sa ´ nchez-Villagra et al., 2008; Weisbecker et al., 2008). However, the goal of these studies was not to characterise ossification sequence changes between closely related taxa, but to highlight large sequence changes over distantly related taxa. Furthermore, the number of elements in each of the previous ARTICLE IN PRESS Contents lists available at ScienceDirect journal homepage: www.elsevier.de/zool Zoology 0944-2006/$ - see front matter & 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.zool.2009.06.002 n Corresponding author. Tel.: + 780 492 3458; fax: + 780 492 9234. E-mail address: emaxwell@ualberta.ca (E.E. Maxwell). Zoology 113 (2010) 57–66