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 difcult. 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 protocolwhereby event-pair transformations along agiven branch areanalyzed en bloc. Ourmethod 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 identied by previous authors. [Character mapping; development; event-pairing; evolutionary transformations; heterochrony; phylogenetic framework. ] The evolution of developmental mechanisms is a key eld of research in modern biology (Holland, 1999; Raff, 2000). Change in developmental timing (heterochrony) is often cited as a potential mechanism of evolutionary change (Gould, 1977, 1982; McKinney and McNamara, 1991; Raff, 1996) and usually is discussed in the context of large-scale timing changes that affect the entire organism (e.g., as formalized as the processes of neotony or paedomorphosis). However, the full extent of heterochrony as an evolutionary mechanism may be underestimated, particularly with respect to smaller-scale heterochronies affecting single organs. This potential underestimation is related in part to the methodological problems in analyzing heterochrony under a phylogenetic framework (Alberch, 1985; Mabee and Trendler, 1996; Mabee et al., 2000). Recently, there has been renewed interest in the analysis of developmental sequences, or the order in which events occur during development (for an excellent summary, see Smith, 2001). This interest derives from two 4Corresponding author. sources. First, the debate concerning the existence of a conserved, “phylotypic” stage in vertebrate development depends critically on how much developmental events change their times of appearance (sequence or event heterochrony) during this period (reviewed in Richardson, 1995, 1999; Smith, 2001). Second, the development of “eventpairing” (Mabee and Trendler, 1996; Smith, 1996), which compares the relative timing of developmental events in a pairwise fashion, has revolutionized the analysis of developmental sequences. Using this method, several studies have demonstrated sequence heterochrony by optimizing event-paired data onto existing phylogenetic trees (e.g., Smith, 1996, 1997; Velhagen, 1997; Jeffrey et al., in press). In this paper, we examine the issue of analyzing developmental sequences within a phylogenetic framework to elucidate instances of sequence heterochrony. In particular, we review general analytical problems inherent to heterochronic data and the advantages that event-pairing provides in comparison with other methods. Although event-pairing represents an