PAPER Paleogenomics of echinoids reveals an ancient origin COLLOQUIUM for the double-negative specification of micromeres in sea urchins Jeffrey R. Thompsona,1, Eric M. Erkenbrackb, Veronica F. Hinmanc, Brenna S. McCauleyc,d, Elizabeth Petsiosa, and David J. Bottjera aDepartment of Earth Sciences, University of Southern California, Los Angeles, CA 90089; bDepartment of Ecology and Evolutionary Biology, Yale University, New Haven, CT 06511; cDepartment of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213; and dHuffington Center on Aging, Baylor College of Medicine, Houston, TX 77030 Edited by Douglas H. Erwin, Smithsonian National Museum of Natural History, Washington, DC, and accepted by Editorial Board Member Neil H. Shubin January 31, 2017 (received for review August 2, 2016) Establishing a timeline for the evolution of novelties is a common, methods thus provide a rigorous methodology in which to examine unifying goal at the intersection of evolutionary and developmental gene expression datasets, and ultimately animal body plan evolu- biology. Analyses of gene regulatory networks (GRNs) provide the tion (11), within the context of evolutionary time. After genomic ability to understand the underlying genetic and developmental novelties underlying differential body plan development have been mechanisms responsible for the origin of morphological structures identified, we can then consider the rates at which these novelties both in the development of an individual and across entire evolution- arise, and the rates at which GRNs evolve. Achieving this end ary lineages. Accurately dating GRN novelties, thereby establishing requires an explicit timeline in which to explore GRN evolution. a timeline for GRN evolution, is necessary to answer questions In a phylogenetically informed, comparative framework, it is about the rate at which GRNs and their subcircuits evolve, and to possible to infer where on a phylogenetic tree and when, in deep tie their evolution to paleoenvironmental and paleoecological time, GRN innovations are likely to have first arisen. Using a changes. Paleogenomics unites the fossil record and all aspects EVOLUTION paleogenomic approach (12, 13), it is possible to incorporate deep of deep time, with modern genomics and developmental biology time into an analysis of when GRN novelties arose and to infer the to understand the evolution of genomes in evolutionary time. Recent work on the regulatory genomic basis of development in regulatory interactions directing the development of extinct organ- cidaroid echinoids, sand dollars, heart urchins, and other nonmodel isms, thereby bringing forth a unique understanding of the evolution echinoderms provides an ideal dataset with which to explore GRN of GRNs and the body plans that they encode. Paleogenomics al- evolution in a comparative framework. Using divergence time lows for the dating of the appearance of apomorphic GRNs, their estimation and ancestral state reconstructions, we have determined subcircuits, and particular network linkages using a combination of the age of the double-negative gate (DNG), the subcircuit which the fossil record, statistically derived divergence dates, and com- specifies micromeres and skeletogenic cells in Strongylocentrotus parative analyses of robust experimental data from extant organ- purpuratus. We have determined that the DNG has likely been used isms. With reliable dates in hand, the task of determining rates of for euechinoid echinoid micromere specification since at least the GRN evolution is not far off. We set out to establish a rigorous Late Triassic. The innovation of the DNG thus predates the burst of framework for determining the timeline of GRN evolution, and to post-Paleozoic echinoid morphological diversification that began in test a recently proposed hypothesis (8) concerning the timing of the the Early Jurassic. Paleogenomics has wide applicability for the in- evolution of a key sea urchin GRN novelty, the double-negative tegration of deep time and molecular developmental data, and has specification of micromeres. wide utility in rigorously establishing timelines for GRN evolution. Echinoids, or sea urchins, represent an ideal model system for understanding the mechanistic basis of GRNs in development and evolution | evo-devo | euechinoid | cidaroid | gene regulatory networks for studying the evolution of development (14–16). Research on the early embryonic development of echinoids has revealed the regula- he investigation of gene regulatory networks (GRNs) in modern tory interactions that compose the circuitry of developmental GRNs Ttaxa allows for the understanding of evolutionary changes in the driving early development of the purple sea urchin Strongylocentrotus regulatory genome that have underpinned the evolution of new purpuratus (14). Importantly, echinoids also have an excellent fossil – morphological structures in deep time (1 4). Establishing a timeline record that dates back to Ordovician strata, more than 400 Mya (17). for the rates at which these novel structures arise, and the rate at The combination of a robust fossil record and detailed under- which the developmental GRNs that encode them evolve, lies at the standing of the early developmental GRNs in numerous species heart of evolutionary developmental biology (5). In recent years, identifying genetic regulatory differences between diverse organisms has become more feasible with broader phylogenetic sampling of This paper results from the Arthur M. Sackler Colloquium of the National Academy of developmental and gene expression data across Metazoa (6, 7). Sciences, “Gene Regulatory Networks and Network Models in Development and Evolu- These new data provide insight into genomically encoded develop- tion,” held April 12–14, 2016, at the Arnold and Mabel Beckman Center of the National mental programs and the species-specific GRNs that direct animal Academies of Sciences and Engineering in Irvine, CA. The complete program and video recordings of most presentations are available on the NAS website at www.nasonline.org/ development in previously unexplored branches of the tree of life. Gene_Regulatory_Networks. Thus, studies comparing GRN subcircuit wiring in distantly diverged Author contributions: J.R.T. designed research; J.R.T. and B.S.M. performed research; J.R.T., taxa (8, 9) are paving the way for the study of GRN evolution. E.M.E., V.F.H., and E.P. analyzed data; and J.R.T., E.M.E., V.F.H., and D.J.B. wrote the paper. The arrival of these new data has introduced new problems, The authors declare no conflict of interest. however. Importantly, as comparative studies of developmental This article is a PNAS Direct Submission. D.H.E. is a guest editor invited by the GRNs are becoming more commonplace, it is critical to keep in Editorial Board. mind that simple pairwise comparisons between taxa violate sta- 1To whom correspondence should be addressed. Email: [email protected]. tistical assumptions of independence and must be carried out in an This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. explicit phylogenetic framework (10). Phylogenetic comparative 1073/pnas.1610603114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1610603114 PNAS Early Edition | 1of8 Downloaded by guest on September 25, 2021 makes echinoids an opportune group in which to implement inte- phylogenetic sampling of data indicating the presence or absence of grated approaches to understanding GRN evolution. the DNG in numerous euechinoid and cidaroid echinoids, as well as The echinoid crown group comprises two clades, the euechinoids in other echinoderms, makes it an ideal candidate subcircuit to study and the cidaroids (18). The adult body plans of these two clades the tempo of GRN evolution. provide prime examples of differential morphological disparity Previously, the initial evolution of the DNG was associated with (19). Euechinoids have evolved numerous diverse morphologies the divergence of cidaroid and euechinoid echinoids, at least 268 throughout their evolutionary history and include morphologi- Mya (8, 21). However, this conclusion was reached outside the cally distinct clades, such as the bilaterally symmetrical sand context of a phylogenetic comparative framework and was based dollars and heart urchins. In contrast, cidaroids have shown re- on a comparison of the differential GRN circuitry responsible for markable morphological conservation, and the earliest fossil micromere specification in regular euechinoid and cidaroid echi- cidaroids are almost morphologically identical to cidaroids living noids given the most recent date at which euechinoid and cidaroid in the oceans today (20, 21). Comparative studies of GRN ar- echinoids could have diverged (21). There are multiple evolu- chitecture between cidaroids and euechinoids have revealed ex- tionary scenarios pertaining to the timing of the evolution of the tensive differences in the wiring of their early developmental DNG that could explain the presence of this circuitry in regular GRNs (8, 22, 23), and have served to inform our understanding euechinoids like S. purpuratus and its absence in cidaroids. By of the genomic underpinning of the myriad morphological dif- analyzing the presence or absence of the DNG within an explicit ferences in their embryonic and larval development (24, 25). phylogenetic framework, it is possible to estimate the age of particular nodes in deep time. We can then estimate the proba- The Double-Negative Gate bility that the DNG