Latent regeneration abilities persist following recent evolutionary loss in asexual annelids Alexandra E. Bely1 and James M. Sikes2 Biology Department, University of Maryland, College Park, MD 20742 Edited by Douglas Futuyma, Department of Ecology and Evolution, State University of New York, Stony Brook, NY, and approved November 5, 2009 (received for review July 15, 2009) Regeneration abilities have been repeatedly lost in many animal Naidine annelids represent a promising model for investigat- phyla. However, because regeneration research has focused almost ing regeneration loss. Naidines sensu lato (Naidinae, Pristininae, exclusively on highly regenerative taxa or on comparisons between and close relatives) are a group of small aquatic oligochaetes, regenerating and nonregenerating taxa that are deeply diverged, many of which reproduce asexually by fission (13–16). The most virtually nothing is known about how regeneration loss occurs. Here, common mode of fission in this group is paratomic fission, in we show that, following a recent evolutionary loss of regeneration, which a new head and tail are intercalated in the middle of the regenerative abilities can remain latent and still be elicited. Using body within a region referred to as the fission zone, thus forming comparative regeneration experiments and a molecular phylogeny, transiently linked individuals (Fig. 1, A and B). Regenerative we show that ancestral head regeneration abilities have been lost abilities have previously been investigated in only a few species, three times among naidine annelids, a group of small aquatic worms but available data reveal important variation. Several naidine that typically reproduce asexually by fission. In all three lineages species possess excellent regenerative abilities, being capable of incapable of head regeneration, worms consistently seal the wound forming a new head and tail from just a small fragment of the butfailtoprogresstothefirst stage of tissue replacement. However, original animal (17, 18), and it is likely that regenerative ability is despite this coarse-level convergence in regeneration loss, further ancestral for naidines, as it is for annelids more broadly (3). We investigation of two of these lineages reveals marked differences in previously identified one species, however, that is incapable of how much of the regeneration machinery has been abolished. Most regenerating any anterior segments (19), suggesting at least one notably, in a species representingoneofthesetwo lineages, butnotin loss within the group. a representative of the other, amputation within a narrow prolifer- We investigated the pattern and process of regeneration fi ative region that forms during ssion can still elicit regeneration of an evolution among closely related species of naidine annelids. essentially normal head. Thus, the presence at the wound site of Comparative regeneration experiments and a molecular phy- elements characteristic of actively growing tissues, such as activated logeny together indicate multiple losses of head regeneration stem cells or growth factors, may permit blocks to regeneration to be within this group. To investigate the process of regeneration loss, circumvented, allowing latent regeneration abilities to be manifested. we assessed the developmental capabilities of two species rep- resenting independent losses of head regeneration, focusing on regeneration loss | Annelida | asexual reproduction | evolution the ability to regenerate segmental and asegmental tissue, to initiate cell proliferation after amputation, and to regenerate egeneration is a process by which many animals can replace following amputation within the fission zone. Our investigations Rlost body parts. Although basal animal lineages and some reveal a phenomenon, fission-zone regeneration, by which an bilaterians are excellent regenerators, numerous animal lineages, otherwise nonregenerating species can still regenerate, a finding including representatives of all three major bilaterian clades, have that has important implications for understanding how regen- incurred severe reductions in the ability to regenerate body parts eration abilities are lost. This study demonstrates that com- (1). Large groups such as nematodes, birds, mammals, and leeches, parative developmental investigations of regeneration among for example, are largely or entirely incapable of regenerating any close relatives represent a powerful approach for providing body part, indicating relatively old losses of regeneration. Many insight into the evolution of regeneration. other, more recent losses of regeneration have also occurred, for example, among annelids, arthropods, planarians, fishes, and liz- Results – ards (2 6). Despite longstanding interest in the process of animal We performed comparative head and tail regeneration experi- regeneration (7, 8), over a century of speculation on the root ments on 19 naidine species, representing 13 genera spanning causes of variation in this feature (1, 4, 9), and recent advances in the group, as well as two outgroups (see Table S1 for species understanding its developmental and molecular basis (10, 11), we acquisition information). Although all naidines in our trials can still know little about the evolutionary and developmental pro- reproduce by fission, we found that nearly one-third of these cesses involved in regeneration loss. species (6/19) are incapable of head regeneration (Fig. 1). For Understanding the pattern and process of regeneration loss these trials, we removed the cephalic segments, a set of anterior- requires a comparative approach that focuses on species that have most segments that are morphologically distinct [typically lacking recently lost regeneration and their regenerating close relatives. dorsal chaetae (bristles) and pigmented gut cells] and that in However, with few exceptions (e.g., 4, 6, 12), regeneration research has focused almost exclusively on regenerating species or on broad comparisons between distantly related regenerating and non- Author contributions: A.E.B. designed research; A.E.B. and J.M.S. performed research; regenerating groups (e.g., amphibians vs. mammals) too deeply di- A.E.B. analyzed data; A.E.B. wrote the paper. verged to reveal much about the mechanism of regeneration loss. The authors declare no conflict of interest. What types of changes are correlated with regeneration loss? Are This article is a PNAS Direct Submission. some aspects of regeneration loss predictable? Can regenerative 1To whom correspondence should be addressed. E-mail: [email protected]. abilities re-evolve after being lost? Important questions such as 2Present address: Department of Cell and Developmental Biology, University of Illinois at these remain largely unanswered, in part because the currently Urbana-Champaign, Urbana, IL 61801. available animal regeneration models are largely inadequate for This article contains supporting information online at www.pnas.org/cgi/content/full/ addressing them. 0907931107/DCSupplemental. 1464–1469 | PNAS | January 26, 2010 | vol. 107 | no. 4 www.pnas.org/cgi/doi/10.1073/pnas.0907931107 Downloaded by guest on September 26, 2021 Fig. 1. Fission and comparative regeneration experiments in naidine annelids. In this and subsequent figures, anterior is left; dark-green and light green mark new head and tail tissue, respectively, formed by fission; dark-gray bars mark the original body region remaining after amputation; light-gray bars mark amputated tissue; orange bars mark regenerated tissue; dashed lines indicate plane of amputation. (A) Naidine paratomic fission occurs by intercalation of new head and tail tissue in the middle of the body (top: prefission; middle: fission; bottom: post-fission). (B) Pa. litoralis individual with a late-stage fission zone, showing the characteristically clear tissue of this zone. Original head is at top, pointing left. (Scale bar, 250 μm.) (C–F) Pr. leidyi can regenerate anteriorly and posteriorly (C and D: 4 days dpa), whereas Pa. litoralis can regenerate posteriorly but not anteriorly (E and F: 7 dpa). (G) Results from comparative re- generation experiments (19 naidine species, top; 2 outgroups, bottom). See Table S2 for amputation locations for each species. Color codes are based on the stage of the most advanced three individuals, scored at the end of that time period, with emergence of chaetae from new segments marking regeneration completion. Absence of regeneration scoring indicates that no individuals survived to that time point. naidines correspond to those anterior segments that form during posterior regeneration could be induced. For the remainder of the fission (20). For brevity, we refer to these segments as “head study, we focused specifically on anterior regeneration ability. segments” and refer collectively to the cephalic segments plus To place the comparative regeneration experiments in a phy- the anterior asegmental tip of the body as the “head.” Following logenetic context, we reconstructed a molecular phylogeny for the amputation of the head (two to eight segments, depending on naidines. We infer that head regeneration ability is ancestral for the species; Table S2), most species regenerated fully within 2–6 naidines and that species incapable of head regeneration stem days (Fig. 1, C and G; Table S2; Table S3), proceeding through from three independent losses (Fig. 2). Bayesian analysis of a five- stages typical of annelid regeneration: wound healing occurred gene data set (Table S4) produced a well-resolved and strongly EVOLUTION by contraction