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Proc Natl Acad Sci U S A. Jan 26, 2010; 107(4): 1464–1469.
Published online Dec 4, 2009. doi:  10.1073/pnas.0907931107
PMCID: PMC2824374
Evolution

Latent regeneration abilities persist following recent evolutionary loss in asexual annelids

Abstract

Regeneration abilities have been repeatedly lost in many animal phyla. However, because regeneration research has focused almost exclusively on highly regenerative taxa or on comparisons between regenerating and nonregenerating taxa that are deeply diverged, virtually nothing is known about how regeneration loss occurs. Here, we show that, following a recent evolutionary loss of regeneration, regenerative abilities can remain latent and still be elicited. Using comparative regeneration experiments and a molecular phylogeny, we show that ancestral head regeneration abilities have been lost three times among naidine annelids, a group of small aquatic worms that typically reproduce asexually by fission. In all three lineages incapable of head regeneration, worms consistently seal the wound but fail to progress to the first stage of tissue replacement. However, despite this coarse-level convergence in regeneration loss, further investigation of two of these lineages reveals marked differences in how much of the regeneration machinery has been abolished. Most notably, in a species representing one of these two lineages, but not in a representative of the other, amputation within a narrow proliferative region that forms during fission can still elicit regeneration of an essentially normal head. Thus, the presence at the wound site of elements characteristic of actively growing tissues, such as activated stem cells or growth factors, may permit blocks to regeneration to be circumvented, allowing latent regeneration abilities to be manifested.

Keywords: regeneration loss, Annelida, asexual reproduction, evolution

Regeneration is a process by which many animals can replace lost body parts. Although basal animal lineages and some bilaterians are excellent regenerators, numerous animal lineages, including representatives of all three major bilaterian clades, have incurred severe reductions in the ability to regenerate body parts (1). Large groups such as nematodes, birds, mammals, and leeches, for example, are largely or entirely incapable of regenerating any body part, indicating relatively old losses of regeneration. Many other, more recent losses of regeneration have also occurred, for example, among annelids, arthropods, planarians, fishes, and lizards (2 6). Despite longstanding interest in the process of animal regeneration (7, 8), over a century of speculation on the root causes of variation in this feature (1, 4, 9), and recent advances in understanding its developmental and molecular basis (10, 11), we still know little about the evolutionary and developmental processes involved in regeneration loss.

Understanding the pattern and process of regeneration loss requires a comparative approach that focuses on species that have recently lost regeneration and their regenerating close relatives. 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 nonregenerating groups (e.g., amphibians vs. mammals) too deeply diverged to reveal much about the mechanism of regeneration loss. What types of changes are correlated with regeneration loss? Are some aspects of regeneration loss predictable? Can regenerative abilities re-evolve after being lost? Important questions such as these remain largely unanswered, in part because the currently available animal regeneration models are largely inadequate for addressing them.

Naidine annelids represent a promising model for investigating regeneration loss. Naidines sensu lato (Naidinae, Pristininae, and close relatives) are a group of small aquatic oligochaetes, many of which reproduce asexually by fission (13 16). The most common mode of fission in this group is paratomic fission, in which a new head and tail are intercalated in the middle of the body within a region referred to as the fission zone, thus forming transiently linked individuals (Fig. 1, A and B). Regenerative abilities have previously been investigated in only a few species, but available data reveal important variation. Several naidine species possess excellent regenerative abilities, being capable of forming a new head and tail from just a small fragment of the original animal (17, 18), and it is likely that regenerative ability is ancestral for naidines, as it is for annelids more broadly (3). We previously identified one species, however, that is incapable of regenerating any anterior segments (19), suggesting at least one loss within the group.

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 ...

We investigated the pattern and process of regeneration evolution among closely related species of naidine annelids. Comparative regeneration experiments and a molecular phylogeny together indicate multiple losses of head regeneration within this group. To investigate the process of regeneration loss, we assessed the developmental capabilities of two species representing independent losses of head regeneration, focusing on the ability to regenerate segmental and asegmental tissue, to initiate cell proliferation after amputation, and to regenerate following amputation within the fission zone. Our investigations reveal a phenomenon, fission-zone regeneration, by which an otherwise nonregenerating species can still regenerate, a finding that has important implications for understanding how regeneration abilities are lost. This study demonstrates that comparative developmental investigations of regeneration among close relatives represent a powerful approach for providing insight into the evolution of regeneration.

Results

We performed comparative head and tail regeneration experiments on 19 naidine species, representing 13 genera spanning the group, as well as two outgroups (see Table S1 for species acquisition information). Although all naidines in our trials can reproduce by fission, we found that nearly one-third of these species (6/19) are incapable of head regeneration (Fig. 1). For these trials, we removed the cephalic segments, a set of anterior-most segments that are morphologically distinct [typically lacking dorsal chaetae (bristles) and pigmented gut cells] and that in naidines correspond to those anterior segments that form during fission (20). For brevity, we refer to these segments as “head segments” and refer collectively to the cephalic segments plus the anterior asegmental tip of the body as the “head.” Following amputation of the head (two to eight segments, depending on the species; Table S2), most species regenerated fully within 2–6 days (Fig. 1, C and G; Table S2; Table S3), proceeding through stages typical of annelid regeneration: wound healing occurred by contraction of the severed edges of the body wall and formation of an intact epithelium covering the wound; the severed gut edges sealed into a blind tube; a regeneration blastema (an unpigmented mass of undifferentiated cells) formed at the wound site; and this blastema grew in size and ultimately differentiated to replace the structures that had been lost, including new segments. However, two species of Paranais (Pa. litoralis and Pa. frici), two species of Chaetogaster (C. diastrophus and C. diaphanus), and two species of Amphichaeta (A. “raptisae” B and A.raptisae” C) sealed the wound but did not form a blastema or replace any missing structures (Fig. 1, E and G; Table S2; Table S3). Follow-up experiments performed under a broad array of conditions all confirmed the inability of these species to regenerate the head. Some anteriorly amputated individuals from all three genera continued to undergo fission during our trials, demonstrating that under the experimental conditions these species could form the head by fission, even though they could not regenerate this region. Nearly all species, including four of the six species incapable of head regeneration, could regenerate posteriorly and did so in a time frame similar to that of anterior regeneration (Fig. 1, D, F, and G; Table S2; Table S3). Posterior regeneration failure in the two remaining species was not further explored in this study, and the possibility remains that under different conditions (e.g., with food provided after amputation) posterior regeneration could be induced. For the remainder of the study, we focused specifically on anterior regeneration ability.

To place the comparative regeneration experiments in a phylogenetic context, we reconstructed a molecular phylogeny for the naidines. We infer that head regeneration ability is ancestral for naidines and that species incapable of head regeneration stem from three independent losses (Fig. 2). Bayesian analysis of a five-gene data set (Table S4) produced a well-resolved and strongly supported tree in which Paranais, Chaetogaster, and Amphichaeta are not each other’s closest relatives. [Previous analyses, based on more limited data sets subsumed within ours, have sometimes recovered a clade composed of Chaetogaster and Amphichaeta, but never one composed of Paranais, Chaetogaster, and Amphichaeta (14, 22).] Our inference that each of these genera represents an independent loss of head regeneration is based on the assumption that regeneration losses are more likely than gains. This is a strong expectation, given that numerous regeneration losses are evident among metazoans, including among annelids, but no clear gains of regeneration have yet been documented (1, 3).

Fig. 2.
Phylogenetic distribution of head regeneration ability in naidines. Open circles mark species that can regenerate a head; solid circles mark species that cannot. Head regeneration appears to have been lost three times within one of the two naidine clades ...

To determine whether species that independently lost head regeneration have converged in other measures of regenerative potential, we further investigated Pa. litoralis and C. diaphanus, representing two of the three lineages incapable of head regeneration, and Pristina leidyi, a fully regenerating outgroup. We performed two minimal anterior amputations, removing either just the asegmental tip (prostomium and peristomium) or this tip plus one anterior segment (Fig. 3). We found that neither Pa. litoralis nor C. diaphanus can regenerate following the removal of even a single head segment: as when all head segments are removed, all individuals wound healed (10/10 for each species), but none formed a blastema (0/10 for each species) (Fig. 3, G and H). However, Pa. litoralis, although not C. diaphanus, can still regenerate the anterior asegmental tip if only this region is removed (6/7 Pa. litoralis regenerated; 0/7 C. diaphanus regenerated; Fig. 3, D and E). The regenerating species Pr. leidyi could regenerate following either type of minimal amputation (10/10 for each amputation treatment; Fig. 3, F and I). For brevity, in the remainder of this article we refer to both Pa. litoralis and C. diaphanus as “non-anteriorly regenerating” because neither species can regenerate anterior segments, even though Pa. litoralis can still regenerate the anterior asegmental tip.

Fig. 3.
Minimal amputation experiments and post-amputation cell proliferation. Body region indicators are as in Fig. 1; arrows mark the prostomium; asterisks mark the mouth. (AC) Head morphology of uncut worms. (DI) Following amputation of the ...

We also asked whether the block to regeneration in Pa. litoralis and C. diaphanus occurs before or after the initiation of post-amputation cell proliferation, as assayed by BrdU incorporation during DNA replication. In the fully regenerating species Pr. leidyi (and other regenerating naidines), cell proliferation increases markedly in the epidermis near the wound site after amputation, peaking 2–3 days post amputation (dpa) (Fig. 3, L and O). Although neither Pa. litoralis nor C. diaphanus forms a visible blastema following head amputation, we found that Pa. litoralis, but not C. diaphanus, consistently exhibits persistent low-level cell proliferation at the wound site for at least 3 days after amputation (Fig. 3, J, K, M, and N). Some early cell proliferation at the wound site could conceivably result from wound-healing processes. However, cell proliferation in Pa. litoralis persists long after the epithelium appears intact, which usually occurs within one day after amputation as in other naidines, and wound healing in C. diaphanus shows no comparable cell proliferation. Amputated C. diaphanus continue to proliferate cells at the fission zone and posterior growth zone (as do amputated Pa. litoralis), demonstrating that the failure to proliferate at the wound site in this species is not simply a consequence of energetic limitations (Fig. 3, K and N). Together, these findings suggest that the block to regeneration may occur before blastema initiation in C. diaphanus but after blastema initiation in Pa. litoralis.

We investigated the possibility that head regeneration might be elicited in non-anteriorly regenerating species by amputating within the new tissue of the fission zone (Fig. 4, A and A’). We found that, following careful bisection of a developing fission zone head (removing the asegmental tip and one to two head segments), Pa. litoralis, but not C. diaphanus, can indeed regenerate a largely normal head through a process we call fission-zone-regeneration (FZ-regeneration) (Fig. 4, B and C). After such cuts, ~73% (16/22) of Pa. litoralis developed a blastema that actively proliferated and grew in size, and nearly half of these individuals (7/16) differentiated most or all of the structures that had been amputated, including the asegmental tip and one to two new segments (Fig. 4, DH; Table S5). Although FZ-regeneration in Pa. litoralis progressed at a slower and more variable rate than is typical for regular naidine regeneration (first sign of blastema 3–7 dpa; first segmental chaetae 4–11 dpa) and sometimes produced a head with some abnormal or missing elements (Fig. 4H), FZ-regeneration clearly resulted in the replacement of structures that had been amputated. Regeneration and fission are similar processes, but FZ-regeneration is not merely a continuation of fission. The contexts of FZ-regeneration and fission differ markedly because FZ-regeneration (and regular regeneration) begins with a wound, involves terminal addition of tissue, and necessitates perforation of a new mouth, whereas fission begins without a wound, involves intercalation of tissue, and involves modification of the original gut to form the mouth. Furthermore, new head tissue formed by FZ-regeneration (e.g., prostomium, segment 1, segment 2) is added anterior to the remaining fission zone segment (i.e., segment 3), whereas, during fission, development always proceeds from anterior to posterior (i.e., prostomium and segments 1 and 2 always form before segment 3). Pa. litoralis that were amputated at the base of the fission zone head, rather than within it, consistently wound healed but failed to regenerate (10/10), suggesting that the nature of the wound stump (the original tissue immediately adjacent to the wound site), rather than merely close proximity to a fission zone, is the critical factor allowing FZ-regeneration to proceed in this species. C. diaphanus wound healed but failed to regenerate whether amputation occurred within or adjacent to the fission zone (12/12 per amputation type), whereas Pr. leidyi regenerated fully following either of these cuts (12/12 per amputation type).

Fig. 4.
FZ-regeneration following fission-zone head bisection. Body region indicators are as in Fig. 1; dark-green and light-green bars mark new head and tail tissue, respectively, formed in the fission zone; paired vertical dashes mark the boundary between original ...

To determine whether FZ-regeneration recovers gene expression characteristic of regular regeneration, we investigated the expression of a homolog of nanos, a gene expressed in somatic stem cells and the posterior growth zone in annelids (23, 24). We found that nanos in Pa. litoralis is undetectable at the (nonregenerating) anterior wound site of head-amputated individuals (Fig. 4J), yet is expressed in the FZ-regeneration blastema (Fig. 4, K and L) in a pattern very similar to that in the regular regeneration blastema of Pr. leidyi (Fig. 4I). In both species, nanos is expressed broadly within the internal mesenchymal cells of the blastema as well as faintly in the overlying epidermis (although in Pa. litoralis expression appears less strong and does not extend to the distal tip). Importantly, during blastema stages of FZ-regeneration, Pa. litoralis nanos expression is weak or undetectable in the remaining fission-zone segment that comprises the stump (Fig. 4, K and L), contrasting with the higher expression within the blastema. Although nanos is expressed in early- to midstage fission zones (in a pattern similar to that seen in the FZ-regeneration blastema), including in this future stump segment, by the time the FZ-regeneration blastema is developing, the remaining fission zone segment has differentiated and nanos expression within it disappears (Fig. 4, K and L). Thus, nanos expression provides evidence that the process of FZ-regeneration is comparable to that of regular regeneration and that the FZ-regeneration blastema represents a developmental field distinct from that of the stump tissue.

Discussion

Multiple losses of head regeneration have occurred among naidines, making this group a useful model in which to investigate regeneration loss. The recent losses revealed within this group challenge several widespread views regarding regeneration evolution. For example, naidines provide the only known natural examples of animals capable of agametic asexual reproduction (e.g., fission, budding) in which regenerative and asexual capabilities are decoupled, countering the common assumption that all agametically reproducing animals can also regenerate. Although many animals capable of agametic reproduction have extraordinary regenerative abilities (21), and asexual and regenerative developmental processes can overlap substantially (1, 11, 25), naidines clearly demonstrate that regeneration is far from universal among agametically reproducing species, even in species, such as naidines, in which agametic reproduction is derived from regeneration (26). Furthermore, naidines join the small but growing list of examples showing that loss of regenerative abilities is not necessarily accompanied by large-scale changes in morphological complexity (2, 4, 5, 12). Naidines all share a similar body plan, and species that cannot regenerate do not differ in any consistent way from fully regenerating species. Although increases in morphological complexity may well entrain a decreased regenerative ability (although even this remains speculative), regeneration losses can clearly occur without any such changes.

This study demonstrates that the superficial similarity of post-amputation phenotypes can mask important differences in regenerative potential. An emerging trend regarding regeneration loss, and one further supported by our data, is that there is a strong phylogenetic component to how regeneration tends to fail. Evolved blocks to normal regeneration tend to result in blastema failure in annelids (this study; ref. 3), malformed regenerates in fish fins (4), and hypomeric, nonfunctional outgrowths in amphibians (12). However, we found that species with superficially similar amputation outcomes can vary dramatically in the extent to which regeneration has been abrogated. Naidines incapable of regenerating head segments all converge on a common post-amputation phenotype, wound healing without forming a detectable blastema, yet whereas one lineage (represented by Pa. litoralis) retains the capability for anterior asegmental regeneration, cell proliferation initiation, and FZ-regeneration, a second lineage (represented by C. diaphanus) has lost all of these capabilities. These differences may be due to the latter lineage having accumulated either a greater number of blocks or more severe blocks to regeneration.

A major conclusion from this study is that regenerative abilities can remain latent for a considerable period of evolutionary time following their effective loss. Both Paranais species that we studied have lost head regeneration ability, suggesting that this loss dates back at least to the time of their common ancestor. However, we demonstrate that in Pa. litoralis, at least, replacement of head structures can still occur by FZ-regeneration. Why has FZ-regeneration ability been retained when regular head regeneration has been effectively lost? FZ-regeneration is almost surely ecologically irrelevant. Successful amputation within the fission zone head is difficult to achieve, both because this region is a very narrow amputation target and because stress near a fission zone (especially at mid- to late-fission stages) tends to cause a premature detachment of the posterior individual (by muscular contraction at the fission plane) without accompanying tissue loss for either individual. Thus, it is implausible that fission zone amputation is more common than regular amputation. Instead, we propose that amputation within the fission zone yields a more permissive context for regeneration that allows the evolved blocks in this lineage to be circumvented. In some vertebrates, regenerative abilities are greater in younger individuals than in older ones (21, 27), and FZ-regeneration in Pa. litoralis represents a new example of developmental youth conferring greater regenerative potential. These findings suggest the intriguing possibility that there may be an ontogenetic pattern to regeneration loss, with regenerative ability becoming restricted to the early stages of development before being lost completely.

That FZ-regeneration can occur in Pa. litoralis, which is otherwise incapable of head regeneration, demonstrates that the morphogenetic process of head regeneration has not itself been abolished. Instead, the block to regular regeneration appears to reside in the potential of the stump, a region that in other regenerating animals is known to be an important source of signals and undifferentiated cells critical to blastema formation (11). In Pa. litoralis, regeneration does not proceed when the stump is composed of fully developed tissue, but can be elicited when the distal part of the stump includes even just a single developing segment of the fission zone. Factors that may be required for regeneration, such as activated stem cells and growth factors, are likely to be already present in developing fission tissue but not within fully grown heads. Thus, we suggest that the block to regeneration in Pa. litoralis lies in the activation of stem cells and/or growth factors in the stump and that when these are provided at the wound site (i.e., by amputating within the fission zone), regeneration can still proceed.

What can explain the persistence of latent regenerative abilities in a lineage? We propose that even after the effective loss of anterior regeneration from a lineage, much of the anterior regeneration process remains intact because of pleiotropies with other developmental phenomena. There is growing evidence that regeneration employs developmental processes shared with other phenomena, such as fission and budding, post-embryonic growth, and embryogenesis (1, 11). Pleiotropies are also expected between regenerative processes that restore different parts of the same animal, such as head regeneration and tail regeneration. In naidines, regeneration and fission are clearly closely related processes (18, 26), and posterior regeneration can persist in non-anteriorly regenerating taxa. Thus, the head regeneration machinery in Paranais may remain largely functional because much of it is shared with posterior regeneration and anterior development by fission.

A fundamental question regarding the evolution of animal regeneration is whether regenerative abilities that have been lost can later re-evolve. Although no examples of this have yet been documented, there is accumulating evidence that lost morphological traits can subsequently re-evolve, presumably by the re-expression of latent potential for producing those traits (28, 29). Our study provides evidence that the potential for re-evolution of regenerative abilities is real: regenerative abilities can remain latent after being effectively lost and can be re-elicited through entirely natural means. Regeneration has been at least partly rescued in Hydra and vertebrates through experimental manipulations, for example, by activating Wnt signaling, ectopically expressing an ion pump, or suppressing the immune response (25, 3032). Our study complements these experimental rescues by demonstrating the possibility for a “natural rescue”: by controlling only the position and developmental timing of amputation, we achieved an essentially complete recovery of normal regeneration, a process likely not seen by selection for millions of years. Thus, a simple morphological change in the Paranais lineage, such as an increase in the relative length of the head region that develops within a fission zone, could be all that is required to make FZ-regeneration sufficiently prevalent in the wild that head regeneration could once again be favored by selection. The re-expression of retained latent regenerative abilities provides a viable mechanism by which regenerative abilities that have been effectively lost from a lineage might subsequently re-evolve.

Materials and Methods

Animal Acquisition.

Species were obtained through field collections or other sources (Table S1). Most species were cultured in the lab to generate material for experiments (SI Methods); field-collected individuals were used directly in a few cases.

Regeneration Experiments.

Comparative regeneration experiments consisted of an anterior amputation treatment, a posterior amputation treatment, and an uncut control (n ≥ 10 individuals/treatment for most species; Table S3). Amputation positions were tailored for each naidine species to represent a comparable minimum amputation challenge, specifically removing body regions formed through fission in each species (Table S2, SI Methods). For minimal anterior amputation experiments, we removed either just one head segment and the asegmental tip or only the asegmental tip. For fission-zone amputations, either the developing head of the fission zone was bisected, removing the asegmental tip and one or more developing head segments (leaving a stump composed of at least one segment from the fission zone), or the cut was made at the base of the head, removing the asegmental tip and all developing head segments (leaving a stump consisting entirely of old segments of the original worm). For these amputations, we used animals at mid- to late-stage fission, when segments have formed (as evidenced by the emergence of segmental chaetae) but the head is still actively growing. See SI Methods for details of amputation procedures.

Sequencing and Phylogenetic Analysis.

We obtained a 5228-bp nucleotide data set composed of fragments of five genes, two mitochondrial ribosomal genes (12S, 16S), one mitochondrial protein-coding gene (cytochrome oxidase I), and two nuclear ribosomal genes (18S, 28S). The data set included all taxa included in our comparative regeneration experiments and several additional taxa. DNA extraction, PCR amplification, and sequencing were performed using standard methods and primers (available from the authors upon request). GenBank accession numbers for all sequences included in our data matrix are listed in Table S4, with new sequences deposited under GQ355365-GQ355466. Sequences were aligned and analyzed using both Bayesian and maximum likelihood approaches with methods described in SI Methods.

BrdU Incorporation, Tissue Labeling, and nanos Expression.

BrdU labeling (0.1 mg/mL; 18-h pulse) and acetylated α-tubulin, serotonin, and phalloidin labeling were performed using standard methods (SI Methods). nanos was isolated from Pa. litoralis and Pr. leidyi using degenerate PCR, 5′ RACE, and 3′ RACE (GenBank accession nos. GQ369728 and GQ369729). Phylogenetic analysis was performed to confirm the identity of the recovered sequences as nanos-class genes (SI Methods; Fig. S1). Expression was investigated through in situ hybridization using a previously published protocol (26).

Imaging.

Samples were imaged with a Zeiss Axioplan 2 epifluorescence microscope or a Zeiss LSM-510 confocal microscope. Confocal image capture and processing are described in SI Methods.

Supplementary Material

Supporting Information:

Acknowledgments

We thank K. Avila, S. Desiraju, D. Freund, L. Krass, M. Hertz, E. Milne, L. Potts, B. Walker, and C. Ward for assistance with comparative regeneration experiments; L. Shapiro for assistance with phylogenetic analyses; E. Zattara for tubulin–serotonin–phalloidin labeling and imaging; D. Sanger and F. Holland for Monopylephorus samples; and L. Shapiro, E. Abouheif, K. Nyberg, E. Zattara, and two anonymous reviewers for comments on the manuscript. This work was funded by National Science Foundation grant IOB-0520389 (A.E.B.) and a University of Maryland General Research Board Award (A.E.B).

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0907931107/DCSupplemental.

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