Phylogenetic Distribution of Regeneration and Asexual Reproduction in Annelida: Regeneration Is Ancestral and ﬁssion Evolves in Regenerative Clades

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Invertebrate Biology 135(4): 400–414. © 2016 The Authors. Invertebrate Biology published by Wiley Periodicals, Inc. on behalf of American Microscopical Society. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modiﬁcations or adaptations are made. DOI: 10.1111/ivb.12151

Phylogenetic distribution of regeneration and asexual reproduction in Annelida: regeneration is ancestral and ﬁssion evolves in regenerative clades

Eduardo E. Zattara,1,2,3,a and Alexandra E. Bely3

1Department of Invertebrate Zoology, National Museum of Natural History, Smithsonian Institution, Washington, District of Columbia 20560-0163, USA 2Department of Biology, Indiana University, Bloomington, Indiana 47405-7107, USA 3Department of Biology, University of Maryland, College Park, Maryland 20742, USA

Abstract. Regeneration, the ability to replace lost body structures, and agametic asexual reproduction, such as fission and budding, are post-embryonic developmental capabilities widely distributed yet highly variable across animals. Regeneration capabilities vary dramat- ically both within and across phyla, but the evolution of regeneration ability has rarely been reconstructed in an explicitly phylogenetic context. Agametic reproduction appears strongly associated with high regenerative abilities, and there are also extensive developmental simi- larities between these two processes, suggesting that the two are evolutionarily related. However, the directionality leading to this relationship remains unclear: while it has been proposed that regeneration precedes asexual reproduction, the reverse hypothesis has also been put forward. Here, we use phylogenetically explicit methods to reconstruct broad patterns of regeneration evolution and formally test these hypotheses about the evolution of fission in the phylum Annelida (segmented worms). We compiled from the literature a large dataset of information on anterior regeneration, posterior regeneration, and fission abilities for 401 species and mapped this information onto a phylogenetic tree based on recent molecular studies. We used Markovian maximum likelihood and Bayesian MCMC methods to evaluate different models for the evolution of regeneration and fission and to estimate the likelihood of each of these traits being present at each node of the tree. Our results strongly support anterior and posterior regeneration ability being present at the basal node of the annelid tree and being lost 18 and 5 times, respectively, but never regained. By contrast, the ability to fission is reconstructed as being absent at the basal node and being gained at least 19 times, with several possible losses. Models assuming independent evolution of regeneration and fission yield significantly lower likelihoods. Our findings suggest that anterior and posterior regeneration are ancestral for Annelida and are consistent with the hypothesis that regenerative ability is required to evolve fission. Additional key words: Annelida, regeneration, asexual reproduction, fission, phylogenetic methods, evolution, post-embryonic development, ancestral state reconstruction

In animals, several possible developmental trajec- are highly variable across animal phylogeny (Ghis- tories can generate the adult phenotype, including elin 1987; Adiyodi & Adiyodi 1994; Brockes & embryogenesis, regeneration, and agametic asexual Kumar 2008; Bely & Nyberg 2010), indicating that reproduction (e.g., fission, budding). While embryo- these trajectories have been evolutionarily labile. genesis is clearly a shared, ancestral feature of ani- Elucidating the pattern of evolution of regeneration mals, both regeneration and agametic reproduction and agametic reproduction can thus provide insights into how developmental trajectories evolve. aAuthor for correspondence. Although regeneration and agametic reproduction E-mail: [email protected] have distinct functions (repair and restoration in the Regeneration and fission in Annelida 401 former; reproduction in the latter) and distinct required to evolve agametic reproduction, then potential adaptive values, they have often been clades with agametic reproduction are expected to viewed as largely equivalent processes (Bourne 1891; be nested within clades that can regenerate, and Galloway 1899; Dehorne 1916; Berrill 1952; Gibson gains of agametic reproduction are expected to fall & Paterson 2003). This is because regeneration and on branches inferred to have sufficient regenerative agametic reproduction tend to co-occur across the abilities. If regeneration evolves as an exaptation animal phylogeny (Vorontsova & Liosner 1960; of agametic reproduction, then regenerating clades Hughes 1989), with organisms that are capable of should nest within clades with agametic reproduc- agametic reproduction typically having high regener- tion, and gains of regeneration are expected to fall ative abilities, and because the developmental on branches inferred to have agametic reproduc- processes underlying these two trajectories within a tion. These two hypotheses, along with the null given organism tend to be extremely similar hypothesis that regeneration and agametic repro- (Adiyodi & Adiyodi 1994; Rinkevich & Matranga duction evolve independently of each other, can 2009; Zattara & Bely 2011). Recent studies demon- be tested by mapping regeneration and fission strate, however, that these developmental trajecto- abilities onto a phylogenetic tree and reconstruct- ries can be decoupled (Bely 1999a; Brockes & ing ancestral states (Pagel et al. 2004; Paradis Kumar 2008; Bely & Sikes 2010) and that, although 2011). very similar, they are not developmentally equiva- The phylum Annelida, the segmented worms, is a lent: differences are evident in the extent and timing group that is particularly well suited for investigat- of tissue remodeling as well as gene expression (Hori ing the evolutionary patterns of regeneration and & Kishida 1998, 2001; Bely & Wray 2001; Martinez agametic reproduction. The relatively uniform body et al. 2005; Reitzel et al. 2007; Burton & Finnerty plan of annelids (Fig. 1A) facilitates comparisons 2009; Lengfeld et al. 2009; Zattara & Bely 2011). of regenerative potential and fission modes, and Thus, regeneration and agametic reproduction variation in both regenerative and fission abilities is appear to be evolutionarily related but not equiva- well documented in the phylum (Bely 1999b, 2006, lent trajectories. 2010; Zattara 2012; Bely et al. 2014). Following But which trajectory evolves first, and which fol- transverse amputation of the body, species vary in lows, remains debated. The most widely held view is their ability to regenerate anterior structures from that regenerative capabilities are a pre-requisite for an anterior wound surface (anterior regeneration) the evolution of agametic reproduction (Morgan as well as their ability to regenerate posterior struc- 1901; Vorontsova & Liosner 1960; Schroeder & tures from a posterior wound surface (posterior Hermans 1975; Ghiselin 1987), and thus that regeneration) (Fig. 1B). Although rare overall in regeneration evolves first. However, it has also been the phylum, agametic reproduction by fission proposed that regeneration evolves as an epiphe- occurs in many groups of annelids (Schroeder & nomenon of agametic reproduction (Darwin 1868; Hermans 1975). Two main types of fission have Sanchez Alvarado 2000; Giangrande & Licciano been described (Fig. 1C): architomy (also known as 2014), and thus that agametic reproduction evolves fragmentation or scissiparity), in which physical first. Arguments in support of the “regeneration separation precedes the development of new tissues; first” hypothesis are based mostly on the phyloge- and paratomy, in which new tissues develop prior netic distribution of these trajectories, with regenera- to physical separation. Much progress has recently tion having a much broader distribution than does been made in clarifying deep level relationships agametic reproduction, and the suggestion that the among major annelid clades (Struck et al. 2011; evolution of agametic reproduction would seem to Weigert et al. 2014; Andrade et al. 2015; Weigert & require a pre-existing ability to regrow the relevant Bleidorn 2016), providing enough phylogenetic con- body parts. Arguments in support of the “agametic text for interpreting variation in regeneration and reproduction first” hypothesis are typically based on fission. the prevalence of agametic reproduction among In this study, we generate the first phylum-wide, basal animals, suggesting it could be ancestral for species-specific mapping of presence/absence of animals, and on the expectation that high regenera- regeneration and fission ability. We estimate ances- tion ability would be an exaptation of an organism’s tral character states for nodes within the annelid ability to reproduce agametically. tree, allowing us to infer the most likely capabilities These two scenarios make different predictions for the last common ancestor of Annelida, and test regarding the expected phylogenetic patterns of hypotheses regarding the evolutionary relationship these two trajectories. If regenerative ability is between regeneration and fission.

Invertebrate Biology vol. 135, no. 4, December 2016 402 o.15 o ,Dcme 2016 December 4, no. 135, vol. Biology Invertebrate ability. regenerative of presence as scored as section were one scored regeneration least were axial at III I but section and type II, no II and type but regeneration types (like regenerated, axial regeneration work; II axial be of axial this type I can Lack III (3) in type segments individuals). type these), regeneration (2) complete more (4) of of two and regenerate), or removal absence in individuals), tissue one partial result complete and terminal can after two zones of plane in each regenerated growth portions result be between caps, small can can located asegmental even plane pygidium are (the (not or zones regeneration regeneration prostomium axial growth axial asegmental (anterior Two no (the main ends. (1) the regeneration segment. terminal as along adjacent segments the here of its categorized cap series and pygidium, a region and of asegmental composed prostomium is regions, individual asegmental the of Most panels. 1. Fig. ie niaewudsrae B o) sinzns(,tpo rhtm lc n o fprtm lc) n out- and block), paratomy of top and dashed block and architomy physical zones; of to growth elsewhere). top prior represent C, (C, bands zone and zones transverse fission (B dark fission characteristic tissues shaded; top), new a of are of (B, formation in tissues line surfaces precedes formed developed wound separation are newly physical indicate structures C, which lines new and in which B architomy, In in annelids: separation. paratomy, in and known structures, are missing reproduction agametic of modes A PRESENT ABSENT B tissues re-grow only terminal prostomial one segmentre-grow prostomium and atleast segmental reg.: asegmental reg.: small prostomial ends failure even to re-grow no regeneration: (asegmental) there isatleastoneamputationplanedividingtwo parts prostomium segmental reg. from atleasttwo fragments: each capable of re-growing intoacomplete individual each capableofre-growing ye frgnrto n sini annelids. in fission and regeneration of Types growth zone regeneration anterior anterior

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segments A. Type III axial reg. Type II axial reg. Type I axial reg. No axial reg. eeaie nei oypa.Atro st h eti all in left the to is Anterior plan. body annelid Generalized C PARATOMY ARCHITOMY agametic reproduction by fission – otro)bd xs Two axis. body posterior) development development separation separation growth zone posterior atr Bely & Zattara (asegmental) C. pygidium w main Two Regeneration and ﬁssion in Annelida 403

Methods one segment (along with associated terminal asegmental tissue), either anteriorly or posteriorly. Scoring for the presence or absence of fission pre- Literature survey and character coding sents a different set of challenges. The presence of We conducted extensive literature searches to col- paratomy is unambiguous, based on the presence of lect records of the success or failure of anterior and characteristic fission zones within individuals and posterior regeneration, and records of the presence the formation of worm chains (Fig. 1C). However, or absence of fission (architomy or paratomy). New ascertaining the presence of architomy can be more records were appended to previous compilations of difficult, especially from field observations, because such data (Bely 1999b, 2006, 2010; Zattara 2012). it can be confounded both with non-reproductive To avoid duplications and confusions due to autotomy and post-injury regeneration. We scored nomenclature changes, all species names were reports of spontaneous fragmentation followed by checked and updated using the World Record of regrowth of missing parts as well as field observa- Marine Species database (WoRMS Editorial Board tions reporting roughly similar frequencies of anteri- 2016). orly and posteriorly regenerating fragments as Based on literature descriptions, we classified the architomy present when the study’s author(s) sus- regenerative abilities of species into the discrete pected architomy. Assessing absences is inherently regeneration categories of Zattara (2012). We more difficult; we typically scored fission as being focused specifically on regeneration of the main absent when a report stated explicitly the suspicion body axis; data on regeneration of appendages (e.g., of a lack of agametic reproduction based on multi- parapodia, palps, tentacles) are limited (Bely et al. ple field observations, or when comparative studies 2014) and were not included in our analysis. In that provided evidence for the presence of fission in annelids, the main body axis is composed primarily certain species did not indicate fission in other spe- of a series of segments, with a small asegmental ter- cies with similar ecologies (e.g., Franke 1999; Peter- minal tip at the anterior end (the prostomium) and sen 1999; Tovar-Hernandez & Dean 2014). We also at the posterior end (the pygidium) (Fig. 1A). A considered blanket statements made about the pres- posterior growth zone, within which new segments ence or absence of fission in particular groups that form during normal growth, is located just anterior have had a history of intense comparative study to the pygidium in most annelid species (Zattara & (e.g., Berrill 1952; Lasserre 1975; Schroeder & € Bely 2013; Ozpolat & Bely 2016); similarly, an ante- Hermans 1975). rior growth zone located between the anterior-most segment and the prostomium has also been Tree construction described (Zattara & Bely 2013). We focused on four types of regeneration along The maximum likelihood ancestral character esti- the main body axis (Fig. 1B), scoring species based mation method we used (see below) requires a fully on the maximum regenerative abilities reported: no resolved rooted phylogenetic tree with complete axial regeneration—even small portions of terminal branch length information (Paradis et al. 2004). tissue along the main body axis fail to be regener- Because no single phylum-wide phylogenetic dataset ated; type I axial—asegmental regeneration occurs available contained most of the species in our data- after partial amputation of the prostomium or base, we generated a phylogeny by first compiling a pygidium; type II axial—asegmental and segmental reference topology based on recent phylogenetic regeneration occurs after removal of the complete studies and then using publicly available molecular prostomium or pygidium and one or more segments, sequence data to estimate branch lengths and but there is no transverse amputation plane that will resolve polytomic nodes. result in regeneration of two complete individuals; We searched the GenBank nr database for avail- and type III axial—at least one transverse plane can able sequence information for each species in our separate the animal into two parts that are each dataset, specifically retrieving data for fragments of capable of regenerating complete individuals. mitochondrial (12S rRNA, 16S rRNA, cytochrome Because many reports were not specific enough to oxidase subunit I) and nuclear (18S rRNA, 28S distinguish between type II and type III, we further rRNA) markers. Wherever possible, we used simplified our criteria and scored no regeneration sequences from the same species for which regenera- and type I as regeneration absent and types II and tion/fission data were scored; when such sequences III as regeneration present (Fig. 1B). In effect, then, were not available, we used sequences from a close our scoring reflects the ability to regenerate at least relative as a proxy. Species in our dataset for which

Invertebrate Biology vol. 135, no. 4, December 2016 404 Zattara & Bely we found no specific or suitable proxy sequences estimation, ape package), which uses maximum like- were excluded from further analysis. All sequences lihood to fit a Markovian model and estimates the were grouped by marker and aligned using MAFFT parameters of a transition rate matrix and the likeli- v7.017 (Katoh & Standley 2013). Alignments were hood for each character state at every node of the inspected and corrected by eye, and then concate- tree, including the basal node. nated. We used TreeGraph2 v.2.7.1 (Stover€ & Muller€ 2010) to place all species in a reference tree Model testing by Bayesian inference derived from recent phylum to family level phylogenetic studies (Bely & Wray 2004; Sjolin et al. 2005; The same species tree and data matrix were also Bely & Sikes 2010; Erseus et al. 2010; Kvist et al. analyzed with the package BayesTraits v2.0 (Pagel 2010; Martin et al. 2010; Capa et al. 2011; Struck et al. 2004, 2006) to perform independent maximum et al. 2011; Aguado et al. 2012; James & Davidson likelihood (ML) and Monte Carlo (MCMC) analy- 2012; Weigert et al. 2014; Mejlon et al. 2015). Sets ses comparing different models of trait evolution. of species for which no prior phylogenetic data were Following the testing framework proposed by available, or which presented no significant internal Keever & Hart (2008), we compared four models variation in regenerative and fission abilities and each for anterior regeneration, posterior regenera- thus would not influence ancestral trait estimations, tion, and fission. The four models we compared were assigned to polytomies in the tree. We then were: (i) a null model with a single transition rate used a maximum likelihood algorithm constrained q01=q10 (where q01 represents the rate of gaining to use this reference tree to estimate all branch the trait and q10 represents the rate of losing the lengths and resolve any existing polytomies. For this trait); (ii) a free model allowing q01 and q10 to be purpose, we ran our multiple sequence alignment in estimated independently; (iii) a no-losses model in RAxML v7.2.8 (Stamatakis 2014), partitioning the which q10=0; and (iv) a no-gains model in which dataset by marker, using a GTRGAMMA substitu- q01=0. For each trait and model, we performed a tion model, and constraining the analysis to use the MultiState analysis running an MCMC chain for reference tree. The ribbon worm Lineus viridis 300 million iterations, sampling every 1000 steps (Nemertea: Lineidae) was used as an outgroup to after an initial burn-in period of 1 million steps. We root the tree. Sequence retrieval, sequence align- used a gamma-distributed hyperprior set between 0 ment, and phylogenetic analyses were performed and 1, and estimated the marginal likelihood for using Geneious R8.1 and its plug-ins (Kearse et al. each model using the stepping stone sampler imple- 2012). The complete database (including species, mented in BayesTraits v2.0. The entire set of runs taxon codes, character states for regeneration and was repeated a second time to verify consistency of fission, and literature references), the multiple results. We tested against our null model each of the sequence alignments, and our complete tree can be other three models using a Bayes factor test (Pagel found in the online Supporting Information and et al. 2006). have been deposited in the Zenodo repository (Zattara & Bely 2016). Contingency testing for regeneration and fission To assess the correlation between the evolution of Ancestral trait estimation by maximum likelihood regeneration and fission, we used the “Discrete” The fully resolved rooted phylogenetic tree was methods implemented in BayesTraits v2.0. These analyzed in the R computing environment (R Devel- methods work as described above, but assume either opment Core Team 2011) using the ape (Paradis independent evolution by estimating four rate et al. 2004; Paradis 2011) and diversitree (FitzJohn parameters (gain and loss rate for each trait) or con- 2012) packages. We coded each of the three charac- tingent evolution, allowing up to eight rate parameters (anterior regeneration, posterior regeneration, ters where probability of gains or losses of one trait and fission) as binary variables (absent or present). can be different depending on the state of the other We also coded fission type as a character with three trait. We performed two sets of comparisons, one in possible states: none, architomy, or paratomy. We which all parameters were freely estimated, and specified a two-parameter model allowing for sepa- another in which we enforced the parameter restric- rate calculation of the rate of gain (0?1) and loss tions of the best model from the original Bayesian (1?0). Each character was then analyzed separately, analysis (see previous section). We calculated mar- removing all species missing data for that trait, and ginal likelihoods and used Bayes factors as described run through the ace function (ancestral character above to test for contingency.

Invertebrate Biology vol. 135, no. 4, December 2016 Regeneration and ﬁssion in Annelida 405

estimating branch lengths based on aligned sequences Results and discussion for several commonly used molecular markers. We were able to retrieve and align sequence data for 243 Knowledge about regeneration and fission covers only of the 401 species in our dataset (Fig. 2B). a small fraction of annelid diversity Mapping the assembled regeneration and fission Regeneration and asexual agametic reproduction data in a phylogenetic context reveals considerable in annelids have long been under study, starting variability in these traits across the phylum (Figs. 3, with the first experimental report in the 18th century 4, S1). Some broad patterns emerge from our map- of a water worm (likely Lumbriculus variegatus) cap- pings, such as that posterior regeneration is very able of growing missing parts after amputation broadly distributed across the phylum (Figs. 3B, 4, (Bonnet 1745). Despite close to three centuries of S1); that anterior regeneration is widely distributed study, however, the phylogenetic distribution of but appears to be absent in certain groups such as these phenomena, and their relationships to each nereids, capitellids, and echiurids (Figs. 3A, 4, S1); other, are only now becoming clarified. that architomic fission occurs in many groups across After a thorough literature search and building the phylum but paratomic fission is restricted to a on previous efforts (Bely 1999b, 2006; Zattara 2012; few specific clades including sipunculans, serpulids, Bely et al. 2014), we compiled a dataset of annelid aeolosomatids, and naidids (Figs. 3A, 4, S1); and species for which presence or absence of anterior that both axial regenerative ability (both anterior regeneration, posterior regeneration, or fission was and posterior) and fission are absent in hirudines explicitly reported (see Appendix S1 for full refer- (Figs. 3, 4, S1). Another important result is that ences, Appendix S2 for the complete database, and many groups in our dataset have missing data for Appendix S3 for description of database fields). regeneration and/or fission and, more broadly, that After evaluating species nomenclature and correct- many annelid groups (including some common and ing for synonymies, our dataset includes 401 species widespread ones) are not represented at all in our for which data were available for at least one of the dataset. Given the apparent bias against reporting traits, and 154 species for which data were available negative results (regeneration failure) (Bely 2010), for all three traits (Fig. 2A). Considering that there we anticipate that many of these blocks of missing are over 17,000 known species of Annelida (Zhang data will ultimately be filled with absence of regen- 2013; Weigert & Bleidorn 2016), our current knowl- eration (i.e., being found unable to regenerate). edge of the distribution of regeneration and fission A common challenge in the analysis of presence/ is based on only ~2% of the total diversity of the absence datasets is detection bias and strength of phylum. evidence. Detection bias results when certain charac- To provide the phylogenetic framework for inter- ter states are more likely to be detected and scored preting our regeneration and fission dataset, we gen- than others. Because absence of evidence is not evi- erated a phylogeny by specifying a topology based on dence of absence, the strength of evidence needed to recent phylogenomic studies (see Methods) and by evaluate different characters can thus vary mark- edly. With respect to our traits of interest, presence of regeneration can be determined based on a single A B fixed specimen showing clear evidence of regeneration, whereas absence of regeneration requires g Po Po re ste reg st r r r er experimental testing; it is easier to detect anterior o io o i ri i o r r r e than posterior regeneration from fixed and field-col- r e t 58 r t 40 e e

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g lected specimens, because anteriorly regenerating A 154 A 119 structures tend to show a more obvious size mis- 57 17 match relative to the stump than do individuals with 153 62 posterior regeneration; and the presence of paratomic fission can be ascertained even from a single, Fission Fission fixed individual, while the presence of architomy Fig. 2. Representation of regeneration (reg) and fission requires either direct live observation of the fission data in our database. A. Venn diagram showing number process, or inference from a considerable number of of species with data (presence or absence) for each of the field-collected specimens. three traits in the complete database. B. Venn diagram as The dataset we have assembled and analyzed here in A but including only species for which sequence data covers annelids broadly and provides the most com- were available. plete and rigorous phylum-wide view of the

Invertebrate Biology vol. 135, no. 4, December 2016 406 Zattara & Bely

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N e TE N N S SER JOSma SA N pa N Y u S N pu SYL SY p R FI S SY AMmuYz a SE ER se SYL YL Ls SYL SY YL R R S pu S YL L RA On F p S SA ER SPR Lim S B gi SAB F X YL IL SY L DOfu ct S EX X B ILstX el Y L DO O l S SAB PM SYL T b AB F S Y L O S EG ABs Isp S Y OD la pX S YL O EU SA MY spX S IO i SAB S t L B M Xs Er SYL RCsOp S SY L P R S A SABY pX e L P P Rpl AB B X SY Y B in S YL MI P RA sp pi S AR S YL MIRedIR A b S L pa B SA a a Y M R SP bX S YL SP I Bal S L MI PIal o I S S PY PIf SY L E MBf be SPI BOCPI G i SY L A R B el PA al S OC SY AR PI p li DOR P SPI BOCh rX OR One a D D U DI SP YPca a IOmX I ON U D p SP POLc X ON INs I P N D Rco SE o DI EU CAP C paX P uX AP AM n ECH te P SIP el ECH BO SI ASP Nvi SIP IbeX URE A SP ECH E ca CH so CHe HY MAL CL c CHA P Ico A Yto CHA SP pr EO PO A PHY AEO Tvi CH a AEOhe CHAv AE CHA Xz O AEOsp OWE MYRsp AEO AEOvi OWE OWEfu AEO RHEne OWE GALoc TUB BRAso LIN LINvi TUB TUBtu GLS HAEde TUB AULp l BRA CIRsaX TUB ILY te BRA BRAit mo BRA B TUB POT RApaX Tha BRA TUB PO slr(0)=0.95 XIRvi Tbe BRA XIRin TUB PO BRA PSAba XIR TUB iX BRA oc ISOg ANKl TUB Big slr(1)=0.05 GLS a TU XI UB CA Rf T Mud M o CA OEDma LIM LILIMcl CAMM CA ho PTE LIM LIM C an IM ve AM M L BOT CAM CA me B RIae C Mfa TU I P Ile AM CCA o CAM M Nemertea (outgroup) PR CAM A ch PRI PR o C Mp RIc u BRA AMpa h PRI IPRIl T B SA ONr o D M Ta OP Yc a G E B A t H v LMBLS H G HY M R R di D ob Basal clades R E R LMB S Ei HY IR l E Rfu L L me R L M UMv NAI DAI D Lin MB B T N I DE r LU L Y A L AMstXhe a N li U M R ALLpa R LU H NAI AL PARf LU M EI Errantia AI Iel M Y I o E N A c r L S te N I t LU U M A I fe NA AI PA Ib M San Is LU NICmP N AI A L M L Oca N p L U M L AI NA NA a G U UM G M D N A E LO M U L E LO Mh o Non-clitellate Sedentaria NAI N e EN N A E te X EJsp NAI S Ejo s ENC N E IGmi lo E C L N o ENC P E O L V f E C e C E N CR v EN C I E ME E ONc chX e I P N NC PmeX a NAI UNCun N a C L IPpa a CO NC E COG NAI N

j TY C NA AR C I AI SP R C M C l NA C S a FR o N EN F EN B U STYla A AI CHAli N G p AI E Scaled likelihood of presence R Aeolosomatidae CHAdp U M R sp e NAI OPH N E NC I N Is gl N AMPs Ir C c NAI S Cb li NAI Ccr C a t C oX NAI a MARspB NA AI a

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Clitellata ENC Scaled likelihood of absence Fig. 3. Phylogenetic distribution and ancestral state estimation of anterior regeneration (A), posterior regeneration (B), and fission (C, presence/absence only) in Annelida. Node likelihoods are shown as pie charts in these radial trees; species character states are shown as circles at the tips. Values in the center show scaled likelihood at the root (slr) for each character state. Colored arcs surrounding the diagrams indicate major annelid groups. A lookup table for taxon codes is provided in Appendix S4: taxa were coded as “FAM GENsp,” where FAM and GEN, respectively, indicate the first three letters of the family and genus, and sp indicates the first two letters of the species name. An X at the end of the name indicates a proxy species that was used to estimate branch lengths for the analysis; in those cases, the indicated trait states correspond to the species indicated in Appendix S4, not the proxy species. Note that branch lengths in all diagrams are arbitrary and do not represent substitution rates or any other value. Full species names, taxonomy, trait states, literature data sources, and sequence accession numbers are provided in the online supporting information and are also available at the Zenodo repository (Zattara & Bely 2016). distribution of regeneration and fission to date. evidence make our results necessarily preliminary. However, the large amount of missing data and Fortunately, increased interest in regeneration issues surrounding detection bias and strength of biology and awareness of the importance of

Invertebrate Biology vol. 135, no. 4, December 2016 Regeneration and fission in Annelida 407 comparative regeneration datasets with fine taxo- depth from fairly deep in the tree (suggesting old nomic sampling have spawned new research efforts losses; e.g., hirudines, nereids) to fairly shallow in that exemplify how these challenges can be over- the tree (suggesting recent losses; e.g., Americonuphis come to gain valuable insight into the evolution of magna [Onuphidae], Myxicola infundibulum [Sabelli- regenerative abilities (Franke 1999; Williams 2000; dae], Paranais spp. [Naididae]). Lindsay et al. 2007; Berke et al. 2009; Bely & Sikes The analysis for posterior regeneration, based on 2010; Licciano et al. 2012, 2015; Pires et al. 2012; 237 species, reconstructs the last common ancestor Murray et al. 2013; Aguado et al. 2014; Goncßalves of annelids as also being capable of posterior regen- et al. 2016). Large-scale coordinated efforts to fill in eration, defined as reconstructing a lost pygidium, the annelid tree for regeneration and fission ability posterior growth zone, and one or more posterior should be encouraged, as these will be particularly segments (Fig. 3B). In our maximum likelihood valuable, especially if they employ standardized pro- analysis, the scaled likelihood of presence at the tocols (e.g., consistent amputation positions), to root is 1, the estimated tree-wide rate of loss is generate comparable data in a broad range of spe- 0.0046Æ0.0022, and the estimated rate of gain is cies. 0Æ0.0365. Note that the estimated rate of loss of posterior regeneration is considerably lower than for anterior regeneration. The Bayesian analysis found Ancestral character estimation strongly supports that the best model of character evolution was the presence of anterior and posterior regeneration at the null model in which gains and losses are equally base of Annelida, with subsequent losses likely (q0?1=q1?0=0.005; Fig. S2B); only the alter- We used the fully resolved phylogenetic tree with native model which forbids gains had a better mar- branch lengths based on molecular data and our ginal likelihood, but the improvement was not character matrix to estimate the likelihood for ante- enough to reject the null model (Bayes factor rior regeneration, posterior regeneration and fission Bf=1.2). Both of these models give a posterior prob- to be present or absent for each node in the tree ability of 1 for posterior regeneration being present (Appendices S4, S5). We analyzed each of the three at the root of the annelid tree, consistent with our traits separately, removing for each analysis those maximum likelihood analysis. Posterior regeneration species with missing data and running a maximum is inferred to have been lost at least five times and likelihood algorithm to independently estimate the never regained (Figs. 4, S1); similar to anterior most likely rates of trait gain and loss. We then regeneration, losses range from relatively deep in the reanalyzed the data using a Bayesian framework to tree (suggesting old losses; e.g., hirudines) to fairly test alternative models of character evolution. shallow in the tree (suggesting recent losses; e.g., Our analysis of anterior regeneration, based on Chaetogaster diastrophus [Naididae]). 163 species, strongly supports the hypothesis that Our analyses provide important first estimates of anterior regeneration (defined as axial type II or III) the number of transitions in regeneration ability in was present at the basal node of Annelida and was annelids. However, it is important to keep in mind subsequently lost multiple times (Fig. 3A). In our that the specific number of reconstructed transitions maximum likelihood analysis, the scaled likelihood is strongly dependent on both taxon sampling and of presence at the root is 1, the estimated mean tree- phylogenetic topology and to note that many of wide rate of loss and its standard error is the regeneration losses map to shallow regions of the 0.0259Æ0.0050, and the estimated rate of gain is tree, where bipartition support is lower than in the 0Æ0.0147. The Bayesian analysis gave a similar deeper regions of the tree due to smaller datasets. result: the best model of character evolution was Another caveat worth noting is that although the that in which no gains are allowed (q0?1=0; maximum likelihood and Bayesian methods we used q1?0=0.03; Fig. S2A), and this model gives a poste- do not incorporate a priori expectations about the rior probability of 1 for anterior regeneration being likelihood of gaining versus losing regeneration and present at the root of the annelid tree. Thus, based fission, they make the somewhat unrealistic assump- on both maximum likelihood and Bayesian analyses, tion that transition rates do not change over time or the last common ancestor of all annelids is inferred across lineages. For these reasons, the reconstructed to have been capable of reconstructing a lost pros- number of regeneration losses should be taken as use- tomium and one or more anterior segments. This ful first estimates rather than as definitive. The recon- ability is inferred to have been subsequently lost at struction of deeper nodes in the tree is less affected by least 18 times across Annelida but never regained the issues just described. Indeed, the basal node of (Figs. 4, S1). Anterior regeneration losses range in annelids is reconstructed by maximum likelihood and

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AP F BRA

Anterior regeneration (A) CAM absent loss (x18) present gain (x0) LMB

Posterior regeneration (P) LUM absent loss (x5)

present gain (x0) Clitellata ENC Fission (F) absent loss (x8) architomy gain (x20) paratomy NAI S e d e n

PRI t a r i a TUB/LIM

AEO

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SPI

SAB

SER

CIR

ORB

NER E r r SYL a n t i a

DOR ancestral annelid node ONU AP F EUN

AMP b a s

CHA a l OWE

Bayesian methods (Fig. 3) as having both anterior In summary, our analyses provide the ﬁrst formal and posterior regeneration present, indicating that reconstruction of regenerative abilities for any phy- this result is robust to reconstruction methods. lum and suggest that annelids have an ancestral

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Fig. 4. Distribution of regeneration and fission across Annelida. Depicted is a dendrogram combining results from the individual trait analyses for anterior regeneration, posterior regeneration, and fission. Observed character states are shown next to the tips; branches are colored according to estimated presence (green) or absence (orange) of anterior regeneration; and dashed branches indicate low confidence reconstructions due to missing data at the tip. The most likely character states at the basal annelid node are indicated near the root of the tree. Also marked are branches along which anterior regeneration (circles), posterior regeneration (bars), and fission (triangles) are inferred to have been lost (orange) or gained (green or blue). Note that branch lengths are arbitrary and do not represent substitution rates or any other value. Gray vertical lines indicate some (but not all) of the groups (usually families) represented as follows: AEO, Aeolosomatidae; AMP, Amphinomiidae; BRA, Branchiobdellidae; CAM, Cambarinicolidae; CAP, Capitellidae; CHA, Chaetopteridae; CIR, Cirratulidae; DOR, Dorvilleidae; ECH, Urechidae+Bonelliidae; ENC, Enchytraeidae; EUN, Eunicidae; LIM, Limnodrilidae; LMB, Lumbriculidae; NAI, Naidinae; NER, Nereididae; ONU, Onuphiidae; ORB, Orbinidae; OWE, Oweniidae; PRI, Pristininae; SAB, Sabellidae; SER, Serupulidae; SPI, Spionidae; SYL, Sylli- dae; TUB, Tubificinae. Brackets indicate higher level clades. Full species names, taxonomy, trait states, literature data sources, and sequence accession numbers are provided in the online supporting information and available at the Zenodo repository (Zattara & Bely 2016). ability to regenerate both anteriorly and posteriorly. reconstructed, although this finding may be the result Specifically, we infer that the ancestral annelid could of sparse sampling of taxa between closely related regenerate—both anteriorly and posteriorly—one or species in which individuals reproduce by fission. more segments, the asegmental caps (prostomium While the same caveats stated above for the regen- and pygidium), and associated growth zones. These eration analysis also apply to this analysis, it is worth abilities have been subsequently reduced or lost noting that both maximum likelihood and Bayesian independently in several lineages, with anterior methods support similar rates of gains and losses of regeneration losses being much more frequent than fission, although a cursory examination of the fre- posterior regeneration losses. Furthermore, there is quency and distribution of fission suggests that gain- significant variation in the age of regeneration ing the ability to fission would be much more likely losses. Since lineages showing relatively recent losses than losing it. Indeed, while several gains of fission are particularly useful for investigating the mecha- have been previously suggested across annelids nisms underpinning loss of regeneration (Bely 2010), (Schroeder & Hermans 1975; Bely & Sikes 2010), our results highlight Annelida as a promising phy- clear and well-supported examples of fission loss have lum in which to study this evolutionary process. yet to be reported. Furthermore, it should be kept in mind that when independent evolutionary changes occur among close relatives, these changes are less Agametic reproduction by fission is inferred to have likely to be correctly reconstructed by methods that been absent in the last common ancestor of Annelida assume fixed rate parameters across the whole tree. and subsequently gained many times independently For example, among the naidid clitellates, paratomic In contrast to our findings for anterior and poste- fission occurs in two closely related clades, the Pristin- rior regeneration, our ancestral character estimation inae and the Naidinae. In our analyses, fission is analyses of fission, based on 189 species, strongly reconstructed as having been gained at the base of the supports the absence of fission in the basal node of common ancestor of the Pristininae and Naidinae the annelid tree (Fig. 3C). In our maximum likeli- and subsequently lost in the lineages intervening hood analysis, the scaled likelihood of absence at the between these two clades (e.g., Rhyacodrilus and root is 0.95, the estimated tree-wide rate of gain of Monopylephorus), yet inclusion of additional interme- fission is 0.0434Æ0.0076, and the estimated rate of diate lineages (which exist but were not present in our loss is 0.0402Æ0.0125. Consistent with these results, dataset) in the analysis could change this reconstruc- the Bayesian analysis found that the best model of tion from a scenario of one gain followed by losses to character evolution was the null model in which a scenario of two independent gains. Indeed, based gains and losses are equally likely (q0?1= on morphological and developmental data it has been q1?0=0.05; Fig. S2C) and the posterior probability argued that these two clades have most likely gained of fission being absent at the root of the annelid tree fission independently (Envall et al. 2006; Zattara is 0.95. Based on our maximum likelihood analysis, 2012) and thus that there have not been any losses. fission is reconstructed as having been subsequently Interestingly, this scenario of a dual origin of fission gained at least 19 times (Figs. 4, S1). Our analysis is recovered when the maximum likelihood analysis is also suggests losses of fission, with eight losses run separately on data for the Naididae clade only

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(unpubl. data), demonstrating how global and local different models that assume either independence parameters can differ in their optima. Cirratulids, ser- or contingency between two binary traits; for this pulids, and sabellids are other groups in which our analysis, we considered anterior regeneration and analysis suggests potential losses of fission; broader fission as the two traits (since posterior regenera- sampling, local character estimation analyses, and tion is nearly always present). In independent mod- developmental studies should be performed in repre- els, the rates of gain and loss are estimated sentatives of these families to obtain stronger data independently for each trait (four parameters in regarding the pattern of fission evolution in these total); in dependent models, rates for a given trait groups. are separately estimated for each of the two possi- Whether agametic reproduction is ancestral for ble states of the other trait (eight parameters in certain bilaterian phyla has long been debated total). We made two sets of comparisons between (Rieger 1986; Janssen et al. 2015) but has not previ- independent and dependent models: in the first, we ously been addressed using comparative methods. allowed all parameters to be freely estimated; in Our analysis represents the first phylum-wide recon- the second, we imposed restrictions based on a sce- struction of agametic reproduction and strongly sup- nario in which anterior regeneration cannot be ports the premise that fission was absent in the basal gained, and fission can only evolve when anterior node of the annelid tree. While this result is sensitive regeneration is present. In both cases, we found to presence of fission in the outgroup (switching the that the dependent models had significantly higher outgroup from fission-absent to fission-present likelihoods than the independent models (Fig. S2D; changes the scaled likelihood of absence at the root Bf=15.7 for free models, Bf=11.6 for restricted from 0.95 to 0.60), there is no strong support for models). agametic reproduction being present in the last com- Based on these results, we reject the null hypothe- mon ancestor of either Mollusca (Haszprunar & sis that regeneration and fission are independent. Wanninger 2012) or Nemertea (Zattara, Norenburg, Furthermore, comparison between the free and and Bely, unpubl. data), the two phyla most closely restricted versions of the dependent model indicates related to Annelida (Kocot 2016). Thus, based on that the latter has significantly higher likelihood the currently available data, we conclude that the (Bf=8.1). This finding supports the hypothesis that last common ancestor of Annelida most likely did evolving fission is contingent on the ability to regen- not reproduce asexually by fission, but its descen- erate anteriorly. Thus, the results of this analysis of dants independently evolved this ability many times. the relationship between fission and anterior regeneration, coupled with the fact that posterior regeneration is present almost throughout the annelids, Fission has evolved in clades with high anterior and suggest that fission evolves specifically in lineages posterior regeneration ability that already have the ability to regenerate anteriorly Is there an evolutionary relationship between and posteriorly (more specifically, in lineages that regeneration and fission in annelids? If there is, what already can regenerate asegmental caps and one or is the polarity of this relationship? Our results indi- more segments at both body ends). This conclusion cate a strong tendency for fission to co-occur with is consistent with the nesting of lineages character- both anterior regeneration and posterior regenera- ized by fission within clades characterized by regen- tion. Across the entire dataset, all but one of the spe- eration that is evident in the annelid tree (Fig. 4). cies that can fission can also regenerate posteriorly, We reject the hypothesis that regeneration evolved and only 5 (7%) of the 68 species characterized by fis- subsequent to the evolution of agametic reproduc- sion also lack anterior regeneration (by contrast, of tion and instead contend that regenerative capabili- 87 species that lack fission, 68 (78%) can regenerate ties are a pre-requisite for the evolution of agametic posteriorly and 43 (49%) can also regenerate anteri- reproduction in annelids. orly). These five species belong to three naidid genera Paranais Chaetogaster, Amphichaeta ( , ) that have been Conclusions previously suggested to represent at least two losses of regeneration nested within a clade in which fission We performed a comparative analysis of a litera- was an ancestral trait (Bely & Sikes 2010). ture-based dataset of the capacity for regeneration To investigate the correlation between regenera- and agametic reproduction by fission within the tion and fission we used the model selection frame- phylum Annelida. We inferred that the last common work implemented in BayesTraits (Pagel et al. 2006). ancestor of annelids was able to regenerate both This method compares the marginal likelihood of anterior and posterior ends but could not reproduce

Invertebrate Biology vol. 135, no. 4, December 2016 Regeneration and ﬁssion in Annelida 411

by fission. Our analyses suggest that regenerative References abilities have been lost several times but not regained. By contrast, they suggest that fission has Adiyodi KG & Adiyodi RG eds. 1994. Reproductive been gained many independent times. Finally, our Biology of Invertebrates. Volume VI, Part B. Asexual Propagation and Reproductive Strategies. John Wiley comparison of models assuming dependent or inde- & Sons, Chichester, UK. 432 pp. pendent evolution of anterior regeneration and fis- Aguado MT, San Martın G, & Siddall ME 2012. System- sion indicates that presence of anterior regeneration atics and evolution of syllids (Annelida, Syllidae). is necessary to evolve fission. This strongly supports Cladistics 28: 234–250. a model in which fission has repeatedly evolved by Aguado MT, Helm C, Weidhase M, & Bleidorn C 2014. co-option of regenerative abilities. 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Evol. 25: point for further analysis. Given the renewed inter- 161–170. est in the phylogenetic distribution of regenerative Bely AE & Sikes JM 2010. Latent regeneration abilities and agametic reproductive abilities, we hope our persist following recent evolutionary loss in asexual work will stimulate more detailed and systematic annelids. Proc. Natl Acad. Sci. 107: 1464–1469. comparative studies among closely related species Bely AE & Wray GA 2001. Evolution of regeneration both in Annelida and other phyla. Such studies and fission in annelids: insights from engrailed- and could offer new insights into the proximate and orthodenticle-class gene expression. Dev. Camb. Engl. ultimate mechanisms for the evolution of regenera- 128: 2781–2791. tion and agametic reproduction throughout the ———— 2004. Molecular phylogeny of naidid worms Metazoa. (Annelida: Clitellata) based on cytochrome oxidase I. Mol. Phylogenet. Evol. 30: 50–63. Bely AE, Zattara EE, & Sikes JM 2014. Regeneration in Acknowledgments. This article grew from the seeds spiralians: evolutionary patterns and developmental originally planted in the introductory chapter of the processes. Int. J. Dev. Biol. 58: 623–634. respective doctoral dissertations of both authors, and was Berke SK, Cruz V, & Osman RW 2009. Sublethal preda- presented as part of the “Annelids: In Memory of Kris- tion and regeneration in two onuphid polychaetes: pat- tian Fauchald” Special Session of the 2016 Annual Meet- terns and implications. Biol. Bull. 217: 242–252. ing of the Society for Integrative & Comparative Biology Berrill NJ 1952. Regeneration and budding in worms. in Portland, OR. The authors wish to thank Damhnait Biol. Rev. 27: 401–438. McHugh and Bruno Pernet for organizing this session, to Bonnet C 1745. Traite d’insectologie. Seconde partie. the many participants who gave us enthusiastic feedback Observations sur quelques especes de vers d’eau douce, about this work, and to the editors of Invertebrate Biol- qui coupes par morceaux, deviennent autant d’animaux ogy for putting together this special issue on segmented complets. A Paris, Chez Durand. 264 pp. worms. We also thank Carson Keever and an anonymous Bourne AG 1891. Notes on the naidiform oligochaeta; reviewer who made important suggestions to improve the containing a description of new species of the genera analyses. This article is dedicated to the memory of Kris- Pristina and Pterostylarides, and remarks upon cephal- tian Fauchald. The authors declare that they have no ization and gemmation as generic and specific charac- competing interests. ters in the group. Q. J. Microsc. Sci. s2-32: 335–356.

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(orange) of anterior regeneration; and dashed branches is indicated with darker shading. (A–C) Path diagrams indicate low confidence reconstructions due to missing for models of anterior regeneration (A), posterior regener- data at the tip. The most likely character states at the ation (B), and fission (C). The null model assumes equal basal annelid node are indicated near the root of the tree. rates of gains and losses, the free model allows separate Also marked are branches along which anterior regenera- estimation of gains and losses, and the no gains and no- tion (circles), posterior regeneration (bars), and fission losses models impose unidirectional transitions. D. Path (triangles) are inferred to have been lost (orange) or diagrams for models assuming either independent (top) or gained (green). Note that branch lengths are arbitrary dependent (bottom) evolution of anterior regeneration and do not represent substitution rates or any other and fission. In the free models (left), all rates are freely value. A lookup table for taxon codes is provided in estimated, while in the constrained models (right), no Appendix S4: taxa were coded as “FAM GENsp,” where gains of regeneration are allowed and gains of fission FAM and GEN, respectively, indicate the first three letters require that regeneration is present. of the family and genus, and sp indicate the first two let- Appendix S1. Literature references for regeneration and ters of the species name. An X at the end of the name asexual reproduction in Annelida. This document lists the indicates a proxy species that was used to estimate branch literature sources used to assign presence/absence charac- lengths for the analysis; in those cases, the indicated trait ter states to all species in this study. The document con- states correspond to the species indicated in Supporting tains an initial group of citations for each group and Document 4, not the proxy species. Gray vertical bars family (order approximately as they are seen in the anne- indicate some (but not all) of the groups (usually families) lid phylogeny used in this study), and a numbered list of represented and brackets indicate higher level clades. For full reference information, ordered alphabetically. The FAM codes, note that most codes represent single formal numbers in the fields Ref_ID_Reg and Ref_ID_fission of families, but a few include multiple families, subfamilies Appendix S2 refer to the numbered reference list in this or informal names (informal names are given in quotes): document. AEO, Aeolosomatidae; AMP, Amphinomiidae; ARE, Appendix S2. Annelid regeneration and fission database. Arenicolidae; BDE, Bdellodrilidae; BRA, Branchiobdelli- This spreadsheet contains the full species database dae; CAM, Cambarincolidae; CAP, Capitellidae; CHA, described in this article, including those species for which Chaetopteridae; CIR, Cirratulidae; CTE, “Ctenodrilidae”; no adequate sequence data were found (and which were DIN, Dinophilidae; DOR, Dorvilleidae; ECH, Urechi- therefore not included in the ancestral trait estimation dae+Bonelliidae; ENC, Enchytraeidae; EUN, Eunicidae; analyses). A description of the contents for each column GLO, Glossoscolecidae; GLS, Glossiphoniidae+Xiron- of the spreadsheet is provided in Appendix S3. References odrilidae+Piscicolidae; GLY, Glyceridae; HES, Hesion- in this database are provided as numbers; these reference idae; LIM, “Limnodrilinae”; LIN, Lineidae (Nemertea: numbers correspond to the numbered list of references Pilidiophora); LMB, Lumbriculidae; LUM, Lumbricidae; from Appendix S1 (not to the references in the Main MAL, Maldanidae; MEG, Megascolecidae; NAI, Naidi- Text). nae; NER, Nereididae; ONU, Onuphiidae; OPH, Opheli- Appendix S3. Field descriptions for annelid regeneration idae; OPI, “Opistocystinae”; ORB, Orbiniidae; OWE, and fission database file. This metadata document Oweniidae; PHY, Phyllodocidae; PLG, Polygordiidae; describes the content of each field (column) of PRI, Pristininae; PRO, Protodrilidae+Saccocirridae; Appendix S2. RHY, “Rhyacodrilinae”; SAB, Sabellidae; SER, Serupuli- Appendix S4. Taxon code lookup table for Figs. 2 and dae; SIP, Sipunculidae+Aspidosiphonidae; SPI, Spionidae; S1. This spreadsheet contains the full list of species codes SYL, Syllidae; TER, Terebellidae; TUB, Tubificinae. used in Figs. 2 and S1, along with corresponding infor- Fig. S2. Path diagrams highlighting comparisons between mation on the phylum, group, order, family, genus, and competing models of evolution of regeneration and fis- species from which sequence data were used. The order in sion, as evaluated by BayesTraits. Boxes represent models which species are listed in this spreadsheet corresponds to differing in how transition between states are constrained; the exact order of species (from top to bottom) in the the marginal log likelihood of each model is shown next phylogeny of Figs. 4 and S1. to the model name on top, the estimated transition rates Appendix S5. NEXUS formatted Multiple Sequence are shown in the middle, and the posterior probability of Alignment and Tree. This text file contains the NEXUS the trait being present at the root is shown at the bottom. standard formatted data for the final multiple sequence Values over arrows indicate the Bayes factor of compar- alignment and the topology of the tree used for the analy- isons between null and alternate models. The best model ses presented in this paper. for each trait, as defined by a Bayes factor of 2 or higher,

Invertebrate Biology vol. 135, no. 4, December 2016