Chaetal arrangement and chaetogenesis of hooded hooks in Lumbrineris ( Scoletoma ) fragilis and...

Chaetal arrangement and chaetogenesis of hooded hooks inLumbrineris (Scoletoma) fragilis and Lumbrineris tetraura (Eunicida, Annelida)

Ekin Tilic,1,a Harald Hausen,2 and Thomas Bartolomaeus1

1 Institute of Evolutionary Biology and Ecology, University of Bonn, 53121 Bonn, Germany2 Sars International Centre for Marine Molecular Biology, N-5008 Bergen, Norway

Abstract. As the structure and arrangement of chaetae are highly specific for annelid speciesand higher taxonomic entities, we assume that rather conservative information guaranteesformation of specific chaetae. Each chaeta of an annelid is formed within an ectodermalinvagination, and the modulation of the apical microvilli pattern of the basalmost cell ofthis invagination determines the structure of the chaeta. Any hypothesis of the homology ofchaetae could thus be tested by examining the process of chaetal formation. Investigationsinto the ultrastructure and formation of hooded hooks in different capitellids and spionidsrevealed that these chaetae can be homologized. The hood of each of their hooded hooks isformed by elongation of two rings of microvilli peripheral to the chaetal anlage, which giverise to the inner and outer layers of the hood. The hood layers are well separated andsurround an empty space. Superficially similar hooded hooks are described for certain Euni-cida. Presently available cladistic analyses suggest that the hooded hooks of eunicidansevolved independently of those in Capitellidae and Spionidae. Compared with the latter twofamilies, we therefore expected to find differences in chaetogenesis of the hooded hooks inthe eunicids Lumbrineris (Scoletoma) fragilis and Lumbrineris tetraura (Lumbrineridae). Thiswas the case. In these eunicidans, the hood was formed by the bisected apical wall of thechaetoblast right after the mid-apical section of the chaeta had been sunk deeply into thechaetoblast during its formation. The apical wall generated a brush of microvilli that pre-formed the hood. Because the microvilli of the hood showed some accelerated differentia-tion, they soon merged with those of the slowly growing setal shaft to form the broadmanubrium of the hooded hook in lumbrinerids. Our study confirms the predicted differ-ences in chaetogenesis of the superficially similar hooded hooks of capitellids and spionidscompared with those of eunicids.

Additional key words: Capitellidae, chaetae, Lumbrineridae, Spionidae, ultrastructure

Chaetae of Annelida are chitinous extracellularstructures that are formed within an ectodermalpouch or chaetal follicle (Bouligand 1967; Schroeder1984; Specht & Westheide 1988; Hausen 2005). Thechaetal follicle consists of a few follicle cells and asingle basal chaetoblast. The pattern of the apicalmicrovilli of the chaetoblast is modified constantly,while N-acetyl-glucosamine is released between thebases of the microvilli. This material polymerizesextracellularly, forming the chaeta; thus, the defini-tive structure of the chaeta is primarily caused bychanges in the pattern of chaetoblast microvilli, i.e.,by dynamic microvilli (O’Clair & Cloney 1974).

Chaetae are considered an autapomorphy of anne-lids, and reconstructions of the ancestral annelidpattern (bauplan) concur that early annelids boresimple capillary chaetae (Struck 2011; Struck et al.2011). The dynamic developmental mode of chaetaeallows for the formation of a plethora of differentchaetal types, and these different chaetae are ofimmense functional importance, e.g., in defense,locomotion, burrowing, and anchoring of the bodyto the tube (Merz & Woodin 2006). Arrangementand structure of the chaetae are so conserved inannelid species and supraspecific taxa that chaeta-tion has become an important source of diagnosticcharacters for taxonomists. As the dynamic struc-ture, orientation, and number of microvilli of thechaetoblast determine chaetal structure, these factors

aAuthor for correspondence.E-mail: [email protected]

Invertebrate Biology x(x): 1–17.© 2014, The American Microscopical Society, Inc.DOI: 10.1111/ivb.12066

must be strictly regulated to guarantee that chaetaeare identical within an individual, a population, or aspecies. Regulation of these factors appears to beconservative enough that certain chaetae, onceevolved, can be passed on to descendants and thusbecome characteristic of supraspecific taxa. Studiesinto chaetogenesis of uncini in certain “sedentary”polychaetes revealed that the structure of these chae-tae results from a uniform process of chaetogenesis(Arenicolidae and Maldanidae: Bartolomaeus &Meyer 1997; Bobin 1944; Tilic, unpubl. data; Psam-modrilidae and Oweniidae: Meyer & Bartolomaeus1996; Pectinariidae; Terebellidae, Serpulidae, Sabelli-dae: Bartolomaeus 1995, 1998, 2002); thus, chaeto-genesis in these cases seems to contain somephylogenetic signal. Studies of chaetogenesis alsosupport the hypothesis of homology between unciniand the hooded hooks of capitellids and spionids(Hausen 2001, 2005; Bartolomaeus et al. 2005). Inmembers of these two families, hooded hooks arefound in the abdominal chaetigers. These hoodedhooks resemble uncini whose apical portion isenclosed by a hood, consisting of an outer lamellaand an inner lamella. Studies of the formation ofthe hood support the assumption of homology ofspionid and capitellid hooded hooks (Hausen &Bartolomaeus 1998; Hausen 2005).

Similar hooded hooks, however, have also beendescribed in Lumbrineridae, a taxon that clearlybelongs to a clade called the Errantia or Aciculata,which consists of at least Eunicida and Phyllodocida(Rouse & Fauchald 1997; Struck et al. 2014;Weigert et al. 2014). Both in a molecular phyloge-netic analysis of Eunicidae (Zanol et al. 2010) andin a recent revision of the taxonomy of Eunicidaebased on morphological characters (Zanol et al.2013), lumbrinerids are considered basally branchingEunicida and therefore taken as outgroups forunraveling the phylogeny of the Eunicidae. On thelight microscopic level, the hooded hooks of Lum-brineridae appear to be similar to those of Capitelli-dae and certain Spionidae, as they also consist of alarge spine that is surmounted by smaller ones. Themain axis of the larger spine is perpendicular to themanubrium, and a large hood surrounds the spines(Hilbig 1989). The structural similarities between thehooded hooks of Capitellidae and Spionidae tothose of Lumbrineridae are either the result of con-vergent evolution, or the result of an early originduring annelid evolution, with subsequent loss ormodification in many different lineages. As the latterscenario appears to be unlikely, we expected strikingdifferences in the mode of chaetogenesis that reflectthe assumed convergent evolution of hooded hooks

in Lumbrineridae on the one hand, and Capitellidaeand Spionidae on the other. Recognizable differ-ences in mode of chaetogenesis would also supportthe value of chaetogenesis as a phylogenetic charac-ter. To test this idea, we analyzed chaetogenesis inLumbrineris (Scoletoma) fragilis M €ULLER 1766 andLumbrineris tetraura SCHMARDA 1861. Details ofchaetal arrangement and the position of the growthzone were used to corroborate the hypothesis of aconvergent evolution of hooded hooks in Lumbri-neridae, Spionidae, and Capitellidae.

Methods

Transmission and scanning electron microscopy

Specimens of Lumbrineris tetraura and Lumbrinerisfragilis were collected from sandy areas underneathand between smaller stones at the rocky shore closeto Plozevet and in Concarneau (Brittany, France).For fixation for transmission electron microscopy(TEM), we used 2.5% glutaraldehyde buffered in0.1 mol L�1 sodium cacodylate at room temperaturefor 1 h. Ruthenium red was added to the fixative,and the animals were dissected prior to fixation.After having been rinsed in the same buffer, the ani-mals were postfixed in 1% OsO4 in 0.1 mol L�1

sodium cacodylate for 1 h, dehydrated in an acetoneseries, and embedded in Araldite. The chaetal follicleand the formative sites of six chaetigers of a youngindividual of L. tetraura were sectioned into a com-plete series of silver-interference colored sections,stained with uranyl acetate and lead citrate, andanalyzed using Zeiss EM 10B and Hitachi H500transmission electron microscopes. The course of for-mation of the hooded hooks was reconstructed fromelectron microscopical data from different formativesites of L. tetraura using Fiji (1.45b) (Schindelin et al.2012)/TrakEM (Cardona et al. 2012) for cell identifi-cation, and Amira (4.0) for 3D visualization.

For scanning electron microscopy (SEM), speci-mens of L. tetraura and L. fragilis were fixed in Bou-in’s fluid, dehydrated in an alcohol series (during thisstep, the animals were sonicated to remove debris andsand particles from the chaetae), and critical pointdried with CO2 in a Balzers critical point dryer. Afterdehydration, they were sputter-coated with gold(Balzers Sputter Coater) and examined using Novo-scan and Hitachi scanning electron microscopes.

Confocal laser scanning microscopy

Specimens used for confocal laser scanningmicroscopy (CLSM) were fixed in 4% paraformal-

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2 Tilic, Hausen, & Bartolomaeus

dehyde. The chaetigers were dissected to separatesingle parapodia or a single segment. Isolated para-podia and segments were permeabilized in four5-min changes of phosphate buffered saline (PBS)with 0.1% Triton X-100 (Fisher Scientific). Theparapodia were then stained overnight at 4°C withTRITC–phalloidin at a dilution of 1:100. Afterstaining, parapodia were rinsed in three quickchanges and subsequently in two 10-min changes ofPBS with 0.1% Triton, and one 10-min rinse in PBSwithout Triton. Smaller parapodia were mounted onpoly-l-lysine-coated coverslips, and larger ones weredirectly placed in hollow-ground slides. The sampleswere quickly dehydrated in isopropanol (2 min eachin 70%, 85%, 95%, 100%, 100%), cleared in three15-min changes of Murray Clear, and mounted inMurray Clear. Slide preparations were sealed withnail polish.

Light microcopy, histology, and 3D reconstruction

Chaetae were isolated from pieces of living speci-mens of L. tetraura by incubation in 5% NaOH for2–3 h. The chaetae were rinsed in distilled water,mounted on slides, and examined using Nomarskidifferential interference contrast with an OlympusBX-51 microscope. Their structure was digitallyrecorded with an Olympus camera (Olympus cc12).

For reconstruction of the chaetal sac, specimensof both species were relaxed for 1.5–2 h in a 1:1mixture of 7% MgCl2 and seawater. They were sub-sequently fixed in 1.25% glutaraldehyde buffered in0.05 mol L�1 phosphate buffer with 0.3 mol L�1

NaCl for 1.5–2 h. Afterward, the specimens werepostfixed in 1% OsO₄ in a 0.05 mol L�1 phosphatebuffer. Before embedding, the specimens were dehy-drated via an ascending acetone series. They werethen transferred via propylene oxide to Araldite.The specimens were fragmented into smaller pieceswithin the resin. Polymerization was started withbenzyldimethylamine (BDMA). Serial 1-lm sectionswere prepared using a diamond knife (DiatomeHisto Jumbo) on a Leica Ultracut S ultramicro-tome, following the method described by Blumeret al. (2002). The sections were stained with tolui-dine blue (1% toluidine, 1% sodium tetraborate,and 20% saccharose) and mounted in Araldite. Thesemithin sections were analyzed with an OlympusBX-51 and photographed with an Olympus camera(Olympus cc12) equipped with the dot slide system(2.2 Olympus, Hamburg). The images were alignedwith IMOD (Boulder Laboratories: Kremer et al.1996) and IMOD-align (http://www.evolution.uni-bonn.de/mitarbeiter/bquast/software). The software

3ds max 13.0 was used for 3D modeling of the chae-tae. The histological images were imported as sur-face materials (discrete) and the chaetae weremodeled using standard cylindrical or conic objects.When necessary, these were modified as NURBS(Nonuniform rational B-Splines)-surfaces. The out-line of the chaetal follicle was created usingNURBS-curves on selected section planes.

Data repository and voucher material

To allow data transparency, all of the alignedserial sections used for 3D modeling are freely acces-sible in the morphological database, MorphDBase(Grobe & Vogt 2009). Series of semithin sections forL.tetraura can be found at https://www.morphdbase.de?E_Tilic_20131009-M-10.1, and for L. fragilisat https://www.morphdbase.de?E_Tilic_20131009-M-12.1 and https://www.morphdbase.de?E_Tilic_20131009-M-11.1. Aligned TEM sections showing chaeto-genesis can be found at https://www.morphdbase.de?E_Tilic_20131009-M-13.1, https://www.morphdbase.de?E_Tilic_20131009-M-14.1, and https://www.morphdbase.de?E_Tilic_20131009-M-15.1. Conspe-cific individuals of both species studied werecollected at the same sampling site and deposited atthe Zoological Museum of the University of G€ottin-gen as voucher material.

Results

Parapodial structure and the arrangement of chaetae

Each parapodium has a small and rounded pre-chaetal lobe, and a larger postchaetal lobe. Macro-scopically, the parapodia of both species studied areuniramous, but series of semithin sections and sub-sequent 3D-reconstruction of the chaetal sac ofLumbrineris fragilis (Fig. 1A–G) and Lumbrineristetraura (Fig. 2A–G) revealed that their notopodiaare extremely vestigial: a dorsal group of small chae-tae, embedded inside the parapodia in both species(Figs. 1G, 2G), originate from their own chaetalsac, independent of that of the neuropodia. Due totheir position and their origin we regard them as no-topodial chaetae, which have been internalized.Although the notopodial chaetae were much smallerand thinner than the neuropodial chaetae, theycould be seen by confocal laser scanning microscopy(CLSM: Fig. 3A–C). As only the neuropodial chae-tae protrude to the outside of the body, the follow-ing description refers to the neuropodial chaetae.

In L. fragilis, the neuropodial chaetation was con-sistent throughout the body; all chaetigers only bore


Chaetogenesis in lumbrinerid annelids 3

Fig. 1. Lumbrineris fragilis. A. 3D reconstruction of the chaetal arrangement. B–F. Aligned semithin sections of thechaetal follicle used to generate the 3D reconstruction. Corresponding section planes are marked in A. G. Semithinsection of the parapodium. The black arrow indicates the rudimentary notopodial chaetal elements. a, anterior; ac,acicula; d, dorsal; Dhh, developing hooded hooks; hh, hooded hooks; p, posterior; v, ventral.



Fig. 2. Lumbrineris tetraura. A. 3D reconstruction of the chaetal arrangement. B–F. Aligned semithin sections of thechaetal follicle used to generate the 3D reconstruction. Corresponding section planes are marked in A. G. Semithin sec-tion of the parapodium. The black arrow indicates the rudimentary notopodial chaetal elements. a, anterior; ac, acicu-la; cc, capillary chaetae; d, dorsal; Dcc, developing capillary chaetae; hh, hooded hooks; p, posterior; v, ventral.



Fig. 3. Lumbrineris tetraura. A. Confocal z-projection of a phalloidin-stained cross section of an anterior segment. Thewhite arrows indicate the rudimentary notopodial chaetal elements. B. Confocal z-projection of a phalloidin-stainedposterior parapodium without capillary chaetae. The white arrow indicates the rudimentary notopodial chaetal ele-ments. C. Confocal z-projection of a phalloidin-stained anterior parapodium with capillary chaetae. Chaetae are auto-fluorescent. The white arrow indicates the rudimentary notopodial chaetal elements. D. Posterior chaetigers withhooded hooks only. E. Anterior parapodia bearing capillary chaetae. a, anterior; ac, aciculae; cc, capillary chaetae; d,dorsal; hh, hooded hooks; p, posterior; v, ventral.



~4 hooded hooks. In L. tetraura, anterior and pos-terior parapodia differed in this respect. Anteriorparapodia bore seamed capillary chaetae as well ashooded hooks, whereas hooded hooks were the onlychaetae in posterior parapodia (Fig. 3B,C). In bothspecies, the chaetae arose from a small rim that wastransverse to the longitudinal body axis (Fig. 3D,E).Internally the structure of the chaetal sac was influ-enced by the insertion of parapodial and chaetalmuscles (Fig. 3A–C). The outline of the chaetal sacthus became extremely irregular and folded a fewmicrometers below the parapodial surface, so thatgroups of chaetae appeared to be isolated withinthe parapodium (Figs. 1C, 2C–E). Nonetheless, allchaetae of one parapodium originated from a singlechaetal sac; within each chaetal sac, hooded hooks,capillary chaetae, and acicula were aligned in a sin-gle dorsoventrally oriented row, sometimes in a zig-zag pattern. The aciculae did not protrude from thecuticle, were inserted much more deeply in the para-podia than the remaining chaetae, and were alwayslocated posterior to the row of capillary chaetae andhooded hooks (Figs. 1A,B, 2A,B, 3). In both spe-cies, new chaetae were formed continuously in eachchaetiger, irrespective of the position and thus ofthe age of the parapodium. Each chaetal sac con-tained two formative sites. In L. fragilis, newlyforming hooded hooks are shown in the 3D recon-struction of chaetae in a frontal segment (Fig. 1A).In a 3D reconstruction of a corresponding segment,L. tetraura only shows newly forming capillary chae-tae, due to the heterogenous chaetation of the ante-rior segments (Fig. 2A). The number of aciculagradually increased during the chaetogenesis(Fig. 4A–C). In L. tetraura, the anteriormost seg-ments sometimes contained up to five acicula, whilein L. fragilis, there were maximally two acicula inone parapodium (Figs. 1A, 2A).

Serial semithin sections of the posteriormost seg-ments in L. tetraura allowed a better insight intochaetogenesis, as the specimen studied still had anactive growth zone, so that the course of chaetalformation could be followed by studying the devel-oping segments anterior to the growth zone(Fig. 4A–E). Both neuropodial and notopodialchaetae could be seen from the youngest segmentonward, although chaetogenesis in notopodiaappeared to be a little bit delayed. Chaetogenesisalways started with the formation of an aciculainside a small basiepidermal chaetal sac. Duringfurther development, the chaetal sac extended intodeeper tissue layers and enlarged while additionalchaetae were formed inside. This was especially evi-dent in neuropodia, while in the notopodia, the

chaetal sac remained small and the tiny aciculatherein remained the only chaeta for a long time.Later, a few additional small chaetae were added. Inneuropodia, the acicula increased rapidly in diame-ter and the first hooded hooks started to developinside the chaetal sac (Fig. 4A,B). Their formationtook place dorsally and ventrally to the acicula, sothat each chaetal sac contained two formative sites.The dorsal formative site was always next to theacicula, and the ventral one clearly offset at the ven-tral edge of the chaetal sac. Chaetogenesis alternatedbetween both sites and finally gave rise to a row ofchaetae. During this process, different stages ofchaetogenesis could be found in a single chaetal sac(Figs. 1A, 2A, 4C,D). Depending on the position ofthe formative site, the oldest chaetae were found indifferent positions relative to the aciculae. Thoseformed by the dorsal formative site were located atthe dorsal edge; those formed by the ventral forma-tive site were found next to the aciculae and thus ina central position in each row of chaetae. In general,the diameter of each acicula was much larger thanthat of the hooded hooks (Fig. 5A,B). The posteriorten chaetal sacs did contain a single acicula, butfurther anteriorly a chaetal sac could contain up tofour aciculae (Fig. 2A).

Ultrastructure of hooded hooks

The hooded hooks possessed a large spine ormain tooth that was almost perpendicular to themain axis of the manubrium or shaft. Lightmicroscopy showed several small canals inside themain tooth and their characteristic bending towardthe manubrium (Fig. 5A). Several smaller spines orteeth surmounted the main tooth adrostrally; theywere aligned in a single row (Fig. 5C,D). A largehood surrounded these apical structures; its largerportion was rostral, and its smaller portion wasadrostral (Fig. 5C,E). A small apical slit allowed aview of the tip of the main tooth and some of thesmaller teeth by SEM. Rostrally this slit endedbeneath the main tooth, whereas it was much deeperon the adrostral side. The hood was not empty; itwas filled with several irregularly arranged rods(Fig. 5E) that were embedded in an amorphousmatrix. Each rod consisted of an electron-dense walland an electron-lucent core. Toward the peripherytheir arrangement was more regular. Here severalrows of electron-dense rods formed the outer surface(layer) and the inner surface (layer next to the shaft)of the hood. As they extended beyond the outer surfaceand inner surface, the hood was covered by denselyarranged knobs (Fig. 5C,F). The rods become less



regularly arranged toward the center of the hood.During preparation for SEM, the inner matrixshrank, so that the space between the rods appearedempty (Fig. 5E). The inner layer of the hood alsoconsisted of densely arranged electron-dense rods.This inner layer was isolated from the manubriumjust below the rostrum, and merged with the outerlayer on either side of the rostro-adrostral axis ofthe chaeta. With Nomarski interference contrastoptics, the margin of the inner layer of the hoodwas clearly visible beneath the rostrum (Fig. 5A).

Hartmann-Schr€oder (1996; p. 268) misinterpretedthis margin as subdistal tooth.

Chaetogenesis

Chaetogenesis is a continuous process. Due to ourmethods (TEM of fixed material), this process wasinferred from a series of different stages. Three ofthem are illustrated in this article, one showing theformation of the teeth (Fig. 6A–D), the secondshowing the beginning of the formation of the hood

Fig. 4. Lumbrineris tetraura. A. Graphical illustration of chaetal development in the posteriormost chaetigers. Aciculaeare shown in black and hooded hooks in gray. B–C. Semithin sections showing developing hooded hooks and aciculaein the posterior segments. White arrows and the asterisks mark the aciculae, and black arrows indicate rudimentarynotopodial chaetae. D–E. Semithin sections showing developing hooded hooks at higher magnification. ac, aciculae;Dhh, developing hooded hooks; hh, hooded hooks.



(Fig. 6E–H), and a third showing completion of thehood (Fig. 7A–D). For comparative reasons, forma-tion of an acicula (Fig. 7E) and a reconstruction oflumbrinerid hooded hook chaetogenesis (Fig. 8A–F)are shown as separate figures.

Chaetogenesis of hooded hooks can be dividedinto two steps, because the formation of the hood isspatially and temporarily separated from the devel-opment of the remaining apical section of thechaeta. In the following description, chaetogenesis

Fig. 5. Lumbrineris tetraura. A. Apical portion of a hooded hook with hood (h), main tooth (mt), smaller teeth(arrows), and shaft (sh). The large arrow marks the inner margin of the hood. B. An acicula. C. Tip of a hooded hookwith small spines surrounded by the large hood. D. Spines are aligned in a row and consist of a large tooth and smal-ler teeth (t). E. Partly opened hood. Note the numerous internal rod-like elements. F. The internal rod-like elementspierce the surface of the hood to form a dense layer of knob-like elements. h, hood; mt, main tooth; sh, shaft; t, smal-ler teeth.



of the apical teeth and the adjacent part of themanubrium will, thus, be separated from the forma-tion of the hood. In terms of topology, we thereforewill distinguish an inner anlage that gives rise to thespines and apical manubrium from an outer anlagethat gives rise to the hood.

Formation of hooded hooks occurred within anectodermal invagination that was continuous withthe exterior and filled with some extracellular mate-rial. This compartment was surrounded by five pro-spective follicle cells; a single large cell was at thebase of this cavity. All cells were interconnected byadluminal adhaerens junctions that linked an innersubapical net of actin filaments (Fig. 6A). Chaeto-genesis started when a few small, ellipsoid clustersof microvilli appeared on the surface of the chaeto-blast. These microvilli extended into the extracellu-lar compartment surrounded by the follicle cells;each cluster of microvilli was isolated from the adja-cent one by protrusions of the first basalmost folliclecells. While chaetal material that was releasedbetween the bases of the microvilli polymerized,additional clusters of microvilli were generated thatwere composed of a lesser number of microvilli thanthe previous ones. These were added to the existingclusters along a rostro-adrostral gradient, so thatthe oldest clusters were situated rostrally. Duringthis process, additional microvilli arose peripherallyto each cluster and enlarged it until it consisted of2–5 rows of 9–11 mv. Each cluster gave rise to asingle apical spine. As the initially formed clusterswere always larger than the subsequently formedones, the apical spines decreased in size toward theadrostral edge of the chaetal anlage (Fig. 6B). Afterthe final number of apical spines was achieved, addi-tion of further microvilli reduced the space betweenthe clusters and led to a merger of all microvilli(Fig. 6C,D). When formation of the apical spineswas completed, all spines were aligned along the

rostro-adrostral axis of the anlage. By that timemicrovilli had begun to withdraw from the spines,and the canals left by them were refilled by electron-dense material. Formation of the inner anlage,which would become the apicalmost hooked part ofthe chaeta, was now complete. Additional electron-dense material released from vesicles of the first twofollicle cells formed an enamel that covered theirregular surface of the chaeta (Figs. 6C, 7D). Thismaterial was produced inside Golgi stacks. The firsttwo follicle cells and the chaetoblast interdigitatedsuch that a protrusion of the follicle cells deeplyextended into the chaetoblast and surrounded theinner anlage (Figs. 6C,D, 8A,D).

During this initial phase of chaetogenesis, theinner anlage continuously sank into the chaetoblast.First, second, and third follicle cells that enwrappedthe inner anlage during formation followed thismovement and formed a small cytoplasmic ring thatsurrounded the anlage and separated it from thechaetoblast (Fig. 6C,G). This ring was connected tothe follicle cells with small rostral and adrostralcytoplasmic bridges that pierced the chaetoblast(Fig. 8B,E). The chaetoblast thus formed lateralwalls on either side of the rostro-adrostral axis ofthe inner anlage; the walls surmounted the inneranlage. The chaetoblast now started to generatelarge clusters of microvilli on top of these lateralwalls, and initiated formation of the outer anlagethat would become the hood (Figs. 6E, 7D). Asthese microvilli extended beyond the inner anlage,chaetal material released between the bases of thenewly formed microvilli formed a cap-like structurethat extended beyond the inner anlage on either sideof its rostro-adrostral axis (Fig. 8B,E). Both cap-likestructures remained adrostrally separated by thebridge-like connection between the cytoplasmic ringand perikarya of the first, second, and third folliclecells (Fig. 6F). The rostral gap was caused by the

Fig. 6. Lumbrineris tetraura. Chaetogenesis of hooded hooks. A–D. Serial sections showing formation of the teeth pre-ceding formation of the hood. Note the interaction of follicle cells 1 and 2 (FC1, FC2) with the chaetoblast (CB),which influences the prospective structure of the chaeta. Large arrows mark adhaerens junctions; small arrows labelbundles of actin filaments. A. Electron-dense tips of small teeth (t) and main tooth (mt). Note several canals insideeach, filled with electron-dense material. B. The teeth are preformed by several microvilli and are empty after the with-drawal of the microvilli. C. Microvilli (mv) fuse to preform the shaft. D. The chaetoblast interdigitates with the folliclecells. Distances between serial sections are A–B, 1.7 lm; B–C, 2.2 lm; and C–D, 2 lm. E–H. Serial sections showingformation of the hood. Note interactions between the chaetoblast and various follicle cells (FC1-FC4). Morphogeneticchanges of cells are inferred from a high density of actin filaments (af). E. The hood is preformed by microvilli (mv) oftwo latero-apical lobes of the chaetoblast. The central part of the chaeta has already been completed. F. Main tooth(mt) indicates reorientation of the anlage. G. FC1 forms a cytoplasmic ring around the chaetal anlage. H. Microvillipreforming the shaft sunk deeply into chaetoblast. An asterisk indicates the coelom. Distances between serial sectionsare E–F, 3.5 lm; F–G, 7.4 lm; and G–H, 2.6 lm. af, actin filaments; CB, chaetoblast; ECM, extracellular matrix; FC,follicle cell; mt, main tooth; mv, microvilli; n, nucleus; t, small teeth.



position of the third follicle cell in a similar manner(Fig. 6E). In fully differentiated hooded hooks, thus,an apical slit indicated the former position of thefollicle cells (Fig. 5C). At this stage, an inner clusterof microvilli forming the inner anlage and two

groups of outer microvilli forming the outer anlagecould be discriminated (Figs. 7C, 8E,F).

During further development, additional outer mi-crovilli were formed to broaden the outer anlage ofthe hood. Finally, both microvilli clusters fused on

Fig. 7. Lumbrineris tetraura. Chaetogenesis of hooded hooks. A–C. Serial sections showing formation of the hood. A.Completed hood with central teeth (t). Note the electron-bright filling between the electron-gray chitinous rods andtheir dark filling. Large arrows mark adhaerens junctions. B. Protrusion of follicle cells (FC) preforms a gap betweenhood and shaft (sh) of the chaeta. Entire anlage is surrounded by chaetoblast (CB). Large arrows mark adhaerensjunctions. C. Merger of microvilli preforming the shaft and the hood (h). Distances between serial sections are A–B,15 lm; and B–C, 3 lm. D. Semisagittal section of hooded hook anlage. Follicle and chaetoblast interact during forma-tion of shaft (sh) and hood (h). White arrows mark the canals filled with electron-dense material. E. Transverse sectionof aciculum-forming chaetoblast. CB, chaetoblast; ECM, extracellular matrix; EMC, epitheliomuscular cells of coelo-mic lining; en, enamel; FC, follicle cell; h, hood; n, nucleus; sh, shaft; t, teeth.



the rostral side, forming a horseshoe-shaped patchof microvilli (Fig. 7A,B). The adrostral cleft wascaused by the position of the first and the third folli-cle cells (Fig. 7B). Compared with the inner anlage,release and polymerization of chaetal material wereaccelerated in the outer anlage. This led to structuraldifferences between hood and shaft in the fullydifferentiated hooded hook. The latter was morecompact and stained much more electron denselythan the hood, where electron-dense fibers, whichmarked the former position of the microvilli,appeared loosely connected by electron-lucent mate-rial (Fig. 7A,B,D). Merging of microvilli of theinner anlage reduced its diameter and initiated for-mation of the chaetal shaft or manubrium (Fig. 8C,

F). When by differential deposition of chaetalmaterial the outer and inner group of microvilliwere finally on the same level, the first and secondfollicle cells began to retract (Fig. 7B). Thus, a largegap remained between the hood and the hookedpart, which is characteristic for fully differentiatedhooded hooks.

The microvilli of the outer anlage were now onthe same level as those of the inner anlage, and sur-rounded them (Fig. 7C). Both merged duringfurther development, although both could still bediscerned for a while as their microvilli differed indiameter. Deposition of chaetal material betweenthe bases of the microvilli and their continuouswithdrawal proceeded, and the manubrium became

Fig. 8. Schematic representation of chaetogenesis as series of sagittal (A–C) and transverse (D–F) sections of subse-quent representative stages of the anlage to illustrate the interaction between the chaetoblast and the follicle cells. Thefollicle cells are numbered along the baso-apical axis of the anlage. A,D. Earliest stage with apical teeth. B,E. Forma-tion of the hood by lateral lobes of the chaetoblast. C,F. Final stage of hood formation. Asterisks mark the cytoplas-mic ring around the chaetal anlage. CB, chaetoblast; FC, follicle cell.



longer. No significant modification of the microvillipattern occurred in this phase of chaetogenesis.During elongation, the newly formed seta waspushed toward the surface, and finally becamevisible externally.

Formation of aciculae was much simpler. Thebasal chaetoblast continuously added microvilli to acentral core group of microvilli. While chaetal mate-rial was continuously released between the bases ofthe microvilli, the diameter of the aciculumenlarged. The small tip and the rapid increase indiameter can be seen in isolated aciculae (Fig. 5B).When the final diameter was reached, no furtherchange in the microvilli pattern of the chaetoblastoccurred; the diameter of the chaeta remained con-stant for its entire length. Electron-dense vesiclesreleased their contents into the small gap betweenchaetoblast and chaetal anlage to form an electron-dense enamel around the acicula (Fig. 7E).

Discussion

In Eunicida, the notopodia are either partially orcompletely reduced (Beesley et al. 2000: 89; Tilic &Bartolomaeus 2014). In both of the analyzed speciesof Lumbrineris, chaetal elements of the rudimentarynotopodia were still present. In Eunice torquataQUATREFAGES 1866, the notopodium is reduced to adorsal cirrus, which still contains chaetal elements.In Lysidice ninetta AUDOUIN & MILNE-EDWARDS

1833, however, no reduced chaetal elements of therudimentary notopodia are present (Tilic & Bartolo-maeus 2014). All species of the Eunicida possess atleast hooded hooks in addition to seamed capillarychaetae; in addition, other kinds of chaetae haveevolved within this group (Tilic & Bartolomaeus2014).

Hooded hooks are also described from spionid,magelonid, and capitellid polychaetes (Hausen2005). The hooded hooks in species of all threegroups are basically arranged in double rows, butalways possess a single formative site within a para-podium. This formative site is always located at theventral edge of the notopodial chaetal sac and atthe dorsal edge of the neuropodial chaetal sac(Hausen & Bartolomaeus 1998 and literaturetherein). In both lumbrinerid species studied here,hooded hooks and capillary chaetae are aligned in asingle row, which may show a zig-zag pattern. Eachrow and each chaetal sac contains a ventral and adorsal formative site. To find out whether thispattern is characteristic for all Eunicida will be partof our ongoing studies into chaetogenesis in thisgroup.

Hooded hooks are described from all lumbrineridspecies (Carrera-Parra 2006). It is remarkable thatin Lumbrineris tetraura and Lumbrineris fragilisthere are two formative sites in each chaetal sac.Although the process of chaetogenesis has not beendescribed in other lumbrinerids, the processdescribed here for L. tetraura and L. fragilis isassumed to be characteristic of Lumbrineridae.Provided this assumption is true, the similaritiesbetween lumbrinerid hooded hooks and those ofspionids, magelonids, and capitellids (Hausen 2005)result from different processes (Table 1). After sum-marizing the course of hooded hook chaetogenesisin both groups as well as that of hooked chaetaeand uncini in related taxa, we will return to thispoint.

Hooded hooks, hooked chaetae, and uncini in other

annelids

In a series of papers, it was argued that the courseof chaetogenesis substantiates the hypothesis of ahomology of hooked chaetae and uncini in arenico-lids, maldanids, terebellids, and sabellids andhooded hooks in spionids, magelonids, and capitel-lids (summarized in Hausen 2005). In arenicolids,maldanids, terebellids, and sabellids, the uncini orhooked chaetae consist of a large spine or rostrum(main fang, main tooth) that is surmounted byseveral smaller spines (teeth) that together comprisethe capitium (Bartolomaeus 2002; Hausen 2005).The main axis of the shaft or manubrium is alwaysperpendicular to that of the rostrum. Below the ros-trum the manubrium is enlarged to form a subros-tral process. In hooded hooks of capitellids andspionids, this apical section is enwrapped by a hoodconsisting of two chitinous lamellae. Uncini, hookedchaetae, and hooded hooks are aligned in a singleor double row within a chaetal sac. In all species ofthese two taxa studied, chaetogenesis occurs in asingle formative site in each sac. Chaetogenesis ofuncini and hooked chaetae always starts with agroup of microvilli that are a template for the ros-trum. Continuous release of N-acetyl-glucosamine atthe base of the microvilli forms the rostrum. Eachof the subsequently formed larger microvilli is atemplate for a tooth of the capitium. During forma-tion of these larger microvilli, the axis of the chaeto-blast shifts so that the spines of the capitiumsurmount the rostrum after uncini formation hasbeen completed. When the axis of the chaetoblast isshifting, additional microvilli are generated belowthe rostrum; these fuse with those that formed therostrum and capitium. Merging of microvilli gener-



ates the shaft of the chaeta. In capitellids, magelo-nids, and certain spionids, this structure is altered byan apical hood enwrapping the rostrum, capitium,and the apical portion of the uncini (Hausen 2005).These chaetae are traditionally called hooded hooks.Microvilli that appear on the surface of the chaeto-blast after the axis of the chaetoblast begins to shiftare the template for the hood. As the main tooth isperpendicular to these microvilli, they form an ad-rostral, horseshoe-shaped group so that a rostralpore (Spionidae) or slit (Capitellidae) remains afterthe hood is completed. During formation, the mi-crovilli split into two layers, an inner and an outerone. These layers are templates for the inner lamellaand the outer lamella of the hood that surroundsrostrum and capitium in Capitella capitata (FABRI-

CIUS 1780) (Capitellidae) and the bifid tip of theScolelepis squamata M €ULLER 1806 (Spionidae). Merg-ing of the inner layer and the manubrium precedesfusion of the outer layer and the shaft. Due to devel-opmental correspondences, a general homologybetween uncini and hooded hooks of capitellid spe-cies has been proposed (Hausen & Bartolomaeus1998; Schweigkofler et al. 1998; Bartolomaeus et al.2005; Hausen 2005). Subsequent studies into thechaetogenesis of additional spionids and magelonidsshowed some differences, but largely confirmed thispattern (Hausen 2001, 2005) (Table 1).

Conclusions

There are several structural and developmentaldifferences between the hooded hooks in the lumb-rinerid species studied and those of spionids andcapitellids. These concern the formation of the api-cal portion of the chaetae and the formation of thehood (summarized in Table 1). While in Capitellacapitata and certain spionids only the rostralmosttooth of the hooded hook, the rostrum, is pre-formed by a group of microvilli, those teeth thatsurmount the rostrum are preformed by a singlelarge microvillus each. In Lumbrineris fragilis andLumbrineris tetraura, all teeth of the hooded hookare formed at the same time; there is no delay intheir appearance. Their hood is formed after theapical portion of the hooded hook has been com-pleted and it is formed by a homogeneous group ofmicrovilli that arise from that part of the chaeto-blast which extends beyond the apical inner part ofthe chaeta. There are no outer and inner lamellaeformed from different groups of microvilli as inspionids and capitellids. An adrostral slit in thehood is caused by follicle cells during the course offormation, while the rostral cleft or pore in capitel-lid and spionids is caused by the position of the ros-trum. Formation of the hood then is acceleratedrelative to the remaining chaeta so that both inner

Table 1. Structure and chaetogenesis of hooded hooks in capitellid, spionid, and lumbrinerid species.

Lumbrineris spp.(Lumbrineridae, Eunicida)

Capitella capitata(Capitellidae)

Scolelepis squamata(Spionidae, Spionida)

Hooded hook substructuresMain tooth Present Present PresentSmaller teeth Surmount main tooth Surmount main tooth Surmount main toothSmaller teeth number At least ten More than ten OneSmaller teeth position Single adrostral row Several adrostral rows Adrostral to main toothShaft Present Present PresentHood Peripherally condensed

rodsInner and outer lamellae Inner and outer lamellae

Hood compartment Discontinuously filledwith chitinous rods

Electron-lucent (empty)with a few fibrils

Electron-lucent (empty)

Hood surface Electron-dense knobs Electron-dense knobs Electron-dense knobsHood opening Adrostral slit Rostral slit Rostral pore

Hooded hook formationMain tooth Group of microvilli Group of microvilli Group of microvilliSmaller teeth Group of microvilli Single microvillus each Group of microvilliShaft Group of microvilli Group of microvilli Group of microvilliHood Two lateral groups

of microvilliSeveral crescent rows ofmicrovilli

Two crescent rows ofmicrovilli

Hood compartment Absent Between rows of microvilli Between rows of microvilliFusion with shaft Entire hood Inner and outer hood

lamellae sequentiallyInner and outer hoodlamellae sequentially



apical portion and hood finally merge and form themanubrium in lumbrinerids. Merging of the innerhood and the outer hood in spionids, magelonids,and capitellids, however, is sequential (Hausen &Bartolomaeus 1998; Schweigkofler et al. 1998; Hau-sen 2005). These differences argue for a convergentevolution of hooded hooks in the lumbrinerids andthe other taxa mentioned.

Differences between similar structures either resultfrom transformations of an ancestral structure, orfrom convergent evolution. Differences alone, thus,do not decisively indicate convergent evolution; thelatter must be the most parsimonious explanation inthe context of a phylogenetic tree. According tomorphological data, Eunicida, Phyllodocida, andAmphinomida form a monophyletic taxon calledErrantia or Aciculata, characterized by internalchaetae (aciculae) and antennae among other traits(Rouse & Fauchald 1997; Bartolomaeus 1998). Ataxon Errantia consisting of Phyllodocida, Eunicida,Amphinomida, and Orbiniidae is monophyletic inrecent molecular analyses by Struck and colleagues(Struck 2011; Struck et al. 2011, 2014). Weigertet al. (2014) could confirm the monophyly of Erran-tia, if Amphinomida were excluded. Hooded hooksare absent in species of Phyllodocida, Orbiniida,and Amphinomida, but present in species of theEunicida. Provided that the mode of formation ofhooded hooks of L. fragilis and L. tetraura is char-acteristic of all lumbrinerids and also for eunicids,the most recent tree in Weigert et al. (2014), as wellas the trees of Rouse & Fauchald (1997) and Strucket al. (2011, 2014), clearly favors the assumptionthat hooded hooks in Eunicida are not homologousto those in spionids and capitellids. According tothese trees, it is more likely that hooded hooksevolved at least twice in annelids, corroborating ourconclusion that the differing course of chaetogenesisof lumbrinerid hooded hooks reflects convergentevolution with eunicidan hooded hooks.

Acknowledgments. We thank Renate Feist, ChristinaF€orster, Claudia M€uller, and Tatjana Bartz for technicalassistance. We also thank Bj€orn Quast, Patrick Beckers,and J€orn von D€ohren for their support. Our thanks arealso due to the Laboratoire de Biologie Marine inConcarneau for hospitality during our collection trips.The study was financially supported by the Germanresearch foundation (DFG BA 1520/2).

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