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The neuroanatomy of the siboglinid Riftia pachyptila highlights sedentarian annelid nervous system evolution

Tracing the evolution of the siboglinid group, peculiar group of marine gutless annelids, requires the detailed study of the fragmentarily explored central nervous system of vestimentiferans and other siboglinids. 3D reconstructions of the neuroanatomy of Riftia revealed that the “brain” of adult vestimentiferans is a fusion product of the supraesophageal and subesophageal ganglia. The supraesophageal ganglion-like area contains the following neural structures that are homologous to the annelid elements: the peripheral perikarya of the brain lobes, two main transverse commissures, mushroom-like structures, commissural cell cluster, and the circumesophageal connectives with two roots which give rise to the palp neurites. Three pairs of giant perikarya are located in the supraesophageal ganglion, giving rise to the paired giant axons. The circumesophageal connectives run to the VNC. The subesophageal ganglion-like area contains a tripartite ventral aggregation of perikarya (= the postoral ganglion of the VNC) interconnected by the subenteral commissure. The paired VNC is intraepidermal, not ganglionated over most of its length, associated with the ciliary field, and comprises the giant axons. The pairs of VNC and the giant axons fuse posteriorly. Within siboglinids, the vestimentiferans are distinguished by a large and considerably differentiated brain. This reflects the derived development of the tentacle crown. The tentacles of vestimentiferans are homologous to the annelid palps based on their innervation from the dorsal and ventral roots of the circumesophageal connectives. Neuroanatomy of the vestimentiferan brains is close to the brains of Cirratuliiformia and Spionida/Sabellida, which have several transverse commissures, specific position of the giant somata (if any), and palp nerve roots (if any). The palps and palp neurite roots originally developed in all main annelid clades (basally branching, errantian and sedentarian annelids), show the greatest diversity in their number in sedentarian species. Over the course of evolution of Sedentaria, the number of palps and their nerve roots either dramatically increased (as in vestimentiferan siboglinids) or were lost.

The nervous systems of the vestimentiferans and the remaining siboglinids were studied by various methods and different levels of accuracy, making them difficult to compare. Vestimentiferan neuroanatomy was studied in some lamellibrachids (Lamellibrachia barhami [4], L. luymesi [16,17], L. satsuma [18]) and tevniids (Riftia pachyptila [19][20][21], Ridgeia piscesae [22], Oasisia alvinae [23]). The ventral nerve cords and brains, including the positions of the perikarya and neuropile, were studied in larvae [24,25] and adults [16,18,[21][22][23]26] using light microscopy and histological techniques. Electron microscopical studies of the neural structures revealed the presence of sensory cells and of glial cells surrounding the neuropile and forming a myelin sheath around the giant axons [21]. The architecture of the frenulate central nervous system is known based on histological and histochemical studies of the ventral nerve cords, neuropile rings and brain area of the supposedly early-branching species Siboglinum caulleryi, S. fiordicum and Nereilinum murmanicum, and derived ones such as Polybrachia annulata and Spirobrachia grandis [7,[27][28][29][30][31][32]. Electron microscopy revealed the presence of glial and sensory elements in the epidermis of frenulates [33]. The structure of the central nervous system of females and dwarf males of seven species of Osedax was described by combining immunohistochemistry with confocal microscopy. This approach revealed numerous commissures and connectives in the brain and trunk nervous system [34,35]. Histological studies of the brain of Sclerolinum contortum revealed layers of apical perikarya and a basal neuropile [36]. Nonetheless, our anatomical knowledge of the organization of the nervous system in vestimentiferans and other siboglinids remains fragmentary. Among siboglinids, the neuroanatomy of Osedax has been studied in the greatest detail. Reconstructions of the neural architecture of the other siboglinids are therefore crucial in tracing the evolution of the key neuroanatomical features of siboglinids.
We currently lack comparisons of the organizations of the central nervous systems of siboglinids and supposedly closely-related annelid groups [11,21,26,37,38]. First attempts have been made on selected vestimentiferans on smaller sizes [16,18,20] and Osedax [34,35]. Miyamoto [18] suggested that the vestimentiferan brain is a simple structure resembling the brain organization of some sedentarian species. The other authors discussed the possible annelid structure of the ventral nerve cord but did not analyze the brain configuration in detail. This restricts the application of the comparative anatomy approach to study the evolution of the siboglinids and annelids.
In the gutless siboglinids the brain occupies an annelid-atypical antero-ventral position. Based on the fact that a coelomic channel passes through the brain, i.e. a rudimentary gut is present in young specimens, Jones and Gardiner [20] suggested that the vestimentiferan brain is a result of the fusion of the supra-and subesophageal ganglia and the circumesophageal connectives. However, fully visualizing the disposition of the transverse commissures of the supraesophageal ganglion and circumesophageal connectives remains to be done. This would highlight not only the brain structure but also help reconstruct the ventral brain formation in vestimentiferans.
Thus, the present study reconstructs the organization of the central nervous system, including the brain and nerve cord, of the large vestimentiferan tubeworm Riftia pachyptila. The focus is on brain reconstruction. Among siboglinids, only vestimentiferan juveniles preserve gut rudiments. This makes them a key group to homologize the brain parts of the ventral brain of siboglinids and annelids. The overall aim is to examine the structures of the supra-and subesophageal ganglia and of the circumesophageal connectives in order to provide a comparative neuroanatomical analysis of the vestimentiferans versus other annelid groups. This calls for comprehensive data on the brain and nerve cord structures to determine the ancestral features of the siboglinid nervous system and to reveal the evolution of neural characters within sedentarian annelids.

Collection and fixation
Five specimens of Riftia pachyptila Jones, 1981 [19] were collected at different latitudes of the East Pacific Rise (EPR), including the Guaymas Basin, Gulf of California, by the Pisces manned submersible during the 12th cruise of the RV Akademik Mstislav Keldysh in 1986 and by Mir-1 & 2 manned submersibles during their 49th cruises in 2003. Lengths of examined specimens range from 8 to 808 mm. For data on collection sites, sexes and fixation of specimens see Table 1.

Histology and LM photography
Animals used for histological analysis were fixed in Bouin's solution, rinsed three times in 70% ethanol and stored in 70% ethanol prior the histological processing. The material was processed by the standard histological procedure: dehydration in series of alcohol and xylene (80% ethanol, 96% ethanol, mixture of ethanol and buthanol (1:1), buthanol, mixture of buthanol and xylene, xylene (2 changes), 1 hour each change, room temperature), and embedding in paraplast or histowax [39]. Transverse sections (5 and 7 μm) were produced with a Leica RM 2125 microtome (Leica Microsystems, Wetzlar, Germany), stained with Caracci hematoxylin, and examined under a Zeiss Axioplan2 microscope equipped with an AxioCam HRm camera (Carl Zeiss Microscopy, LLC, United States) as well as a Leica DM5000 B microscope equipped with a Leica DFC425 C camera. Microscopic images were optimized for contrast and level using Adobe Photoshop 7.0 (Adobe Systems, San Jose, CA, USA). Drawings were done with Adobe Illustrator CC 2014. To visualize the anastomosing neurites in the trunk epidermis, a 808-mm-long specimen was photographed with a Canon Power Shot S90 camera. For better understanding the trasverse sections, the dorsal sides are always at top. In case of sagittal sections, anterior ends to left.

3D modeling
The arrangement of neurite bundles in the brain and the anteriormost ventral nerve cord were visualized with the software 3D-DOCTOR 3.5.040724 (Able Software Corporation of Lexington, USA). Alignment was performed using the same software by comparing the sections of adjacent planes. Image series of 77 cross sections of the 78-mm-long specimen were used tp model the brain organization. 19 objects were traced inside the brain, including the boundary of the brain. Photos were saved in JPEG format with a resolution of 3900 x 3090 pixels and 8 bits / pixel. The field of view was 2812 μm, the parameters of the voxels of the images are 0.721 x 0.721 x 15 μm3. The outlined boundaries yielded 3D models. The implemented smoothing tool was used for natural perception of object surfaces. Interactive features as well as a transparency filter, different colours and lighting effects were applied to show complex and hidden objects. Three-dimensional images under appropriate angles were processed in Adobe Photoshop 7.0.

Gross anatomy of nervous system
Riftia's central nervous system is composed of a ventral brain and ventral nerve cord (B, VNC, Figs 1A, 2A, 3A and 4A).
The ventral brain lies in the anteriormost vestimentum (Figs 1A, 4A and 4B). Two brain lobes form a heart-like structure in transverse sections (Figs 5-7, S1-S3 Figs). A dorsal furrow between the brain lobes encloses the bases of the obturacules (OBL, Figs 4A, 5 and 6, S1 Fig). Posteriorly, the excretory tree adjoins the brain (ET, Fig 4B). The whole brain lies inside the epithelium, and no basal laminae separate the brain from the epidermis (EP, Figs 4-7, S1-S4 Figs). A thick layer of cuticle (schield, or plate) protects the apical surface of the brain (CUP, Fig 4B, S1-S3 Figs). Furthermore, a collar of vestimental wings encloses the ventral brain (VWF, Figs 1B and 4). The brain of the 80-mm-long specimen is 1 mm high, 1 mm long, and 2 mm wide.
Tentacle lamellae originate at the brain periphery of the dorsal, lateral and ventrolateral sides of the brain (Figs 5-7, S1-S3A and S5 Figs). The posterior brain gives rise to lamellae only on the dorsal surface (Figs 6 and 7, S3A Fig), whereas on the anterior brain the tentacle lamellae occupy dorsal, lateral and ventrolateral surfaces (Fig 5, S1 and S2 Figs). Lamellae appear on the dorsal side of the brain. Here, they are the least differentiated (S5 Fig). The ventrolateral lamellae are bilayered (read below about the epidermis of the external and internal walls) and extend towards the anterior end of the tentacle crown. They remain undifferentiated over part of their length and then separate into individual tentacles. Thus, the undifferentiated tentacle lamellae (LR) are lamellae that are not divided into separate tentacles (LR, Figs 5-7, S1-S3A and S5 Figs).
Three coelomic channels pass through the brain tissue: one pair of obturacular coeloms with blood vessels and an unpaired enteral coelom (OBC, EC, S6 Fig). In juvenile individuals the enteral coelom comprises the gut rudiment (G, Fig 4A). In larger specimens the enteral coelom is occupied by mesenchymal cells (Fig 6). In the anterior brain the enteral coelom has a «Λ» shape when viewed in the transverse profile ( Fig 5). The oral siphon is preserved in juvenile Riftia (34 mm long), and in 79-mm-long individuals the intestine rudiment remains in the coelomic channel running through the brain. In larger individuals, only the coelomic channel remains.   The ventral nerve cord (VNC) connects to the brain via longitudinal nerve tracts (LNT, Fig 1B-1E). Anteriorly to the ventral ciliary field (CF) the VNC splits into a pair of strands (PNC) connected to each other with transverse neurite bundles (Fig 2A-2D). The strands surround the ventral ciliary field (Fig 2B and 2C) and fuse into a single VNC at the border of the vestimentum and trunk before they extend along ventral midline to the end of the body ( Fig  3A). The width of the prominent VNC can reach up to 1 mm in an 808-mm-long specimen ( Fig 3B). Notably, its width decreases towards the posterior trunk (Fig 3C and 3D).
The VNC lies inside the epidermis (Fig 1E). The epidermal cells have a wide apical part underlying the cuticle and a basal process running towards the ECM (Fig 1C).
Apically, a thick cuticular layer (CU) protects the VNC, especially in the anteriormost part ( Fig 1C). In the opisthosome the cuticle protecting the VNC makes folds between the apical parts of epidermal cells (arrows, Fig 3F).  Most of the dorsal brain is represented by a large paired neuropile of the lateral brain lobes (NE, Numerous tentacle neurite bundles (TEN) extend radially from the NE to the bases of the tentacles (Figs 5 and 6, S1-S3 and S5 Figs). The latter are arranged into rows with the fused bases, so-called tentacle lamellae. Each tentacle lamella represents a thin fold of the epidermis and closely adjoins the next lamella (S5A- S5D Fig). The epidermis of the external lamellae     The dorsalmost side of the midbrain (close to ECM layer) bears a pair of dorsal longitudinal bundles (DLN, Figs 4A, 5, 6, 8A-8D and 10A, S1-S3A and S9A-S9C Figs). They start from a dorsal aggregation of perikarya (dop) in the midrain (Figs 5 and 10A, S1 and S2 Figs) and run along the dorsal groove of the brain.
Short anterior vertical median bundles (VAN) pass between the obturacular coeloms in the midbrain (Fig 10B-10D, S10 Fig). They run in the ventro-dorsal direction and connect the dorsal and supraenteral commissures. The neurites of the anterior vertical median bundles cross: for example, the neurites from the right root of the supraenteral commissure extend to the left roots of the dorsal commissure ( Fig 10D, S10B Fig).
Posterior vertical median bundles (VPN, Figs 6, 8B and 10E, S3A and S10 Figs) are located posterior to the anterior vertical median bundles. They do not contain any crossing bundles and connect the supraenteral commissure and the posterior dorsal commissure.
Peripheral perikarya of the lateral brain lobes (nep, Figs 4-7 and 8A-8D, S1-S3, S5, S6, S7I and S7J Figs) are represented by two layers: inner layer of small perikarya, 5 μm (ps), and outer layers of big ones (pl), 20 μm (Fig 7, S3A and S5A Figs). In juvenile specimens with fewer tentacle lamellae, the small perikarya are grouped into distinct lobules which correspond to the tentacle lamellae. In bigger specimens with more tentacle lamellae, the arrangement of small perikarya is more regular. Accordingly, the nep does not represent a true cluster or aggregation, but rather the layer of the somata spreading over the dorsal and lateral surface of the brain. In the anterior part of the brain the peripheral zone of perikarya expands significantly and covers laterally a tripartite ventral aggregation of perikarya (vtp, Figs 5-7, S1-S3 and S6-S8 Figs, more about vtp read below).

Ventral brain structures
Most of the neuropile of the ventral brain is occupied by paired prominent longitudinal nerve tracts (LNT, Figs 1B-1D, 6, 7, 8B-8D, 9 and 10A, S1-S3, S7 and S9B-S9J Figs), which are continuations of nerve fibers from the ventral nerve cord (Fig 1B-1D). As the longitudinal nerve tracts enter the brain, each of them runs along the lateral sides of the obturacle and enteral coelomic channels and gradually rises to the anterior dorsal side of the brain (Figs 5-7).

Giant perikarya and axons in the brain
In adult specimens, the giant axons are present in the midbrain originating from at least two pairs of the giant perikarya (GA, gap, Figs 6, 7, 8E-8H and 9, S3, S9G-S9L and S10B-S10G Figs). One pair starts in the dorsal commissure, runs inside the crossing anterior vertical median bundles (VAN) between the obturacle coeloms ( Fig 10D and 10F, S9G-S9L and S10E-S10G  (Fig 1A and 1B).
Notably, in adult specimens, the dorsal commissure has only one pair of the giant perikarya, whose nuclei and nucleoli are degraded (Fig 10F) The neuroanatomy of Riftia pachyptila dorsal giant perikarya (Fig 10B and 10C). Considering the number of the giant perikarya in juveniles and that the giant perikarya in adult animals degrade, we assume at least 3 pairs of giant perikarya for the vestimentiferans.
In the posterior brain the giant axons extend to the ventral nerve cord (Fig 9, S9G-S9J Fig).

Ventral nerve cord
Within the vestimentum, the neuropile of the paired ventral nerve cord (VNC) consists of two lateral longitudinal nerves (LNT, Fig 1B-1D) connected via transverse (commissural) neurite bundles (CNC, Figs 1B, 1D and 2B). A pair of giant axons lies in the central part of the VNC (Fig 1A-1D). Numerous small perikarya form two lateral and one central accumulation (lpnc, cpnc, Fig 1C and 1D) which are continuations of the ventral tripartite aggregation of the ventral brain (vvtp, Fig 1B and 1C).
Around the ventral ciliary field, each strand of PNC contains the epidermal cells, basal neuropile, apical perikarya, and a single fiber representing the giant axon envelopped by the coating cells (ega) (Fig 2D). Most perikarya lie externally to the giant axon in each strand. The ciliary field consists of columnar ciliary epidermal cells (Fig 2C). Their basal parts contain commissural neurite bundles (CNC) which form a net-like structure and connect the strands with each other (Fig 2A, 2B and 2D).
In the trunk the VNC has an invariable diameter-the neuropile has no swellings and is separated by the giant axon into two longitudunal strands (Fig 3A-3D). The epidermal cell processes extend to the ECM inside the neuropile (EXP, Fig 3B). The VNC perikarya spread along the left and right sides of the giant axon (Fig 3B-3D). Both small (3.5 μm) and big (20 μm) perikarya (ps, pl, Fig 3B) are present. The giant axon extends to the border of the trunk and opisthosome (Fig 3D).
In the opisthosome, the arragement of the apical somata and the basal neuropile of the VNC is comparable to the rest of the body (Fig 3F and 3G). There is no giant axon; all perikarya are small.

Segmental nerve bundles
In the anteriormost vestimentum, several thick transverse lateral neurite bundles part off the ventral nerve cord (LN, Fig 1A). Three pairs were present in a 16-mm-long specimen. The first, most prominent pair extends to the anterior collar and forms neurites of the vestimental wings (VWN, Fig 1A and 1B). At the level of the ciliary field, many irregular bundles branch off the lateral neuropile of the VNC strands and extend into the epidermis of the vestimental wings (Fig 2A and 2E). Transverse neurite bundles branch off from the single VNC in the trunk. They ramify and anastomose (Fig 3E). In a 79-mm-long specimen, lateral bundles part off the cord, yielding 350-360 pairs of such bundles in the trunk. In each opisthosomal segment, a pair of lateral bundles leaves the neuropile of the VNC (Fig 3A, compare F&G).

Ventral nerve cord in vestimentifera
All described species of vestimentiferans have a uniform structure of the ventral nerve cord (VNC), with variations evident only in the length of giant axons and the organization of perikarya aggregations within the trunk [16,18,19,22,23,26,40]. The VNC in Ridgeia piscesae and Lamellibrachia satsuma consist of a central neuropile and two lateral rows of perikarya, thus showing a paired structure [18,22], whereas Riftia (present study) and Oasisia alvinae exhibit single layers of apical perikarya and a basal neuropile [23]. Furthermore, a median groove runs along the midline of the VNC in the opisthosome of O. alvinae [23].
A pair of giant axons extends from the pair of giant perikarya reported in the vestimentiferans Ridgeia, Riftia, Oasisia, and Lamellibrachia [17,[22][23][24]. Notably, the giant axons terminate at different levels within the trunk VNC: in L. luymesi, giant axons terminate in the anterior part of the trunk [16], in L. barhami the latter extend somewhat further back [41], and in R. piscesae, O. alvinae and Riftia they extend up to the border between trunk and the first opistosome segment [22,23]. Previous studies reported that a pair of giant perikarya is retained in juvenile R. piscesae and O. alvinae in the mid-dorsal part of the brain [22][23][24]26]. In the present analysis, we found two pairs of the giant neurons in the dorsal commissure of juvenile Riftia (Fig 10B and 10C). Moreover, the lateral branches of giant axons (S10E-S10G Fig
All siboglinids have a paired VNC. First, the VNC is paired in the anterior segment of the vestimentiferans (the vestimentum), frenulates (the forepart) and Osedax priapus (the trunk), which were suggested to be homologous to each other [35]. Second, vestimentiferans and large frenulates have a pair of giant axons (absent in Osedax and Sclerolinum). Third, in Sclerolinum, with the single nerve cord, the VNC bifurcates into two strands around the ventral ciliated field. Fourth, Osedax species (females and males) exhibit a distinct pair of widely separated strands of the VNC in the trunk [21,27,28,30,[34][35][36].
The ventral ciliary field, a structure conserved in most siboglinids, lies in the anterior body part: in the trunk of frenulates, in the vestimentum of vestimentiferans, within the forepart of Sclerolinum and within the anterior trunk of female O. priapus [21,30,35,36]. Although the ciliary field in frenulates and in both the vestimentiferans and Sclerolinum lies in different regions, it always originates from the larval neurothroch [42,43]. In the developing larvae of the frenulate Siboglinum fiordicum, the anterior part of the neurotroch extends to the future forepart, whereas the posterior part of the neurotroch extends to the future trunk. In developing S. fiordicum, only the posterior part of the neurotroch remains in the trunk of adults [42,43]. In adult vestimentiferans the ciliary field (= the anterior part of the neurotroch) remains in the vestimentum [21,38]. We assume that in adult frenulates and vestimentiferans, different parts of the neurotroch remain, possibly due to different larval life modes. Vestimentiferan larvae swim for a long time in the water, whereas in frenulates they settle and simultaneously undergo metamorphosis. Among the questions for future studies are the functions of the ciliary fields in siboglinids as well as whether the lateral ciliary bands of Osedax females originate from the neurotroch or not.
Perikarya do not form accumulations along most of the length of the VNC, i.e. in forepart/ vestimentum and trunk, but their number increases in the region of annular chaetae, as reported in the frenulate Lamellisabella zachsi [27,28]. In short opisthosomal segments of the frenulate Siboglinum fiordicum the perikarya possibly form the ganglionic masses [29,30]. In contrast to the vestimentiferans' anchoring opisthosome, the frenulate opisthosome is designed to protrude out of the posterior tube opening and to dig into the sediment [30]. Due to this high mobility, the VNC in the frenulate opisthosome forms three strands with paired somata masses in each segment in Siboglinum fiordicum [29,44].
Giant axons in the vestimentiferans Ridgeia, Riftia and Oasisia [22][23][24] were found to extend up to the posterior end of the trunk. In large frenulates such as Spirobrachia and Lamelisabella, a pair of giant axons extend from the giant unipolar perikarya located in the brain [28,30]. In small frenulates such as Nereilinum there is only one giant axon, and it runs only along one side of the ventral ciliary field [30]. In frenulates, the giant axons extend only until the girdle of hook-shaped chaetae located approximately in the mid-trunk, whereas in the vestimentiferans the latter structure is detectable until the end of the trunk. The giant axons support a rapid contraction of the longitudinal musculature, serving the so-called "flight response"-in the frenulates and vestimentiferans it is the retraction of the body deep into the tube when threatened (i.e. by claws of crabs). To do so, frenulates are anchored to the wall of the tube with their girdle chaetae, whereas the vestimentiferans use chaetae of the opisthosome for this purpose. That may explain why the giant axons extend only to the girdle in the frenulates, and to the opisthosome in the vestimentiferans. Interestingly, Osedax and Sclerolinum lack giant axons.
Thus, the VNC in siboglinids is arranged in a very similar way. In the anterior part of the body the paired strands are associated with the ventral ciliary field. In all groups, the VNC lies entirely within the epidermis and contains the giant axons. The VNC is not ganglionated over most of its length. The question for future studies is whether the frenulates and Sclerolinum have a ganglionized VNC (includingthe opisthosome).

Ventral nerve cord in siboglinids and annelids
The siboglinids have an intraepidermal paired medullary ventral nerve cord (VNC) containing giant axons (except Sclerolinum and Osedax) and associated with the ventral ciliary field. The intraepidermal position of the VNC is also known in species of Opheliidae, Spionidae, Syllidae, Maldaniidae, Cossuridae, Polygordiidae and Protodrillidae as well as in the basally branching Chaetopteridae, Magelonidae and Oweniidae [45][46][47][48]. Siboglinids are one more annelid group with the intraepidermal VNC, additionally underlining that such a condition might be part of the annelid ground pattern.
The arrangement of the segmental neurite bundles in vestimentiferans is similar to that in oweniids [61,66,67]: numerous and anastomosing neurites in the long anterior segments, and condensed single bundles in the short posterior segments. This convergent pattern may reflect segment elongation.
Giant axons and giant perikarya are common among annelids, especially in large forms [64,68]. A common feature of most annelids is the multicellular or unicellular giant fibres extending from the giant somata. The latter are usually situated in subesophageal ganglia and/ or other segmental ganglia. In vestimentiferans, one pair of giant perikarya (as opposed to the at least three pairs we detected in Riftia) are located within the supraesophageal ganglion. Among annelids, only in sabellids such as large Myxicola infundibulum and Sabella pavonina, and spionids, such as Prionospio steenstru, do the giant perikarya lie in the supraesophageal ganglion. In the others they lie in the VNC [68].
Vestimentiferans together with most remaining siboglinids have the ventral ciliary field bordered by a pair of VNC strands. The ciliary field is not common in sexually mature annelids. It is known in presumably progenetic Dinophilus gyrociliatus [69,70], where it is used for gliding. Note that the tiny frenulate Nereilinum murmanicum uses this ciliary field to glide vertically along the tube as well [31]. Other functions of this structure in siboglinids remain theoretical [17].
Riftia pachyptila's brain is heart-shaped in transverse section, with significantly developed dorso-lateral lobes (Figs 5 and 8A-8D). In Ridgeia piscesae it is triangular in transverse section and has a wide ventral side [22]. In contrast, this organ is oval-transverse in Lamellibrachia luymesi [16]. The two latter species have less developed dorso-lateral lobes than Riftia (S5 Fig). Rifia possesses 340 tentacles per lamellae and 335 lamellae on each side of the obturaculum, whereas 70 lamellae in Escarpia seem to be the maximum value in other vestimentiferans [19,71,72]. This helps explain the enlarged dorso-lateral lobes in Riftia's brain. Importantly, despite the brain shape differences, tentacle nerves originate from the same dorso-lateral areas of the brain neuropile in Riftia and all other vestimentiferans.
Finally, a prominent cuticle shield protects the ventral side of the brain. This shield has direct contact with the tube or ambient environment in all studied vestimentiferans (Fig 10G) [16,22,23,26]. The dorsal and frontal sides of the brain are covered by tentacles and obturacules (Figs 4 and 5). Moreover, the brain can be penetrated by cuticle rods and plates extending from the cuticle of tentacle lamellae, as observed in L. luymesi, R. piscesae, O. alvinae, but not in Riftia [16,23,26].

Brain anatomy of vestimentiferans and annelids
The vestimentiferan brain lies completely within the epidermis at the anteriormost part of the vestimentum. It comprises the large and dense mass of the neuropile and perikarya, giving the impression of a single entity. The "brain" was once suggested to be a product of the fusion of supra-and subesophageal ganglia [20]. The juvenile vestimentiferans exhibit the gut rudiment, which can be used to help homologize the brain parts of the gutless siboglinids with the relevant parts of the annelid brain (supraesophageal ganglion) and postoral segmental paired ganglion of the ventral nerve cord (subesophageal ganglion). Below we provide detailed homologization.
Longitudinal nerve tracts (LNT) can be homologized with the circumesophageal connectives of annelids (Figs 9 and 11). First, like annelid circumesophageal connectives [45,73], the LNT are somata-free nerve bundles interconnecting the postoral segmental paired ganglia of the ventral nerve cord (VNC) and the supraesophageal ganglion in the anteriormost part of the brain in Riftia (Figs 5-7; S1-S3 Figs). Second, like annelid circumesophageal connectives [47,49,[74][75][76], the LNT run ventrally to the the enteral coelomic channel within the VNC and the posterior brain, extend from the VNC anteriorly, run along the lateral sides of the gut, and rise to the dorsal "brain" in Riftia. Third, like annelid circumesophageal connectives, the LNT give rise to two pairs of roots: dorsal and ventral ones (Fig 6; S3A Fig). The dorsal roots are interconnected via the dorsal commissures, the ventral roots via the supraenteral commissure (Fig 9). Fourth, in annelids, the lateral and ventral sides of the circumesophageal connectives and the ventral nerve cord are associated with ganglia cells of the postoral ganglia of the VNC [49]. In Riftia, both the LNT and VNC are surrounded laterally and ventrally by the ventral tripartite aggregation of the perikarya (vtp).
The part of the vestimentiferan "brain" lying dorsally to the enteral coelomic channel can be homologized with the supraesophageal ganglion (Figs 4, 8A-8D and 11), the part of the brain ventral to the enteral coelomic channel with the subesophageal ganglion (Figs 4, 8E-8H  and 11).
The supraesophageal ganglion in Riftia, like in other annelids (and certain other taxa), is the most prominent anterior condensation of neurons [73]. This is the first reason. Second, in Riftia this ganglion has the peripheral perikarya (nep) surrounding the neuropile of the lateral  [47] showed the general presence of 4 transverse commissures in the brain (a.k.a. supraesophageal ganglion) of numerous annelids (A), which join through their roots and circumesophageal connectives to the ventral nerve cords. The palp neurites extend from the dorsal root of the circumesophageal connectives in annelids. In vestimentiferans (C), the "brain" is a fusion of the supra-and subesophageal ganglia. Longitudinal nerve tracts (LNT), as circumesophageal connectives, connect the ganglia. The dorsal (DC) and supraenteral (SPC) commissures connect transversely the paired structures of the supraesophageal ganglion, whereas the subenteral commissure (SBC) connects transversely the structures of the subesophageal ganglion. The innervation of tentacles from the DC makes them homologous to peristomial palps of other annelids. Hypothetical ancestral state of vestimentiferan brain (B) is transitional between annelid brain (A) and vestimentiferan brain (C). Dorsal side to the top. A-supra-and subesophageal ganglia in annelids (after [47]). Bhypothetical transitional state. C-vestimentiferan brain. APN-neurite bundles of palps, B-brain, C-commissure of sbg, CC-circumesophageal connectives, DC-dorsal commissure, DRCC-dorsal (posterior) root of the CC, EC-enteral coelom, LNT-longitudinal nerve tracts projecting from ventral nerve cord into brain, sbgsubesophageal ganglion, SBC-subenteral commissure, spg-supraesophageal ganglion (the brain in annelids), SPC-supraenteral commissure, VRCC-ventral (anterior) root of CC, TEN-neurite bundles of tentacles (palps). brain lobes (NE) (Fig 5; S1 Fig). Third, like in various other annelids [47], in the dorsal part of the brain (which we homologize with the supraesophpageal ganglion) we found two of the most prominent commissures (dorsal and supraenteral) connected via dorsal and ventral roots to the circumesophageal connectives (= longitudinal nerve tracts, LNT). These commissures are also present in the vestimentiferans Oasisia and Ridgeia (Fig 7 in [22], Fig 4c in [23]). Fourth, in annelids, the somata of the brain may be arranged in distinct groups around the central neuropile, with a few somata usually located ventrally [45]. In Riftia's supraesophageal ganglion, most somata groups are located dorsally and laterally; the ventral side is almost free of somata, except for the anterior median aggregation of perikaria (amp, Figs 5-7; S1-S3 Figs).
Commissural cell clusters at the junction of the dorsal and ventral roots of the circumoesophageal connectives are widespread among "polychaetes" (including most sedentarian species) [47,49] and are suggested to be a ground pattern character of annelids [64]. In the present analysis, we hypothesize that the lateroventral perikarya of the tripartite ventral aggregation (lvtp), which are situated in the posterior brain in front of the junction of the dorsal and supraenteral commissures into the LNT (= circumesophageal connectives), are the commissural cluster of somata (Fig 7; S3 Fig). Here, some neurite bundles branch off and extend from the LNT to the small perikarya of the lvtp. Moreover, in annelids, the commissural cell cluster very often fuses with the first ventral ganglion [49,64]. In Riftia, as well as in Oasisia and Ridgeia [22,23], the lvtp is always associated with the vtp, which is the first ventral ganglion.
Mushroom bodies, or the "corpora pedunculata" known in arthropods, annelids and possibly in some flatworms and molluscs, are the stalk-like neuropil forming several lobes and surrounded by a cap of small neuronal somata termed globuli cells [45,63,64,[77][78][79][80][81][82]. The globuli cells possess a minute amount of cytoplasm and are especially rich in chromatin. They form columns comprising from ten to thousands of cells [49,63,64,73]. Accordingly, the mushroom-like structures in Riftia could be a complex formed by the anterior pair of the median aggregation of perikarya (amp) and two pairs of supraenteral longitudinal neurite bundles (SLN). The SLN could be homologous to a stalk-like neuropile of the linear neurite bundles. This is surrounded distally by amp, which could be homologous to the small globuli cells arranged in rows (Fig 5; S1 and S2 Figs). The whole complex of amp and SLN adjoins the supraesophageal ganglion. In the vestimentiferan Oasisia, the amp and SLN were also detected as P-medN and vCS, respectively (Fig 5a in [23]). Mushroom bodies are found in many errant and in some sedentary annelids such as the serpulid Serpula, the alvinellid Paralvinella hessleri, and sabellids, including Chone and Euchone species [49,64,80,83]. As in P. hessleri [83] and E. papillosa [49], the mushroom-like structures in Riftia do not occupy the typical dorso-lateral position in the brain, but instead extend anteriorly. Interestingly, the chemosynthetic P. hessleri exhibits theses structures, which potentially play a specific chemosensory role in the vent environment [83].
Most annelid palps (in 22 out of the 32 polychaete families) are innervated from the roots of the circumesophageal connectives; single palps are innervated from the dorsal commissures (see Fig 2 in [47]) [49]. The innervation of the numerous tentacles of R. pachyptila is provided by the tentacle neurite bundles (TEN) radially emanating from the neuropiles of the lateral brain lobes (NE), which in turn receive the neurites from both roots of the longitudinal nerve tracts, dorsal and supraenteral (LNT, Figs 7, 8A-8D and 11C). The latter are homologous to the dorsal and ventral roots of circumesophageal connectives. The palps are the only appendages of annelids to receive the neurites from both the ventral and dorsal roots. The conclusion is that the tentacles of Riftia are innervated from both roots of the circumesophageal connectives as are most annelid palps. Why do vestimentiferans have palps but no other annelid anterior appendages or organs? Various antennae in annelids are innervated only from certain parts of the dorsal root of connectives [47]. The stomatogastric neurites in annelids extend from the commissures and not from connective roots [47]. No oral filaments, buccal appendages, nuchal organ or eyes have been detected in adult vestimentiferans.
Previously, vestimentiferan tentacles were homologised with polychaete palps [11]. Nonetheless, this homology was considered doubtful based on differences in the external and internal structures (lack of ciliated grooves, absence of longitudinal support rods and the presence of the afferent and efferent blood vessels inside each tentacle) [38]. Our data on the innervation of Riftia tentacles proves the annelid palp hypothesis of vestimentiferan tentacles (Fig 11). In vestimentiferans (especially Riftia), parts of the longitudinal nerve tracts and neuropile of the lateral brain lobes are incomparably larger than the corresponding neural structures in annelids. This is because the tentacle apparatus of vestimentiferans is highly developed. A similar correlation between the sizes of tentacle crowns and brains is clearly evident in oweniids and sabellids. In oweniids, featuring simple gill tentacles and pigmented eyes, the brain is a simple transverse commissure followed by the layer of perikarya passing in the epidermis dorsal to the digestive tract [66,67]. In contrast, in sabellids-featuring a large tentacle crown serving for food collection, pigmented eyes and statocysts-the brain consists of four main transverse commissures, dorsal and ventral roots of the circumesophageal connectives, an additional four commissures, anterior loops of the dorsal root, blood vessel neurites, giant perikarya and axons, nuchal nerves, neurites of statocysts and eyes, palp nerves, two pairs of mushroom bodies, as well as lateral and median clusters of brain [49].
We agree with Jones and Gardiner [20] that the brain is a result of the union of the supraand subesophageal ganglia. For the first time, in the dorsal part of the vestimentiferan brain (= supraesophageal ganglion), we found homologues of the dorsal and ventral pairs of the transverse commissures, mushroom bodies, commissural cell clusters, palp neurite bundles, and giant somata. Furthemore, we found homologues of circumesophageal connectives and the subesophageal ganglion. We have found possible homologs that were considered to be absent. Thus, we do not confirm the hypothesis on the simplicity of the vestimentiferan brains [18].
Our comparative anatomical approach shows that the structure of the vestimentiferan brain and VNC does not go beyond the diversity of neuronal structures in Annelida. Within Sedentaria, vestimentiferans have complex brains, which are comparable to brains of their close sister-clades, Cirratuliformia and Spionida/Sabellida, as well as to representatives of possible basally branching sedentarians, like Orbiniidae [47]. Whereas siboglinid brains (with several transverse commissures and palp neurites) do not resemble brains of more distant sedentarian clades like terrebellids and pectinariids [13]. Brains of the latters comprise a single transverse commissure that gives rise to the neurites of buccal appendages and to the stomatogastric neurites [47].
We suggest a homologization of the dorsal commissure of Riftia (DC) and anterior commissure in Osedax (ACBR, [35]). They are situated in the anteriormost parts of the brains and also give rise to the neurite bundles of the homologous anterior structures (Fig 12). First, palps are innervated by the palp neurites branching off the roots of the circumesophageal connectives in both Riftia (TEN) and Osedax (PN). Second, the obturacule neurites in vestimentiferans (OBN) and the antero-dorsal nerves or the anterior nerve net in Osedax (ADN and ANN) give rise from the middle parts of the dorsal commissures (Fig 12; see Fig 2 in [35]). A future task will be to trace the vertical bundles branching from the dorsal commissure in other siboglinid species as well as to find homologous neuronal structures among siboglinids and annelids.
We suggest that the anterior and posterior bundles of the posterior commissure in Osedax (PCBR) are homologous to the bundles of the supraenteral and subenteral commissures in Riftia (SPC, SBC, Fig 12). Osedax lacks a gut, we use the remaining intracerebral neural structures for this homologization. First, crossing neurites connect the dorsal commissure to anterior bundles of the PCBR in Osedax (MCC, [35]) and to the SPC in Riftia (VAN). Second, the VNC is interconnected by the posterior bundles of the PCBR in Osedax and the SBC in Riftia.
We consider the lateral longitudinal bundles in Osedax (LLN) and the longitudinal nerve tracks in Riftia (LNT) to be homologous to each other and to the annelid circumesophageal connectives (Fig 12). Both the LLN in Osedax and the LNT in Riftia are connected by the main transverse commissures, their roots give rise palp neurites, and they are continuations of the VNC.
The brain organization in frenulates, sister group to the remaining siboglinids [2,86], is important in analyising the ancestral state of the siboglinid brain, but remains to be studied in detail. Notably, studied frenulates have commissures in the epidermis of the dorsal body side: a single dorsal commissure in Polybrachia annulata, Siboglinum caulleryi [27,28], and two dorsal commissures in Nereilinum murmanicum, S. modestum and S. subligatum [31]. These commissures in frenulates give rise the neurite bundles to anterior tentacles, which we preliminarily homologise with the annelid palps based on the origin from the dorsal root of the circumesophageal connectives (Fig 12).
The Sclerolinum brain is a very simple structure lying entirely in the ventral epidermis and having two layers: apical perikarya and basal neuropile [36]. We currently lack sufficient details to homologize it with other siboglinids and annelids.

Conclusions
A comparative neuroanatomical analysis of the siboglinids and the annelid sister clades enables us to hypothesize that the last common ancestor of siboglinids had separate supra-and subesophageal ganglia, two roots of the circumesophageal connectives giving rise to neurite bundles to numerous palps, a commissural cell cluster, a paired ventral nerve cord, and had giant perikarya in the supraesophageal ganglion with paired giant axons running within the paired nerve cord (Fig 12). The strands of the VNC and the giant axons probably fused posteriorly. Notably, within Sedentaria siboglinids form the sister clade with the Cirratuliformia, and Spionida/Sabellida having the complex brain with the similar structures, like several transverse commissures and palp neurites. Siboglinids do not exhibit reduction in neuroanatomical complexity, like terebellids and pectinariids, which have no traces of commissures of the circumesophageal roots as well as no palp neurites. Future neuroanatomical studies should reveal if within the Sedentaria the simplification of the brain was the one of the trends of their evolution.
Our study provides definitive closure in the dispute on the origin of the siboglinid tentacles and proves them to be palps based on their innervation, as originally proposed by Orrhage [47]. This conclusion was supported by tracing the direction of evolution of palp nerve roots within Annelida in light of current phylogenetic analyses [13,87]. The palps and their nerve roots are a recognised ground pattern of the annelids [47,64,88,89]. Indeed, palp-bearing species are present in all three major groups. Therefore, we assume that palp-bearing species have palp neurites. Within the basally branching groups and Errantia, each clade comprises palpbearing species, including the Amphinomidae, Chaetopteridae, Magelonidae [47,90], Eunicida, Phyllodocidae, and Protodriliformia [91][92][93][94][95][96]. Within Sedentaria, only the basally branching lineages have palps, such as the Orbiniida (Nerellidae; in Orbinidae the palp nerves are present [95,97]), Spionida/ Sabellida [47,49,64], and Siboglinidae/ Cirratuliformia (within the Cirratuliformia: Flabelligeridae and Acrocirridae [47]. More distant sedentarian clades are palp-less, e.g. Clitellata, Terebelliformia/ Arenicolidae, and Opheliidae/ Capitellidae/ Echiura (Ophelia has palp nerve roots though). The sedentarian groups are distinguished by a high variety in the number of palp nerve roots: from zero to 335 pairs; whereas this variety is lower in Errantia and basally branching groups. There are 0, or 3-4 pairs of palp roots within basally branching species versus 1-6 within the errantian species [64]. Accordingly, sedentarian species exhibit the greatest variety in the number of palp nerve roots. Their number changed from several to many palps (as in vestimentiferans) or was reduced completely (as in clitellates). Future research should examine if and how the heterogeneity of palp nerve roots arrangements in annelids is based on their functionality. Numerous radial tentacle neurite bundles (TEN) extend from neuropile of lateral brain lobes to bases of tentacle lamellae (A). Each lamella is a fold of epidermis represented by two layers: thin external lamellae wall (OEP) and thick internal lamella wall (IEP) containing basiepithelial tentacle neurites (TEN). Tentacle lamellae originate at brain periphery of dorsal, lateral and ventrolateral sides of brain (B-D). Dorsalmost lamellae are least differentiated (B). Ventrolateral lamellae extend toward anterior end of tentacle crown (D). They remain undifferentiated over a certain part of their length, and then separate into individual tentacles. A-scheme of neural elements of undifferential tentacle lamellae: perikarya and neurite bundles. B-D-tentacle lamellae bases on dorsal, lateral and ventrolateral sides of brain surface, respectively. ECMextracellular matrix, EP-epidermis, IEP-epidermis of the internal lamellae wall, OEP-epidermis of external lamellae wall, LR-undifferentiated tentacle lamellae, NE-neuropile of lateral brain lobes, nep-peripheral perikarya of lateral brain lobes, NB-neurite bundles, OB-obturaculum, pl-large perikarya, ps-small perikarya, TEN-neurite bundles of tentacles (palps). (TIF) S6 Fig. Coelomic channels running through the brain. Three coelomic channels pass through brain: a single enteral coelom (EC) and a pair of obturacular channels (OBC). The EC encompassing the gut rudiment marks area of peripheral perikarya of lateral brain lobes (nep, a.k.a. supraesophageal ganglion) and tripartite aggregation of perikarya (vtp, a.k.a. subesophageal ganglion). Obturacular coelomic channels encompassing obturacular blood vessels make an S-like loop in brain tissue. 3D models of Riftia brain. A, C, E-peripheral perikarya of lateral brain lobes (nep) are on dorsal side of brain (purple). B, D, F-tripartite aggregation of perikarya (vtp) is on ventral side and under obturacular and enteral coeloms (blue). View sides shown at right lower corners of each image. Cube side 255 μm.