DOI: 10.1111/ede.12231

RESEARCH PAPER

Ground plan of the larval nervous system in : Evidence from larvae of viviparous

Elena N. Temereva

Department of Invertebrate Zoology, Biological Faculty, Moscow State Nervous system organization differs greatly in larvae and adults of many , but University, Moscow, Russia has nevertheless been traditionally used for phylogenetic studies. In phoronids, the organization of the larval nervous system depends on the type of development. With Correspondence Elena N. Temereva, Department of the goal of understanding the ground plan of the nervous system in phoronid larvae, the Invertebrate Zoology, Biological Faculty, development and organization of the larval nervous system were studied in a viviparous Moscow State University, Moscow 119991, phoronid species. The ground plan of the phoronid larval nervous system includes an Russia. Email: [email protected] apical organ, a continuous nerve tract under the preoral and postoral ciliated bands, and two lateral nerves extending between the apical organ and the nerve tract. A bilobed Funding information Russian Foundation for Basic Research, with such an organization of the nervous system is suggested to be the primary Grant numbers: 15-29-02601, 17-04- larva of the taxonomic group Brachiozoa, which includes the phyla Brachiopoda and 00586; Russian Science Foundation, Phoronida. The ground plan of the nervous system of phoronid larvae is similar to that Grant number: 14-50-00029 of the early larvae of annelids and of some deuterostomians. The - and deuterostome-like features, which are characteristic of many organ systems in phoronids, were probably inherited by phoronids from the last common bilaterian ancestor. The information provided here on the ground plan of the larval nervous system should be useful for future analyses of phoronid phylogeny and evolution.

KEYWORDS development, evolution, larvae, nervous system, Phoronida, phylogeny

1 | INTRODUCTION molecular phylogeny, the phoronids are regarded as proto- stomian , which are close relatives of annelids (Kocot Information on nervous system development and organiza- et al., 2017). These data contradict the traditional view, which tion has traditionally been considered as phylogenetically is based on development and anatomy, that phoronids are informative and has sometimes been used to determine the deuterostomian animals with a radial type of the egg cleavage, relationships among higher taxa of invertebrates (Hay- a multicellular (in some cases entercoelic) formation of the Schmidt, 2000; Wanninger, 2008, 2009). Obtaining informa- coelomic mesoderm, and three coelomic compartments tion about nervous system organization is particularly (Emig, 1982; Ruppert, Fox, & Barnes, 2004). To date, important for taxa with unstable phylogenetic positions morphological features have not been identified that support such as phoronids, which are worm-like marine invertebrates the molecular-based inference that phoronids are protosto- with a biphasic life cycle (Emig, 1982). The position of mian animals. The inconsistency between molecular and phoronids in the phylogenetic tree of the Bilateria has been morphological/embryological data might be clarified by new changed greatly based on molecular phylogenetic data information on nervous system organization and development (Halanych et al., 1995; Peterson & Eernisse, 2001). In recent in phoronids.

Evolution & Development. 2017;19:171–189. wileyonlinelibrary.com/journal/ede © 2017 Wiley Periodicals, Inc. | 171 172 | TEMEREVA

New information on nervous system organization and animals were separated from sediment by washing on a sieve development could also provide insights into the evolution of with 2-mm openings. Late gastrulae and early larvae were the phoronid life cycle (Arendt, Denes, Jekely, & Tessmar- collected from the trunk coelom of mothers via dissection. Raible, 2008; Nomaksteinsky et al., 2009). For example, the Different stages of advanced larvae were collected with a deep information could help answer a basic question: Which came net. The larvae were identified based on previous descriptions first—larvae or adults (Nielsen, 2013; Sly, Snoke, & Raff, (Temereva & Malakhov, 2004; Temereva & Neretina, 2013). 2003; Temereva & Malakhov, 2015; Temereva & Tsitrin, Consecutive stages of larval development were observed with 2014a)? Although adult phoronids have a nerve plexus that an Olympus XI83 microscope, photographed, and fixed for lacks prominent nerve centers (Bullock & Horridge, 1965; light microscopy, TEM, and immunocytochemistry combined Fernández, Pardos, Benito, & Roldan, 1996; Silén, 1954b; with laser confocal microscopy. See Temereva and Chichvar- Temereva, 2015; Temereva & Malakhov, 2009), phoronid khin (2017) for information on the location of larvae in the larvae have a complex nervous system with two or one nerve mother's trunk and for a detailed description of the morphology centers and prominent nerve tracts (Santagata & Zimmer, of the newly identified phoronid species, P. embryolabi. 2002; Temereva & Tsitrin, 2014a,b; Zimmer, 1964). The plexus-like organization of the nervous system in adult 2.2 | Light microscopy and transmission phoronids is traditionally regarded as a primitive feature of electron microscopy their organization (Mamkaev, 1962; Schmidt-Rhaesa, 2007). At the same time, this plexus-like structure of the adult For light microscopy and TEM, advanced larvae and adult nervous system can be caused by a sessile life style (Temereva animals containing early embryos were fixed at 4°C in 2.5% & Malakhov, 2009). glutaraldehyde in 0.05 M cacodylate buffer containing NaCl Phoronids have four types of development, which are and were then postfixed in 1% osmium tetroxide in the same characterized by differences in egg cleavage, morphology of buffer. The specimens were dehydrated in ethanol followed the first swimming stage and of competent larvae, duration of by an acetone series and then embedded in EMBed-812. the larval stage, and organization of the larval nervous system Semi-thin sections were cut with a Leica UC6 ultramicrotome (Emig, 1977; Santagata & Zimmer, 2002; Silén, 1954a; (Leica Microsystems, Wetzlar, Germany). Semi-thin sections Temereva, 2009; Temereva & Malakhov, 2007, 2012, 2016). were stained with methylene blue, observed with a Zeiss These differences in development should be considered in any Axioplan2 microscope, and photographed with an AxioCam reconstruction of the ground plan of the phoronid nervous HRm camera. Ultrathin sections were mounted on slot grids system. In other words, the reconstruction should be based on and mesh grids, contrasted with 2% uranyl acetate- and 4% data obtained from species with all four types of development. lead citrate-solution, and examined by TEM (Jeol JEM 1011, The investigation of species with different types of develop- Jeol Ltd., Tokyo, Japan). ment may also answer a fundamental question about the evolution of the phoronid life cycle, that is, did planktotrophy 2.3 | Immunohistochemistry precede lecithotrophy or vice versa? This report describes for the first time the development For cytochemistry, early, advanced, and competent larvae and organization of the larval nervous system in a phoronid were narcotized in MgCl2, fixed overnight in a 4% species (Phoronis embryolabi) with a recently discovered paraformaldehyde solution in phosphate buffer (pH 7.4) unusual type of development, that is, viviparity of larvae (Fisher Scientific, Pittsburgh, PA, USA), and washed (two (Temereva & Chichvarkhin, 2017; Temereva & Malakhov, times) in phosphate buffer with Triton X-100 (1%) (Fisher 2016). The main goal of this study was to provide new data on Scientific) for a total of 2 hr. Nonspecific binding sites were the ground plan of the larval nervous system in phoronids. blocked with 1% normal donkey serum (Jackson Immuno The data obtained will be useful for future analysis of Research, Newmarket, Suffolk, UK) in PBT overnight at phoronid phylogeny and of phoronid life cycle evolution. 4°C. Subsequently, the larvae were transferred into primary antibody, which was a mixture of alpha acetylated-tubulin 2 | MATERIALS AND METHODS (1:700) and anti-serotonin (1:1,000) (ImmunoStar, Hudson, WI, USA) in PBT. After the larvae were incubated with 2.1 | Sampling of animals primary antibody for 24 hr at 4°C and with gentle rotation, they were washed for 8 hr at 4°C (at least three times) in PBT and Adult phoronids and larvae were collected in July 2015 in then exposed to the secondary antibody: 532-Alexa-Rabbit Vostok Bay, Sea of Japan. Adult phoronids with sediment were (1:2,000) and 635-Alexa-Mouse (1:2,000) (Invitrogen, Grand removed from the burrows of the shrimp Nihonotrypaea Island, NY, USA) in PBT for 24 hr at 4°C. After the larvae were japonica with a vacuum pump. The pump was driven into the incubated with secondary antibody for 24 hr at 4°C and with burrow holes, which are located at a depth of 60 cm. Tubes with gentle rotation, they were washed in PBS (three times for TEMEREVA | 173

40 min each time), mounted on a cover glass covered with gastrula, early larva, young larva, precompetent larva, and poly-L-lysine (Sigma-Aldrich, St. Louis, MO, USA), and competent larva. Although the larval stages of this viviparous embedded in Murray Clear. Specimens were viewed with a phoronid were described in detail in a previous paper Nikon Eclipse Ti confocal microscope (Moscow State (Temereva & Neretina, 2013), it is necessary to give short University, Moscow, Russia). Z-projections were generated description of different stages, which were investigated here. using the program ImageJ version 1.43 (Schneider, Rasband, & The late gastrula has a body length of 80 μm (Figure 1a). The Eliceiri, 2012). Three-dimensional reconstructions were body is subdivided into two regions: the anterior, which is that generated using Amira version 5.2.2 software (Bitplane, anlage of the preoral lobe, and the posterior, which is the Zurich, Switzerland). collar region (Figure 2a). The anterior part bears the apical plate, which is a thick epithelium with long cilia (Figure 1a). 3 | RESULTS In the later gastrula, the digestive tract is closed; it begins with a wide mouth and consists of a short esophagus, a voluminous 3.1 | Morphology of P. embryolabi larvae sack-like stomach, and the anlage of the proctodaeum (Figures 1a, 2a, and 2b). The early larva has a body length The organization of the nervous system of P. embryolabi was of 90 μm (Figure 1b). The body consists of two parts: the investigated at the following stages of development: late preoral lobe and the collar region. The preoral lobe is well

FIGURE 1 Consecutive stages of larval development for Phoronis embryolabi. Photographs of live animals. The apical is at the top in all photographs. (a) Late gastrula from the mother's trunk coelom: the digestive tract is closed. (b) Early larva from the mother's trunk coelom: the digestive tract is complete, the preoral lobe is well developed, and the protonephridia are forming. (c and d) Young larvae from plankton samples: the preoral lobe is large, and the tentacles are developed. (e) Precompetent larva from plankton samples. (f) Competent larva. ap, apical plate; apr, anlage of proctodaeum; bm, blood mass; es, esophagus; m, mouth; ms, metasomal sac; pcb, postoral ciliated band; pl, preoral lobe; prn, protonephridium; sd, stomach diverticulum; st, stomach; t, tentacle; tr, trunk; tt, telotroch 174 | TEMEREVA

FIGURE 2 Organization of the nervous system at early stages of development in Phoronis embryolabi. In all photographs, the apical is at the top, except in (g), where the apical is at the upper left corner. (a) Late gastrula viewed from the left; SEM. (b) Late gastrula, sagittal semi- thin section. (c) Z-projections of late gastrula after staining against alpha acetylated-tubulin (gray) and serotonin (yellow). (d) Late gastrula:3D reconstruction combined with volume renderings based on confocal image stacks; staining against alpha acetylated-tubulin. (e) Early larva: sagittal semi-thin section. (f) Early larva: 3D reconstruction combined with volume renderings based on confocal image stacks; staining against alpha acetylated-tubulin. (g) Portion of the apical organ in late gastrula; a perikaryon with a long basal process (shown by arrowheads) is visible. (h) Two 5ht-lir perikarya of the apical organ in the late gastrula. an, anterior nerve; apl, anlage of the preoral lobe; cc, coelomic cavity; dc, the dorsal commissure; ds, distal swell; es, esophagus; in, intestine; ln, lateral nerves of preoral lobe; mc, muscle cells; m, mouth; mi, microvilli; pao, perikarya of the apical organ; pcb, postoral ciliated band; pe, perikarya; pes, perikarya around esophagus; pl, preoral lobe; pn, posterior nerve; prn, protonephridium; st, stomach; vlb, ventro-lateral branches developed and hangs over the mouth, forming the vestibulum. protonephridia have begun to develop in the early larva and In the early larva, the digestive tract is complete and consists consist of the common base and two lateral branches of the esophagus, stomach, and proctodaeum (Figure 2e). The (Figure 2f). The body of the young larva is 200–260 μm early larva has a developed postoral ciliated band, which is long (Figures 1c and 1d) and has three parts: the preoral lobe, formed by columnar cells with long cilia (Figure 2e). The the collar region, and the trunk. The preoral lobe is large and TEMEREVA | 175 covers the oral field, which is the upper portion of the collar preoral lobe (Figure 4b). Each group consists of three to four region (Figure 1d). The collar region bears two median 5ht-lir perikarya, which seem to be monopolar and do not tentacles and two short anlagen of lateral tentacles. The trunk contact the surface of the epithelium (Figure 4c). In the bears the terminal telotroch. The precompetent larva has a middle, each thick, lateral, radial nerve branches and gives body length of 370–390 μm (Figure 1e). The comparative rise to the main tentacular ring (Figure 4c). The main volume of the preoral body and the trunk decreases several tentacular ring is a thin neurite bundle extending under the times because of the increasing length of the trunk. The base of the tentacles. A large portion of the tentacular nerve competent larva has a body length of 400–430 μm (Figure 1f). ring is represented by 5ht-lir neurites, which give rise to the The competent larvae are characterized by the presence of latero-abfrontal nerves of the tentacles (Figure 4d). Each eight short tentacles, three blood masses (two dorso-lateral tentacle has two latero-abfrontal nerves, which contact each and one ventral), a long trunk with a large telotroch, and a other forming a terminal loop with numerous varicosities spacious dark-pigmented ventral stomach diverticulum. (Figure 4d). The two 5ht-lir branches of the tentacular main ring connect via the dorsal commissure (Figure 4d). The 3.2 | Development and organization of the preoral lobe contains many thin radial neurites that start from nervous system in embryolabi larvae the apical organ and pass to the edge of the preoral lobe (Figures 4e and 3f). Some of these neurites exhibit serotonin- In the late gastrula, tubulin-like immunoreactivity is ex- like immunoreactivity (Figure 4g). Numerous radial neurites pressed in two main nerves and in the epithelium of the apical form the median nerve of the preoral lobe. This nerve consists plate (Figure 3a). The posterior nerve forms an open circle, of two lateral 5ht-lir neurite bundles, each of which bends which extends along the anlage of the preoral lobe and along near the edge of the preoral lobe and extends along it the ventro-lateral sides of the collar region. The ventro-lateral (Figure 4g). Two thick marginal nerves extend along the edge branches of the posterior nerve connect via the dorsal of the preoral lobe: anterior (corresponding to the anterior commissure (Figures 2d and 3a). The anterior nerve repeats nerve in the late gastrula) and posterior (corresponding to the the shape of the posterior nerve, but the anterior nerve is posterior nerve in the late gastrula) (Figure 4h). The anterior closed in the collar region (Figure 3a). The apical plate marginal nerve branches into numerous thin neurites that contains a horseshoe-shaped neuropil. Serotonin-like immu- extend along the oral field and penetrate into each tentacle noreactivity is found in the epithelium of the apical plate as forming the medio-frontal tentacular nerve (Figure 4e). The well as in several perikarya around the esophagus (Figure 3e). medio-frontal tentacular nerve does not exhibit serotonin-like In the apical plate, the neuropil and five to seven perikarya immunoreactivity and can be visualized by staining against exhibit serotonin-like immunoreactivity (Figure 2c). These alpha acetylated-tubulin (Figures 4e and 4f). form the apical organ; perikarya appear to be sensory in that In precompetent and competent larvae (Figure 5a), they are flask-like, they contact the surface of the epithelium, perikarya, which are located along the edge of the preoral and they have a thin basal projection (Figure 2h). According lobe, increase in number. At these stages, perikarya form two to transmission electron microscopy (TEM), the epithelium of or three pairs of groups at the base of the preoral lobe (Figures the apical plate contains cells with electron-dense cytoplasm 5b, 5d, and 5e). Flask-shaped perikarya are scattered along the and a lobed nucleus (Figure 2g). These cells contact the medio-lateral sides of the edge of the preoral lobe, forming a surface of the epithelium and have a long basal projection. sensory field (Figure 5b). In competent larva, the perikarya of Around the esophagus, there are four to five perikarya, which the apical organ increase in number. They are arranged in two are roundish and do not contact the surface of the epithelium horseshoe-shaped groups: anterior and posterior (Figures (Figures 2c and 3e). 5g and 5h). These groups are separated from each other by a In the early larva, all nerve elements except the perikarya part of the neuropil. around the esophagus remain (Figures 3b and 3f). The The apical organ of competent larvae is located between anterior nerve forms paired ventro-lateral branches, each of large glandular cells, which contain electron-lucent, mucus- which bears a distal swelling (Figures 2f and 3b). Two lateral like material (Figure 6a). The apical organ has two types of serotonin-like immunoreactive (5ht-lir) neurite bundles perikarya (Figures 6a, 6b, and 6d). Perikarya of the first type extend along the lateral sides of the preoral lobe from the are located anteriorly and have roundish nuclei with a few neuropil of the apical organ to the posterior nerve of the diffuse chromatin (Figure 6d). In some perikarya of the first preoral lobe (Figures 2f and 3f). type, the basal portion, which contains the nucleus, is These lateral radial neurite bundles become prominent in submerged under the epithelium (Figures 6a and 6b). the next stage. In the young larva (Figure 4a), thick lateral Perikarya of the second type are located posteriorly and radial neurite bundles start from the dorsal horns of the U- have elongated nuclei with much dense chromatin shaped apical organ and extend to the edge of the preoral lobe, (Figure 6d). All perikarya of the apical organ are apparently where two groups of perikarya are located on both sides of the sensory: each flask-shaped perikaryon bears the cilium, 176 | TEMEREVA

FIGURE 3 Schemes of nervous system organization in consecutive stages of larval development in Phoronis embryolabi. The apical is at the top; the ventral side is to the left in all photographs. Organization of the nervous system as indicated by staining against alpha acetylated-tubulin (a–d) and serotonin (e–h). (a and e) Late gastrula. (b and f) Early larva. (c and g) Young larva. (d and h) Competent larva. am, anterior marginal nerve of preoral lobe; an, anterior nerve; an(=am), portion of anterior nerve passing along edge of preoral lobe; ao, apical organ; dc, the dorsal commissure; ds, distal swell; fo, frontal organ; la, latero-abfrontal nerve of tentacle; lf, latero-frontal nerve of tentacle; lgp, lateral group of perikarya; lmn, lateral branches of median nerve of preoral lobe; ln, lateral nerves of preoral lobe; mf, medio-frontal nerve of tentacle; mn, median nerve of preoral lobe; ms, metasomal sac; msf, median sensory field; nao, neurites of apical organ; oms, opening of metasomal sac; pes, perikarya around esophagus; pm, posterior marginal nerve of preoral lobe; pms, prerikarya of metasomal sac; pn, posterior nerve; pn(=pm), portion of posterior nerve passing along edge of preoral lobe; tn, tentacular main nerve ring; trn, trunk nerve; ttn, telotroch nerves; tv, terminal varicose; vlb, ventro-lateral branches which can be found by staining against serotonin—it is so immunoreactivity (Figures 5f–5h). Near the apical organ, called serotonin cilium (Figure 5i). According to TEM and to three neurite bundles pass close to each other and connect immunocytochemical staining, the apical organ lacks a basal with the group of flask-like 5ht-lir perikarya, which contact layer of perikarya (Figure 6d). The neuropil of the apical the surface of the epithelium and form the frontal organ organ contains large-diameter basal cavities filled with (Figure 5b). Two lateral 5ht-lir neurite bundles bend along the electron-lucent material. These cavities are clearly visible lateral edges of the preoral lobe (Figure 5h). The space in semi-thin sections (Figures 6a and 6b). The neuropil between the two lateral neurite bundles is occupied by a appears disorganized, and neurites are separated from each median neurite bundle, which can be recognized by staining other by large electron-lucent spaces. Neurites contain against alpha acetylated-tubulin (Figure 5f). The nerve bulb synaptic vesicles with different contents (Figure 6c). forms where the median and marginal nerves contact each The median nerve of the preoral lobe consists of other. According to TEM, this bulb is formed by numerous three neurite bundles; two of these exhibit serotonin-like neurites of different diameter (Figure 7a). In some cases, TEMEREVA | 177

FIGURE 4 Organization of the nervous system in young larvae of Phoronis embryolabi. The apical is at the top; the ventral side is to the left in all photographs except (h), where the ventral side is to the front. 3D reconstruction (b) and Z-projections (c–h) after staining against alpha acetylated-tubulin (gray) and serotonin (glow and green). (a) Photograph of live, young larva. (b) 3D reconstruction combined with volume renderings based on confocal image stacks; staining against alpha acetylated-tubulin (gray) and serotonin (glow). (c) 5ht-lir elements of preoral lobe. (d) 5ht-lir elements of whole larva. (e) Alpha acetylated-tubulin-like immunoreactive elements of the left body side. (f) Peculiarities of innervation of tentacles. (g) Two lateral branches of median nerve of the preoral lobe. (h) Marginal nerves of the preoral lobe. am, anterior marginal nerve of preoral lobe; ao, apical organ; dc, the dorsal commissure; la, latero-abfrontal nerve of tentacle; lf, latero-frontal nerve of tentacle; lgp, lateral group of perikarya; lmn, lateral branches of median nerve of preoral lobe; ln, lateral nerves of preoral lobe; mf, medio-frontal nerve of tentacle; nao, neurites of apical organ; pm, posterior marginal nerve of preoral lobe; rnpl, radial nerves of preoral lobe; tn, tentacular main nerve ring; tv, terminal varicose; vlb, ventro-lateral branches neurites of large diameter, which are filled with transparent anterior marginal neurite bundle is much thicker than at material, are located around the periphery and surround a earlier stages. The posterior marginal neurite bundle exhibits mass of central neurites of small diameter (Figure 7b). serotonin-like immunoreactivity and connects with lateral The marginal nerve consists of anterior and posterior groups of 5ht-lir sensory cells (Figure 3h). According to neurite bundles (Figure 3d). In the competent larva, the TEM, the anterior marginal neurite bundle is associated with a 178 | TEMEREVA

FIGURE 5 Organization of the nervous system in competent larvae of Phoronis embryolabi. The apical is at the top; the ventral side is to the left in all photographs. Z-projections (b–c, i) and 3D reconstruction (d–h, j) after staining against alpha acetylated-tubulin (gray) and serotonin (glow and green). (a) Photograph of live larva. (b) 5ht-lir elements of whole larva. (c) Anterior and posterior marginal nerves of the preoral lobe give rise to the ventro-latearl branches and latero-frontal nerve tract, respectively. (d) 3D reconstruction of serotonin-like nervous system of whole larva, viewed from the left; the lateral nerve of the collar is evident. (e) 3D reconstruction of serotonin-like nervous system of whole larva, viewed from the right; trunk nerves are evident. (f) 3D reconstruction of median nerve of preoral lobe, which consists of two thick lateral nerves and one thin median nerve. (g) 3D reconstruction of the apical organ: two rows of flask-shaped perikarya are visible. (h) 5ht-lir elements of the preoral lobe. (i) Some perikarya of the apical organ: flask-like cells with apical serotonin cilium. (j) 5ht-lir elements of the metasomal sac and telotroch. am, anterior marginal nerve of preoral lobe; ao, apical organ; ap, apical plate; bm, blood mass; clm, lateral nerve of collar; dc, the dorsal commissure; fo, frontal organ; la, latero-abfrontal nerve of tentacle; lf, latero-frontal nerve of tentacle; lgp, lateral group of perikarya; lmn, lateral branches of median nerve of preoral lobe; ln, lateral nerves of preoral lobe; mf, medio-frontal nerve of tentacle; mg, midgut; mmn, median branch of median nerve of preoral lobe; ms, metasomal sac; nao, neurites of apical organ; pm, posterior marginal nerve of preoral lobe; pms, prerikarya of metasomal sac; rnpl, radial nerves of preoral lobe; t, tentacle; tn, tentacular main nerve ring; tt, telotroch; ttn, telotroch nerves; v, vestibulum; vlb, ventro-lateral branches TEMEREVA | 179

FIGURE 6 Organization of the apical organ in competent larvae of Phoronis embryolabi; TEM. The apical is at the top; the ventral side is to the left in all photographs. (a) Semi-thin sagittal cross-section of whole larva. (b) Semi-thin sagittal cross-section of the preoral lobe. (c) Basal portion of the neuropil. (d) Thin parasagittal section of the apical organ. ao, apical organ; bc, blastocoel; bl, basal lamina; c3–trunk coelom; els, electron-lucent space; es, esophagus; gc, gland cell; mc, muscle cells; ms, metasomal sac; msf, median sensory field; nf, nerve fiber; np, neuropil; pkI, perikarya of first type; pkII, perikarya of second type; pr, proctodaeum; sd, stomach diverticulum; st, stomach; sve, synaptic vesicles; tt, telotroch; v, vestibulum specialized cell that has a large vacuole containing many Each ventro-lateral nerve branches into many neurites, some bacteria (Figures 7a and 7c). Cells with bacteria were found in of which penetrate into tentacles and pass along their frontal several competent larvae and were always located near the side (Figures 3d, 3h, and 5c). The posterior marginal neurite anterior marginal neurite bundle. bundle comes to nerve, which passes under the postoral The anterior marginal neurite bundle extends from the ciliated band. In each tentacle, this nerve extends along the preoral edge to the ventro-lateral sides of the oral field, where latero-frontal sides (Figure 5c). it forms thick nerves (Figure 5c). According to TEM, these The apical organ gives rise to the 5ht-lir lateral nerve, nerves consist of 20–25 neurites, which are separated by which extends along the lateral side of the collar and connects electron-lucent spaces of 4–6 μm in diameter (Figure 7d). to the main tentacular nerve ring (Figures 5d and 5e). The 180 | TEMEREVA

FIGURE 7 Ultrastructure of nerve elements in competent larvae of Phoronis embryolabi. Sagittal sections of larvae. (a) The median sensory field at the edge of the preoral lobe is represented by a large bulb of numerous neurites. (b) Details of organization of the median sensory field, which contains large-diameter neurites (lan) with electron-lucent cytoplasm. (c) The portion of the epithelium of the edge of the preoral lobe with specialized cells bearing bacteria in a large vacuole. (d) Portion of the epithelium of the oral field with neurites of ventro-lateral branches. (e) Organization of the tentacular main nerve ring, which consists of numerous neurite bundles. (f) Perikaryon in the tentacular main nerve ring. (g) A few neurites (indicated by a circle) at the base of the epithelium of the telotroch. ba, bacteria; bc, blastocoel; bl, basal lamina; c3–trunk coelom; er, erythrocyte; mc, muscle cells; lan, neurites of large diameter; mm, marginal muscle of preoral lobe; msf, median sensory field; my, myofilaments; nf, nerve fiber; ntn, neurites of tentacular main nerve ring; nttn, neurites of telotroch nerve; pk, perikaryon; rm, radial muscles of preoral lobe; sd, stomach diverticulum; va, vacuole containing bacteria main tentacular nerve exhibits serotonin-like immunoreac- The apical organ gives rise to two dorso-lateral 5ht-lir tivity; it passes under the tentacles and gives rise to the latero- nerves that extend to the trunk and branch into several neurite abfrontal 5ht-lir tentacular nerves (Figure 5d). The main bundles that connect with several perikarya and contribute to tentacular nerve consists of numerous neurite bundles, which the innervation of the larval trunk and the telotroch (Figures form a thick net under the tentacle base (Figure 7e). Some 3d, 3h, and 5e). The telotroch is innervated by a thick net of perikarya are scattered among the neurites of the main 5ht-lir neurites and perikarya, which form two circles around tentacular nerve (Figure 7f). the ciliated band (Figure 5j). According to TEM, the telotroch TEMEREVA | 181 nerve ring is formed by a few thin neurites, which extend at 4.2 | Organization of the nervous system in the base of the telotroch epithelium (Figure 7g). competent phoronid larvae The metasomal sac, which is well developed in competent larvae, is innervated by many 5ht-lir neurites and perikarya Previous descriptions of competent actinotroch nervous (Figure 5j). These neurites form a thick net around the systems defined that the organization of the nervous system terminal portion of the metasomal sac, which will become the differs among competent phoronid larvae (Santagata & ampulla of the adult. Zimmer, 2002; Temereva & Tsitrin, 2014b). Two types of nervous systems in several advanced phoronid larvae were defined (Santagata & Zimmer, 2002). 4 | DISCUSSION Differences concern the innervation of tentacles, presence or absence of lateral nerves of the preoral lobe, and the 4.1 | Development of the nervous system in connection between the minor nerve ring and other nerve phoronid larvae elements (Table 1). Thus, large planktotrophic larvae have the The development of the nervous system has been previously type 1 nervous system (Santagata & Zimmer, 2002) described in two phoronid species: Phoronis ijimai, which (Figure 8a). In large phoronid larvae, there are lateral nerves broods its embryos in masses between tentacles (Hay- passing from the apical organ and giving rise to the minor Schmidt, 1990a), and Phoronopsis harmeri, whose develop- nerve ring. Tentacles are innervated mostly from the main ment completely occurs in the sea water (Temereva & nerve ring; frontal and latero-frontal tentacle nerves are Wanninger, 2012). The present report describes the nervous absent. In small planktotrophic larvae (the type 2 nervous system in the larvae of the viviparous phoronid P. embryolabi. system), radial nerves of the preoral lobe are absent; the minor The current report cannot be easily compared with the nerve ring connects with the marginal nerve of the preoral previous reports because the latter lack detailed information lobe; tentacles are innervated mostly from the minor nerve about tubulin-like immunoreactive nerve elements. ring and have frontal and later-frontal nerves (Santagata & In all phoronid larvae studied to date, the first 5ht-lir Zimmer, 2002) (Figure 8b). As shown in a previous paper perikarya appear at the apical pole of the embryo. This pole (Temereva & Tsitrin, 2014b), the organization of the nervous then becomes the apical organ of the larva (Hay-Schmidt, system in competent larvae of P. harmeri differs from both of 1990a; Temereva & Wanninger, 2012). During development, these types (Table 1 and Figure 8c). the perikarya increase in number, and early larvae without The nervous system of competent larvae of P. embryolabi tentacles have about 10–12 5ht-lir perikarya (Hay-Schmidt, (Figure 8d) differs from both types described by Santagata 1990a; Temereva & Wanninger, 2012). Some of these and Zimmer (2002) as well as from the type detected in larvae perikarya then submerge into the epithelium and give rise to of P. harmeri (Temereva & Tsitrin, 2014b) and P. muelleri the multipolar non-sensory perikarya (Sonnleitner, Schwaha, (Hay-Schmidt, 1990b; Sonnleitner et al., 2014). The nervous & Wanninger, 2014; Temereva & Wanninger, 2012). system of larvae of P. embryolabi has a lot in common with The first 5ht-lir neurites extend along the lateral nerves of that of larvae of P. vancouverensis Hay-Schmidt, 1990a; the preoral lobe and the minor nerve ring in P. ijimai, along Lacalli, 1991). First of all, competent larvae of P. embryolabi the main nerve ring in P. harmeri, and along the lateral nerves lack the minor nerve ring—the circle nerve tract above of the preoral lobe in P. embryolabi. Thus, P. ijimai and tentacles, but have nerve tract under the postoral ciliated band P. embryolabi are partly similar in location of the first 5ht-lir (Table 1 and Figure 8). neurites. Moreover, in the early gastrula of both P. ijimai and Innervation of tentacles in phoronid larvae is controver- P. embryolabi, the group of 5ht-lir perikarya appears along sial and has been discussed in several papers (Hay-Schmidt, the base of the esophagus. The immunoreactivity to these 1989, 1990b; Santagata & Zimmer, 2002; Sonnleitner et al., cells is no longer detectable in late stages in both species. 2014; Temereva & Wanninger, 2012). In phoronid larvae, These similarities in the development of the nervous system each tentacle has several ciliated zones that are innervated by reflect similarities in the morphology of larvae of both neurite bundles arising from different main nerves (Table 1). species. In contrast, the morphology of P. harmeri larvae The difference in innervation of tentacles in phoronid larvae differs greatly from the morphology of P. ijimai and reflects the different fates of larval tentacles in metamorpho- P. embryolabi larvae. The early appearance of the main sis: in some phoronids, larval tentacles are directly remodeled nerve in the development of P. harmeri may indicate that it into the juvenile tentacles; in other phoronids, juvenile has an important role in pelagic life of larvae. This early tentacles arise from basal thickenings underneath and appearance of the main nerve may also correlate with the attached to the larval tentacles; and in yet other phoronids, presence of numerous tentacles, whose abundance is larval tentacles are completely consumed by the juvenile, and characteristic of larvae of P. harmeri and other phoronid the definitive tentacles arise de novo (Santagata & Zimmer, species with large planktotrophic larvae. 2002; Temereva & Malakhov, 2015; Zimmer, 1964). 182 | TEMEREVA

TABLE 1 Some features of organization of the nervous system in larvae of phoronid species Type 1 Type 2 Phoronis Actinotrocha D, pallida, P. P. muelleri Phoronopsis Actinotrocha C, vancouverensis, P. (Hay- harmeri Phoronis hippocrepia P. vancouverensis Schmidt, (Temereva & architecta (Santagata, 2002; (Hay-Schmidt, 1990b; Tsitrin, 2014b; Species (Santagata & Santagata & Zimmer, 1990a; Lacalli, Sonnleitner Temereva & P. embryolabi Features Zimmer, 2002) 2002) 1991) et al., 2014) Wanninger, 2012) (herein) I. Circle nerve tract above the tentacles Presence + + −−+ − Originates from Lateral Marginal nerve of the −−Marginal nerve of − serotonergic preoral lobe the preoral lobe processes Presence of + −−−−− serotonin- like neurites II. Nerve tract −− ++++ under the postoral ciliated band III. Innervation of tentacles Frontal neurite − + + + + + bundle Originates from the Originates from Originates Originates from Originates circle nerve tract the marginal from the the circle nerve from the above the tentacles nerve marginal tract above the anterior nerve tentacles marginal nerve Latero-frontal − + + + + + neurite Originate from the Independent nerve Originate Independent nerve Originate from bundles circle nerve tract tract extending from the tract extending the posterior above the tentacles under the marginal under the marginal postoral ciliated nerve postoral ciliated nerve band band Abfrontal + + − + −− neurite Originates from Originates from the Originates bundle the main nerve main nerve ring from the ring marginal nerve Latero- + + − + + + abfrontal Originate from the Originate from the Originate Originate from the Originate from neurite minor nerve circle nerve tract from the main nerve ring the main bundles ring above the tentacles main nerve ring nerve ring VI. Addition nerve centers Frontal organ + + + + + + Outwardly Outwardly invisible Outwardly Outwardly Outwardly Outwardly prominent invisible prominent invisible invisible Median sensory −− − ++ + field Dorsolateral −− − −−+ sensory fields V. Organization of the apical organ Number of 44 2 33–42 types of perikarya (Continues) TEMEREVA | 183

TABLE 1 (Continued) Type 1 Type 2 Phoronis Actinotrocha D, pallida, P. P. muelleri Phoronopsis Actinotrocha C, vancouverensis, P. (Hay- harmeri Phoronis hippocrepia P. vancouverensis Schmidt, (Temereva & architecta (Santagata, 2002; (Hay-Schmidt, 1990b; Tsitrin, 2014b; Species (Santagata & Santagata & Zimmer, 1990a; Lacalli, Sonnleitner Temereva & P. embryolabi Features Zimmer, 2002) 2002) 1991) et al., 2014) Wanninger, 2012) (herein) Presence of ++ − ++ − basal multipolar perikarya Presence of −− − −−+ second row of sensory flask-shaped perikarya VI. Presence of + − ++− + the lateral nerves between the apical organ and the marginal nerve VII. Innervation ? ? ? ? From the main From the of the larval nerve ring apical organ trunk

“+” indicates the presence of the feature; “−” indicates its absence; “?” indicates that information is lacking.

The structure of the apical organ in phoronid larvae of the marginal nerves. The second additional nerve center is several species has been studied by TEM and immunocyto- associated with specialized cells that contain bacteria. The chemistry (Hay-Schmidt, 1989; Lacalli, 1991; Sonnleitner function of these bacteria is unclear. et al., 2014; Temereva, 2012; Temereva & Tsitrin, 2014b; Some bacteria that were isolated from sediments from Temereva & Wanninger, 2012). According to TEM, the the adult habitat induced metamorphosis of larvae of P. apical organ of most phoronid larvae consists of four types of muelleri (Herrmann, 1976, 1995), that is, these bacteria perikarya (Hay-Schmidt, 1989; Lacalli, 1991; Santagata, trigger metamorphosis. The same role for bacteria from the 2002; Temereva & Tsitrin, 2014b). The organization of the substratum has been described for some other invertebrates apical organ in P. embryolabi larvae is unique among (Hadfield, 2011; Huggett, Williamson, de Nys, Kjelleberg, phoronid larvae because of three features. First, the apical & Steinberg, 2006; Tran & Hadfield, 2011; Unabia & organ of Hadfield, 1999). There is, however, a great difference P. embryolabi includes only two types of perikarya. Second, between the bacteria in the substratum and those in the multipolar perikarya, which are typically located under the specialized cells in larvae of P. embryolabi and other sensory perikarya, are absent. Third, flask-like shaped organisms. In some bryozoan larvae, bacteria occur in sensory perikarya are arranged in two rows: anterior and epidermal cells (Woollacott, 1981). These bacteria are posterior. transferred from the mother to the larvae, and they protect An additional sensory organ—the frontal organ (hood the embryo during its pelagic life (Sharp, Davidson, & sense organ)—is located at the midline of the preoral lobe Haygood, 2007). In adult phoronids, specialized cells that and can be outwardly prominent or invisible in larvae of contain bacteria are found in the epithelium of the different phoronid species (Santagata & Zimmer, 2002; lophophoral organs (Temereva & Malakhov, 2006). Temereva, 2009; Temereva & Tsitrin, 2014b). According to These bacteria may produce specific substances required many suppositions, competent phoronid larvae use the by their host. frontal organ to select the substratum for settlement (Herrmann, 1976, 1979, 1995; Santagata, 2004; Temereva 4.3 | Ground plan of the larval nervous system & Tsitrin, 2014b). in phoronids Larvae of P. embryolabi have several additional nerve centers. The first additional center is the frontal organ, which The apical organ in most phoronid larvae has two groups of is outwardly invisible. The second center is located at the multipolar perikarya, which give rise to the main nerve ring midline of the edge of the preoral lobe and is associated with (Temereva & Tsitrin, 2014b). The absence of these groups 184 | TEMEREVA

FIGURE 8 Schemes of nervous system organization in competent larvae of phoronids. The apical is at the top; the ventral side is to the left in all photographs. (a) The type I nervous system has been described in larvae of Phoronopsis harmeri, Actinotrocha D, and Actinotrocha C. The scheme is based on Santagata and Zimmer (2002). (b) The type II nervous system has been described in larvae of Phoronis pallida, P. vancouverensis, and P. hippocrepia. The scheme is based on Lacalli (1991), Santagata (2002), and Santagata & Zimmer (2002). (c) Organization of the nervous system in competent larvae of Phoronopsis harmeri according to Temereva and Tsitrin (2014b) and Zimmer (1964). (d) Organization of the nervous system in competent larvae of Phoronis embryolabi according to the current study. ao, apical organ; fo, frontal organ; la, latero-abfrontal nerve of tentacle; lf, latero-frontal nerve of tentacle; ln, lateral nerves of preoral lobe; lsf, lateral sensory field; ma, marginal nerve(s) of the preoral lobe; mf, medio-frontal nerve of tentacle; mn, median nerve of preoral lobe; mnr, minor nerve ring; msf, median sensory field; tn, tentacular main nerve ring; vlb, ventro-lateral branches of perikarya makes the main nerve thinner in larvae of system in larvae of P. ovalis (Grobe, 2007), larvae of P. embryolabi than in most other phoronid larvae studied to phoronids with lecithotrophic development also lack the main date. Larvae of P. vancouverensis, which also lack a basal nerve (Figure 9c). In all phoronid larvae, the main nerve does layer of perikarya in the apical organ (Lacalli, 1991), lack a not contribute to the innervation of the postoral ciliated band. main nerve (Hay-Schmidt, 1990a). Thus, the reduction of the Because all adult phoronids have the tentacular nerve ring main nerve ring is evident in the following line: large (Fernández et al., 1996; Silén, 1954b; Temereva, 2015; planktotrophic larvae—larvae of viviparous phoronids— Temereva & Malakhov, 2009), whose origin in the larval larvae of brooding species (Figures 9a and 9b). According to main nerve has been detected in some phoronids (Santagata, the little that is known about the organization of the nervous 2002; Temereva & Tsitrin, 2014a) and whose location TEMEREVA | 185

FIGURE 9 Schemes of organization of the nervous system in larvae of phoronids with different types of development. Structures on the sideline are shown by dashed line. (a) The organization of the larval nervous system in large planktotrophic phoronid larvae (based on Temereva & Tsitrin, 2014b). (b) The organization of the larval nervous system in some brooding species of phoronids (based on Hay-Schmidt, 1990a; Lacalli, 1991; Santagata & Zimmer, 2002). (c) The organization of the nervous system in lecithotrophic larvae of Phoronis ovalis (the rough scheme is based on the 3D reconstruction from Grobe, 2007). (d) The hypothetical ground plan of the nervous system in phoronid larvae. (e) Typical trilobed competent lava of (Rhynchonelliformea) (based on Santagata, 2011). (f) Planktotrophic larva (based on Hay-Schmidt, 1992; Santagata, 2011). (g) Organization of the nervous system in early trochophore (based on Nezlin, 2010; Voronezhskaya, 2016). (h) Organization of the nervous system in early dipleurula (based on Nielsen & Hay-Schmidt, 2007; Nezlin & Yushin, 2004). ao, apical organ; con, circumoral nerve; dn, dorsal nerve; inr, inner nerve ring; ln, lateral nerves; mtn, nerve of the metatroch; onr, outer nerve ring; pen, nerve underlies the preoral ciliated band; pon, nerve underlies the postoral ciliated band; ptn, nerve of the prototroch; tn, tentacular main nerve ring; vg, ventral ganglion; vln, ventral lophophoral nerve corresponds to that of the larval main nerve, the presence of Thus, the ground plan of the nervous system in larvae of the main nerve in larvae should be regarded as the juvenile phoronids includes the apical organ, single nerve tract characteristic (Figure 9a). That competent phoronid larvae underlining both the preoral and the postoral ciliated bands, have juvenile characteristics has been previously discussed and the lateral nerves passing from the apical organ to the (Temereva & Malakhov, 2000, 2015). nerve tract of the preoral ciliated band (Figure 9d). 186 | TEMEREVA 4.4 | Larval nervous system in Brachiozoa

According to recent results, phoronids and brachiopods form a united clade called the Brachiozoa (Cohen, 2013; Santagata & Cohen, 2009). Like phoronids, brachiopods exhibit several types of development: planktotrophy, brooding, lecytotrophy, and viviparity (Williams, James, Emig, Mackay, & Rhodes, 1997). The organization of the nervous system differs in lectotrophic and planktotrophic brachiopod larvae. The first difference concerns organization of the apical organ: lecititrophic larvae have a few 5ht-lir flask-shaped perikarya (Altenburger, Martinez, & Wanninger, 2011; Altenburger & Wanninger, 2010) (Figure 9e), whereas planktotrophic larvae FIGURE 10 Types of development within the Brachiozoa. The have a multicellular apical organ (Hay-Schmidt, 1992; phylogenetic tree is based on the most recent analysis of brachiozoan Santagata, 2011) (Figure 9f). In addition to the apical organ, phylogeny (Cohen, 2013). Gray indicates lecithotrophy and black planktotrophic larvae have large ventral ganglion (Santagata, indicates planktotrophy. The bilobed stage has been described in all 2011). The second difference concerns the presence and groups of the Brachiozoa and can be regarded as having the ground plan of larvae in this clade. Planktotrophic larvae with tentacles occur location of the main nerves, which emanate from the apical in the Linguliformea and in most phoronids. The typical competent organ. In planktotrophic larvae, both ventral and dorsal stage of the Rhynchonelliformea is trilobed lophophoral nerves project from the neuropil of the apical ganglion (Figure 9f). The ventral lophophoral nerves make both inner and outer nerve rings extending above the Among the Brachiozoa, lecithotrophy occurs in three tentacles. The dorsal lophophoral nerves extend under the groups (the Rhinchonelliformea, Phoronida, and Craniifor- tentacles and make connections between the ventral ganglion mea), and planktorophy occurs in two groups (the Phoronida and the apical ganglion (Figure 9f). Lecititrophic brachiopod and Linguliformea) (Figure 10). According to this analysis, larvae have (i) anterior nerve ring that underlies the transverse the bilobed larval stage occurs in all groups of the Brachiozoa ciliated band of the apical lobe, (ii) medial, and (iii) lateral (Figure 10). The bilobed stage is the first larval stage of many nerve tracts branch out from either side of the apical neuropil brachiopods (Kuzmina, Temereva, & Malakhov, 2016; (Figure 9e). Williams et al., 1997) and phoronids (Emig, 1977; Temereva The difference in the organization of the nervous system & Malakhov, 2007; Temereva & Tsitrin, 2013; Zimmer, of the two kinds of brachiopod larvae reflects differences in 1964). The primary larva of the Brachiozoa seems to be a their morphology: lecithotrophic larvae have a prominent bilobed larva with two continuous ciliated bands, which are apical lobe and lack tentacles, whereas planktotrophic larvae underlined by a united nerve tract that extends from the apical lack an apical lobe but have tentacles. The inner and outer organ. It is difficult to determine whether this primary larva circumoral nerves of planktotrophic brachiopod larvae and was planktotrophic or lecithotrophic. the anterior nerve ring in lecithotrophic larvae seem to be the lower and upper portions, respectively, of a united nerve tract, which underlines the ciliated bands of tentacles and the apical 4.5 | New results in the light of phoronid lobe. Because in larvae of both types different portion of phylogeny larvae are absent (apical lobe or tentacles), the united nerve tract is partly represented. In recent molecular phylogeny, the phoronids are regarded as If we assume the homology of tentacles in planktotrophic protostomian animals, which are close relatives of annelids larvae of both phoronids and brachiopods, the main nerve ring and mollusks (Kocot et al., 2017). These data contradict the of phoronid larvae seems to be homologous with the dorsal traditional view, which is based on development and nerves of brachiopod larvae. The presence of the ventral anatomy, that phoronids are deuterostomian animals with a ganglion and dorsal nerves in planktotrophic brachiopod radial type of egg cleavage, a multicellular (in some cases larvae should be regarded as the juvenile characteristic of the entercoelic) formation of the coelomic mesoderm, and three larval nervous system. This is consistent with the view that coelomic compartments. This contradiction has been some- Glottidia larvae, as well as planktotrophic larvae of all what resolved by novel data on phoronid development and lingulid brachiopods, are juveniles (Long & Stricker, 1991). It anatomy. Thus, some protostomian features were discovered follows that large, advanced planktotrophic larvae of both in phoronid egg cleavage (Pennerstorfer & Scholtz, 2012), in phoronids and brachiopods should be regarded as juveniles the organization of the coelom in phoronid larvae (Bartolo- with some juvenile features of the nervous system. maeus, 2001) and adults (Gruhl, Grobe, & Bartolomaeus, TEMEREVA | 187

2005), and in the anatomy of the phoronid neuro-muscular ACKNOWLEDGMENTS system (Santagata & Zimmer, 2002). At the same time, most Author is thankful to anonymous reviewer, whose suggestion of these phoronid features depend on the type of development about Brachiozoa phylogeny is used in this paper. I am also (Temereva & Malakhov, 2012) and cannot be regarded as extremely grateful to Dr. Wallace Arthur, whose help typical protostomian characteristics of phoronids. Further- provided the great improvement of the manuscript, especially more within Brachiopoda, both protostomic and deuteros- the abstract of the paper. The Russian Foundation for Basic tomic developmental modes are present in relatively Research provided support for the collection of material (#15- closely related genera of brachiopods (e.g., Martín-Durán, 29-02601) and for the TEM studies (#17-04-00586). The Passamaneck, Martindale, & Hejnol, 2016) in stark contrast Russian Science Foundation provided support for the to the traditional division of phylogeny based on this cytochemical investigations and for the 3D reconstructions feature of early development, highlighting the probable (#14-50-00029). The research was performed at the User homoplastic nature of development within this group of Facilities Center of M.V. Lomonosov Moscow State organisms and thus consequently caution against the use University with financial support from the Ministry of of this character as phylogenetically informative at the level Education and Science of the Russian Federation. of resolving deep animal phylogenetic relationships as also pointed out to be the case by the other reviewer with regard to neural development. ORCID Comparative analysis of the organization of the larval nervous system should include data on the earliest stages Elena N. Temereva http://orcid.org/0000-0001-7791- (Voronezhskaya, 2016) but should not include data on 0553 competent larvae, as was erroneously done in our previous paper (Temereva & Tsitrin, 2014b). Because of the great variations, it is difficult to suggest a ground plan of the REFERENCES larval nervous system in all protostomians and even in annelids. According to a review of annelid development Altenburger, A., Martinez, P., & Wanninger, A. (2011). Homeobox gene expression in Brachiopoda: The role of Not and Cdx in bodyplan (Nezlin, 2010; Voronezhskaya, 2016), the ground plan of patterning, neurogenesis, and germ layer specification. Gene the larval nervous system in annelids includes the apical Expression Patterns, 11, 427–436. organ, the nerve tract under the prototroch, lateral nerves Altenburger, A., & Wanninger, A. (2010). Neuromuscular development between the apical organ and the prototroch nerve tract, and in anomala: Evidence for the presence of serotonin and a the circumoral ring extending from the apical organ spiralian-like apical organ in lecithotrophic brachiopod larvae. (Figure 9g). The organization of the nervous system differs Evolution and Development, 12,16–24. among ciliated deuterostomian larvae (Beer, Moss, & Arendt, D., Denes, A. S., Jekely, G., & Tessmar-Raible, K. (2008). Thorndyke, 2001; Byrne, Nakajima, Chee, & Burke, The evolution of nervous system centralization. Philosophical Transactions of the Royal Society B: Biological Sciences, 363, 2007; Nezlin & Yushin, 2004; Nielsen & Hay-Schmidt, 1523–1528. 2007) but generally includes the apical organ and a Bartolomaeus, T. (2001). Ultrastructure and formation of the body cavity continuous nerve tract, which underlines the preoral and lining in Phoronis muelleri (Phoronida, ). Zoomor- postoral ciliated bands (Figure 9h). phology, 120, 135–148. The organization of the nervous systems is similar in early Beer, A.-J., Moss, C., & Thorndyke, M. (2001). Development of phoronid larvae, annelids, and some deuterostomians (Figures serotonin-like and SALMFamide-like immunoreactivity in the 9d, 9g, and 9h). Thus, the presence of the prominent nerve, nervous system of the sea urchin . Biological – which underlies the preoral ciliated band and is connected to Bulletin, 200, 268 280. Bullock, H., & Horridge, G. A. (1965). Structure and function in the the apical organ by lateral nerves, makes the nervous system of nervous system of invertebrates (Vol. 1). San Francisco; London: phoronid larvae similar to that of annelid larvae (Figures 9d and W.H. Freeman, 9g). On the other hand, the presence of the continuous nerve Byrne, M., Nakajima, Y., Chee, F. C., & Burke, R. D. (2007). Apical that underlies both preoral and postoral ciliated bands makes organs in echinoderm larvae: Insights into larval evolution in the the nervous system of phoronid larvae similar to that of Ambulacraria. Evolution and Development, 9, 432–445. deuterostomian larvae (Figures 9d and 9h). The combination Cohen, B. L. (2013). Rerooting the rDNA gene tree reveals phoronidsto ‘ ’ protostome- and deuterostome-like features was mentioned be brachiopods without shells ; dangers ofwide taxon samples in metazoan phylogenetics (Phoronida; Brachiopoda). Zoological before in phoronids and allowed researchers to suggest that Journal of the Linnean Society, 167,82–92. phoronids (and some other bilaterians) have maintained some Emig, C. C. (1977). Embryology of Phoronida. American Zoologist, 17, ancestral features that were inherited from the last common 21–37. bilaterian ancestor (Peterson & Eernisse, 2001; Temereva & Emig, C. C. (1982). The biology of Phoronida. Advances in Marine Malakhov, 2012). Biology, 19,2–90. 188 | TEMEREVA

Fernández, I., Pardos, F., Benito, J., & Roldan, C. (1996). Ultrastructural Mamkaev, Y. V. 1962. About Phoronids of Far Eastern seas. Researches observation on the phoronid nervous system. Journal of Morphol- of Far Eastern Seas USSR, 8, 219–237 (in Russian). ogy, 230, 265–281. Martín-Durán, J. M., Passamaneck, Y. J., Martindale, M. Q., & Hejnol, Grobe, P. (2007). Larval development, the origin of the coelom and the A. (2016). The developmental basis for the recurrent evolution of phylogenetic relationships of the Phoronida (Doctoral dissertation). 1–91. deuterostomy and protostomy. Nature Ecology & Evolution, 1:1–10. Gruhl, A., Grobe, P., & Bartolomaeus, T. (2005). Fine structure of the Nezlin, L. P. (2010). The golden age of comparative morphology: laser epistome in Phoronis ovalis: Significance for the coelomic scanning microscopy and neurogenesis in trochophore animals. organization in Phoronida. Invertebrate Biology, 124, 332–343. Russian Journal of Developmental Biology, 41(6), 381–390. Hadfield, M. G. (2011). Biofilms and marine invertebrate larvae: What Nezlin, L. P., & Yushin, V. V. (2004). Structure of the nervous system in bacteria produce that larvae use to choose settlement sites. Annual the tornaria larva of Balanoglossus proterogonius (Hemichordata: Review of Marine Science, 3, 453–470. Enteropneusta) and its phylogenetic implications. Zoomorphology, Halanych, K. M., Bacheller, J. D., Aguinaldo, A. M., Liva, S. M., Hillis, 123,1–13. D. M., & Lake, J. A. (1995). Evidence from 18S ribosomal DNA that Nielsen, C. (2013). Life cycle evolution: Was the eumetazoan ancestor a the lophophorates are protostome animals. Science, 267, 1641–1643. holopelagic, planktotrophic gastraea? BMC Evolutionary Biology, Hay-Schmidt, A. (1989). The nervous system of the actinotroch larva of 13, 171. Phoronis muelleri (Phoronida). Zoomorphology, 108, 333–351. Nielsen, C., & Hay-Schmidt, A. (2007). Development of the Enter- Hay-Schmidt, A. (1990a). Catecholamine-containing, serotonin-lake opneust Ptychodera flava: Ciliary bands and nervous system. and FMRFamide-like immunoreactive neurons and processes in the Journal of Morphology, 268, 551–570. nervous system of the early actinotroch larva of Phoronis Nomaksteinsky, M., Roettinger, E., Dufour, H. D., Chettouh, Z., Lowe, vancouverensis (Phoronida): Distribution and development. Cana- C. J., Martindale, M. Q., & Brunet, J.-F. (2009). Centralization of the dian Journal of Zoology, 68(7), 1525–1536. deuterostome nervous system predates chordates. Current Biology, Hay-Schmidt, A. (1990b). Distribution of catecholamine containing, 19, 1264–1269. serotonin-like and neuropeptide FMRFamide-like immunoreactive Pennerstorfer, M., & Scholtz, G. (2012). Early cleavage in Phoronis neurons and processes in the nervous system of the actinotroch larva muelleri (Phoronida) displays spiral features. Evolution and of Phoronis muelleri (Phoronida). Cell and Tissue Research, 259, Development, 14(6), 484–500. 105–118. Peterson, K. J., & Eernisse, D. J. (2001). Animal phylogeny and the Hay-Schmidt, A. (1992). Ultrastructure and immunocytochemistry of ancestry of bilaterians: Inferences from morphology and 18S rDNA the nervous system of the larvae of Lingula anatina and Glottidia sp. gene sequences. Evolution and Development, 3, 170–205. (Brachiopoda). Zoomorphology, 112, 189–205. Ruppert, E. E., Fox, R. S., & Barnes, R. D. (2003). Invertebrate zoology: Hay-Schmidt, A. (2000). The evolution of the serotonergic nervous A functional evolutionary approach (7th ed). Belmont, CA: Brooks/ system. Philosophical Transactions of the Royal Society B: Cole. Biological Sciences, 267, 1071–1079. Santagata, S. (2002). Structure and metamorphic remodeling of the Herrmann, K. (1976). Untersuchungen über Morphologie, Physiologie, larval nervous system and musculature of Phoronis pallida und Ökologie der Metamorphose von Phoronis muelleri (Phoronida). (Phoronida). Evolution and Development, 4,28–42. Zoologische Jahrbücher Anatomie, 95, 354–426. Santagata, S. (2004). A waterborne behavioral cue for the actinotroch Herrmann, K. (1979). Larval development and metamorphosis of larva of Phoronis pallida (Phoronida) produced by Upogebia Phoronis psammophila (Phoronida, Tentaculata). Helgol Wiss pugettensis (Decapoda: Thalassinidea). Biological Bulletin, 207, Meeresunters, 32, 550–581. 103–115. Herrmann, K. (1995). Induction and regulation of metamorphosis in Santagata, S. (2011). Evaluating neurophylogenetic patterns in the larval planktonic larvae—Phoronis muelleri (Tentaculata) as archetype. nervous systems of brachiopods and their evolutionary significance Helgol Wiss Meeresunters, 49, 255–281. to other bilaterian phyla. Journal of Morphology, 272, 1153–1169. Huggett, M. J., Williamson, J. E., de Nys, R., Kjelleberg, S., & Steinberg, Santagata, S., & Cohen, B. (2009). Phoronid phylogenetics (Brachio- P. D. (2006). Larval settlement of the common Australian sea urchin poda; Phoronata): Evidence from morphological cladistics, small Heliocidaris erythrogramma in response to bacteria from the surface and large subunit rDNA sequences, and mitochondrial cox1. of coralline algae. Oecologia, 149(4), 604–619. Zoological Journal of the Linnean Society, 157,34–50. Kocot, K. M., Struck, T. H., Merkel, L., Waits, D. S., Todt, C., Brannock, Santagata, S., & Zimmer, R. L. (2002). Comparison of the neuromuscu- P. M., ...Halanych, K. M. (2017). Phylogenomics of lophotrocho- lar system among actinotroch larvae: Systematic and evolutionary zoa with consideration of systematic error. Systematic Biology, implication. Evolution and Development, 4,43–54. 66(2), 256–282. https://doi.org/10.1093/sysbio/syw079 Schmidt-Rhaesa, A. (2007). The evolution of organ systems. Oxford: Kuzmina, T. V., Temereva, E. N., & Malakhov, V. V. (2016). Larval Oxford University Press. development of brachiopod Coptothyris grayi (Davidson, 1852) Schneider, C. A., Rasband, W. S., & Eliceiri, K. W. (2012). NIH Image (Brachiopoda, Rhynchonelliformea). Doklady Biological Sciences, to ImageJ: 25 years of image analysis. Nature Methods, 9(7), 471, 258–260. 671–675. Lacalli, T. C. (1991). Structure and organization of the nervous system in Sharp, K. H., Davidson, S. K., & Haygood, M. G. (2007). Localization of the actinotroch larva of Phoronis vancouverensis. Philosophical ‘Candidatus Endobugula sertula’ and the bryostatins throughout the Transactions of the Royal Society of London, 327, 655–685. life cycle of the bryozoan Bugula neritina. ISME Journal, 1, Long, J. A., & Stricker, S. A. (1991). Brachiopoda. In A. J. Giese, J. S. 693–702. Pearse, & V. B. Pearse (Eds.), Reproduction of marine invertebrates Silén, L. (1954a). Developmental biology of Phoronidea of the Gullmar (Vol. 6, pp. 47–84). California: Blackwell Scientific. Fiord area (West coast of Sweden). Acta Zoologica, 35, 215–257. TEMEREVA | 189

Silén, L. (1954b). On the nervous system of Phoronis. Ark Zool Nye Ser, plan and life cycle. BMC Evolutionary Biology, 15,1–28. https://doi. 6,1–40. org/10.1186/s12862-015-0504-0 Sly, B. J., Snoke, M. S., & Raff, R. A. (2003). Who came first—Larvae or Temereva, E. N., & Neretina, T. V. (2013). A distinct phoronid larva: adults? Origins of metazoan bilaterian larvae. International Journal Morphological and molecular evidence. Invertebrate Systematics, of Developmental Biology, 47, 623–632. 27(6), 622–633. Sonnleitner, B., Schwaha, T., & Wanninger, A. (2014). Inter- and Temereva, E. N., & Tsitrin, E. B. (2013). Development, organization, intraspecific plasticity in distribution patterns of immunoreactive and remodeling of phoronid muscles from embryo to metamorphosis compounds in actinotroch larvae of Phoronida (Lophotrochozoa). (Lophotrochozoa: Phoronida). BMC Developmental Biology, 13, 14. Journal of Zoological Systematics and Evolutionary, 52(1), 1–14. https://doi.org/10.1186/1471-213X-13-14 Temereva, E. (2015). Phoronida. In A. Schmidt-Rhaesa, S. Harzsch, & Temereva, E. N., & Tsitrin, E. B. (2014a). Organization and G. Purschke (Eds.), Structure and evolution of invertebrate nervous metamorphic remodeling of the nervous system in juveniles of systems (pp. 351–359). London, UK: Oxford University Press. Phoronopsis harmeri (Phoronida): Insights into evolution of the Temereva, E., & Wanninger, A. (2012). Development of the nervous bilaterian nervous system. Frontiers in Zoology, 11,1–25. system in Phoronopsis harmeri (Lophotrochozoa, Phoronida) Temereva, E. N., & Tsitrin, E. B. (2014b). Development and organization reveals both deuterostome- and trochozoan-like features. BMC of the larval nervous system in Phoronopsis harmeri: New insights Evolutionary Biology, 12, 121. into phoronid phylogeny. Frontiers in Zoology, 11(1), 3, https://doi. Temereva, E. N. (2009). New data on distribution, morphology and org/10.1186/1742-9994-11-3 of phoronid larvae (Phoronida, Lophophorata). Inverte- Tran, C., & Hadfield, M. G. (2011). Larvae of Pocillopora damicornis brate Zoology, 6(1), 47–64. (Anthozoa) settle and metamorphose in response to surface-biofilm Temereva, E. N. (2012). Ventral nerve cord in Phoronopsis harmeri bacteria. Marine Ecology Progress Series, 433,85–96. larvae. Journal of Experimental Zoology. Part B, Molecular and Unabia, C. R. C., & Hadfield, M. G. (1999). Role of bacteria in larval Developmental Evolution, 318B,26–34. settlement and metamorphosis of the Hydroides elegans. Temereva, E. N., & Chichvarkhin, A. (2017). A new phoronid species, Marine Biology, 133,55–64. Phoronis embryolabi, with a novel type of development, and Voronezhskaya, E. E. (2016). Early events in neurogenesis of mollusks consideration of phoronid taxonomy and DNA barcoding. Inverte- and annelids: What is the important for comparative analysis and brate Systematics, 31,65–84. phylogeny. Materialy shkoly dlya molodyh specialistov. Sain- Temereva, E. N., & Malakhov, V. V. (2000). The circulatory system of Petersburg, Zoological Museum,13–15. (in Russian). phoronid larvae. Doklady Biological Sciences, 375(5), 712–714. Wanninger, A. (2008). Comparative lophotrochozoan neurogenesis and Temereva, E. N., & Malakhov, V. V. (2004). Key to the phoronid larvae larval neuroanatomy: Recent advances from previously neglected (Phoronida, Lophophorata) from the Sea of Japan. Zoologicheskii taxa. Acta Biologica Hungarica, 59(Suppl), 127–136. – Zhurnal, 83(9), 1115 1126. Wanninger, A. (2009). Shaping the things to come: Ontogeny of Temereva, E. N., & Malakhov, V. V. (2006). Microscopical anatomy and lophotrochozoan neuromuscular systems and the Tetraneuralia ultrastructure of the lophophoral organs and adjacent epitheliums of concept. Biological Bulletin, 216, 293–306. the lophophoral concavity and anal papilla of Phoronopsis harmeri Williams, A., James, M. A., Emig, C. C., Mackay, S., & Rhodes, M. C. Pixell, 1912 (Lophophorata, Phoronida). Russian Journal of Marine (1997). Brachiopod anatomy. In R. L. Kaesler (Ed.), Treatise on – Biology, 32(3), 134 141. invertebrate paleontology. Part H, Brachiopoda V 1 Introduction Temereva, E. N., & Malakhov, V. V. (2007). Embryogenesis and larval (pp. 7–189). Boulder, Colorado and Lawrence, Kansas: The development of Phoronopsis harmeri Pixell, 1912 (Phoronida): Dual Geological Society of America Inc and the University of Kansas. origin of the coelomic mesoderm. Invertebrate Reproduction and Woollacott, R. M. (1981). Association of bacteria with bryozoan larvae. – Development, 50,57 66. Marine Biology, 65, 155–158. Temereva, E. N., & Malakhov, V. V. (2009). Microscopic anatomy and Zimmer, R. L. (1964). Reproductive biology and development of ultrastructure of the nervous system of Phoronopsis harmeri Pixell, Phoronida. Ann Arbor: University Microfilm. 1912 (Lophophorata: Phoronida). Russian Journal of Marine Biology, 35(5), 388–404. Temereva, E. N., & Malakhov, V. V. (2012). Embryogenesis in phoronids. Invertebrate Zoology, 9(1), 1–39. How to cite this article: Temereva EN. Ground plan Temereva, E. N., & Malakhov, V. V. (2016). Viviparity of larvae, a new of the larval nervous system in phoronids: Evidence type of development in phoronids (Lophophorata: Phoronida). from larvae of viviparous phoronid. Evolution & Doklady Biological Sciences, 467(3), 362–364. Development. 2017;19:171–189. Temereva, E. N., & Malakhov, V. V. (2015). Metamorphic remodeling of morphology and the body cavity in Phoronopsis harmeri https://doi.org/10.1111/ede.12231 (Lophotrochozoa, Phoronida): The evolution of the phoronid body