Invertebrate Reproduction and Development, 50:2 (2007) 57–66 57 Balaban, Philadelphia/Rehovot 0168-8170/07/$05.00 © 2007 Balaban

Embryogenesis and larval development of Phoronopsis harmeri Pixell, 1912 (Phoronida): dual origin of the coelomic

ELENA N. TEMEREVA* and VLADIMIR V. MALAKHOV Biological Faculty, Invertebrate Zoology, Moscow State University, Moscow 119992, Russia Tel. and Fax: +8 (495) 939-4495; email: [email protected]

Received 12 December 2006; Accepted 26 March 2007

Summary are marine invertebrates which great significance from the perspective of comparative anatomy. Phoronids have traditionally been described as because they exhibit developmental and larval traits similar to those traits found in echinoderms and hemichordates. However, molecular phylogenetic evidence has consistently identified the phoronids and as a monophyletic group within the lophotrochozoan . The nature of egg cleavage, and the origin of coelomic mesoderm can help to resolve this contradiction. These questions were investigated with Phoronopsis harmeri. and actinotroch larvae were cultivated in the laboratory, and embryonic and larval development was followed with SEM, light and video microscopy. Egg cleavage is radial, although the furrows of the 4th and succeeding divisions are oblique, which allows for blastula formation by the 16-cell stage. The ciliated blastula hatches 10 h after the onset of cleavage and acquires the apical tuft. Gastrulation proceeds by invagination. Coelomic mesoderm derives from two, the anterior and posterior, precursors. The anterior precursor forms by cell migration from the anterior wall of the , the posterior one by enterocoelic outpouching of the midgut. The lining of preoral and tentacle coelom is derived from the anterior precursor, whilst the lining of trunk coelom originates from the posterior one. The intestine forms as an ectodermal invagination of the posterior part of larval body. Protonephridial canals form as ectodermal dorso-lateral furrows on both sides of the anus. A typical actinotroch is formed 100 h after the onset of cleavage. Larvae are described at the stages of 8, 12, 20 and 24 tentacles.

Key words: Phoronida, actinotroch, , larval development, coelomic mesoderm

Introduction Siewing, 1980; Temereva and Malakhov, 2006), and as Phoronids are marine invertebrates which have great deuterostomes because they exhibit developmental and significance from the perspective of comparative larval traits similar to those found in echinoderms and anatomy. Traditionally phoronids were considered as hemichordates (Nielsen, 2002). Phoronids, like all archicoelomate , because larvae and deuterostomes, possess the radial type of egg cleavage adults have a trimeric organization of the coelom and mesoderm formation by cell migration (Zimmer, (Masterman, 1898; Remane, 1949; Zimmer, 1978; 1964; Herrmann, 1986; Siewing, 1974; Emig, 1977;

*Corresponding author. 58 E.N. Temereva and V.V. Malakhov / IRD 50 (2007) 57–66

Freeman, 1991; Malakhov and Temereva, 1999; started at a density of two larvae per 3 to 4 ml of filtered Santagata, 2001, 2004) or by (Malakhov seawater. Larvae were fed mixtures of Rhodomonas lens and Temereva, 1999). and Chaetoceros calcitrans. Cultures were fed and However, molecular phylogenetic evidence has given a 75% water change and a vessel change every consistently identified the phoronids and brachiopods as 2 days. a monophyletic group within the lophotrochozoan Different developmental stages were observed and protostomes (Halanych et al., 1995; Erber et al., 1998; sketched with a light microscope. Competent larvae de Rosa et al., 1999; Cohen et al., 1998; Cohen, 2000; were prepared for scanning electron microscopy (SEM). Cohen, Weydmann, 2005). These molecular phylo- They were fixed in 4% formalin then dehydrated in an genetic data are supported by other investigations. For ethanol series of increasing concentration, dried at a example, according to Bartolomaeus (2001) phoronid critical point, mounted on stubs with double-stick tape, larvae have a single coelomic compartment instead of sputter-coated with platinum–palladium and examined three; the mesoderm forms on the border between with a JSM 63LA. and (Freeman and Martindale, 2002); structure of neuromuscular system is not typical Results for deuterostomes (Santagata and Zimmer, 2002). Thus, there is contradiction between the traditional Embryonic development view based on , morphology and compara- Fertilization of oocytes occurs in the female trunk tive anatomy and recent data provided by new methods coelom, so that spawned eggs are already fertilized. of molecular and experimental biology. The question of Most of them are at the stage of extrusion of the first the phylogenetic position of Phoronida thus remains polar body; however, some contain two polar bodies and open. even embryos with 2–4 blastomeres were found. The In this study our primary goal is to provide a clear polar bodies are superimposed transparent vesicles of description of egg cleavage, gastrulation and the origin 8–10 µm in cross-section, with the first polar body being of coelomic mesoderm during development of Phoro- somewhat larger (Fig. 1A). nopsis harmeri. Freshly spawned eggs are 90–100 µm in diameter and enclosed in a slightly wrinkled transparent envelope which is 7–9 µm thick (Fig. 1A). After 10–15 min the wrinkles smoothen. Material and Methods The fist division occurs in the meridional plane Specimens of P. harmeri were collected in Vostok (Fig. 1B) and is initiated 1 h after extrusion of the Bay of the Sea of Japan from sandy sediments at 3–4 m second polar body. It results in one blastomere being depth. Spawning and subsequent development of slightly larger than the other (Fig. 1B). The stage lasts fertilized eggs was observed in the laboratory in August until the blastomeres separate, distorting the elastic 1996 and September–November 1999–2002. To induce envelope. The smaller blastomere forms a cytoplasmic spawning, the following procedure was applied. protrusion towards the larger one after separation Several specimens were drawn from their tubes and (Fig. 1C). The process of division and blastomere dissected in the posterior region of the trunk to allow motion lasts for 15 min. sperm to be released into the water. Several ml of water The second division of the egg cleavage starts 20– with sperm was added to the medium containing intact 24 min after the completion of the first in most embryos. specimens, which triggered spawning. Cleavage furrows also form in the meridional plane Fertilized eggs were maintained in a 20 ml Petri dish (Fig. 1D). Upon completion of the second division the with a perforated floor sealed with a cover glass at a resulting 4 cells differ in size: the two descendants of the density of about 3–5 cells per ml. The dish was placed in larger blastomere are larger than the other two (Fig. 1D). a thermostatic chamber on an inverted microscope MBI- The third division starts 26–30 min after completion 13 at 15EС and recorded with 4 snapshots per min for of the previous stage. The grooves are oriented in the 10 h. The experiment was designed to track about 5 equatorial plane. The 8-blastomere pattern is typical for embryos on average at a time, and those developing radial cleavage with four vegetal blastomeres strictly normally to the stage of a mobile blastula were then juxtaposed with four blastomeres (Fig. 1E). analyzed. Later stages (gastrulae and young larvae) were The fourth division begins 32–36 min after com- filmed selectively under similar experimental pletion of the previous stage, i.e. 2 h after the onset of conditions. cleavage; the cleavage furrow forms in the meridional Developing embryos and larvae were kept in two plane. During this division blastomeres separate to form vessels with filtered sea water. Larval cultures were an internal cavity in the (Fig. 1F). The embryo E.N. Temereva and V.V. Malakhov / IRD 50 (2007) 57–66 59

diameter is now 150–170 µm. The cleavage furrows between sister blastomeres are oblique with respect to the animal-vegetal axis, and the regular radial cleavage pattern is thus distorted (Fig. 1F). The presence of the cavity distinguishes the 16-celled stage of the P. harmeri embryo as a blastomeric blastula. The fifth division starts 32–34 min after completion of the previous one (about 2 h 45 min after the onset of the cleavage). The grooves of the division are latitu- dinal, but blastomeres move apart forming a spacious blastocoel (Fig. 1G). Later in development the animal pole cells divide faster than those of the vegetal pole. As a result, the latter consists of larger cells, and the corresponding wall of the blastula is noticeably thicker than its animal pole counterpart (Fig. 1H). Six hours after the beginning of cleavage the embryo contains about 1,000 cells. At this stage, cells of the blastula become ciliated, although it starts swimming actively only 10 hrs after the onset of cleavage. The blastula is a spheroid with a diameter of 260– 270 µm. The animal pole wall is 30–40 µm thick, the vegetal pole wall is 100–110 µm (Fig. 1I); 12 h after the onset of cleavage, embryos form a tuft at the animal pole, the precursor of the apical tuft (Fig. 2A). Gastrulation begins 13–14 h after the onset of cleavage with flattening of the vegetal pole and forma- tion of the vegetal plateau (Fig. 1J, 2B). Some cells of the plate floor migrate into the blastocoel and later settle on its inner walls (Fig. 1J, 2B). Gastrulation occurs via invagination of the vegetal plate inside the embryo to form a shallow depression, the archenteron (Fig. 1K, 2C). Simultaneously the form of the embryo changes. The apical tuft shifts anteriorly, and the apical pole Fig. 1. Embryonic development of Phoronopsis harmeri. Photos from inverted microscope MBI-13. AV axis indicates flattens forming the neural plate (Fig. 1K, 2C). Thus, the animal and vegetal poles of embryo. A: Egg. Polar body is AV axis is bent (Fig. 1K, 2C). indicated by arrow. B: Two-cell stage 15 min after the onset The primary blastopore looks like a small round of cleavage. C: Formation of the smaller blastomere cyto- orifice in the center of the vegetal pole. After 25–28 h, it plasmic projection (arrow). D: Four-cell stage about 40 min elongates along the anterior-posterior axis (Fig. 1L). after the onset of cleavage. E: The 8-cells stage about 1 h The blastopore has a broader anterior, a narrow middle after the onset of cleavage. F: Fourth division 2 h after the and slightly wider and elongated posterior (Fig. 1L). onset of cleavage. G: Embryo about 2 h 45 min after the The blastopore lips gradually close in at its middle and onset of cleavage (64-cell stage). H: Embryo about 3 h 30 min after the onset of the cleavage (120-cell stage). later at the posterior region. The remnant of the I: Blastula. J: Formation of the vegetal plate. Primary blastopore anteriorly forms the mouth (Fig. 2E). mesenchyme cells are indicated by arrows. K: Gastrulation. At the elongated blastopore stage (about 34–36 h Neural plate is indicated by arrow. L: Stage of an inverted after the onset of cleavage), a horseshoe-shaped bulge skittle-shaped blastopore (indicated by arrows). Cells of forms at its anterior edge and protrudes into the blasto- anterior mesoderm precursor are indicated by arrowheads. coel (Fig. 1M, 2D). With time, the constituent cells M: Formation of the anterior mesoderm precursor (indicated become bulb-shaped and their wholescale migration into by arrows). N: Embryo with bubble-shaped front part which the blastocoel of the anterior part of the larva is initiated will become the larval preoral lobe. O: Young larva 110 h after onset of cleavage with preoral (indicated by two (Fig. 1L, 2E). They assemble a horseshoe-shaped body arrows) and postoral (indicated by arrowheads) ciliated enveloping the anterior portion of the archenteron bands and neural plate (indicated by arrow). Scale bars: frontally and laterally (Fig. 2E). This body constitutes A,C–E = 10 µm; B,F,G = 20 µm; H–J = 30 µm; K–O = the anterior mesoderm precursor and stays in long-term 50 µm. contact with the archenteron. 60 E.N. Temereva and V.V. Malakhov / IRD 50 (2007) 57–66

Fig. 2. Embryonic and early larval development of Phoronopsis harmeri. Drawings of live animals. AV axis indicates animal and vegetal poles of embryo. A: Blastula. B: Formation of the vegetal plate and appearance of primary mesenchyme cells. C, D: Formation of the anterior mesoderm precursor (C: lateral view; D: frontal view). F: Formation of the preoral coelom cavity. G: Formation of two tiers of mesoderm cells stretching into the postoral region of the embryo. H: Formation of the cylindrical preoral coelom and start of intestine formation. Ectodermal invagination is indicated by arrow. I: Formation of the preoral lobe and the canal of the protonephridium. J: Young larva with two ciliated bands. K: Formation of the posterior mesoderm precursor. L: Young larva with tentacle precursors and horseshoe-shaped posterior mesoderm precursor. Larvae in Figs. J–L are somewhat compressed between cover glass and slide so that we can see sagittal view of preoral lobe and parafrontal view of collar and trunk. am, anterior mesoderm precursor; ar, archenteron; np, neural plate; at, apical tuft; bl, blastocoel; bp, blastopore; c1, preoral coelom; i, intestine; mc, mesenchyme cells; mg, midgut; mt, postoral ciliated band; es, esophagus; pc, preoral coelom; pm, posterior mesoderm precursor; pi, precursor of the intestine; ppn, precursor of the protonephridium; pt, preoral ciliated band; tmc, tier of mesoderm cells; s, stomach; sc, star-shaped cells; v, vestibulum. Scale bars in A–L = 50 µm. E.N. Temereva and V.V. Malakhov / IRD 50 (2007) 57–66 61

Initially, the anterior mesoderm precursor consists of tightly packed cells (Fig. 2E). After the complete closing of the blastopore (about 44 h after the onset of cleavage), a cavity appears between these cells, which have separated from each other. This cavity, the preoral coelom, forms by (Fig. 2F). Two tiers of cells stretch from the wall of the new cavity along the side of the stomodeum into the postoral region of the larva (Fig. 2G). The front part of the larva inflates and becomes bubble shaped (Fig. 1N, 2G). The preoral part of the body now hangs over the mouth aperture frontally, thus forming the embryonic preoral lobe (Fig. 1N, 2H). The front surface of the lobe becomes impressed into the vestibulum and forms its upper wall, the preoral lobe subumbrella (Fig. 2I). The bulk of preoral mesoderm becomes tied with the inner surface of the ectoderm of the preoral lobe’s undermost surface (Fig. 2H, I). Sixty hours after the onset of cleavage a portion of meso- dermal cells moves upwards forming the head cylinder under the neural plate (Fig. 2H). The head cylinder is lined by mesodermal cells (Fig. 2I), and its internal cavity can thus be considered coelomic (Temereva and Malakhov, 2006). The ectodermal portion of the gut is basically sac shaped. The rear part of the sac then forms a bulge directed towards the ectoderm of the hinder end of the Fig. 3. Photos of young (A), 12-tentacle (B) and 18-tentacle body (Fig. 2H). A counter-directed shallow ectodermal larvae of the Phoronopsis harmeri. Young larva with neural depression forms concurrently. The two structures plate (np), protonephridia (pn) and metacoel mesoderm merge to make up a through intestine 58–60 h after the (indicated by arrowheads). 12-tentacle larva possesses tenta- onset of cleavage (Fig. 2I). cles (t), telotroch (tt) and two masses of blood corpuscles The embryonic protonephridial system is also (bc). 18-tentacle larva with cylindrical preoral coelom (c1), formed at this stage as paired dorso-lateral depressions large stomach diverticulum (sd), long metasomal sac (ms), spacious balstocoel (bl) under tentacles between the body in then ectoderm on both sides of the anus (Fig. 2I) that wall and trunk coelom wall (wc3). Scale bars: A = 50 µm; are precursors of the protonephridial canals. Initially B, C = 100 µm. they take the form of short tubules with slightly swollen rounded distal parts (Fig. 2I). 65–67 h after the onset of cleavage, individual spindle-like cells bud off into the lumen. Then the precursor buds off and becomes a blastocoel cavity from the epithelium of the distal tips horseshoe-shaped body which embraces the intestine and later give rise to flame cells (Fig. 2J, 3A). dorsally and on both sides (Fig. 2L, 3A). Later in By the stage of anus formation the larva has two development, two branches of the metacoel horseshoe ciliated bands (Fig. 2J, K). The preoral band is meet on the ventral side, thus forming the ventral horseshoe-shaped and extends along the edge of the mesenterium (Fig. 4B). preoral lobe (Fig. 2J, K). The horseshoe-shaped postoral At this stage the larva becomes Г-shaped. Its body band grows dorsolaterally in the anterior region of the length (~300 µm) approximates to the diameter of the body and obliquely descends along the body sides, preoral lobe (260–270 µm) (Fig. 1O). The postoral running very low and frontally to the anus in young ciliated band forms two symmetric outgrowths, the larvae (Fig. 2J, K). predecessors of the larval tentacles of the Actinotrocha About 90–100 h after the onset of cleavage, the (Fig. 2L). formation of the posterior mesoderm precursor begins (Fig. 2K). The posterior precursor has the shape of a minor dorsal prominence at the junction of the midgut Larval development and the proctodeum (Fig. 2K). The prominence grows A 4-tentacle pair larva is about 450 µm in length, with time and acquires a cavity connected with the gut with the preoral lobe diameter being ~320 µm (Fig. 4A). 62 E.N. Temereva and V.V. Malakhov / IRD 50 (2007) 57–66

Fig. 5. Details of the larval anatomy with SEM. A: Longi- tudinal section through the tentacle with radial coelomic channel (c2) and muscular cells (mc) in the blastocoel. B: Parafrontal section through the preoral lobe (pl; bl, blasto- coel; bm, blood mass; eso, esophagus). C: Sagittal section through the 12-tentacle larva (bl, blastocoel; eso, esophagus; ms, metasomal sac; fo, frontal organ; s, two septum inside Fig. 4. Larval development of Phoronopsis harmeri. the stomach diverticulum (sd); tt, telotroch; v, vestibulum). Drawings of live animals. A: 8-tentacle stage; B: 12-tentacle D: Sagittal section through the preoral coelom (c1) adjacent stage; C: 20-tentacle stage; D: 24-tentacle stage. np, neural to esophagus wall (eso) and neural plate (np). E: General plate; bc, blood corpuscles; bc1, border of the preoral view of the competent larva (pl, preoral lobe; t, tentacles; tt, coelom; c1, preoral coelom; c2, tentacle coelom; c3, trunk telotroch). Scale bars: A,D = 40 µm; B = 120 µm; C,E = coelom; dv, dorsal blood vessel; i, intestine; mg, midgut; ms, 300 µm. metasomal sac; pl, preoral lobe; pms, pore of the metasomal sac; pn, protonephridium; fo, frontal organ; s, stomach; sd, stomach diverticulum; tt, telotroch; v, vestibulum; vm, ven- tral mesentery. Scale bars: A–D = 100 µm. of blood corpuscles are observed (Fig. 3B). They lie dorso-laterally in the blastocoel (Fig. 5B). With time, the number of constituent cells increases (Fig. 3C). Late At this stage a well defined telotroch is distinguished in development (in the 10-tentacle pair larva) another around the anus. pair of erythrocyte aggregations appears ventrolaterally At the 6-tentacle pair stage the larva is 500–600 µm (Fig. 4C). long; it grows a yet larger trunk, whereas the preoral The 6-tentacle pair larva develops an early meta- lobe undergoes no visible change and remains 300 µm somal sac (Fig. 4B). A ventral outgrowth develops on in diameter (Fig. 4B). Mesodermal cells underlying the the stomach wall, the so-called stomach diverticulum postoral band form a horseshoe-shaped coelomic cavity (Fig. 4B). ). The nephridial canals elongate and become of the tentacle coelom (Fig. 4B). Muscle cells inside the U-shaped. blastocoel of tentacles run along the frontal side and The 12-tentacle pair larva possesses a distinct dorsal form longitudinal muscles in each tentacle (Fig. 5A). At blood vessel and six or eight masses of red erythrocytes this stage (6 tentacle pairs), in the preoral lobe of the (Fig. 4D). The stomach diverticulum protrudes signi- larva at the level of the pharynx, two symmetric masses ficantly to reach the ectoderm of the oral field (Fig. 5C). E.N. Temereva and V.V. Malakhov / IRD 50 (2007) 57–66 63

Throughout larval development, the neural plate is well According to some views, phoronids possess an endo- defined in the preoral lobe (Fig. 4). It constitutes a dermal proctodeum (Emig, 1977; Herrmann, 1986). Our thickening of ectoderm with the preoral coelomic studies of P. harmeri show that here the proctodeum cylinder attached to it (Fig. 5D). The preoral lobe larva forms by ectodermal invagination. The ectodermal with 12 pairs of tentacles possesses the frontal organ origin of the proctodeum was demonstrated experi- which situated above the preoral ciliated band (Figs. 4D, mentally in Phoronis vancouverensis (Freeman and 5C). Martindale, 2002). Thus, the competent larva (Fig. 5E) is 1400– Most investigators suggest that the mesoderm in 1500 µm long, possesses 12 tentacle pairs, 4, 6 or 8 phoronids arises by migration of cells from the walls of blood masses, one stomach diverticulum and a frontal the archenteron and that the coelomic cavity forms by organ. Primordia of definitive tentacles are absent. The schizocoely (Zimmer, 1964, 1980; Emig, 1974; Herr- live larva is transparent and pinkish in color. mann, 1986, Santagata 2004). Our investigations have revealed that the mesoderm in phoronids has a dual origin, anteriorly and posteriorly. The mesodermal Discussion lining of preoral and tentacle originates from anterior mesoderm precursor. These are cells originating Modern investigators showed that phoronids under- from the anterior wall of the archenteron. The lining of go radial cleavage of the egg (Zimmer, 1964; Emig, the trunk coelom forms from the posterior mesoderm 1979; Herrmann, 1986; Freeman, 1991; Malakhov and precursor and its cavity arises by enterocoely. Our Temereva, 1999; Santagata, 2001). According to our results agree with those of Freeman and Martindale data observations, cleavage in P. harmeri follows the radial (2002), according to which trunk mesoderm of P. pattern. In this species, cleavage furrows from the 4th vancouverensis forms at the border of ectoderm and division on are tilted against the animal-vegetal pole of endoderm. the egg, a trait sometimes observed in animal groups Two mesoderm origins (anterior and posterior) are with classic radial cleavage (e.g., in sea urchins, see characteristic of other groups among Bilateria. Thus, in Boveri, 1901; Horstadius, 1928). This cleavage furrow Spiralia the precursor of the posterior mesoderm is orientation allows P. harmeri to develop a spherical quadrant D, or more precisely, the 4d blastomere, blastula by the 16-cell stage. In some other species, the whereas anteriorly mesoderm originates from the grooves remain regularly transverse for a longer time descendants of blastomeres 2a, 2b, 2c or 3a, 3b and (e.g., in P. psammophila, Emig, 1977; Freeman, 1991), gives rise to pharynx muscles (Boyer et al., 1998; in which case the blastomeric blastula preserves an Henry, Martindale, 1998; Lartillot et al., 2002a). In opening at the animal pole. chordates the posterior mesoderm is mesoderm of the A peculiar property of cleavage in P. harmeri is the tail bud, and the anterior mesoderm represents the so- inequality of the first division, as observed in several called prechordal mesoderm (Seifert et al., 1993; other phoronid species (Foettinger, 1882; Emig, 1974, Kiecker and Niehrs, 2001). Two origins also occur in 1977). Recent experimental studies showed that the first crustaceans, where the bulk of mesoderm comes from groove is usually transverse to the anterior-posterior posterior portion of the embryonic plate (Weygoldt, axis of the larval body and that one of the first two 1960, 1961; Anderson, 1967; Behesch, 1969). On the blastomeres gives rise to anterior part of the larval body other hand, many species have the so-called preantennal and other gives rise to posterior part (Freeman, 1991). mesoderm which segregates anteriorly in the embryo The blastocoel cavity in phoronids forms at the 16- (Nair, 1939, 1949; Weygoldt, 1960, 1961; Benesch, cell stage (Emig, 1977; Freeman, 1991). According to 1969) and gives rise to the muscles of upper labium, the published evidence, gastrulation occurs through pharynx and eye-stalks. invagination which is preceded by formation of a gas- The anterior and posterior mesoderm precursors are trula plateau at the vegetal pole (Zimmer, 1964, 1980; topologically associated with mouth and anus, respec- Emig, 1977, 1982; Herrmann, 1986; Freeman, 1991; tively, which within the framework of comparative Malakhov and Temereva, 1999). anatomy are products of the slit blastopore segregation The blastopore closes from the posterior to the of a radially symmetric ancestor (Sedgwick, 1884; anterior end of the gastrula leaving an opening, which Beneden, 1891; Beklemishev, 1944; Jagersten, 1955). will become the definitive mouth (Hyman, 1959; Emig, Interestingly, in both chordates and invertebrates, the 1974). The anus in phoronids does not form in direct regions of anterior and posterior mesoderm precursors association with the blastopore (Shearer, 1906; Emig, express the homeobox genes brachiury, goosecoid and 1974). Whether the proctodeum originates from forkhead (Bassham, Postlethwait, 2000; Tagawa et al., ectoderm or endoderm is disputed in the literature. 2001; Technau, 2001; Takade et al., 2002). Homologous 64 E.N. Temereva and V.V. Malakhov / IRD 50 (2007) 57–66 genes express in Cnidaria circularly around the mouth Russian Foundation for Basic Research (project 05-04- opening (Technau and Bode, 1999; Broun et al., 1999; 49272), and the Federal Agency of Sciences and Inno- Scholz and Technau, 2003). This may suggest that the vations (project 02.442.11.7393). two precursors associated with anterior and posterior ends of the slit blastopore descend from a primarily circular region of mesoderm formation of a radial- References symmetric ancestor that split apart in evolution of Bilateria (Lartillot et al., 2002b). Anderson, D., Larval development and segment formation in the Branchiopod crustaceans. Ibid, 15 (1967) 47–91. The traditional interpretation of phoronid regiona- Bartolomaeus T., Ultrastructure and formation of the body tion is that there are three coelom compartments called cavity lining in Phoronis muelleri (Phoronida, Lopho- preoral, tentacle and trunk, respectively. This view has phorata). Zoomorph, 120 (2001) 135–148. been criticized by several authors (Bartolomaeus, 2001; Bassham, S. and Postlethwait, J., Brachiury (T) expression in Gruchl et al., 2005). Thus, according to the TEM studies embryos of a larvacean urochordate, Oikopleura dioica, of the former, larvae possess only trunk coelom, preoral and the ancestral role of T. Dev. Biol, 220 (2000) and tentacle coeloms being absent (Bartolomaeus, 322–332. 2001). However, our new data show that P. harmeri Beklemishev, W., Principles of Comparative Anatomy of Invertebrates. Sovetskaya Nauka, Moscow, 1944 (in larvae have all three coelomic compartments (Teme- Russian). reva and Malakhov, 2006). Furthermore, Santagata Beneden, E. Van., Recherches sur le development des (2002) showed that P. pallida larvae have tentacular Arachnactis. Contribution a la morphologie de Ceri- coelomic canals which are retained at metamorphosis, anthides. Arch. Biol. Paris, 11 (1891) 114–146. albeit with some remodeling of the neuromuscular Benesch, R., Zur Ontogenie und Morphologie von Artemia system. salina. Zool. Jahrb. Anat, 86 (1969) 307–458. The early actinotrocha possesses two ciliated bands, Boveri, T., Di Polarität der Oocyte, Ei und Larve von preoral and postoral (Nielsen, 1987). We consider that, Strongylocentrotus lividus. Zool. Jb. Anat, 14 (1901) 630–651. topologically, the bands are homologous with ciliated Boyer, B.C., Henry, J.J. and Martindale, M.Q., The cell structures in other bilaterian larvae. The preoral band lineage of a polyclad turbellarian embryo reveals close corresponds to the larval prototroch in Spiralia, and the similarity to coelomic spiralians. Dev. Biol, 204 (1998) postoral to the larval metatroch. The pre- and postoral 111–123. bands of actinotrocha can also be homologized with Broun, M., Sokol, S. and Bode, H.R., Cngsc, a homologue of corresponding larval bands in Deuterostomia. goosecoid, participates in the patterning of the head, and The size, pigmentation, numbers of tentacles and of is expressed in the organizer region of Hydra. Dev., 126 blood masses, the existence of adult tentacle primordia (1999) 5245–5254. Cohen, B.L., Gawthrop, A. and Cavalier-Smith, T., Mole- and a frontal organ are very important for identification cular phylogeny of brachiopods and phoronids based on of phoronid larvae. We suggest that the form of the nuclear–encoded small subunit ribosomal RNA gene preoral coelom can also be used as a diagnostic feature. sequences. Phil. Trans. Roy. Soc. B: Biol. Sci, 353 Larvae belonging to the Phoronopsis species possess (1998) 2039–2061. a cylindrical protocoel (Temereva and Malakhov, 2004), Cohen, B.L., Monophyly of brachiopods and phoronids: whereas Phoronis larvae have a preoral coelom with a reconciliation of molecular evidence with Linnaean different shape. However, application of these diag- classification (the subphylum Phoroniformea nov.). Proc. nostic features of phoronid larvae is beset with difficulty Royal. Soc. Lond. B, 267 (2000) 1–7. 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Nezlin for his help with photos. I 2–90. also thank two anonymous reviewers for their comments Emig, C.C., 2006. http://www.com.univ–mrs.fr/DIMAR/ on the manuscript. The project was financed by the Phoro/. E.N. Temereva and V.V. Malakhov / IRD 50 (2007) 57–66 65

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