DOI: 10.1111/ede.12231 RESEARCH PAPER Ground plan of the larval nervous system in phoronids: Evidence from larvae of viviparous phoronid Elena N. Temereva Department of Invertebrate Zoology, Biological Faculty, Moscow State Nervous system organization differs greatly in larvae and adults of many species, 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, larva 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 protostome- 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 animals, 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,