Bull. Natl. Mus. Nat. Sci., Ser. B, 35(4), pp. 169–182, December 22, 2009

Distribution Patterns of the Radiolarian Nuclei and Symbionts Using DAPI-Fluorescence

Noritoshi Suzuki1*, Kaoru Ogane1,9, Yoshiaki Aita2, Mariko Kato3, Saburo Sakai4, Toshiyuki Kurihara 5, Atsushi Matsuoka6, Susumu Ohtsuka7, Akio Go8, Kazumitsu Nakaguchi8, Shuhei Yamaguchi8, Takashi Takahashi7 and Akihiro Tuji9

1 Institute of Geology and Paleontology, Graduate School of Science, Tohoku University, Aoba 6–3, Aramaki, Aoba-ku, Sendai, 980–8578 Japan 2 Department of Geology, Faculty of Agriculture, Utsunomiya University, Mine-machi 350, Utsunomiya, 321–8505 Japan 3 Plant Science, Graduate School of Agricultural Science, Utsunomiya University, Mine-machi 350, Utsunomiya, 321–8505 Japan 4 Institute of Biogeoscience, JAMSTEC, Natsushima-cho 2–15, Yokosuka, 237–0061 Japan 5 Graduate School of Science and Technology, Niigata University, Niigata, 950–2181 Japan 6 Department of Geology, Faculty of Science, Niigata University, Niigata, 950–2181 Japan 7 Takehara Marine Science Station, Setouchi Field Science Center, Graduate School of Biosphere Science, Hiroshima University, Minato-machi 5–8–1, Takehara, Hiroshima, 725–0024 Japan 8 Faculty of Applied Biological Science, Hiroshima University, Kagamiyama 1–4–4, Higashi-Hiroshima, 739–8528 Japan 9 Department of Botany, National Museum of Nature and Science, Amakubo 4–1–1, Tsukuba, 305–0005 Japan *E-mail: [email protected]

Abstract This study is the first report on the successful application of the DAPI (4Ј,6-diamidino- 2-phenylindole) staining technique to 22 families of five of the six radiolarian orders (Acantharia, Collodaria, Nassellaria, Spumellaria, and Taxopodia, excluding Entactinaria). A total of six Acan- tharian species, three Collodarian species, three Spumellarian species, 18 Nassellarian species, and one Taxopod species emitted blue light under epifluorescence microscopy after DAPI staining. These results and existing data indicated that Acantharia and two collodarian families (Col- losphaeridae and Sphaerozoidae) are multi-nucleated, whereas Nassellaria, Spumellaria, Taxopo- dia, and Thalassosphaeridae form a single-nucleus group. The shape, location, and number of nuclei in Radiolaria were variable not only at the family level but also at lower taxonomic levels. Even within species such as Spirocyrtis scalaris, nuclei can move from the cephalis to intracapsu- lum lobes below the cephalis. Key words : cytology, DAPI, nucleus, Radiolaria.

generally considered single-nucleated Protoctista Introduction (De Wever et al., 2001), but carmine-dying meth- Radiolarians are planktonic marine unicellular ods have indicated that several species of Acan- protoctists that appeared during the Cambrian tharia and Collodaria contain many nuclei in a period and currently consist of six orders: Acan- singl cell (Hertwig, 1879; Brandt, 1885; tharia, Collodaria, Entactinaria, Nassellaria, Schewiakoff, 1926; Febvre, 1989). These studies Spumellaria, and Taxopodia (De Wever et al., put into question their taxonomic relationships of 2001; Kunitomo et al., 2006). Siliceous test-bear- Radiolaria with respect to the evolution of the ing radiolarians belonging to Collodaria, Entacti- variability in nuclear traits. Haeckel (1887) de- naria, Nassellaria, and Spumellaria are tradition- scribed the nuclear characteristics of several radi- ally grouped as polycystine radiolarians or olarian groups, such as uni- versus multi-nuclear, simply polycystines. Polycystine radiolarians are central versus eccentric positions, homogenous 170 Noritoshi Suzuki et al. versus allogenous composition, shape, and differ- were fixed in 2% formaldehyde and kept at 5°C ences in ontogenetic growth. Thus, the Entacti- for 2 weeks, whereas the Sesoko samples were naria, Nassellaria, and Spumellaria each have preserved in 2% glutaraldehyde after substitution single nuclei except during the reproductive of 100% methanol for 6 months at Ϫ10°C. stage. However, these century-old descriptions of The fixed radiolarian samples were distributed nuclear traits require verification using modern onto glass slides, dyed with 1 mg/ml DAPI for techniques. 5–10 min and examined under an epifluorescence The number of nuclei in radiolarian cells is microscope (Olympus AX80 Provis) at the De- thought to remain constant throughout the life partment of Botany, National Museum of Nature cycle, except during the reproductive stage, and and Science, Tokyo. Images were captured using the cytoplasmic trait of whether radiolarians are a single-lens reflex camera (Canon EOS KISS), single- or multi-nucleated is presumed to be an after which deconvolution processing using order-level characteristic. To determine the ve- PopImaging ver. 4.0 for Windows® (Digital racity of these assumptions, we examined the Being Kids, http://www.dbkids.co.jp/) was ap- nuclear status of about 60 species of radiolarians. plied to the images. Cells were dyed with the fluorogenic compound DAPI (4Ј,6-diamidino-2-phenylindole), as the Results position and number of nuclei within a cell are Acantharia easily distinguished with this compound. DAPI, Six Acantharian taxa from the Sesoko sample one of the most common fluorescent compounds harbored numerous small nuclei. Acanthostaurus for the recognition of nuclei, passes through the henseni (Popofsky) (Phyllacanthoidea, Phyl- intact cell membrane of both live and fixed cells lostauridae) had several very small, oblong to and binds with double-stranded DNA. The round nuclei scattered throughout the intracapsu- stained DNA is excited as blue emission (461 nm lum (Table 1). Phyllostaurus cuspidatus (Haeck- at an emission maximum wavelength) with ultra- el) (Phyllacanthoidea, Phyllostauridae) (Fig. 1E, violet (358 nm at a maximum absorption wave- F) and Xiphacantha sp. A (Phyllacanthoidea, length) under epifluorescence microscopy. De- Stauracanthidae) (Fig. 1G, H) had spherical intra- spite the wide application of DAPI, only a few capsulum, in which small spherical nuclei were studies have applied it to radiolarians (e.g., Taka- marginally distributed. The nuclei of Heteracon (?) hashi et al., 2003). We dyed radiolarian species sp. (Chaunacanthoidea, Stauroconidae) (Fig. 1C, that varied in developmental stages from young D) were apparently distributed in a similar man- to mature. ner to P. c u s p i d a t u s . Diploconus fasces Haeckel (Sphaenacanthoidea, Diploconidae) (Fig. 1A, B) Materials and Methods had a spherical to ellipsoidal central shell with Samples were collected from the surface water two characteristic reversed corn (cornets) that of the Pacific Ocean near the Nansei Islands, contained nuclei at both ends. These nuclei were south of mainland Japan, during the No. 2009-02 easily distinguished from algal symbionts, as the cruise of the TRV Toyoshio-Maru of Hiroshima latter emitted a red autofluorescence. The nuclei University from 18 to 28 May, 2009 (hereafter, of D. fasces were large compared to those of Toyoshio sample) and from the surface water off other Acantharians. of Sesoko Island near the Okinawa Island, the Ryukyus, on 1 December 2008 (hereafter, Sesoko Collodaria sample). Both the Toyoshio and Sesoko samples We examined three species of Collodarians were collected during 5-min tows using a 38–43- (Table 1). Collozoum huxleyi Müller (Col- mm mesh plankton net. The Toyoshio samples losphaeridae) (Fig. 1I-K) exhibited a single layer Radiolarian Nuclei 171 Fig. patterns Results on DAPI of central capsule inside the intracapsulum of central capsule of central capsule Haeckel ambigous Haeckel ambigous Ehrenberg conical shape in the cephalis reversed 2O, 2P (Ehrenberg) ambigous Tan et Tchang ambigous Haeckel not emitted 3K, 3L (Haeckel) on the half hemisphere ca. 90 nuclei visible 1E, 1F Haeckel spherical in cephalis (Ehrenberg) cephalis and intracapsulum lobe ? (Ehrenberg) ca. 10–15 nuclei in each central capsule 1L, 1M (Popofsky) numerous along the four longer spines (Popofsky) in cephalis lobe-like (Hollande et Enjumet) spherical in shape the peripherical part 3E, 3F Ehrenberg ambigous 3M, 3N (Haeckel) in cephalis lobe-like (Haeckel) in cephalis lobe-like (Haeckel) lobe-like 3C, 3D Brandt ca. 4–5 nuclei in the each central capsule 1N, 1O Hertwig portion elliptical in the ventral 3G, 3H Hertwig in the intracapsulum periperiphy 3A, 3B Jørgensen half in the intracapsulum lower 2K, 2L (Ehrenberg) spherical in cephalis (Haeckel) spherical in cephalis Hertwig not emitted 3I, 3J Müller on the half hemisphere ca. 100 nuclei vislble 1I, 1K Müller (young cell)Müller (young ambigous 3O, 3P (Popofsky) lobe-like Haeckel (young cell) (young Haeckel spherical in cephalis 2E, 2F Haeckel in both cornets 24 nuclei visible 1A, 1B Ehrenberg (young cell) (young Ehrenberg spherical in cephalis 2I, 2J sp. A (young cell)sp. A (young cell) (young B sp. heteropolar distribution spherical 2A, 2B 2C, 2D sp. Asp. intracapsulum the in half lower 2M, 2N sp. Asp. hemisphere half the on visible nuclei 16 ca. 1G, 1H sp. (young cell)sp. (young spherical in cephalis 2G, 2H (?) sp. in the intracapsulum ca. 60 nuclei visible 1C, 1D Haliommilla capillaceum Tetrapyle octacanthaPyloniidae gen. et sp. indet. tetras tetras Spongaster scalaris Spirocyrtis Theocorythium trachelium Pterocorys bacca Lithopera praetextum Pterocanium productus Zygocircus Acanthocorys castanoides testudus Botryopera spherical in the center buetschli Lophophaena Lophophaena hispida Lophophaena variabilis Peromelissa phalacraPlectacantha oikiskos Plectacantha Psilomelissa thoracites abietina Plagiacantha Pseudocubus obeliscus coarctatum Clathrocanium Tetraphormis dodecaster Spongosphaera streptacantha Spongosphaera Hexalonchetta Hexalonchetta Sphaerozoum haeckeli Sphaerozoum myriacantha Arachnosphaera sphaerica Cenosphaera Disolenia zanguebarica Diploconus fasces Heteracon huxleyi Collosphaera Xiphacantha Sticholonche zancelea Sticholonche Acanthostaurus henseni cuspidatus Phyllostaurus Table 1.Radiolaria to application SummaryDAPI of results Rhizosphaeridae Pterocorythidae Stichocapsidae Plagiacanthidae Sethoperidae Sethophormidae Spongosphaeridae Hexastylidae Sphaerozoidae Ethmosphaeridae Stauracanthidae PylonioideaSpongodiscoidea Pyloniidae Euchitoniidae AcanthodesmoideaPlagiacanthoidea Stephaniidae Lophophaenidae SphaenacanthoideaChaunacanthoidea Diploconidae Stauroconidae Order Superfamily Family name Taxon Nassellaria Eucyrtidioidea Artostrobiidae Spumellaria Actinommoidea Astrosphaeridae Collodaria Collosphaeridae Taxopodia Sticholonchidae Acantharia Pyllacanthoidea Phyllostauridae 172 Noritoshi Suzuki et al.

Fig. 1. Acantharia and Collodaria. A, B: Diploconus fasces (Sesoko sample); C, D: Heteracon (?) sp. (Sesoko sample); E, F: Phyllostaurus cuspidatus (Sesoko sample); G, H: Xiphacantha sp. A (Sesoko sample); I–K: Collosphaera huxleyi (Toyoshio sample); L, M: Disolenia zanguebarica (Toyoshio sample); N, O: Sphaero- zoum haeckeli (Toyoshio sample). Figure 1D, F, H, M, and O show enlarged views of the boxes in Fig. 1C, E, G, L, and N, respectively. Figures with light backgrounds, including Fig. 1N are transmission light microscopy photographs, whereas those with black backgrounds represent epifluorescence microscopy with DAPI. Scale barsϭ50 mm. Abbreviations: cmϭcapsular membrane, Cnϭcornet, Cpϭcentral portion, Csϭcortical shell, cvϭcentral vacuole, ectϭectoplasm, endϭendoplasm, gmϭgelatin matrix, N(r)ϭnucleus of radiolarians, N(s)ϭnucleus of symbionts, Scϭisolated spicules, Syϭsymbionts. Radiolarian Nuclei 173 of small nuclei just beneath the capsular mem- emissions were not distinguishable from blue brane. These nuclei were distributed equidistant autoluminescence. Spongaster tetras tetras from one another. The diameter of each nucleus Ehrenberg (Fig. 3M, N) also appeared ambigu- (6 mm in diameter) was slightly larger than the ously blue, even after splitting the specimen into diameter of the pores (4.9 mm in diameter) on the pieces. cortical shell, and each nucleus was distributed under one pore. An aggregated distribution was Nassellaria detected in the nuclei (9.5 mm in diameter) of All of the Nassellarian species that emitted Disolenia zanguebarica (Ehrenberg) (Fig. 1L, blue light using DAPI were confirmed to be sin- M), but fewer nuclei were present in each cell gle-nucleated (Table 1). One spherical to ellipti- compared to C. huxleyi. Moreover, the pore dis- cal nucleus was observed in Clathrocanium tributions of the cortical shell did not correspond coarctatum Ehrenberg (Fig. 2O, P), Lophophae- to the distribution of nuclei. We also stained Col- na buetschlii (Haeckel), Lophophaena hispida losphaera tuberosa Haeckel with propidium (Ehrenberg), Lophophaena variabilis (Popofsky), iodide (PI), a fluorescent compound, and several Peromelissa phalacra Haeckel, Psilomelissa tho- spheres in the periphery of the intracapsulum racites (Haeckel), and Tetraphormis dodecaster emitted bright red light, indicating multi-nucle- Haeckel. The nuclei of Plagiacantha abietina ation. Hertwig (Fig. 3A, B), Plectacantha oikiskos Sphaerozoum haeckeli Brandt (Sphaerozoidae) Jørgensen (Fig. 2K, L), and Plectacantha sp. A (Fig. 1N, O) contained many loosely scattered (Fig. 2M, N) were irregular in form within the tiny siliceous spicules throughout the cell. The cephalis. Acanthocorys castanoides Tan et colony examined in our study was composed of Tchang, Pseudocubus obeliscus (Haeckel) (Fig. six S. haeckeli cells and numerous algal sym- 3C, D), and Trisulcus testudus Petrushevskaya bionts. Each cell harbored five nuclei, each of each contained one undulated nucleus. Thus, all which was large (10 mm in diameter) compared of the plagiacanthoid species harbored a single to the nuclei of C. huxleyi. nucleus inside the cephalis, the shape of which could be classified into three types: spherical to Entactinaria and Spumellaria elliptical, peripheral, or undulated. We stained nine species of these groups, but Several species belonging to the superfamily only three successfully fluoresced (Table 1). Eucyrtidioidea (sensu Petrushevskaya, 1971) Yo u n g c e l l s o f Hexalonchetta sp. A exhibited were also examined using DAPI. Of these, Lith- blue luminescence along the periphery and in the opera bacca Ehrenberg (young cell; Fig. 2I, J), upper lateral portion between the microsphere Spirocyrtis scalaris Haeckel (young cell; Fig. 2E, and macrosphere (Fig. 2A, B), whereas Hexa- F), Theocorythium trachelium (Ehrenberg) (not lonchetta sp. B emitted spherical blue light out- quite mature cell), Pterocanium charybdeum side of the microsphere (Fig. 2C, D). In charybdeum (Müller) (mature cell), and Pteroco- Cenosphaera sphaerica (Hollande et Enjumet), rys sp. (young cell; Fig. 2G, H) emitted blue light the periphery of the inside of the central capsule to some extent. L. bacca, S. scalaris, and Ptero- emitted blue light, but we could not determine corys sp. emitted blue light inside the cephalis whether the nuclear area was multi-nucleated (4-7 mm in diameter). Two cells of the Zygocircus (Fig. 3E, F). productus Hertwig group (Fig. 3I, J) were also The remaining six species did not clearly emit examined using both DAPI and PI. PI generated blue light. Both Haliommilla capillaceum Haeckel relatively better emission than DAPI, but the (Fig. 3K, L) and Tetrapyle octacantha Müller presence of a nucleus was difficult to verify using (Fig. 3O, P) emitted weak blue light throughout either stain in the species. the entire portion of the central capsule, but the 174 Noritoshi Suzuki et al.

Fig. 2. Spumellaria and Nassellaria. A, B: Young Hexalonchetta sp. A (Sesoko sample); C, D: Young Hexa- lonchetta sp. B (Sesoko sample); E, F: Young Spirocyrtis scalaris (Sesoko sample); G, H: Young Pterocorys sp. (Sesoko sample); I, J: Young Lithopera bacca (Sesoko sample); K, L: Plectacantha oikiskos; M, N. Plec- tacantha sp. A; O, P: Clathrocanium coarctatum. Figure 2B, D, F, H, J, L, N, and P are enlarged views of the boxes in Fig. 2A, C, E, G, I, K, M, and O, respectively. Figures with light background are transmission light microscopy photographs, whereas those with black backgrounds represent those by epifluorescence microscopy using DAPI. Scale barsϭ10 mm. Abbreviations: abϭabdomen, cpϭcephalis, maϭmacrosphere, MBϭmedian bar, miϭmicrosphere, ncϭnuclear capsule, rsϭradial spine, txϭthorax. For all other abbrevia- tions, see Fig. 1.

Taxopodia 3G, H); instead, the ventral side of the cell emit- The one species examined, Sticholonche zan- ted substantially more light. clea Hertwig (Table 1), exhibited very little blue luminescence within the nuclear capsule (Fig. Radiolarian Nuclei 175

Fig. 3. Nassellaria, Taxopodia, and non-emitting Spumellaria. A, B: Plagiacantha abientina (Sesoko sample); C, D: Pseudocubus obeliscus (Sesoko sample); E, F: Cenosphaera sphaerica; G, H: Sticholonche zanclea (Sesoko sample); I, J: Zygocircus productus (Sesoko sample); K, L: Haliommilla capillaceum (Toyoshio sam- ple); M, N: Smashed cell of Spongaster tetras tetras (Toyoshio sample); O, P: Tetrapyle octacantha (Sesoko sample). Figure 3B, D, F, H, J, L, N, and P show enlarged views of the boxes in Fig. 3A, C, E, G, I, K, M, and O, respectively. Figures with light backgrounds are transmission light microscope views, whereas those with black backgrounds represent epifluorescence microscopy using DAPI. Scale barsϭ50 mm. For all abbrevia- tions, see Figs. 1 and 2.

Discussion hibiting the infiltration of DAPI into the intracap- Technical issues sulum. We attempted to expose the intracapsu- The most serious problem encountered with lum within the capsular membrane by strongly DAPI was that nearly all polycystine radiolarians, pushing the cell with a ballpoint pen, but the cap- except for Collodaria, emitted blue light only sular membrane never burst. We also tried to cut very weakly or did not emit. In addition, the cap- the capsular membrane with the edge of a cover sular membrane is thick and chitinous, likely in- glass (0.15–0.19 mm thick), but the membrane 176 Noritoshi Suzuki et al. adhered to the cover glass and the kerf closed The multi-nucleation of Collodaria was initial- soon after cutting. Chemical treatment with ly reported in the late 19th century (Brandt, bleach and an interfacial active agent was not 1885). Dark spheres (Ͻ20 mm in diameter) successful in penetrating the capsular membrane. beneath the capsular membrane (size of intracap- We were, however, able to expose the intracapsu- sulum approximately 100 mm in diameter) of C. lar protoplasm by simply mashing the cell with a huxleyi were first noticed by Cienkowski (1871), ballpoint pen, but the tissues in the intracapsulum but he erroneously assigned the large central vac- were crushed as well. Clearly, a new technique uole (vacuole size approximately 60 mm in diam- or other fluorescent compounds are needed for eter) to be a nucleus. The multi-nuclear habit of further cytological studies of Radiolaria. collodarians has already been recognized in Polysolenia spinosa (Haeckel) (ϭAcrosphaera Acantharia spinosa in the original paper) (Collosphaeridae), Acantharia was recognized as a multi-nucleat- C. huxleyi, Sphaerozoum fuscum Meyen ed Protoctista as early as the 1870s (Hertwig, (ϭSphaerozoum punctatum), and Rhaphidozoum 1879), but the nuclei of Acantharia and algal acuferum (Müller) (ϭSphaerozoum acuferum) symbionts were indistinguishable in the illustra- (Sphaerozoidae) (Brandt, 1885). In contrast, tions of very old studies (Hertwig, 1879; Thalassicolla nucleata Huxley (Thalassicollidae) Schewiakoff, 1926). Although Schewiakoff has a single, large nucleus in the center of the (1926) described the presence of many algal intracapsulum (Huth, 1913). Observations of tis- symbionts in most Acantharian species, only sue sections under TEM revealed that Collozoum Haptophyceae (Pymnesiophyceae) could be iden- species possess multiple nuclei (Anderson, tified in the extracapsulum of Acanthometra pel- 1976c; Swanberg and Anderson, 1981; Anderson lucida Müller (Acanthometridae), Amphilonche et al., 1999), whereas T. nuculeata was con- elongata (Müller) (Acanthometridae), and firmed as having a single nucleus (Anderson, Lithoptera muelleri Haeckel (Lithopteridae) by 1976a). Although multiple nuclei have been transmission sectioned light and transmission observed in the mitotic phase of S. fuscum (ϭS. electron microscopy (TEM) (Febvre and Febvre- punctatum in the original paper) (Anderson, Chevalier, 1979). The taxonomy of the Acanthar- 1976b), mitotic nuclei are easily distinguishable ian symbionts examined here has not yet been from trophont nuclei, because the intracapsulum identified, but the red autofluorescence of Phyl- fills with very small nuclei during mitosis. Thus, lostaurus cuspidus (Fig. 1F), for example, points the multi-nuclear habit of Collosphaeridae and to the presence of algal symbionts, as chlorophyll Sphaerozoidae observed here is part of the a emits bright red light. This unique autofluores- trophont stage. cence of chlorophyll a allowed us to be the first Collosphaera, Disolenia, and Polysolenia to distinguish between the Acantharian nucleus (ϭAcrosphaera) belong to the family Col- and algal symbionts under epifluorescence mi- losphaeridae; Collozoum, Sphaerozoum, and croscopy. Rhaphidozoum belong to Sphaerozoidae; and Thalassicolla belongs to Thalassicollidae, sug- Collodaria gesting a trophont multi-nuclear mode in Col- Our DAPI results indicated multiple nuclei in losphaeridae and Sphaerozoidae and a trophont Collosphaera huxleyi, Disolenia zanguebarica, mono-nuclear mode in Thalassicollidae. The and Sphaerozoum haeckeli. Under PI staining, C. molecular cladograms presented in Yuasa et al. tuberosa also appeared to be multi-nucleated (2005) and Kunitomo et al. (2006) showed three (data not shown). We did not examine whether clades in Collodaria: a Thalassicolla species the number of nuclei in these species was vari- clade, a Collozoum-Sphaerozoum-Rhaphidozoum able across ontogenetic stages. clade, and an Acrosphaera-Collosphaera- Radiolarian Nuclei 177

Fig. 4. Schematic illustration of hypothesized evolution in Collodaria. Schematic dendrogram adapted from the molecular phylogeny in Yuasa et al. (2005) and Kunitomo et al. (2006).

Siphonosphaera clade. These three clades corre- The peripheral nucleus form is illustrated in Figs. spond to the families Thalassicollidae, Sphaero- 2L, 2N, and 3B. As shown in Fig. 2L and N, zoidae, and Collosphaeridae, respectively. The elliptical cytoplasm consisting of endoplasm and molecular cladograms also describe a close phy- the nucleus was located above the median bar logenetic relationship between Collosphaeridae (MB, a cephalic internal spicular system), and and Sphaerozoidae, with both families evolving the lower half of the elliptical cytoplasm con- from Thalassicollidae. This phylogeny suggests tained the light-emitting portions of the nucleus. that a trophont multi-nuclear mode emerged dur- In the upper view of the cephalis (Fig. 3B), the ing the evolution of Collosphaeridae and marginal part just inside of the capsular mem- Sphaerozoidae from the single-nucleated Thalas- brane emitted blue light. sicollidae (Fig. 4). Phylogenetic relationships based on the shape of the nucleus in Plagiacanthoidea need careful Nassellaria discussion because the taxonomy of this group at Here, Plagiacanthoidea was classified as hav- both the family and genus levels has not yet been ing three forms of nuclei. Clathrocanium, settled. Petrushevskaya (1971) thoroughly re- Lophophaena, Peromelissa, Psilomelissa, and vised the classification of Plagiacanthoidea Tetraphormis had one spherical nucleus; Plagia- through a detailed examination of the internal cantha and Plectacantha each had a peripheral structures of the cephalis, and subsequent studies nucleus; and Acanthocorys, Botryopera, and established several genera based on cephalic Pseudocubus contained an undulated nucleus. structure (Nishimura, 1990; Sugiyama, 1993; 178 Noritoshi Suzuki et al.

Funakawa, 1994, 1995). They clearly defined the not always located at the center of the protoplasm classification at the genus level, but their criteria and the sectioned nucleus appeared in sector require examination of fine cephalic structure, form in their periaxoplastid group. Our results which are not easily observable under light indicated that Hexalonchetta sp. A (Fig. 2A, B) microscopy. Due to these uncertainties, phyloge- had a dome-shaped nucleus because the sector netic relationships in Plagiacanthoidea are not form of the nucleus in thin section could only be fully understood. Molecular studies based on derived from a dome shape. In contrast, a taxo- ribosomal DNA sequences support a single clade nomically similar species, Hexalonchetta sp. B among Lophophaena cylindrica (Cleve) (Fig. 2C, D), exhibited a spherical nucleus in the (ϭLithomelissa sp. 2003 in the original paper), center of the test. Our observations demonstrate a Psilomelissa thoracites (ϭLithomelissa sp. variety of nuclei shapes at the species level, 8012), and Pseudocubus obeliscus (Kunitomo rather than at the family level or higher. Hollande et al., 2006). Lophophaena and Psilomelissa be- and Enjumet (1960) emphasized the relationship long to Lophophaenidae, whereas Pseudocubus between the nucleus and the axoplast (the bundle belongs to Plagiacanthidae. The three types of of tubulins inside the intracapsulum) as a family nuclear forms described above overlap across fam- or higher taxonomic-level classification; howev- ilies. For example, families exhibiting a spherical er, our results somewhat contradict this concept. nucleus include Lophophaenidae (Lophophaena, The shape of the nucleus can apparently Peromelissa, Psilomelissa), whereas Plectacan- change at not only the species level but also tha and Botryopera (both Lophophaenidae as within a species. Hollande and Enjumet (1960) well) harbor a peripheral nucleus and an undulat- clearly showed a nearly spherical nucleus in ed nucleus, respectively. If these genera are Cenosphaera sphaerica, but we observed a pe- monophyletic, the shape of the nucleus appears ripheral nucleus just beneath the capsular mem- to have developed independently. brane in the same species (Fig. 3F). Blue fluorescence was observed in the cephalis of Lithopera bacca and Spirocyrtis scalaris, sug- Taxopodia gesting the presence of the nucleus in the Cachon and Cachon (1978) described the cephalis. In contrast to our results for the latter nucleus of Sticholonche zanclea as occurring species, the nucleus of the S. scalaris cell oc- inside the nuclear capsule on the dorsal side of curred below the cephalis in TEM images the cell. However, we observed bright blue light (Sugiyama and Anderson, 1997). Our cell was in the ventral portion of the intracapsulum, out- perhaps too young and contained so little intra- side the nuclear capsule. S. zanclea is often capsulum protoplasm that the nucleus was not infected by a parasitic dinoflagellate, Amoe- yet located below the cephalis. These results sug- bophrya stycholonchae Koeppen, which always gest that the nucleus can change position at dif- remains in the ventral portion of the host (Fol, ferent ontogenetic stages. 1883; Borgert, 1898; Drebes, 1984). Thus, the emitting ventral portion of the examined speci- Entactinaria and Spumellaria men was likely the nucleus of the parasite. According to Hertwig (1879) and Hollande and Enjumet (1960), species of Entactinaria and Types of nuclei Spumellaria contain single nuclei at the center of Based on the present and previous data, the the siliceous skeleton. Hertwig (1879), however, shapes of the radiolarian nucleus can be catego- did not present the entire image of each speci- rized into 11 types (Table 2, Fig. 5). According to men; thus, the species identities were impossible Haeckel (1887, p. 8–16), the typical shape of the to determine from his illustrations. Hollande and nucleus is primarily spherical but can be classi- Enjumet (1960) documented that the nucleus is fied into five types: ellipsoidal (Nassellaria, Lith- Radiolarian Nuclei 179

eloidea, and Pylonioidea in Spumellaria), dis- 5A 5I coidal (Spongodiscoidea in Spumellaria), stel- late (Thalassicollidae), amoeboid (irregular forms of Spumellaria and Acantharia), and lo- bate (cyrtid Nassellaria). We cannot specify type of nuclei between our results and Haeckel’s (Collodaria); 5B (Nassellaria) 5H Xiphacantha (Spumellaria) (Collodaria)

, scheme because we did not examined the radio- larians regarded in Haeckel (1887). Disolenia sp. B (Spumellaria) 5D sp. A (Spumellaria) 5C Sphaerozoum Plagiacantha (Nassellaria) 5J , , Phyllostaurus , (Nassellaria) 5K (Nassellaria) 5G Conclusions (Nassellaria) 5F 1. DAPI application was successful for Acan- (Acantharia); sphaerica Cenosphaera (Nassellaria) tharia, Collodaria, plagiacanthoid Nassellar- Hexalonchetta Hexalonchetta Lithopera Hexalonchetta Hexalonchetta Pterocorys Collosphaera Plectacantha Spirocyrtis Diploconus Eucyrtidium, Spirocyrtis, Clathrocanium Pseudocubus ia, and several young cells of Nassellaria and Spumellaria. In contrast, mature cells of Nassellaria and Spumellaria did not emit MB blue light. 2. The presence of multiple nuclei was con- MB firmed in Acantharia and two Collodarian MB families (Collosphaeridae and Sphaero- zoidae), and whereas the nuclei in Acan- MB tharia were scattered throughout the intra-

, and the lower part nucelus the of lower the and , capsulum, those in Collosphaeridae and MB just above just above Sphaerozoidae were located just beneath the

, but is slightly sliced through , but is slightly capsular membrane. Nassellaria, Spumellar- MB ia, Taxopodia, and Thalassosphaeridae in Collodaria possessed a single nucleus. 3. Based on molecular analyses of Collodarian families (Yuasa et al., 2005; Kunitomo et

MB al., 2006), the evolutionary progression of multiple nuclei in Collodaria was as follows: a single nucleus in Thalassosphaeridae pro- gressed to multiple nuclei in Sphaerozoidae the bottom or upper view is hanged from followed by multiple nuclei in Collosphaeri- In Nassellaria, the nucleus is irregularly shaped shaped irregularly is nucleus the Nassellaria, In In Nassellaria, the nuclues is located on

Table 2.corresponding their and Radiolaria in definitions recognized types Nucleus dae. 4. In the Plagiacanthoidea of Nassellaria, Clathrocanium, Lophophaena, Peromelissa, Psilomelissa, and Tetraphormis each exhib- ited one spherical nucleus; Plagiacantha and Plectacantha had a peripheral nucleus;

median bar, a rod of the cephalic internal spicular system. median bar, and Acanthocorys, Botryopera, and ϭ

MB Pseudocubus contained an undulated nucle-

MB us. In Eucyrtidioidea, Spirocyrtis scalaris

the and Lithopera bacca each harbored a spher- peripheral single nucleusspherical single nucleus in the cephalis In Nassellaria, the nucleus is centered in intracapsulum above positioned in intracapsulum nucleus is eccentrically periaxoplastid Spumellaria and Entactinaria 5E dome-shaped nucleusperipheral single nucleus in the cephalis positioned in the intracapsulum above In Nassellaria, the nucleus is eccentrically spherical from nucleus is dome-shaped so that it seen fan-shaped lateral profile while spherical single nucleusundulated single nucleus nucleus is positioned in the center of central capsule peripheral multiple nuclei nuclei are arraged portion in the exterior in the central capsule lobe-like single nucleus above and below single nucleus above lobe-like nucleus outside of the cephalis In Nassellaria, the nucleus is not positioned inside cephalis scattered multiple nuclei nuclei are scattered throughout intracapsulum lobate nucleusical nucleus located on In Nassellaria, the nucleus is mainly in the cephalis. Abbreviation: Abbreviation: 5. In Spumellaria, Hexalonchetta exhibited a Nucleus type explanation Abbreviated Characteristic features example Typical Fig. Single-nucleus type Multi-nculeus type 180 Noritoshi Suzuki et al.

Fig. 5. Nucleus types in Radiolaria (no scale): (A) scattered multiple nuclei, (B) peripheral multiple nuclei, (C) spherical single nucleus, (D) dome-shaped single nucleus, (E) peripheral single nucleus, (F) spherical single nucleus in the cephalis, (G) peripheral single nucleus in the cephalis, (H) undulated single nucleus, (I) lobe- like single nucleus above and below the MB, (J) lobate nucleus, (K) nucleus outside of the cephalis. Abbreviations: MBϭmedian bar, a rod of the cephalic internal spicular system. Radiolarian Nuclei 181

dome-shaped or spherical nucleus, and menden Parasiten (Spiralkorper Fol, Amoebophyra Cenosphaera sphaerica had a peripheral or Köppen). Zeitschrift für Wissenschaftliche Zoologie 63: spherical nucleus, suggesting that the shape 141–186. Brandt, K. 1885. Die koloniebilden Radiolarien (Sphaero- of the nucleus is variable within these zoeen) des Golfes von Neapel. Fauna und Flora des species of Spumellaria. Golfes von Neapel 13: 1–276. 6. The shape and position of the nucleus have Cachon, J. and Cachon, M. 1978. Sticholonche zanclea historically been considered family-level or Hertwig: A reinterpretation of its phylogenetic position higher taxonomic characteristics of spherical based upon new observations on its ultrastructure. Archiv für Protistenkunde 120: 148–168. radiolarians (Hollande and Enjumet, 1960). Cachon, J. and Cachon, M. 1985. Class Polycystinea. In: In fact, nucleus shape was utilized as such in Lee, J. J., Hutner, S. H. and Bovee, E. C. (eds.), Illus- the well-known comprehensive book, Illus- trated Guide to the Protozoa, pp. 283–295. Society of trated Guide to the Protozoa (Cachon and Protozoologists, Lawrence. Cachon, 1985). However, our results clearly Cienkowski, L. 1871. Über Schwarmer-Bildung bei Radi- indicated that the shape, position, and num- olarien. Archiv für Mikroskopische Anatomie 7: 372– 381. ber of nuclei cannot reliably be used to dis- De Wever, P., Dumitrica, P., Caulet, J. P., Nigrini, C. and tinguish higher taxonomy. Caridroit, M. 2001. Radiolarians in the Sedimentary Record. Gordon and Breach Science Publishers, Ams- terdam. Acknowledgments Drebes, C. 1984. Life cycle and host specificity of marine We thank the staff of both the Sesoko Tropical parasitic dinophytes. Helgoländer Meeresuntersuchun- Biosphere Center, University of the Ryukyus, gen 37: 603–622. Japan (particularly Mr. Shigeo Nakamura), and Febvre, J. 1989. Phylum Actinopoda, Class Acantharia. In: Margulis, L., Corliss, J. O., Melkonian, M. and TRV Toyoshio-Maru. This research is the results Chapman, D. J. (eds.), Handbook of Protoctista, pp. of the 10th Observation Tour of Living Radiolar- 363–379. Jones and Bartlett Publishers, Boston. ians at Sesoko Island, hosted over by Atsushi Febvre, J. and Febvre-Chevalier, C. 1979. Ultrastructural Matsuoka and the No. 2009-02 cruise of the TRV study of zooxanthellae of three species of Acantharia Toyoshio-Maru. (protozoa; Actinopoda), with details of their taxonomic position in the Prymnesiales (Prymnesiophycene, Hib- berd, 1976). Journal of the Marine Biological Associa- References tion of the United Kingdom, N.S. 59: 215–226. Fol, H. 1883. Sur le Sticholonche zanclea et un nouvel Anderson, O. R. 1976a. A cytoplasmic fine-structure ordre de Rhizopodes. Memoires de l’Institut National study of two spumellarian Radiolaria and their sym- Genevois 15: 1–35. bionts. Marine Micropaleontology 1: 81–99. Funakawa, S. 1994. Plagiacanthidae (Radiolaria) from the Anderson, O. R. 1976b. Fine structure of a collodarian Upper Miocene of eastern Hokkaido, Japan. Transac- radiolarian (Sphaerozoum punctatum Müller 1858) and tions and Proceedings of the Palaeontological Society cytoplasmic changes during reproduction. Marine of Japan, New Series (174): 458–483. Micropaleontology 1: 287–297. Funakawa, S. 1995. Lophophaeninae (Radiolaria) from Anderson, O. R. 1976c. Ultrastructure of a colonial radio- the Upper Oligocene to Lower Miocene and intragener- larian Collozoum inerme and a cytochemical determi- ic variation in their internal skeletal structures. Journal nation of the role of its zooxanthellae. Tissue & Cell 8: of Geosciences, Osaka City University 38: 13–61. 195–208. Haeckel, E. 1887. Report on the Radiolaria collected by Anderson, O. R., Gastrich, M. D. and Amarala Zettler, L. H.M.S. Challenger during the years 1873–1876. Report A. 1999. Fine structure of the colonial radiolarian Col- on the Scientific Results of the Voyage of H.M.S. Chal- lozoum serpentinum (Polycystinea: Spumellaria) with a lenger during the year 1873–1876, Zoology 18: reconsideration of its taxonomic status and re-establish- 1–1803. ment of the genus Collophidium (Haeckel). Marine Hertwig, R. 1879. Der Organismus der Radiolarien. Ver- Micropaleontology 36: 81–89. lag von Gustav Fischer, Jena. Borgert, A. 1898. Beiträge zur Kenntnis des in Sti- Hollande, A. and Enjumet, M. 1960. Cytologie, evolution cholonche zanclea und Acanthometridenarten vorkom- et systematique des Sphaeroides (Radiolaires). Archives 182 Noritoshi Suzuki et al.

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