Steven Sadro Fig. I . Generalized life cycles o f typical solitary ascidians (top) and compound ascidians (bottom).The diagrams incorporate characteristics o f many species, not all aspects of which are present in the Widespread interest in the study of tunicates began after Kowalevskys publications (1886-1871) describing the chordate nature of the ascidian tadpole larva. Since then, larvae from ten families worldwide of the class Ascidiacea have been described (Cloney, 1982). There are ca 60 species of tunicates from ten families found in the waters of the Pacific Northwest (see Tables 1 and 2). small, hatching / simple tadpole external embryogenesis \ settlement external fertilization SOLITARY OVIPAROUS URODELE t metamorphosis / , short planktonic large, complex taqpole f \ 1 settlement larval release metamorphosis COLONIAL OVOVIVIPAROUS URODELE internal embryogenesis fusion with seasonal blastozooids life cycle o f any given species. (From Svane and Young, 1989) Urochordata: Ascidiacea Reproduction and Development Ascidian species that reproduce sexually are considered simple ascidians and are solitary. Species that reproduce both sexually and asexually (e.g., through budding) are considered compound ascidians and have a colonial growth form (Berrill, 1975; Strathmann, 1987). Most ascidians are simultaneous hermaphrodites, and examples of both self-fertile species and species that are not self-fertilizing exist (Berrill, 1975). Ascidians may be oviparous (typically solitary forms), ovoviviparous (typically compound forms), or viviparous (Fig. 1). Most solitary oviparous ascidians produce large numbers of relatively small eggs that undergo development into tadpole larvae (Berrill, 1950). Compound ascidians generally produce only a few large eggs that develop into relatively complex tadpole larvae. All ascidian larvae are lecithotrophic, though some are direct-developing and bypass the tadpole stage completely to hatch as small juveniles (e.g., Molgula pacifica and Pelonia corrugata). Ascidian tadpole morphology has received considerable study and review (see Millar, 1971; Berrill, 1975; Cloney, 1978, 1982; Katz, 1983; Svane and Young, 1989; Burighel and Cloney, 1997). There is large variation in ascidian size, color, and complexity of internal structures. Compound ascidians have the largest larvae (to 4.5 mm long) and most complex internal anatomy (Fig. 2). Solitary ascidians typically have smaller larvae (to 1.0 mm long) and possess less complex internal anatomy. The surface of all ascidian larvae is covered by a transparent tunic. branchial sensory siphon ganglion vesicle visceral ganglion Fig. 2. Diagrammat~c compound ascidian larva of Disralpia occidentalis. Details of the atrium, inner cuticular layer; and cells of the tunic are omitted.The length of the trunk is I mm and of the entire larva, including the caudal fin, 3.2 mm. (From Cloney, 1982) Identification Guide t o Larval Marine Invertebrates of the Pacific Northwest Fig. 3. Basic morphological differences between ascidian tadpole larvae and larvaceans. (A) Ascidian tadpole larva. (8) Larvacean Oikopleura sp. Scale = I00 pm (A from Berrill, 1947, Fig. I; B from A Guide to Marine Coastal Plankton and Marine Invertebrate Larvae, Second Edition, by DeBoyd L. Smith and Kevin B. Johnson. Copyright 1996 by DeBoyd L. Smith and Kevin B. Johnson. Reprinted by permission of KendallIHunt Publishing Company) Fig. 4. (A) Corella inpato, swimming larval stage, showing the trunk and part o f the tail.The outer (C I ) and inner (C2) cuticular layers o f the tunic are visible. In this species, C2 terminates at the base of the tail. (Total length 0.28 mm.) (B) Ascidia paratropa, larval trunk with about half o f the tail visible in a lateral view.The outer cuticular layer o f tunic (C I ) is covered with firmly attached test cells (TC). (Total length 0.54 mm.) (From Burighel and Cloney, 1997) Because of the short pelagic duration and small range of dispersal of most tadpole larvae (Svane and Young 1989), the likelihood of encountering them in the plankton is low. Special care should be taken to not confuse them with the more abundant larvaceans, which if superficially examined outside their "houses" are somewhat similar in morphology to tadpole larvae (Fig. 3). The most obvious feature differentiating larvaceans from ascidian tadpole larvae is the position of the tail (S. Bassham, pers. comm.). In ascidian tadpole larvae, the tail protrudes from the posterior end of the trunk; in larvaceans, the tail is shifted to a position about halfway between the mouth and the posterior end of the trunk. Other features present in larvaceans but not tadpole larvae include a mouth at the anterior end of the trunk and gonad masses (in ripe larvaceans). ldentification of Local Taxa Lacking morphological information on most larval ascidian, this chapter presents a compilation of useful diagnostic characteristics with pictures included when available. From this information identification to species level is not possible for most larvae, though in some cases one may identify the Urochordata: Ascidiacea family or genus. At present, the only way to identify a larval type to species is to raise larvae to the adult stage. See Strathmann (1987) for information on culturing larvae. Relatively few characteristics are available to key ascidian larvae without difficult dissections and tissue analysis. Easily observed morphological characteristics such as color, length, and shape of trunk are useful when differentiating between solitary and colonial ascidian larvae. To distinguish among the less differentiated solitary ascidian larvae usually requires closer examination through a compound microscope to examine the cuticular layers of the trunk tunic and the attached test cells. All ascidian larvae have two cuticular layers of tunic (Fig. 4), but the inner cuticular layer is absent in the tail of some species. The larvae of some species of solitary ascidian have prominent, firmly attached test cells on the outer cuticular Fig. 5. Solitary ascidian larvae: (A) Ciona intestinalis. (6) Boltenia villosa. (C) Metandrocarpa taylori. (D) Styela sp. Species o r genera found locally are in bold. Abbreviations: adhesive papillae (p), cerebral vesicle (cv). (A from Berrill, 1947, Fig. I ; B from Cloney, 196 1 , Plate I - I ; C from Abbott. 1955. Fig. I; D from Grave, 1944, Fig. I ) Fig. 6. Larvae o f solitary ascidians (tails not shown). (A) Ciona intestinalis. (6) Perophora listeri. (C) Pyum microcosmus. (D) Molgula citrina. (E) Ascidia mentulo. (F) Styela portita. (G) Dendrodoa grossuloria. Species o r genera found locally in bold. Figures not t o scale. (From Millar; 197 1, Fig. 5) 298 Identification Guide t o Larval Marine Invertebrates of the Pacific Northwest Table 1. Diagnostic information for solitary ascidian species Species Mean Length (mm) Order Pleurogona Suborder Phlebobranchia Family Cionidae Ciona intestinalis I . I Family Perophoridae Perophora annectens Family Corellidae Chelyosoma columbianum Chelyosoma productum 1.2 Corella inflata 0.9 Corella willmeriana 0.9 Family Ascidiidae Ascidia callosa 1.2 Ascidla paratropa 1.2 Ascidia prunum I .2 Ascidia ceretodes I .2 Suborder Stolidobranchia Family Styelidae Cnemidocarpa finmarkensis Dendrodoa abbotti Metandrocarpa dura Metandrocarpa taylor~ Styela clavata Styela coriacea Styela gibbsii Styela montereyensis Styela truncata Styela (barnham) clava Family Pyuridae Boltenia villosa Halocynthio aurant~um Halocynthia igaboja Pyura haustor Pyura mirabilis Family Molgulidae Mo/gula cooper1 Molgula manhattensis Molgula oregonia Molgula pacifica Molgula pugetiensis Color Tail with Attached Inner Cuticular Test Cells1 Layer' Transparent + t Transparent + + Transparent + Transparent + Transparent + Transparent Transparent Transparent -? Transparent -? Pale Rose. Orange + Transparent Vermillion Orange Orange Orange Orange Transparent Pale Yellow Pale Yellow Transparent I Presence o f character: + = yes; - = no. Urochordata: Ascidiacea layer of tunic which are visible under a compound microscope (Burighel and Cloney, 1997). Solitary Ascidians All solitary ascidian larvae are small ( d . 5 mm length) and have relatively simple internal structures (Figs. 5, 6). All but the family Molgulidae have three simple attachment papillae with a coniform shape and a triangular configuration (see Fig. 5). The Molgulidae lack attachment papillae. Ciona intestinalis and all species from the family Ascidiidae have firmly attached test cells on the outer cuticular layer of tunic. The inner cuticular layer of the tunic is absent in the tail of the following species: Corella inflata, Chelyosoma productum, Ascidia callosa, A. paratropa (probably all ascidiids), and Molgula occidentalis (probably all molgulids) (R. A. Cloney, pers. comm.) (see Fig. 4). Corella and Chelyosoma species have trunks compressed in the sagittal plane. Halocynthia species have long trunks that taper down in Fig. 7. Larvae of compound ascidians. (A) Clavelina lepadrforrnis. (6) Pycnoclavella stanleyi. (C) Distaplia rosea. (D) Cystodytes dellechiajei. (E) Eudistoma illoturn. (F) Synoicum georgianurn. (G) Euhedmania claviformis. ( H ) Apidium nordarnanni. (I) Diplosoma listerianum. u) Botrylloides leachi. (K) Sycozoa sigillinoides. (L) Archidistoma aggregaturn. (M) Didemnum albidum. (N) Didemnum helgolandicurn. (0) Ritterela ~ b m . Species o r genera found locally are in bold. Figures A-L, N are not t o scale. (A-K, N from Millar; 197 I. Fig. 5; L from Berrill, 1948, Fig. 2; M from Marks 1996, Fig. 4A; 0 from Abbott andTrason, 1968, Fig. I C) Identification Guide to Larval Marine Invertebrates of the Pacific Northwest diameter toward the papillae. All other species listed have trunks that are round to ovoid in cross section at the middle of the trunk. Additional diagnostic information on other solitary species is compiled in Table 1. Table 2. Diagnostic information for compound ascidian larvae Species Length Color Adhesive Papillae Tail Twisted (mm) Shape1 Configuration Counter- Clockwise Order Enterogona Suborder Aplousobranchia Family Clavelinidae Archid~stoma molle Archidistoma psammion Eudistoma ritteri Eudistoma purpuropunctatum Clavelina huntsmani Clavelina sp. Cystodytes lobatus Distaplia occidentalis Distaplia smithi Pycnoclavella stanleyi Family Polyclinidae Aplidium arenotum Aplidium californicum Aplidium glabrum Apl~dium propinquum Aplidium solidum Aplidium sp. Euherdmania claviformis Ritterella aequalisiphonis Ritterella pulchra Ritterella rubra Synoicum parfistis Synoicum sp. Family Didemnidae Didemnum albidum Didemnum carnulentum Diplosoma listerianum Trididemnum opacum Trididemnum strangulatum Suborder Stolidobranchia Family Styelidae Botrylloides sp. Botryllus sp. SCY Orange Scy Goblet Goblet Goblet Goblet Goblet Goblet T-inv Sagittal Sagittal Triangle Triangle Sagittal Sagittal Sagittal Sagittal Sagittal Sagittal s CY Sagittal Sim SCY Triangle 'Sty = scyphate, the papillae appear to be set in cup-shaped invaginations in the trunk (e.g., Distaplia, see Fig. 2);T-inv = inverted (invaginated) tubular papillae that extend deeply into the body cavity of the trunk (e.g., Pycnoclavella, Fig. 7B); Goblet = goblet-shaped papillae with elongate tubular stalks (e.g., Aplidium, Fig. 7H). Urochordata: Ascidiacea Compound Ascidians All larvae from colonial ascidians are greater than 1.5 mm in length and have relatively complex internal structures. All have three adhesive papillae except Euherdmania claviformis, which has two. Additional diagnostic characteristics of the larvae of local colonial ascidians are compiled in Table 2, and compound ascidian larvae are illustrated in Fig. 7. References Abbott, D. P. (1955). Larval structure and activity in the ascidian Metandrocarpa taylori. J. Morph. 97569-94. Abbott, D. P. and W. Trason (1968). Ritterella rubra and Distaplia smithi: Two new colonial ascidians from the west coast of North America. Bull. So. Calif. Acad. Sci. 67143-53. Berrill, N. J. (1947). The development and growth of Ciona. Jour. Mar. Biol. Assoc. 26:616-25. (1948). Structure, tadpole and bud formation in the ascidian Archidistoma. Jour. Mar. Biol. Assoc. (UK) 27:380-88. (1950). The Tunicata with an account of the British species. London: The Ray Society. (1975). Chordata: tunicata. In: Reproduction of Marine Invertebrates, A. C. Giese and J. S. Pearse (eds.), pp 241-82. Academic Press, New York. Burighel, P. and R. A. Cloney (1997). Urochordata: Ascidiacea. In: Microscopic Anatomy of Invertebrates, F. W. Harrison and F.-S. Chia (eds.), pp. 221-347. Wiley-Liss, Inc., New York. Cloney, R. A. (1961). Observations on the mechanism of tail resorption in ascidians. Amer. Zool. 1:67-87. (1978). Ascidian metamorphosis: review and analysis. In: Settlement and Metamorphosis of Marine Invertebrate Larvae, F.-S. Chia and M. Rice (eds.), pp. 255-82. Elsevier, New York. (1982). Ascidian larvae and the events of metamorphosis. Amer. Zool. 22:817-826. Grave, C. (1944). The larva of Styela (Cynthia) partita: structure, activities and duration of life. J. Morph. 75:173-91. Katz, M. J. (1983). Comparative anatomy of the tunicate tadpole, Ciona intestinalis. Biol. Bull. 1641-27. Marks, J. A. (1996). Three sibling species of didemnid ascidians from northern Norway: Didemnum albidum (Verrill, 1871), Didemnum polare (Hartmeyer, 1903), and Didemnum romssae sp. nov. Can. J. Zool. 74:357-79. Millar, R. H. (1971). The biology of Ascidians. Adv. Mar. Biol., Vol. 9:l-100. Strathmann, M. F. (1987). Reproduction and Development of Marine Invertebrates of the Northern Pacific Coast. University of Washington Press, Seattle. Svane, I. B. and C. M. Young (1989). The ecology and behaviour of ascidian larvae. Oceanogr. Mar. Biol. Annu. Rev. 274.5-90.