Published for The Royal Swedish Academy of Sciences and The Royal Danish Academy of Sciences and Letters Acta Zoologica (Stockholm) 97: 102–116 (January 2016) doi: 10.1111/azo.12109 Early stalked stages in ontogeny of the living isocrinid sea lily Metacrinus rotundus Shonan Amemiya,1,2,3 Akihito Omori,4 Toko Tsurugaya,4 Taku Hibino,5 Masaaki Yamaguchi,6 Ritsu Kuraishi,3 Masato Kiyomoto2 and Takuya Minokawa7 Abstract 1Department of Integrated Biosciences, Amemiya,S.,Omori,A.,Tsurugaya,T.,Hibino,T.,Yamaguchi,M.,Kuraishi,R., Graduate School of Frontier Sciences, The Kiyomoto,M.andMinokawa,T.2016.Earlystalkedstagesinontogenyoftheliving University of Tokyo, Kashiwa, Chiba, isocrinid sea lily Metacrinus rotundus. — Acta Zoologica (Stockholm) 97: 102–116. 277-8526, Japan; 2Marine and Coastal Research Center, Ochanomizu University, The early stalked stages of an isocrinid sea lily, Metacrinus rotundus,wereexam- Ko-yatsu, Tateyama, Chiba, 294-0301, ined up to the early pentacrinoid stage. Larvae induced to settle on bivalve shells 3 Japan; Research and Education Center of and cultured in the laboratory developed into late cystideans. Three-dimensional Natural Sciences, Keio University, Yoko- (3D) images reconstructed from very early to middle cystideans indicated that hama, 223-8521, Japan; 4Misaki Marine 15 radial podia composed of five triplets form synchronously from the crescent- Biological Station, Graduate School of Sci- ence, The University of Tokyo, Misaki, shaped hydrocoel. The orientation of the hydrocoel indicated that the settled Kanagawa, 238-0225, Japan; 5Faculty of postlarvae lean posteriorly. In very early cystideans, the orals, radials, basals and Education, Saitama University, 255 Shim- infrabasals, with five plates each in the crown, about five columnals in the stalk, o-Okubo, Sakura-ku, Saitama City, 338- and five terminal stem plates in the attachment disc, had already formed. In 6 8570, Japan; Division of Life Science, mid-cystideans, an anal plate appeared in the crown. Late cystideans cultured in Graduate School of Natural Science and the field developed into pentacrinoids about 5 months after settlement. These Technology, Kanazawa University, Ka- pentacrinoids shared many crown structures with adult sea lilies. On the other kuma, Kanazawa, 920-1192, Japan; 7Research Center for Marine Biology, To- hand, many features of the stalk differed from those in adult isocrinids, while hoku University, Sakamoto 9, Asamushi, sharing many characteristics with the stalk of feather star pentacrinoids, includ- Aomori,039-3501,Japan ing disc-like proximal columnals, high and slender median columnals, synarthri- al articulations developmentally derived from the symplexial articulations, Keywords: limited formation of cirri only in the proximal columnal(s), and an attachment sea lily, crinoid, isocrinid, echinoderm, disc. On the basis of these findings, phylogenetic relationships among extant cri- Articulata, Metacrinus rotundus, cystidean, noid orders are discussed. pentacrinoid, scanning electron microscopy, 3D reconstruction Shonan Amemiya, Marine and Coastal Research Center, Ochanomizu Univer- sity, Ko-yatsu, Tateyama, Chiba 294-0301, Japan. E-mail: [email protected]. Accepted for publication: ne.jp 04 November 2014 now accepted that the comatulids plus the stalked bourgueti- Introduction crinids are derived within the articulates, there is no consensus Crinoids, comprising the stalked sea lilies and the stalkless co- about relationships among the more basal stalked orders. One matulids, are widely recognized as a sister group to the other of these basal articulate groups is the order Isocrinida, which four classes of extant echinoderms and considered to be the includes Metacrinus rotundus Carpenter, 1885, the most acces- most basal of living echinoderms (Paul and Smith 1984; Lit- sible sea lily known, being relatively abundant at depths of tlewood et al. 1997; Janies 2001; Scouras and Smith 2006; 100–150 m off the southern coast of Japan (Oji 1989). Infor- Rouse et al. 2013). All extant crinoids belong to the subclass mation on the development of isocrinids is important when Articulata, which originated from Palaeozoic sea lilies near the considering evolution within the echinoderms in general and start of the Mesozoic era (Ubaghs 1978b; Hess and Ausich the crinoids in particular, and more arguably for discussing 1999; Hess 2011). At present, some of the relationships evolution of the deuterostomes. among the extant orders of articulate crinoids remain contro- We have previously reported the development of M. rotun- versial (Hess and Messing 2011). For example, although it is dus between fertilization and larval settlement (Nakano et al. 102 © 2014 The Authors. Acta Zoologica published by John Wiley & Sons Ltd on behalf of Royal Swedish Academy of Sciences. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. Acta Zoologica (Stockholm) 97: 102–116 (January 2016) Amemiya et al. Early stalked stages in sea lily 2003). At that time, the swimming larval stages were and the settled postlarvae in the cups were examined on 19 describedindetail,butthesettledstageswerenotcharacter- March and 3 April 2008. ized substantially because the larvae died soon after they had settled.Here,therefore,wedescribe the newly settled stages Preparation of specimens at the early attached stage for scanning and development throughout the early pentacrinoid stage. electron microscopy (SEM) This is the first description of these stages based on living material. We focus on the developing skeletal system, to Living organisms at the early attached stage were fixed over- enable comparison with the adult condition and comparable night in seawater-Bouin’s fluid, dehydrated in a graded etha- developmental stages of feather stars. The axial organization nol series and critical-point-dried in a CP-5A CO2 dryer of the postlarva is discussed based on Carpenter’s system of (Topcon, Tokyo, Japan). The dried specimens were attached the hydrocoel and surrounding skeletal elements. to aluminium stubs via double-sided adhesive tape, coated withgoldinanE-101ionsputtercoater(Hitachi,Tokyo, Japan) and observed with an S-3000N SEM (Hitachi). Materials and Methods Animals and larvae Visualization of the skeleton by SEM and polarizing microscopy Adults of the isocrinid sea lily M. rotundus were collected Postlarvae at various stages were fixed with 70% ethanol using a gill net from a depth of 130 m in the south-eastern (EtOH) after removal from the substrates. The fixed speci- area of the Uraga Channel in the Sagami Sea, Japan, from late mens were rinsed with 70% EtOH and preserved in 70% September to middle October. Eggs and sperm were obtained EtOH at room temperature. The preserved specimens were and fertilized, and developmental stages before settlement hydrated with a graded ethanol series, treated with bleach were cultured as described previously (Amemiya et al. 2013). containing 0.4% sodium hypochlorite for 40 s, rinsed with Occasionally, artificial sea water (Jamarin U) containing or distilled water, dehydrated with a graded ethanol series, air- lacking antibiotics (100 lg/mL streptomycin and 100 unit/ dried and mounted on an aluminium stub with double-sided mL penicillin) was used for larval culture. The use of artificial electrically conductive adhesive tape. The specimens were sea water might potentially delay larval development. The occasionally bleached further with the same or a higher antibiotics were effective for keeping the swimming larvae (0.6–1.2%) concentration of sodium hypochlorite for the healthy, but reversibly prevented their settlement. same period or longer (1–2 min) on the adhesive tape stuck to the aluminium stub. The dried specimens were observed with a JSM-6510LV SEM (JEOL, Tokyo, Japan) in back-scattered Induction of settlement electron mode to examine the skeletal ossicles. Two very early To induce settlement of the swimming larvae of M. rotundus, cystideans were dehydrated with a graded ethanol series after we added shell fragments of Cryptopecten sp. (a pectinid treatment with bleach and photographed in benzyl benzoate bivalve collected using a gill net along with the adult sea lilies) andbenzylalcohol(2:1)(BABB)underaBiophot(Nikon, to the culture dishes. The larvae became attached to the shells Tokyo, Japan) or a BX53F (Olympus, Tokyo, Japan) DIC or, less often, to the inner wall of the culture dishes. The set- microscope. tled postlarvae, which initially did not feed, were maintained in a laboratory incubator in the dark at 14 °C. Occasionally, Three-dimensional (3D) reconstruction middle to late cystideans were cultured at 16 °C, 18 °Cor 20 °C to examine their tolerance to higher temperatures. A very early cystidean, about 8 days after settlement, and a They grew healthily up to 18 °C, but became weakened and mid-cystidean, about 19 days after settlement, were fixed with died at 20 °C. 4% paraformaldehyde for 16 h according to Omori et al. (2011). An early cystidean, about 9 days after settlement, was fixed with seawater-Bouin’s fluid overnight. After dehydration Raising of postlarvae from the late cystidean to pentacrinoid stages in a graded ethanol series and transfer through 100% acetone, Eleven disposable plastic cups with volumes of 350 mL or the specimens were embedded in Technovit 8100 resin (Kul- 500mLwereusedtoraisethepentacrinoids.Asmallfrag-