Zoomorphology (2008) 127:143���159 DOI 10.1007/s00435-008-0059-3 123 ORIGINAL PAPER Muscular systems in gymnolaemate bryozoan larvae (Bryozoa: Gymnolaemata) Alexander Gruhl Received: 8 November 2007 / Accepted: 22 January 2008 / Published online: 26 February 2008 �� Springer-Verlag 2008 Abstract Gymnolaemate bryozoan species exhibit larval forms that can either be planktotrophic cyphonautes or one of the several types of lecithotrophic larvae. Due to the lack of both a phylogenetic system of the Bryozoa and detailed information about larval morphology for many species, it is diYcult to elaborate hypotheses about the evolution of lar- val forms in this group at the moment. In this study, phal- loidin staining and confocal laser scanning microscopy are used to provide detailed morphological descriptions of the three-dimensional structure of muscular systems in the lar- vae of Flustrellidra hispida, Alcyonidium gelatinosum, Membranipora membranacea, Bugula fulva and Bower- bankia gracilis. A comparison to the available data from the literature is added. The results show muscular systems to be much more complex than previously recognized. In all but one species, an anterior median muscle inserts with one end at the basal side of the apical organ and with its other end at the papilla of the pyriform organ. This muscle can be paired or unpaired and was referred to as ���neuro- muscular strand��� in earlier descriptions. All species except Bugula fulva exhibit a coronal ring muscle running concen- tric to the larva���s main locomotory ciliary band. Lateral muscle strands that connect the pallial epithelium to either the corona or oral epithelium are found mainly in the cyph- onautes and pseudocyphonautes larvae and, in modiWed shape though, also in some coronate larvae. Transversal or shell adductor muscles can be shown to co-occur with lat- eral muscles and possibly originate from the same source. The Wndings are in accordance with the idea of a multiple origin of lecithotrophic forms within gymnolaemate Bryozoa. Keywords Bryozoa �� Gymnolaemata �� Larva �� Musculature �� Morphology �� Evolution Introduction Bryozoa or Ectoprocta are colonial, sessile suspension-feed- ers occurring in both marine and limnetic habitats. Com- monly three major subtaxa are recognized: Phylactolaemata, Stenolaemata, and Gymnolaemata. The Phylactolaemata exclusively inhabit freshwater with about 60 species world- wide. Stenolaemata with its only recent subtaxon Cyclosto- mata are caliWed and occur in the oceans. Gymnolaemata consist of Ctenostomata, which are uncalciWed and exhibit marine and a few brackish-water and freshwater species, and the calciWed Cheilostomata representing the dominating group of bryozoans in the oceans. Recent data argue for Cte- nostomata to be a paraphyletic assemblage including possi- ble sister groups of both Cheilostomata and Cyclostomata (Todd 2000 Ernst and Sch��fer 2006). Whereas bryozoan colonies grow through the asexual process of budding, new colonies (genets) arise sexually in most cases. Most bryozoans are colonial hermaphrodites with their reproducing zooids being either gonochorists or proterandrous hermaphrodites. Fertilization seems to be generally internal, as shown at least for gymnolaemates (Temkin 1994, 1996). Zygotes are shed through the supra- neural canal into the water in species with planktotrophic larvae. In species with lecithotrophic larvae, eggs are either placed into external brood chambers or retained in the body cavity, in such cases where extraembryonic nutrition occurs (Str��m 1977). A. Gruhl (&) Systematik und Evolution der Tiere, Institut f��r Biologie, Freie Universit��t Berlin, K��nigin-Luise-Str. 1-3, 14195 Berlin, Germany e-mail: agruhl@zoosyst-berlin.de

144 Zoomorphology (2008) 127:143���159 123 Gymnolaemate larvae are diverse in many aspects but share a general body organization, consisting of a deWned set of organs and tissues: The most prominent structures are the corona, which is the locomotory ciliary band, and a ciliated apical sense organ. The pyriform organ consists of glands and sensory cells and is involved in settlement. The internal sac is an invaginated epithelium that is expelled at the onset of metamorphosis (see Fig. 1 for a brief over- view see Nielsen 1971 Zimmer and Woollacott 1977 Reed 1991 for comprehensive reviews). There have been several attempts to classify the diVerent larval forms, the most well known of which might be from Zimmer and Woollacott (1977). These authors diVerentiate between seven types of gymnolaemate larvae including the well known cyphonautes, shelled lecithotrophic (pseudocyph- onautes) larvae, as well as Wve types of so-called coronate larvae (Type O, E, AE, AEO/ps and AEO/PS). The latter types resemble each other in being lecithotrophic and unshelled. They are distinguished mainly by position and extension of the corona (although the types share more characters): Type O and E have rather narrow coronae that are located either orally (O) or equatorially (E). In type AE, the corona is expanded and covers the equatorial and aboral part of the larva. The aboral epidermis is invaginated and forms a so-called pallial sinus. AEO/ps larvae have expanded coronae that form most of the larval surface and a small pallial sinus, whereas that of the oral-aborally elon- gated AEO/PS larvae is large. When trying to correlate larval types with higher level taxa in traditional bryozoan classiWcation (Boardman et al. 1983 Hayward 1985 McKinney and Jackson 1989 Hay- ward and Ryland 1998), it turns out that many types seem to be ���randomly��� distributed, suggesting that they have arisen independently multiple times within Bryozoa. How- ever, assumptions like these can only be drawn reliably when a suYcient amount of background knowledge is available. This would include a reliable phylogenetic sys- tem, which is unfortunately not available at present. Fur- thermore, suYciently detailed morphological data are required for as many taxa as possible in order to make con- Wdent decisions about primary homology of structures. The currently available descriptions of gymnolaemate larvae diVer tremendously in their level of detail. Therefore a comparative approach on certain larval characters might have the potential to bring new light into an evolutionary discussion. A useful character should be easily accessible, be complex, and show a certain amount of variation in order to be informative for phylogenetic analyses (Jenner and Schram 1999). Musculature has been widely ignored in most studies on larval morphology so far, mainly because of methodic diYculties. However since methods like Xuo- rescence staining and confocal laser scanning microscopy (CLSM) are available, musculature meets these require- ments, as already shown in many other metazoan groups (e.g., Wanninger 2004 M��ller and Worsaae 2005 Wannin- ger et al. 2005 Fuchs et al. 2006 Leasi et al. 2006 Schmidt-Rhaesa and Kulessa 2007). Three-dimensional analyses of muscular anatomy have already contributed to solve evolutionary or phylogenetic problems (Haszprunar and Wanninger 2000 Purschke and M��ller 2006). Fig. 1 General morphology of bryozoan larvae, represented here by two types. Because the terms dorsal and ventral can be misleading in bryozoan larvae, body axes are termed by convention (Zimmer and Woollacott 1977 Reed 1991) aboral or apical for the former animal re- gion of the embryo bearing the apical organ and oral for the opposite side that is derived from the vegetal region. The anterior (ant) region is deWned by the presence of the pyriform organ, the opposite region is posterior (pos). A ciliary band, the corona (co), consisting of multicil- iated cells is the main organ for swimming. All larvae bear an apical organ (ao), that can be either simple, that is, consisting of a small num- ber of cells, or complex. In the latter case it is usually referred to as api- cal disc. The pyriform organ (po), a complex of ciliated and glandular cells is situated anteriorly. At the posterior side, the internal sac (is), an epithelial invagination, functioning as adhesive organ during setttle- ment, can be found. a Coronate larva are lecithotrophic. The corona forms the largest part of the larval surface. The gut is either absent or vestigial. The aboral epithelium is invaginated in some cases, forming the so-called pallial sinus (ps). Between pyriform organ and internal sac a small ciliated cleft (cc) is located. b Cyphonautes larvae are planktotrophic and have a functional digestive tract (in). The aboral epithelium secretes the lateral shells (sh). The oral surface is densely ciliated and invaginated forming the atrium (at) with its ciliated ridges (cr), where plankton particles are captured. an anus, mo mouth

Zoomorphology (2008) 127:143���159 145 123 Morphology of the larval musculature in Bryozoa is closely linked to larval biology and metamorphosis (Reed 1985) so that many diVerences between the larvae should be reX- ected in their musculature. The aim of this study is to compare muscular systems of gymnolaemate larvae. Musculature of Wve gymnolaemate species is examined by means of phalloidin staining and CLSM. Species were chosen, for which detailed ultrastruc- tural information is already available from the literature, if not for the same species, at least for congenerics. This is done to validate comparisons between data obtained by CLSM and transmission electron microscopy (TEM). The obtained results are then extended by the results from the literature and discussed in a phylogenetic context. While Wnishing this manuscript, a partially overlapping study was published by Santagata (2008). Materials and methods Collection of larvae Reproducing colonies of Flustrellidra hispida (Fabricius, 1780) and Alcyonidium gelatinosum (Linnaeus, 1761) were collected in the rocky intertidal of Concarneau (Bretagne/ France) between 2005 and 2006. Both species prefer thalli of Fucus serratus, whereas F. hispida occurs on the lower parts of the phylloids, and A. gelatinosum inhabits the tips. Bugula fulva Ryland, 1960 was collected in the harbour of Brest (Bretagne, France) on the undersides of Xoating docks in May 2007. Bowerbankia gracilis Leidy, 1855 was collected in the harbour of Helgoland (Germany) in June 2006. Cyphonautes larvae of Membranipora membranacea (Linnaeus, 1767) were collected in Laminaria forests in Helgoland (Germany) in June 2006 using a 70- m plankton net. As it is not possible to determine the cyphonautes lar- vae safely, about half of the collected larvae were allowed to settle in glass dishes and observed over a period of 3 days. All formed twin-ancestrulae, which are unique for M. membranacea. Colonies of F. hispida and A. gelatino- sum were kept in vessels with a constant current of fresh seawater entering the vessel at its base. The overXow at the top of the vessel was directed into a chamber with a 100- m plankton net. Shed larvae were obtained from the plankton net twice daily, whereas larval release was highest during the morning hours. Colonies of B. fulva and B. grac- ilis were kept in 300-ml glass dishes. Emerging larvae are strongly phototactic and were attracted to the vessel wall by Wber-optics illumination. Clouds of larvae were then trans- ferred into smaller dishes for Wxation using a glass pipette. Live observations of larvae were accomplished with a Leica MZ 16A dissecting microscope and an Olympus BZ51 compound light microscope. Phalloidin staining Specimens were Wxed in 4% paraformaldehyde in 0.05 M PBS containing 0.33 M NaCl. Duration of Wxation was from 30 min to 6 h at room temperature. Some larvae were relaxed using a 7% MgCl2 solution prior to Wxation. Phal- loidin staining was accomplished by incubating permeabili- zed (0.1 % Triton-X 100 in PBS, 2 h) specimens with Alexa-Flour-568 labeled phalloidin at a Wnal dilution of 1 U/500 l for 6���12 h. Specimens were rinsed in PBS and mounted on slides using CitiXour Glycerol solution. Confo- cal image stacks were taken on a Zeiss LSM 410 and a Leica TCS SPE. Altogether 24 specimens of F.hispida, 11 of A. gelatinosum, 10 of M. membranacea, 14 of B. fulva, and 9 of B. gracilis were examined. ImageJ with WCIF plu- gin bundle was used to process digital image stacks and to generate projection views. Three-dimensional inspection was done by volume rendering using Voxx2. Scanning Electron Microscopy Specimens were Wxed either with 2.5% Glutaraldehyde in 0.05 M PBS containing 0.33 M NaCl (1 h, 4��C) or with 1% OsO4 in seawater. Fixative was removed by washing sev- eral times with PBS. Specimens were dehydrated in a graded ethanol series, critical point dried, mounted, and subsequently sputter-coated with gold. Specimens were examined using a FEI Quanta 200 SEM at 15 kV. Results Flustrellidra hispida (Ctenostomata, Flustrellidridae) The larva of F. hispida is of the shelled lecithotrophic type, sometimes also referred to as pseudocyphonautes, indicat- ing its morphological resemblance to the planktotrophic cyphonautes. Descriptions on its morphology are found in Barrois (1877), Prouho (1890), and Pace (1906). Ultrastruc- tural details have been added by d���Hondt (1977c). The larva (Fig. 2a) is of elongate shape and nearly trian- gular in cross-section. It bears two chitinous shells that completely cover the lateral sides of the body. The oral sur- face is heavily ciliated and a pronounced ciliary band, the corona, runs at the rim of the oral surface. Anteriorly the pyriform organ is located, visible from exterior by the occurrence of several tufts of cilia (collectively referred to as vibratile plume). The larval mouth is situated directly behind the pyriform organ, leading into a lumen-less gut. The internal sac appears as a deep invagination posterior to the larval mouth. It is made up of a thickened glandular epi- thelium that shows clear regionalization. At the posterior end of the larva, a Weld of elongate cilia is present. At the