Biol. Lett. (2005) 1, 178–180 Hereafter, polyembryony refers to the monozygotic doi:10.1098/rsbl.2004.0259 process. The persistence of polyembryony in certain Published online 16 May 2005 taxa has puzzled evolutionary biologists, because it can appear to combine the contrasted fitness disad- vantages of cloning and sexual reproduction, while Paradoxical compromising the respective benefits (Craig et al. 1995, 1997; but see Hardy 1995a). polyembryony? Embryonic Extensive polyembryony, generating copious clone- cloning in an ancient order mates per zygote, is characteristic of rust fungi (Alexopoulos 1952), red algae (Searles 1980), of marine bryozoans encyrtid wasps (Hardy 1995b) and cyclostome bryozoans (Ryland 1996). Some authors also include Roger N. Hughes1,*, M. Eugenia D’Amato2,†, cases where cloning occurs in post-embryonic or John D. D. Bishop3,4, Gary R. Carvalho2, larval stages of the life history, as in endoparasitic Sean F. Craig1,5, Lars J. Hansson3,‡, hydrozoans, flatworms and barnacles and in a few Margaret A. Harley2 and Andrew J. Pemberton3 starfish and brittle stars (reviewed in Craig et al. 1School of Biological Sciences, University of Wales Bangor, (1997); see also Cable & Harris (2002) on gyrodacty- Bangor LL57 2UW, UK lid flatworms). Limited polyembryony, or ‘twinning’, 2Department of Biological Sciences, University of Hull, forms an integral part of the life history in many Hull HU6 7RX, UK 3Marine Biological Association of the United Kingdom, The Laboratory, gymnosperms (Filonova et al. 2002), certain angios- Citadel Hill, Plymouth PL1 2PB, UK perms (Carman 1997) and armadillos (Prodohl et al. 4School of Biological Sciences, University of Plymouth, 1996). Twinning also occurs aberrantly in diverse Drake Circus, Plymouth PL4 8AA, UK mammalian species (Gleeson et al. 1994). 5Department of Biological Sciences, Humboldt State University, Arcata, CA 95521, USA Polyembryony in cyclostome bryozoans was first *Author for correspondence ([email protected]) reported by Harmer (1892, 1898),andalthough † Present address: Department of Genetics, University of Stellenbosch, amended in certain detail, his interpretation of embry- ZA-7602 Stellenbosch, South Africa ‡Present address: Danish Institute for Fisheries & Marine Research, ogenesis in Crisia species, based on microscopy, was Kavalergarden 6, DK-2920 Charlottenlund, Denmark corroborated by Borg (1926) for a range of genera. A Prolific polyembryony is reported in few major syncytial reticulum nourishes the budding embryos, all taxa, but its occurrence has generated theo- enclosed within an expanded chamber. Although this retical debate on potential conflict between brood chamber varies considerably in size among sexual and asexual reproduction. It is, therefore, cyclostome families, it is almost always relatively much important to genetically confirm a widely cited larger than the ovicells of cheilostome bryozoans, inference, based on microscopy, that poly- which typically house single embryos (Ryland 1970). embryony characterizes marine bryozoans of We may, therefore, reasonably infer that the brood the order Cyclostomata. Microsatellite genotyp- chamber, recorded in all living families of cyclostomes ing of brooded embryos and maternal colonies except the Cinctiporidae (Boardman et al.1992), is conclusively demonstrated polyembryony, while genetic variation among broods within colonies associated with embryonic budding (Borg 1926; Stro¨m indicated outcrossing via water-borne sperm, in 1977). Moreover, because most post-Triassic fossil the rocky-shore species Crisia denticulata.The species, again apart from the Cinctiporidae, also characteristically voluminous brood chamber of possess similar brood chambers (McKinney & Taylor cyclostomes is judged to be an adaptation linked 1997) it is probable that embryonic budding is a to larval cloning and hence an indicator of plesiomorphic character of the crown-group Cyclosto- polyembryony. We speculate that although the mata (Taylor 2000; Taylor & Weedon 2000). In almost universal occurrence of polyembryony particular, microscopical evidence of polyembryony in among crown-group Cyclostomata is probably crisiids (above), which have primitive status within attributable to phylogenetic constraint, adaptive consequences are likely to be significant. cyclostome phylogeny, suggests that polyembryony arose basally in the crown group, while similar evidence Keywords: clonal reproduction; cross-fertilization; for lichenoporids (Borg 1926), which are advanced, larvae; microsatellites; phylogenetic constraint suggests that polyembryony was subsequently retained, becoming a general characteristic of cyclos- tomes (Taylor & Weedon 2000; P. D. Taylor, personal 1. INTRODUCTION communication). It would be desirable, nevertheless, Monozygotic polyembryony is a form of asexual to obtain independent confirmation of this inference reproduction that proceeds by division or budding for a more comprehensive range of examples. during post-zygotic or early embryonic stages of the Although the histological patterns described by life history (Hughes 1989). Only monozygotic poly- Harmer (1892, 1898) and Borg (1926) seem clearly embryony occurs in animals, whereas in certain to indicate polyembryony, the possibility of multiple higher plants, especially among angiosperms, fertilization within the brood chamber or of partheno- a second form of ‘polyembryony’ involves somatic genesis cannot be dismissed without knowing the embryogenesis and perhaps represents a maternal corresponding genotypic composition of mother strategy, without equivalents in animals, to counteract and embryos. Accordingly, we used previously iso- brood reduction resulting from sibling rivalry among lated microsatellite markers for Crisia denticulata sexual embryos (Shaanker & Ganeshaiah 1996). (Craig et al. 2001) to genotype mothers and their Received 19 May 2004 178 q 2005 The Royal Society Accepted 1 October 2004 Polyembryony in bryozoans R. N. Hughes and others 179 brooded embryos. Having thus confirmed polyem- 4. DISCUSSION bryonic cloning in C. denticulata, we discuss its Our data conclusively demonstrate polyembryony in evolutionary significance in the crown-group Cyclos- C. denticulata. Fertilization of multiple ova within a tomata and examine adaptive theories of brood chamber can be discounted since brood-mates polyembryony. were always genetically identical. Obligatory partheno- genesis of any type (White 1973; Bell 1982; Hughes 1989) is excluded by the widespread occurrence of 2. MATERIAL AND METHODS paternal alleles that were absent from corresponding Colonies of C. denticulata were collected from the low intertidal and maternal genotypes, verifying cross-fertilization. The shallow subtidal at Wembury, Devon, UK (National Grid reference SX518482; 488190 N4850 W). Independent samples were taken in genetic variation among broods was consistent with the summers of 1998 and 2001, the first being analysed at the open fertilization in a ‘sperm-cast’ mating system University of Hull and the second at The Marine Biological (Pemberton et al. 2003). It is clear, therefore, that Association, Plymouth. each brood chamber of C. denticulata accommodates For the 1998 samples, whole branches bearing brood chambers were preserved frozen in 0.1 M ethylenediaminetetraacetic acid one clone at a time and that different brood chambers (EDTA; pH 8) before the brood chambers were dissected and house different clones derived from separately ferti- embryos preserved individually in 8 ml EDTA in microwell plates. lized ova. Embryos and maternal branches were incubated overnight at 55 8C in 500 ml of a digestion solution containing 0.1 M Tris–HCl pH 8, The typically low frequency of brooding among 1.25% SDS, and 400 mM Proteinase K. After one phenol–chloro- zooids within colonies of cyclostome bryozoans led form and one chloroform extraction, DNA was precipitated over- Ryland (1996) to suggest that polyembryony compen- night at K20 8C in 0.3 M sodium acetate and two volumes of 99% sates for infrequent fertilization, inferred to be associ- ethanol. Samples were washed once in 70% ethanol, dried and resuspended in 50 ml of doubly distilled sterile water. Polymerase ated with limited sperm production of typically small chain reaction (PCR) reactions were performed with 2 ml DNA colonies living at low population density. Contrary to extracted from embryos, or 0.5 ml DNA from maternal branches, in this argument, however, sessile invertebrates that a20ml volume containing 0.3 mM dNTPs for embryos or 0.2 mM fertilize retained eggs by water-borne sperm seem able dNTPs for branches, 2 mM MgCl2, and 0.12–0.3 mM of each primer and 1 U BioLine Taq Polymerase. Optimal cycling con- to reduce sperm limitation through entrainment in the ditions for the primers CD 4b, CD 5, CD 7-1 and CD 17-3 were feeding current, together with prolonged storage of described in Craig et al. (2001). PCR products of the CD 5 end- viable allosperm (Pemberton et al. 2003). Low fre- labelled primers were screened on an ALFexpress automatic sequen- cer (Pharmacia) and run along with size markers obtained as in Van quency of brooding zooids might instead reflect Oppen et al. (1997). One hundred and fifty-seven individuals were limitation of the total maternal energy allocation that genotyped, representing six colonies, 3–8 chambers per colony and can be supported by a colony. Successful brooding in 3–10 embryos per chamber. the cheilostome C. hyalina requires translocation of Year 2001 samples were kept overnight in 100 ml of aged filtered