polyembryony? Embryonic cloning in an ancient order of marine bryozoans

Prolific polyembryony is reported in few majortaxa, but its occurrence has generated theo-retical debate on potential conflict betweensexual and asexual reproduction. It is, therefore,important to genetically confirm a widely citedinference, based on microscopy, that poly-embryony characterizes marine bryozoans ofthe order Cyclostomata. Microsatellite genotyp-ing of brooded embryos and maternal coloniesconclusively demonstrated polyembryony, whilegenetic variation among broods within coloniesindicated outcrossing via water-borne sperm, inthe rocky-shore species Crisia denticulata.Thecharacteristically voluminous brood chamber ofcyclostomes is judged to be an adaptation linkedto larval cloning and hence an indicator ofpolyembryony. We speculate that although thealmost universal occurrence of polyembryonyamong crown-group Cyclostomata is probablyattributable to phylogenetic constraint, adaptiveconsequences are likely to be significant.Keywords: clonal reproduction; cross-fertilization;larvae; microsatellites; phylogenetic constraint1. INTRODUCTIONMonozygotic polyembryony is a form of asexualreproduction that proceeds by division or buddingduring post-zygotic or early embryonic stages of thelife history (Hughes 1989). Only monozygotic poly-embryony occurs in animals, whereas in certainhigher plants, especially among angiosperms,a second form of ‘polyembryony’ involves somaticembryogenesis and perhaps represents a maternalstrategy, without equivalents in animals, to counteractbrood reduction resulting from sibling rivalry amongsexual embryos (Shaanker & Ganeshaiah 1996).Hereafter, polyembryony refers to the monozygoticprocess. The persistence of polyembryony in certaintaxa has puzzled evolutionary biologists, because itcan appear to combine the contrasted fitness disad-vantages of cloning and sexual reproduction, whilecompromising the respective benefits (Craig et al.1995, 1997; but see Hardy 1995a).Extensive polyembryony, generating copious clone-mates per zygote, is characteristic of rust fungi(Alexopoulos 1952), red algae (Searles 1980),encyrtid wasps (Hardy 1995b) and cyclostomebryozoans (Ryland 1996). Some authors also includecases where cloning occurs in post-embryonic orlarval stages of the life history, as in endoparasitichydrozoans, flatworms and barnacles and in a fewstarfish and brittle stars (reviewed in Craig et al.(1997); see also Cable & Harris (2002) on gyrodacty-lid flatworms). Limited polyembryony, or ‘twinning’,forms an integral part of the life history in manygymnosperms (Filonova et al. 2002), certain angios-perms (Carman 1997) and armadillos (Prodohl et al.1996). Twinning also occurs aberrantly in diversemammalian species (Gleeson et al. 1994).Polyembryony in cyclostome bryozoans was firstreported by Harmer (1892, 1898),andalthoughamended in certain detail, his interpretation of embry-ogenesis in Crisia species, based on microscopy, wascorroborated by Borg (1926) for a range of genera. Asyncytial reticulum nourishes the budding embryos, allenclosed within an expanded chamber. Although thisbrood chamber varies considerably in size amongcyclostome families, it is almost always relatively muchlarger than the ovicells of cheilostome bryozoans,which typically house single embryos (Ryland 1970).We may, therefore, reasonably infer that the broodchamber, recorded in all living families of cyclostomesexcept the Cinctiporidae (Boardman et al.1992), isassociated with embryonic budding (Borg 1926; Stro¨m1977). Moreover, because most post-Triassic fossilspecies, again apart from the Cinctiporidae, alsopossess similar brood chambers (McKinney & Taylor1997) it is probable that embryonic budding is aplesiomorphic character of the crown-group Cyclosto-mata (Taylor 2000; Taylor & Weedon 2000). Inparticular, microscopical evidence of polyembryony incrisiids (above), which have primitive status withincyclostome phylogeny, suggests that polyembryonyarose basally in the crown group, while similar evidencefor lichenoporids (Borg 1926), which are advanced,suggests that polyembryony was subsequentlyretained, becoming a general characteristic of cyclos-tomes (Taylor & Weedon 2000; P. D. Taylor, personalcommunication). It would be desirable, nevertheless,to obtain independent confirmation of this inferencefor a more comprehensive range of examples.Although the histological patterns described byHarmer (1892, 1898) and Borg (1926) seem clearlyto indicate polyembryony, the possibility of multiplefertilization within the brood chamber or of partheno-genesis cannot be dismissed without knowing thecorresponding genotypic composition of motherand embryos. Accordingly, we used previously iso-lated microsatellite markers for Crisia denticulata(Craig et al. 2001) to genotype mothers and their