Phylogenetically Widespread Polyembryony in Cyclostome Bryozoans and the Protracted Asynchronous Release of Clonal Brood-Mates.

Polyembryony-the production of multiple cloned embryos from a single fertilised egg-is a seemingly paradoxical combination of reproductive modes that nevertheless persists in diverse taxa. We document features of polyembryony in the Cyclostomata (Bryozoa)-an ancient order of modular colonial marine invertebrates-that suggest a substantial reduction in the paradoxical nature of this enigmatic reproductive mode. Firstly, we provide molecular evidence for polyembryony in three exemplar species, supporting the widely cited inference that polyembryony characterises the entire order. Secondly, genotyping demonstrates protracted release of cloned offspring from the primary embryo in a given gonozooid (chamber for embryonic incubation), thus exposing the same genotype to changing environmental conditions over time. Finally, we confirm that each gonozooid produces a distinct genotype, with each primary embryo being the result of a separate fertilisation event. We hypothesise that the sustained release of one or a few genotypes against varying environmental conditions achieves levels of risk-spreading similar to those in organisms that release multiple, unique genotypes at a single time. We argue that polyembryony, specifically with the production of a large number of progeny per fertilisation event, has been favoured in the Cyclostomata over long geological periods.

Molecular variability in the Celleporella hyalina (Bryozoa; Cheilostomata) species complex: evidence for cryptic speciation from complete mitochondrial genomes.

The bryozoan Celleporella has been shown to be composed of multiple, often cryptic, lineages. We sequenced two complete mitochondrial (mt) genomes of the Celleporella hyalina species complex from Wales, UK and Norway (i) to determine genetic divergence at the complete mt genome level, and (ii) to design new molecular markers for examining the interrelationships amongst the major lineages. In addressing (i), we estimated genetic divergence at three levels: (a) nucleotide diversity (π), (b) genome size, and (c) gene order. Genes nad4L, nad6, and atp8 showed the highest levels of divergence, and rrnL, rrnS, and cox1 showed the lowest levels. Inter-genome nucleotide divergence of protein-coding and ribosomal RNA genes, measured as π, was 0.21. The two genomes differed substantially in size, with the Norwegian genome being 2,573 base pairs (bp) longer than the Welsh genome, 17,265 and 14,692 bp, respectively. This difference in size is attributable to long non-coding regions present in the Norwegian genome. Both genomes exhibit similar gene orders, except for the translocation of one transfer RNA (trnA). Considering the high nucleotide diversity, genome size difference and change in gene order, these mt genomes are considered sufficiently divergent to have originated from two distinct species. In addressing (ii) we designed PCR primers that flank the most conserved regions of the genome: 1,300 bp of cox1 and a contiguous 2,000 bp fragment of rrnL + rrnS. The primers have yielded products for tissue from Wales, Norway, New Zealand, Alaska and Chile and should provide useful tools in establishing species- and population-level diversity within the Celleporella complex.

Mitochondrial DNA phylogeography and mating compatibility reveal marked genetic structuring and speciation in the NE Atlantic bryozoan Celleporella hyalina.

The marine bryozoan Celleporella hyalina is a species complex composed of many highly divergent and mostly allopatric genetic lineages that are reproductively isolated but share a remarkably similar morphology. One such lineage commonly encrusts macroalgae throughout the NE Atlantic coast. To explore the processes leading to geographical diversification, reproductive isolation and speciation in this taxon, we (i) investigated NE Atlantic C. hyalina mitochondrial DNA phylogeography, and (ii) used breeding trials between geographical isolates to ascertain reproductive isolation. We find that haplotype diversity is geographically variable and there is a strong population structure, with significant isolation by distance. NE Atlantic C. hyalina is structured into two main parapatric lineages that appear to have had independent Pleistocene histories. Range expansions have resulted in two contact zones in Spain and W Ireland. Lineage 1 is found from Ireland to Spain and has low haplotype diversity, with closely related haplotypes, suggesting a recent population expansion into the Irish Sea, S Ireland, S England and Spain. Lineage 2 is found from Iceland to Spain and has high haplotype diversity. Complete reproductive isolation was found between some geographical isolates representing both lineages, whereas it was incomplete or asymmetric between others, suggesting these latter phylogeographical groups probably represent incipient species. The phylogeographical distribution of NE Atlantic C. hyalina does not fall easily into a pattern of southern refugia, and we discuss likely differences between terrestrial and marine system responses to Pleistocene glacial cycles.

Mating trials validate the use of DNA barcoding to reveal cryptic speciation of a marine bryozoan taxon.

Despite increasing threats to the marine environment, only a fraction of the biodiversity of the oceans has been described, owing in part to the widespread occurrence of cryptic species. DNA-based barcoding through screening of an orthologous reference gene has been proposed as a powerful tool to uncover biological diversity in the face of dwindling taxonomic expertise and the limitations of traditional species identification. Although DNA barcoding should be particularly useful in the sea, given the prevalence of marine cryptic species, the link between taxa identified through DNA barcodes and reproductively isolated taxa (biological species) has rarely been explicitly tested. Here, we use an integrated framework comparing breeding compatibility, morphology and mitochondrial (cytochrome c oxidase 1) and nuclear (elongation factor-1-alpha) DNA sequence variation among globally distributed samples of the cosmopolitan marine bryozoan Celleporella hyalina (L.). Our results reveal that C. hyalina comprises numerous deep, mostly allopatric, genetic lineages that are reproductively isolated, yet share very similar morphology, indicating rampant cryptic speciation. The close correspondence between genetic lineages and reproductively isolated taxa in the context of minimal morphological change suggests that DNA barcoding will play a leading role in uncovering the hidden biodiversity of the oceans and that the sole use of morphologically based taxonomy would grossly underestimate the number of marine species.

From cells to colonies: at what levels of body organization does the 'temperature-size rule' apply?

An inverse relationship between temperature during ontogeny and final body size is widespread in ectotherms, but poorly understood. Evidence suggests that within organs, this "temperature-size rule" (TSR) may also apply to cell size with no change in numbers. So how closely do reductions in size and number of cells and other repeated structures correlate with size reduction at higher levels of organization? We examine this in the context of a proposal that size and/or number changes at various organizational levels are adaptive responses to temperature- and size-dependent oxygen supply. We subjected two clones of the modular colonial bryozoan, Celleporella hyalina, to orthogonal combinations of two temperatures and two oxygen concentrations during ontogeny, observing effects on sizes of colonies and larvae, and sizes and numbers of cells, tentacles, and modules (autozooids). We found that the size:number responses varied among cell types and among structures at different levels of organization, with the inverse temperature-size relationship applying only to larval parenchymal cells and colony modules. Using our findings and other evidence we propose a unifying adaptive hypothesis that predicts how temperature affects the sizes of mitochondria, cells, organs, modules and organisms, and their relationships with processes that determine the functional capacity of aerobic metabolism.

Using molecular and quantitative variation for assessing genetic impacts on Nucella lapillus populations after local extinction and recolonization.

The dogwhelk Nucella lapillus is a predatory marine gastropod living on rocky shores in the North Atlantic. As with many other gastropod species, Nucella was affected by tributyltin (TBT) pollution during the 1970s and 1980s, and local populations underwent extinction. After a partial ban on TBT in the UK in 1987, vacant sites have been recolonized. Levels of genetic diversity and quantitative genetic variation in shell form were compared between recolonized sites and sites that showed continuous population at three localities across the British Isles. Overall, estimates of genetic diversity were only slightly lower in recolonized populations, suggesting that populations have recovered from previous impacts due to the relatively high levels of migration from non-impacted sites. Molecular and quantitative analyses are broadly concordant and a positive correlation was observed (although not statistically significant) between molecular and quantitative estimates of genetic diversity, indicating the potential usefulness of quantitative methods to complement molecular population genetics analyses.

Paradoxical polyembryony? Embryonic cloning in an ancient order of marine bryozoans.

Prolific polyembryony is reported in few major taxa, but its occurrence has generated theoretical debate on potential conflict between sexual and asexual reproduction. It is, therefore, important to genetically confirm a widely cited inference, based on microscopy, that polyembryony characterizes marine bryozoans of the order Cyclostomata. Microsatellite genotyping of brooded embryos and maternal colonies conclusively demonstrated polyembryony, while genetic variation among broods within colonies indicated outcrossing via water-borne sperm, in the rocky-shore species Crisia denticulata. The characteristically voluminous brood chamber of cyclostomes is judged to be an adaptation linked to larval cloning and hence an indicator of polyembryony. We speculate that although the almost universal occurrence of polyembryony among crown-group Cyclostomata is probably attributable to phylogenetic constraint, adaptive consequences are likely to be significant.

Kin or self-recognition? Colonial fusibility of the bryozoan Celleporella hyalina.

We estimated fusion frequency with respect to coancestry in the bryozoan Celleporella hyalina, whose briefly planktonic sexually produced larvae settle on algal substrata and proceed to form encrusting colonies by iterative budding. Frequency of fusion between paired colonies growing on an artificial substratum was positively correlated with coefficient of relatedness, with allorecognition ability increasing during the early stages of colonial growth after larval settlement. Parents repressed the growth of F1 progeny with which they had fused. The results are concordant with the Feldgarden-Yund model of selection for self-recognition, which regards fusion with kin as an inevitable source of error whose cost diminishes with increasing relatedness. Contrary to fusion compatibility, gametic compatibility is negatively correlated with coancestry, indicating a need for further research on the possibility of common or linked genetic control that has opposite effect at somatic and gametic levels.

Efficient utilization of very dilute aquatic sperm: sperm competition may be more likely than sperm limitation when eggs are retained.

Fertilization success may be severely limited in marine invertebrates that spawn both male and female gametes. In a diverse group of aquatic organisms only sperm are released, with sperm-egg fusion occurring at the mother. Here, we report fertilization kinetics data for two such 'brooding' or 'spermcast' species--representing each major clade of the animal kingdom. High levels of fertilization were achieved at sperm concentrations of two or three orders of magnitude lower than is common with broadcast spawning species. At a concentration of 100 sperm ml(-1), fertilization rates of a bryozoan and colonial ascidian were near maximum, whereas most broadcast spawners would have displayed near complete reproductive failure. A further experiment looked at the rate of uptake of sperm under natural conditions. Results suggested that sperm released at ca. 0.9 m from an acting female could be collected at a rate of 3-12 times greater than the minimum required simply to avoid sperm limitation. Thus, evolutionary pressures on gametic and other reproductive characteristics of many species that release sperm but retain eggs may be quite different from those of broadcast spawners and may confer on the former an enhanced scope for sperm competition and female choice.

Female investment is retarded pending reception of allosperm in a hermaphroditic colonial invertebrate.

Young colonies of the bryozoan Celleporella hyalina are capable of acquiring water-borne allosperm and of using it to fertilize ova for a period of 3-6 weeks after reaching female sexual maturity. In these simultaneous hermaphrodites, early allocation to female modules, but not male, is greatly enhanced by the acquisition of allosperm. The degree of enhancement is inversely proportional to coancestry of the recipient and donor colonies, thus promoting outcrossing. This apparently novel mechanism of adjusting operational sex ratio depends on the uptake and storage of sperm by nonreproductive (somatic) modules and subsequent translocation to females.

Are digestive characteristics important contributors to the profitability of prey? : A study of diet selection in the fifteen-spined stickleback, Spinachia spinachia (L.).

In the field, Spinachia fed on four types of prey; copepods, isopods, mysids and amphipods. As fish size increased, mysids gradually succeeded amphipods as the most important food type in the diet. Prey dimensions and morphometry of the fish's mouth most accurately predicted capture efficiency for amphipods, whereas for mysids capture efficiency was determined by the prey's escape response and the fish's fast-start capability. Responses to model prey revealed the ability of fish to differentiate among contrasting prey characteristics, resulting in the adoption of appropriate predatory tactics. Amphipods were associated with a shorter gut evacuation time than mysids, although approximately equal proportions of energy were absorbed from each. Similar rations of mysids and amphipods were required to satiate fish. The lower energy content per unit dry mass of amphipods was off set by their lower water content. From pre-digestive behaviour, we predicted that mysids were more profitable than amphipods, and this was reflected in the fish's choice. Conversely, incorporating the net rate of energy uptake by the gut led us to predict that amphipods were more profitable. Although physiological constraints clearly influence the net rate of energy uptake, it appears that dietary preferences are based on pre-digestive predatory behaviour and hence on time minimisation.

Reproductive effort of winkles (Littorina spp.) with contrasted methods of reproduction.

Reproductive effort was compared in Littorina rudis (ovoviviparous), L. nigrolineata (benthic eggs with direct development), L. neritoides and L. littorea (planktonic eggs and larvae). Three indices of reproductive effort were used: the proportion of total production committed to reproduction per unit time, the cumulative proportion of total production committed to reproduction up to a given age, and the ratio of reproductive production per unit time to somatic biomass at the beginning of the unit time interval. The indices were plotted against age and the snails ranked in order of their reproductive efforts at equivalent ages. When plotted on axes of absolute time, all three indices ranked the snails in the order L. neritoides