Latitudinal variation of a defensive symbiosis in the Bugula neritina (Bryozoa) sibling species complex.

Mutualistic relationships are beneficial for both partners and are often studied within a single environment. However, when the range of the partners is large, geographical differences in selective pressure may shift the relationship outcome from positive to negative. The marine bryozoan Bugula neritina is a colonial invertebrate common in temperate waters worldwide. It is the source of bioactive polyketide metabolites, the bryostatins. Evidence suggests that an uncultured vertically transmitted symbiont, "Candidatus Endobugula sertula", hosted by B. neritina produces the bryostatins, which protect the vulnerable larvae from predation. Studies of B. neritina along the North American Atlantic coast revealed a complex of two morphologically similar sibling species separated by an apparent biogeographic barrier: the Type S sibling species was found below Cape Hatteras, North Carolina, while Type N was found above. Interestingly, the Type N colonies lack "Ca. Endobugula sertula" and, subsequently, defensive bryostatins; their documented northern distribution was consistent with traditional biogeographical paradigms of latitudinal variation in predation pressure. Upon further sampling of B. neritina populations, we found that both host types occur in wider distribution, with Type N colonies living south of Cape Hatteras, and Type S to the north. Distribution of the symbiont, however, was not restricted to Type S hosts. Genetic and microscopic evidence demonstrates the presence of the symbiont in some Type N colonies, and larvae from these colonies are endowed with defensive bryostatins and contain "Ca. Endobugula sertula". Molecular analysis of the symbiont from Type N colonies suggests an evolutionarily recent acquisition, which is remarkable for a symbiont thought to be transmitted only vertically. Furthermore, most Type S colonies found at higher latitudes lack the symbiont, suggesting that this host-symbiont relationship is more flexible than previously thought. Our data suggest that the symbiont, but not the host, is restricted by biogeographical boundaries

<i>B. neritina</i> sibling species collected by haphazard sampling.

<p>Proportions at each site indicated by blue (Type N) and red (Type S) in charts.</p

Primers and probes used in this study.

<p>Primers and probes used in this study.</p

Molecular characterization of <i>B. neritina</i> and HPLC analysis of crude larval extract.

<p>Agarose gel showing <i>Dde</i>I and <i>Hha</i>I restriction digestion of <i>B. neritina</i> adult (top two samples) or pooled larval (bottom sample) cytochrome <i>c</i> oxidase I; amplification of <i>bryS</i> to demonstrate presence of symbiont; and absorbance at 229 nm (which is diagnostic of bryostatins) of extract collected from larvae represented by this gDNA.</p

Co-occurrence of Type N and Type S <i>B. neritina</i>.

<p>Colonies collected together in (A) Beaufort, NC, and (B) Oyster, VA. Blue arrows indicate Type N colonies; red indicate Type S.</p

Symbiotic status of sampled colonies.

<p>Proportions at each site indicated by black (symbiotic) and gray (aposymbiotic) in charts. Results from both haphazardly collected and targeted samples are included. Number of colonies sampled shown beside each graph.</p

<i>Bugula neritina</i> symbiont frequency along North American Atlantic coast.

<p>Symbiont occurrence was determined both for colonies collected haphazardly and in targeted sampling (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108783#s2" target="_blank">Materials and Methods</a>); not all haphazardly selected colonies were assayed for symbiont presence. Dashes indicate host phylotypes not found in a location during sampling.</p><p><i>Bugula neritina</i> symbiont frequency along North American Atlantic coast.</p

Percent identity of marker sequences among <i>B. neritina</i> sibling species.

<p>Types D and S show the greatest COI identity, but Type D differs from both Types S and N in symbiont markers.</p><p>Percent identity of marker sequences among <i>B. neritina</i> sibling species.</p