Evidence for a clade composed of molluscs with serially repeated structures: Monoplacophorans are related to chitons

An Antarctic Benthic Deep-Sea Biodiversity oceanographic campaign (ANDEEP III) with the RV Polarstern to the Weddell Sea (Antarctica), 3 km southwest of Wegener Canyon at ≈3,100-m depth, yielded one small specimen (1.7-mm shell length) of the monoplacophoran Laevipilina antarctica Warén & Hain, 1992 (19), one of the 26 known species of this group of molluscs (9, 20). The single specimen was obtained from an epibenthic sledge sample that had been fixed with precooled 96% EtOH for molecular studies and stored at −20°C for 48 h. The shell (ZSM Moll 20050866; Fig. 1 A and B) was removed for gross anatomy and SEM examination, the soft body was photographed (Fig. 1 C and D), and half of the specimen was used for molecular work.

Although monoplacophoran DNA was highly degraded, perhaps because of bulk fixation of the sediment performed in the vessel, we were able to amplify and sequence a 1.2-kb fragment of the large nuclear ribosomal subunit (28S rRNA). This gene has proven to be highly informative in recent studies on metazoan and molluscan evolution (17, 21).

Analysis of the data using a single-step phylogenetic approach with direct optimization (Fig. 2) and a two-step approach using Bayesian phylogenetics (Fig. 4, which is published as supporting information on the PNAS web site) exhibited congruent results suggesting monophyly of molluscs as well as that of the molluscan classes Caudofoveata, Solenogastres, Scaphopoda, and Cephalopoda. Resolution with high jackknife support is found mostly within the main clades of Scaphopoda (Dentaliida and Gadilida), Cephalopoda (Nautiloidea, Coleoidea, and the sister group relationship of the vampire squid to decabrachians, which include the giant squid Architeuthis dux and the pygmy squid Idiosepius pygmaeus), as well as within Bivalvia (Palaeoheterodonta and Euheterodonta) and Gastropoda (Patellogastropoda, Neritopsina, Caenogastropoda, and Heterobranchia). However, the available sequence data do not recover monophyly of Gastropoda or Bivalvia, which are both diphyletic, with patellogastropods separated from the other gastropods and heteroconchs separated from the remainder of the bivalves (protobranchs and pteriomorphians).

Nodal support for interclass relationships or for the relationships of the two clades of bivalves and gastropods is low in general, but a clade containing Monoplacophora and Polyplacophora received strong nodal support (90–100% jackknife support value depending on the analysis, as well as 1.0 posterior probability). Interestingly, this clade, which we name “Serialia,” contains the two classes whose members present a variable number of serially repeated gills and eight sets of dorsoventral pedal retractor muscles. This result clearly contrasts with previous cladistic hypotheses suggesting that Monoplacophora constitute the sister group to the remainder of the conchiferans (4, 5, 14), those molluscs with a true shell unlike that of chitons or the vermiform aplacophorans, although it finds no clear support for the exact position of Serialia. To our knowledge, this is also the first published analysis that demonstrates monophyly of the phylum Mollusca using a range of appropriate outgroups, but we caution the reader to consider that jackknife support for molluscan monophyly is low. The results further support a previous study (22) that indicates that Xenoturbella is not a bivalve mollusc.

All analyses (including different optimality criteria and alternative models of indel and base substitutions) support a Polyplacophora plus Monoplacophora clade. However, L. antarctica appears nested within the chiton tree in some analyses, a result that may look suspicious at first. Evidence for including Monoplacophora within Polyplacophora is restricted to one node, which groups nonlepidopleurid chitons with the monoplacophoran species (70% jackknife support; Fig. 1), but this is not the case when considering only the 1.2-kb region of 28S rRNA amplified for Laevipilina (tree not shown). Furthermore, detailed examination of the DNA sequences clearly illustrates that chitons share unambiguous positions in the alignment not found in L. antarctica (Fig. 3). This fact eliminates the possibility of contaminant DNA in our analysis.

Evidence for a clade of serialian molluscs is important for our current understanding of molluscan relationships and may have implications for deeper metazoan evolution. This new evidence may imply that serially repeated structures (e.g., gills and pedal retractor muscles in both monoplacophorans and chitons) are not primitive for molluscs, as was previously thought (9). However, it is fair to mention that additional types of serial repetition of dorsoventral musculature have been reported in other molluscan groups (23), including the eight sets of pedal retractors of the Ordovician lucinoid bivalve Babinka (11), the serially repeated rows of spicules in caudofoveate larvae (3), or the two pairs of gills and nephridia in cephalopods (3). Whether these represent true seriality or not may have profound implications in reconstructing the molluscan common ancestor, but it does not contradict the evidence of our Serialia clade.

The classical hypothesis for the position of monoplacophorans as basal conchiferans relies heavily on the presence of a true dorsal shell with similar mineralogical composition to that of many basal members of each conchiferan class. However, the mode of shell deposition by the mantle edge and the microstructure and composition of the chitinous organic layer in monoplacophorans differ from those of higher conchiferans or polyplacophorans (9, 24, 25), which makes monoplacophorans apomorphic (derived) in the form of shell deposition. The rejection of conchiferan monophyly based on shell deposition would be consistent with our findings, which suggest that serial repetition of anatomical structures such as gills and muscles may have evolved once in the common ancestor of chitons and monoplacophorans. Therefore, serial repetition of these structures could constitute a derived feature that would not support the hypothesis of a segmented ancestral mollusc. Again, other interpretations may exist if the pedal scars of Bibankia were the result of muscles homologous to the serialian dorsoventral pedal muscles.

Molluscs are undoubtedly one of the animal phyla with the largest disparity. Numerous Cambrian forms such as Wiwaxia and Halkieria or the Silurian Acaenoplax have been more or less ambiguously assigned to this animal phylum (26–28). Kimberella is another putative mollusc extending the age of the group back to the Neoproterozoic (29). Although chitons were once thought to have changed little since their first appearance in the Late Cambrian period (30), recent discoveries of articulated polyplacophorans and multiplacophorans from the Ordovician to the Carboniferous (31, 32) suggest that a much larger disparity evolved during the Paleozoic. Perhaps such an episode of diversification is responsible for the two modern anatomies of molluscs with conspicuous serial repetition of organs, but no explanation for their divergent evolution of shell morphologies can be provided at this point. Recognition of a serialian clade comprised of chitons and monoplacophorans broadens our perspective toward new interpretations of molluscan anatomy and once more questions preconceived ideas on molluscan relationships that rely almost entirely on shell morphology.

Here we provide the first molecular test for the phylogenetic position of Monoplacophora by using sequence data from a deep-sea monoplacophoran species from Antarctica. Contrary to all previously published accounts, which placed monoplacophorans as a sister group to higher, i.e., shelled, molluscs, our data strongly support a clade including Monoplacophora and Polyplacophora. This rather surprising result from a conchological perspective is congruent with soft anatomy data. It furthermore reopens the debate about the putative ancestral segmentation of molluscs (3), because serial repetition of gills and pedal retractor muscles may be derived and not primitive features within molluscs. If this were the case, little evidence would remain for the case of homology of segmentation in annelids and serial repetition in molluscs (33), as confirmed in part by recent reevaluation of their early development (34, 35).

We are indebted to the numerous colleagues who supplied tissue samples, without whom this work would not have been possible. Rebecca Budinoff assisted with laboratory work. Angelika Brandt organized the ANDEEP III expedition. Katrin Linse, Enrico Schwabe, and the onboard sorting team provided the monoplacophoran specimen. Greg Edgecombe, Andy Knoll, Sigurd von Boletzky, Claus Nielsen, Jim Valentine, and an anonymous reviewer provided comments that helped to improve this article. Enrico Schwabe provided pictures for Fig. 1 A and B. This material is based on work supported by the National Science Foundation Assembling the Tree of Life Program (Grant 0334932 to G.G.) and Population Biology Program (Grant 0316516 to M.K.N.). Field activities of M.S. and his team were supported by the GeoBioCenterLMU. This article is ANDEEP contribution no. 58.