Evolutionary relationships among the eight major lineages of Mollusca have remained unresolved despite their diversity and importance. Previous investigations of molluscan phylogeny, based primarily on nuclear ribosomal gene sequences1–3 or morphological data4, have been unsuccessful at elucidating these relationships. Recently, phylogenomic studies using dozens to hundreds of genes have greatly improved our understanding of deep animal relationships5. However, limited genomic resources spanning molluscan diversity has prevented use of a phylogenomic approach. Here we use transcriptome and genome data from all major lineages (except Monoplacophora) and recover a well-supported topology for Mollusca. Our results strongly support the Aculifera hypothesis placing Polyplacophora (chitons) in a clade with a monophyletic Aplacophora (worm-like molluscs). Additionally, within Conchifera, a sister-taxon relationship between Gastropoda and Bivalvia is supported. This grouping has received little consideration and contains most (>95%) molluscan species. Thus we propose the node-based name Pleistomollusca. In light of these results, we examined the evolution of morphological characters and found support for advanced cephalization and shells as possibly having multiple origins within Mollusca.

Phylogenomic studies have improved understanding of deep metazoan phylogeny and show promise for resolving incongruences among analyses based on limited numbers of loci. One region of the animal tree that has been especially difficult to resolve, even with phylogenomic approaches, is relationships within Lophotrochozoa (the animal clade that includes molluscs, annelids, and flatworms among others). Lack of resolution in phylogenomic analyses could be due to insufficient phylogenetic signal, limitations in taxon and/or gene sampling, or systematic error. Here, we investigated why lophotrochozoan phylogeny has been such a difficult question to answer by identifying and reducing sources of systematic error. We supplemented existing data with 32 new transcriptomes spanning the diversity of Lophotrochozoa and constructed a new set of Lophotrochozoa-specific core orthologs. Of these, 638 orthologous groups (OGs) passed strict screening for paralogy using a tree-based approach. In order to reduce possible sources of systematic error, we calculated branch-length heterogeneity, evolutionary rate, percent missing data, compositional bias, and saturation for each OG and analyzed increasingly stricter subsets of only the most stringent (best) OGs for these five variables. Principal component analysis of the values for each factor examined for each OG revealed that compositional heterogeneity and average patristic distance contributed most to the variance observed along the first principal component while branch-length heterogeneity and, to a lesser extent, saturation contributed most to the variance observed along the second. Missing data did not strongly contribute to either.Additional sensitivity analyses examined effects of removing taxa with heterogeneous branch lengths, large amounts of missing data, and compositional heterogeneity. Although our analyses do at University of Oslo Library on September 26, 2016 http://sysbio.oxfordjournals.org/ Downloaded from 3 not unambiguously resolve lophotrochozoan phylogeny, we advance the field by reducing the list of viable hypotheses. Moreover, our systematic approach for dissection of phylogenomic data can be applied to explore sources of incongruence and poor support in any phylogenomic dataset.Keywords: Trochozoa, Spiralia, Mollusca, Nemertea, Annelida, Brachiopoda, Phoronida, Entoprocta, Platyzoa, Polyzoa, Bryozoa Understanding of deep phylogeny has improved with the application of phylogenomic approaches (e.g., Philippe et al. 2004Philippe et al. , 2005Matus et al. 2006; Delsuc et al. 2006; Dunn et al. 2008; Hejnol et al. 2009; Kocot et al. 2011;Smith et al. 2011;Struck et al. 2011;Zhong et al. 2011a;Ryan et al. 2013;Moroz et al. 2014, Torruella et al. 2015, Whelan et al. 2015.Nonetheless, some regions of the tree of life with short internodes, probably due to rapid diversification, still lack resolution. Relationships within Lophotrochozoa (Halanych et al. 1995) are one such example. Lophotrochozoa is a well-supported clade of invertebrates that includes Anneli...

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SummaryMollusca is an animal phylum with vast morphological diversity and includes worm-shaped aplacophorans, snails, bivalves, and the complex cephalopods [1]. The interrelationships of these class-level taxa are still contentious [2, 3], but recent phylogenomic analyses suggest a dichotomy at the base of Mollusca, resulting in a monophyletic Aculifera (comprising the shell-less, sclerite-bearing aplacophorans and the eight-shelled polyplacophorans) and Conchifera (all other, primarily univalved groups) [4, 5]. The Aculifera concept has recently gained support via description of the fossil Kulindroplax, which shows both aplacophoran- and polyplacophoran-like features and suggests that the aplacophorans originated from a shelled ancestor [6], but the overall morphology of the last common aculiferan ancestor remains obscure. Here we show that larvae of the aplacophoran Wirenia argentea have several sets of muscles previously known only from polyplacophoran mollusks. Most of these are lost during metamorphosis, and we interpret them as ontogenetic remnants of an ancestor with a complex, polyplacophoran-like musculature. Moreover, we find that the first seven pairs of dorsoventral muscles develop synchronously in Wirenia, similar to juvenile polyplacophorans [7], which supports the conclusions based on the seven-shelled Kulindroplax. Accordingly, we argue that the simple body plan of recent aplacophorans is the result of simplification and does not represent a basal molluscan condition.

BackgroundThe 18S rRNA gene is one of the most important molecular markers, used in diverse applications such as molecular phylogenetic analyses and biodiversity screening. The Mollusca is the second largest phylum within the animal kingdom and mollusks show an outstanding high diversity in body plans and ecological adaptations. Although an enormous amount of 18S data is available for higher mollusks, data on some early branching lineages are still limited. Despite of some partial success in obtaining these data from Solenogastres, by some regarded to be the most "basal" mollusks, this taxon still remained problematic due to contamination with food organisms and general amplification difficulties.ResultsWe report here the first authentic 18S genes of three Solenogastres species (Mollusca), each possessing a unique sequence composition with regions conspicuously rich in guanine and cytosine. For these GC-rich regions we calculated strong secondary structures. The observed high intra-molecular forces hamper standard amplification and appear to increase formation of chimerical sequences caused by contaminating foreign DNAs from potential prey organisms. In our analyses, contamination was avoided by using RNA as a template. Indication for contamination of previously published Solenogastres sequences is presented. Detailed phylogenetic analyses were conducted using RNA specific models that account for compensatory substitutions in stem regions.ConclusionsThe extreme morphological diversity of mollusks is mirrored in the molecular 18S data and shows elevated substitution rates mainly in three higher taxa: true limpets (Patellogastropoda), Cephalopoda and Solenogastres. Our phylogenetic tree based on 123 species, including representatives of all mollusk classes, shows limited resolution at the class level but illustrates the pitfalls of artificial groupings formed due to shared biased sequence composition.