Sónia C. S. Andrade

Resolving the evolutionary relationships of molluscs with phylogenomic tools

Molluscs (snails, octopuses, clams and their relatives) have a great disparity of body plans and, among the animals, only arthropods surpass them in species number. This diversity has made Mollusca one of the best-studied groups of animals, yet their evolutionary relationships remain poorly resolved. Open questions have important implications for the origin of Mollusca and for morphological evolution within the group. These questions include whether the shell-less, vermiform aplacophoran molluscs diverged before the origin of the shelled molluscs (Conchifera) or lost their shells secondarily. Monoplacophorans were not included in molecular studies until recently, when it was proposed that they constitute a clade named Serialia together with Polyplacophora (chitons), reflecting the serial repetition of body organs in both groups. Attempts to understand the early evolution of molluscs become even more complex when considering the large diversity of Cambrian fossils. These can have multiple dorsal shell plates and sclerites or can be shell-less but with a typical molluscan radula and serially repeated gills. To better resolve the relationships among molluscs, we generated transcriptome data for 15 species that, in combination with existing data, represent for the first time all major molluscan groups. We analysed multiple data sets containing up to 216,402 sites and 1,185 gene regions using multiple models and methods. Our results support the clade Aculifera, containing the three molluscan groups with spicules but without true shells, and they support the monophyly of Conchifera. Monoplacophora is not the sister group to other Conchifera but to Cephalopoda. Strong support is found for a clade that comprises Scaphopoda (tusk shells), Gastropoda and Bivalvia, with most analyses placing Scaphopoda and Gastropoda as sister groups. This well-resolved tree will constitute a framework for further studies of mollusc evolution, development and anatomy.

Disentangling Ribbon Worm Relationships: Multi-Locus Analysis Supports Traditional Classification of the Phylum Nemertea

The phylogenetic relationships of selected members of the phylum Nemertea are explored by means of six markers amplified from the genomic DNA of freshly collected specimens (the nuclear 18S rRNA and 28S rRNA genes, histones H3 and H4, and the mitochondrial genes 16S rRNA and cytochrome c oxidase subunit I). These include all previous markers and regions used in earlier phylogenetic analyses of nemerteans, therefore acting as a scaffold to which one could pinpoint any previously published study. Our results, based on analyses of static and dynamic homology concepts under probabilistic and parsimony frameworks, agree in the non‐monophyly of Palaeonemertea and in the monophyly of Heteronemerta and Hoplonemertea. The position of Hubrechtella and the Pilidiophora hypothesis are, however, sensitive to analytical method, as is the monophyly of the non‐hubrechtiid palaeonemerteans. Our results are, however, consistent with the main division of Hoplonemertea into Polystilifera and Monostilifera, the last named being divided into Cratenemertea and Distromatonemertea, as well as into the main division of Heteronemertea into Baseodiscus and the remaining species. The study also continues to highlight the deficient taxonomy at the family and generic level within Nemertea and sheds light on the areas of the tree that require further refinement.

Phylogenomic analyses of deep gastropod relationships reject Orthogastropoda.

Gastropods are a highly diverse clade of molluscs that includes many familiar animals, such as limpets, snails, slugs and sea slugs. It is one of the most abundant groups of animals in the sea and the only molluscan lineage that has successfully colonized land. Yet the relationships among and within its constituent clades have remained in flux for over a century of morphological, anatomical and molecular study. Here, we re-evaluate gastropod phylogenetic relationships by collecting new transcriptome data for 40 species and analysing them in combination with publicly available genomes and transcriptomes. Our datasets include all five main gastropod clades: Patellogastropoda, Vetigastropoda, Neritimorpha, Caenogastropoda and Heterobranchia. We use two different methods to assign orthology, subsample each of these matrices into three increasingly dense subsets, and analyse all six of these supermatrices with two different models of molecular evolution. All 12 analyses yield the same unrooted network connecting the five major gastropod lineages. This reduces deep gastropod phylogeny to three alternative rooting hypotheses. These results reject the prevalent hypothesis of gastropod phylogeny, Orthogastropoda. Our dated tree is congruent with a possible end-Permian recovery of some gastropod clades, namely Caenogastropoda and some Heterobranchia subclades.

A phylogenetic backbone for Bivalvia: an RNA-seq approach.

Bivalves are an ancient and ubiquitous group of aquatic invertebrates with an estimated 10 000–20 000 living species. They are economically significant as a human food source, and ecologically important given their biomass and effects on communities. Their phylogenetic relationships have been studied for decades, and their unparalleled fossil record extends from the Cambrian to the Recent. Nevertheless, a robustly supported phylogeny of the deepest nodes, needed to fully exploit the bivalves as a model for testing macroevolutionary theories, is lacking. Here, we present the first phylogenomic approach for this important group of molluscs, including novel transcriptomic data for 31 bivalves obtained through an RNA-seq approach, and analyse these data with published genomes and transcriptomes of other bivalves plus outgroups. Our results provide a well-resolved, robust phylogenetic backbone for Bivalvia with all major lineages delineated, addressing long-standing questions about the monophyly of Protobranchia and Heterodonta, and resolving the position of particular groups such as Palaeoheterodonta, Archiheterodonta and Anomalodesmata. This now fully resolved backbone demonstrates that genomic approaches using hundreds of genes are feasible for resolving phylogenetic questions in bivalves and other animals.

Phylogenetic analysis of four nuclear protein-encoding genes largely corroborates the traditional classification of Bivalvia (Mollusca)

Revived interest in molluscan phylogeny has resulted in a torrent of molecular sequence data from phylogenetic, mitogenomic, and phylogenomic studies. Despite recent progress, basal relationships of the class Bivalvia remain contentious, owing to conflicting morphological and molecular hypotheses. Marked incongruity of phylogenetic signal in datasets heavily represented by nuclear ribosomal genes versus mitochondrial genes has also impeded consensus on the type of molecular data best suited for investigating bivalve relationships. To arbitrate conflicting phylogenetic hypotheses, we evaluated the utility of four nuclear protein-encoding genes-ATP synthase β, elongation factor-1α, myosin heavy chain type II, and RNA polymerase II-for resolving the basal relationships of Bivalvia. We sampled all five major lineages of bivalves (Archiheterodonta, Euheterodonta [including Anomalodesmata], Palaeoheterodonta, Protobranchia, and Pteriomorphia) and inferred relationships using maximum likelihood and Bayesian approaches. To investigate the robustness of the phylogenetic signal embedded in the data, we implemented additional datasets wherein length variability and/or third codon positions were eliminated. Results obtained include (a) the clade (Nuculanida+Opponobranchia), i.e., the traditionally defined Protobranchia; (b) the monophyly of Pteriomorphia; (c) the clade (Archiheterodonta+Palaeoheterodonta); (d) the monophyly of the traditionally defined Euheterodonta (including Anomalodesmata); and (e) the monophyly of Heteroconchia, i.e., (Palaeoheterodonta+Archiheterodonta+Euheterodonta). The stability of the basal tree topology to dataset manipulation is indicative of signal robustness in these four genes. The inferred tree topology corresponds closely to those obtained by datasets dominated by nuclear ribosomal genes (18S rRNA and 28S rRNA), controverting recent taxonomic actions based solely upon mitochondrial gene phylogenies.

A Transcriptomic Approach to Ribbon Worm Systematics (Nemertea): Resolving the Pilidiophora Problem

Resolving the deep relationships of ancient animal lineages has proven difficult using standard Sanger-sequencing approaches with a handful of markers. We thus reassess the relatively well-studied phylogeny of the phylum Nemertea (ribbon worms)-for which the targeted gene approaches had resolved many clades but had left key phylogenetic gaps-by using a phylogenomic approach using Illumina-based de novo assembled transcriptomes and automatic orthology prediction methods. The analysis of a concatenated data set of 2,779 genes (411,138 amino acids) with about 78% gene occupancy and a reduced version with 95% gene occupancy, under evolutionary models accounting or not for site-specific amino acid replacement patterns results in a well-supported phylogeny that recovers all major accepted nemertean clades with the monophyly of Heteronemertea, Hoplonemertea, Monostilifera, being well supported. Significantly, all the ambiguous patterns inferred from Sanger-based approaches were resolved, namely the monophyly of Palaeonemertea and Pilidiophora. By testing for possible conflict in the analyzed supermatrix, we observed that concatenation was the best solution, and the results of the analyses should settle prior debates on nemertean phylogeny. The study highlights the importance, feasibility, and completeness of Illumina-based phylogenomic data matrices.

Re-evaluating the phylogeny of Sipuncula through transcriptomics.

Sipunculans (also known as peanut worms) are an ancient group of exclusively marine worms with a global distribution and a fossil record that dates back to the Early Cambrian. The systematics of sipunculans, now considered a distinct subclade of Annelida, has been studied for decades using morphological and molecular characters, and has reached the limits of Sanger-based approaches. Here, we reevaluate their family-level phylogeny by comparative transcriptomic analysis of eight species representing all known families within Sipuncula. Two data matrices with alternative gene occupancy levels (large matrix with 675 genes and 62% missing data; reduced matrix with 141 genes and 23% missing data) were analysed using concatenation and gene-tree methods, yielding congruent results and resolving each internal node with maximum support. We thus corroborate prior phylogenetic work based on molecular data, resolve outstanding issues with respect to the familial relationships of Aspidosiphonidae, Antillesomatidae and Phascolosomatidae, and highlight the next area of focus for sipunculan systematics.

Phylogenetic placement of a new hoplonemertean species commensal on ascidians

The hoplonemertean Vieitezia luzmurubeae, gen. et sp. nov. is described from specimens collected in a national park on the north-west Iberian Peninsula, the Parque Nacional Maritimo-Terrestre das Illas Atlanticas de Galicia. The species, previously mistaken as the Mediterranean species Tetrastemma vittigerum (Burger, 1904), is frequently associated with two common species of sea squirt, Phallusia mamillata (Cuvier, 1815) and Ciona intestinalis (Linnaeus, 1767), inside which the nemertean completes its life cycle. Some of the specimens examined were protandrous hermaphrodites. Data on morphology and anatomy are provided with illustrations. Sequences of the nuclear ribosomal gene 18S rRNA and the mitochondrial gene cytochrome c oxidase subunit I were compared with those of other hoplonemertean species and all phylogenetic analyses suggested that Vieitezia is sister to the genus Gononemertes, which parasitises ascidians, within a clade also containing the genera Oerstedia and Nemertellina. In contrast, the morphologically similar genus Tetrastemma appears in a separate clade. This study stresses the need for combining molecular and morphological data when studying nemertean biodiversity.

Redescription of Lineus acutifrons Southern, 1913 (Nemertea: Pilidiophora) and comments on its phylogenetic position

The nemertean Lineus acutifrons Southern (1913) is a species rarely encountered in sandy beaches of Europe and is only really known from its original and short description. This species has been found almost a century later in Galician sandy beaches (northwest Spain) and is re-described here. Data detailing its internal morphology and external anatomy, and digital photographs are included. In addition, sequences of the ribosomal gene 28S and the mitochondrial gene cytochrome oxidase I (CO1) were obtained. These sequences were used in a phylogenetic analysis to test its phylogenetic position with respect to other sequences available for pilidiophorans.

At least some meiofaunal species are not everywhere. Indication of geographic, ecological and geological barriers affecting the dispersion of species of Ototyphlonemertes (Nemertea, Hoplonemertea)

Most meiofaunal species are known to have a broad distribution with no apparent barriers to their dispersion. However, different morphological and/or molecular methods supported patterns of diversity and distribution that may be different among taxa while also conflicting within the same group. We accurately assessed the patterns of geographic distribution in actual genetic species of a marine meiofaunal animal model: Ototyphlonemertes. Specimens were collected from several sites around Europe, Northern and Central America, Southern America, Pacific Islands and Asia. We sequenced regions of two mitochondrial and two nuclear genes. Using single‐gene, a concatenated data set, multilocus approaches and different DNA taxonomy methods, we disentangled the actual diversity and the spatial structures of haplotypes and tested the possible correlation between genetic diversity and geographic distance. The results show (i) the importance of using several genes to uncover both diversity and highlight phylogeographic relationships among species and that (ii) independent genetic evolutionary entities have a narrower distribution than morphological species. Moreover, (iii) a Mantel test supported a positive correlation between genetic and geographical distance. By sampling from the two sides of Isthmus of Panama, we were additionally able to identify lineage divergence times that are concordant with vicariance mechanisms caused by the geological closure of the seaway across the Isthmus. We therefore propose that in addition to distance, other geological and ecological conditions are also barriers to the dispersion of and gene flow in marine meiofaunal organisms.