Phylogenetic tree building in the genomic age.

Knowing phylogenetic relationships among species is fundamental for many studies in biology. An accurate phylogenetic tree underpins our understanding of the major transitions in evolution, such as the emergence of new body plans or metabolism, and is key to inferring the origin of new genes, detecting molecular adaptation, understanding morphological character evolution and reconstructing demographic changes in recently diverged species. Although data are ever more plentiful and powerful analysis methods are available, there remain many challenges to reliable tree building. Here, we discuss the major steps of phylogenetic analysis, including identification of orthologous genes or proteins, multiple sequence alignment, and choice of substitution models and inference methodologies. Understanding the different sources of errors and the strategies to mitigate them is essential for assembling an accurate tree of life.

DNA Sequences Are as Useful as Protein Sequences for Inferring Deep Phylogenies.

Inference of deep phylogenies has almost exclusively used protein rather than DNA sequences based on the perception that protein sequences are less prone to homoplasy and saturation or to issues of compositional heterogeneity than DNA sequences. Here, we analyze a model of codon evolution under an idealized genetic code and demonstrate that those perceptions may be misconceptions. We conduct a simulation study to assess the utility of protein versus DNA sequences for inferring deep phylogenies, with protein-coding data generated under models of heterogeneous substitution processes across sites in the sequence and among lineages on the tree, and then analyzed using nucleotide, amino acid, and codon models. Analysis of DNA sequences under nucleotide-substitution models (possibly with the third codon positions excluded) recovered the correct tree at least as often as analysis of the corresponding protein sequences under modern amino acid models. We also applied the different data-analysis strategies to an empirical dataset to infer the metazoan phylogeny. Our results from both simulated and real data suggest that DNA sequences may be as useful as proteins for inferring deep phylogenies and should not be excluded from such analyses. Analysis of DNA data under nucleotide models has a major computational advantage over protein-data analysis, potentially making it feasible to use advanced models that account for among-site and among-lineage heterogeneity in the nucleotide-substitution process in inference of deep phylogenies.

The ultrastructure of the apical organ of the Müller's larva of the tiger flatworm Prostheceraeus crozieri.

The tiger flatworm Prostheceraeus crozieri (Polycladida) develops via an eight-lobed, and three-eyed planktonic Müller's larva. This larva has an apical organ, ultrastructural details of which remain elusive due to a scarcity of studies. The evolution and possible homology of the polyclad larva with other spiralian larvae is still controversial. Here, we provide ultrastructural data and three-dimensional reconstructions of the apical organ of P. crozieri. The apical organ consists of an apical tuft complex and a dorso-apical tuft complex. The apical tuft complex features a central tuft of five long cilia, which emerge from four or five individual cells that are themselves encircled by two anchor cells. The necks of six multibranched gland cells are sandwiched between ciliated tuft cell bodies and anchor cells. The proximal parts of the ciliated cell bodies are in contact with the lateral brain neuropil via gap junctions. Located dorsally of the apical tuft complex, the dorso-apical tuft complex is characterized by several long cilia of sensory neurons, these emerge from an epidermal lumen and are closely associated with several gland cells that form a crescent apically around the dorsal anchor cell, and laterally touch the brain neuropil. Such ciliated sensory neurons emerging from a ciliated lumen are reminiscent of ampullary cells of mollusc and annelid larvae; a similar cell type can be found in the hoplonemertean decidula larva. We hypothesize that the ampullary-like cells and the tuft-forming sensory cells in the apical organs of these spiralian larvae could be homologous.

CeLaVi: an interactive cell lineage visualization tool.

Recent innovations in genetics and imaging are providing the means to reconstruct cell lineages, either by tracking cell divisions using live microscopy, or by deducing the history of cells using molecular recorders. A cell lineage on its own, however, is simply a description of cell divisions as branching events. A major goal of current research is to integrate this description of cell relationships with information about the spatial distribution and identities of the cells those divisions produce. Visualizing, interpreting and exploring these complex data in an intuitive manner requires the development of new tools. Here we present CeLaVi, a web-based visualization tool that allows users to navigate and interact with a representation of cell lineages, whilst simultaneously visualizing the spatial distribution, identities and properties of cells. CeLaVi's principal functions include the ability to explore and manipulate the cell lineage tree; to visualise the spatial distribution of cell clones at different depths of the tree; to colour cells in the 3D viewer based on lineage relationships; to visualise various cell qualities on the 3D viewer (e.g. gene expression, cell type) and to annotate selected cells/clones. All these capabilities are demonstrated with four different example data sets. CeLaVi is available at http://www.celavi.pro.

OMA standalone: orthology inference among public and custom genomes and transcriptomes.

Genomes and transcriptomes are now typically sequenced by individual laboratories but analyzing them often remains challenging. One essential step in many analyses lies in identifying orthologs-corresponding genes across multiple species-but this is far from trivial. The Orthologous MAtrix (OMA) database is a leading resource for identifying orthologs among publicly available, complete genomes. Here, we describe the OMA pipeline available as a standalone program for Linux and Mac. When run on a cluster, it has native support for the LSF, SGE, PBS Pro, and Slurm job schedulers and can scale up to thousands of parallel processes. Another key feature of OMA standalone is that users can combine their own data with existing public data by exporting genomes and precomputed alignments from the OMA database, which currently contains over 2100 complete genomes. We compare OMA standalone to other methods in the context of phylogenetic tree inference, by inferring a phylogeny of Lophotrochozoa, a challenging clade within the protostomes. We also discuss other potential applications of OMA standalone, including identifying gene families having undergone duplications/losses in specific clades, and identifying potential drug targets in nonmodel organisms. OMA standalone is available under the permissive open source Mozilla Public License Version 2.0.

A software tool 'CroCo' detects pervasive cross-species contamination in next generation sequencing data.

BACKGROUND: Multiple RNA samples are frequently processed together and often mixed before multiplex sequencing in the same sequencing run. While different samples can be separated post sequencing using sample barcodes, the possibility of cross contamination between biological samples from different species that have been processed or sequenced in parallel has the potential to be extremely deleterious for downstream analyses. RESULTS: We present CroCo, a software package for identifying and removing such cross contaminants from assembled transcriptomes. Using multiple, recently published sequence datasets, we show that cross contamination is consistently present at varying levels in real data. Using real and simulated data, we demonstrate that CroCo detects contaminants efficiently and correctly. Using a real example from a molecular phylogenetic dataset, we show that contaminants, if not eliminated, can have a decisive, deleterious impact on downstream comparative analyses. CONCLUSIONS: Cross contamination is pervasive in new and published datasets and, if undetected, can have serious deleterious effects on downstream analyses. CroCo is a database-independent, multi-platform tool, designed for ease of use, that efficiently and accurately detects and removes cross contamination in assembled transcriptomes to avoid these problems. We suggest that the use of CroCo should become a standard cleaning step when processing multiple samples for transcriptome sequencing.

Regulatory circuit rewiring and functional divergence of the duplicate admp genes in dorsoventral axial patterning.

The spatially opposed expression of Antidorsalizing morphogenetic protein (Admp) and BMP signals controls dorsoventral (DV) polarity across Bilateria and hence represents an ancient regulatory circuit. Here, we show that in addition to the conserved admp1 that constitutes the ancient circuit, a second admp gene (admp2) is present in Ambulacraria (Echinodermata+Hemichordata) and two marine worms belonging to Xenoturbellida and Acoelomorpha. The phylogenetic distribution implies that the two admp genes were duplicated in the Bilaterian common ancestor and admp2 was subsequently lost in chordates and protostomes. We show that the ambulacrarian admp1 and admp2 are under opposite transcriptional control by BMP signals and knockdown of Admps in sea urchins impaired their DV polarity. Over-expression of either Admps reinforced BMP signaling but resulted in different phenotypes in the sea urchin embryo. Our study provides an excellent example of signaling circuit rewiring and protein functional changes after gene duplications.

Acoelomorph flatworms are deuterostomes related to Xenoturbella.

Xenoturbellida and Acoelomorpha are marine worms with contentious ancestry. Both were originally associated with the flatworms (Platyhelminthes), but molecular data have revised their phylogenetic positions, generally linking Xenoturbellida to the deuterostomes and positioning the Acoelomorpha as the most basally branching bilaterian group(s). Recent phylogenomic data suggested that Xenoturbellida and Acoelomorpha are sister taxa and together constitute an early branch of Bilateria. Here we assemble three independent data sets-mitochondrial genes, a phylogenomic data set of 38,330 amino-acid positions and new microRNA (miRNA) complements-and show that the position of Acoelomorpha is strongly affected by a long-branch attraction (LBA) artefact. When we minimize LBA we find consistent support for a position of both acoelomorphs and Xenoturbella within the deuterostomes. The most likely phylogeny links Xenoturbella and Acoelomorpha in a clade we call Xenacoelomorpha. The Xenacoelomorpha is the sister group of the Ambulacraria (hemichordates and echinoderms). We show that analyses of miRNA complements have been affected by character loss in the acoels and that both groups possess one miRNA and the gene Rsb66 otherwise specific to deuterostomes. In addition, Xenoturbella shares one miRNA with the ambulacrarians, and two with the acoels. This phylogeny makes sense of the shared characteristics of Xenoturbellida and Acoelomorpha, such as ciliary ultrastructure and diffuse nervous system, and implies the loss of various deuterostome characters in the Xenacoelomorpha including coelomic cavities, through gut and gill slits.

Light-sheet microscopy for everyone? Experience of building an OpenSPIM to study flatworm development.

BACKGROUND: Selective plane illumination microscopy (SPIM a type of light-sheet microscopy) involves focusing a thin sheet of laser light through a specimen at right angles to the objective lens. As only the thin section of the specimen at the focal plane of the lens is illuminated, out of focus light is naturally absent and toxicity due to light (phototoxicity) is greatly reduced enabling longer term live imaging. OpenSPIM is an open access platform (Pitrone et al. 2013 and OpenSPIM.org) created to give new users step-by-step instructions on building a basic configuration of a SPIM microscope, which can in principle be adapted and upgraded to each laboratory's own requirements and budget. Here we describe our own experience with the process of designing, building, configuring and using an OpenSPIM for our research into the early development of the polyclad flatworm Maritigrella crozieri - a non-model animal. RESULTS: Our OpenSPIM builds on the standard design with the addition of two colour laser illumination for simultaneous detection of two probes/molecules and dual sided illumination, which provides more even signal intensity across a specimen. Our OpenSPIM provides high resolution 3d images and time lapse recordings, and we demonstrate the use of two colour lasers and the benefits of two color dual-sided imaging. We used our microscope to study the development of the embryo of the polyclad flatworm M. crozieri. The capabilities of our microscope are demonstrated by our ability to record the stereotypical spiral cleavage pattern of M. crozieri with high-speed multi-view time lapse imaging. 3D and 4D (3D + time) reconstruction of early development from these data is possible using image registration and deconvolution tools provided as part of the open source Fiji platform. We discuss our findings on the pros and cons of a self built microscope. CONCLUSIONS: We conclude that home-built microscopes, such as an OpenSPIM, together with the available open source software, such as MicroManager and Fiji, make SPIM accessible to anyone interested in having continuous access to their own light-sheet microscope. However, building an OpenSPIM is not without challenges and an open access microscope is a worthwhile, if significant, investment of time and money. Multi-view 4D microscopy is more challenging than we had expected. We hope that our experience gained during this project will help future OpenSPIM users with similar ambitions.

The origin and evolution of arthropods.

The past two decades have witnessed profound changes in our understanding of the evolution of arthropods. Many of these insights derive from the adoption of molecular methods by systematists and developmental biologists, prompting a radical reordering of the relationships among extant arthropod classes and their closest non-arthropod relatives, and shedding light on the developmental basis for the origins of key characteristics. A complementary source of data is the discovery of fossils from several spectacular Cambrian faunas. These fossils form well-characterized groupings, making the broad pattern of Cambrian arthropod systematics increasingly consensual.

Improving animal phylogenies with genomic data.

Since the first animal genomes were completely sequenced ten years ago, evolutionary biologists have attempted to use the encoded information to reconstruct different aspects of the earliest stages of animal evolution. One of the most important uses of genome sequences is to understand relationships between animal phyla. Despite the wealth of data available, ranging from primary sequence data to gene and genome structures, our lack of understanding of the modes of evolution of genomic characters means that using these data is fraught with potential difficulties, leading to errors in phylogeny reconstruction. Improved understanding of how different character types evolve, the use of this knowledge to develop more accurate models of evolution, and denser taxonomic sampling, are now minimizing the sources of error. The wealth of genomic data now being produced promises that a well-resolved tree of the animal phyla will be available in the near future.

Phylogenomic analysis of echinoderm class relationships supports Asterozoa.

While some aspects of the phylogeny of the five living echinoderm classes are clear, the position of the ophiuroids (brittlestars) relative to asteroids (starfish), echinoids (sea urchins) and holothurians (sea cucumbers) is controversial. Ophiuroids have a pluteus-type larva in common with echinoids giving some support to an ophiuroid/echinoid/holothurian clade named Cryptosyringida. Most molecular phylogenetic studies, however, support an ophiuroid/asteroid clade (Asterozoa) implying either convergent evolution of the pluteus or reversals to an auricularia-type larva in asteroids and holothurians. A recent study of 10 genes from four of the five echinoderm classes used 'phylogenetic signal dissection' to separate alignment positions into subsets of (i) suboptimal, heterogeneously evolving sites (invariant plus rapidly changing) and (ii) the remaining optimal, homogeneously evolving sites. Along with most previous molecular phylogenetic studies, their set of heterogeneous sites, expected to be more prone to systematic error, support Asterozoa. The homogeneous sites, in contrast, support an ophiuroid/echinoid grouping, consistent with the cryptosyringid clade, leading them to posit homology of the ophiopluteus and echinopluteus. Our new dataset comprises 219 genes from all echinoderm classes; analyses using probabilistic Bayesian phylogenetic methods strongly support Asterozoa. The most reliable, slowly evolving quartile of genes also gives highest support for Asterozoa; this support diminishes in second and third quartiles and the fastest changing quartile places the ophiuroids close to the root. Using phylogenetic signal dissection, we find heterogenous sites support an unlikely grouping of Ophiuroidea + Holothuria while homogeneous sites again strongly support Asterozoa. Our large and taxonomically complete dataset finds no support for the cryptosyringid hypothesis; in showing strong support for the Asterozoa, our preferred topology leaves the question of homology of pluteus larvae open.

MicroRNAs and phylogenomics resolve the relationships of Tardigrada and suggest that velvet worms are the sister group of Arthropoda.

Morphological data traditionally group Tardigrada (water bears), Onychophora (velvet worms), and Arthropoda (e.g., spiders, insects, and their allies) into a monophyletic group of invertebrates with walking appendages known as the Panarthropoda. However, molecular data generally do not support the inclusion of tardigrades within the Panarthropoda, but instead place them closer to Nematoda (roundworms). Here we present results from the analyses of two independent genomic datasets, expressed sequence tags (ESTs) and microRNAs (miRNAs), which congruently resolve the phylogenetic relationships of Tardigrada. Our EST analyses, based on 49,023 amino acid sites from 255 proteins, significantly support a monophyletic Panarthropoda including Tardigrada and suggest a sister group relationship between Arthropoda and Onychophora. Using careful experimental manipulations--comparisons of model fit, signal dissection, and taxonomic pruning--we show that support for a Tardigrada + Nematoda group derives from the phylogenetic artifact of long-branch attraction. Our small RNA libraries fully support our EST results; no miRNAs were found to link Tardigrada and Nematoda, whereas all panarthropods were found to share one unique miRNA (miR-276). In addition, Onychophora and Arthropoda were found to share a second miRNA (miR-305). Our study confirms the monophyly of the legged ecdysozoans, shows that past support for a Tardigrada + Nematoda group was due to long-branch attraction, and suggests that the velvet worms are the sister group to the arthropods.

Dollo Parsimony Overestimates Ancestral Gene Content Reconstructions.

Ancestral reconstruction is a widely used technique that has been applied to understand the evolutionary history of gain and loss of gene families. Ancestral gene content can be reconstructed via different phylogenetic methods, but many current and previous studies employ Dollo parsimony. We hypothesize that Dollo parsimony is not appropriate for ancestral gene content reconstruction inferences based on sequence homology, as Dollo parsimony is derived from the assumption that a complex character cannot be regained. This premise does not accurately model molecular sequence evolution, in which false orthology can result from sequence convergence or lateral gene transfer. The aim of this study is to test Dollo parsimony's suitability for ancestral gene content reconstruction and to compare its inferences with a maximum likelihood-based approach that allows a gene family to be gained more than once within a tree. We first compared the performance of the two approaches on a series of artificial data sets each of 5,000 genes that were simulated according to a spectrum of evolutionary rates without gene gain or loss, so that inferred deviations from the true gene count would arise only from errors in orthology inference and ancestral reconstruction. Next, we reconstructed protein domain evolution on a phylogeny representing known eukaryotic diversity. We observed that Dollo parsimony produced numerous ancestral gene content overestimations, especially at nodes closer to the root of the tree. These observations led us to the conclusion that, confirming our hypothesis, Dollo parsimony is not an appropriate method for ancestral reconstruction studies based on sequence homology.

Single cell atlas of Xenoturbella bocki highlights limited cell-type complexity.

Phylogenetic analyses over the last two decades have united a few small, and previously orphan clades, the nematodermatids, acoels and xenoturbelids, into the phylum Xenacoelomorpha. Some phylogenetic analyses support a sister relationship between Xenacoelomorpha and Ambulacraria (Xenambulacraria), while others suggest that Xenacoelomorpha may be sister to the rest of the Bilateria (Nephrozoa). An understanding of the cell type complements of Xenacoelomorphs is essential to assessing these alternatives as well as to our broader understanding of bilaterian cell type evolution. Employing whole organism single-cell RNA-seq in the marine xenacoelomorph worm Xenoturbella bocki, we show that Xenambulacrarian nerve nets share regulatory features and a peptidergic identity with those found in cnidarians and protostomes and more broadly share muscle and gland cell similarities with other metazoans. Taken together, these data are consistent with broad homologies of animal gland, muscle, and neurons as well as more specific affinities between Xenoturbella and acoel gut and epidermal tissues, consistent with the monophyly of Xenacoelomorpha.

Single-cell atlases of two lophotrochozoan larvae highlight their complex evolutionary histories.

Pelagic larval stages are widespread across animals, yet it is unclear whether larvae were present in the last common ancestor of animals or whether they evolved multiple times due to common selective pressures. Many marine larvae are at least superficially similar; they are small, swim through the beating of bands of cilia, and sense the environment with an apical organ. To understand these similarities, we have generated single-cell atlases for marine larvae from two animal phyla and have compared their cell types. We found clear similarities among ciliary band cells and between neurons of the apical organ in the two larvae pointing to possible homology of these structures, suggesting a single origin of larvae within Spiralia. We also find several clade-specific innovations in each larva, including distinct myocytes and shell gland cells in the oyster larva. Oyster shell gland cells express many recently evolved genes that have made previous gene age estimates for the origin of trochophore larvae too young.

Deuterostome phylogeny reveals monophyletic chordates and the new phylum Xenoturbellida.

Deuterostomes comprise vertebrates, the related invertebrate chordates (tunicates and cephalochordates) and three other invertebrate taxa: hemichordates, echinoderms and Xenoturbella. The relationships between invertebrate and vertebrate deuterostomes are clearly important for understanding our own distant origins. Recent phylogenetic studies of chordate classes and a sea urchin have indicated that urochordates might be the closest invertebrate sister group of vertebrates, rather than cephalochordates, as traditionally believed. More remarkable is the suggestion that cephalochordates are closer to echinoderms than to vertebrates and urochordates, meaning that chordates are paraphyletic. To study the relationships among all deuterostome groups, we have assembled an alignment of more than 35,000 homologous amino acids, including new data from a hemichordate, starfish and Xenoturbella. We have also sequenced the mitochondrial genome of Xenoturbella. We support the clades Olfactores (urochordates and vertebrates) and Ambulacraria (hemichordates and echinoderms). Analyses using our new data, however, do not support a cephalochordate and echinoderm grouping and we conclude that chordates are monophyletic. Finally, nuclear and mitochondrial data place Xenoturbella as the sister group of the two ambulacrarian phyla. As such, Xenoturbella is shown to be an independent phylum, Xenoturbellida, bringing the number of living deuterostome phyla to four.

TranslatorX: multiple alignment of nucleotide sequences guided by amino acid translations.

We present TranslatorX, a web server designed to align protein-coding nucleotide sequences based on their corresponding amino acid translations. Many comparisons between biological sequences (nucleic acids and proteins) involve the construction of multiple alignments. Alignments represent a statement regarding the homology between individual nucleotides or amino acids within homologous genes. As protein-coding DNA sequences evolve as triplets of nucleotides (codons) and it is known that sequence similarity degrades more rapidly at the DNA than at the amino acid level, alignments are generally more accurate when based on amino acids than on their corresponding nucleotides. TranslatorX novelties include: (i) use of all documented genetic codes and the possibility of assigning different genetic codes for each sequence; (ii) a battery of different multiple alignment programs; (iii) translation of ambiguous codons when possible; (iv) an innovative criterion to clean nucleotide alignments with GBlocks based on protein information; and (v) a rich output, including Jalview-powered graphical visualization of the alignments, codon-based alignments coloured according to the corresponding amino acids, measures of compositional bias and first, second and third codon position specific alignments. The TranslatorX server is freely available at http://translatorx.co.uk.

Genome Assembly of the Polyclad Flatworm Prostheceraeus crozieri.

Polyclad flatworms are widely thought to be one of the least derived of the flatworm classes and, as such, are well placed to investigate evolutionary and developmental features such as spiral cleavage and larval diversification lost in other platyhelminths. Prostheceraeus crozieri, (formerly Maritigrella crozieri), is an emerging model polyclad flatworm that already has some useful transcriptome data but, to date, no sequenced genome. We have used high molecular weight DNA extraction and long-read PacBio sequencing to assemble the highly repetitive (67.9%) P. crozieri genome (2.07 Gb). We have annotated 43,325 genes, with 89.7% BUSCO completeness. Perhaps reflecting its large genome, introns were considerably larger than other free-living flatworms, but evidence of abundant transposable elements suggests genome expansion has been principally via transposable elements activity. This genome resource will be of great use for future developmental and phylogenomic research.