Leonid L. Moroz

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.

The ctenophore genome and the evolutionary origins of neural systems

The origins of neural systems remain unresolved. In contrast to other basal metazoans, ctenophores (comb jellies) have both complex nervous and mesoderm-derived muscular systems. These holoplanktonic predators also have sophisticated ciliated locomotion, behaviour and distinct development. Here we present the draft genome of Pleurobrachia bachei, Pacific sea gooseberry, together with ten other ctenophore transcriptomes, and show that they are remarkably distinct from other animal genomes in their content of neurogenic, immune and developmental genes. Our integrative analyses place Ctenophora as the earliest lineage within Metazoa. This hypothesis is supported by comparative analysis of multiple gene families, including the apparent absence of HOX genes, canonical microRNA machinery, and reduced immune complement in ctenophores. Although two distinct nervous systems are well recognized in ctenophores, many bilaterian neuron-specific genes and genes of ‘classical’ neurotransmitter pathways either are absent or, if present, are not expressed in neurons. Our metabolomic and physiological data are consistent with the hypothesis that ctenophore neural systems, and possibly muscle specification, evolved independently from those in other animals.

Phylogenomics reveals deep molluscan relationships

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 sequences or morphological data, have been unsuccessful at elucidating these relationships. Recently, phylogenomic studies using dozens to hundreds of genes have greatly improved our understanding of deep animal relationships. 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.

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.

Neuronal Transcriptome of Aplysia: Neuronal Compartments and Circuitry

Molecular analyses of Aplysia, a well-established model organism for cellular and systems neural science, have been seriously handicapped by a lack of adequate genomic information. By sequencing cDNA libraries from the central nervous system (CNS), we have identified over 175,000 expressed sequence tags (ESTs), of which 19,814 are unique neuronal gene products and represent 50%-70% of the total Aplysia neuronal transcriptome. We have characterized the transcriptome at three levels: (1) the central nervous system, (2) the elementary components of a simple behavior: the gill-withdrawal reflex-by analyzing sensory, motor, and serotonergic modulatory neurons, and (3) processes of individual neurons. In addition to increasing the amount of available gene sequences of Aplysia by two orders of magnitude, this collection represents the largest database available for any member of the Lophotrochozoa and therefore provides additional insights into evolutionary strategies used by this highly successful diversified lineage, one of the three proposed superclades of bilateral animals.

Modulation of ion channels in rod photoreceptors by nitric oxide

Subcellular compartments in the outer retina of the larval tiger salamander were identified as likely sites of production of nitric oxide (NO), a recently recognized intercellular messenger. NADPH diaphorase histochemistry and NO synthase immunocytochemistry labeled photoreceptor ellipsoids and the distal regions of bipolar and glial cells apposing photoreceptor inner segments, suggesting a role for NO in visual processing in the outer retina. We investigated the actions of NO on several rod photoreceptor ion channels. Application of the NO-generating compound S-nitrosocysteine increased Ca2+ channel current and a voltage-independent conductance, but had no affect on voltage-gated K+ or nonspecific cation currents. Given the steep relation between voltage-dependent Ca2+ influx and photoreceptor synaptic output, these results indicate that NO could modulate transmission of the photoresponse to second order cells.

Interfering with Nitric Oxide Measurements 4,5-DIAMINOFLUORESCEIN REACTS WITH DEHYDROASCORBIC ACID AND ASCORBIC ACID

4,5-Diaminofluorescein (DAF-2) is widely used for detection and imaging of NO based on its sensitivity, noncytotoxicity, and specificity. In the presence of oxygen, NO and NO-related reactive nitrogen species nitrosate 4,5-diaminofluorescein to yield the highly fluorescent DAF-2 triazole (DAF-2T). However, as reported here, the DAF-2 reaction to form a fluorescent product is not specific to NO because it reacts with dehydroascorbic acid (DHA) and ascorbic acid (AA) to generate new compounds that have fluorescence emission profiles similar to that of DAF-2T. When DHA is present, the formation of DAF-2T is attenuated because the DHA competes for DAF-2, whereas AA decreases the nitrosation of DAF-2 to a larger extent, possibly because of additional reducing activity that affects the amount of available N2O3 from the NO. The reaction products of DAF-2 with DHA and AA have been characterized using capillary electrophoresis with laser-induced fluorescence detection and electrospray mass spectrometry. The reactions of DAF-2 with DHA and AA are particularly significant because DHA and AA often colocalize with nitric-oxide synthase in the central nervous, cardiovascular, and immune systems, indicating the importance of understanding this chemistry.

Error, signal, and the placement of Ctenophora sister to all other animals

Significance Traditional interpretation of animal phylogeny suggests traits, such as mesoderm, muscles, and neurons, evolved only once given the assumed placement of sponges as sister to all other animals. In contrast, placement of ctenophores as the first branching animal lineage raises the possibility of multiple origins of many complex traits considered important for animal diversification and success. We consider sources of potential error and increase taxon sampling to find a single, statistically robust placement of ctenophores as our most distant animal relatives, contrary to the traditional understanding of animal phylogeny. Furthermore, ribosomal protein genes are identified as creating conflict in signal that caused some past studies to recover a sister relationship between ctenophores and cnidarians. Elucidating relationships among early animal lineages has been difficult, and recent phylogenomic analyses place Ctenophora sister to all other extant animals, contrary to the traditional view of Porifera as the earliest-branching animal lineage. To date, phylogenetic support for either ctenophores or sponges as sister to other animals has been limited and inconsistent among studies. Lack of agreement among phylogenomic analyses using different data and methods obscures how complex traits, such as epithelia, neurons, and muscles evolved. A consensus view of animal evolution will not be accepted until datasets and methods converge on a single hypothesis of early metazoan relationships and putative sources of systematic error (e.g., long-branch attraction, compositional bias, poor model choice) are assessed. Here, we investigate possible causes of systematic error by expanding taxon sampling with eight novel transcriptomes, strictly enforcing orthology inference criteria, and progressively examining potential causes of systematic error while using both maximum-likelihood with robust data partitioning and Bayesian inference with a site-heterogeneous model. We identified ribosomal protein genes as possessing a conflicting signal compared with other genes, which caused some past studies to infer ctenophores and cnidarians as sister. Importantly, biases resulting from elevated compositional heterogeneity or elevated substitution rates are ruled out. Placement of ctenophores as sister to all other animals, and sponge monophyly, are strongly supported under multiple analyses, herein.

On the Independent Origins of Complex Brains and Neurons

Analysis of the origin and evolution of neurons is crucial for revealing principles of organization of neural circuits with unexpected implications for genomic sciences, biomedical applications and regenerative medicine. This article presents an overview of some controversial ideas about the origin and evolution of neurons and nervous systems, focusing on the independent origin of complex brains and possible independent origins of neurons. First, earlier hypotheses related to the origin of neurons are summarized. Second, the diversity of nervous systems and convergent evolution of complex brains in relation to current views about animal phylogeny is discussed. Third, the lineages of molluscs and basal metazoans are used as illustrated examples of multiple origins of complex brains and neurons. Finally, a hypothesis about the independent origin of complex brains, centralized nervous systems and neurons is outlined. Injury-associated mechanisms leading to secretion of signal peptides (and related molecules) can be considered as evolutionary predecessors of inter-neuronal signaling and the major factors in the appearance of neurons in the first place.

Nitric oxide synthase-immunoreactive cells in the CNS and periphery of Lymnaea

The presence and distribution of nitric oxide synthase (NOS) in the CNS and peripheral organs (buccal muscles, oesophagus, salivary glands, foot, mantle and pneumostome) of the pulmonate mollusc, Lymnaea stagnalis were studied using an antiserum developed against rat cerebellar NOS. NOS-immunopositive neurones in Lymnaea were localized predominantly in the buccal ganglia as well as in distinct areas of the cerebral and suboesophageal ganglia. NOS-immunoreactive terminals were also found on the somata of some central neurones. In the periphery, NOS-immunostaining was detected only in a few neurones in the pneumostome area and in the osphradial ganglion. In addition, approximately 100 NOS-immunopositive cells have been found in the salivary glands. Our data supports other recent reports indicating that NO may be a signal molecule in the CNS of molluscs.