Masa-aki Yoshida

Genome structure analysis of molluscs revealed whole genome duplication and lineage specific repeat variation.

Comparative genome structure analysis allows us to identify novel genes, repetitive sequences and gene duplications. To explore lineage-specific genomic changes of the molluscs that is good model for development of nervous system in invertebrate, we conducted comparative genome structure analyses of three molluscs, pygmy squid, nautilus and scallops using partial genome shotgun sequencing. Most effective elements on the genome structural changes are repetitive elements (REs) causing expansion of genome size and whole genome duplication producing large amount of novel functional genes. Therefore, we investigated variation and proportion of REs and whole genome duplication. We, first, identified variations of REs in the three molluscan genomes by homology-based and de novo RE detection. Proportion of REs were 9.2%, 4.0%, and 3.8% in the pygmy squid, nautilus and scallop, respectively. We, then, estimated genome size of the species as 2.1, 4.2 and 1.8 Gb, respectively, with 2× coverage frequency and DNA sequencing theory. We also performed a gene duplication assay based on coding genes, and found that large-scale duplication events occurred after divergence from the limpet Lottia, an out-group of the three molluscan species. Comparison of all the results suggested that RE expansion did not relate to the increase in genome size of nautilus. Despite close relationships to nautilus, the squid has the largest portion of REs and smaller genome size than nautilus. We also identified lineage-specific RE and gene-family expansions, possibly relate to acquisition of the most complicated eye and brain systems in the three species.

Genetic mechanisms involved in the evolution of the cephalopod camera eye revealed by transcriptomic and developmental studies

BackgroundColeoid cephalopods (squids and octopuses) have evolved a camera eye, the structure of which is very similar to that found in vertebrates and which is considered a classic example of convergent evolution. Other molluscs, however, possess mirror, pin-hole, or compound eyes, all of which differ from the camera eye in the degree of complexity of the eye structures and neurons participating in the visual circuit. Therefore, genes expressed in the cephalopod eye after divergence from the common molluscan ancestor could be involved in eye evolution through association with the acquisition of new structural components. To clarify the genetic mechanisms that contributed to the evolution of the cephalopod camera eye, we applied comprehensive transcriptomic analysis and conducted developmental validation of candidate genes involved in coleoid cephalopod eye evolution.ResultsWe compared gene expression in the eyes of 6 molluscan (3 cephalopod and 3 non-cephalopod) species and selected 5,707 genes as cephalopod camera eye-specific candidate genes on the basis of homology searches against 3 molluscan species without camera eyes. First, we confirmed the expression of these 5,707 genes in the cephalopod camera eye formation processes by developmental array analysis. Second, using molecular evolutionary (dN/dS) analysis to detect positive selection in the cephalopod lineage, we identified 156 of these genes in which functions appeared to have changed after the divergence of cephalopods from the molluscan ancestor and which contributed to structural and functional diversification. Third, we selected 1,571 genes, expressed in the camera eyes of both cephalopods and vertebrates, which could have independently acquired a function related to eye development at the expression level. Finally, as experimental validation, we identified three functionally novel cephalopod camera eye genes related to optic lobe formation in cephalopods by in situ hybridization analysis of embryonic pygmy squid.ConclusionWe identified 156 genes positively selected in the cephalopod lineage and 1,571 genes commonly found in the cephalopod and vertebrate camera eyes from the analysis of cephalopod camera eye specificity at the expression level. Experimental validation showed that the cephalopod camera eye-specific candidate genes include those expressed in the outer part of the optic lobes, which unique to coleoid cephalopods. The results of this study suggest that changes in gene expression and in the primary structure of proteins (through positive selection) from those in the common molluscan ancestor could have contributed, at least in part, to cephalopod camera eye acquisition.

Loss of the six3/6 controlling pathways might have resulted in pinhole-eye evolution in Nautilus

Coleoid cephalopods have an elaborate camera eye whereas nautiloids have primitive pinhole eye without lens and cornea. The Nautilus pinhole eye provides a unique example to explore the module of lens formation and its evolutionary mechanism. Here, we conducted an RNA-seq study of developing eyes of Nautilus and pygmy squid. First, we found that evolutionary distances from the common ancestor to Nautilus or squid are almost the same. Although most upstream eye development controlling genes were expressed in both species, six3/6 that are required for lens formation in vertebrates was not expressed in Nautilus. Furthermore, many downstream target genes of six3/6 including crystallin genes and other lens protein related genes were not expressed in Nautilus. As six3/6 and its controlling pathways are widely conserved among molluscs other than Nautilus, the present data suggest that deregulation of the six3/6 pathway led to the pinhole eye evolution in Nautilus.

Phylogeny of Selected Sepiidae (Mollusca, Cephalopoda) on 12S, 16S, and COI Sequences, with Comments on the Taxonomic Reliability of Several Morphological Characters

Abstract Phylogenetic relationships among 11 species of sepiids from Japanese waters and Sepia officinalis from Mediterranean were studied using partial sequences of the mitochondrial 12S rRNA, 16S rRNA, and cytochrome c oxidase subunit I genes. These three genes had been analyzed in an Atlantic species S. elagans and was obtained from database. In the two-gene set analysis (16S+COI), sequence data of another 4 species were added from database. We also studied morphological characters of radulae, tentacular clubs, and cuttlebones. The molecular phylogeny was not congruent with relationships detected by the number of rows in radulae and the arrangement of suckers on the tentacular club. As to the cuttlebone shape, the molecular phylogeny suggests the separation of two groups, Doratosepion species with a lanceolate cuttlebone and the others with a broad cuttlebone. Our molecular phylogenetic study revealed these sepiids are separated into four clades. The first clade includes Sepia officinalis, S. hierrendda, S. bertheloti, S. pharaonis and Sepiella japonica. The second clade consists of S. latimanus and Metasepia tullbergi from sub-tropical waters. The third clade includes Sepia esculenta, S. madokai, S. aculeata and S. lycidas, which have a cuttlebone with a prominent spine. The fourth clade consists of Doratosepion species complex, S. kobiensis, S. lorigera, S. pardex, S. peterseni, and S. sp., which are characterized by a narrow cuttlebone with a distinct outer cone at the posterior end. The lack of membranous structures in the cuttlebone is a synapomorphy for this clade. S. elegans did not clearly belong to any of these clades and might represent the fifth clade.

Mitochondrial Genome of a Japanese Placozoan

Placozoans are marine invertebrates found in tropical and subtropical waters. Their body plan is among the simplest of free-living animals. The present study determined the mitochondrial genome sequence of a placozoan collected on the coast of Shirahama, Wakayama, Honshu, Japan, and compared it with those of Trichoplax adhaerens from the Red Sea and of three strains from the Caribbean Sea. The sequences of mitochondrial respiratory chain of the Japanese placozoan genes are very similar to those of the BZ49 strain from the Caribbean Sea. However, there are distinct differences in gene arrangement, such as the location of two open reading frames. This Japanese placozoan is therefore distinguishable from the other strains. Based on current knowledge of placozoan 16S diversity our ‘Shirahama’ strain most likely represents the H15 lineage, known from the Philippines. In the mitochondrial genome of placozoans, substitution rates are slower than in bilaterians, whereas the rate of rearrangements is faster.

In vitro homology search array comprehensively reveals highly conserved genes and their functional characteristics in non-sequenced species

BackgroundWith the increase in genomic and transcriptomic data produced by the recent advancements in next generation sequencers and microarrays, it is now easier than ever to conduct large-scale comparative genomic studies for familiar species. However, there are more than ten million species on earth, and the study of all remaining species is not realistic in terms of cost and time. There have been a number of attempts at using microarrays for cross-species hybridization; however, those approaches only utilized the same probes for each species or different probes designed from orthologous genes. To establish easier and cheaper methods for the large-scale comparative genomic study of non-sequenced species, we developed an in vitro homology search array with the aid of a bioinformatic approach to probe design.ResultsTo perform large-scale genomic comparisons of non-sequenced species, we chose squid, one of the most intelligent species among Protostomes, for comparison with human genes. We designed a microarray using human single copy genes and conducted microarray experiments with mRNAs extracted from the squid. Multi-copy genes could not be detected using the microarray in this study because their sequence similarity caused cross-hybridization. A search for squid homologous genes among human genes revealed that 68% of the human probes tested showed the expression of squid homolog genes and 95 genes were confirmed to be expressed highly in squid. Functional classification analysis showed that these highly expressed genes comprise DNA binding proteins, which are under pressure of DNA level mutation and, consequently, show high similarity at the nucleotide level.ConclusionsOur array could detect homologous genes in squids and humans in spite of the distant phylogenic relationships between the species. This experimental method will be useful for identifying homologs in non-sequenced species, for the development of genetic resources and for the collection of information on biodiversity, particularly when using the genome of sibling or closely related species.

Venous branching asymmetry in the pygmy squid Idiosepius (Cephalopoda: Idiosepiida) with reference to its phylogenetic position and functional significance

The Japanese pygmy squid, Idiosepius paradoxus (Ortman, 1888), is one of the smallest cephalopods in the world. Their fully described developmental stages and the ready availability of eggs also make pygmy squids suitable material for cephalopod developmental studies. However, their phylogenetic position among the Decabrachia is unclear. We investigated the vascular anatomy of the Japanese pygmy squid using serial sections of the adult. Their venous system is different from that of teuthoids with respect to the presence of the vena cava sinistra. The branching pattern of the vena cava is similar to that of the sepiolids in having two pathways, but different in that it lacks a connection above the hind gut. The Idiosepius heart is also similar to that of sepiids in having the genital artery located dorsally. These features of Idiosepius agree with molecular phylogenies suggesting they are related to the sepiolids. We discuss the venous branching asymmetry with reference to its phylogenetic and functional significance.