Hidden genetic history of the Japanese sand dollar Peronella (Echinoidea: Laganidae) revealed by nuclear intron sequences.

The marine environment around Japan experienced significant changes during the Cenozoic Era. In this study, we report findings suggesting that this dynamic history left behind traces in the genome of the Japanese sand dollar species Peronella japonica and P. rubra. Although mitochondrial Cytochrome C Oxidase I sequences did not indicate fragmentation of the current local populations of P. japonica around Japan, two different types of intron sequence were found in the Alx1 locus. We inferred that past fragmentation of the populations account for the presence of two types of nuclear sequences as alleles in the Alx1 intron of P. japonica. It is likely that the split populations have intermixed in recent times; hence, we did not detect polymorphisms in the sequences reflecting the current localization of the species. In addition, we found two allelic sequences of theAlx1 intron in the sister species P. rubra. The divergence times of the two types of Alx1 intron sequences were estimated at approximately 14.9 and 4.0 million years ago for P. japonica and P. rubra, respectively. Our study indicates that information from the intron sequences of nuclear genes can enhance our understanding of past genetic events in organisms.

A New Species of Sexually Dimorphic Brittle Star of the Genus Ophiodaphne (Echinodermata: Ophiuroidea).

We describe a new species of sexually dimorphic brittle star, Ophiodaphne spinosa, from Japan associated with the irregular sea urchin, Clypeaster japonicus based on its external morphology, and phylogenetic analyses of mitochondrial COI (cytochrome c oxidase subunit I). Females of this new species of Ophiodaphne are characterized mainly by the presence of wavy grooves on the surface of the radial shields, needle-like thorns on the oral skeletal jaw structures, and a low length-to-width ratio of the jaw angle in comparison with those of type specimens of its Ophiodaphne congeners: O. scripta, O. materna, and O. formata. A tabular key to the species characteristics of Ophiodaphne is provided. Phylogenetic analyses indicate that the new species of Ophiodaphne, O. scripta, and O. formata are monophyletic. Our results indicate that the Japanese Ophiodaphne include both the new species and O. scripta, and that there are four Ophiodaphne species of sexually dimorphic brittle stars with androphorous habit.

DNA barcoding and morphological analyses revealed validity of Diadema clarki Ikeda, 1939 (Echinodermata, Echinoidea, Diadematidae).

A long-spined sea urchin Diadema-sp reported from Japanese waters was genetically distinct from all known Diadema species, but it remained undescribed. Extensive field surveys in Japan with molecular identification performed in the present study determined five phenotypes (I to V) in Diadema-sp according to the presence and/or shape of a white streak and blue iridophore lines in the naked space of the interambulacral area. All phenotypes were distinct from Diadema setosum (Leske, 1778) and Diadema savignyi (Audouin, 1829), of which a major type (I) corresponded to Diadema clarki Ikeda, 1939 that was questioned and synonymized with Diadema setosum by Mortensen (1940). The holotype of Diadema clarki has not been found, but three unlabeled dried tests of Diadema were found among Ikeda's original collection held in the Kitakyushu Museum of Natural History and Human History, Fukuoka, Japan. A short mtDNA COI fragment (ca. 350bp) was amplified from one of the tests, and the nucleotide sequence determined (275bp) was nearly identical with that of Diadema-sp. Arrangements of the primary tubercles on the coronal plates in Diadema-sp and the museum specimen also conformed with Diadema clarki, indicating that Diadema-sp is identical to Diadema clarki and a valid species. Narrow latitudinal distribution (31°N to 35°N) of Diadema clarki in Japan was observed, where it co-existed with abundant Diadema setosum and rare Diadema savignyi. No Diadema clarki was found in the southern islands in Japan, such as Satsunan Islands to Ryukyu Islands and Ogasawara Island, where Diadema setosum and Diadema savignyi were commonly observed.

Peltogasterella sensuru n. sp. (Crustacea: Cirripedia: Rhizocephala) from off Okinawa Island (Ryukyu Archipelago, Japan) with remarks on its single brood externae.

Peltogasterella sensuru n. sp. infests Pagurixus hermit crabs inhabiting rocky shores off Okinawa Island (Ryukyu Archipelago, Japan). This species is clearly distinguishable from Peltogasterella gracilis (Boschma, 1927): the stalk emerges from the middle part of the externa in the present species and P. sulcata (Lilljeborg, 1859), while the stalk in P. gracilis emerges from the posterior end of the externae. The new species differs from P. sulcata based on the morphology of the mantle aperture. Peltogasterella sensuru n. sp. repeatedly produces single brood externae that have not been previously observed in species belonging to the suborder Kentrogonida Delage, 1884. We also determined partial sequences of the COI gene and 16S rRNA gene of the new species for use as molecular markers for species identification.

A new genus and two new species of Peltogastridae (Crustacea: Cirripedia: Rhizocephala) parasitizing hermit crabs from Okinawa Island (Ryukyu Archipelago, Japan), and their DNA-barcodes.

A new genus and two new species of Peltogastridae, Peltogaster postica sp. nov. and Ommatogaster nana gen. et sp. nov., are described from Okinawa Island, Ryukyu Islands, southwestern Japan. The two new rhizocephalans were found to be parasitic on the estuarine hermit crabs, Pagurus minutus Hess, 1865 and Diogenes leptocerus Forest, 1956, respectively. Peltogaster postica sp. nov. is allied to P. curvata Kossmann, 1874, P. paguri Rathke, 1842 , and P. reticulata Shiino, 1943 , but is distinguished by its relative length and internal and external structures of the mature externa. Ommatogaster gen. nov. is established for the present new species O. nana based on the morphologies of the visceral mass of the externa and the presence of a nauplius eye in the larvae. Partial COI sequences were obtained from the two new species and one known species, Dipterosaccus indicus Van Kampen and Boschma, 1925, to test the possible usefulness of the sequences as tags for species identification.

A new didemnid ascidian Lissoclinum midui sp. nov. from Kumejima Island (Okinawa, Japan), with remarks on the absence of a common cloacal system and the presence of an unknown organ.

A new photosymbiotic didemnid, Lissoclinum midui sp. nov., is described from coral reefs in the Ryukyu Archipelago, Japan. Colonies of the didemnid are green due to Prochloron algal symbionts, which are distributed solely in the tunic. The new species is placed in Lissoclinum because of its uncoiled vas deferens and the presence of globular spicules. However, two unique characters distinguish this species from all other didemnid ascidians: the absence of a common cloacal system, and the presence of an unknown organ in the bottom wall of the branchial sac. In the phylogenetic trees inferred from partial sequences of cytochrome c oxidase subunit I (COI) gene, the new species diverged at the basal point of the clade of four photosymbiotic Lissoclinum species analyzed here.

Timing of spawning and early development of Palythoa tuberculosa (Anthozoa, Zoantharia, Sphenopidae) in Okinawa, Japan.

The spawning behavior and early embryogenesis of Palythoa tuberculosa (Anthozoa, Zoantharia) were observed in August 2009 off Okinawa Island, Japan. P. tuberculosa released zygotes just after high tide around new moon nights. The mean diameter of zygotes was 365.6 ± s.d.14.8 µm, and zygotes did not contain any symbiotic algae (zooxanthellae). About 2 h after spawning, the first cleavage furrow appeared on one side of the zygotes, although it was uncertain when eggs were fertilized. After second cleavage, the arrangement of blastomeres was pseudospherical. At 9 h after spawning, the embryo became a concave-convex dish shape, then gastrulation occurred and the blastopore was formed. Seven-day old larvae were ellipsoid and about 700 µm long, with an open mouth at one end. Two weeks after spawning, the larvae developed a longitudinal band of long cilia (= ventral ciliate band) that is characteristic of zoanthella larvae. In P. tuberculosa, larvae show a non-radial body plan and then metamorphose to almost-radial (in outward appearance) polyps after settlement. These results may support a hypothesis that a common ancestor of Cnidaria had a bilateral body plan that has been secondarily lost in some extant cnidarians.

Genetic isolation among morphotypes in the photosymbiotic didemnid Didemnum molle (Ascidiacea, Tunicata) from the Ryukyus and Taiwan.

Didemnum molle, a colonial ascidian that harbors the symbiotic cyanophyte Prochloron spp., is distributed throughout the coral reefs of the Indo-West Pacific Ocean. Several morphotypes of D. molle are characterized by the color and size of their colonies. Previous molecular phylogeny inferred from gene sequences for the cytochrome oxidase subunit I (COI) identified four morphotypes (i.e., gray, brown, white, and large) from several sites in the Ryukyu Islands, Japan. With the addition of 17 specimens, including another morphotype (small), from several collection sites (Taiwan and the Ryukyus), the present report demonstrates genetic separation among the five morphotypes based on COI sequences. A number of sexually mature specimens of the different morphotypes were collected at the same times and sites, indicating reproductive isolation among morphotypes.

Epizoanthus spp. associations revealed using DNA markers: a case study from Kochi, Japan.

Zoanthids (Cnidaria, Hexacorallia) of the genus Epizoanthus are often found in association with other marine invertebrates, including gastropods and hermit crabs. However, little information exists on the specificity and nature of these associations due to a lack of investigation into Epizoanthus species diversity, and the taxonomy of Epizoanthus is therefore confused. In this study, analyses of morphological data (tentacle number, polyp size, etc) and molecular data (mitochondrial cytochrome oxidase subunit 1 = COI, 16S ribosomal DNA = 16S rDNA) were used to examine Epizoanthus specimens from Tosa Bay, Kochi, Japan. The Epizoanthus specimens were found on both live gastropods (Gemmula unedo) and hermit crabs (Paguristes palythophilus) inhabiting G. unedo and G. cosmoi shells. While morphological analyses did not show clear differences between examined specimens, both COI and mt 16S rDNA clearly divided the specimens into two groups, one associated only with hermit crabs (= Epizoanthus sp. C), and another associated only with living gastropods (= Epizoanthus sp. S). Unexpectedly, DNA sequences from both groups did not match with two previously reported Epizoanthus species from Japan (E. indicus, E. ramosus), indicating they both may be undescribed species. These results highlight the utility of DNA "barcoding" of unknown zoanthids, and will provide a foundation for re-examinations of Epizoanthus species diversity and specificity, which will be critical in understanding the evolution of these unique marine invertebrates.

The first internal molecular phylogeny of the animal phylum Entoprocta (Kamptozoa).

This article provides the first molecular phylogenetic study of the enigmatic invertebrate phylum Entoprocta and was designed to resolve the internal phylogenetic relationships of the taxon. The study is based on partial and combined analyses of the mitochondrial gene cytochrome c oxidase subunit I (COI), as well as the nuclear ribosomal genes 28S rDNA and 18S rDNA. A short morphological character matrix was constructed to trace character evolution along the combined molecular phylogenetic tree. The combined analyses of all three genes strongly support the monophyly of the phylum Entoprocta and a sister group relationship of Entoprocta and Cycliophora, a result which is consistent with a number of previous morphological and molecular assessments. We find evidence for two separate lineages within the Entoprocta, one lineage leading to all recent colonial taxa, Coloniales, another representing the clade of solitary entoprocts, Solitaria. Our study suggests that Loxosomella is a paraphyletic assembly with regard to the genera Loxomitra, Loxosoma, and Loxocorone. The results imply that the ancestral entoproct was a solitary, marine organism with an epizoic life style. The groundplan of the entoproct adult stage probably included a bilobed centralized nervous system, and the larva was assumedly planktonic, with a gut and a ciliated creeping sole.

The dynamic genome of Hydra.

The freshwater cnidarian Hydra was first described in 1702 and has been the object of study for 300 years. Experimental studies of Hydra between 1736 and 1744 culminated in the discovery of asexual reproduction of an animal by budding, the first description of regeneration in an animal, and successful transplantation of tissue between animals. Today, Hydra is an important model for studies of axial patterning, stem cell biology and regeneration. Here we report the genome of Hydra magnipapillata and compare it to the genomes of the anthozoan Nematostella vectensis and other animals. The Hydra genome has been shaped by bursts of transposable element expansion, horizontal gene transfer, trans-splicing, and simplification of gene structure and gene content that parallel simplification of the Hydra life cycle. We also report the sequence of the genome of a novel bacterium stably associated with H. magnipapillata. Comparisons of the Hydra genome to the genomes of other animals shed light on the evolution of epithelia, contractile tissues, developmentally regulated transcription factors, the Spemann-Mangold organizer, pluripotency genes and the neuromuscular junction.

Using hydrofluoric acid for morphological investigations of Zoanthids (Cnidaria: Anthozoa): a critical assessment of methodology and necessity.

Zoanthids comprise an order of benthic, generally colonial cnidarians, which can usually be distinguished from other hexacorallians by embedded sand and detritus in their mesoglea to help strengthen their structure. These animals are becoming increasingly important research subjects in biochemistry and other research fields. Their inclusion of both calcium and silica results in the need for both decalcification and desilification for internal morphological examinations. Since the methodology of hydrofluoric acid (HF) desilification has rarely been documented in zoanthids, histological surveys for zoanthid taxonomy have often been abandoned and their taxonomy is often problematic. Recent investigations utilizing molecular methods have brought a clearer understanding of zoanthid diversity, but standardization of HF treatments are still needed to provide a link between molecular and more traditional techniques, and to properly examine specimens for which molecular methods may not be an option (e.g., formalin-preserved specimens, etc.). Here, we use both "straight" HF and, for the first time with zoanthids, buffered HF (BHF) treatments at different treatment lengths (1-48 h) on polyps from three different species of zoanthids for histological examination. Section conditions were judged based on the presence/absence of embedded detritus, drag marks, and tissue condition. Results show that the BHF treatment resulted in slightly better tissue conditions for all specimens, and suggest that desilification works well regardless of treatment time for species with smaller (polyp diameter <0.5 cm), less heavily encrusted polyps. Desilification of heavily encrusted Palythoa mutuki polyps were still problematic, with at least 24 h treatment needed. To aid future research, we provide guidelines for HF treatments of zoanthid specimens.

DNA barcoding in photosymbiotic species of Diplosoma (Ascidiacea: Didemnidae), with the description of a new species from the southern Ryukyus, Japan.

Partial sequences of the cytochrome c oxidase subunit I (COI) gene were determined for six species of the genus Diplosoma (Ascidiacea, Didemnidae) to develop tools for species identification. Because each Diplosoma species has distinctly different COI haplotype(s), the gene sequence seems to be usable for species discrimination in this ascidian genus. The phylogenetic hypothesis supported by the COI data is congruent with the distribution of character states of the retractor muscle. in this paper, we describe a new Diplosoma species harboring symbiotic cyanophytes, found on Miyakojima Island, Ryukyu Archipelago, Japan. Diplosoma aggregatum sp. nov. forms mosaic-like aggregates of small colonies. Although the zooids of D. aggregatum are similar to those of D. virens, these species are differentiated by colony form and COI sequences.

Two new species of photosymbiotic ascidians of the genus Diplosoma from the Ryukyu Archipelago, with partial sequences of the COI gene.

Two new species of Diplosoma are described from coral reefs in the Ryukyu Archipelago, Japan: D. watanabei sp. nov. and D. gumavirens sp. nov. Colonies of both species are green due to algal symbionts of Prochloron, which are distributed in the common cloacal cavity. Both species are characterized by the point of emergence of the retractor muscle and a unique combination of stigma numbers among the four rows of the branchial sac. There are three adhesive papillae in the embryos of D. watanabei, as in most ascidian embryos and larvae, whereas the pre-hatching embryos of D. gumavirens sp. nov. posses 12-16 adhesive papillae. Partial sequences of the cytochrome c oxidase subunit I (COI) gene discriminated the new species from each other and from congeners.

Body colors and algal distribution in the acoel flatworm Convolutriloba longifissura: histology and ultrastructure.

Convolutriloba longifissura is a red flatworm with white dots that harbors unicellular green algae within its body. The red pigment of the flatworm that is present in round cells is soluble in ethanol or acetone, whereas the white pigment contained in the crystalline (retractile) platelets of amoeboid-shaped cells is soluble in 1% NH4OH. These two types of pigment cells form the body coloration and are probably involved in light protection of the algal symbionts, as many algal cells are distributed beneath the body wall and some are in the highly vacuolated parenchyma. The ultrastructural features of these cells suggest a close relationship with Tetraselmis spp. The morphology of sagittocysts within the mantle is also described by means of scanning and transmission electron microscopy.

Early development of zooxanthella-containing eggs of the corals Porites cylindrica and Montipora digitata: The endodermal localization of zooxanthellae.

We studied the early development of zooxanthellae-containing eggs of the scleractinian corals Porites cylindrica and Montipora digitata to elucidate how zooxanthellae become localized to the endoderm of planulae during the course of development. In both species, zooxanthellae were distributed evenly in the oocytes and delivered almost equally to the blastomeres during cleavage. In P. cylindrica, gastrulation occurred via delamination or ingression, and blastomeres containing zooxanthellae dropped into the blastocoel during gastrulation. Thus, zooxanthellae were restricted to the endodermal cells at the gastrula or early planula stage in P. cylindrica. In M. digitata, gastrulation occurred by a combination of invagination and epiboly to form a somewhat concave gastrula. Zooxanthellae were present in both endodermal and ectodermal cells of early planulae, but they disappeared from the ectoderm as the planulae matured. In our previous study on two species of Pocillopora, we found that zooxanthellae were localized in eggs as well as in embryos, and that blastomeres containing zooxanthellae later dropped into the blastocoel to become restricted to the endoderm (Hirose et al., 2000). The timing and mechanism of zooxanthella localization and types of gastrulation differed among species belonging to the three genera. These results suggest that zooxanthella localization in the embryos reflects the timing of the determination of presumptive endoderm cells and/or specificity of zooxanthellae toward presumptive endoderm cells.

Localization of symbiotic cyanobacteria in the colonial ascidian Trididemnum miniatum (Didemnidae, Ascidiacea).

Trididemnum miniatum is a colonial ascidian harboring the photosymbiotic prokaryote Prochloron sp. These bacterial cells are located in the tunic of the host animal. The present study revealed, by ultrastructural analysis, that the Prochloron cells were exclusively distributed and proliferated in the tunic. They were shown to be embedded in the tunic matrix and to have no direct contact with ascidian cells. Some tunic cells of the ascidians, however, did phagocytize and digest the symbiont. Round cell masses were sometimes found in the tunic and appeared to consist of disintegrating cyanobacterial cells. The thoracic epidermis of ascidian zooids was often digitated, and the epidermal cells extended microvilli into the tunic. Since there were no Prochloron cells in the alimentary tract of the ascidian zooids, the photosymbionts would not be considered part of the typical diet of the host ascidians. Thin layer chromatography showed that the symbionts possessed both chlorophyll a and b, while a 16S rRNA gene phylogeny supported the identification of the photosymbiont of T. miniatum as Prochloron sp.