Ecology and colonial dynamics of some Pacific reef flat Didemnidae (Ascidiacea)

Non-cryptic didemnid ascidians containing the prokaryote Prochloron are abundant on the intertidal flat of many Pacific reefs. Species with sheet-like colonies (e.g. Diplosoma similis, Trididemnum paracyclops ) are characteristic of high wave energy situations and clean hard surfaces; didemnids in which the colony divides repeatedly into small cormomeres (e.g. Diplosoma virens, D. multipapillata, Lissoclinum voeltzkowi ) are in less exposed situations, often on algae or unstable substrata. The unusual back-reef cascade habitat of D. multipapillata on a Fijian fringing-reef is described. In Fiji, replicative division leads to population build-up between June and October, with a hot season decline in November-December. The effects of torrential rain in April 1980 are described. Studying L. voeltzkowi in permanent quadrats has shown that the populations of cormomeres are dynamic, with much movement occurring. The process of colony division is described in Diplosoma virms from the Great Barrier Reef. Possible advantages of didemnid dynamism discussed include maximization of periphery to area (perhaps increasing the efficiency of feeding and/or growth), the exclusion of competitors, and the mingling of clones to facilitate cross-fertilization.     

Transmission of cyanobacterial symbionts during embryogenesis in the coral reef ascidians Trididemnum nubilum and T. clinides (Didemnidae, Ascidiacea, Chordata)

Vertical transmission of cyanobacterial symbionts occurs in didemnid ascidians harboring Prochloron as an obligate symbiont; the photosymbionts are transferred from the parental ascidian colony to the offspring in various ways depending on host species. Although several didemnids harbor non-Prochloron cyanobacteria in their tunics, few studies have reported the processes of vertical transmission in these didemnids. Here we describe the histological processes of the transmission of cyanobacteria in two didemnids, Trididemnum nubilum harboring Synechocystis and T. clinides harboring three cyanobacterial species. In both species, the photosymbionts in the tunic of the parent colony were apparently captured by the tunic cells of the host and transferred to the embryos brooded in the tunic. The symbiont cells were then incorporated into the inner tunic of the embryo. This mode of transmission is essentially the same as that of T. miniatum harboring Prochloron in the tunic, although there are some differences among species in the timing of the release of the symbionts from the tunic cells. We suggest that the similar modes of vertical transmission are an example of convergent evolution caused by constraints in the distribution patterns of symbiont cells in the host colony.     

Ovulation and egg segregation in the tunic of a colonial ascidian,Diplosoma listerianum (Tunicata,Ascidiacea)

Summary The process of egg segregation in the tunic of the ovoviviparous ascidian Diplosoma listerianum was studied by light and electron microscopy. One egg at a time was seen to mature in each zooid. The eggs had large yolk and grew on the ovary wall enveloped in four layers: (1) outer follicle cells (OFC), long and rich in RER (rough endoplasmic reticulum) and with dense granules in the Golgi region; (2) flat inner follicle cells (IFC); (3) a loosely fibrillar vitelline coat (VC); (4) test cells encased on the egg surface. The growing egg protrudes from the ovary wall and presses on the contiguous epidermis. Granulocytes enter the space between the epidermis and the egg and insinuate cytoplasmic protrusions, disrupting the continuity of the OFC layer. At ovulation, OFC and IFC are discharged and form a post-ovulatory follicle (corpus luteum). The epidermis shrinks and closes, possibly by activation of microfilaments, causing the egg to be completely surrounded by the tunic. In the zooid, the wound caused by the passage of the egg is repaired both by contraction of the epidermis and by phagocytic activity. Altered spermatozoans are found in phagocytosing cells in the lumen of the ovary. These are presumably remnants of those which entered to fertilize the egg before segregation.     

An atrial membrane in the colonial ascidian, Ritterella tokioka Kott, 1992 (Urochordata: Ascidiacea) from Sagami Bay

An atrial membrane, similar to that previously reported (as a placental membrane) in Placentela crystallina Redikorzev, 1913, has been found in the holotype and other specimens of another aplousobranch ascidian, Ritterella tokioka Kott, 1992 (=R. pedunculata Tokioka, 1953). In serial sections, the membrane is seen to be an extensive fold of the atrial epithelium over the rectum that projects into the atrial cavity and supports developing embryos over its outer surface. The similar states in the atrial membrane seen in the two species are attributable to homoplasy, rather than homology.     

Ovulation in Diplosoma (Tunicata, Ascidiacea, Didemnidae): a light microscopical study

Serial sections of non-ovulated and ovulated eggs of Diplosoma migrans (Tunicata, Ascidiacea, Didemnidae) were compared in order to study the process of ovulation on the level of light microscopy. Fully grown eggs are surrounded by an outer follicular epithelium consisting of cubic cells, and an inner follicular epithelium consisting of flat cells. Egg release is initiated by a fusion of the epidermis with the outer follicular epithelium at the distal pole of the egg. A contraction of the outer follicular epithelium leads to an opening of both epidermis and outer follicular epithelium, and their removal from the egg. The envelope of ovulated eggs corresponds to the former inner follicular epithelium. Mainly due to a contraction of the epidermis, the outer follicular epithelium is shifted into the abdomen and separates from the epidermis. It transforms into a vestigial body (corpus luteum) and finally atrophies. The epidermis flattens and resumes its typical appearance. On the basis of these observations, the results of studies on the closely related Diplosoma listerianum are discussed and questioned. Electronic Publication     

Polymorphic microsatellite loci isolated from the Atlanto‐Mediterranean colonial ascidian Pycnoclavella sp. (Ascidiacea,Tunicata)

Eight microsatellite loci were characterized in the colonial ascidian Pycnoclavella sp. from an enriched library of genomic DNA. Most microsatellites were large and compound. Allelic variation was assessed in 30 individuals collected from Cerro Gordo (SW Mediterranean Sea). The number of alleles per locus ranged from one to nine, observed heterozygosity from 0.05 to 0.29 and expected heterozygosity from 0.15 to 0.39. No significant linkage disequilibrium between pairs of loci was detected, but five loci showed significant heterozygote deficiency that may be explained either by inbreeding, population substructure or the presence of null alleles.     

Sensory endings of the ascidian static organ (Chordata,Ascidiacea)

Summary The ultrastructure of the static organ is examined in larvae of Diplosoma macdonaldi, a colonial ascidian, and Styela plicata, a solitary ascidian; the results are similar. As previous workers found, the cell body of a unicellular statocyte lies in the lumen of the sensory vesicle and contains the statolith. A narrow neck connects the cell body to an anchoring foot in the floor of the sensory vesicle. Two previously undescribed sensory endings project into the lumen just to the left of the statocyte, one anterior and one posterior to the neck. A network of fine processes from each ending contacts the statocyte body. It is proposed that movements of the statocyte cell body are detected by these endings. They arise from neurons in the ventral wall of the sensory vesicle that project axons to the visceral ganglion. The placement of the sensory endings may allow discrimination of the directon of statocyte deflection.Abbreviations ax axons - bb ciliary basal body - bl basal lamina - c cilium - cr striated ciliary rootlet - ec ependymal cells - en endoderm - h hemocoel - ly lysosome - mv microvilli - n neuron - nf neurofilaments - ns neck of the statocyte - sb statocyte cell body - sd sensory dendrite - sn sensory neuron - sp sensory processes - stf statocyte foot - svl sensory vesicle lumen - zo zonula occludens     

Two new species of diplosoma (Ascidiacea: Didemnidae) bearing prokaryotic algae prochloron from Okinawajima (Ryukyu Archipelago, Japan)

Two new species of didemnid ascidians, Diplosoma ooru sp. nov. and Diplosoma simileguwa sp. nov., are described from coral reefs on Okinawajima (Ryukyu Archipelago, Japan). These two species form green colonies, having a symbiotic association with a prokaryotic alga Prochloron sp. The former species was found at the reef edges in the subtidal zone and the latter was found in a shallow reef lagoon. In these species, the colonies are thinner and the zooids are smaller than those of any other Prochloron-bearing Diplosoma species so far described. Moreover, each of the present new species has a unique combination of stigmatic numbers: 5 stigmata in the first and third rows, 6 in the second row, and 4 in the fourth in D. ooru; 4 stigmata in the first and third rows, 5 in the second row, and 3 in the fourth in D. simileguwa. In both of the new species, the retractor muscle emerges from the underside of the thorax. Larval morphology of D. ooru is also described.     

Distribution and abundance of the ascidian Ecteinascidia turbinata (Ascidiacea: Perophoridae) in Cuba

Permanently submerged mangrove roots (Rhizophora mangle) are the main habitat of the ascidian Ecteinascidia turbinata in Cuba. It was occasionally found on black coral (Antiphates caribeana) between 22 and 38 meters deep. This species exhibits a wide distribution in all the mangrove keys surrounding the Island of Cuba but does not occur in riparian or fringing mangroves. Populations of this species are abundant in Cuba: in 75% of the 58 localities sampled the species was present and in 57% more than 50% of the roots held at least one colony. The highest colony densities were found in the northern coast of Pinar del Rio province with values near one colony per lineal meter of mangrove root. We found the highest density (1.46 col/m) and greatest biomass at Jutías Key, with values between 25 and 660 g/m. The average of wet biomass in the studied mangroves was 73.63 g/m.     

Occurrence of different secretin-like cells in the digestive tract of the ascidian Styela plicata (Urochordata,Ascidiacea)

Summary Secretin-like cells have been detected in the digestive tract of the ascidian Styela plicata by means of immunofluorescent and immunocytochemical methods.Especially, in the esophageal epithelium there are immunoreactive cells (S₂) in which a biogenic amine (5-HT) and a regulatory peptide (secretin) occur together. In the gastric epithelium only secretin-like cells (S₁) are present.Tests of cross-reactivity performed with glucagon, GIF and VIP, have confirmed the presence of a secretin-like molecule only in the S₁ and S₂ cells.     

Tunic phagocytes are involved in allorejection reaction in the colonial tunicate Aplidium yamazii (Polyclinidae, Ascidiacea)

The colonial ascidian Aplidium yamazii exhibited an allorejection reaction when two allogeneic colonies were brought into contact at their growing edges or at artificial cut surfaces. This species has no vascular network in the tunic, unlike the botryllid ascidians, which have a vascular network throughout the colony's common tunic. In the allorejection reaction induced by contact at the growing edges, some small, hard-packed tunic masses were formed at the contact points. Histological and electron microscopic investigation of these tunic masses revealed that they contained aggregates of tunic cells, with tunic phagocytes being the major cell type present. Some of the tunic phagocytes in these tunic masses appeared to be disintegrating. When allogeneic colonies were placed in contact at their artificial cut surfaces, the colonies partially fused, then separated. In this allorejection reaction, some loosely packed tunic masses remained in the gap between the two withdrawn colonies. These results strongly suggest that the tunic phagocytes are likely to be the major effector cells in the allorejection reaction. We also propose that the tunic phagocytes are not only the effector cells in the allorejection reaction but also bear the sites of allorecognition.     

Genetic diversity of symbiotic cyanobacteria in Cycas revoluta (Cycadaceae)

The diversity of cyanobacterial species within the coralloid roots of an individual and populations of Cycas revoluta was investigated based on 16S rRNA gene sequences. Sixty-six coralloid roots were collected from nine natural populations of cycads on Kyushu and the Ryukyu Islands, covering the entire distribution range of the species. Approximately 400?bp of the 5'-end of 16S rRNA genes was amplified, and each was identified by denaturing gradient gel electrophoresis. Most coralloid roots harbored only one cyanobiont, Nostoc, whereas some contained two or three, representing cyanobiont diversity within a single coralloid root isolated from a natural habitat. Genotypes of Nostoc within a natural population were occasionally highly diverged and lacked DNA sequence similarity, implying genetic divergence of Nostoc. On the other hand, Nostoc genotypes showed no phylogeographic structure across the distribution range, while host cycads exhibited distinct north-south differentiation. Cycads may exist in symbiosis with either single or multiple Nostoc strains in natural soil habitats.     

Stem cells in asexual reproduction of the colonial ascidian <Emphasis Type="Italic">Botryllus tubaratus</Emphasis> (Tunicata: Ascidiacea)

Histological, cytochemical and ultrastructure research on the budding of the colonial ascidian Botryllus tuberatus aimed at searching for stem cells was performed. A dense mass of undifferentiated cells and the connection of the outer epidermal and inner atrial epithelia were revealed for the first time in the early buds of B. tuberatus. Undifferentiated cells revealed in the early buds and vascular system of the colony had morphological features of the stem and primary germ cells of metazoans. Intensive expression of alkaline phosphatase, the cytochemical marker of embryonal stem and primary germ cells of vertebrate animals, was revealed in developing buds and in some cells of the hematocyte population. Based on the literature and the author’s data it is hypothesized that the self-renewing pool of stem cells of the colonial ascidian B. tuberatus is the cellular basis of its reproductive strategy, including both sexual and asexual reproduction.     

Degenerative regression of the digestive tract in the colonial ascidian Botryllus schlossen (Pallas)

Summary Degenerative changes in the digestive tract of zooids of Botryllus schlosseri were studied by light and electron microscopy. Three main processes occurred in the tissues: contraction, involution and phagocytosis. The contraction of epidermis and peribranchial epithelium in which cytoplasmic microfilaments probably participate, seemed to have a special role in compressing the underlying organs. During contraction most of the body cavities collapsed, the branchial walls disintegrated and the fragments were rapidly taken up by large phagocytes. The gut epithelium retained its apparent continuity longer, though isolated phagocytes infiltrated it to eliminate single cells. Cell degeneration came about chiefly either through swelling and lysis of cells or through loss of water and condensation of cytoplasm and nucleus.The fate of all regressed tissues was to be engulfed and digested by wandering phagocytes. However, it was also observed that numerous cells of different epithelia could act as fixed phagocytes by engulfing cell debris and entire cells into heterophagic vacuoles.     

The alien ascidian Styela clava now invading the Sea of Marmara (Tunicata: Ascidiacea)

During the implementation of a large project aimed to investigate the benthic community structures of the Sea of Marmara, specimens of the invasive ascidian species Styela clava were collected on natural substrata (rocks) at 10 m depth at one locality (Karamürsel) in İzmit Bay. The specimens were mature, containing gametes, indicating that the species had become established in the area. The Sea of Marmara seems to provide suitable conditions for this species to survive and form proliferating populations.     

Cryptic divergence and strong population structure in the colonial invertebrate Pycnoclavella communis (Ascidiacea) inferred from molecular data

We studied sequence variation in the mitochondrial gene cytochrome c oxidase subunit I (COI) for 135 individuals from eight Mediterranean populations of the colonial ascidian Pycnoclavella communis across most of its presently known range of distribution in the Mediterranean. Three haplotypes from Atlantic locations were also included in the study. Phylogenetic, phylogeographic and population genetic analyses were used to unravel the genetic variability within and between populations. The study revealed 32 haplotypes for COI, 29 of them grouped within two Mediterranean lineages of P. communis (mean nucleotide divergence between lineages was 8.55%). Phylogenetic and network analyses suggest the possible existence of cryptic species corresponding to these two lineages. Population genetic analyses were restricted to the five populations belonging to the main genetic lineage, and for these localities we compared the information gleaned from COI sequence data and from eight microsatellite loci. A high genetic divergence between populations was substantiated using both kinds of markers (COI, global Fst=0.343; microsatellite loci, global Fst=0.362). There were high numbers of private haplotypes (COI) and alleles (microsatellites) in the populations studied. Restricted gene flow and inbreeding occur in the present range of distribution of the species. Microsatellite loci showed a strong incidence of failed amplifications, which we attribute to the marked intraspecies variability that hampered the application of these highly specific markers. Our results show important genetic variability at all levels studied, from within populations to between basins, possibly coupled to speciation processes. This variability is attributable to restricted gene flow among populations due to short-distance dispersal of the larvae.     

The postbranchial digestive tract of the ascidian, Polyandrocarpa misakiensis (Tunicata: Ascidiacea). 1. Oesophagus

The organization of the oesophagus in the budding styelid ascidian, Polyandrocarpa misakiensis, is described. The oesophagus consists of external and internal epithelium, and there are loose connective tissue, blood sinuses, and a muscular layer between them. The internal epithelium is simple columnar, except for the bottom of three folds. The external epithelium is simple squamous. The internal epithelium contains four cell types, i.e., ciliated mucous cells, band cells, endocrine cells, and undifferentiated cells. The ciliated mucous cells have apical cilia and microvilli, and two types of mucous vesicle. The band cells also have apical cilia and electron-dense granules in the apical cytoplasm. The endocrine cells are bottle-shaped, and have electron-dense granules both above and below the nucleus. The undifferentiated cells form pseudostratified epithelium at the bottom of each fold, and they have nuclei with prominent nucleoli. One type of coelomic cell, which has retractile cytoplasm, often migrates in the internal epithelium. Near the stomach, there are many darkly stained round cells clustered around the posterior end of the oesophagus. These two types of coelomic cells may be involved in the defense mechanism against the invasion of foreign organisms. The basic organization of the oesophagus of P. misakiensis is similar to those of other ascidians. However, the presence of three folds is a characteristic of a solitary species, rather than of a colonial species. Although ascidians are chordate invertebrates, the organization of their oesophagus is not very complex, which might reflect their life style.     

Rhopalaea cloneyi (Ascidiacea, Cionidae), a new deep-water ascidian from the coastal waters of British Columbia

A new species of solitary ascidian from the British Columbia fjords, Rhopalaea cloneyi is described. R. cloneyi is characterized by its strong musculature, consisting of 14-15 longitudinal bands which are branched several times on the ventral side, converging to the endostylar band of the thorax. Its differences with the seven species of the genus Rhopalaea described up to the present are discussed. A checklist of the associated benthos found with the nine deep ascidians from this area observed with the PISCES IV submersible shows that R. cloneyi and Bathypera feminalba are found associated with the brachiopods Crania and Laqueus , the asteroid Mediaster aequalis and the anthozoid Stomphia .