Multiple origins of the ascidian-Prochloron symbiosis: molecular phylogeny of photosymbiotic and non-symbiotic colonial ascidians inferred from 18S rDNA sequences

In the tropics, certain didemnid ascidians harbor the prokaryotic photosymbiont Prochloron. To date, this photosymbiosis has been found in four didemnid genera that include non-symbiotic species. Here, we report the molecular phylogeny of symbiotic and non-symbiotic didemnids based on their 18S rDNA sequences. The data cover all four genera containing symbiotic species and one other genus comprised of only non-symbiotic species. Near-complete nucleotide sequences of 18S rDNAs were determined for four non-didemnid species and 52 didemnid samples (five genera), including 48 photosymbiotic samples collected from the Ryukyu Archipelago, the Great Barrier Reef, Hawaii, and Bali. Our phylogenetic trees indicated a monophyletic origin of the family Didemnidae, as well as each of the didemnid genera. The results strongly support the hypothesis that establishment of the ascidian-Prochloron symbiosis occurred independently in the Didemnidae lineage at least once in each of the genera that possess symbiotic species.     

Photosymbiotic ascidians in Singapore: turbid?waters?may reduce living space

The photosymbiotic ascidian fauna at Changi Beach, Pulau Semakau, Sentosa and St. John’s Island, Singapore were surveyed. A total of five species, Diplosoma simile, Lissoclinum bistratum, Lissoclinum punctatum, Lissoclinum timorense and Trididemnum cyclops, were recorded, with Lissoclinum timorense and Trididemnum cyclops being newly recorded in Singapore. However, no photosymbiotic species were found at Changi Beach probably due to the polluted waters in the region. Coastal development has caused Singapore waters to become turbid, leading to decrease in suitable habitats for photosymbiotic ascidians. Clean waters in Pulau Semakau probably provide a better environment for the growth of photosymbiotic ascidians and this area has a greater variety of these ascidians than the other areas in Singapore. Each of the five species has also been recorded in the Ryukyu Archipelago (Japan) and three species (Diplosoma simile, Lissoclinum bistratum and Trididemnum cyclops) have also been recorded in Taiwan.     

Convergent evolution of the vertical transmission mode of the cyanobacterial obligate symbiont Prochloron distributed in the tunic of colonial ascidians

In chordates, obligate photosynthetic symbiosis has been reported exclusively in some colonial ascidians of the family Didemnidae. The vertical transmission of the symbionts is crucial in establishing the obligate symbiosis between the cyanobacteria and the host ascidians. The results of comparative surveys on the morphological processes of cyanobacterial transmission suggest the occurrence of convergent evolution of the vertical transmission in the host species harboring symbionts in the cloacal cavity. In Trididemnum species harboring cyanobacterial cells in the tunic, the symbiont cells are transported by the tunic cells to the tunic of embryos brooded in the tunic of the parent colony. The present study examined whether the mode of symbiont transmission is the same in host species harboring the symbionts in the tunic, regardless of host genera, or whether non-Trididemnum hosts have a different vertical transmission mode. Our results showed that the vertical transmission process in Lissoclinum midui was almost the same as in the Trididemnum species, supporting the occurrence of convergent evolution in the two distinct didemnid genera, that is, Trididemnum and Lissoclinum. High plasticity of the embryogenic process in didemnid ascidians may be important in developing the mechanism of vertical transmission; this assumption may also explain why the obligate cyanobacterial symbiosis has been exclusively established in didemnid ascidians among chordates.     

The relatedness of Prochloron sp. Isolated from different didemnid ascidian hosts

The relationship of Prochloron sp. isolated from four different didemnid ascidian hosts, namely Lissoclinum patella, Lissoclinum voeltzkowi, Diplosoma virensand Trididemnum cyclops was elucidated by comparative analysis of their 16S ribosomal ribonucleic acid (RNA). The oligonucleotide catalogues of the 16S rRNA obtained are almost identical, indicating a very close relationship among the prochlorophytes investigated. Phylogenetically Prochloron is a member of Cyanobacteriales.     

Host specificity and phylogeography of the prochlorophyte Prochloron sp., an obligate symbiont in didemnid ascidians

Prochloron is an oxygenic photosynthetic bacterium that lives in obligate symbiosis with didemnid ascidians, such as Diplosoma spp., Lissoclinum spp. and Trididemnum spp. This study investigated the genetic diversity of the genus Prochloron by constructing a phylogenetic tree based on the 16S rRNA gene sequences of 27 isolates from 11 species of didemnid ascidians collected from Japan, Australia and the USA. The 27 isolates formed three phylogenetic groups: 22 of the samples were identified to be closely related members of Prochloron. Two samples, isolated from Trididemnum nubilum and Trididemnum clinides, were found to belong to the species Synechocystis trididemni, the closest relative of Prochloron. Three isolates formed a separate group from both Prochloron sp. and S. trididemni, potentially indicating a new symbiotic phylotype. Genomic polymorphism analysis, employing cyanobacterium-specific highly iterative palindrome 1 repeats, could not delineate the isolates further. For the Prochloron sp. isolates, the phylogenetic outcome was independent of host species and geographic origin of the sample indicating a low level of host specificity, low genetic variation within the taxon and possibly a lack of a host-symbiont relationship during reproductive dispersal. This study contributes significantly to the understanding of Prochloron diversity and phylogeny, and implications for the evolutionary relationship of prochlorophytes, cyanobacteria and chloroplasts are also discussed.     

Cyanobacterial diversity and a new acaryochloris-like symbiont from Bahamian sea-squirts

Symbiotic interactions between ascidians (sea-squirts) and microbes are poorly understood. Here we characterized the cyanobacteria in the tissues of 8 distinct didemnid taxa from shallow-water marine habitats in the Bahamas Islands by sequencing a fragment of the cyanobacterial 16S rRNA gene and the entire 16S-23S rRNA internal transcribed spacer region (ITS) and by examining symbiont morphology with transmission electron (TEM) and confocal microscopy (CM). As described previously for other species, Trididemnum spp. mostly contained symbionts associated with the Prochloron-Synechocystis group. However, sequence analysis of the symbionts in Lissoclinum revealed two unique clades. The first contained a novel cyanobacterial clade, while the second clade was closely associated with Acaryochloris marina. CM revealed the presence of chlorophyll d (chl d) and phycobiliproteins (PBPs) within these symbiont cells, as is characteristic of Acaryochloris species. The presence of symbionts was also observed by TEM inside the tunic of both the adult and larvae of L. fragile, indicating vertical transmission to progeny. Based on molecular phylogenetic and microscopic analyses, Candidatus Acaryochloris bahamiensis nov. sp. is proposed for this symbiotic cyanobacterium. Our results support the hypothesis that photosymbiont communities in ascidians are structured by host phylogeny, but in some cases, also by sampling location.     

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.     

Stable photosymbiotic relationship under CO₂-induced acidification in the acoel worm Symsagittifera roscoffensis

As a consequence of anthropogenic CO? emissions, oceans are becoming more acidic, a phenomenon known as ocean acidification. Many marine species predicted to be sensitive to this stressor are photosymbiotic, including corals and foraminifera. However, the direct impact of ocean acidification on the relationship between the photosynthetic and nonphotosynthetic organism remains unclear and is complicated by other physiological processes known to be sensitive to ocean acidification (e.g. calcification and feeding). We have studied the impact of extreme pH decrease/pCO? increase on the complete life cycle of the photosymbiotic, non-calcifying and pure autotrophic acoel worm, Symsagittifera roscoffensis. Our results show that this species is resistant to high pCO? with no negative or even positive effects on fitness (survival, growth, fertility) and/or photosymbiotic relationship till pCO? up to 54 K μatm. Some sub-lethal bleaching is only observed at pCO? up to 270 K μatm when seawater is saturated by CO?. This indicates that photosymbiosis can be resistant to high pCO?. If such a finding would be confirmed in other photosymbiotic species, we could then hypothesize that negative impact of high pCO? observed on other photosymbiotic species such as corals and foraminifera could occur through indirect impacts at other levels (calcification, feeding).     

Their Day in the Sun: molecular phylogenetics and origin of photosymbiosis in the 'other' group of photosymbiotic marine bivalves (Cardiidae: Fraginae)

The subfamily Fraginae (Cardiidae) is a morphologically diverse group of small-bodied marine clams inhabiting shallow seas worldwide. Like the exclusively photosymbiotic giant clams (Cardiidae: Tridacninae), some fragines are known to host zooxanthellae photosymbionts. However, surveys to widely determine photosymbiotic status and the lack of a comprehensive phylogeny have hindered attempts to track the evolution of photosymbiosis in the group. Worldwide sampling of all fragine genera and subgenera with phylogenetic reconstructions based on four gene regions [nuclear (28S) and mtDNA (16S, cytochrome oxidase I, cytochrome b )] does not support a monophyletic Fraginae. Sampled taxa form four restructured clades: (1) the ' Fragum ' group, (2) the ' Trigoniocardia ' and ' Ctenocardia ' groups, (3) the ' Parvicardium ' group and (4) the ' Papillicardium ' group. Maximum likelihood analyses strongly support a clade of European cardiids uniting species from three subfamilies. Live examination of > 50% of species reveals that less than half of derived genera and subgenera host photosymbionts, supporting a single and relatively late origin of photosymbiosis in the Fraginae. The evolutionary implications for a small and little modified earliest diverging photosymbiotic lineage are discussed.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 97 , 448–465.     

Stable Photosymbiotic Relationship under CO2-Induced Acidification in the Acoel Worm Symsagittifera Roscoffensis

As a consequence of anthropogenic CO₂ emissions, oceans are becoming more acidic, a phenomenon known as ocean acidification. Many marine species predicted to be sensitive to this stressor are photosymbiotic, including corals and foraminifera. However, the direct impact of ocean acidification on the relationship between the photosynthetic and nonphotosynthetic organism remains unclear and is complicated by other physiological processes known to be sensitive to ocean acidification (e.g. calcification and feeding). We have studied the impact of extreme pH decrease/pCO₂ increase on the complete life cycle of the photosymbiotic, non-calcifying and pure autotrophic acoel worm, Symsagittifera roscoffensis. Our results show that this species is resistant to high pCO₂ with no negative or even positive effects on fitness (survival, growth, fertility) and/or photosymbiotic relationship till pCO₂ up to 54 K µatm. Some sub-lethal bleaching is only observed at pCO₂ up to 270 K µatm when seawater is saturated by CO₂. This indicates that photosymbiosis can be resistant to high pCO₂. If such a finding would be confirmed in other photosymbiotic species, we could then hypothesize that negative impact of high pCO₂ observed on other photosymbiotic species such as corals and foraminifera could occur through indirect impacts at other levels (calcification, feeding).     

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.     

Planktonic foraminiferal response to the latest Maastrichtian abrupt warm event: a case study from South Atlantic DSDP Site 525A

An abrupt global warming of 3–4°C occurred near the end of the Maastrichtian at 65.45–65.10 Ma. The environmental effects of this warm event are here documented based on stable isotopes and quantitative analysis of planktonic foraminifera at the South Atlantic DSDP Site 525A. Stable isotopes of individual species mark a rapid increase in temperature and a reduction in the vertical water mass stratification that is accompanied by a decrease in niche habitats, reduced species diversity and/or abundance, smaller species morphologies or dwarfing, and reduced photosymbiotic activity. During the warm event, the relative abundance of a large number of species decreased, including tropical–subtropical affiliated species, whereas typical mid-latitude species retained high abundances. This indicates that climate warming did not create favorable conditions for all tropical–subtropical species at mid-latitudes and did not cause a massive retreat in the local mid-latitude population. A noticeable exception is the ecological generalist Heterohelix dentata Stenestad that dominated during the cool intervals, but significantly decreased during the warm event. However, dwarfing is the most striking response to the abrupt warming and occurred in various species of different morphologies and lineages (e.g. biserial, trochospiral, keeled globotruncanids). Dwarfing is a typical reaction to environmental stress conditions and was likely the result of increased reproduction rates. Similarly, photosymbiotic activity appears to have been reduced significantly during the maximum warming, as indicated by decreased δ¹³C values. The foraminiferal response to climate change is thus multifaceted resulting in decreased species diversity, decreased species populations, increased competition due to reduced niche habitats, dwarfing and reduced photosymbiotic activity.     

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.     

Microbial diversity of biofilm communities in microniches associated with the didemnid ascidian Lissoclinum patella

We assessed the microbial diversity and microenvironmental niche characteristics in the didemnid ascidian Lissoclinum patella using 16S rRNA gene sequencing, microsensor and imaging techniques. L. patella harbors three distinct microbial communities spatially separated by few millimeters of tunic tissue: (i) a biofilm on its upper surface exposed to high irradiance and O₂ levels, (ii) a cloacal cavity dominated by the prochlorophyte Prochloron spp. characterized by strong depletion of visible light and a dynamic chemical microenvironment ranging from hyperoxia in light to anoxia in darkness and (iii) a biofilm covering the underside of the animal, where light is depleted of visible wavelengths and enriched in near-infrared radiation (NIR). Variable chlorophyll fluorescence imaging demonstrated photosynthetic activity, and hyperspectral imaging revealed a diversity of photopigments in all microhabitats. Amplicon sequencing revealed the dominance of cyanobacteria in all three layers. Sequences representing the chlorophyll d containing cyanobacterium Acaryochloris marina and anoxygenic phototrophs were abundant on the underside of the ascidian in shallow waters but declined in deeper waters. This depth dependency was supported by a negative correlation between A. marina abundance and collection depth, explained by the increased attenuation of NIR as a function of water depth. The combination of microenvironmental analysis and fine-scale sampling techniques used in this investigation gives valuable first insights into the distribution, abundance and diversity of bacterial communities associated with tropical ascidians. In particular, we show that microenvironments and microbial diversity can vary significantly over scales of a few millimeters in such habitats; which is information easily lost by bulk sampling.     

Pattern of cell proliferation during budding in the colonial ascidian Diplosoma listerianum

Many invertebrates reproduce asexually by budding, but morphogenesis and the role of cell proliferation in this diverse and nonconserved regeneration-like process are generally poorly understood and particularly little investigated in didemnid ascidians. We here analyzed cell proliferation patterns and telomerase activity during budding in the colonial didemnid ascidian Diplosoma listerianum, with special focus on the thoracic bud where a new brain develops de novo. To help define developmental stages of the thoracic bud, the distribution of acetylated tubulin was also examined. We found extensive cell proliferation in both the thoracic and abdominal buds of D. listerianum as well as higher telomerase activity in bud tissue compared to adult tissues. In the parent adult, proliferation was found in various tissues, but was especially intense in the adult esophagus and epicardial structures that protrude into the proliferating and developing buds, confirming these tissues as the primary source of the cells that form the buds. The neural complex in the thoracic bud forms from a hollow tube that appears to separate into the neural gland and the cerebral ganglion. Whereas most of the bud undergoes proliferation, including the hollow tube and the neural gland, the cerebral ganglion shows little or no proliferation. Pulse-chase labeling experiments indicate that the ganglion, as well as the myocardium, in adult zooids are instead composed of postmitotic cells.     

Cytotoxic labdane alkaloids from an ascidian Lissoclinum sp.: isolation, structure elucidation, and structure-activity relationship

Four labdane alkaloids, haterumaimides N-Q (1-4), were isolated from an ascidian Lissoclinum sp. and their structures were elucidated by chemical and spectral analyses. Investigation of the structure-activity relationships of haterumaimides J-K, N-Q, and 14 related compounds suggested that the presence of hydroxyl groups at C-6, C-7, C-12, and C-18, a chlorine atom at C-2, and an imido NH in ring C should be essential for cytotoxicity against P388 cells.     

Symbiont bleaching in fossil planktonic foraminifera

Size restricted carbon isotopes (δ¹³C) are used to track changes in the ontogenetic life strategies of two species of extinct planktonic foraminifera and demonstrate that the species Morozovelloides crassatus lost their photosymbiotic association prior to their extinction in the latest middle Eocene. M. crassatus exhibit a strong positive correlation between test size and δ¹³C between 39.5 Ma and 38.7 Ma and a Δδ¹³C shift of 1.0‰/100 μm, this is analogous with modern species that possess an association with algal photosymbionts. Turborotalia cerroazulensis is interpreted as an asymbiotic, thermocline dweller and consistently shows no size related δ¹³C trends and greater δ¹⁸O values in comparison to Morozovelloides. We show a long-term (1.5 million year) deterioration of Morozovelloides ecology that culminated in their extinction at 38.021 Ma. The Δδ¹³C /100 μm in M. crassatus is dramatically reduced from 1.0‰ at 39.53 to only 0.2‰ at 38.026 Ma, 5 kyr before their extinction. The decline in ontogenetic δ¹³C suggests diminished photosymbiotic activity (bleaching) and disruption of foraminiferal ecology in the interval preceding their extinction. We conclude that the demise of Morozovelloides was directly related to the deterioration of photosymbiotic partnerships with algae.     

Discovery of new pyridoacridine alkaloids from Lissoclinum cf. badium that inhibit the ubiquitin ligase activity of Hdm2 and stabilize p53

Compounds that stabilize p53 could suppress tumors providing a additional tool to fight cancer. Mdm2, and the human ortholog, Hdm2 serve as ubiquitin E3 ligases and target p53 for ubiquitylation and degradation. Inhibition of Hdm2 stabilizes p53, inhibits cell proliferation and induces apoptosis. Using HTS to discover inhibitors, we identified three new alkaloids, isolissoclinotoxin B, diplamine B, and lissoclinidine B from Lissoclinum cf. badium. Lissoclinidine B inhibited ubiquitylation and degradation of p53, and selectively killed transformed cells harboring wild-type p53, suggesting this compound could be used to develop new treatments.     

Occurrence,structure and synthesis of 3-(N-Methylamino)glutaric acid,an amino acid from Prochloron didemnii

3-(N-Methylamino)glutaric acid has been identified as a new free amino acid in extracts from Prochloron didemnii (Lewin), a unique prokaryotic algal symbiont associated with certain didemnid ascidians. Its structure was established by elucidation of the mass spectra of its TMSi and other derivatives and confirmed by synthesis.     

Identifying bacterial genes and endosymbiont DNA with Glimmer

MOTIVATION: The Glimmer gene-finding software has been successfully used for finding genes in bacteria, archaea and viruses representing hundreds of species. We describe several major changes to the Glimmer system, including improved methods for identifying both coding regions and start codons. We also describe a new module of Glimmer that can distinguish host and endosymbiont DNA. This module was developed in response to the discovery that eukaryotic genome sequencing projects sometimes inadvertently capture the DNA of intracellular bacteria living in the host. RESULTS: The new methods dramatically reduce the rate of false-positive predictions, while maintaining Glimmer's 99% sensitivity rate at detecting genes in most species, and they find substantially more correct start sites, as measured by comparisons to known and well-curated genes. We show that our interpolated Markov model (IMM) DNA discriminator correctly separated 99% of the sequences in a recent genome project that produced a mixture of sequences from the bacterium Prochloron didemni and its sea squirt host, Lissoclinum patella. AVAILABILITY: Glimmer is OSI Certified Open Source and available at http://cbcb.umd.edu/software/glimmer.