Strategies for Amplification by Polymerase Chain Reaction of the Complete Sequence of the Gene Encoding Nuclear Large Subunit Ribosomal RNA in Corals

The nearly complete nuclear large subunit ribosomal RNA (LSU rRNA) gene in corals was amplified by primers designed from polymerase chain reaction (PCR) strategies. The motif of the putative 3′-terminus of the LSU rRNA gene was sequenced and identified from intergenic spacer (IGS) clones obtained by PCR using universal primers designed for corals. The 3′-end primer was constructed in tandem with the universal 5′-end primer for the LSU rRNA gene. PCR fragments of 3500 bp were amplified for octocorals and non-Acropora scleractinian corals. More than 80% of the Acropora LSU rRNA gene (3000 bp) was successfully amplified by modification of the 5′-end of the IGS primer. Analysis of the 5′-end of LSU rDNA sequences, including the D1 and D2 divergent domains, indicates that the evolutionary rate of the LSU rDNA differs among these taxonomic groups of corals. The genus Acropora showed the highest divergence pattern in the LSU rRNA gene, and the presence of a long branch of the Acropora clade from the other scleractinian corals in the phylogenetic tree indicates that the evolutionary rate of Acropora LSU rDNA might have accelerated after divergence from the common ancestor of scleractinian corals. Received February 17, 2000; accepted June 12, 2000.     

A new sequence data set of SSU rRNA gene for Scleractinia and its phylogenetic and ecological applications

Scleractinian corals (i.e. hard corals) play a fundamental role in building and maintaining coral reefs, one of the most diverse ecosystems on Earth. Nevertheless, their phylogenies remain largely unresolved and little is known about dispersal and survival of their planktonic larval phase. The small subunit ribosomal RNA (SSU rRNA) is a commonly used gene for DNA barcoding in several metazoans, and small variable regions of SSU rRNA are widely adopted as barcode marker to investigate marine plankton community structure worldwide. Here, we provide a large sequence data set of the complete SSU rRNA gene from 298 specimens, representing all known extant reef coral families and a total of 106 genera. The secondary structure was extremely conserved within the order with few exceptions due to insertions or deletions occurring in the variable regions. Remarkable differences in SSU rRNA length and base composition were detected between and within acroporids (Acropora, Montipora, Isopora and Alveopora) compared to other corals. The V4 and V9 regions seem to be promising barcode loci because variation at commonly used barcode primer binding sites was extremely low, while their levels of divergence allowed families and genera to be distinguished. A time‐calibrated phylogeny of Scleractinia is provided, and mutation rate heterogeneity is demonstrated across main lineages. The use of this data set as a valuable reference for investigating aspects of ecology, biology, molecular taxonomy and evolution of scleractinian corals is discussed.     

Analysis of the mitochondrial 12S rRNA gene supports a two-clade hypothesis of the evolutionary history of scleractinian corals

Scleractinian corals have long been assumed to be a monophyletic group characterized by the possession of an aragonite skeleton. Analyses of skeletal morphology and molecular data have shown conflicting patterns of suborder and family relationships of scleractinian corals, because molecular data suggest that the scleractinian skeleton could have evolved as many as four times. Here we describe patterns of molecular evolution in a segment of the mitochondrial (mt) 12S ribosomal RNA gene from 28 species of scleractinian corals and use this gene to infer the evolutionary history of scleractinians. We show that the sequences obtained fall into two distinct clades, defined by PCR product length. Base composition among taxa did not differ significantly when the two clades were considered separately or as a single group. Overall, transition substitutions accumulated more quickly relative to transversion substitutions within both clades. Spatial patterns of substitutions along the 12S rRNA gene and likelihood ratio tests of divergence rates both indicate that the 12S rRNA gene of each clade evolved under different constraints. Phylogenetic analyses using mt 12S rRNA gene data do not support the current view of scleractinian phylogeny based upon skeletal morphology and fossil records. Rather, the two-clade hypothesis derived from the mt 16S ribosomal gene is supported.     

Symbiont diversity in scleractinian corals from tropical reefs and subtropical non-reef communities in Taiwan

We examined zooxanthellae diversity in scleractinian corals from southern Taiwan and the Penghu Archipelago, a tropical coral reef and a subtropical non-reefal community, respectively. Zooxanthellae diversity was investigated in 52 species of scleractinian corals from 26 genera and 13 families, using restriction fragment length polymorphism (RFLP), and phylogenetic analyses of the nuclear small-subunit ribosomal DNA (nssrDNA) and large-subunit ribosomal DNA (nlsrDNA). RFLP and phylogenetic analyses of nuclear-encoded ribosomal RNA genes showed that Symbiodinium clade C was the dominant zooxanthellae in scleractinian corals in the seas around Taiwan; Symbiodinium clade D was also found in some species. Both Symbiodinium clade C and D were found in colonies of seven species of scleractinian corals. Symbiodinium clade D was associated with corals that inhabit either shallow water or the reef edge in deep water, supporting the hypothesis that Symbiodinium clade D is a relatively stress-tolerant zooxanthellae found in marginal habitats.Communicated by Biological Editor H.R. Lasker     

Molecular evolution of a portion of the mitochondrial 16S ribosomal gene region in scleractinian corals

Relationships among families and suborders of scleractinian corals are poorly understood because of difficulties 1) in making inferences about the evolution of the morphological characters used in coral taxonomy and 2) in interpreting their 240-million-year fossil record. Here we describe patterns of molecular evolution in a segment of the mitochondrial (mt) 16S ribosomal gene from taxa of 14 families of corals and the use of this gene segment in a phylogenetic analysis of relationships within the order. We show that sequences obtained from scleractinians are homologous to other metazoan 16S ribosomal sequences and fall into two distinct clades defined by size of the amplified gene product. Comparisons of sequences from the two clades demonstrate that both sets of sequences are evolving under similar evolutionary constraints: they do not differ in nucleotide composition, numbers of transition and transversion substitutions, spatial patterns of substitutions, or in rates of divergence. The characteristics and patterns observed in these sequences as well as the secondary structures, are similar to those observed in mt 16S ribosomal DNA sequences from other taxa. Phylogenetic analysis of these sequences shows that they are useful for evaluating relationships within the order. The hypothesis generated from this analysis differs from traditional hypotheses for evolutionary relationships among the Scleractinia and suggests that a reevaluation of evolutionary affinities in the order is needed. Received: 4 September 1996 / Accepted: 7 April 1997     

The mitochondrial genome of the plant bug Apolygus lucorum （Hemiptera： Miridae）＂ Presently known as the smallest in Heteroptera

The complete mitochondrial （mt） genome of the plant bug, Apolygus lucorum, an important cotton pest, has been sequenced and annotated in this study. The entire circular genome is 14 768 bp in size and represents the smallest in presently known heteropteran mt genomes. The mt genome is encoding for two ribosomal RNA （rRNA） genes, 22 transfer RNA （tRNA） genes, 13 protein coding genes and a control region, and the order, content, codon usage and base organization show similarity to a great extent to the hypothetical ancestral model. All protein coding genes use standard initiation codons ATN. Conventional stop codons TAA and TAG have been assigned to the most protein coding genes; however, COIII, ND4 and ND5 genes show incomplete terminator signal （T）. All tRNA genes possess the typical clover leaf structure, but the dihydrouridine arm of tRNAser（A6N） only forms a simple loop. Secondary structure models of rRNA genes are generally in accordance with the former models, although some differences exist in certain parts. Three intergenic spacers have never been found in sequenced mt genomes of Heteroptera. The phylogenetic study based on protein coding genes is largely congruent with previous phylogenetic work. Both Bayesian inference and maximum likelihood analyses highly support the sister relationship ofA. lucorum and Lygus lineolaris, and Miridae presents a sister position to Anthocoridae.     

Amplification of mitochondrial small subunit ribosomal DNA of polypores and its potential for phylogenetic analysis

There has been a systematic need to seek adequate phylogenetic markers that can be applied in phylogenetic analyses of fungal taxa at various levels. The mitochondrial small subunit ribosomal DNA (mt SSU rDNA) is generally considered to be one of the molecules that are appropriate for phylogenetic analyses at a family level. In order to obtain universal primers for polypores of Hymenomycetes, mt SSU rRNA genes were cloned from Bjerkandera adusta, Ganoderma lucidum, Phlebiopsis gigantea, and Phellinus laevigatus and their sequences were determined. Based on the conserved sequences of cloned genes from polypores and Agrocybe aegerita, PCR primers were designed for amplification and sequencing of mt SSU rDNAs. New primers allowed effective amplification and sequencing of almost full-sized genes from representative species of polypores and related species. Phylogenetic relationships were resolved quite efficiently by mt SSU rDNA sequences, and they proved to be more useful in phylogenetic reconstruction of Ganoderma than nuclear internal transcribed spacer (ITS) rDNA sequences.     

The genetic identity of dinoflagellate symbionts in Caribbean octocorals

Many cnidarians (e.g., corals, octocorals, sea anemones) maintain a symbiosis with dinoflagellates (zooxanthellae). Zooxanthellae are grouped into clades, with studies focusing on scleractinian corals. We characterized zooxanthellae in 35 species of Caribbean octocorals. Most Caribbean octocoral species (88.6%) hosted clade B zooxanthellae, 8.6% hosted clade C, and one species (2.9%) hosted clades B and C. Erythropodium caribaeorum harbored clade C and a unique RFLP pattern, which, when sequenced, fell within clade C. Five octocoral species displayed no zooxanthella cladal variation with depth. Nine of the ten octocoral species sampled throughout the Caribbean exhibited no regional zooxanthella cladal differences. The exception, Briareum asbestinum, had some colonies from the Dry Tortugas exhibiting the E. caribaeorum RFLP pattern while elsewhere hosting clade B. In the Caribbean, octocorals show more symbiont specificity at the cladal level than scleractinian corals. Both octocorals and scleractinian corals, however, exhibited taxonomic affinity between zooxanthella clade and host suborder.Communicated by R.C. Carpenter     

A phylogenetic reconsideration of suctorian ciliates (Protista,Ciliophora, Phyllopharyngea) based on small subunit rRNA gene sequences

Compared with other ciliated protozoa, molecular studies of phylogenetic relationships within the subclass Suctoria are rare. In this work, phylogenetic analyses focusing on this group were performed based on all data available. In addition, the small subunit ribosomal RNA (SSU rRNA) genes of three suctorian ciliates (Acineta compressa, Acineta tuberosa and Paracineta limbata) were newly sequenced. Furthermore, the putative secondary structures of the variable region 2 of the SSU rRNA gene were predicted and compared within the Suctoria. Our results show that (i) there is support for the monophyly of the subclass Suctoria, which is a sister clade to the cyrtophorids; (ii) based on combined morphologic and molecular features, we propose the following evolutionary routine within the Suctoria: Exogenina – Evaginogenina – Endogenina; (iii) the similarities of the secondary structures of the V2 region and the SSU rRNA gene sequences within the subclass Suctoria are consistent with the branching of the phylogenetic lineages.     

Genotypic diversity and distribution of Ostreobium quekettii within scleractinian corals

The green filamentous endolithic alga Ostreobium quekettii resides inside skeletons of scleractinian corals in close proximity with their tissue and plays a role in the viability of the coral and its associates. This study examined the distribution and diversity of O. quekettii within scleractinian corals from the Red Sea (Eilat, Gulf of Aqaba), using a molecular phylogenetic marker. The massive coral species Porites lutea and Goniastrea perisi were sampled from a depth range of 6–55 m, and ribulose 1,5-bisphosphate carboxylase large subunit gene (rbcL) DNA sequence of the alga was amplified and analyzed for diversity and distribution of ecological patterns. This work reveals that O. quekettii has at least seven different clades distributed along a depth gradient in the examined scleractinian corals. Among the seven identified clades, four were found only in P. lutea, while the other two clades are found in both P. lutea and G. perisi. Goniastrea perisi colonies at depth of 30 m had a distinct O. quekettii clade that was absent in P. lutea. It is obvious from this study that the green endolithic alga O. quekettii is not a single genotype as previously considered but a complex of genotypes and that this differentiation is of ecological significance.     

124 Evidence for Lateral Transfer of a IE Intron between Fungal and Red Algal Small Subunit Ribosomal RNA Genes

In a recent study of the North American biogeography of the red algae genus Hildenbrandia, the presence of group I introns were noted in the nuclear SSU rRNA gene of the marine species H. rubra (Hildenbrandiales). Group I introns in the nuclear encoded rRNAs have been previously reported in the Hildenbrandiales as well as the Bangiales. All reported introns within the red algae have been identified as belonging to the IC1 subclass and occur at two insertion sites in the nuclear small subunit rRNA (516 and 1506). However, an unclassified intron was discovered at position 989 in the nuclear SSU rRNA gene of a collection of H. rubra from British Columbia, Canada. We have determined that the intron is a member of the IE subclass and this is the first report of an IE intron and an intron in position 989 in the red algae. Phylogenetic analyses of the intron sequences reveal a close relationship between this group IE intron and similar ascomycete and basidiomycete fungal IE introns in the nuclear SSU rRNA genes at positions 989 and 1199. In addition, a common unique helix (structural signature) in the P13 domain of the Hildenbrandia intron and those of the fungi at the 989 and 1199 IE positions in the nuclear SSU rRNA gene also indicates a close relationship. Hence, this study provides evidence for a possible lateral transfer of the IE intron in position 989 between fungal and red algal nuclear SSU rRNA genes.     

Uncovering Cryptic Diversity in Two Amoebozoan Species Using Complete Mitochondrial Genome Sequences

The Amoebozoa are a major eukaryotic lineage that encompasses a wide range of amoeboid organisms. The group is understudied from a systematic perspective: molecular tools have only been applied in the last 15 yr. Hence, there is an undersampling of both genes and taxa in the group especially compared to plants, animals, and fungi. Here, we present the complete mitochondrial genomes of two ubiquitous and abundant morpho‐species (Acanthamoeba castellanii and Vermamoeba vermiformis). Both have mitochondrial genomes of close relatives previously available, enabling insights into recent divergences at a genomic scale, while simultaneously offering comparisons with divergence estimates obtained from traditionally used single genes, SSU rDNA and cox1. The newly sequenced mt genomes are significantly divergent from their previously sequenced conspecifics (A. castellannii 16.4% divergence at nucleotide level and 10.4% amino acid; V. vermiformis 21.6% and 13.1%, respectively), while divergence at the small subunit ribosomal DNA is below 1% within both species. Morphological analyses determined that these lineages are indistinguishable from their previously sequenced counterparts. Phylogenetic reconstructions using 26 mt genes also indicate a level of divergence that is comparable to divergence among species, while reconstructions using the small subunit ribosomal DNA (SSU rDNA) do not. In addition, we demonstrate that between closely related taxa, there are high levels of synteny, which can be explored for primer design to obtain larger fragments than the traditional barcoding genes. We conclude that, although most systematic work has relied on SSU, this gene alone can severely underestimate diversity. Thus, we suggest that the mt genome emerges as an alternative for unraveling the lower level phylogenetic relationships of Amoebozoa.     

Mitochondrial Genome of <Emphasis Type="Italic">Ciona savignyi</Emphasis> (Urochordata,Ascidiacea, Enterogona): Comparison of Gene Arrangement and tRNA Genes with <Emphasis Type="Italic">Halocynthia roretzi</Emphasis> Mitochondrial Genome

The complete nucleotide sequence of the urochordate Ciona savignyi (Ascidiacea, Enterogona) mitochondrial (mt) genome (14,737 bp) was determined. The Ciona mt genome does not encode a gene for ATP synthetase subunit 8 but encodes an additional tRNA^Gly gene (anticodon UCU), as is the case in another urochordate, Halocynthia roretzi (Ascidiacea, Pleurogona), mt genome. In addition, the Ciona mt genome encodes two tRNA^Met genes; anticodon CAT and anticodon TAT. The tRNA^Cys gene is thought to lack base pairs at the D-stem. Thus, the Ciona mt genome encodes 12 protein, 2 rRNA, and 24 tRNA genes. The gene arrangement of the Ciona mt genome differs greatly from those of any other metazoan mt genomes reported to date. Only three gene boundaries are shared between the Halocynthia and the Ciona mt genomes. Molecular phylogenetic analyses based on amino acid sequences of mt protein genes failed to demonstrate the monophyly of the chordates.     

Phylogenetic Screening of Ribosomal RNA Gene-Containing Clones in Bacterial Artificial Chromosome (BAC) Libraries from Different Depths in Monterey Bay

Marine picoplankton are central mediators of many oceanic biogeochemical processes, but much of their biology and ecology remains ill defined. One approach to better defining these environmentally significant microbes involves the acquisition of genomic data that can provide information about genome content, metabolic capabilities, and population variability in picoplankton assemblages. Previously, we constructed and phylogenetically screened a Bacterial Artificial Chromosome (BAC) library from surface water picoplankton of Monterey Bay. To further describe niche partitioning, metabolic variability, and population structure in coastal picoplankton populations, we constructed and compared several picoplankton BAC libraries recovered from different depths in Monterey Bay. To facilitate library screening, a rapid technique was developed (ITS-LH-PCR) to identify and quantify ribosomal RNA (rRNA) gene-containing BAC clones in BAC libraries. The approach exploited natural length variations in the internal transcribed spacer (ITS) located between SSU and LSU rRNA genes, as well as the presence and location of tRNA-alanine coding genes within the ITS. The correspondence between ITS-LH-PCR fragment sizes and 16S rRNA gene phylogenies facilitated rapid identification of rRNA genes in BAC clones without requiring direct DNA sequencing. Using this approach, 35 phylogenetic groups (previously identified by cultivation or PCR-based rRNA gene surveys) were detected and quantified among the BAC clones. Since the probability of recovering chimeric rRNA gene sequences in large insert BAC clones was low, we used these sequences to identify potentially chimeric sequences from previous PCR amplified clones deposited in public databases. Full-length SSU rRNA gene sequences from picoplankton BAC libraries, cultivated bacterioplankton, and nonchimeric RNA genes were then used to refine phylogenetic analyses of planktonic marine gamma Proteobacteria, Roseobacter, and Rhodospirillales species.(M.T. Suzuki and C.M. Preston) These authors contributed equally to this paper.     

Competitive strategies of soft corals (Coelenterata: Octocorallia). II. Variable defensive responses and susceptibility to scleractinian corals

Interactions involving competition for space between several species of alcyonacean and scleractinian corals were assessed experimentally on Britomart Reef, central region of the Great Barrier Reef, Australia. Colonies of three soft coral species, Sarcophyton ehrenbergi Marenzeller, Nephthea brassica Kukenthal, and Capnella lacertiliensis Macfayden Forskal (Coelenterata:Alcyonacea) were relocated within stands of two scleractinian corals, Parités andrewsi Vaughan (= P. cylindrica Dana) and Pavona cactus Förskal (Coelenterata:Scleractinia). Undisturbed scleractinian and relocated alcyonacean controls were also monitored.Alcyonacean corals induced necrosis of tissue in scleractinian corals. Necrosis was significantly more pronounced when colonies were in contact but was also observed in the absence of contact, implicating the presence of active allelopathic agents. Scleractinian coral species varied in their susceptibility to the ill effects of alcyonaceans, with Pontes andrewsi being more susceptible than Pavona cactus. Of the soft corals, Nephthea caused the highest degree of mortality in the two scleractinian corals examined and Sarcophyton the least. Some soft corals appear to retain their toxins while others release them, implying a combination of anti-predatory and anti-competitor roles for the secondary metabolites. Scleractinian corals were often overgrown by soft corals.Both species of scleractinian corals were found to cause approximately equal amounts of tissue necrosis in alcyonaceans. These effects were more pronounced when colonies were in direct contact. The necrotic effects among alcyonacean corals were species-specific. Alcyonaceans also overgrew scleractinian corals and secreted a protective polysaccharide layer in areas proximal to scleractinians. Secretion of this layer was stimulated differentially by the two scleractinian species and also varied in frequency of occurrence among the alcyonaceans.High levels of tissue necrosis were observed in both groups of organisms within 3 wk of initiation of the experiment. Necrosis increased with time in the scleractinian corals and decreased in the alcyonaceans. The development of a protective polysaccharide layer in the alcyonaceans increased with time.