Genetic variation of the scleractinian coral Stylophora pistillata, from western Pacific reefs

Most scleractinian coral species are widely distributed across the tropical and subtropical Indo-Pacific. However, the genetic connectivity between populations of corals separated by large distances (thousands of kilometers) is not well known. We analyzed variability in the nucleotide sequence of the internal transcribed spacer-1 (ITS-1) of the nuclear ribosomal gene unit in the ubiquitous coral Stylophora pistillata, across the western Pacific Ocean. Eight populations from Japan, Malaysia, and the northern and southern Great Barrier Reef (GBR) were studied. Phylogenetic analyses and analysis of molecular variance (AMOVA) clearly revealed that there is panmixia among these coral populations. AMOVA showed that ITS-1 sequence variability was greater within populations (78.37%) than among populations (12.06%). These patterns strongly suggest high levels of connectivity across the species' latitudinal distribution range in the western Pacific, as is seen in many marine invertebrates.

The use and implications of ribosomal DNA sequencing for the discrimination of digenean species

In just over a decade, the use of molecular approaches for the recognition of parasites has become commonplace. For trematodes, the internal transcribed spacer region of ribosomal DNA (ITS rDNA) has become the default region of choice. Here, we review the findings of 63 studies that report ITS rDNA sequence data for about 155 digenean species from 19 families, and then review the levels of variation that have been reported and how the variation has been interpreted. Overall, complete ITS sequences (or ITS1 or ITS2 regions alone) usually distinguish trematode species clearly, including combinations for which morphology gives ambiguous results. Closely related species may have few base differences and in at least one convincing case the ITS2 sequences of two good species are identical. In some cases, the ITS1 region gives greater resolution than the ITS2 because of the presence of variable repeat units that are generally lacking in the ITS2. Intraspecific variation is usually low and frequently apparently absent. Information on geographical variation of digeneans is limited but at least some of the reported variation probably reflects the presence of multiple species. Despite the accepted dogma that concerted evolution makes the individual representative of the entire species, a significant number of studies have reported at least some intraspecific variation. The significance of such variation is difficult to assess a posteriori, but it seems likely that identification and sequencing errors account for some of it and failure to recognise separate species may also be significant. Some reported variation clearly requires further analysis. The use of a yardstick to determine when separate species should be recognised is flawed. Instead, we argue that consistent genetic differences that are associated with consistent morphological or biological traits should be considered the marker for separate species. We propose a generalised approach to the use of rDNA to distinguish trematode species.

Digenean trematodes infecting the tropical abalone Haliotis asinina have species-specific cercarial emergence patterns that follow daily or semilunar spawning cycles

Approximately 1-2% of the tropical abalone Haliotis asinina inhabiting Heron Island Reef are infected with opecoelid digeneans. These largely inhabit the haemocoel surrounding the cerebral ganglia and digestive gland-gonad complex, and infected abalone typically have significantly reduced or ablated gonads. Observations of infected abalone reveal two distinct cercarial emergence patterns, one which correlates tightly with the abalone's highly regular and synchronous fortnightly spawning cycle, and the other which occurs in a circadian pattern. The former appears to be a novel emergence strategy not previously observed in digeneans. While the cercariae in all abalone are morphologically indistinguishable, comparison of sequences from the internal transcribed spacer 2 (ITS 2) region of the ribosomal DNA reveals a 5.7% difference between cercariae displaying different emergence patterns, indicating these are two distinct species that probably belong to the same genus. The ITS 2 sequences of the species with the daily emergence pattern are identical to that of an undescribed adult opecoelid from the gut of the barramundi cod, Cromileptes altivelis. Combined molecular, morphological and emergence data suggest that while these opecoelid cercariae use the same first intermediate host and are closely related species-members of the genus Allopodocotyle-they fill different ecological niches that are likely to include different definitive hosts.

Sensitive and specific detection of Clavibacter xyli subsp. xyli, causal agent of ratoon stunting disease of sugarcane, with a polymerase chain reaction-based assay

A sensitive, specific polymerase chain reaction-based assay was developed for the detection of the causal agent of ratoon stunting disease of sugarcane, Clavibacter xyli subsp. xyli. This assay uses oligonucleotide primers derived from the internal transcribed spacer region between the 16S and 23S rRNA genes of the bacterial rRNA operon. The assay is specific for C. xyli subsp. xyli and does not produce an amplification product from the template of the closely related bacterium C. xyli subsp. cynodontis, nor from other bacterial species. The assay was successfully applied to the detection of C. xyli subsp. xyli in fibrovascular fluid extracted from sugarcane and was sensitive to approximately 22 cells per PCR assay. A multiplex PCR test was also developed which identified and differentiated C. xyli subsp. xyli and C. xyli subsp. cynodontis in a single PCR assay.

A DNA-based demonstration of a three-host life-cycle for the Bivesiculidae (Platyhelminthes : Digenea)

Immature bivesiculid trematodes collected from the intestine of Thalassoma lunare (Labridae) are shown to be morphologically consistent with adults of Bivesicula claviformis from Epinephelus fasciatus (Serranidae). In addition, the immature bivesiculids have the same sequence for the second internal transcribed spacer of the ribosomal DNA. Comparison with three other species of Bivesiculidae showed differences of between 23% and 30%. These results show that bivesiculids may have three-host life-cycles in addition to the two-host life-cycles that have been demonstrated previously. The three-host life-cycle enables bivesiculids to infect large carnivorous fishes. (C) 1998 Australian Society for Parasitology. Published by Elsevier Science Ltd. All rights reserved.

Determination of Ralstonia (Pseudomonas) solanacearum rDNA subgroups by PCR tests

Rapid and sensitive polymerase chain reaction (PCR) methods ape described for determination of the two 16 S rDNA subgroups of Ralstonia solanacearum, the causal agent of bacterial wilt. A third subgroup consisting of Indonesian R. solanacearum isolates belonging to Division II, the blood disease bacterium and Pseudomonas syzygii can also be identified. Primers were designed to sequences within R, solanacearum 16 S rDNA (equivalent to Escherichia coli 16 S rDNA positions 74-97, 455-475, 1454-1474), and the internal transcribed spacer region between the 16 S and 23 S rDNA genes. Different combinations of forward and reverse primers allowed selective PCR amplification of (a) R. solanacearum Division I (biovars 3, 4 and 5), (b) Division TI (biovars 1, N2, and 2) including the blood disease bacterium and P. syzygii, or (c) amplification of Division II only except for five biovar 1, 2 or N2 isolates of R. solanacearum from Indonesia, P. syzygii and the BDB. A total of 104 R. solanacearum, 14 blood disease bacterium and 10 P. syzygii isolates were tested. Simultaneous detection of species and subdivision was achieved by designing a multiplex PCR test in which a 288-base pair (bp) band is produced by all R. solanacearum isolates, and an additional 409-bp band in Division I strains.

Low symbiont diversity in southern Great Barrier Reef corals, relative to those of the Caribbean

The specific identity of endosymbiotic dinoflagellates (Symbiodinium spp.) from most zooxanthellate corals is unknown. In a survey of symbiotic cnidarians from the southern Great Barrier Reef (GBR), 23 symbiont types were identified from 86 host species representing 40 genera. A majority (>85%) of these symbionts belong to a single phylogenetic clade or subgenus (C) composed of closely related (as assessed by sequence data from the internal transcribed spacer region and the ribosomal large subunit gene), yet ecologically and physiologically distinct, types. A few prevalent symbiont types, or generalists, dominate the coral community of the southern GBR, whereas many rare and/or specific symbionts, or specialists, are found uniquely within certain host taxa. The comparison of symbiont diversity between southern GBR and Caribbean reefs shows an inverse relationship between coral diversity and symbiont diversity, perhaps as a consequence of more-rapid diversification of Caribbean symbionts. Among clade C types, generalists C1 and C3 are common to both Caribbean and southern GBR symbiont assemblages, whereas the rest are regionally endemic. Possibly because of environmental changes in the Caribbean after geographic isolation through the Quaternary period, a high proportion of Caribbean fauna associate with symbiont taxa from two other distantly related Symbiodinium clades (A and B) that rarely occur in Pacific hosts. The resilience of Porites spp. and the resistance of Montipora digitata to thermal stress and bleaching are partially explained by their association with a thermally tolerant symbiont type, whereas the indiscriminant widespread bleaching and death among certain Pacific corals, during El Nino Southern Oscillation events, are influenced by associations with symbionts possessing higher sensitivity to thermal stress.

Ecological distribution of mosquito larvae of the Anopheles punctulatus group on Niolam (Lihir) Island, Papua New Guinea

We surveyed the larval habitats of member,, of the Anopheles punctulatus group of mosquitoes on Niolam (Lihir) Island. Papua New Guinea. Identification of this group was undertaken by polymerase chain reaction-restriction fragment length polymorphism analysis of the amplified internal transcribed spacer unit 2 of rDNA. because morphologic separation of member species is unreliable. The most widespread malaria vector species and their most common larval habitats identified to aid source-reduction programs for malaria control. The most ubiquitous species was An. punctulatus, followed by An. farauti no. 2. then An. farauti s.s. Anopheles punctulatus has increased relative to An.farauti s.l. since the start of development projects on Lihir Island. The most common larval habitats were shallow temporary pools with clay substrate and with plants or floatage. These habitats. mostly encountered alongside poorly drained roads, may be increased by development projects.

Identical digeneans in coral reef fishes from French Polynesia and the Great Barrier Reef (Australia) demonstrated by morphology and molecules

Three coral reef fish species, Zanclus cornutus, Chaetodon vagabundus and Naso lituratus, were collected in French Polynesia and on the Great Barrier Reef, Queensland. These fish species were each infected by one morphologically similar digenean species in both localities; Schistorchis Zancli Hanson, 1953 was found in Zanclus cornutus. Preptetos laguncula Bray and Cribb, 1996 in Naso lituratus and Neohypocreadium dorsoporum Machida and Uchida, 1987 in Chaetodon vagabundus. In addition, on the Great Barrier Reef P. laguncula was also found in Naso unicornis and N. dorsoporum in Chaetodon ephippium and Chaetodon flavirostris. Morphometric differences between the species from the two sites were only slight. Sequences from the second internal transcribed spacer of the ribosomal DNA of each worm revealed total homology or negligible divergence between samples from hosts caught in French Polynesia and on the Great Barrier Reef. These results show that across more than 6000 km these digeneans are similar in morphology and genotype. Some species of fishes and molluscs a-re considered to have distributions that encompass the entire tropical Indo-West Pacific. These findings suggest that at least some of their parasites have similarly broad distributions. (C) 2001 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

A total-evidence phylogeny of ticks provides insights into the evolution of life cycles and biogeography

We inferred the phylogeny of 33 species of ticks from the subfamilies Rhipicephalinae and Hyalomminae from analyses of nuclear and mitochondrial DNA and morphology. We used nucleotide sequences from 12S rRNA, cytochrome c oxidase I, internal transcribed spacer 2 of the nuclear rRNA, and 18S rRNA. Nucleotide sequences and morphology were analyzed separately and together in a total-evidence analysis. Analyses of the five partitions together (3303 characters) gave the best-resolved and the best-supported hypothesis so far for the phylogeny of ticks in the Rhipicephalinae and Hyalomminae, despite the fact that some partitions did not have data for some taxa. However, most of the hidden conflict (lower support in the total-evidence analyses compared to that in the individual analyses) was found in those partitions that had taxa without data. The partitions with complete taxonomic sampling had more hidden support (higher support in the total-evidence analyses compared to that in the separate-partition analyses) than hidden conflict. Mapping of geographic origins of ticks onto our phylogeny indicates an African origin for the Rhipicephalinae sensu lato (i.e., including Hyalomma spp.), the Rhipicephalus-Boophilus lineage, the Dermacentor-Anocentor lineage, and the Rhipicephalus-Booophilus-Nosomma-Hyalomma-Rhipicentor lineage. The Nosomma-Hyalomma lineage appears to have evolved in Asia. Our total-evidence phylogeny indicates that (i) the genus Rhipicephalus is paraphyletic with respect to the genus Boophilus, (ii) the genus Dermacentor is paraphyletic with respect to the genus Anocentor, and (iii) some subgenera of the genera Hyalomma and Rhipicephalus are paraphyletic with respect to other subgenera in these genera. Study of the Rhipicephalinae and Hyalomminae over the last 7 years has shown that analyses of individual datasets (e.g., one gene or morphology) seldom resolve many phylogenetic relationships, but analyses of more than one dataset can generate well-resolved phylogenies for these ticks. (C) 2001 Academic Press.

The phylogeography and connectivity of the latitudinally widespread scleractinian coral Plesiastrea versipora in the Western Pacific

Whereas terrestrial animal populations might show genetic connectivity within a continent, marine species, such as hermatypic corals, may have connectivity stretching to all corners of the planet. We quantified the genetic variability within and among populations of the widespread scleractinian coral, Plesiastrea versipora along the eastern Australian seaboard (4145 km) and the Ryukyu Archipelago (Japan, 681 km) using sequences of internal transcribed spacers (ITS1-2) from ribosomal DNA. Geographic patterns in genetic variability were deduced from a nested clade analysis (NCA) performed on a parsimony network haplotype. This analysis allowed the establishment of geographical associations in the distribution of haplotypes within the network cladogram, therefore allowing us to deduce phylogeographical patterns based under models of restricted gene flow, fragmentation and range expansion. No significant structure was found among Ryukyu Archipelago populations. The lack of an association between the positions of haplotypes in the cladogram with geographical location of these populations may be accounted for by a high level of gene flow of P. versipora within this region, probably due to the strong Kuroshio Current. In contrast, strong geographical associations were apparent among populations of P. versipora along the south-east coast of Australia. This pattern of restricted genetic connectivity among populations of P. versipora on the eastern seaboard of Australia seems to be associated with the present surface ocean current (the East Australian Current) on this side of the south-western Pacific Ocean.

Symbiont diversity within the widespread Scleractinian coral Plesiastrea versipore across the northwestern Pacific

The molecular diversity of symbiotic dinoflagellates associated with the widespread western Pacific coral Plesiastrea versipora was explored in order to examine if associations between reef-building corals and symbiotic dinoflagellates change with environment. Several ribosomal DNA genes with different evolutionary rates were used.. including the large subunit (28S), the 5.8S region and the internal transcribed spacers (ITS). The phylogenetic analysis of the 28S and 5.8S rDNA regions indicated that a single endosymbiont species, highly related to one of the species of Symbiodinium in clade C (=Synbiodinium goreaui, Trench et Blank), associates with P. versipora along the Ryukyu Archipelago. The persistence of the same endosymbiont within P. versipora across this wide array of latitudes may be a result of such features as the Kuroshio Current, which brings tropical temperatures as far north as Honshu, Japan. Analysis of the faster evolving ITS rDNA region revealed significant genetic variability within endosymbionts from different populations. This variation was due to a high degree of interpopulation variability, based on the proportion of pairwise variation detected among the populations (0.95% approximately). By comparison with other studies, the results also indicate that some ITS1 haplotypes from P. versipora endosymbionts seem to be widely distributed within the western Pacific Ocean, ranging from the Great Barrier Reef to the northeast of the China Sea.

Two new blood flukes (Digenea: Sanguinicolidae) from epinephelinae (Perciformes: Serranidae) of the Pacific Ocean

Pearsonellum pygmaeus n. sp. is described from Cromileptes altivelis (Serranidae), the Barramundi Cod, from Heron Island (southern Great Barrier Reef) and Lizard Island (northern Great Bat-Her Reef). This new species differs from Pearsonellum eorventum (type and only species) in the combination of smaller overall body size, the relative distance of the brain from the anterior end, the relative lengths of both the oesophagus and the testis, the degree to which the testis extends outside the intercaecal field, the shape of the testis, the shape and size of the ovary and the extent to which the uterzus loops around the ovary. There are in addition, 20 base pair differences between the ITS2 rDNA sequence of P. pygmaeus n. sp. and that of P corventum. Three new host records for P. corventum are reported. Adelomyllos teenae n. g., n. sp. is described from Epinephelus coioides (Serranidae), the Estuary Cod, from Moreton Bay, southeast Queensland. The new genus differs from the 22 other sanguinicolid genera in the combined possession of two testes, a cirrus-sac, separate genital pores, a post-ovarian uterus and an H-shaped intestine. A. teenae n. sp. is the third sanguinicolid described from the Epinephelinae. Sanguinicolids have now been reported from 11 species of Serranidae. (C) 2004 Elsevier Ireland Ltd. All rights reserved.

An exceptionally rich complex of Sanguinicolidae von Graff, 1907 (Platyhelminthes : Trematoda) from Siganidae, Labridae and Mullidae (Teleostei : Perciformes) from the Indo-West Pacific Region

We describe an unprecedented radiation of sanguinicolid blood flukes ( Digenea: Sanguinicolidae) from two species of Labridae (Choerodon venustus and C. cauteroma), seven species of Mullidae (Mulloidichthys vanicolensis, Parupeneus barberinoides, P. barberinus, P. bifasciatus, P. cyclostomus, P. indicus and P. multifasciatus) and ten species of Siganidae (Siganus argenteus, S. corallinus, S. doliatus, S. fuscescens, S. lineatus, S. margaritiferus, S. puellus, S. punctatus, S. virgatus and S. vulpinus) from sites off Australia and Palau. The flukes were morphologically similar in having the combination of a long thread-like body, tegumental spines in lateral transverse rows, a vestigial oral sucker bearing concentric rows of fine spines, an H-shaped intestine, a cirrussac, a notch level with the male genital pore, a lateral or post-ovarian uterus, a uterine chamber and separate genital pores. These species are divided into two genera on the basis of testis number. Sanguinicolids from Siganus fuscescens have a single large testis between the intestinal bifurcation and the ovary and are placed in Ankistromeces Nolan & Cribb, 2004. Species from the remaining nine species of Siganidae, Labridae and Mullidae are placed in Phthinomita n. g.; these species have two testes, the anterior testis being large and between the intestinal bifurcation and the ovary whereas the small posterior testis is at the posterior end of the body and appears rudimentary or degenerate and probably non-functional. The second internal transcribed spacer (ITS2) of ribosomal DNA ( rDNA) from 29 host/parasite/location combinations (h/p/l) was sequenced together with that of Ankistromeces mariae Nolan & Cribb, 2004 for comparison. From 135 samples we found 19 distinct genotypes which were interpreted as representing at least that many species. Replicate sequences were obtained for 25 of 30 h/p/l combinations ( including A. mariae); there was no intraspecific variation between replicates sequences for any of these. Interspecific variation ranged from 1 - 41 base differences (0.3 - 12.7% sequence divergence). The 19 putative species were difficult to recognise by morphological examination. We describe 13 new species; we do not describe (= name) six species characterised solely by molecular sequences and three putative species for which morphological data is available but for which molecular data is not. We have neither morphological nor molecular data for sanguinicolids harboured in five hosts species ( Siganus margaritiferus, S. puellus, Choerodon cauteroma, Parupeneus indicus and P. multifasciatus) in which we have seen infections. Where host species were infected in different localities they almost always harboured distinct species. Some host species ( for example, S. argenteus and S. lineatus from Lizard Island) harboured two or three species in a single geographical location. This suggests that, for parts of this system, parasite speciation has outstripped host speciation. Distance analysis of ITS2 showed species from each host family ( Siganidae, Mullidae and Labridae) did not form monophyletic clades to the exclusion of species from other host families. However, a host defined clade was formed by the sequences from sanguinicolids from S. fuscescens.