Molecular systematic and phylogenetic assessment of 34 calanoid copepod species of the Calanidae and Clausocalanidae

DNA sequences for a 639 bp region of mitochondrial cytochrome oxidase I (mtCOI) were determined for 34 species of ten genera in two families of calanoid copepods, including: Calanoides, Cosmocalanus, Meoscalanus, Nannocalanus, Neocalanus, and Undinula (family Calanidae); and Clausocalanus, Ctenocalanus, Drepanopus, and Pseudocalanus (family Clausocalanidae). MtCOI gene sequences proved to be diagnostic molecular systematic characters for accurate identification and discrimination of the species. Levels of mtCOI variation within species (range: 1-4%) were significantly less than those between species (9-25%). Higher levels of intraspecific variation (>2%) usually resulted from comparisons between ecologically distinct or geographically isolated populations. MtCOI sequence variation resolved evolutionary relationships among species of Clausocalanus, Neocalanus, and Pseudocalanus, although there was evidence of saturation at some variable sites. Phylogenetic relationships among 11 copepod genera (adding Calanus to the list above) were reconstructed using a 660 bp region of nuclear small-subunit 18S rRNA, a slowly evolving gene that showed no variability within a species and differed by <1-6% among the genera. The 18S rRNA molecular phylogeny was consistent with the accepted limits of the Calanidae and Clausocalanidae and clearly resolved relationships among genera within each family. This molecular systematic and phylogenetic study was part of the ZooGene project, an international partnership to create a DNA sequence database as a tool for uniform, molecularly based species identification of planktonic calanoid copepods and euphausiids.     

Chromosomal evolution in Mugilidae: karyotype characterization of Liza saliens and comparative localization of major and minor ribosomal genes in the six Mediterranean mullets

This study continues a comparative cytogenetic analysis of the fish family Mugilidae, reporting the karyotype characterization of the leaping mullet, Liza saliens, by C-banding, Ag- and fluorochrome-staining, and completing the fluorescence in situ hybridization (FISH)-mapping of the 18S and 5S rRNA genes (rDNA) to the chromosomes of the six Mediterranean mullets, namely L. saliens, L. ramada, L. aurata, Mugil cephalus, Chelon labrosus and Oedalechilus labeo. In all species, except M. cephalus, the 5S rDNA sites were localized on a medium-sized acrocentric chromosome pair, which was considered homeologous in all of them. In L. saliens, an additional 5S rDNA site was detected in a location close to the one shown by major ribosomal genes in M. cephalus, i.e. the subtelomeric region of chromosome pair 1. The 5S rDNA site in M. cephalus is located on the smallest chromosome pair of the complement, which, on the other hand, though on a different position, bears 18S rDNA in all the species of Liza and Chelon examined. The heterochromatin composition and the major and minor ribosomal gene locations suggest that the karyotype of L. saliens (subgenus Protomugil) can be considered intermediate between the karyotype of the more primitive M. cephalus and those of the other Liza (subgenus Liza) species and of the representatives of the more derived genera Chelon and Oedalechilus.     

Phylogeny and biogeography of Sicydiinae (Teleostei: Gobiidae) inferred from mitochondrial and nuclear genes

In the Indo-Pacific area, the Caribbean region and West Africa, insular systems are colonised by particular Gobiids of the Sicydiinae subfamily. These species spawn in freshwater, the free embryos drift downstream to the sea where they undergo a planktonic phase, before returning to rivers to grow and reproduce; an amphidromous lifestyle. These gobies are the biggest contributors to the diversity of fish communities in insular systems and have the highest levels of endemism, yet their phylogeny has not been explored before with molecular data. To understand the phylogeny and the biogeography of this subfamily, sequences from the mitochondrial 16S rDNA and cytochrome oxidase I and from the nuclear rhodopsin gene were obtained for 50 Sicydiinae specimens of seven genera. Our results support the monophyly of the subfamily and of all the genera except Sicyopus, which is polyphyletic. Five major clades were identified within this subfamily. One clade clusters Sicyopterus and Sicydium as sister genera, one contains the genus Stiphodon split into two different groups, two other clades include only Sicyopus (Smilosicyopus) and Cotylopus, respectively, and the last clade groups Akihito, Lentipes and Sicyopus (Sicyopus). As a result, the subgenus Smilosicyopus is elevated herein as a genus. A molecular dating approach helps the interpretation of these phylogenetic results in terms of amphidromy and biogeographical events that have allowed the Sicydiinae to colonise the Indo-Pacific, West African and Caribbean islands.     

Extreme mitochondrial DNA divergence within populations of the deep-sea gastropod Frigidoalvania brychia

The deep sea supports a diverse and highly endemic invertebrate fauna, the origin of which remains obscure. Little is known about geographic variation in deep-sea organisms or the evolutionary processes that promote population-level differentiation and eventual speciation. Sequence variation at the 16 S rDNA locus was examined in formalin-preserved specimens of the common upper bathyal rissoid Frigidoalvania brychia (Verrill, 1884) to examine its population genetic structure. The specimens came from trawl samples taken over 30 years ago at depths of 457-1,102 m at stations in the Northwest Atlantic south of Woods Hole, Massachusetts, USA. Near the upper boundary of its bathymetric range (500 m), extremely divergent haplotypes comprising three phylogenetically distinct clades (average uncorrected sequence divergence among clades ~23%, ~3% within clades) were found at stations separated by a maximum distance of ~80 km, suggesting the presence of high levels of intraspecific divergence or the possibility of morphologically cryptic species. Only one of these clades was found at two stations in the mid- to lower part of F. brychia's depth distribution (800-1,100 m), suggesting lower clade diversity with increasing depth, although among-sample divergence, with a single exception, was minimal. One station was genetically divergent from all others sampled, containing a unique suite of haplotypes including two found only at this site. Steep vertical selective gradients, major oceanographic changes during the late Cenozoic, and habitat fragmentation by submarine canyons might have contributed to an upper bathyal region that is highly conducive to evolutionary change.     

Interspecific infection of aposymbiotic juveniles of <Emphasis Type="Italic">Codakia</Emphasis><Emphasis Type="Italic">orbicularis</Emphasis> by various tropical lucinid gill-endosymbionts

Previous molecular phylogenetic analyses have shown that five tropical lucinid species living in or near Thalassia testudinum seagrass beds are colonized by the same bacterial symbiont species. In addition, a new lucinid species belonging to the genus Anodontia, which inhabits reducing sediment found near seagrass beds and in mangrove swamps, has been included in the present study. Endosymbiosis in Anodontia alba was examined according to symbiont phylogenetic and gill ultrastructural analysis. Phylogenetic analysis showed that partial 16S rDNA sequences of A. alba- and Codakia orbicularis-symbionts were 100% identical at all nucleotide positions determined, suggesting that A. alba also harbors the same symbiont species as C. orbicularis (and, consequently, as C. orbiculata, C. pectinella, Linga pensylvanica and Divaricella quadrisulcata). Based on light and electron microscopy, the cellular organization of the gill filament appeared similar to those already described in other lucinids. The most distinctive feature is the lack of "granule cells" in the lateral zone of A. alba gill filaments. In order to confirm the single-species hypothesis, purified fractions of gill bacterial symbionts obtained from the gills of each of the six tropical lucinids cited above were used to infect aposymbiotic juveniles of C. orbicularis. In each case, aposymbiotic juvenile batches were successfully infected by the gill-endosymbiont fractions, whereas, during the experiments, juveniles from the negative control were still uninfected. These experimental data confirm the phylogenetic data and also demonstrate that chemoautotrophic bacterial endosymbionts from their host cells can colonize aposymbiotic juveniles. The conclusion also follows that intracellular gill-endosymbionts still have the capacity to recognize and colonize new host generations. Lucinids provide a unique model for the study of sulfide-oxidizing symbiosis, even if symbionts remain unculturable.     

Host specificity of the symbiotic cyanobacterium<Emphasis Type="Italic"> Oscillatoria spongeliae</Emphasis> in marine sponges,<Emphasis Type="Italic"> Dysidea</Emphasis> spp.

Marine sponges can host a variety of cyanobacterial and bacterial symbionts, but it is often unclear whether these symbionts are generalists that occur in many host species or specialists that occur only in certain species or populations of sponges. The filamentous cyanobacterium Oscillatoria spongeliae is found in the sponges Dysidea n. sp. aff. herbacea 1A and 1B, and similar cyanobacteria are found in D. n. sp. aff. granulosa. We amplified and sequenced sponge nuclear ribosomal DNA (rDNA) and cyanobacterial 16S rDNA from specimens of these three sponges. We then used these sequences to construct phylogenies for host sponges and their symbiotic cyanobacteria. Each of these three sponge species hosts a distinct cyanobacterial clade, suggesting a high degree of host specificity and potential coevolution between symbiotic cyanobacteria and their host sponges.     

Development of a PCR strategy for thraustochytrid identification based on 18S rDNA sequence

The identification and characterization of the thraustochytrids, an emerging economically and biotechnologically important group of marine heterotrophic protists, is usually based on morphological characters. In this research we used molecular markers to identify thraustochytrids. We designed three sets of primers based on the 18S rDNA sequence alignment of known strains and employed a PCR (polymerase chain reaction) strategy to identify unknown thraustochytrid isolates. DNA from 26 thraustochytrids (three isolated from primary cell cultures of the tunicate Botryllus schlosseri and 23 from a coral holding aquarium) were amplified by these primers, revealing 21 isolates with three bands each, which were assigned to two groups according to PCR fragment sizes. Taxonomic characterizations were deduced by comparing with GenBank data. Four isolates were further studied by sequencing their 18S rDNA. Sequence alignments and phylogenetic analysis revealed that isolates from the coral aquarium (7-5 and 8-7) were highly similar to each other and 95.0-97.0% similar to Thraustochytrium multirudimentale and Schizochytrium minutum. Isolates from the tunicate primary cell cultures (BS1 and BS2) were also closely related to each other and 84.3-86.0% similar to labyrinthulid quahog parasite and Thraustochytrium pachydermum. AFLP (amplified fragment-length polymorphism) analysis revealed 2.5-3.6% differences within the genomic DNA of each group, showing that each isolate is different, although isolates within each group may belong to the same species, in spite of differences found in the general morphology.     

A molecular phylogeny of the Patellogastropoda (Mollusca: Gastropoda)

Phylogenetic analyses of partial 18S rDNA sequences from species representing all living families of the order Patellogastropoda, most other major gastropod groups (Cocculiniformia, Neritopsina, Vetigastropoda, Caenogastropoda, Heterobranchia, but not Neomphalina), and two additional classes of the phylum Mollusca (Cephalopoda, Polyplacophora) confirm that Patellogastropoda comprises a robust clade with high statistical support. The sequences are characterized by the presence of several insertions and deletions that are unique to, and ubiquitous among, patellogastropods. However, this portion of the 18S gene is insufficiently informative to provide robust support for the monophyly of Gastropoda, or to address the division of the Gastropoda into the subclasses Eogastropoda (= Patellogastropoda + hypothetical coiled ancestors) and Orthogastropoda. These sequence data invariably group Patellogastropoda in a weakly supported clade with cocculiniform limpets, despite greater sequence divergences between Patellogastropoda and “Cocculiniformia” than between the Patellogastropoda and Orthogastropoda. Partial 18S sequences support the inclusion of the family Neolepetopsidae within the superfamily Acmaeoidea, and refute its previously hypothesized position as sister group to the remaining living Patellogastropoda. This region of the 18S rDNA gene diverges at widely differing rates, spanning an order of magnitude among patellogastropod lineages, and therefore does not provide meaningful resolution of the relationships among higher taxa of patellogastropods. Data from one or more genes that evolve more uniformly and more rapidly than the 18S rDNA gene (possibly one or more of the mitochondrial genes) seem more likely to be informative about relationships within Patellogastropoda. Received: 5 February 1999 / Accepted: 16 May 2000     

Phylogeography of the pipefish, <Emphasis Type="Italic">Urocampus carinirostris</Emphasis>, suggests secondary intergradation of ancient lineages

We assayed the pattern of mitochondrial DNA evolution in the live bearing, seagrass specialist pipefish, Urocampus carinirostris, in eastern Australia. These life history attributes were predicted to result in strong phylogeographic structure in U. carinirostris. Phylogenetic analysis of cytochrome b sequences detected two monophyletic mtDNA clades that differed by 8.69% sequence divergence - a large level of intraspecific divergence for a marine fish. The geographical distribution of clades was non-random and resembled clinal secondary intergradation over a 130-km stretch of coastline. Contrary to phylogeographic predictions, this large phylogeographic break does not occur across a traditionally recognised biogeographic boundary. Analyses of historical demography suggested that individuals belonging to the most widespread clade underwent a population expansion from a small refuge population during the Pleistocene.     

DNA sequence evidence for speciation,paraphyly and a Mesozoic dispersal of cancellothyridid articulate brachiopods

Because the classification of extant and fossil articulate brachiopods is based largely upon shell characters observable in fossils, it identifies morphotaxa whose biological status can, in practice, best be inferred from estimates of genetic divergence. Allozyme polymorphism and restriction fragment length polymorphism of mitochondrial DNA (mtDNA RFLP) have been used to show that nuclear and mitochondrial genetic divergence between samples of the cancellothyridid brachiopods Terebratulina septentrionalis from Canada and T. retusa from Europe is compatible with biological speciation, but the genetic distances obtained were biased by methodological limitations. Here, we report estimates of divergence in 12S rDNA mitochondrial sequences within and between samples of these brachiopods. The sequence-based genetic distance between these samples (5.98ǂ.07% SE) is at least 10 times greater than within them and, since they also differ in a complex life-history trait, their species status is considered to be securely established. Divergence levels between 12S rDNA genes of three other cancellothyridids, T. unguicula from Alaska, T. crossei from near Japan, and Cancellothyris hedleyi from near Australia are higher than between the two North Atlantic species, and the mean nucleotide distance between all these cancellothyrids is similar to the mean distance between species of Littorina (Mollusca: Gastropoda). Sequences of both 12S and 16S genes from cancellothyridids and other short-looped brachiopod species show neither saturation nor lineage-specific rate differences and, when analysed with different outgroups, either separately or together, yield one unexpected, but well-supported, tree with Alaskan T. unguicula basal and C. hedleyi nested within Terebratulina, i.e. these genera are paraphyletic. A geologically dated divergence between Antarctic and New Zealand species of the short-looped brachiopod Liothyrella is used to calibrate the rate of 12S divergence at ca. 0.1% per million years (MY), and this rate is used to infer that T. septentrionalis and T. retusa have been diverging for ca. 60 MY and that they and T. unguicula have been diverging from their last common ancestor for ca. 100 MY. This indicates a Mesozoic origin for the present-day distribution of cancellothyridids and the basal position of T. unguicula suggests a possible North Pacific centre of origin, with separate Atlantic and Pacific radiations. The inclusion of Cancellothyris within Terebratulina also shows that adult shell characters such as umbo, foramen and symphytium shape, whilst probably indispensible for the practical classification of fossils, are not reliable guides to genealogy.     

Phylogenetic relationships within the Octocorallia (Cnidaria: Anthozoa) based on nuclear 18S rRNA sequences

We determined the nuclear 18S rRNA sequences for 41 species of octocorals and used these to address the validity of the historical ordinal divisions and the current subordinal divisions within the subclass Octocorallia. We also explored the phylogenetic affinities of the species Dendrobrachia paucispina, which was originally classified in the order Antipatharia (subclass Ceriantipatharia) although polyp structure indicates it belongs in the subclass Octocorallia. Trees constructed using maximum likelihood techniques are incongruent with the current and historical taxonomy of the Octocorallia. There appeared to be three major clades of octocorals. The first clade included most, but not all, pennatulaceans as a monophyletic group. The second clade contained 21 species, representing all major octocoral groups other than pennatulaceans. The third clade contained members from three suborders of the Alcyonacea and one member of the Pennatulacea. These data could not be used to distinguish the branching order of the three major clades. The species D. paucispina had a close affinity with the genera Corallium and Paragorgia (Alcyonacea: Scleraxonia), although its morphology suggests it is more similar to the genus Chrysogorgia (Alcyonacea: Calcaxonia). The morphological character of dimorphism (the presence of both autozooids and siphonozooids within a single colony) corresponded loosely with the topology of the most likely trees, and a single origin of dimorphism could not be rejected. Despite sampling from the majority of families within the Octocorallia, many of the relationships within this group remain ambiguous. Received: 16 June 2000 / Accepted: 14 September 2000     

Mitochondrial DNA variation in Eritrean hamadryas baboons (Papio hamadryas hamadryas): life history influences population genetic structure

The hamadryas baboon, Papio hamadryas hamadryas, represents a rare exception from the pattern of female philopatry and male-biased dispersal predominant in mammals including primates. To elucidate the possible consequences of the dispersal pattern on the population genetic structure of hamadryas baboons, we sequenced the maternally transmitted mitochondrial hypervariable region I of 74 individuals from ten sampling locations in different ecogeographic zones of Eritrea. To this end, individual fecal samples were collected at sleeping cliffs. Upon comparing the individual sequences by means of phylogenetic tree reconstructions and AMOVA, we could not detect a population genetic structure corresponding to a geographic pattern. Tree reconstructions revealed the existence of two profoundly different lineages both present at most of the sampling locations. These findings and Mantel correlations of genetic distances and the frequency of shared haplotypes to geographic distances point to the presence of female dispersal. Female-mediated gene flow is detectable over geographic distances exceeding those between neighboring subpopulations. Our study therefore corroborates local behavioral observations on a broad geographic scale. After inclusion of geographically closely situated olive baboons, P. h. anubis, in the analyses, all anubis sequences fell within one hamadryas clade. Possible scenarios leading to this situation including long-term hybridization processes are discussed.     

Crinoid phylogeny: a preliminary analysis (Echinodermata: Crinoidea)

We describe the first molecular and morphological analysis of extant crinoid high-level inter-relationships. Nuclear and mitochondrial gene sequences and a cladistically coded matrix of 30 morphological characters are presented, and analysed by phylogenetic methods. The molecular data were compiled from concatenated nuclear-encoded 18S rDNA, internal transcribed spacer 1, 5.8S rDNA, and internal transcribed spacer 2, together with part of mitochondrial 16S rDNA, and comprised 3,593 sites, of which 313 were parsimony-informative. The molecular and morphological analyses include data from the bourgueticrinid Bathycrinus; the antedonid comatulids Dorometra and Florometra; the cyrtocrinids Cyathidium, Gymnocrinus, and Holopus; the isocrinids Endoxocrinus, and two species of Metacrinus; as well as from Guillecrinus and Caledonicrinus, whose ordinal relationships are uncertain, together with morphological data from Proisocrinus. Because the molecular data include indel-rich regions, special attention was given to alignment procedure, and it was found that relatively low, gene-specific, gap penalties gave alignments from which congruent phylogenetic information was obtained from both well-aligned, indel-poor and potentially misaligned, indel-rich regions. The different sequence data partitions also gave essentially congruent results. The overall direction of evolution in the gene trees remains uncertain: an asteroid outgroup places the root on the branch adjacent to the slowly evolving isocrinids (consistent with palaeontological order of first appearances), but maximum likelihood analysis with a molecular clock places it elsewhere. Despite lineage-specific rate differences, the clock model was not excluded by a likelihood ratio test. Morphological analyses were unrooted. All analyses identified three clades, two of them generally well-supported. One well-supported clade (BCG) unites Bathycrinus and Guillecrinus with the representative (chimaeric) comatulid in a derived position, suggesting that comatulids originated from a sessile, stalked ancestor. In this connection it is noted that because the comatulid centrodorsal ossicle originates ontogenetically from the column, it is not strictly correct to describe comatulids as unstalked crinoids. A second, uniformly well-supported clade contains members of the Isocrinida, while the third clade contains Gymnocrinus, a well-established member of the Cyrtocrinida, together with the problematic taxon Caledonicrinus, currently classified as a bourgueticrinid. Another cyrtocrinid, Holopus, joins this clade with only weak molecular, but strong morphological support. In one morphological analysis Proisocrinus is weakly attached to the isocrinid clade. Only an unusual, divergent 18S rDNA sequence was obtained from the morphologically strange cyrtocrinid Cyathidium. Although not analysed in detail, features of this sequence suggested that it may be a PCR artefact, so that the apparently basal position of this taxon requires confirmation. If not an artefact, Cyathidium either diverged from the crinoid stem much earlier than has been recognised hitherto (i.e., it may be a Palaeozoic relic), or it has an atypically high rate of molecular evolution.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by J.P. Thorpe, Port Erin     

Phylogeny of the order Zoantharia (Anthozoa,Hexacorallia) based on the mitochondrial ribosomal genes

Zoantharia (or Zoanthidea) is the third largest order of Hexacorallia, characterised by two rows of tentacles, one siphonoglyph and a colonial way of life. Current systematics of Zoantharia is based exclusively on morphology and follows the traditional division of the group into the two suborders Brachycnemina and Macrocnemina, each comprising several poorly defined genera and species. To resolve the phylogenetic relationships among Zoantharia, we have analysed the sequences of mitochondrial 16S and 12S rRNA genes obtained from 24 specimens, representing two suborders and eight genera. In view of our data, Brachycnemina appears as a monophyletic group diverging within the paraphyletic Macrocnemina. The macrocnemic genus Epizoanthus branches as the sister group to all other Zoantharia that are sampled. All examined genera are monophyletic, except Parazoanthus, which comprises several independently branching clades and individual sequences. Among Parazoanthus, some groups of species can be defined by particular insertion/deletion patterns in the DNA sequences. All these clades show specificity to a particular type of substrate such as sponges or hydrozoans. Substrate specificity is also observed in zoantharians living on gorgonians or anthipatharians, as in the genus Savalia (Gerardia). If confirmed by further studies, the substrate specificity could be used as reliable character for taxonomic identification of some Macrocnemina.     

Geographic clines and stepping-stone patterns detected along the East Pacific Rise in the vetigastropod Lepetodrilus elevatus reflect species crypticism

Three different molecular markers (i.e. seven allozyme loci, two nuclear gene loci and, mtCOI DNA sequences) were used to assess the genetic structure of the vent gastropod Lepetodrilus elevatus collected from three vent fields along the East Pacific Rise (13°N, 9°50′N and 17°S). While allozymes and nuclear loci suggested a strong stepping-stone pattern, a multivariate analysis performed on allozymic frequencies showed the presence of two distinct evolutionary lineages: the first situated in the north from 13°N to 9°50′N and the second in the south from 9°50′N to 17°S. The analysis of mitochondrial DNA sequences confirmed the separation of L. elevatus into two distinct clades with a divergence of 6.5%, which is consistent with the interspecific level of sequence variation in other vent species. A divergence time of 6–14 Mya was estimated between the two clades from previous clock calibrations. Our results suggest that these taxa followed an allopatric speciation between the northern and southern parts of the EPR with a recent demographic expansion of the southern clade to the north and a subsequent secondary contact (clade hybridisation). This speciation was probably reinforced by a habitat specialisation of the two cryptic species because the southern clade was mainly found associated with mussel-dominated communities and the northern clade with tubeworm-dominated communities. However, the analysis of shell morphology failed to separate the two cryptic species based on this sole criterion although they differed from Lepetodrilus elevatus galriftensis (Galapagos population) by a higher shell elevation. Within each clade, genetic differentiation was not related to the distance across populations and could be within vent field as important as between fields. While both clades appear to be in expansion since their speciation, significant excesses in heterozygotes suggest a very recent and local bottleneck at 17°S, probably due to massive site extinction in this region.     

Genus-specific associations of marine sponges with group I crenarchaeotes

Sponges have rich and diverse associated microbial communities, which may have important functions in their metabolism. A survey of the archaeal communities of 23 poriferan species, focusing on the family Axinellidae, was conducted over the period 2002–2004 using 16S rDNA gene libraries created with archaeal-specific primers. The 28S rDNA sequences of the sponge hosts were also obtained. Of 23 species, 19 showed evidence of archaeal communities from group C1a (marine group I; Crenarchaeota), with three of these also showing evidence of Archaea from group E2 (marine group II; Euryarchaeota). Within the Crenarchaeota, two strongly supported sponge-specific clades were identified corresponding to the sponge family Axinellidae, and a novel sponge clade denoted clade C. These findings suggest that these archaea have evolved closely with their sponge hosts and are likely to play an important role in their metabolism.

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Predominance of clade D <Emphasis Type="Italic">Symbiodinium</Emphasis> in shallow-water reef-building corals off Kish and Larak Islands (Persian Gulf,Iran)

Scleractinian coral species harbour communities of photosynthetic taxa of the genus Symbiodinium. As many as eight genetic clades (A, B, C, D, E, F, G and H) of Symbiodinium have been discovered using molecular biology. These clades may differ from each other in their physiology, and thus influence the ecological distribution and resilience of their host corals to environmental stresses. Corals of the Persian Gulf are normally subject to extreme environmental conditions including high salinity and seasonal variation in temperature. This study is the first to use molecular techniques to identify the Symbiodinium of the Iranian coral reefs to the level of phylogenetic clades. Samples of eight coral species were collected at two different depths from the eastern part of Kish Island in the northern Persian Gulf, and Larak Island in the Strait of Hormuz. Partial 28S nuclear ribosomal (nr) DNA of Symbiodinium (D1/D2 domains) were amplified by polymerase chain reaction (PCR). PCR products were analyzed using single stranded conformational polymorphism and phylogenetic analyses of the LSU DNA sequences from a subset of the samples. The results showed that Symbiodinium populations were generally uniform among and within the populations of eight coral species studied, and there are at least two clades of Symbiodinium from Kish and Larak islands. Clade D was detected from eight of the coral species while clade C was found in two of species only (one species hosted two clades simultaneously). The dominance of clade D might be explained by high temperatures or the extreme temperature variation, typical of the Persian Gulf. Publication of this article was held up owing to technical problems. The publisher apologizes sincerely for this lengthy delay.     

Genetic structure of round scad mackerel <Emphasis Type="Italic">Decapterus macrosoma</Emphasis> (Carangidae) in the Indo-Malay archipelago

Round scad mackerel sampled in 1995-1998 were analysed for genetic variation using mitochondrial-DNA and nuclear-DNA markers. Sequence variation for a fragment of the cytochrome b gene (mitochondrial), amplified by polymerase chain reaction, was screened across individuals using single strand conformation polymorphism (SSCP). Sequence analysis of all SSCP haplotypes indicated two mitochondrial clades separated by, on average, 2.3% nucleotide divergence. The geographic distribution of haplotypes was homogeneous (Weir and Cockerham's [^(q)] \hat \theta =-0.002). Also, no geographic heterogeneity was detected for length polymorphism of Intron 1 of the gene encoding aldolase B ([^(q)] \hat \theta =0.005). Although homogeneity in allele frequencies throughout the Indo-Malay archipelago conformed to the expectations for a widely distributed pelagic fish in a highly connected habitat, this was at variance with the sharp geographic structure previously uncovered in Indian scad mackerel, Decapterus russelli, a fish with life-history characteristics similar to D. macrosoma. A remarkable similarity, however, was the occurrence of two similarly distinct clades within each species, suggesting a common history of geographic isolation. Low sea levels in the Pleistocene might have caused the separation and vicariance of populations within both D. macrosoma and D. russelli. Subsequent genetic exchange between populations would then have erased allele-frequency differences at the cytochrome b and aldolase B loci in D. macrosoma while some barrier to gene flow was maintained in D. russelli.     

A systematic study of some Black Corals species (Antipatharia,Hexacorallia) based on rDNA internal transcribed spacers sequences

A phylogenetic analysis based on rDNA internal transcribed spacers (ITS) sequences was performed on 15 species of black corals assigned to the genera Antipathes, Stichopathes, Cirrhipathes, Rhipidipathes, Antipathella, Myriopathes and Cupressopathes recorded from the Messina Strait (Mediterranean Sea) and the Bunaken Marine Park (Celebes Sea, Indonesia). The phylogenetic analysis shows that the examined species are grouped in two main branches corresponding to the families Antipathidae-Aphanipathidae and Myriopathidae. While among the Myriopathidae species there is a very small genetic distance, the Antipathidae-Aphanipathidae clade shows a high degree of divergence between different genera. According to this study, the taxon Antipathes? sp. 3 characterised by a bush-like corallum without a well-defined axis, probably belongs to a new undescribed genus of the family Antipathidae.     

Development of clade-specific Symbiodinium primers for quantitative PCR (qPCR) and their application to detecting clade D symbionts in Caribbean corals

We developed quantitative PCR (qPCR) assays to distinguish each of the four clades (A–D) of dinoflagellate endosymbionts (genus Symbiodinium) commonly found in Caribbean corals. We applied these primer sets, which target portions of the multi-copy ribosomal DNA (rDNA) gene family, to assess the presence/absence of symbionts in clade D (as indicated by the detection of clade D DNA). We detected these symbionts in five of six Caribbean host species/genera (21% of samples analyzed, N = 10 of 47 colonies), from which clade D had rarely or never been observed. This suggests that Symbiodinium in clade D are present in a higher diversity of coral species than previously thought. This qPCR-based approach can improve our understanding of the total microbial diversity associated with corals, particularly in hosts thought to be relatively specific, and has many other potential applications for studies of coral reef ecology and conservation.