Going where traditional markers have not gone before: utility of and promise for RAD sequencing in marine invertebrate phylogeography and population genomics.

Characterization of large numbers of single-nucleotide polymorphisms (SNPs) throughout a genome has the power to refine the understanding of population demographic history and to identify genomic regions under selection in natural populations. To this end, population genomic approaches that harness the power of next-generation sequencing to understand the ecology and evolution of marine invertebrates represent a boon to test long-standing questions in marine biology and conservation. We employed restriction-site-associated DNA sequencing (RAD-seq) to identify SNPs in natural populations of the sea anemone Nematostella vectensis, an emerging cnidarian model with a broad geographic range in estuarine habitats in North and South America, and portions of England. We identified hundreds of SNP-containing tags in thousands of RAD loci from 30 barcoded individuals inhabiting four locations from Nova Scotia to South Carolina. Population genomic analyses using high-confidence SNPs resulted in a highly-resolved phylogeography, a result not achieved in previous studies using traditional markers. Plots of locus-specific FST against heterozygosity suggest that a majority of polymorphic sites are neutral, with a smaller proportion suggesting evidence for balancing selection. Loci inferred to be under balancing selection were mapped to the genome, where 90% were located in gene bodies, indicating potential targets of selection. The results from analyses with and without a reference genome supported similar conclusions, further highlighting RAD-seq as a method that can be efficiently applied to species lacking existing genomic resources. We discuss the utility of RAD-seq approaches in burgeoning Nematostella research as well as in other cnidarian species, particularly corals and jellyfishes, to determine phylogeographic relationships of populations and identify regions of the genome undergoing selection.

Spatial and temporal patterns of genetic variation in the widespread antitropical deep-sea coral Paragorgia arborea.

Numerous deep-sea species have apparent widespread and discontinuous distributions. Many of these are important foundation species, structuring hard-bottom benthic ecosystems. Theoretically, differences in the genetic composition of their populations vary geographically and with depth. Previous studies have examined the genetic diversity of some of these taxa in a regional context, suggesting that genetic differentiation does not occur at scales of discrete features such as seamounts or canyons, but at larger scales (e.g. ocean basins). However, to date, few studies have evaluated such diversity throughout the known distribution of a putative deep-sea species. We utilized sequences from seven mitochondrial gene regions and nuclear genetic variants of the deep-sea coral Paragorgia arborea in a phylogeographic context to examine the global patterns of genetic variation and their possible correlation with the spatial variables of geographic position and depth. We also examined the compatibility of this morphospecies with the genealogical-phylospecies concept by examining specimens collected worldwide. We show that the morphospecies P. arborea can be defined as a genealogical-phylospecies, in contrast to the hypothesis that P. arborea represents a cryptic species complex. Genetic variation is correlated with geographic location at the basin-scale level, but not with depth. Additionally, we present a phylogeographic hypothesis in which P. arborea originates from the North Pacific, followed by colonization of the Southern Hemisphere prior to migration to the North Atlantic. This hypothesis is consistent with the latest ocean circulation model for the Miocene.

Comparative phylogeography among hydrothermal vent species along the East Pacific Rise reveals vicariant processes and population expansion in the South.

The use of sequence polymorphism from individual mitochondrial genes to infer past demography has recently proved controversial because of the recurrence of selective sweeps acting over genes and the need for unlinked multilocus data sets. However, comparative analyses using several species for one gene and/or multiple genes for one species can serve as a test for potential selective effects and clarify our understanding of historical demographic effects. This study compares nucleotide polymorphisms in mitochondrial cytochrome oxidase I across seven deep-sea hydrothermal vent species that live along the volcanically active East Pacific Rise. Approximate Bayesian Computation (ABC) method, developed to trace shared vicariant events across species pairs, indicates the occurrence of two across species divergence times, and suggests that the present geographical patterns of genetic differentiation may be explained by two periods of significant population isolation. The oldest period dates back 11.6 Ma and is associated with the vent limpet Lepetodrilus elevatus, while the most recent period of isolation is 1.3 Ma, which apparently affected all species examined and coincides with a transition zone across the equator. Moreover, significant negative Tajima's D and star-like networks were observed for all southern lineages, suggesting that these lineages experienced a concomitant demographic and geographical expansion about 100 000-300 000 generations ago. This expansion may have initiated from a wave of range expansions during the secondary colonization of new sites along the Southern East Pacific Rise (founder effects below the equator) or recurrent bottleneck events because of the increase of eruptive phases associated with the higher spreading rates of the ridge in this region.

Development and characterization of 12 microsatellite markers from the deep-sea hydrothermal vent siboglinid Riftia pachyptila.

Ecological processes at deep-sea hydrothermal vents on fast-spreading mid-ocean ridges are punctuated by frequent physical disturbance, often accompanied by a high occurrence of population turnover. To persist through local extinction events, sessile invertebrate species living in these geologically and chemically dynamic habitats depend on larval dispersal. We characterized 12 polymorphic microsatellite loci from one such species, the siboglinid tubeworm Riftia pachyptila. All loci conformed to Hardy-Weinberg expectations without linkage (mean H(E)  = 0.9405, mean N(A)  = 20.25). These microsatellites are being employed in the investigation of spatial and temporal population genetic structure in the eastern Pacific Ocean.

A sea-floor spreading event captured by seismometers.

Two-thirds of Earth's surface is formed at mid-ocean ridges, yet sea-floor spreading events are poorly understood because they occur far beneath the ocean surface. At 9 degrees 50'N on the East Pacific Rise, ocean-bottom seismometers recently recorded the microearthquake character of a mid-ocean ridge eruption, including precursory activity. A gradual ramp-up in activity rates since seismic monitoring began at this site in October 2003 suggests that eruptions may be forecast in the fast-spreading environment. The pattern culminates in an intense but brief (approximately 6-hour) inferred diking event on 22 January 2006, followed by rapid tapering to markedly decreased levels of seismicity.

Biogeography and ecological setting of Indian Ocean hydrothermal vents.

Within the endemic invertebrate faunas of hydrothermal vents, five biogeographic provinces are recognized. Invertebrates at two Indian Ocean vent fields (Kairei and Edmond) belong to a sixth province, despite ecological settings and invertebrate-bacterial symbioses similar to those of both western Pacific and Atlantic vents. Most organisms found at these Indian Ocean vent fields have evolutionary affinities with western Pacific vent faunas, but a shrimp that ecologically dominates Indian Ocean vents closely resembles its Mid-Atlantic counterpart. These findings contribute to a global assessment of the biogeography of chemosynthetic faunas and indicate that the Indian Ocean vent community follows asymmetric assembly rules biased toward Pacific evolutionary alliances.

Chemical speciation drives hydrothermal vent ecology.

The physiology and biochemistry of many taxa inhabiting deep-sea hydrothermal vents have been elucidated; however, the physicochemical factors controlling the distribution of these organisms at a given vent site remain an enigma after 20 years of research. The chemical speciation of particular elements has been suggested as key to controlling biological community structure in these extreme aquatic environments. Implementation of electrochemical technology has allowed us to make in situ measurements of chemical speciation at vents located at the East Pacific Rise (9 degrees 50' N) and on a scale relevant to the biology. Here we report that significant differences in oxygen, iron and sulphur speciation strongly correlate with the distribution of specific taxa in different microhabitats. In higher temperature (> 30 degrees C) microhabitats, the appreciable formation of soluble iron-sulphide molecular clusters markedly reduces the availability of free H2S/HS- to vent (micro)organisms, thus controlling the available habitat.

Miocene radiation of deep-sea hydrothermal vent shrimp (Caridea: Bresiliidae): evidence from mitochondrial cytochrome oxidase subunit I.

The evolutionary history of deep-sea shrimp (Caridea: Bresiliidae) inhabiting deep-sea hydrothermal vent and hydrocarbon seep environments was assessed using the mitochondrial Cytochrome c Oxidase subunit I (COI) gene (600 bp). Phylogenetic analyses (parsimony, likelihood, and neighbor-joining) recovered three distinct clades (A, Rimicaris/Chorocaris/Opaepele; B, Alvinocaris; and C, Mirocaris) consistent with higher level taxonomy based on morphology. However, robust phylogenetic results suggested that Chorocaris is paraphyletic and that Mirocaris fortunata and M. keldyshi may not be genetically distinct. A Kishino-Hasegawa likelihood approach was used to test alternative phylogenetic hypotheses based on biogeography and morphology. Evolutionary relationships of vent-endemic shrimp species did not appear to be correlated either with their extant biogeographic distribution or with the history of sea floor spreading. Additionally, COI data suggested that these vent-endemic organisms are not remnants of a Mesozoic vent assemblage; instead, they radiated in the Miocene.

Molecular systematics of shrimp (Decapoda: Bresiliidae) from deep-sea hydrothermal vents, I: Enigmatic "small orange" shrimp from the Mid-Atlantic Ridge are juvenile Rimicaris exoculata.

Independent species descriptions of a "small orange" caridean shrimp found at deep-sea hydrothermal vents along the Mid-Atlantic Ridge have created the synonymous names Iorania concordia Vereshchaka 1996b and Rimicaris aurantiaca Martin et al. 1997. Our genetic analyses involving allozymes and mitochondrial DNA sequences reveal that the "small orange" shrimp described in these studies are a juvenile form of Rimicaris exoculata Williams and Rona, a species commonly found at these sites. In light of this result, we reconsider the life history and ecologic characteristics of juvenile and adult stages of Rimicaris exoculata.

Vestimentiferan on a whale fall.

Discovery of chemosynthetic communities associated with whale bones led to the hypothesis that whale falls may serve as stepping-stones for faunal dispersal between disjunct hydrothermal vents and cold seeps on the ocean floor (1). The initial observation was followed by a faunal inventory that revealed a diverse assemblage of microbes and invertebrates, supported by chemoautotrophic production, living in close proximity to whale remains (2, 3). To date, the conspicuous absence from whale falls of vestimentiferan tubeworms (a predominant constituent of eastern Pacific vent and seep habitats) has been a major objection to the stepping-stone hypothesis (4-5). We report the first evidence of a vestimentiferan tubeworm associated with a whale fall (Fig. 1). The tubeworm, Escarpia spicata, was identified by morphological criteria and DNA sequence data from a portion of the mitochondrial cytochrome oxidase C subunit I (COI) gene. Additionally, the bacterial endosymbiont in the tubeworm possessed a 16S rRNA gene that was similar to that of endosymbionts from vestimentiferans in sedimented cold-seep environments.

Ultrasound guided biopsy of nonpalpable and difficult to palpate thyroid masses.

We reviewed our experience with ultrasound-guided biopsies of masses of the thyroid gland that were either nonpalpable or difficult to localize by palpation to evaluate the technique and correlate the results. Thirty-two biopsies were performed upon 25 patients whose clinical presentations were palpable nodule (six patients), throat discomfort (two patients), postpartial thyroidectomy follow-up evaluation (two patients), incidental discovery of a mass--by ultrasound of the neck (two patients), roentgenogram of the chest (two patients), computed tomography of the chest (one patient) and during tracheostomy placement (one patient). Other presentations were eliminate infection (one patient), odynophagia (one patient), hoarseness (one patient), cold nodule on a nuclear medicine study (one patient), hyperparathyroidism (one patient), rule out metastasis from carcinoma of the colon (one patient), persistent cough (one patient), enlarged thyroid gland (one patient) or family history of carcinoma of the thyroid gland (one patient). Fifteen patients had nuclear medicine studies showing either a cold nodule (ten patients), multinodular goiter (one patient), normal examination (two patients), hot nodule (one patient) or no thyroid gland activity (one patient). The ultrasound examinations showed either a hypoechoic nodule (25 patients), inhomogeneous or mixed echogenic nodule (six patients) or a hyperechoic nodule with hypoechoic rim (one patient). The nodules ranged in size from 3 milliliters to 7 centimeters. Twenty-six lesions were less than 3 centimeters in diameter; of the other six, four were substernal goiters. Six patients had a previous nondiagnostic biopsy directed by palpation only. Biopsy was performed using real-time ultrasound guidance with various needles. One patient had a small hematoma, which was the only complication in the study. The results of the biopsies were diagnostic in 26 of 32 patients. The final diagnosis was benign follicular cells (ten patients), adenomatous nodule (seven patients), follicular neoplasm (three patients), colloid cyst (two patients), aspergillus (two patients), fibrosis (one patient) and papillary carcinoma (one patient). Six of the biopsies yielded unsatisfactory specimens. One of the patients with a diagnosis of benign follicular cells on biopsy had a follicular carcinoma after surgical pathologic factors were obtained; that was the only false-negative result. We conclude that ultrasound-guided biopsy of the thyroid is a safe and useful method of evaluating nonpalpable and difficult to palpate thyroid masses.

Detection and comparison of DNA adducts after in vitro and in vivo diesel emission exposures.

Development of methods to evaluate certain classes of polycyclic aromatic compounds (PAC) detected in complex mixtures to which humans are exposed would greatly improve the diagnostic potential of 32P-postlabeling analysis. Identification of DNA adduct patterns or specific exposure-related marker adducts would strengthen associations between observed DNA adducts and exposures to different environmental pollutants (e.g., kerosene, cigarette smoke, coke oven, and diesel). We have compared diesel-modified DNA adduct patterns in various in vitro and in vivo rodent model systems and compared them to DNA reactive oxidative and reductive metabolites of 1-nitropyrene. The formation of nitrated polycyclic aromatic hydrocarbon (nitrated PAH) DNA adducts, derived from the metabolism of diesel extract constituents, was enhanced relative to other PAH-derived DNA adducts via xanthine oxidase-catalyzed nitroreduction. These adducts were detectable only by the butanol extraction version of the postlabeling analysis. Five major DNA adducts were detected in human lymphocytes treated in vitro with diesel extract. A major adduct detected in human lymphocytes treated in vitro with diesel extract comigrated with a major adduct detected in lymphocyte DNA treated with benzo[a]pyrene (BaP) alone. Other adducts that co-migrated with the major BaP-derived adducts were detected in skin and lung DNA isolated from rodents topically treated with (50 mg) diesel extract and the major adduct detected in calf thymus DNA treated with rat liver S9 and diesel particle extract. Postlabeling of lung DNA isolated from rodents exposed via lung inhalation for 24 months to diesel combustion emissions resulted in the formation of a major nuclease-P1-sensitive DNA adduct that did not co-migrate with the major BaP-diol epoxide adduct.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA adduct profiles and levels in placenta, blood and lung in relation to cigarette smoking and smoky coal emissions.

Tobacco smoking and indoor smoky coal combustion emissions lead to exposures to complex environmental mixtures that have been associated with increased lung cancer mortality rates in the USA and Xuan Wei County, China, respectively. Human exposures to benzo[a[pyrene (BaP) present in smoky coal emissions are 20-200 times greater than exposure to BaP from smoking 1-2 packs of cigarettes per day. We compared DNA adducts resulting from cigarette smoking and coal smoke exposures in blood cells, placental syncytial nuclei, whole placental tissue homogenates and lung cells. Postlabelling analysis of DNA isolated from placental tissue of smokers revealed multiple adducts as a diagonal radioactive zone. DNA adduct levels for both white blood cells (WBC) and lymphocyte DNA were approximately 2.5-fold higher in smokers than in non-smokers. One smoking-related adduct was detected in both WBC and lymphocyte DNA, but no exposure-related adducts were detected in either WBC DNA or placental tissue DNA isolated from coal smoke-exposed individuals, despite higher exposure to BaP and aromatic organics. In contrast, discrete adducts unrelated to smoking status were detected, by 32P-postlabelling analysis, in DNA from syncytial nuclei isolated from placental villus samples from smokers and non-smokers. One major placental syncytial adduct co-migrated with a major BaP-derived DNA adduct. DNA adduct levels were approximately four-fold higher in bronchoalveolar lavage (BAL) cells from coal smoke-exposed individuals compared to unexposed individuals and suggest that DNA-reactive intermediates concentrate in the respiratory tract and may not be detectable in whole WBC or placental tissue homogenates. Our findings also suggest that the distribution and concentration of placental DNA adducts may be dependent on the site at which the placenta is sampled.

Relative sensitivity of 32P-postlabelling of DNA and the autoradiographic UDS assay in the liver of rats exposed to 2-acetylaminofluorene (2AAF).

Groups of male Alderley Park rats were dosed concomitantly with 2-acetylaminofluorene (2AAF) by gavage at doses between 0.01 mg/kg and 40 mg/kg, and livers sampled 2-72 h later. The liver of one group of animals was perfused to yield hepatocytes which were assayed in vitro for unscheduled DNA synthesis (UDS) via incorporation of tritiated thymidine and autoradiography. DNA was extracted from the livers of the other group and DNA adduct levels determined using the 32P-postlabelling technique. The major C-8 2-aminofluorene/guanosine adduct and 3 minor adducts were quantitated, enabling the relative sensitivity of the 2 techniques to be compared. A dose- and time-related UDS response was observed, which, at the most sensitive time-point (12 h) enabled DNA repair to be discerned at a dose level of 0.1-1 mg/kg of 2AAF, a response classified as formally positive at 5 mg/kg 2AAF. Only the C-8 adduct, as determined by 32P-postlabelling, was discernible at 0.01 mg/kg of 2AAF, although other adducts were visible on autoradiograms at higher dose levels. It is concluded that as part of a well-defined dose response, UDS can be discerned with confidence for doses of 2AAF between approximately 0.1 and 5 mg/kg, and DNA adducts for doses of 2AAF between approximately 0.01 and 1 mg/kg. Discernible UDS for 2AAF in the rat liver is apparent at approximately 13 DNA (total) adducts/10(8) nucleotides, or approximately 8 DNA (C-8) adducts/10(8) nucleotides. The presumed C-8 2-acetylaminofluorene/guanosine adduct, prepared by reaction of 2-acetoxy-2-acetylaminofluorene (2AAAF) with DNA, was a significant but unreliable marker of 2AAF/DNA adducts in the rat liver in vivo. DNA repair did not appear to remove DNA adducts selectively, and adducts remained in DNA when discernible DNA repair had ceased.

Relative sensitivity of 32P-postlabelling of DNA and the autoradiographic UDS assay in the liver of mice exposed to 2-acetylaminofluorene (2AAF).

In contrast to earlier studies conducted at lower dose levels, 2AAF is shown to induce a positive UDS response in the liver of mice dosed orally at dose levels between 500 and 1000 mg/kg. Similarly exposed mice had low levels of 2AAF-related hepatic DNA adducts at dose levels in the range 10-1000 mg/kg 2AAF, as determined by 32P-postlabelling analysis. It is concluded that the attenuated UDS response observed in the mouse liver, as compared to the rat liver, is due primarily to metabolic differences between these two species, coupled to a reduced capacity for UDS in the mouse liver for a given level of total 2AAF-related adducts per unit of DNA. These observations are compared and contrasted with identical studies conducted in the rat and reported in the preceding paper (Gallagher et al., 1991).