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2017
Hansman, RL, Thurber AR, Levin LA, Aluwihare LI.  2017.  Methane fates in the benthos and water column at cold seep sites along the continental margin of Central and North America. Deep-Sea Research Part I-Oceanographic Research Papers. 120:122-131.   10.1016/j.dsr.2016.12.016   AbstractWebsite

The potential influence of methane seeps on carbon cycling is a key question for global assessments, but the study of carbon cycling in surface sediments and the water column of cold seep environments is complicated by the high temporal and spatial variability of fluid and gas fluxes at these sites. In this study we directly examined carbon sources supporting benthic and planktonic food webs at venting methane seeps using isotopic and molecular approaches that integrate this variability. At four seep environments located along North and Central America, microorganisms from two size fractions were collected over several days from 2800 to 90501 of seawater to provide a time-integrated measure of key microbial groups and the carbon sources supporting the overall planktonic microbial community. In addition to water column measurements, the extent of seafloor methane release was estimated at two of the sites by examining the stable carbon isotopic signature (delta C-13) of benthic metazoan infauna. This signature reveals carbon sources fueling the base of the food chain and thus provides a metric that represents a time-integrated view of the dominant microbial processes within the sediment. The stable carbon isotopic composition of microbial DNA (delta C-13-DNA), which had values between -17.0 and -19.5%(0), indicated that bulk planktonic microbial production was not ultimately linked to methane or other C-13-depleted seep-derived carbon sources. Instead these data support the importance of organic carbon derived from either photo- or chemoautotrophic CO2 fixation to the planktonic food web. Results of qPCR of microbial DNA sequences coding for a subunit of the particulate methane monooxygenase gene (pmoA) showed that only a small percentage of the planktonic microbial community were potential methane oxidizers possessing pmoA (< 5% of 16S rRNA gene copies). There was an overall decrease of C-13-depleted carbon fueling the benthic metazoan community from 3 to 5 cm below the seafloor to the sediment surface, reflecting limited use of isotopically depleted carbon at the sediment surface. Rare methane emission as indicated by limited aerobic methane oxidation acts to corroborate our findings for the planktonic microbial community.

2016
Pasulka, AL, Levin LA, Steele JA, Case DH, Landry MR, Orphan VJ.  2016.  Microbial eukaryotic distributions and diversity patterns in a deep-sea methane seep ecosystem. Environmental Microbiology. 18:3022-3043.   10.1111/1462-2920.13185   AbstractWebsite

Although chemosynthetic ecosystems are known to support diverse assemblages of microorganisms, the ecological and environmental factors that structure microbial eukaryotes (heterotrophic protists and fungi) are poorly characterized. In this study, we examined the geographic, geochemical and ecological factors that influence microbial eukaryotic composition and distribution patterns within Hydrate Ridge, a methane seep ecosystem off the coast of Oregon using a combination of high-throughput 18S rRNA tag sequencing, terminal restriction fragment length polymorphism fingerprinting, and cloning and sequencing of full-length 18S rRNA genes. Microbial eukaryotic composition and diversity varied as a function of substrate (carbonate versus sediment), activity (low activity versus active seep sites), sulfide concentration, and region (North versus South Hydrate Ridge). Sulfide concentration was correlated with changes in microbial eukaryotic composition and richness. This work also revealed the influence of oxygen content in the overlying water column and water depth on microbial eukaryotic composition and diversity, and identified distinct patterns from those previously observed for bacteria, archaea and macrofauna in methane seep ecosystems. Characterizing the structure of microbial eukaryotic communities in response to environmental variability is a key step towards understanding if and how microbial eukaryotes influence seep ecosystem structure and function.

2015
Case, DH, Pasulka AL, Marlow JJ, Grupe BM, Levin LA, Orphan VJ.  2015.  Methane seep carbonates host distinct, diverse, and dynamic microbial assemblages. Mbio. 6   10.1128/mBio.01348-15   AbstractWebsite

Marine methane seeps are globally distributed geologic features in which reduced fluids, including methane, are advected upward from the subsurface. As a result of alkalinity generation during sulfate-coupled methane oxidation, authigenic carbonates form slabs, nodules, and extensive pavements. These carbonates shape the landscape within methane seeps, persist long after methane flux is diminished, and in some cases are incorporated into the geologic record. In this study, microbial assemblages from 134 native and experimental samples across 5,500 km, representing a range of habitat substrates (carbonate nodules and slabs, sediment, bottom water, and wood) and seepage conditions (active and low activity), were analyzed to address two fundamental questions of seep microbial ecology: (i) whether carbonates host distinct microbial assemblages and (ii) how sensitive microbial assemblages are to habitat substrate type and temporal shifts in methane seepage flux. Through massively parallel 16S rRNA gene sequencing and statistical analysis, native carbonates are shown to be reservoirs of distinct and highly diverse seep microbial assemblages. Unique coupled transplantation and colonization experiments on the seafloor demonstrated that carbonate-associated microbial assemblages are resilient to seep quiescence and reactive to seep activation over 13 months. Various rates of response to simulated seep quiescence and activation are observed among similar phylogenies (e.g., Chloroflexi operational taxonomic units) and similar metabolisms (e.g., putative S oxidizers), demonstrating the wide range of microbial sensitivity to changes in seepage flux. These results imply that carbonates do not passively record a time-integrated history of seep microorganisms but rather host distinct, diverse, and dynamic microbial assemblages. IMPORTANCE Since their discovery in 1984, the global distribution and importance of marine methane seeps have become increasingly clear. Much of our understanding of methane seep microorganisms-from metabolisms to community ecology-has stemmed from detailed studies of seep sediments. However, it has become apparent that carbonates represent a volumetrically significant habitat substrate at methane seeps. Through combined in situ characterization and incubation experiments, this study demonstrates that carbonates host microbial assemblages distinct from and more diverse than those of other seep habitats. This emphasizes the importance of seep carbonates as biodiversity locales. Furthermore, we demonstrate that carbonate-associated microbial assemblages are well adapted to withstand fluctuations in methane seepage, and we gain novel insight into particular taxa that are responsive (or recalcitrant) to changes in seep conditions.

Burkett, AM, Rathburn AE, Perez ME, Levin LA, Cha H, Rouse GW.  2015.  Phylogenetic placement of Cibicidoides wuellerstorfi (Schwager, 1866) from methane seeps and non-seep habitats on the Pacific margin. Geobiology. 13:44-52.   10.1111/gbi.12118   AbstractWebsite

Benthic foraminifera are among the most abundant groups found in deep-sea habitats, including methane seep environments. Unlike many groups, no endemic foraminiferal species have been reported from methane seeps, and to our knowledge, genetic data are currently sparse for Pacific deep-sea foraminifera. In an effort to understand the relationships between seep and non-seep populations of the deep-sea foraminifera Cibicidoides wuellerstorfi, a common paleo-indicator species, specimens from methane seeps in the Pacific were analyzed and compared to one another for genetic similarities of small subunit rDNA (SSU rDNA) sequences. Pacific Ocean C. wuellerstorfi were also compared to those collected from other localities around the world (based on 18S gene available on Genbank, e.g., Schweizer et al., 2009). Results from this study revealed that C. wuellerstorfi living in seeps near Costa Rica and Hydrate Ridge are genetically similar to one another at the species level. Individuals collected from the same location that display opposite coiling directions (dextral and sinstral) had no species level genetic differences. Comparisons of specimens with genetic information available from Genbank (SSU rDNA) showed that Pacific individuals, collected for this study, are genetically similar to those previously analyzed from the North Atlantic and Antarctic. These observations provide strong evidence for the true cosmopolitan nature of C. wuellerstorfi and highlight the importance of understanding how these microscopic organisms are able to maintain sufficient genetic exchange to remain within the same species between seep and non-seep habitats and over global distances.

2013
Levin, LA, Ziebis W, Mendoza GF, Bertics VJ, Washington T, Gonzalez J, Thurber AR, Ebbed B, Lee RW.  2013.  Ecological release and niche partitioning under stress: Lessons from dorvilleid polychaetes in sulfidic sediments at methane seeps. Deep-Sea Research Part Ii-Topical Studies in Oceanography. 92:214-233.   10.1016/j.dsr2.2013.02.006   AbstractWebsite

Organisms inhabiting methane seep sediments are exposed to stress in the form of high levels of hydrogen sulfide, which result mainly from sulfate reduction coupled to anaerobic methane oxidation. Dorvilleidae (Polychaeta) have successfully invaded this ecosystem, and multiple species in divergent genetic clades co-occur at high densities. At methane seeps in the NE Pacific off California and Oregon, the genera Ophryotrocha, Parougia and Exallopus are especially well represented. To test the hypothesis that dorvilleid coexistence is facilitated by niche partitioning through sulfide tolerance and trophic patterns, we examined dorvilleid species-specific patterns of occurrence and nutrition at methane seeps off Eel R. [ER] on the Californian continental slope and at Hydrate Ridge [HR] on the Oregon continental slope, and in two habitats (clam bed and microbial mat) characterized by lower and higher hydrogen sulfide levels, respectively. Microelectrode measurements of hydrogen sulfide enabled characterization of environmental sulfide levels for species sampled in background sediment cores and in colonization trays. Dorvilleids tolerated H2S levels from 10 mu M to over 2.6 mM, with the majority of species inhabiting sediments with similar environmental H2S concentrations (median 85-100 mu M). Dorvilleid species richness was greater at HR than ER, but did not differ between clam bed and microbial mat habitats. Species distribution patterns reflected preferences for ER clam bed (lower sulfide levels), ER mat and HR clam bed (moderate sulfide levels), or HR mat (very high sulfide levels). Nutritional patterns, including trophic diversity and functional similarity, were examined using community stable isotope metrics based on delta N-15 and delta C-13. Within each region, dorvilleid species exhibited multiple trophic strategies. Co-existing congeners typically exhibited distinct isotope signatures, suggesting trophic partitioning. Trophic diversity and delta N-15 range for whole assemblages (measured by Total Hull Area and Standard Elliptical Area using species averages) and functional redundancy or species packing (measured as distance to nearest neighbor) among species and individuals were generally higher at ER, where sulfide levels were lower than at HR. In contrast, average trophic diversity among individuals within a species was greater at HR than ER. In colonization experiments involving agar-based manipulations of sulfide in tray sediments that mimicked clam bed and mat conditions, dorvilleids comprised 68% and 48% of colonists at ER and HR, respectively. Dorvilleid species richness was higher in trays that were initially more sulfidic. However, habitat exerted stronger influence on the composition of colonizing dorvilleids than did sulfide additions. In the NE Pacific, regional, habitat and vertical (down-core) variation in hydrogen sulfide creates complex environmental heterogeneity at methane seeps, promoting high diversity of stress-tolerant taxa such as dorvilleid polychaetes. (C) 2013 Elsevier Ltd. All rights reserved.

2012
Thurber, AR, Levin LA, Orphan VJ, Marlow JJ.  2012.  Archaea in metazoan diets: implications for food webs and biogeochemical cycling. ISME Journal. 6:1602-1612.   10.1038/ismej.2012.16   AbstractWebsite

Although the importance of trophic linkages, including 'top-down forcing', on energy flow and ecosystem productivity is recognized, the influence of metazoan grazing on Archaea and the biogeochemical processes that they mediate is unknown. Here, we test if: (1) Archaea provide a food source sufficient to allow metazoan fauna to complete their life cycle; (2) neutral lipid biomarkers (including crocetane) can be used to identify Archaea consumers; and (3) archaeal aggregates are a dietary source for methane seep metazoans. In the laboratory, we demonstrated that a dorvilleid polychaete, Ophryotrocha labronica, can complete its life cycle on two strains of Euryarchaeota with the same growth rate as when fed bacterial and eukaryotic food. Archaea were therefore confirmed as a digestible and nutritious food source sufficient to sustain metazoan populations. Both strains of Euryarchaeota used as food sources had unique lipids that were not incorporated into O. labronica tissues. At methane seeps, sulfate-reducing bacteria that form aggregations and live syntrophically with anaerobic-methane oxidizing Archaea contain isotopically and structurally unique fatty acids (FAs). These biomarkers were incorporated into tissues of an endolithofaunal dorvilleid polychaete species from Costa Rica (mean bulk delta C-13 = -92 +/- 4 parts per thousand; polar lipids -116 parts per thousand) documenting consumption of archaeal-bacterial aggregates. FA composition of additional soft-sediment methane seep species from Oregon and California provided evidence that consumption of archaeal-bacterial aggregates is widespread at methane seeps. This work is the first to show that Archaea are consumed by heterotrophic metazoans, a trophic process we coin as 'archivory'. The ISME Journal (2012) 6, 1602-1612; doi:10.1038/ismej.2012.16; published online 8 March 2012

2010
Levin, LA, Mendoza GF, Gonzalez JP, Thurber AR, Cordes EE.  2010.  Diversity of bathyal macrofauna on the northeastern Pacific margin: the influence of methane seeps and oxygen minimum zones. Marine Ecology-an Evolutionary Perspective. 31:94-110.   10.1111/j.1439-0485.2009.00335.x   AbstractWebsite

The upper continental slope in the northeastern Pacific Ocean is intercepted by a deep oxygen minimum zone (OMZ; 650-1100 m) and punctuated by conduits of methane seepage. We examined the effects of these two dominant sources of heterogeneity on the density, composition and diversity of heterotrophic macrofauna off Hydrate Ridge, Oregon (OR; 800 m water depth), where the seeps co-occur within an OMZ, and off the Eel River, Northern California (CA; 500 m), where seeps are overlain by better oxygenated waters. We hypothesized that seeps (containing clam beds and microbial mats) should contribute a suite of distinct species to the regional margin species pool but that OMZ-associated hypoxia would dampen seep-related heterogeneity. Macrofaunal densities were highest (23,000-33,510 ind.m(-2)) in the CA seep sediments and in the OR near-seep samples, intermediate in the OR seep, CA near seep and CA and OR 500-m margin sediments (10,05419,777 ind.m(-2)), and lowest in the CA and OR OMZ habitats at 800 m (42697847 ind.m(-2)). Annelids constituted over 50% of the taxa in all but the CA clam bed and OR microbial mat sediments, where mollusks were abundant. Approximately 50% of seep species appeared to be habitat endemic; species present in microbial mats largely formed a subset of those present in the clam beds. Dorvilleid and ampharetid polychaetes were dominant in the seep sediments; non-seep margin sediments at 500 and 800 m were populated heavily by branckiate polychaetes including cossurids and paraonids. Alpha diversity (Es[20] calculated per core) was lowest and rank 1 dominance was highest in the CA and OR microbial mat habitats. Pooled analyses of Es[100] revealed highest species richness in the CA clam bed and near-seep habitats (30.3 and 29.6, respectively), and lowest species richness in the OR microbial mat and near-seep habitats (16.5 and 17.9, respectively). Non-seep sediments (500 and 800 m) off both CA and OR were more homogeneous (55-57% within-habitat similarity) than clam bed and microbial mat sediments (only 32-37% within-habitat similarity). CA sediment macrofauna generally exhibit higher alpha diversity, and as habitats are combined, a higher rate of increase in the slope of the species accumulation curves than do OR margin macrofauna. Methane seeps in the NE Pacific introduce significant heterogeneity that increases margin biodiversity at multiple spatial scales. However, our hypothesis that the OMZ would lessen the seep contributions to diversity was not supported. The better oxygenated CA seeps at 500 in shared more of the background margin fauna (at 500 m) than did the OR seeps at 800 m (with OMZ fauna at 800 in). Geographical differences in the fluxes of methane-rich fluids and the increased reliance on chemosynthetic food sources with increased depth could explain these results.

2007
Levin, LA, Mendoza GF.  2007.  Community structure and nutrition of deep methane-seep macrobenthos from the North Pacific (Aleutian) Margin and the Gulf of Mexico (Florida Escarpment). Marine Ecology-an Evolutionary Perspective. 28:131-151.   10.1111/j.1439-0485.2006.00131.x   AbstractWebsite

Methane seeps occur at depths extending to over 7000 m along the world's continental margins, but there is little information about the infaunal communities inhabiting sediments of seeps deeper than 3000 m. Biological sampling was carried out off Unimak Island (3200-3300 m) and Kodiak Island (4500 m) on the Aleutian margin, Pacific Ocean and along the Florida Escarpment (3300 m) in the Gulf of Mexico to investigate the community structure and nutrition of macrofauna at these sites. We addressed whether there are characteristic infaunal communities common to the deep-water seeps or to the specific habitats (clam beds, pogonophoran fields, and microbial mats) studied here, and ask how these differ from background communities or from shallow-seep settings sampled previously. We also investigated, using stable isotopic signatures, the utilization of chemosynthetically fixed and methane-derived organic matter by macrofauna from different regions and habitats. Within seep sites, macrofaunal densities were the greatest in the Florida microbial mats (20,961 +/- 11,618 ind(.)m(2)), the lowest in the Florida pogonophoran fields (926 +/- 132 ind(.)m(2)), and intermediate in the Unimak and Kodiak seep habitats. Seep macrofaunal densities differed from those in nearby non-seep sediments only in Florida mat habitats, where a single, abundant species of hesionid polychaete comprised 70% of the macrofauna. Annelids were the dominant taxon (> 60%) at all sites and habitats except in Florida background sediments (33%) and Unimak pogonophoran fields (27%). Macrofaunal diversity (H') was lower at the Florida than the Alaska seeps, with a trend toward reduced richness in clam bed relative to pogonophoran field or non-seep sediments. Community composition differences between seep and non-seep sediments were evident in each region except for the Unimak margin, but pogonophoran and clam bed macrofaunal communities did not differ from one another in Alaska. Seep VC and delta N-15 signatures were lighter for seep than non-seep macrofauna in all regions, indicating use of chemosynthetically derived carbon. The lightest delta C-13 values (average of species' means) were observed at the Florida escarpment (-42.87 parts per thousand). We estimated that on average animal tissues had up to 55% methane-derived carbon in Florida mats, 3144% in Florida clam beds and Kodiak clam beds and pogonophoran fields, and 9-23% in Unimak seep habitats. However, some taxa such as hesionid and capitellid polychaetes exhibited tremendous intraspecific 613C variation (> 307.0) between patch types. Overall we found few characteristic communities or features common to the three deep-water seeps (> 3000 m), but common properties across habitats (mat, clam bed, pogonophorans), independent of location or water depth. In general, macrofaunal densities were lower (except at Florida microbial mats), community structure was similar, and reliance on chemosynthesis was greater than observed in shallower seeps off California and Oregon.

2006
Levin, LA, Ziebis W, Mendoza GF, Growney-Cannon V, Walther S.  2006.  Recruitment response of methane-seep macrofauna to sulfide-rich sediments: An in situ experiment. Journal of Experimental Marine Biology and Ecology. 330:132-150.   10.1016/j.jembe.2005.12.022   AbstractWebsite

Hydrodynamically unbiased colonization trays were deployed for 6 months (Oct. 2000 to April 2001) on the northern California margin (Eel R. region; 525 m) to examine macrofaunal colonization rates at methane seeps. The influence of sulfide on recruitment and survival was examined by deploying sediments with and without sulfide added; effect of seep proximity was evaluated by placing trays inside and outside seeps. The trays contained a two-layer system mimicking vesicomyid clam bed habitat geochemistry, with 89 9 mM sulfide in a lower agar layer at the start of the experiment. After 6 month on the seabed, the lower agar layer contained 2-4 mM H2S. We observed rapid macrofaunal colonization equivalent to 50% of initial non-seep ambient densities. There was no difference in total colonizer densities, number of species, or rarefaction diversity among 3 treatments: (1) controls (no sulfide added) placed outside seeps, (2) trays with sulfide added placed outside seeps and (3) trays with sulfide added placed inside seep patches. Colonization trays with sulfide placed at seeps had different species composition from trays without sulfide place outside seeps; there were more amphipods (non-ampeliscid) and cumaceans in the seep/sulfide treatment and more nemerteans, Nephtys cornuta and tanaids in the non-seep/no-sulfide treatment. Outside seeps, annelids comprised <15% of tray colonists; within seep patches, annelids comprised 5 of the top 10 dominant colonizing taxa (24% of the total). The polychaetes Mediomastus sp., Aphelochaeta sp., Paraonidae sp., and Nerillidae sp. exhibited significantly higher densities in sulfide additions. Tanaids, echinoderms, and N. cornuta exhibited sulfide avoidance. At least 6 dorvilleid polychaete species colonized the experiments. Of these, 4 species occurred exclusively in trays with sulfide added and 80% of all dorvilleid individuals were found in trays with sulfide placed inside seep sediments. Counts of large sulfur bacterial filaments were positively correlated with maximum sulfide concentration in each tray, and with proximity of sulfide to the sediment surface. However, total macrofaunal densities were not correlated with tray sulfide concentrations. As a group, tray assemblages achieved some but not all characteristics of ambient seep assemblages after 6-month exposure on the sea floor. Distinctive colonization patterns at methane seeps contribute to the dynamic mosaic of habitat patches that characterize the eastern Pacific continental margin. Overall, proximity of seep habitats had at least as great an influence on macrofaunal colonization as tray sulfide concentrations. Taxa characteristic of seep sediments were more likely to settle into trays placed inside rather than outside seep patches. Whether this is due to limited dispersal ability or local geochemical cues remains to be determined. (C) 2005 Elsevier B.V. All rights reserved.