Larvae from deep-sea methane seeps disperse in surface waters

Many species endemic to deep-sea methane seeps have broad geographical distributions, suggesting that they produce larvae with at least episodic long-distance dispersal. Cold-seep communities on both sides of the Atlantic share species or species complexes, yet larval dispersal across the Atlantic is expected to take prohibitively long at adult depths. Here, we provide direct evidence that the long-lived larvae of two cold-seep molluscs migrate hundreds of metres above the ocean floor, allowing them to take advantage of faster surface currents that may facilitate long-distance dispersal. We collected larvae of the ubiquitous seep mussel “Bathymodiolus” childressi and an associated gastropod, Bathynerita naticoidea, using remote-control plankton nets towed in the euphotic zone of the Gulf of Mexico. The timing of collections suggested that the larvae might disperse in the water column for more than a year, where they feed and grow to more than triple their original sizes. Ontogenetic vertical migration during a long larval life suggests teleplanic dispersal, a plausible explanation for the amphi-Atlantic distribution of “B.” mauritanicus and the broad western Atlantic distribution of B. naticoidea. These are the first empirical data to demonstrate a biological mechanism that might explain the genetic similarities between eastern and western Atlantic seep fauna.     

Twelve microsatellites for two deep sea polychaete tubeworm species,Lamellibrachia luymesi and Seepiophila jonesi,from the Gulf of Mexico

The vestimentiferan tubeworms Lamellibrachia luymesi and Seepiophila jonesi are found at hydrocarbon seeps in the Gulf of Mexico. Primers for polymorphic microsatellite loci were developed from genomic libraries of L. luymesi (five loci) and from S. jonesi tissue (eight loci) and were used to screen individuals collected from nine northern Gulf of Mexico hydrocarbon seep sites. Loci had from four to more than 50 alleles with high expected levels of heterozygosity. Cross‐species amplification, tested on seven vestimentiferan species including both hydrothermal vent and cold seep species, was generally strong in similar species but weak in more genetically distant species.     

High connectivity across the fragmented chemosynthetic ecosystems of the deep Atlantic Equatorial Belt: efficient dispersal mechanisms or questionable endemism?

Chemosynthetic ecosystems are distributed worldwide in fragmented habitats harbouring seemingly highly specialized communities. Yet, shared taxa have been reported from highly distant chemosynthetic communities. These habitats are distributed in distinct biogeographical regions, one of these being the so‐called Atlantic Equatorial Belt (AEB). Here, we combined genetic data (COI) from several taxa to assess the possible existence of cryptic or synonymous species and to detect the possible occurrence of contemporary gene flow among populations of chemosynthetic species located on both sides of the Atlantic. Several Evolutionary Significant Units (ESUs) of Alvinocarididae shrimp and Vesicomyidae bivalves were found to be shared across seeps of the AEB. Some were also common to hydrothermal vent communities of the Mid‐Atlantic Ridge (MAR), encompassing taxa morphologically described as distinct species or even genera. The hypothesis of current or very recent large‐scale gene flow among seeps and vents was supported by microsatellite analysis of the shrimp species Alvinocaris muricola/Alvinocaris markensis across the AEB and MAR. Two nonmutually exclusive hypotheses may explain these findings. The dispersion of larvae or adults following strong deep‐sea currents, possibly combined with biochemical cues influencing the duration of larval development and timing of metamorphosis, may result in large‐scale effective migration among distant spots scattered on the oceanic seafloor. Alternatively, these results may arise from the prevailing lack of knowledge on the ocean seabed, apart from emblematic ecosystems (chemosynthetic ecosystems, coral reefs or seamounts), where the widespread classification of endemism associated with many chemosynthetic taxa might hide wider distributions in overlooked parts of the deep sea.     

Community structure of vestimentiferan-generated habitat islands from Gulf of Mexico cold seeps

Biologically generated structures create habitat and influence the distribution and abundance of species in many marine systems. In the rather monotonous and nutrient-poor environment of the deep-sea, cold seep environments and their associated chemosynthetic communities offer islands of primary production and habitat to a generally sparsely distributed macrofauna. In this study, we investigate the structure of macrofaunal assemblages associated with vestimentiferan aggregations on the upper Louisiana slope of the Gulf of Mexico and the relationships between assemblage composition and the size and complexity of the vestimentiferan-generated habitat. Using custom-designed and custom-built devices, we collected seven whole vestimentiferan aggregations along with their associated fauna during the summers of 1997 and 1998. Sixty-five species were found associated with the four vestimentiferan aggregations collected in 1998, more than doubling the number of species previously reported for seeps in this region. Individual aggregations contained between 23 and 44 different non-vestimentiferan species. General trends of increasing species richness with increasing habitat size and increasing faunal density with increasing habitat complexity were identified, but substantial variability suggested other factors also control the composition of faunal associates. Faunal abundances decreased with increasing aggregation age. Seep endemics dominated the communities of younger aggregations, but non-endemic species dominated communities of older aggregations. Relative dominance of the heterotrophic community by primary consumers decreased, while predatory secondary and higher-order consumers increased with increasing aggregation age. These trends are discussed in terms of successional changes in aggregation structure, habitat heterogeneity and environmental conditions.     

Long-term history of chemoautotrophic clam-dominated faunas of petroleum seeps in the Northwestern Gulf of Mexico

Summary Chemoautotrophic clam-dominated assemblages are commonly associated with petroleum seepage on the continental slope of the Gulf of Mexico. We examine the persistence and resilence of these communities by evaluating downcore trends in abundance, biomass, and trophodynamics in communities from four separate petroleum seep sites on the Louisiana continental slope. Some petroleum seep sites retained optimal habitat for some species continuously over geologically-relevant periods of time. More commonly, however, habitat optimality varied substantially over time scales of hundreds of years. Thus, one important characteristic of these sites was the degree of persistence of the chemoautotrophic biota. A fauna typically was persistent over a time span of a few hundred years, but was typically not persistent over a longer time span. The mechanisms producing local extinction remain unclear, however temporal variations in juvenile survivorship seem to be substantially larger than temporal variations in larval settlement, to the extent that the heavily taphonomically-biased record of juvenile individuals permits such a conclusion. When local extinctions occurred in the chemoautotrophic biota, the biota was replaced by a normal slope biota or a mixture of a normal slope biota and the juveniles of chemoautotrophic species that failed to survive to adulthood. Thus, the only faunal transitions were between specific chemoautotrophic faunas and the non-chemoautotrophic fauna. Not one distinctive faunal transition between two chemoautotrophic faunas was observed. Accordingly, each discrete chemoautotrophic fauna was resilient over long time scales; time scales of geological importance.     

Deep-water chemosynthetic ecosystem research during the census of marine life decade and beyond: a proposed deep-ocean road map

The ChEss project of the Census of Marine Life (2002-2010) helped foster internationally-coordinated studies worldwide focusing on exploration for, and characterization of new deep-sea chemosynthetic ecosystem sites. This work has advanced our understanding of the nature and factors controlling the biogeography and biodiversity of these ecosystems in four geographic locations: the Atlantic Equatorial Belt (AEB), the New Zealand region, the Arctic and Antarctic and the SE Pacific off Chile. In the AEB, major discoveries include hydrothermal seeps on the Costa Rica margin, deepest vents found on the Mid-Cayman Rise and the hottest vents found on the Southern Mid-Atlantic Ridge. It was also shown that the major fracture zones on the MAR do not create barriers for the dispersal but may act as trans-Atlantic conduits for larvae. In New Zealand, investigations of a newly found large cold-seep area suggest that this region may be a new biogeographic province. In the Arctic, the newly discovered sites on the Mohns Ridge (71 °N) showed extensive mats of sulfur-oxidisng bacteria, but only one gastropod potentially bears chemosynthetic symbionts, while cold seeps on the Haakon Mossby Mud Volcano (72 °N) are dominated by siboglinid worms. In the Antarctic region, the first hydrothermal vents south of the Polar Front were located and biological results indicate that they may represent a new biogeographic province. The recent exploration of the South Pacific region has provided evidence for a sediment hosted hydrothermal source near a methane-rich cold-seep area. Based on our 8 years of investigations of deep-water chemosynthetic ecosystems worldwide, we suggest highest priorities for future research: (i) continued exploration of the deep-ocean ridge-crest; (ii) increased focus on anthropogenic impacts; (iii) concerted effort to coordinate a major investigation of the deep South Pacific Ocean - the largest contiguous habitat for life within Earth's biosphere, but also the world's least investigated deep-ocean basin.     

Cold Seep Epifaunal Communities on the Hikurangi Margin,New Zealand: Composition,Succession, and Vulnerability to Human Activities

Cold seep communities with distinctive chemoautotrophic fauna occur where hydrocarbon-rich fluids escape from the seabed. We describe community composition, population densities, spatial extent, and within-region variability of epifaunal communities at methane-rich cold seep sites on the Hikurangi Margin, New Zealand. Using data from towed camera transects, we match observations to information about the probable life-history characteristics of the principal fauna to develop a hypothetical succession sequence for the Hikurangi seep communities, from the onset of fluid flux to senescence. New Zealand seep communities exhibit taxa characteristic of seeps in other regions, including predominance of large siboglinid tubeworms, vesicomyid clams, and bathymodiolin mussels. Some aspects appear to be novel; however, particularly the association of dense populations of ampharetid polychaetes with high-sulphide, high-methane flux, soft-sediment microhabitats. The common occurrence of these ampharetids suggests they play a role in conditioning sulphide-rich sediments at the sediment-water interface, thus facilitating settlement of clam and tubeworm taxa which dominate space during later successional stages. The seep sites are subject to disturbance from bottom trawling at present and potentially from gas hydrate extraction in future. The likely life-history characteristics of the dominant megafauna suggest that while ampharetids, clams, and mussels exploit ephemeral resources through rapid growth and reproduction, lamellibrachid tubeworm populations may persist potentially for centuries. The potential consequences of gas hydrate extraction cannot be fully assessed until extraction methods and target localities are defined but any long-term modification of fluid flow to seep sites would have consequences for all chemoautotrophic fauna.     

A Review on the Phylogeography of Potentially Chemoautotrophic Bacteria from Major Vent and Seep Fauna and Their Contribution to Primary Production

Though geochemically and microbially well-defined, the phylogeographic data of microbial symbionts in these highly productive vent and seep systems require a closer examination and synthesis. QIIME analysis of 16S rDNA of bacterial associates of major fauna from 1995 to 2015 was thus undertaken to examine phylogeography of their microbial symbionts along with host specificity. While phylotypes were generally unrelated, bivalve Calyptogena exhibited vertical transmission sharing similar symbionts in geographically separated geosystems. Different species of tubeworms possessed identical symbionts through horizontal acquisition at geographically distinct Guaymas basin vent and the Arctic seep. Vents were more versatile with both mobile and sessile fauna hosting ecto- and endo-symbionts. Comparatively, seeps were more specialized with sessile animal hosts with endosymbionts. C-fixation rate measurements are still scanty for sediments, bedrocks and serpentine systems; vent, seep, anoxic and oxic basins were shown to fix up to 22, 325, 96, and 37,400 g C m^?3 y^?1, respectively. Estimation of chemosynthetic primary production rates in chemoautotrophic ecosystems could endeavor to improve existing biogeographic models by coupling volcanism and plate-tectonics to global climate and phylogeography.     

A hierarchical classification of freshwater mussel diversity in North America

Aim North America harbours the most diverse freshwater mussel fauna on Earth. This fauna has high endemism at the continental scale and within individual river systems. Previous faunal classifications for North America were based on intuitive, subjective assessments of species distributions, primarily the occurrence of endemic species, and do not portray continent‐wide patterns of faunal similarity. The aim of this study is to provide an analytical portrayal of patterns of mussel diversity in a hierarchical framework that informs the biogeographical history of the fauna. Location The study considered the mussel fauna of North America from the Rio Grande system northwards. Methods Patterns of mussel faunal similarity in 126 river systems or lake watersheds across North America were examined. The dataset was developed from the literature and consisted of recent species presence/absence (282 species) in each drainage unit; subspecies were not included. Patterns of mussel diversity were examined with hierarchical cluster analysis, based on a pairwise distance matrix between all drainage units. Results Cluster analysis revealed 17 faunal provinces within four major faunal regions: Mississippian, Atlantic, Eastern Gulf and Pacific. The Mississippian Region dominates the North American fauna with 11 provinces, including five not recognized by previous classifications: Mississippi Embayment, Upper Mississippi, Great Plains, Ohioan and Pontchartrain–Pearl–Pascagoula. Within the Eastern Gulf Region (containing three provinces), the Escambia–Choctawhatchee Province is distinctive from the Apalachicolan Province, under which it was previously subsumed. Patterns of diversity in the Atlantic Region (two provinces) and Pacific Region (one province) were similar to previous classifications. Main conclusions The classification proposed in this study largely corroborates earlier schemes based on the occurrence of endemic species but identifies additional heterogeneity that reflects unique assemblages of widely distributed species. The study proposes a hierarchical structure that illustrates relationships among these provinces. Although some provinces in the Mississippian Region have high endemism, all Mississippian provinces share a group of widely distributed species. The Atlantic and Eastern Gulf regions have distinctive, endemic faunas suggesting limited past connectivity with the Mississippian Region. The Pacific Region is the most distinct fauna in North America and bears close affinity to the Eurasian mussel fauna.     

Aspects of the Ecology of the Deep-water Fauna of the Gulf of Mexico

Recent transects across the continental slope off western Louisiana,the Mississippi River delta, and the Florida peninsula in thegeneral depth range of 300–3,000 m have provided informationon habitat variables and on faunal composition, density, anddepth zonation. In the meiofauna (retained by 63µm screens)nematodes, harpacticoid copepods, nauplii, polychaetes, ostracods,and kinorynchs were numerically dominant, in that order, andtogether these groups made up 98% of the fauna. The macrofauna(retained by 0.3 mm screens) was dominated by polychaetes, ostracods,bivalves, tanaids, bryozoans, and isopods in that order, andtogether these made up 86% of the fauna. Densities of both groupswere highest on the Central Transect, and densities of bothtended to decrease with depth. Between the depths of 300 m and3,000 m there was a threefold decrease in meiofaunal and a twofolddecrease in macrofaunal density. Among the megafauna (collectedby otter trawl) invertebrate densities, dominated by crustaceans,were four to five times as great as fish densities at all depthsand on all transects. Densities were greatest on the Easternand least on the Central Transect, and on all transects theydecreased with depth. On the slope off Louisiana and East Texas,in the depth range of 400–900 m, dense biological communitieshave been encountered at about 40 locations aggregated aroundoil and gas seeps. These organisms include clusters of largetube worms (vestimentiferans), vesicomyid clams, mussels, galatheidcrabs, bresiliid shrimps, neogastropods, limpets, and fishes.This community is trophically dependent upon chemoautotrophicbacteria (which utilize hydrogen sulfide), although some musselsdirectly utilize methane as a carbon source. This communityis closely related to that of the hydrothermal vent systemsof the East Pacific Rise and to the seep communities at thebase of the Florida escarpment. The megafauna of the northernand eastern Gulf of Mexico falls naturally into the followingdepth distribution pattern: Shelf/Slope Transition Zone (118–475m), Archibenthal Zone—Horizon A (500–775 m), ArchibenthalZone—Horizon B (800–975 m), Upper Abyssal Zone (1,000–2,275m), Mesoabyssal Zone (2,300–3,225 m), and Lower AbyssalZone (3,250–3,850 m). Biological characteristics of eachzone are discussed.     

Ecology and Biogeography of Free-Living Nematodes Associated with Chemosynthetic Environments in the Deep Sea: A Review

Background

Here, insight is provided into the present knowledge on free-living nematodes associated with chemosynthetic environments in the deep sea. It was investigated if the same trends of high standing stock, low diversity, and the dominance of a specialized fauna, as observed for macro-invertebrates, are also present in the nematodes in both vents and seeps.

Methodology

This review is based on existing literature, in combination with integrated analysis of datasets, obtained through the Census of Marine Life program on Biogeography of Deep-Water Chemosynthetic Ecosystems (ChEss).

Findings

Nematodes are often thriving in the sulphidic sediments of deep cold seeps, with standing stock values ocassionaly exceeding largely the numbers at background sites. Vents seem not characterized by elevated densities. Both chemosynthetic driven ecosystems are showing low nematode diversity, and high dominance of single species. Genera richness seems inversely correlated to vent and seep fluid emissions, associated with distinct habitat types. Deep-sea cold seeps and hydrothermal vents are, however, highly dissimilar in terms of community composition and dominant taxa. There is no unique affinity of particular nematode taxa with seeps or vents.

Conclusions

It seems that shallow water relatives, rather than typical deep-sea taxa, have successfully colonized the reduced sediments of seeps at large water depth. For vents, the taxonomic similarity with adjacent regular sediments is much higher, supporting rather the importance of local adaptation, than that of long distance distribution. Likely the ephemeral nature of vents, its long distance offshore and the absence of pelagic transport mechanisms, have prevented so far the establishment of a successful and typical vent nematode fauna. Some future perspectives in meiofauna research are provided in order to get a more integrated picture of vent and seep biological processes, including all components of the marine ecosystem.     

The palaeoecology of the latest Jurassic–earliest Cretaceous hydrocarbon seep carbonates from Spitsbergen,Svalbard

Latest Jurassic–earliest Cretaceous hydrocarbon seeps from Spitsbergen, Svalbard, are known to contain unusual fauna, lacking most of the species characteristic for roughly coeval seep deposits. This study summarizes and analyses the fauna from 16 seep carbonate bodies from Spitsbergen to explain its composition. The seeps formed in a shallow epicontinental sea with widespread deposition of fine‐grained, organic‐rich sediments. They are spread over a relatively large area and are positioned roughly in the same interval, indicating seepage over extensive areas of the palaeo‐Barents Sea. The seep fauna is very species rich and with low dominance, comprising 54 species, with a composition similar to that of Jurassic–Cretaceous normal‐marine environments of other Boreal seas. Seep‐restricted fauna is not abundant and is represented by four species only. Hokkaidoconchids and possible siboglinid worm tubes characteristic for high sulphide fluxes are rare. Apart from seep‐restricted sulphide‐mining lucinid and thyasirid bivalves, chemosymbiosis was also a source of nourishment for background solemyid and nucinellid bivalves, all of which take sulphide from infaunal sources. This all suggests a relatively weak sulphide flux. The high diversity and low dominance of the fauna and significant richness and abundance of background species is typical for shallow water seeps.     

A hydrothermal seep on the Costa Rica margin: middle ground in a continuum of reducing ecosystems

Upon their initial discovery, hydrothermal vents and methane seeps were considered to be related but distinct ecosystems, with different distributions, geomorphology, temperatures, geochemical properties and mostly different species. However, subsequently discovered vents and seep systems have blurred this distinction. Here, we report on a composite, hydrothermal seep ecosystem at a subducting seamount on the convergent Costa Rica margin that represents an intermediate between vent and seep ecosystems. Diffuse flow of shimmering, warm fluids with high methane concentrations supports a mixture of microbes, animal species, assemblages and trophic pathways with vent and seep affinities. Their coexistence reinforces the continuity of reducing environments and exemplifies a setting conducive to interactive evolution of vent and seep biota.     

Specific and genetic diversity at deep-sea hydrothermal vents: an overview

Hydrothermal vent communities are ancient (i.e. early Mesozoic) and characterized by high biomasses, low number of species and high levels of endemism. However, little is known about the ecology and behaviour of the vent macro-and megafauna. Data on the biology and the life-history of hydrothermal-vent organisms are scarce and lead us to hypothesize various ways in which such species disperse and colonize their habitat. Such biological and ecological patterns are important for assessing both the spatial and temporal distribution of the vent fauna and the evolution of such peculiar species with geological times and, therefore need to be reviewed. Scattered information referring to vent-site distribution, bottom currents, temporal evolution of the vent emissions and their implication on the related fauna have been accumulated over the last decade. To date, several ecological and genetic studies have attempted to analyse vent fauna structures to understand how populations and communities evolve with time in such a patchy and unstable environment. They also provide faunistic comparisons across the vent communities discovered so far on well-separated oceanic ridges. This article provides a synthetic overview on biodiversity in deep-sea hydrothermal vents, genetic diversity of hydrothermal-vent species and factors responsible for similarities or differences among the vent fauna within and between well-separated venting areas of the Atlantic and Pacific ridges.     

Chemosymbiotic bivalves from the mud volcanoes of the Gulf of Cadiz, NE Atlantic, with descriptions of new species of Solemyidae, Lucinidae and Vesicomyidae

The chemosymbiotic bivalves collected from the mud volcanoes of the Gulf of Cadiz are reviewed. Of the thirteen species closely associated with chemosynthetic settings two Solemyidae, Solemya (Petrasma) elarraichensissp. n. and Acharax gadiraesp. n., one Lucinidae, Lucinoma asapheussp. n., and one Vesicomyidae, Isorropodon megadesmussp. n. are described and compared to close relatives of their respective families. The biodiversity and distribution of the chemosymbiotic bivalves in the Gulf of Cadiz are discussed and compared to the available information from other cold seeps in the Eastern Atlantic and Mediterranean. Although there is considerable similarity at the genus level between seep/mud volcano fields in the Eastern Atlantic and Mediterranean, there is little overlap at the species level. This indicates a high degree of endemism within chemosymbiotic bivalve assemblages.     

Cryptic biodiversity and phylogeographical patterns in a snapping shrimp species complex

Recent investigations suggest that marine biodiversity may be much higher than earlier estimates, and an important hidden source of diversity in marine systems is the phenomenon of cryptic species complexes. Such complexes are informative models for research into the evolutionary processes that govern species compositions of marine fauna. The snapping shrimp genera Alpheus and Synalpheus are known to harbour large numbers of cryptic species; here, I characterize the genetic structure of the Alpheus armillatus species complex in the northern Caribbean, west Atlantic, and Gulf of Mexico using mitochondrial and nuclear sequence data. Over this geographical region, the complex harbours at least three lineages that are probable reproductively isolated species; all major lineages diverged subsequent to the close of the Isthmus of Panama. Only one lineage was present in the Gulf of Mexico, whereas outside the Gulf of Mexico there was no clear tendency for lineage dominance by geographical region, as most sites were populated by shrimp from at least two lineages. However, within each lineage, there was strong evidence of population genetic differentiation between geographical regions. All lineages showed strong signals of demographic expansion, and one lineage showed sharply reduced genetic diversity, suggestive of past population bottlenecks or recently founded populations with low gene flow from other sites. These results show that evolutionary processes leading to divergence and speciation have been common and recent in the snapping shrimp, and suggest that connectivity among shrimp populations may be limited.     

Shell nacre ultrastructure and depressurisation dissolution in the deep-sea hydrothermal vent mussel Bathymodiolus azoricus

This study describes the micro-morphological features of the shell nacre in the vent mytilid Bathymodiolus azoricus collected along a bathymetric gradient of deep-sea hydrothermal vents of the mid-Atlantic ridge (MAR). Pressure-dependent crystallisation patterns were detected in animals subjected to post-capture hydrostatic simulations. We provide evidence for the following: (1) shell micro morphology in B. azoricus is similar to that of several vent and cold-seep species, but the prismatic shell layers may vary among bathymodiolids; (2) nacre micro-morphology of mussels from three vent sites of the MAR did not differ significantly; minor differences do not appear to be related to hydrostatic pressure, but rather to calcium ion availability; (3) decompression stress may cause drop off in pH of the pallial fluid that damages nascent crystals, and in a more advanced phase, the aragonite tablets as well as the continuous layer of mature nacre; and (4) adverse effects of decompression on calcium salt deposition in shells was diminished by re-pressurisation of specimens. The implications of the putative influence of hydrostatic pressure on biomineralisation processes in molluscs are discussed. An erratum to this article can be found at     

Shell nacre ultrastructure and depressurisation dissolution in the deep-sea hydrothermal vent mussel <Emphasis Type="Italic">Bathymodiolus azoricus</Emphasis>

This study describes the micro-morphological features of the shell nacre in the vent mytilid Bathymodiolus azoricus collected along a bathymetric gradient of deep-sea hydrothermal vents of the mid-Atlantic ridge (MAR). Pressure-dependent crystallisation patterns were detected in animals subjected to post-capture hydrostatic simulations. We provide evidence for the following: (1) shell micro morphology in B. azoricus is similar to that of several vent and cold-seep species, but the prismatic shell layers may vary among bathymodiolids; (2) nacre micro-morphology of mussels from three vent sites of the MAR did not differ significantly; minor differences do not appear to be related to hydrostatic pressure, but rather to calcium ion availability; (3) decompression stress may cause drop off in pH of the pallial fluid that damages nascent crystals, and in a more advanced phase, the aragonite tablets as well as the continuous layer of mature nacre; and (4) adverse effects of decompression on calcium salt deposition in shells was diminished by re-pressurisation of specimens. The implications of the putative influence of hydrostatic pressure on biomineralisation processes in molluscs are discussed.