Widespread occurrence of nitrate storage and denitrification among Foraminifera and Gromiida Elisa Piña-Ochoaa, Signe Høgslunda, Emmanuelle Geslinb,c, Tomas Cedhagend, Niels Peter Revsbeche, Lars Peter Nielsene, Magali Schweizerf, Frans Jorissenb,c, Søren Rysgaardg, and Nils Risgaard-Petersena,1 aCenter for Geomicrobiology, Department of Biological Sciences, Aarhus University, DK-8000 Aarhus C, Denmark; bLaboratory of Recent and Fossil Bio- Indicators, Angers University, 49045 Angers Cedex, France; cLEBIM, 85350 Ile d’Yeu, France; dSection of Marine Ecology, Department of Biological Sciences, Aarhus University, DK-8200 Aarhus N, Denmark; eSection of Microbiology, Department of Biological Sciences, Aarhus University, DK-8000 Aarhus C, Denmark; fGeological Institute, ETH Zurich, 8092 Zurich, Switzerland; and gGreenland Climate Research Centre, 3900 Nuuk, Greenland Edited by Donald E. Canfield, University of Southern Denmark, Odense M, Denmark, and approved November 30, 2009 (received for review July 31, 2009) Benthic foraminifers inhabit a wide range of aquatic environments sampled from different marginal marine environments: Aiguillon including open marine, brackish, and freshwater environments. Bay (France), Bay of Biscay (France), Disko Bay (Greenland), Here we show that several different and diverse foraminiferal Gullmars Fjord (Sweden), Limfjorden (Denmark), the North Sea, groups (miliolids, rotaliids, textulariids) and Gromia, another taxon the Peruvian–Chilean OMZ, the Rhône prodelta (France), and also belonging to Rhizaria, accumulate and respire nitrates through the Skagerrak. In addition, 55 specimens belonging to the genus denitrification. The widespread occurrence among distantly re- Gromia were tested for nitrate content. This aquatic ameboid lated organisms suggests an ancient origin of the trait. The diverse protist genus bears an organic test and is thought to be the sister metabolic capacity of these organisms, which enables them to group of foraminifers within Rhizaria (10). respire with oxygen and nitrate and to sustain respiratory activity even when electron acceptors are absent from the environment, Results and Discussion may be one of the reasons for their successful colonization of Taxonomic Diversity Among Nitrate-Storing Foraminifers. Up to 48 diverse marine sediment environments. The contribution of eukar- specimens were analyzed individually from each foraminiferal yotes to the removal of fixed nitrogen by respiration may equal the species to determine nitrate accumulation. A species was con- ECOLOGY importance of bacterial denitrification in ocean sediments. firmed as a nitrate collector if the intracellular nitrate concen- tration was 100 μM or more than 10-fold the maximum eukaryotic nitrate respiration | nitrogen cycle concentration in the environment (Table S1). More than half of the tested foraminiferal species and all of the gromiids were fi nly two eukaryotes, the marine benthic foraminiferal species con rmed as nitrate collectors (Table 1). Globobulimina turgida* and Nonionella cf. stella (1, 2), are Nitrate-collecting species were found within the miliolids, O rotaliids, and textulariids. However, none of the six tested known to carry out complete denitrification of nitrate to N2. They accumulate intracellular nitrate to millimolar concen- allogromiids or the single tested lagenid contained measurable trations, which they subsequently respire in the absence of oxy- nitrate. All of the tested gromiids contained nitrate with average gen. It has been uncertain whether this is a previously internal concentrations ranging from 53 to 566 mM (Table 1). undescribed evolutionary trait found only in a few closely related Nitrate collection capacity was broadly distributed in genera within all three rotaliid clades proposed by Schweizer et al. (4) species that share the same mode of life or an old or coevolved (Table 1): Bolivina, Cassidulina, Globobulimina, and Uvigerina widespread trait in Foraminifera and other eukaryotes. Our (clade 1); Hyalinea (clade 2); Bulimina, Chilostomella, Melonis, finding of denitrification via nitrate pools in mobile eukaryotic Nonionella, and Stainforthia (clade 3); and also in genera not yet organisms adds a previously undescribed dimension to the represented in phylogenetic analyses such as Cancris, Gyroidina, marine nitrogen cycle. Because benthic foraminifers inhabit a and Valvulineria. Within textulariids, nitrate collection was found wide range of aquatic environments and can be found in den- in Cyclammina, Labrospira, Pseudoclavulina, Textularia, and sities of up to several million individuals per square meter (3), members of the Valvulinidae family, and, in miliolids, Pyrgo they may play an important role in temporal nitrate sequestering, elongata displayed the trait (Table 1). fi nitrate transport, and nitrogen removal through denitri cation. Nitrate-collecting species within a genus showed variable G. turgida and Nonionella cf. stella belong to the foraminiferal nitrate content, and the magnitude of intracellular nitrate con- order Rotaliida, inside which they group into different clades tent generally did not map onto the foraminiferal phylogeny. The according to molecular phylogeny (4). Both taxa thrive in oxy- nitrate content seemed to reflect different physiological and – gen-free sediment environments (5 7) where alternative electron environmental conditions because considerable intraspecific acceptors such as nitrate are required for respiration. Many variation in intracellular nitrate concentration was observed other genera have been observed in such environments (e.g., among the species confirmed as nitrate collectors. The coef- Chilostomella, Stainforthia, Bolivina, Uvigerina, Bulimina, and Reophax) (8), and recently published data suggest that the use of fi nitrate is not con ned to only two genera. In a Swedish fjord, Author contributions: E.P.-O., S.H., E.G., T.C., N.P.R., L.P.N., and N.R.-P. designed research; nitrate detected in G. turgida accounted for only about 20% of E.P.-O., S.H., E.G., T.C., N.P.R., L.P.N., S.R., and N.R.P. performed research; E.P.-O., S.H., E.G., the total cell-bound nitrate pool in the sediment (2), suggesting T.C., N.P.R., L.P.N., M.S., F.J., S.R., and N.R.-P. analyzed data; and E.P.-O., S.H., E.G., T.C., N. that other foraminifers might also accumulate nitrate. Stainfor- P.R., L.P.N., M.S., F.J., S.R., and N.R.-P. wrote the paper. fl thia sp. from the oxygen minimum zone (OMZ) of the con- The authors declare no con ict of interest. tinental shelf off Chile (1) and Uvigerina akitaensis, Bolivina This article is a PNAS Direct Submission. spissa, and Textularia sp. from Japanese deep-ocean margin 1To whom correspondence should be addressed at: Center for Geomicrobiology, Depart- ment of Biological Sciences, Ny Munkegade 114-116, Building 1540, Aarhus University, sediment (9) also have been shown to accumulate nitrate. DK-8000 Aarhus C, Denmark. E-mail: [email protected]. To evaluate the evolutionary origin, environmental affiliation, *After an in-depth taxonomic analysis, we decided that Globobulimina pseudospinescens, and biogeochemical importance of denitrification in foraminifers, described in ref. 2, should be considered as and named G. turgida. fi we have determined the denitri cation rates of seven more species This article contains supporting information online at www.pnas.org/cgi/content/full/ and measured the nitrate content of 67 foraminiferal species 0908440107/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.0908440107 PNAS Early Edition | 1of6 Downloaded by guest on September 24, 2021 Table 1. Intracellular nitrate content and concentration in foraminifers and gromiids − NO3 (pmol per cell) 3 − Species Location n Mean (±SEM) Range Volume (mm )* NO3 (mM)* Allogromiids Agglutinated sp. Rhône Delta 1 0 0 0 Bathysiphon cf. argenteus OMZ-Perú 2 0 0 0 Bathysiphon minutus Skagerrak 2 0 0 0 Crithionina hispida OMZ-Perú 4 0 0 0 Hippocrepinella alba Skagerrak 6 5 (1) 1–6 2.0E−01 (5.0E−02) 0 Komokiacea OMZ-Perú 3 0 0 0 Pelosina variabilis Skagerrak 3 50 (25) 6–92 2.2E−01 (2.2E+02) 0 Saccammina sp. Bay of Biscay 2 0 0 0 Technitella legumen Skagerrak 13 5 (1) 1–10 4.0E−01 (4.4E−02) 0 Miliolids Biloculinella depressa North Sea 1 0 0 0 Pyrgo elongata Rhône Delta 9 43 (14) 19–139 4.7E−02 (5.8E−03) 0.8 (0.2) Pyrgo williamsoni North Sea 1 5 5 4.7E−02 0.10 Pyrgoella sphaera North Sea 2 6 (1) 5–7 4.7E−02 (5.8E−03) 0.1 (0.02) Quinqueloculina sp. OMZ-Perú 2 0 0 0 Quinqueloculina Skagerrak 2 0 0 0 seminulum Quinqueloculina Bay of Biscay 10 0 0 0 seminulum Quinqueloculina Rhône Delta 2 0 0 0 seminulum Triloculina tricarinata North Sea 1 0 0 0 Lagenids Dentalina sp. Rhône Delta 1 0 0 0 Rotaliids Unknown clade Bolivinita quadrilatera Bay of Biscay 1 0 0 0 Cancris inflatus OMZ-Perú 18 262,877 (4,253) 3,920–76,475 1.2E−01 (2.4E−02) 262 (37) Gyroidina altiformis Bay of Biscay 1 0 0 0 Gyroidina neosoldanii OMZ-Perú 6 13,190 (480) 25–3,375 2.7E−02 (1.2E−02) 241 (46) Nonion scaphum Rhône Delta 9 0 0 0 Nonion scaphum Bay of Biscay 2 0 0 0 Valvulineria bradyana Rhône Delta 16 1,268 (164) 176–2,541 1.5E−02 (1.4E−03) 95 (15) Valvulineria cf. laevigata OMZ-Perú 17 865 (640) 55–1,093 1.9E−02 (3.7E−03) 25 (12) Clade 1 Bolivina alata Bay of Biscay 6 615 (154) 188–1,266 1.7E−02 (1.1E−03) 37 (12) Bolivina cf. abbreviata OMZ-Perú 14 1,081 (368) 26–4,934 1.2E−02 (2.7E−03) 153 (49) Bolivina cf. skagerrakensis North Sea 1 83 83 1.7E−02 5 Bolivina plicata OMZ-Perú 24 478 (72) 59–1,037 7.5E−03 (9.5E−04) 79 (15) Bolivina seminuda OMZ-Perú 10 564 (135) 187–807 5.2E−03 (1.8E−03) 118 (18) Bolivina subaenariensis Bay of Biscay 47 285 (46) 43–1,023 2.5E−02 (4.3E−03) 44 (9) Cassidulina carinata Rhône Delta 23 3(1) 0–21 4.1E−03 (2.2E−04) 1 (0.5) Cassidulina cf.