Widespread Occurrence of Nitrate Storage and Denitrification Among

Home , Ammonia (genus), Ammonia tepida, Gromia, Quinqueloculina, Rotaliida, Textulariida

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- importance of bacterial denitrification in ocean sediments. firmed as a nitrate collector if the intracellular nitrate concentration 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.

1148–1153 | PNAS | January 19, 2010 | vol. 107 | no. 3 www.pnas.org/cgi/doi/10.1073/pnas.0908440107 Downloaded by guest on October 2, 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 ECOLOGY 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. laevigata North Sea 1 21 21 4.1E−03 5 Cassidulina cf. laevigata OMZ-Perú 6 523 (289) 194–1,388 1.2E−02 (3.6E−03) 41 (12) Globobulimina affinis OMZ-Perú 10 1,298 (2,320) 784–24,853 5.7E−02 (1.0E−02) 310 (97) Globobulimina affinis Bay of Biscay 1 3,290 3,29,018 9.3E−02 (2.0E−02) 292 G. auriculata cf. arctica Greenland 11 10,624 (3,555) 0–34,902 1.0E−01 (1.7E−02) 113 (43) Globobulimina cf. ovula OMZ-Perú 5 3,369 (1,602) 633–7,600 1.0E−03 (2.3E−03) 375 (174) Globobulimina turgida Gullmar fjord 20 18,000 (4,852) 0–72,000 5.0E−01 (3.6E+00) 10 (2) Globobulimina turgida Skagerrak 17 8,192 (1,497) 15–15,982 1.0E−01 (1.7E−02) 71 (13) Uvigerina elongatastriata Bay of Biscay 4 274 (244) 0–1,003 5.1E−03 (5.7E−04) 60 (55) Uvigerina elongatastriata Rhône Delta 2 5,389 (687) 4,703–6,077 2.4E−02 (9.4E−03) 286 (143) Uvigerina mediterranea Bay of Biscay 8 101 (66) 0–537 2.0E−02 (6.6E−03) 6 (4) Uvigerina peregrina Bay of Biscay 2 0 0 0 Uvigerina peregrina North Sea 5 332 (184) 17–792 2.0E−02 (6.6E−03) 16 (9) Uvigerina phlegeri Rhône Delta 48 444 (44) 29–1,092 8.4E−03 (1.8E−04) 209 (48) Clade 2 Ammonia beccarii Rhône Delta 6 0 0 0

Piña-Ochoa et al. PNAS | January 19, 2010 | vol. 107 | no. 3 | 1149 Downloaded by guest on October 2, 2021 Table 1. Cont. − NO3 (pmol per cell)

3 − Species Location n Mean (±SEM) Range Volume (mm )* NO3 (mM)*

Ammonia beccarii Bay of Biscay 9 0 0 0 Pseudoeponides falsobeccarii Rhône Delta 1 0 0 0 Ammonia sp. Limfjorden 14 1 (0.5) 0–5 2.3E−01 (1.7E−02) 0 Ammonia tepida Aiguillon Bay 15 13 (4) 0–58 2.3E−01 (1.7E−02) 0 Haynesina germanica Aiguillon Bay 13 0 0 0 Hyalinea balthica North Sea 14 8 (2) 0–30 8.0E−03 (1.2E−01) 1 (0.3) Clade 3 Bulimina aculeata Rhône Delta 7 0 0–2 6.0E−03 (4.3E−04) 0 Bulimina aculeata Bay of Biscay 6 19 (12) 0–63 7.4E−03 (3.8E−04) 3 (2) Bulimina cf. elongata OMZ-Perú 5 817 (287) 263–1,812 7.9E−03 (1.2E−03) 116 (43) Bulimina marginata Skagerrak 4 5 (2) 0–9 1.1E−03 (1.1E−02) 0.5 (0.2) Bulimina marginata Bay of Biscay 14 40 (4) 40–60 3.2E−02 (1.1E−03) 4 (1) Bulimina marginata Rhône Delta 1 0 0 0 Chilostomella oolina Bay of Biscay 4 1,124 (520) 185–2,611 2.0E−02 (2.0E−03) 65 (36) Cibicidoides pachyderma Bay of Biscay 1 0 0 0 Epistominella exigua OMZ-Perú 2 0 0 0 Melonis barleeanus North Sea 7 9 (3) 1–27 1.4E−02 (2.0E−02) 0.6 (0.2) Melonis barleeanus Rhône Delta 2 0 0 0 Nonionella cf. stella OMZ-Chile 43 186 (24) 8–794 5.2E−03 (7.1E−04) 35 (5) Stainforthia sp. var. I OMZ-Chile 26 60 (46) 0–172 3.3E−04 (2.1E−05) 180 (29)

Textulariids Clavulina cylindrica Bay of Biscay 6 1,941 (314) 568–2,651 3.7E−02 (5.8E−03) 61 (12) Clavulina cylindrica Rhône Delta 3 2,202 (480) 840–3,672 3.5E−02 (9.8E−04) 48 (13) Cypris subglobosus Bay of Biscay 1 0 0 0 Cyclammina cancellata OMZ-Perú 2 45,563 (45,563) 209–90,915 3.8E−01 (3.1E−03) 119 (118) Cyclammina cancellata Bay of Biscay 1 0 0 0 Goesella ﬂintii OMZ-Perú 6 459 (424) 0–2,575 1.0E−01 (2.7E−02) 24 (23) Labrospira cf. kosterensis OMZ-Perú 6 3,139 (845) 474–5,016 5.1E−02 (1.2E−02) 57 (12) Labrospira cf. subglobosa OMZ-Perú 2 0 0 0 Nouria polymorphinoides Bay of Biscay 2 0 0 0 Pseudoclavulina crustata Bay of Biscay 1 598 598.00 2.90E−01 2 Reophax micaceus Bay of Biscay 3 0 0 0 Reophax sp. OMZ-Perú 1 0 0 0 Rhabdammina inaequalis North Sea 1 0 0 0 Textularia cf. tenuissima OMZ-Perú 2 450 (432) 406–493 1.1E−02 (2.9E−03) 43 (7)

Other Rhizaria Gromia sp. North Sea 11 14,682 (4,649) 158–47,526 1.6E−01 (3.5E+00) 140 (46) Gromia sp. Skagerrak 12 35,277 (16,546) 158–2,04,000 5.1E−01 (1.1E−01) 53 (19) Gromia sp. OMZ-Perú 4 29,328 (7,589) 13,723–50,000 1.0E−01 (4.0E−02) 567 (283) Gromia sp. Bay of Biscay 2 2,846 (1,275) 1,571–4,121 9.3E−02 (2.0E−02) 35 (21) Gromia sp. Rhône Delta 13 3,889 (1,024) 576–12,676 1.6E−01 (1.1E−01) 91 (26) Gromia sp. Greenland 13 12,997 (2,954) 1,205–35,911 8.0E−02 (2.3E−02) 163 (54)

Species with a intracellular nitrate concentration > 0.1 mM are those conﬁrmed as nitrate collectors. The taxonomic classiﬁcation accommodates tradi- tional and molecular phylogenetic systems: (i) allogromiids sensu Cedhagen et al. (11)—unilocular foraminifers with an organic or agglutinated test (tradi- tional orders Allogromiida and Astrorhizida), (ii) miliolids—multilocular foraminifers with a calcitic porcelaneous test (order Miliolida), (iii) lagenids— multilocular foraminifers with a monolamellar perforated calcitic test (order Lagenida), (iv) rotaliids sensu lato—multilocular foraminifers with a bilamellar perforated calcitic test (orders Globigerinida, Rotaliida and Buliminida), and (v) textulariids sensu lato—multilocular foraminifers with an agglutinated test (orders Lituolida, Trochamminida and Textulariida). *Values are mean values. SEM is given in parentheses.

ficient of variation was 48–160% for species where n > 10, and Among the species with at least 10 replicates measured, only some individuals were completely void of nitrate. This intra- Ammonia sp., Ammonia tepida, Haynesina germanica, Quinque- specific variation indicates that a dynamic intracellular nitrate loculina seminulum, Nonion scaphum, and Technitella legumen pool is replenished and depleted, depending on the history of showed a consistent absence of nitrate in their cells. We cannot nitrate exposure, growth, starvation, and dormancy of the conclude whether the lack of measurable nitrate accumulation organisms. Exposure to oxygen might also lead to reduced results from the absence of a denitrification pathway or whether nitrate content. Melonis barleeanus sampled in the oxic zone of the species might accumulate nitrate under other environmental the Rhône Delta sediment did not contain nitrate whereas the conditions. This applies even more to taxa that are poorly same species sampled in the nitrate reduction zone in the North replicated in the present study (Biloculinella depressa, Bolivinita Sea contained nitrate. quadrilatera,Cibicidoides pachyderma, Cribrostomoides sub-

1150 | www.pnas.org/cgi/doi/10.1073/pnas.0908440107 Piña-Ochoa et al. Downloaded by guest on October 2, 2021 globosus, Dentalina sp., Rhabdamina inequalis, and Triloculina intertidal mudflats such as A. tepida and Haynesina germanica did tricarinata; n = 1). Thus, nitrate storage could be even more not store nitrate, and so far this environment does not seem to widely distributed within Foraminifera than indicated by the house nitrate-storing foraminifers (Table 1). present data set. Nitrate collection capacity is also found for foraminifers occupying various microhabitats. Some of the nitrate-collecting Nitrate Respiration. In previous studies the ability of foraminifers taxa prefer oxygen-depleted environments such as the sediments to store intracellular nitrate was found to be associated with the within the OMZs (see Table 1 and ref. 8) or layers in the sedi- ability of these organisms to respire nitrate through complete ment where oxygen and even nitrate are absent from the pore fi denitri cation to N2 (1, 2). Using a subset of the species analyzed water [e.g., the deep-living sedimental infaunal genera Globo- fi for intracellular nitrate, we measured denitri cation as N2O bulimina and Chilostomella (7, 8)]. Other nitrate-storing species, production after acetylene inhibition of N2O reduction to N2. such as Bolivina subaenariensis and Uvigerina mediterranea, are This method may underestimate but will never overestimate opportunistic with respect to oxygen and may be found in both fi denitri cation (12), and rates should therefore be considered as oxic and oxygen-free environments (5, 14, 15). Species that are minimum estimates. mainly found in oxic microhabitats, such as Cassidulina carinata Denitrification capacity was found for all analyzed species that fi and P. elongata (16, 17), can, however, also store nitrate. contained intracellular nitrate (Table 2), thus con rming that the This almost ubiquitous presence of nitrate-collecting fora- intracellular nitrate pool was used for respiration. Denitrification −1 −1 minifers suggests that the trait is one of the reasons for their rates ranged between 45 and 248 pmol nitrogen individual d , successful colonization of marine sediments. As facultative which is close to the activities measured previously in G. turgida anaerobes, the nitrate-storing foraminifers can use either oxygen and Nonionella cf. stella (1, 2). There was no N O production 2 or nitrate in the environment for respiration. The combination of associated with the nonnitrate-accumulating foraminifer A. tepida. nitrate storage and nitrate respiration enables the organisms to Interestingly, four species collected in the Peruvian OMZ sustain respiratory activity in shorter or longer periods when produced N O in the absence of acetylene (Bolivina plicata, 2 oxygen or/and nitrate is absent from the environment. This Bolivina seminuda, Valvulineria cf. laevigata, Stainforthia sp.), suggesting a lack of nitrous oxide reductase in these organisms allows them to explore food resources and to periodically seek shelter from predation in deeper sediment layers and also to and thus making them greenhouse gas sources. fl All tested denitrifying species could also respire with oxygen survive exposure to environmental uctuations such as passive (Table 2)—even those collected in oxygen-free environments transport into anoxic environments caused by sediment resus- such as the Peruvian OMZ. The denitrifying foraminifers should pension and macrofauna-mediated bioturbation events. therefore be regarded as facultative anaerobes. The oxygen Molecular Phylogeny and Evolutionary Implications. The molecular respiration rates were generally about 3–13 times higher than the denitrification rates and, given the generally higher energy yield phylogeny of foraminifers based on partial small subunit (SSU) from oxic respiration (13), this could indicate that denitrification ribosomal DNA (rDNA) sequences inferred here (Fig. S1)is is an auxiliary metabolism used for cell maintenance, food col- congruent with previous studies. Allogromiids form a para-

lection, and locomotion during temporary stays in oxygen-free phyletic group at the base of the tree, whereas miliolids and ECOLOGY environments, whereas oxygen might be required for growth and rotaliids/textulariids (together with allogromiids of clades A and reproduction. C) form two separated clades arising from allogromiids (18, 19). At the moment, there is no published sequence of lagenids, but Ecological Diversity of Nitrate-Storing Foraminifers. Nitrate storage this group branches as a sister group of the textulariid/rotaliid was common in foraminifers from very diverse benthic marine clade according to previous results (20). The polyphyly of tex- environments. In the OMZ, 16 of 23 tested species stored tulariids (18) as well as the monophyly of rotaliids sensu lato, nitrate. More widespread species such as Uvigerina peregrina, with Buliminida and Globigerinida included (4, 21) can also been Valvulineria bradyana, and Clavulina cylindrica from continental observed. There is a lack of taxonomic sampling to determine the slopes, shelves, and coastal sediments (3) also store nitrate. molecular phylogenies of miliolids or textulariids. However, most Additionally, nitrate collectors were found in bathyal sediments of the morphological superfamilies of the rotaliids have been (e.g., Bulimina marginata), and several of the species sampled in sampled, and for these we now have a good overview (Fig. S1 the Rhône Delta also accumulated nitrate. Species typically in and ref. 22).

Table 2. Denitriﬁcation and oxygen respiration rates of various foraminifers Denitriﬁcation Oxygen respiration −1 −1 † −1 −1 Species Individuals* (pmol nitrogen individual d ) Individuals (pmol O2 individual d )

Ammonia tepida (Aiguillon Bay) 2 0 (n = 1) 2 2030 ± 72 (n =4) Bolivina subaenariensis (B.Biscay) 10–12 78 ± 2(n = 2) 8 252 ± 50 (n =3) Uvigerina phlegeri (Rhône) 10 46 ± 2(n =1) 5–890± 25 (n =3) Valvulineria bradyana (Rhône) 10 183 ± 10 (n =2) 2–4 759 ± 288 (n =3) ‡ Nonionella cf. stella (OMZ, Chile) 3–584± 33 (n =3) 3–5 760 ± 87 (n =3)§ ‡ Globobulimina turgida (Gullmar fjord) 3 565 Bolivina plicata (OMZ, Perú) 3 79 (n =1) Valvulineria cf. laevigata (OMZ, Perú) 10 248 ± 180 (n = 2) 7 754 ± 146 (n =2) Bolivina seminuda (OMZ, Perú) 3 216 (n = 1) 3 368 (n =1) Stainforthia sp. (OMZ, Perú) 4 70 (n = 1) 4 822 (n =1)

Rates are given as the mean (±SEM); n designates the number of replicates. *Individuals in the measuring chamber during denitriﬁcation measurements. † Individuals in the measuring chamber during oxygen respiration measurements. ‡Data are from ref. 2. §Data are from ref. 1.

Piña-Ochoa et al. PNAS | January 19, 2010 | vol. 107 | no. 3 | 1151 Downloaded by guest on October 2, 2021 Nitrate-collecting rotaliids belong mainly to clades 1 and 3. of Uvigerina phlegeri, G. turgida, and Valvulineria bradyana in the Hyalinea balthica is the only nitrate-collecting foraminifer within Tagus prodelta (32), and their cell-specific denitrification rate, clade 2, which primarily encompasses shallow water and epi- foraminiferal denitrification rates range between 72 and 240 μmol − − faunal/planktic species. Nitrate collection was also found among nitrogen m 2·d 1. Denitrification measured there in July–Octo- the textulariids and miliolids and even among the gromiids (Fig. ber, which corresponds to the period when the above-mentioned − − S1), which probably share a common ancestor with Foraminifera species were enumerated (32), is 480–960 μmol nitrogen m 2·d 1 (10). Species that may lack the ability to accumulate nitrate are (33). The foraminifers might thus contribute 8–50% of the likewise represented within all major groups (Fig. S1), suggesting measured denitrification activity. However, there are also sites a complex evolutionary history of nitrate accumulation and where the abundance of denitrifying foraminifers is too low to denitrification within Foraminifera. contribute significantly to nitrogen loss via denitrification. In the Assuming that none of the nitrate-collecting species depend on Sagami Bay (Japan), the small population of foraminifers appa- fi symbiotic bacteria for denitri cation (2), two hypotheses arise rently contributes only 4% to benthic N2 production (9). concerning their evolutionary history. Either the trait appeared Foraminiferal denitrification challenges measurements of during the Neoproterozoic in a common ancestor of foraminifers marine denitrification. Methods that rely on, for example, 15N and gromiids and was possibly followed by several losses in separate tracer additions (e.g., the method used in refs. 27, 29, and 33) or on phylogenetic lineages or it was acquired several times independ- modeling of nitrate pore water profiles may underestimate deni- − ently in different lineages in more recent history. In other words, trification of intracellular NO3 pools and will consequently acquisition of denitrifying genes among foraminifers occurred underestimate true denitrification (1). Methods that rely on incu- either through the primitive endosymbiosis leading to denitrifying bations, during which nitrate decreases or the N2/Ar ratio increases mitochondria or through subsequent lateral gene transfer(s). in the overlying waters (e.g., the methods used in ref. 31), on the Denitrification relies on a large cluster of genes (23), and even other hand, may capture foraminiferal denitrification (1). Sig- when found in eukaryotic fungi, most of these genes apparently nificant foraminifer-mediated denitrification may therefore call have a bacterial origin (24). If the same holds true for the fora- for revisions of current estimates of nitrogen loss from the marine minifers and gromiids, it is likely that denitrification was incorpo- environment and the methodologies used to quantify this loss. rated with the protomitochondrion in the very first eukaryotes and In the past 10 years, our understanding of nitrogen-cycling pro- that more eukaryotic phyla could have retained the trait. cesses and the microorganisms that mediate these processes has advanced significantly. The microbiology of this cycle has been Nitrate-Storing Foraminifers: Implications for the Marine Nitrogen fi fi signi cantly revised with therecognition that key processesare more Cycle. Prokaryotic denitri cation and anaerobic ammonium oxi- broadly distributed among the primary domains of life than predation are considered to be the only processes returning combined viously thought (25, 34). Until recently, bacterial nitrification cou- nitrogen to the atmosphere from the sea (25). The widespread pled with denitrification was considered the only process directing occurrence of nitrate storage and denitrification among fora- fixed nitrogen back to the atmosphere as N2. Today it seems that, in minifers demonstrated here indicates that eukaryotes may also the ocean, anaerobic ammonium oxidation (anammox) could be as play a role. fi fi important as bacterial denitri cation for N2 formation (35, 36). We Total foraminifer-mediated denitri cation in various envi- can now supplement the enormous phylogenetic and metabolic ronments can be estimated by combining the abundance of living biodiversity that is hidden in the microbial world with our identi- nitrate-storing foraminifers and denitrification rates measured fication of phylogenetically and geographically widespread nitrate- on isolated specimens, assuming that the rates estimated here storing and denitrifying foraminifers and gromiids. are representative of the organisms in their natural habitat. Corliss and Weering enumerated the foraminiferal population Materials and Methods in the shelf sediments from the Skagerrak (26). Among the taxa fi Sites Description and Sampling. Live specimens of benthic foraminifers were that they found, three have a documented denitri cation capacity: collected during different cruises in 2006–2008 from marginal marine envi- Bolivina sp., Globobulimina sp., and Uvigerina sp. (Table 2). Using ronments and open-sea localities (Table S1). Sediment samples were taken the abundance data for these taxa at site A84-1 in ref. 26 and their either by hand with a scraper at the shallow sites or by multicoring at deeper cell-specific denitrification rate (Table 2), foraminiferal deni- sites. The top 10 cm of the sediment was collected and immediately sieved − − trification is about 720 μmol nitrogen m 2·d 1. For comparison, (fractions 63 and 150 μm) using water at in situ temperature and oxygen the denitrification rate measured close to this site is 1030 μmol concentration. The different fractions were stored at in situ temperature. − − nitrogen m 2·d 1 (site BB12 in ref. 27), suggesting that foraminifers are responsible for up to 70% of the measured benthic Specimen Documentation. Specimens were identified using a stereo- denitrification activity. Likewise, in a canyon of the Bay of Biscay, microscope (Leica MZ 12.5 or Wild Heerbrugg M3) or, when necessary, air 2 dried, coated with gold and examined in a LEO 1450VP scanning electron B. subaenariensis alone with 82 cells/cm (15) would produce fi μ – −2· −1 fi microscope. Species were identi ed by reference to the taxonomical liter- about 64 mol N2 nitrogen m d . Total denitri cation esti- ature (Table S2). mated from the nitrate pore water profiles measured at the site μ −2· −1 (28) is around 76 mol nitrogen m d , suggesting that fora- Isolation of Living Foraminifers and Gromiids. Individual live specimens, minifers are quantitatively important denitrifiers at this location selected for nitrate analysis, were cleaned with a brush in low-oxygen, too. In part of the OMZ off Chile where foraminiferal abundance nitrate-free artificial seawater, transferred to sealed PCR tubes, and ana- −2 is high [205 cells cm (1)], foraminifer-mediated denitrification is lyzed immediately or stored at –20°C until analysis. Denitrification meas- − 173 μmol N d 1 (1). Total benthic denitrification reported for this urements were carried out immediately on live specimens harvested directly − − site is around 250 μmol N m 2·d 1 (29), indicating that fora- from the sediment. Viability of each specimen was assessed by color, the minifers may account for almost 70% of the nitrogen loss from the amount of cell cytoplasm, and the gathering of organic matter around the sediment. In the Arabian Sea OMZ, however, the foraminifers aperture or pseudopodial movement. contribute less to denitrification. With a density of 48 denitrifying foraminifers/cm2 (30), foraminifer-based denitrification accounts Measurement of Nitrate Content. The intracellular nitrate content of indi- μ −2· −1 – vidual species was analyzed using the VCl3 reduction method (37) as for 78 mol nitrogen m d , which corresponds to 9 15% of the fi fi – μ −2· −1 described previously (1, 2) with the following modi cation: Nitrate was measured benthic denitri cation [510 840 mol nitrogen m d extracted from individual specimens with 10 μl of NaOH (10%) solution in (31)]. In estuaries, foraminiferal denitrification may also play a the PCR tubes and was measured in 10-μl subsamples. This procedure avoids significant role in the loss of combined nitrogen, and it may disruption of the foraminiferal test, so that shell volume and species identity thereby mitigate coastal eutrophication. Using the standing stock could be determined after nitrate measurements. Empty foraminiferal tests

1152 | www.pnas.org/cgi/doi/10.1073/pnas.0908440107 Piña-Ochoa et al. Downloaded by guest on October 2, 2021 and sediment corresponding to the volume of the foraminifers were used as were performed with PhyML (42) under the HKY+I+Γmodel (43). The main contamination controls. alignment is based on a region of SSU situated at the 3′-end (fragment s14F-sB) and includes published sequences from all available genera (45 allogromiids, Denitrification Capacity. Foraminiferal cells were placed in 300 nl chambers 19 miliolids, 14 textulariids, and 42 rotaliids) and Gromiida as an out-group. with 5 mM Hebes buffer media and nitrate respiration rates were determined fi from N2Opro les after acetylene inhibition of N2O reduction (1, 2, 38, 39). ACKNOWLEDGMENTS. We thank P. Sørensen for making microsensors. We Oxygen in the headspace above the measuring chamber was trapped in an are grateful to crew and organizers of cruises with Vædderen, R/V Maria S. alkaline 0.1 M ascorbate solution, separated from the headspace by a silicon Merian, the Côte de la Manche, the R/V Tethys II, and R/V G.M. Dannevig. We membrane. Oxygen respiration rates were determined using a Clark-type thank B .B. Jørgensen and two anonymous reviewers for valuable comments oxygen microsensor (1, 40). and A. Winter and A. Haxen for checking the English. This research was financially supported by the European Union Marie Curie Fellowship (FP7- IEF-220894), the Danish National Science Research Council (Grant 272-06- Molecular Phylogenetic Analysis. To infer the molecular phylogeny of nitrate- 0504), the Danish National Research Foundation, the German Max Plank storing foraminifers, complete and partial SSU rDNA sequences were taken Society, the Commission for Scientific Research in Greenland, the Aarhus from the EMBL/GenBank database (access numbers indicated in Table S3) and University Research Foundation, and the Danish Expedition Foundation. This aligned with Clustal X (41) with manual corrections. The phylogenetic analyses is Galathea 3 contribution no. P52.

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