Physiological and morphological plasticity in response to nitrogen availability of a yeast widely distributed in the open ocean

Abstract Yeasts are prevalent in the open ocean, yet we have limited understanding of their ecophysiological adaptations, including their response to nitrogen availability, which can have a major role in determining the ecological potential of other planktonic microbes. In this study, we characterized the nitrogen uptake capabilities and growth responses of marine-occurring yeasts. Yeast isolates from the North Atlantic Ocean were screened for growth on diverse nitrogen substrates, and across a concentration gradient of three environmentally relevant nitrogen substrates: nitrate, ammonium, and urea. Three strains grew with enriched nitrate while two did not, demonstrating that nitrate utilization is present but not universal in marine yeasts, consistent with existing knowledge of nonmarine yeast strains. Naganishia diffluens MBA_F0213 modified the key functional trait of cell size in response to nitrogen concentration, suggesting yeast cell morphology changes along chemical gradients in the marine environment. Meta-analysis of the reference DNA barcode in public databases revealed that the genus Naganishia has a global ocean distribution, strengthening the environmental applicability of the culture-based observations. This study provides novel quantitative understanding of the ecophysiological and morphological responses of marine-derived yeasts to variable nitrogen availability in vitro, providing insight into the functional ecology of yeasts within pelagic open ocean environments.


Introduction
Marine yeasts are a group of heterotrophic microbial eukaryotes activ e thr oughout div erse marine envir onments including the open ocean (Grossart et al. 2019 ).The term yeast unites fungi capable of unicellular growth, which occur exclusively within the phyla Ascomycota and Basidiomycota, but do not form a monophyletic group (El Baidouri et al. 2021 ).Environmental DN A-based surveys sho w that fungal community composition varies between marine ecosystems (Amend et al. 2019 ).Open ocean fungal communities are dominated by the Ascomycota and Basidiomycota at the surface (Wang et al. 2014 ) and in the deep ocean (Bass et al. 2007 ).The Chytridiomycota show high r elativ e contributions in coastal waters (Taylor andCunliffe 2016 , Debeljak andBaltar 2023 ), in association with sea ice in the Arctic Ocean (Hassett and Gradinger 2016 ), and with low salinity in the Baltic Sea (Rojas-Jimenez et al. 2019 ).Earl y div er ging gr oups also r epr esent a major fr action of fungal div ersity in coastal sediments (Picard 2017 ) and deep-sea sediments (Nagahama et al. 2011 ).Despite ric h mor phological and taxonomic div ersity across marine ecosystems, yeasts dominate planktonic fungal diversity at the global scale (Hassett et al. 2020 ).
Yeasts are saprotrophs, releasing extracellular enzymes to break down high molecular weight organic matter into smaller compounds, which can then be taken up by osmotrophy, a nutritional str ategy shar ed with marine bacteria (Worden et al. 2015 ).Cunliffe et al. ( 2017 ) identified active saprotrophs of phytoplankton-deriv ed or ganic carbon in a coastal ecosystem, including Malassezia (Basidiomycota), which contains unicellular yeast species found throughout pelagic marine environments (Amend 2014, Boekhout et al. 2022 ).In addition to sa pr otr ophy, marine yeasts are associated with other trophic interactions, including predation by zooplankton (Cleary et al. 2016 ), and parasitism of phytoplankton (Li et al. 2016 ) and zooplankton (Seki and Fulton 1969 ).Marine yeast abundances have been shown to fluctuate by se v er al orders of magnitude over relativ el y short time-scales during a spring phytoplankton bloom (Priest et al. 2021 ), while inter annual r ecurr ence patterns of yeast-containing taxa further suggest a dynamic role for yeasts in marine plankton communities (Taylor andCunliffe 2016 , Chrismas et al. 2023 ).Absolute abundances of marine yeast taxa have been significantly positively correlated with envir onmental concentr ations of nitr ogen-containing compounds (Taylor andCunliffe 2016 , Priest et al. 2021 ), but quantitative data describing marine yeast responses to nitrogen availability ar e curr entl y lac king in contr ast with other major plankton groups.
Ph ytoplankton ecoph ysiology and comm unity composition ar e driven by the concentration and chemical forms of nitrogen available (Glibert et al. 2016 ).Large diatoms dominate productiv e, nitr ate-ric h surface waters (e.g.Kudela and Dugdale 2000 ), whereas smaller phytoplankton such as Cyanobacteria dominate when reduced forms including ammonium and urea are the primary nitrogen sources (e.g.Berg et al. 2003 ).Despite strong links to seasonal bloom e v ents, nitr ate utilization is not a universal trait in marine phytoplankton, e.g.some ecotypes of the globally abundant picoc y anobacterium Proc hlorococcus lac k the ca pacity for nitrate uptake (Moore et al. 2002 ).Nitrate assimilation is also restricted to a subset of yeast genera not including the model fission yeast genus Sc hizosacc harom yces (Siv erio 2002 ), whic h had the highest r elativ e sequence abundance of identified fungal gener a acr oss most oceanogr a phic r egions in a global meta genomic dataset (Hassett et al. 2020 ).In the marine water column, nitrate utilization could offer some yeasts a distinct adv anta ge, especially when high-nitrate conditions trigger rapid diatom growth, pr oviding r eady supplies of both nitrogen and a suitable diatomderiv ed or ganic carbon substr ate (Cunliffe et al. 2017 ).Efficient growth on ammonium and/or urea could sustain marine yeasts that are (a) unable to take up nitrate, or (b) in low-nitrate conditions, wher e micr obial r eminer alization of r educed nitr ogen fuels micr obial pr oduction (Dugdale andGoering 1967 , Glibert et al. 2016 ).To date, it has been difficult to address ecological hypotheses regarding marine yeast nitrogen dynamics, due to insufficient knowledge of their nitrogen uptake capabilities, specificall y whic h nitr ogen substr ates can be used and whether nitrogen limits y east gro wth at concentr ations r ele v ant to pela gic marine en vironments .
Nitrate (NO 3 − ) is typically the most abundant form of fixed nitrogen in the ocean and nitrate concentration increases with depth in near-surface waters (Gruber 2008 ).Ammonium (NH 4 + ) tends to be present at much lo w er apparent concentrations due to r a pid micr obial c ycling in the w ater column (Glibert and Goldman 1981 ).Across the Atlantic Ocean, euphotic zone nitrate fluctuates by se v er al orders of magnitude (0.005-33 μmol l −1 ), while ammonium is m uc h less v ariable (0.109 ± 0.150 μmol l −1 , maximum 5.2 μmol l −1 ) (Rees et al. 2006 ).Ur ea concentr ations in the euphotic Atlantic are typically 0.15-0.35μmol l −1 , sometimes r eac hing 1.72 μmol l −1 (Painter et al. 2008 ), representing an important source of dissolved nitrogen for phytoplankton, particularly Prochlorococcus in the subtropical North Atlantic (Casey et al. 2007, Painter et al. 2008 ).Phytoplankton nutrient utilization traits exhibit allometric scaling relationships across species and across major taxonomic groups , e .g. biomass-scaled nutrient affinities tend to decrease with increasing cell volume (Edwards et al. 2012 ).As cell size constr ains micr obial physiology and micr obe-micr obe inter actions, it is considered a k e y functional trait regulating marine plankton biogeogr a phy (Barton et al. 2013 ).Allometric relationships have been investigated among filamentous marine fungi (Fuentes et al. 2015, Aguilar-Trigueros et al. 2017 ) and between major cell types of marine fungi (Thomas et al. 2022 ), but trait variations across marine yeast taxa, or within taxa under changing conditions are not w ell-documented.Tw o Pacific Ocean y east isolates sho w ed v ariable gr owth r ates and cell volumes along a salinity gradient and the responses were strain-specific (Hernandez-Saavedra et al. 1995 ).Ecophysiolog ical and morpholog ical tr ait data cov ering a wider range of taxa and environmental conditions are needed to explore possible size-scaling relationships in marine yeasts and mor e br oadl y ho w y east tr aits r elate to their ecological function within marine plankton communities.
Cultur e-based a ppr oac hes r e v eal that marine-deriv ed yeast gr owth r esponds to abiotic envir onmental par ameters suc h as temper atur e, salinity, and pH (Norkr ans 1966, Hernandez-Saav edr a et al. 1995, Breyer et al. 2023 ) and that carbon substr ate pr efer ences v ary between marine fungal taxa (Thomas et al. 2022, Breyer et al. 2023 ).These studies have begun to describe the physiolog ical and morpholog ical c har acteristics of marine yeast growth in vitro , but trait-based data remain scarce, particularly responses to nutrient availability and in relation to open ocean ecosystems.To address these gaps in understanding of planktonic marine yeast ecophysiology, this study quantifies the growth of open ocean yeast isolates under variable nutrient conditions in labor atory cultur e. Fiv e str ains isolated fr om the North Atlantic as part of the Marine Biological Association Fungal Culture Collection, were assessed with a phenotype microarray for the ability to use a diversity of nitrogen-containing compounds as the primary nitrogen source.One model strain, Naganishia diffluens MB A_F0213, w as here exposed to nitrogen concentrations spanning se v er al orders of ma gnitude in thr ee envir onmentall y r ele v ant c hemical forms: ammonium, nitr ate, and ur ea.Observ ations of cell density, gr owth substr ate uptake, and cell morphology are used to describe N. diffluens MBA_F0213 responses to nitrogen form and concentr ation.Meta-anal ysis of the r efer ence DNA barcode in public databases is used to explore the marine biogeogr a phic distribution of the genus Naganishia , to contextualize the nitr ogen dynamics observ ed in vitro with N. diffluens MBA_F0213 cultures.

Cultures and growth media
Five yeast and yeast-like cultur es wer e selected from the Marine Biological Association Fungal Culture Collection (Table 1 ).All str ains wer e isolated fr om the North Atlantic Ocean at the Porcupine Abyssal Plain Sustained Observatory (PAP-SO) ( Supplementary material ) in June 2019.Cultur es wer e maintained on Wickerham's yeast malt medium (WYM) or potato dextrose medium (PDM) agar plates at either 10 • C or 15 • C depending on the isolation depth (Table 1 ).
For all experiments, liquid cultures were prepared in seawater f/2 medium (Guillard 1975 ) with minor modifications as follo ws.Glucose w as added to filter ed natur al seawater at 1.8 g l −1 and autoclaved.Sodium phosphate (NaH 2 PO 4 •H 2 O), trace metals, and vitamin solutions were filter-sterilized (0.22 μm) and added to the autocla ved sea water at concentrations in line with the standard f/2 protocol (Guillard 1975 ).Chloramphenicol was added at 0.2 g l −1 .The form and concentration of nitr ogen substr ates added varied depending on experimental treatment (see below).As natural seawater forms the base for this growth medium, background dissolv ed nutrient concentr ations depend on the envir onmental source .Autocla v ed filter ed natur al seawater as used here was shown to contain ∼3.4 μmol l −1 nitrate and ∼0.9 μmol l −1 ammonium ( Supplementary material ).Urea concentration could not be determined in this anal ysis.An y nitr ogen substr ate additions to the f/2 medium are therefore considered nitrogen enrichments to these bac kgr ound concentr ations.

Di v ersity of nitrogen substr a te use
The diversity of nitrogen substrate use was screened using 96well culture plates containing 95 different nitrogen sources and a negativ e contr ol (BIOLOG PM3B Phenotype Micr oArr a y).T his commerciall y pr oduced assay is designed to test for microbial utiliza-T able 1. Y east strains used in this study.Marine Biological Association Fungal Culture Collection strain codes with species identification, water depth of isolation, agar media formulation of isolation/maintenance, and temperature of culture maintenance.

Isolation depth (m) Media
Temper a ture ( tion of a diverse array of nitrogen sources, including ammonia, nitrate and urea, as well as various amino acids, amines and nucleobases (Fig. 1 A).Nitr ogen enric hments ar e between 1 and 5 mM, except amino acids , purines , and pyrimidines , whic h ar e included at 20-200 μM (BIOLOG, personal communication).Late exponential phase cultures w ere w ashed and resuspended in modified f/2 medium (see above) with no nitr ogen enric hment.Washed cell suspensions were diluted to ∼10 4 cells ml −1 , added at 100 μl per well and incubated at 15 • C for 4 da ys .Triplicate plates were inoculated for each strain.Optical density at 600 nm (OD 600 ) was measur ed dail y using a micr oplate r eader (CLARIOstar) and used to calculate growth rates.Relative growth rate is the growth rate on eac h substr ate normalized by the maxim um gr owth r ate of that str ain on an y substr ate as measur ed in the BIOLOG micr oarr ay ( Supplementary material ).A substr ate div ersity index was used to quantify the range of substrates used by each strain based on the number of substrates yielding positive growth rates (Thomas et al. 2022 ) ( Supplementary material ).

Nitrogen concentr a tion r ange
Gr owth r esponses of N. diffluens MBA_F0213 to ammonium ((NH 4 ) 2 SO 4 ), nitrate (NaNO 3 ), or urea (CO(NH 2 ) 2 ) were assessed in batc h cultur e experiments with initial media enrichments of 0.1, 1, 10, 100, 1000, or 9000 μmol l −1 N. The dissolved carbon source of 1.8 g l −1 glucose corresponds to 10 mmol l −1 glucose or 60 mmol l −1 carbon.The uppermost N addition of 9 mmol l −1 was deriv ed fr om this carbon concentr ation using a C:N r atio of ∼108:16 r epr esenting the av er a ge composition of marine or ganic matter (Redfield 1934 ).The lo w est initial N concentration tested here (with 0.1 μmol l −1 added N) is ∼4.6 μmol l −1 N, accounting for dissolv ed inor ganic nitr ogen pr esent in the natur al seawater ( Supplementary material ).The lo w er [N] r ange, ther efor e encompasses nitrogen concentrations relevant in the open ocean e.g.surface [NO 3 − ] < 10 μmol l −1 at PAP-SO (Hartman et al. 2015 ).
Liquid batch cultures of 40 ml in vented polystyrene flasks (Sarstedt T-25) were incubated at 15 • C, orbitally shaken at 100 r/m and lit by a 14:10 h light-dark cycle.Naganishia diffluens MBA_F0213 cells were acclimated to each treatment condition for at least three subcultures (each ∼5 days) prior to experiments.Fr esh gr owth media were inoculated in triplicate with lateexponential phase cultures to a cell density of ∼10 4 cells ml −1 to initiate experiments.Experiments were terminated when incubations r eac hed stationary phase, indicated by no significant difference in cell density between consecutive daily time points.
Cell density was monitored with daily cell counts on an Impr ov ed Neubauer hemocytometer (Hawksley) and Leica DM1000 LED micr oscope.Specific gr owth r ate v alues wer e obtained in triplicate for each treatment.A modified logistic model was fit to logtransformed cell density data (Zwietering et al. 1990 ) or where the data fit the model poorly, estimated as the maximum gradient between individual time points in the exponential phase ( Supplementary material ).A growth rate value of 0 was assigned when there was no significant difference in means ( n = 3) between the final and initial cell densities.
Media subsamples of 1 ml were taken daily, centrifuged at 16 000 × g for 5 min and the supernatant stored at −20 • C. dglucose concentr ations wer e determined using a glucose oxidase assa y (In vitrogen Amplex Red A222189).Glucose uptake was calculated as the difference between the final and initial media glucose concentrations.
At each time point, at least 10 cells per sample were imaged using Leica LAS EZ softwar e. Cell mor phometry was quantified using ima ge anal ysis softwar e (Ima geJ).Ellipses wer e manuall y fit to 10 cells per sample, outputting mean measurements of the major and minor elliptical axes.Cell dimensions were used to estimate cell eccentricity [a measure of ellipsoidal shape elongation i.e. how m uc h the cell de viates fr om being circular, which could have a role in the efficiency of nutrient uptake (Yan et al. 2021 )] and cell surface area to volume ratio (equations in Supplementary material ).

Naganishia DNA barcode meta-analysis
Strain MB A_F0213 w as pr e viousl y identified as the basidiomycete yeast N. diffluens (order Filobasidiales ) by DNA sequencing of the ITS region.According to an integrated phylogenetic classification (Liu et al. 2015 ), all type strains of species within the Filobasidiales were queried against the NCBI nucleotide database (Sayers et al. 2022 ) to identify all available 18S rRNA encoding gene r efer ence sequences.A phylogenetic tree was generated by pairwise 18S sequence alignments using the NCBI BLAST Tr ee Vie w tool (Altsc hul et al. 1990 ). AliVie w softwar e was used to gener ate a MAFFT alignment of the r efer ence sequences, fr om whic h the 18S rRNA gene hypervariable V9 regions were identified ( Supplementary material ).The V9 regions were compared to assess the taxonomic resolution of potential metabarcode sequences from the Filobasidiales in the Tara Oceans eukaryote 18S V9 database (De Vargas et al. 2015 ).The 18S rRNA gene r efer ence sequence NG_062944.1 from N. diffluens CBS 160 type material was queried against the Tara Oceans database using the Ocean Barcode Atlas web portal (Vernette et al. 2021 ).Operational taxonomic units (OTUs) with > 97% identity to r efer ence sequence NG_062944.1 were selected to represent Naganishia at the genus level.The marine geographic distribution of Naganishia OTUs was analysed using the Ocean Barcode Atlas.Relative abundance data represent OTU counts as a fraction of total metabarcode reads in each sample.

Sta tistical anal yses and da ta av ailability
Gro wth data w ere processed and visualized using Gr a phP ad Prism 9.5.1.DNA sequences were aligned and visualized using AliVie w.Statistical anal yses wer e carried out in Minitab 20.3.The Kruskal-Wallis test was used to test for differences between treatments with significance reported at P < .05unless otherwise stated.Gr a ph err or bars r epr esent the standard de viation.All raw data used in this study are available as a spreadsheet file ( Supplementary material ).

Nitrogen substr a te use di v ersity
The five marine yeast and yeast-like fungal isolates can grow using a range of nitrogen-containing compounds as the primary nitrogen source (Fig. 1 A).All strains grew with enriched ammonia and urea, but only N. diffluens MBA_F0213, Aureobasidium pullulans MBA_F0181 and Sporobolomyces roseus MBA_F0295 gr e w with enric hed nitr ate (Fig. 1 B).Ther e wer e str ain-specific differ ences in nitr ogen substr ate use div ersity ( P = .047)(Fig. 1 C).Naganishia diffluens MBA_F0213 gr e w on the broadest diversity of nitrogen substrates, yielding the highest substrate diversity index (2.96± 0.19) of the five strains.

Physiological responses of N. diffluens to nitrogen substr a te and concentr a tion
The final cell density (i.e.yield) of N. diffluens MBA_F0213 increased with increasing nitrogen enrichment between 0.1 and 9000 μmol l −1 for all thr ee nitr ogen substr ates ( P ≤ .007)(Fig. 2 A) and peaked at 9.67 ± 0.64 × 10 6 cells ml −1 (9000 μmol l −1 added ammonium treatment).Below 10 μmol l −1 added N, final cell density w as lo w er on nitrate than on urea ( P = .027),suggesting that N. diffluens MBA_F0213 population size is controlled by nitrogen speciation under low nitrogen conditions.
Specific growth rate increased between 1 and 100 μmol l −1 added nitrate ( P = .004)but was unaffected by the concentration of ammonium or urea (Fig. 2 B).Growth rates w ere belo w detection at 0.1-1 μmol l −1 added nitrate (no significant difference between final and initial cell densities).At 9000 μmol l −1 added N, specific gr owth r ate was higher on ammonium (3.02 ± 0.13 d −1 ) than on nitrate (1.92 ± 0.02 d −1 ) ( P = .007),suggesting N. diffluens MB A_F0213 gro ws mor e efficientl y on r educed inor ganic nitr ogen than on oxidized inorganic nitrogen at high concentrations.
Cell surface area to volume ratio decreased with increasing nitr ogen enric hment between 1 and 100 μmol l −1 nitrate ( P < .001)and 1-100 μmol l −1 ammonium ( P < .001),but increased slightly between 1 and 100 μmol l −1 added urea ( P = .004)(Fig. 2 F).The minimum cell surface area to volume ratio of 1.31 ± 0.06 μm −1 , observed in the 1000 μmol l −1 added ammonium treatment, was lo w er than for nitrate or urea at the same concentration ( P < .001).At 0.1-10 μmol l −1 added N, cell surface area to volume was higher on nitrate than on ammonium or urea ( P ≤ .003),indicating that the changes in cell length and shape under low nitrate enrichment (cells became smaller and more elongated) had the combined result of increasing cell surface area to volume.In combination with the physiological data (i.e.no growth detected below 10 μmol l −1 added nitrate), this suggests a morphological stress response to nitrogen limitation in N. diffluens MBA_F0213.
Gr owth r ate, cell length and cell surface area to volume are all affected by nitrogen enrichment in the form of nitrate, which could be explained by allometric relationships between physiological processes and cell size in N. diffluens MB A_F0213, whereb y decreasing size would be expected to increase nutrient affinity.In contrast with nitrate, urea enrichment does not affect growth rate, nor does it result in decreasing cell surface area to volume, despite increasing cell length.The changes in cell shape but lack of gr owth r ate r esponse to ur ea or ammonium enric hment suggest that N. diffluens MBA_F0213 adapts cell morphology while maintaining physiological rates across variable nutrient conditions in some cases.

Distribution of Naganishia in the global ocean
Ther e ar e 19 species within order Filobasidiales with a type strain 18S rRNA encoding gene r efer ence sequence av ailable on NCBI, including five from the genus Naganishia (Fig. 3 A).Using the V9 region of the 18S rRNA gene type strain reference sequence alone, N. diffluens is not distinguishable from two other Naganishia species ( N .adeliensis and N .uzbekistanensis ) but differs from all other Filobasidiales tested (Fig. 3 B).Querying the Tara Oceans eukaryote 18S V9 metabarcode database yielded five OTUs with > 97% (genus le v el) identity to the N. diffluens r efer ence sequence, totalling 15 659 reads (Fig. 4 , inset).Pooled relative abundances of the five selected OTUs demonstrate that genus Naganishia is widely distributed in the global ocean, including at open ocean locations (Fig. 4 ).

Discussion
Here we have shown that marine yeasts can use a broad diversity of nitrogen substrates, that varies between strains, possibly due to differing enzymatic capabilities in different species.It is important to r eiter ate that the concentrations used in the BIOLOG assay (Fig. 1 ) are higher than observed in the open ocean and, therefore we are not considering any concentration dependent factors in this part of our study.Some of the nitrogen substrates in the BI-OLOG assay could also have potential toxic effects on some yeasts, such as nitrite (Kubisi et al. 1996 ).Yeasts use membrane transporters (permeases) to take up nitrogenous compounds and additional enzymes to conv ert differ ent molecules into a form that is r eadil y assimilated, commonl y ammonium (Villers et al. 2017 ).All marine yeasts in this study were able to grow with ammonium or urea as the primary nitr ogen source.Although ur ea (CO(NH 2 ) 2 ) is a reduced form of nitrogen, cells must catalyse the hydrolytic cleav a ge of ur ea to r elease ammonium.Some ascomycete yeasts and unicellular green algae use ATP-dependent urea amidolyase to degr ade ur ea, while other fungi, microalgae, and bacteria use nic kel-containing ur eases (Bekheet andSyr ett 1977 , Nav ar athna et al. 2010 ).The only ascom ycete yeast-lik e fungus in this study, A. pullulans MBA_F0181, has been shown to produce urease (Federici 1982 ), while the four other strains are basidiomycetes, which univ ersall y possess genes encoding urease and an associated nickel transporter (Zhang et al. 2009, Navarathna et al. 2010 ).Marine yeast assimilation of urea using urease would impose a nickel dependency, whic h could potentiall y become nic kel-nitr ogen colimitation, as demonstrated in cultures of the marine diatom Thalassiosira weissflogii (Price and Morel 1991 ) and marine c y anobacterium Synechococcus (Dupont et al. 2008 ), as well as natural phytoplankton communities in offshore Pacific surface waters (Dupont et al. 2010 ).Urea-utilizing marine yeasts may therefore contribute to the biological drawdown of nic kel, whic h r esults in c har acteristic n utrient de pth profiles of marine nic kel concentr ations (Sclater et al. 1976 ).
To convert nitrate to ammonium, some yeasts use nitrate and nitrite reductases (Siverio 2002 ), with an analogous pathway in photosynthetic eukaryotes (Sanz-Luque et al. 2015 ).Here, we found that marine isolates N. diffluens MBA_F0213, A. pullulans MBA_F0181, and S. roseus MBA_F0295 are able to grow with nitrate as the primary nitrogen source, in agreement with previous studies on nonmarine isolates of these species (Ali and Hipkin 1985, Zheng et al. 2008, Fotedar et al. 2018 ).Rhodotorula mucilaginosa MBA_F0294 and Holtermanniella festucosa MBA_F0175 did not grow with enriched nitrate, in agreement with evidence for an incomplete nitrate assimilation pathway in R. mucilaginosa (Sen et al. 2019 ) and the absence of nitrate assimilation throughout genus Holtermanniella (Wuczkowski et al. 2011 ).Sporobolomyces and Rhodotorula (phylum Basidiom ycota, subdi vision Pucciniom ycotina) were both identified by 18S rRNA gene tag sequencing  during a coastal spring phytoplankton bloom, with cell counts and biomass of Pucciniomycotina significantl y positiv el y corr elating with nitrate concentration (Priest et al. 2021 ), suggesting related marine y east gro wth r esponds to nitr ate av ailability in pela gic marine en vironments .
Ammonium is the ener geticall y favour ed nitr ogen source within cells given its direct synthesis into glutamate and subsequentl y macr omolecules (Sanz-Luque et al. 2015, Villers et al. 2017 ), but dissolved ammonium is typically a scarce nutrient in marine surface waters due to r a pid micr obial assimilation (Glibert and Goldman 1981 ).The ability to use nitrate could give some yeasts a competitive adv anta ge ov er micr obes r eliant on r educed nitr ogen, particularl y in nitr ate-ric h marine waters found commonly at depth and sporadically in the surface open ocean due to upwelling or vertical mixing events .T his potential benefit could be amplified where high nitrate coincides with an abundance of bioav ailable or ganic carbon, suc h as the biogeoc hemicall y important phytoplankton-derived polysaccharide laminarin (Becker et al. 2020 ), which fungal plankton including basidiomycete yeasts assimilate in coastal ecosystems (Cunliffe et al. 2017 ).Initial evidence for nitrate-supported marine yeast growth comes from Helgoland Roads in the German Bight, where peak yeast abundance (3.3 × 10 5 cells l −1 ) coincided with r elativ el y high nitrate (23.02 μmol l −1 ) compared to ammonia (1.26 μmol l −1 ) during a spring phytoplankton bloom dominated by pennate diatoms (Priest et al. 2021 ).At concentrations below 10 μmol l −1 nitrate [0.1 and 1 μmol l −1 nitr ate enric hments equate to media concentrations of ∼3.5 μmol l −1 and ∼4.4 μmol l −1 nitr ate, r espectiv el y ( Supplementary material )], which are typical in the North Atlantic Ocean (Hartman et al. 2015 ), the lack of detectable population growth in N. diffluens MBA_F0213 suggests a possible surviv al tr ade-off between cellular ener gy inv estments in nitr ate uptake versus reproduction by cell division.
It should be noted that the upper range of ammonium, nitrate and ur ea concentr ations explor ed in the N. diffluens MBA_F0213 culture experiments (100-9000 μmol l −1 N) are unrealistic in the marine environmental context.Also, because we used natural seawater containing ∼3.4 μmol l −1 nitrate and ∼0.9 μmol l −1 ammonium, nitr ogen substr ate additions ar e enric hments.Acr oss the euphotic Atlantic Ocean, peak concentrations of 5.2 μmol l −1 ammonium, 33 μmol l −1 nitrate, and 1.72 μmol l −1 ur ea hav e been observed (Rees et al. 2006, Painter et al. 2008 ).The lo w est media glucose concentration across the experiment was still > 1 mmol l −1 , corresponding to > 6 mmol l −1 DOC, in excess of typical envir onmental DOC concentr ations.Annual mean DOC in the Western English Channel off Plymouth is ∼0.07 μmol l −1 (Hochman et al. 1995 ), which is ∼10 5 times lo w er than the most glucosedepleted media at the end of this experiment.Despite low av er a ge concentrations , nutrient a vailability in the marine water column is heterogeneous at the microscale (Stocker 2012 ), with nitrogen substr ate concentr ations sometimes orders of magnitude higher around marine aggregates (Shanks and Trent 1979 ).T herefore , the responses of N. diffluens MBA_F0213 to high nitrogen could bear some r ele v ance to marine yeast ecophysiology at the micr oscale.
Pela gic osmotr ophs face a tr ait tr ade-off between nutrient uptake and predator defence, with smaller cells having a competiti ve ad vantage in diffusi ve n utrient uptak e , while larger cells a void predation by small, abundant, and fast-growing zooplankton (e.g.Ace v edo-Tr ejos et al. 2015 ).The smaller cell sizes of N. diffluens MB A_F0213 under lo w nitr ogen enric hment could be explained by size-de pendent n utrient acquisition in marine yeasts.Applying allometric parameters empirically determined in phytoplankton by Edw ar ds et al. ( 2012), a 40% decrease in cell length (as observed in N.diffluens MBA_F0213) would increase biomass-scaled nitrogen affinity 2.6-fold.Under high nutrient conditions, larger cell sizes could reduce grazing pressure in pelagic ecosystems (Lürling 2021 ).Larger marine yeasts may also have greater capacity for intr acellular stor a ge of nonlimiting r esour ces, as has been sho wn in heter otr ophic bacteria, with excess glucose causing carbonrich cell inclusions (Thingstad et al. 2005 ).Div erse micr obial eukaryotes are capable of intracellular nitrogen storage (Kamp et al. 2015 ) including yeasts, which sustain growth using internally accum ulated nitr ogen when external nitr ogen is depleted (Gutiérr ez et al. 2016 ).Incr eased internal stor a ge of glucose or nitrogen could explain why N. diffluens MBA_F0213 increases cell size with incr easing nitr ogen concentr ations but this would r equir e v alidation through macromolecular analysis.
Cell surface area to volume ratio (a product of cell size and shape), affects phytoplankton nutrient uptake potential and sinking (Lewis Jr 1976 ).Yeasts have low surface area to volume ratios r elativ e to filamentous fungi, reducing contact with the extracellular environment and potentially affecting yeast nutrient uptake and sedimentation rates in aquatic ecosystems (El Baidouri et al. 2021 ).By increasing cell surface area to volume under low nitrogen availability, N. diffluens MBA_F0213 could theor eticall y ac hie v e more efficient dissolved n utrient uptak e per unit cell volume, as described in phytoplankton ecology (Edw ar ds et al. 2012 ).Again, it is important to state the higher nitrate concentrations used in this study when the surface area-to-volume ratio stops changing (i.e.> 100 μmol l −1 ) are unrealistic in the marine environmental context.There was no response in the surface area-to-volume ratio of N. diffluens MBA_F0213 under increasing ammonium and urea concentrations.It is possible that neither of these two reduced dissolv ed nitr ogen species ar e common sources of nutrient for the yeast in the open ocean, perhaps because other plankton such as bacteria are better adapted for ammonium and urea assimilation.Future studies could consider nutrient uptake competition experiments between marine yeasts and bacteria.
According to a size-based classification of all pelagic marine life (Andersen et al. 2016 ), at ∼3-6 μm cell length, N. diffluens MBA_F0213 falls within the c har acteristic size r ange of unicellular phototrophs.Considering trophic strategies, marine yeasts are mor e closel y compar able to osmo-heter otr ophic bacteria, whic h also use organic carbon but are typically an order of magnitude smaller in size (Andersen et al. 2016 ).The common trophic strategies and distinct cell sizes of marine yeasts and heter otr ophic bacteria could have important implications for understanding the fate of organic carbon in marine ecosystems.If marine yeasts are trul y functionall y distinct fr om other plankton, the y lik ely mediate flows of energy and nutrients not currently represented in ecolog ical models, g iv en that cell size and tr ophic mode ar e k e y traits used to define plankton functional groups (Barton et al. 2013 ).
Based on Tara Oceans sampling locations, the yeast genus Naganishia has a wide marine distribution, suggesting species within this genus are adapted for survival in a broad spectrum of oceanogr a phic conditions .T his evidence adds weight to the idea that the mor phology, r epr oduction, and dispersal strategies of unicellular budding yeasts make them well suited to a planktonic mode of life (El Baidouri et al. 2021 ).Sampling of Naganishia -assigned OTUs at low-latitude open ocean locations where surface nitrogen concentr ations commonl y limit micr obial pr oductivity (Moor e et al. 2013 ), suggests Naganishia species encounter low nitrogen availability in natura , but OTU counts alone do not allow the environmental trait variability of this genus to be explored.Given its isolation from the surface North Atlantic, the ada ptiv e physiological and morphological responses of N. diffluens MBA_F0213 to low nitr ogen av ailability in vitro pr ovide insight into the possible mechanisms of yeast survival in surface ocean nutrient conditions.We postulate that marine yeast tr aits, and ther efor e ecological functions, vary across environmental gradients such as dissolv ed nitr ogen av ailability in the open ocean.
To de v elop a functional understanding of marine yeast ecology, futur e r esearc h will need to incor por ate observ ations of cell physiology and morphology in natura , to complement cultureand DNA-based a ppr oac hes.Quantification of yeast population/biomass dynamics in open ocean en vironments , as previousl y demonstr ated for a coastal ecosystem (Priest et al. 2021 ), would impr ov e the scalability of yeast tr ait observ ations to pela gic ecosystems more broadly.We advocate for the application of traitbased a ppr oac hes to c har acterizing marine-occurring yeasts and marine fungi in general, as a way to unify principles from fungal and plankton ecology and to advance our understanding of the structure and function of pelagic ecosystems.

F
igure 1. (A) Gro wth r ates of fiv e marine y east and y east-like fungal str ains on 95 nitr ogen sources, normalized to the maxim um gr owth r ate ac hie v ed by each strain.Colour indicates relative growth rate.White space indicates no significant growth above the non-nitrogen-enriched f/2 medium control.Nitr ogen enric hments ar e between 1 and 5 mM, except amino acids, purines, and pyrimidines, whic h ar e included at 20-200 μM (BIOLOG, personal comm unication).(B) Gr owth r ates with enric hment of ammonia, nitr ate, or ur ea (expanded fr om A). (C) Substr ate div ersity index based on the number of nitrogen substrates yielding positive growth rates.Plotted values and labels indicate the mean ( n = 3).Error bars indicate the standard deviation.

Figure 2 .
Figure 2. Physiological and morphological responses of N. diffluens MBA_F0213 to nitrogen enrichment in three different forms: ammonium, nitrate and urea.(A) Cell density at the final time point.(B) Specific growth rate based on cell density measurements.(C) Total glucose uptake from culture media.(D) Cell size (major elliptical axis).(E) Cell shape (elliptical eccentricity).(F) Estimated cell surface area to volume ratio based on measurements of major and minor elliptical axes.Morphological data (D)-(F) include measurements from all time points before stationary phase (5-9 days).Plotted values indicate the mean ( n = 3 for A-C, n ≥15 for D-F).Error bars indicate standard deviation.

Figure 3 .
Figure 3. (A) Phylogenetic tree of the 19 species within order Filobasidiales, which have a type strain 18S rRNA encoding gene reference sequence available on NCBI.Tree generated using pairwise 18S sequence alignments on BLAST Tree View tool, NCBI.(B) Visualization of partial V9 regions of MAFFT aligned 18S rRNA encoding gene r efer ence sequences of Filobasidiales.

Figure 4 .
Figure 4. Distribution of Naganishia operational taxonomic units (OTUs) in the global ocean.OTUs from the Tara Oceans eukaryote 18S V9 database selected due to > 97% (genus le v el) similarity to N. diffluens (inset table).Circle area is proportional to relative OTU abundance, i.e. pooled counts of the five selected O TUs , as a fraction of total metabarcode reads.Crosses mark sampling sites.Plotted using Ocean Barcode Atlas web tool.